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11857588 | V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Extracts obtained from the genusHippophaeor other plant sources or compositions produced by chemical or molecular biological techniques each having in common certain moieties in amounts effective when combined with reproductive cells to reduce the loss of function (collectively referred to as “extracts”). Definitions. The term “plant source” means a plant or plants from one or more plant family, genus or species, individually or in combination, from which an extract as defined herein can be obtained or produced including without limitationHippophae(sea buckthorn),Vitis(grape),Prunus Padus(chokecherry or chokeberry),Punica(pomegranate),Vaccinium(blueberries),Prunus(plum)Prunus(cherry),Rubus(raspberry or blackberry),Euterpe(acai),Glycine(soybeans),Lycium(gogi),Carya(pecan),Rosacea(strawberry),Litchi(lychee),Amelanchier(saskatoon), orOlea(Olive). The term “Hippophae” refers to the genus of plants which, without limiting the plant members of the genus, includes the speciesrhamnoides, salicifolia, tibetana, gvantsensisandneurocarpaand plants identified by the common names Sea Buckthorn, Sandorn, Espino Falso, Oblebicha, Olivella Spinosa, Sallow Thorn, or Duindoorn. The term “extract or extracts” refers to any moiety or moieties isolated from a plant source or a combination of plant source(s) of one or more family, genus or species including without limitation a portion of a plant such as the leaves, fruit, or seeds (for example a plant product ofHippophae) whether from one member of the genus or a combination of members of the genus, regardless as to whether such moiety or moieties are isolated singularly, or in multiple simultaneous fashion, or is a product of combining such moiety or moieties, or produced by molecular biology techniques or chemical synthesis techniques, and specifically includes, without limiting the forgoing, Extracts 1-9, as described below, or any combination of such Extracts as compositional equivalents of such Extracts individually or in various combinations or as compositions prepared by molecular biology techniques or chemical synthesis techniques which contain moieties or combinations of such moieties identified within such extracts of plant origin as being effective in reducing loss of function of reproductive cells in vitro as described herein and without limitation includes compositions which comprise at least an amount of antioxidant activity of at least 90 micromolar trolox equivalent per gram, an amount of fatty acids of at least 3 percent (such as palmitic acid, linoleic acid, stearic acid and mixtures thereof), an amount of polyphenolic compounds of at least 275 mg gallic acid equivalents per 100 grams, or as otherwise specifically described herein. Extracts can further include tocopherols of about 60 mg/100 g and can further include and polar lipids of about 10% to about 15%. The term “reproductive cell” refers to one or a plurality of oocytes, sperm cells, embryos, embryonic stem cells, or any other cells or cell lines, obtained from any species of animal useful for reproductive purposes including without limitation artificial insemination, in vitro fertilization, intra cytoplasmic sperm injection, or cloning and without limiting the forgoing encompasses mammalian reproductive cells obtained without limitation from bovine, equine, ovine, or porcine species or breed, or avian reproductive cells obtained without limitation from chickens, ducks, geese, turkeys, pheasants, and quail. The term “diluent” refers to any solution that comes in contact with a reproductive cell such as buffered solutions which contain one or more of sodium citrate, Tris[hydroxymethyl]aminomethane, TES (N-Tris [Hydroxymethyl]methyl-2-aminoethanesulfonic acid), monosodium glutamate, HEPES; medium such as HEPES buffered medium, HEPES buffered bovine gamete medium and particularly HBGM3 as described by J. J. Parish “Capacitation of Bovine Sperm By Heparin” 38 Biology of Reproduction 1171 (1988); cell culture media; extenders such as E-Z Mixin, E-Z Freezin-MFRS or E-Z Freezin-LE (Animal Reproduction Systems, 14395 Ramona Avenue, Chino, Calif.); TALP or Fert-TALP or egg-yolk citrate which contain Calcium chloride, potassium chloride, magnesium chloride, sodium phosphate, lactic acid, sodium pyruvate and which can further contain cryoprotectants such as glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol; other organic substances such as egg yolk, an egg yolk extract, milk, a milk extract, casein, albumin, lecithin, bovine serum albumin, cholesterol; sugars such as the monosacharides, glucose, fructose, or mannose; detergents such as sodium dodecyl sulfate; antioxidants such butylated hydroxytoluene; capacitation facilitators such as alpha amylase, beta amylase, or beta glucuronidase; antibiotics such as tylosin, gentamicin, lincomycin, spectinomycin, linco-spectin (a combination of lincomycin and spectinomycin), penicillin, streptomycin, and ticarcillin; flow cytometer sheath fluids such as 98.6 mM sodium citrate dihydrate or 197 mM Tris(hydroxymethyl) aminomethane, 55.4 mM citric acid monohydrate and 47.5 mM fructose each adjusted to pH 6.8, which may be utilized for any purpose such as washing, culturing, handling, or cryopreserving the reproductive cell. For purposes of the present invention, “embryo” refers to the stages of development whereby a conceptus develops into a fetus. The term “combination or combining” refers to any method of putting two or more materials together. Such methods include, but are not limited to, mixing, blending, commingling, concocting, homogenizing, ultrasonic homogenizing, incorporating, intermingling, fusing, joining, shuffling, stirring, coalescing, integrating, confounding, joining, uniting, creating a stable suspension of two immiscible liquids via any number of means such as membrane emulsions, or the like. The term “reduces loss of function” or “reducing the loss of function” refers to inhibiting events or decreasing the likelihood of events in vitro which damage, impair, diminish, or decrease the in vivo cellular characteristics of a reproductive cell such as events which: increase reproductive cell death, decrease reproductive cell fertility, decrease reproductive cell viability, decrease sperm motility, increase premature acrosome reactions, damage the lipid bilayer, damage cellular DNA, damage mitochondrial DNA, damage cellular organelles, initiate or result in apoptosis, initiate or result in necrotic cells, generate reactive oxygen species (ROS), react reactive oxygen species with the reproductive cell, or the like, thereby, the in vivo cellular characteristics, or the cellular characteristics of newly collected or harvested reproductive cells, may be retained or retained to a greater degree in vitro as assessed by one or more assays when treated with extracts as compared to control reproductive cells (not treated with extracts), and can maintain such a state over an extended period of time. Measures of “in vivo cellular characteristics” may include, for example, motility, capacitation, cell leakage, DNA fragmentation, necrosis, apoptosis, or the like. The term “emulsifier” refers to a substance having the capacity to maintain a dispersion or suspension of two immiscible substances such as oil in water, and specifically includes, without limitation, egg yolk, orvus paste, lecithin, cyclodextrin, or the like. The term “membrane integrity” refers to the ability of an intact membrane to exclude propidium iodide from being incorporated by a reproductive cell including without limitation sperm cells (43). Incorporation of propidium iodide by a reproductive cell correlates with a loss of membrane integrity of the cell in which the membrane is no longer intact. The loss of membrane integrity correlates with a loss or reduced function of the reproductive cell. The term “sperm cell or sperm cells (43)” generally refers to sperm cells (43) from any genus or species of animal including without limitation avian sperm cells of chickens, ducks, geese, turkeys, pheasants, quail or the like, and mammalian sperm cells of bovids, ovids, porcine, canids, felids, whales, porpoises, or the like, unless otherwise indicated. For the purposes of the present invention, ranges may be expressed herein as from “about” one particular value to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Moreover, for the purposes of the present invention, the term “a” or “an” entity refers to one or more of that entity; for example, “a protein” or “an peptide” refers to one or more of those compounds or at least one compound. As such, the terms “a” or “an”, “one or more” and “at least one” can be used interchangeably herein. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including combinations of two or more of the compounds. According to the present invention, an isolated or biologically pure bioactive agent is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis. Preparation of Extracts. Now referring primarily toFIG.1, a variety of extracts can be obtained from plant sources, as described herein. The preparation of extracts from plant sources is illustrated by the genusHippophae(1) useful in reducing the loss of function in reproductive cells. Pulp oil extract (including the non-limiting example of Extract 1) (2) can be prepared from the pulp and skin (3) of the fruit (or berries) (4) obtained from one or more species ofHippophae(1). The fruit (4) can be broken up, cut in pieces, or otherwise reduced as necessary (hereinafter “comminuted”(16) to allow separation of the juice (6) and whole seeds (9) from the fruit (4). Typically, the comminuted fruit (4) is pressed (7) to obtain the juice (6) and a press cake (8) of the pulp and skins (3) (and other solids which may be carried with the pulp and skins) and whole seeds (9). The whole seeds (9) can be separated from the pulp and skins (3). The pulp and skins (3) can be mixed and milled (10) and extracted (11) with hypercritical carbon dioxide (or with other solvents such as hexane) to obtain a pulp oil (including the non-limiting example of Extract 1)(2). Alternately, pulp oil (2) ofHippophaesuitable for use as pulp oil extract (2) in the invention can be obtained from Leinig Wildfrucht-Verrarbeitung, Markische Str./Gewerbegebiet, DE-15806 Dabendorf, Germany. Similarly, the pulp oil (2) of other plant sources can be obtained without undue experimentation. The whole seeds (9) obtained in the above-described method for producing pulp oil extract (2) can be cleaned (12), ground (13) and extracted (14) with hypercritical carbon dioxide (or extracted with other solvents such as hexane) to generate seed oil extract (including the non-limiting example of Extract 2)(15). Alternately, seed oil (15) ofHippophaesuitable for use in the invention can be obtained from Leinig Wildfrucht-Verrarbeitung, Markische Str./Gewerbegebiet, DE-15806 Dabendorf, Germany. The seed oil (15) of other plant sources can be similarly obtained without undue experimentation. A carotenoid-lipoprotein complex extract (including the non-limiting example of Extract 5) (16) can also be derived from the pulp and skin solids (3) of the Hippophe fruit and obtained commercially from SC Proplanta, S.A., Str. Seslului 2, 400372, Cluj-Napoca, Romania, or other commercial sources. The carotenoid-lipoprotein complex (16) of other plant sources can be similarly obtained without undue experimentation. A fruit tincture extract (including the non-limiting example of Extract 6) (17) can further be obtained by centrifugation (18) of the pulp and skins (3) at about 200 rounds per minute (rpm) to generate a lipoproteic fraction (LPF) (19). About five grams of the lipoproteic fraction (LPF) can be combined with about 200 mL of a 1:1 solution of 98 percent (%) ethanol (EtOH):distilled water (20). The mixture can be incubated (21) in the dark for five days at about 25 degrees centigrade (° C.). The use of particular numbers of particles, amounts, volumes, weights, or other measures throughout this description is not intended to limit the scalability of the invention, but rather to provide non-limiting examples of how to make and use particular embodiments of the invention. The mixture can then be filtered (22) to generate an amount of the fruit tincture extract (including the non-limiting example of Extract 6) (23). Alternately, a fruit tincture extract ofHippophaefruit suitable for use in the invention can be obtained from SC Proplanta, S.A., Str. Seslului 2, 400372, Cluj-Napoca, Romania. The fruit tincture (17) of other plant sources can be similarly obtained without undue experimentation. Again referring toFIG.1, a juice extract (including the non-limiting example of Extract 3) (24) of one or more species ofHippophaefruit or berries (4) suitable for use in the invention can be prepared by removing the vegetal portion and other debris from the fruit (4). The fruit (4) can be comminuted (16) sufficiently to allow the removal of the whole seeds (25) from the remaining fruit solids (26). The remaining fruit solids (26) can then be combined with about three volumes of ice cold phosphate buffered saline pH 7.4 (27). The resulting combination can be homogenized at about 0° C. until a homogeneous mixture (28) is obtained. The homogeneous mixture (28) can be transferred to a centrifuge container and centrifuged (29) at 2500×g at about 4° C. for about 10 minutes (min). The supernatant (including the non-limiting example of Extract 3) (24) can be utilized directly in embodiments of the invention or can be stored at about −70° C. The centrifugation pellet obtained as above-described containing pulp and skin solids (along with other solids carried with the pulp and skin solids) can be utilized in various embodiments of the invention as berry sediment extract (including the non-limiting example of Extract 4) (25). Alternately, a juice extract ofHippophaefruit suitable for use in the invention can be obtained from Seabuckthorn International Inc., 4154 Ponderosa Drive, Peachland, BC VOH 1X5. The juice extract (24) of other plant sources can be similarly obtained without undue experimentation. The leaves of plants of the genusHippophae(“leaves”)(26) can be separated from the stems and other debris. Fifty grams of the leaves (26) can be comminuted (27) and combined with 500 mL of a 1:2 solution of 98% EtOH:distilled water (28). The mixture can be incubated (29) in the dark for five days at about 25° C. The mixture can be filtered (30) to provide a leaf tincture (including the non-limiting example of Extract 7) (31). Alternately, a leaf tincture extract suitable for use in the invention can be obtained from SC Proplanta, S.A., Str. Seslului 2, 400372, Cluj-Napoca, Romania. The leaf tincture (31) of other plant sources can be similarly obtained without undue experimentation. Similarly, fifty grams of the leaves (26) can be comminuted (27) then combined with 670 mL paraffin oil (32). The mixture can be heated (33) for 5 min and incubated (34) in the dark for a period of about five days at about 25° C. The mixture can be filtered (35) to provide a leaf paraffin oil (including the non-limiting example of Extract 8) (36). Alternately, a suitable leaf paraffin oil extract (36) suitable for use in the invention can be obtained from SC Proplanta, S.A., Str. Seslului 2, 400372, Cluj-Napoca, Romania. The leaf paraffin oil (36) of other plant sources can be similarly obtained without undue experimentation. Again, fifty grams of the leaves (26) can be comminuted (27) then combined with about 500 mL 1:1 glycerol:distilled water (37). The mixture can be heated (38) for 5 min and incubated (39) in the dark for a period of about five days at about 25° C. The mixture can be filtered (40) to provide a leaf hydroglycerin extract (including the non-limiting example of Extract 9)(41). Alternately, a suitable leaf hydroglycerin extract (41) for use in the invention can be obtained from SC Proplanta, S.A., Str. Seslului 2, 400372, Cluj-Napoca, Romania. The leaf hydroglycerin extract (41) of other plant sources can be similarly obtained without undue experimentation. General Use Of Extracts. In general the invention encompasses combining an amount of one or more extracts from one or more plant sources with reproductive cells directly or indirectly sufficient to reduce loss of function of one or more in vivo reproductive cell characteristics. Extract(s) can be combined directly with reproductive cells contained in an amount of biological fluid, such as sperm cells contained in an amount of seminal fluid, and which may thereafter be combined with an amount of one or more diluents in a single or a plurality of steps. Alternately, reproductive cells can be combined with one or more diluents and an amount of extract added to the combination of reproductive cells and diluents. Similarly, an amount of one or more extracts can be added to the diluents and the diluents added to reproductive cells. Additionally, an amount of one or more extracts can be transferred to a container or vessel to which diluents or reproductive cells are thereafter transferred. The extracts may in certain instances be transferred to the container or vessel and an amount of the solvent in the extract removed to allow the extract to adhere to the container or vessel wall surface. Additionally, extracts can be utilized in vivo to condition the reproductive tract for artificial insemination or natural insemination, in vivo fertilization of oocytes, embryo implantation, or the like. As to these embodiments of the invention, the extracts can be administered vaginally in the form of inserts, gels, foams, pessaries, suppositories, or the like. In an alternative or complimentary embodiment, extracts may be administered as a cream or gel on the male genitalia. Compositions for vaginal administration may contain gelatinizing agents, lubricating agents or other additives compatible with extracts and the reproductive environment. Extracts can also be utilized on resulting zygotes or embryos during in vitro development or prior to intrauterine transfer. Particular embodiments of the invention include the combination of an effective amount of one or more extracts into any conventional method in which reproductive cells are used. While specific examples of combining reproductive cells with one or more extracts have been described, these particular examples are not intended to be limiting but rather illustrative of the numerous and varied methods by which an effective amount of one or more extracts can be combined with an at least one or a plurality of reproductive cells to reduce loss of function of in vivo characteristics. Collection of Reproductive Cells. As to particular embodiments of the invention, extracts (including without limitation Extracts 1-9) described below, can be combined with collected reproductive cells from a female or a male donor animal. Specifically, with respect to semen collected from cattle or horses via artificial vagina, or manual or electrical manipulation of the penis or otherwise obtained, or avian species by abdominal massage or otherwise obtained, extracts can be combined directly with the seminal plasma of the freshly collected semen or indirectly by combination of the extract with a diluent which can then be combined with the collected semen. An effective amount of the extract can also be combined with sperm cells (43) in which the seminal plasma is lacking or has been removed (whether to the sperm cells (43) directly, after being combined with a diluent, or to the diluent then combined with such sperm cells (43)). The effective amount of extract combined with the sperm cells can vary and be adjusted dependant upon the application, species, volume of ejaculate, or like parameters. In another embodiment of the present invention, extracts can be combined with the diluent into which freshly harvested eggs are transferred. Alternately, freshly harvested eggs can be transferred to the diluent in a petri dish or other container and then one or more extracts thereafter combined with the diluent in the petri dish, or other container into which the egg(s) have been transferred. Similarly, an effective amount of one or more extracts can be added as an ingredient to the media in which oocytes are bathed upon collection. Processing of Reproductive Cells. In another particular embodiment of the invention, extracts can be utilized to reduce loss of function of reproductive cells during the various steps of a process preparing the reproductive cells for use. An amount of one or more extracts can be combined with the reproductive cells in one or more steps of the process in sufficient amount to reduce the loss of function, or otherwise mitigate damage to the reproductive cell, without compromising the integrity of the solution(s) used in the modified procedural step. The term “reduced loss of function” refers to a measurable characteristic of reproductive cells such as motility, membrane integrity, acrosome damage, DNA damage, or the like compared between reproductive cells treated with one or more extracts and reproductive cells which have not been treated with one or more extracts under controlled conditions expressed as a percentage of the respective populations of reproductive cells analyzed. Specifically in regard to processing sperm cells, multi-step processing steps often cause significant damage to sperm, especially in the cases of multi-step processes like flow analysis or flow sorting of sperm cells used to obtain sex selected inseminates used for inseminations to achieve offspring of a specified, or pre-selected sex. During sperm cell sexing processes sperm cells can be exposed to reactive oxygen species (ROS), and extracts can be useful in reducing loss of function of the sperm cells by combination with the initial ejaculate, to extended sperm cells, the sheath fluid used during the analysis and sorting process, to the DNA staining solution, to the collection fluid, to the sperm post-sort and post-centrifugation, in the diluents used for freezing the sperm, or in the vessel used for freezing sperm cells. Extracts utilized in such key steps can substantially reduce the loss of motility of the sex selected inseminates at the time of artificial insemination and can increase the likelihood of pregnancy of the female recipient. In another embodiment of the invention, extracts can be adhered to beads used for separation of sperm cells (43) during centrifugation, gravity sedimentation, or column elution to prevent oxidation during events which expose sperm cells (43) to oxygen. Specific examples include Percoll gradients commonly used to isolate sperm cells (43) from seminal plasma. Extracts can be attached to Percoll, or similar beads using knowledge commonly known in the art. Further processing through gradients such as percoll or a similar column separation can cause damage which may include premature acrosome reactions. Addition of extracts to sperm cells (43) prior to gradient processing can protect them from damage. In another embodiment of the invention, an effective amount of one or more extracts can be combined to previously disclosed sperm cell extenders or diluents. The amount of extract combined or the concentration of extract in the extender or diluent achieved can be adjusted to provide an effective amount of extract depending on the species or subspecies from which the reproductive cells are derived, or the individual animal from which the reproductive cells are collected. Extracts can be combined with the extender or diluent in concentrations ranging from nanogram concentrations to milligram concentrations. Such addition can occur as the extender or diluent is being made, or just prior to use. Examples of such solutions include egg yolk TALP, skim or whole milk extender, or the like. In yet another embodiment of the invention, damage to other attributes of sperm cells (43) can be mitigated by use of Extracts in steps of dilution. Such cellular attributes include the acrosome, the DNA of the cell, the mitochondrial DNA, the mitochondria, the outer membrane and the like. Extracts utilized to reduce damage to these attributes could increase the likelihood of pregnancy and full term development of the embryo. In another embodiment of the invention, extracts can be combined to the media used over a centrifugation gradient, or column, to help limit the amount of oxidants in the solution and thus limit sperm cell exposure to oxidants. Examples of such media include Biggers, Whitten and Whittingham (BWW), and Earle's media. In yet another embodiment of the invention, extracts can be combined with diluents or culture media utilized to incubate oocytes until they mature and can be utilized in diluents or media utilized during fertilization or for freezing. Such culture media include Irvine's media or the like. Extracts can be combined in concentrations sufficient to reduce external ROS, and reduce oxidized lipids in the oocyte membrane, and reduce damage from damaging compounds in the environment, yet low enough to avoid affecting the pH and osmotic potential of the solution. Cooling or Freezing of Reproductive Cells. In a particular embodiment of the present invention, extracts can be utilized to reduce loss of function of reproductive cells during the steps of cooling or freezing of diluents for reproductive cell storage and during the steps of warming the diluents for subsequent use of the reproductive cells in various applications. During extended storage, gamete membranes are can be most susceptible to damage by free radicals, internally or externally generated, ice crystals, bacterial damage, and damage from other substances in the diluents. The assessment of sperm cell motility can provide a model for assessing the effectiveness of extracts obtained from plant sources or chemical synthesis, or molecular biological techniques including without limitation plants of the genusHippophaeto reduce loss of function of reproductive cells, to reduce the loss of in vivo characteristics of reproductive cells, to reduce the loss of in vivo characteristics of newly harvested reproductive cells, and specifically, to reduce the loss of fertility of sperm cells. As to each, the assessment of sperm cell motility can be less costly, less difficult, and less time consuming, than assessment of the effectiveness of extracts based on other measures of reproductive cells such as assessment of membrane damage. Recently, Kirk, et al. “Comparison of In Vitro Laboratory Analyses with the Fertility of Cryopreserved Stallion Spermatozoa”, Theriogenology 64: 1422-1439 (2005) studied the correlation of several inexpensive, rapid assays to the fertility of stallion sperm cells. The best one-variable model for predicting fertility was the percentage motile sperm cells at about 90 minutes post-thaw (r2=0.60)(“90 minute motility”). Interestingly, the best two, and three variable models also included 90 minute motility. The best four variable model included 90 minute motility, as well as 0 minute motility, percentage of live sperm cells and the percentage of live-acrosome intact sperm cells (r2=0.79). Therefore, assessing the effectiveness of extracts ofHippophaeto reduce loss of motility in sperm cells using a 90 minute motility post-thaw assessment, or two or three hours post-warm motility assessment, plus acrosome intactness can provide a measure of the effectiveness of extracts of plants in the genusHippophaeto reduce loss of function of sperm cells, or the loss of fertility of sperm cells, or both, which may be useful in assessing the effectiveness ofHippophaeextracts in reducing the loss of function of other types of reproductive cells. EXAMPLE 1 In a first experiment (data set out to the right of Exp 1 in Tables 1-6), semen was collected from two stallions via artificial vagina. The motility of each ejaculate was determined subjectively via light microscope and the semen was diluted to a final concentration of about 5×108motile sperm cells/ml with E-Z freezin LE (Animal Reproduction Systems, Chino, Calif.) and combined to one each of thirteen 0.25 mL semen samples was an amount of a corresponding one of thirteen adjuvants: (1) no addition-control (“untreated control semen sample); 2) about 5% cyclodextrin; 3) about 2.5% cyclodextrin; 4) about 5% pulp oil in cyclodextrin; 5) about 2.5% pulp oil in cyclodextrin; 6) about 5% seed oil in cyclodextrin; 7) about 2.5% seed oil in cyclodextrin; 8) emulsion of about 5% seed oil, 9) emulsion of about 2.5% seed oil, 10) emulsion of about 5% pulp oil, 11) emulsion of about 2.5% pulp oil, 12) about 5% juice, and 13) about 2.5% juice. Throughout this description the amount of extract is expressed as a percentage volume to volume (v/v) unless otherwise indicated. Following dilution of each semen sample with E-Z freezin LE and combining the diluted semen with the corresponding adjuvant (“treated semen samples”), the treated semen samples were cooled by reducing the temperature at a controlled rate to about 5° C. Treated semen samples were then frozen by conventional procedures on racks in static liquid nitrogen vapor. Frozen treated semen samples were subsequently thawed in a water bath at about 37° C. and estimates of motility were made after incubating samples at about 37° C. for 0 and 2 hours (h) post-thawing. Cyclodextrin proved to be spermicidal in this study, and therefore motility data is not shown for those treatments that contain cyclodextrin alone or in combination with the oils. Now referring to Tables 1-6 below, the results show that in general the treated semen samples containing adjuvants with pulp oil extract, seed oil extract, or juice extract at either 2.5% or 5% v/v (Extracts 1, 2, or 3 (2)(15)(24) at either 2.5% or 5% v/v) exhibited a reduced loss of motility as compared with the untreated control semen sample. EXAMPLE 2 In a second experiment (data set out to the right of Exp 2 in Tables 5-6), semen was collected from one stallion via artificial vagina. The motility of the ejaculate was determined objectively using a Hamilton-Thorn sperm analyzer with a 37° C. stage warmer. The assessed data included percentage of motile sperm cells, progressive motile sperm cells, as well as average path velocity, straight line velocity, and straightness of motile sperm cells in accordance with conventional operating procedures for the instrument. The semen was diluted to a final concentration of about 5×108motile cells/ml with E-Z Mixin CST (Animal Reproduction Systems, Chino, Calif.) and to each of fifteen aliquots of 0.25 mL one each of fifteen adjuvants was combined: (1) no addition-control, 2) about 5% cyclodextrin control, 3) about 2.5% cyclodextrin, 4) about 5% pulp oil in cyclodextrin, 5) about 2.5% pulp oil in cyclodextrin, 6) about 5% seed oil in cyclodextrin, 7) about 2.5% seed oil in cyclodextrin, 8) emulsion of about 5% seed oil, 9) emulsion of about 2.5% seed oil, 10) emulsion of about 5% pulp oil, 11) emulsion of about 2.5% pulp oil, 12) about 5% juice, 13) about 2.5% juice 14) about 5% berry sediment, and 15) about 2.5% berry sediment. Following dilution of each semen sample with E-Z Mixin CST and the corresponding adjuvant, the treated semen samples were cooled by reducing the temperature at a controlled rate to about 4° C. Motility was evaluated subjectively via light microscope immediately at 24, 38, and 72 h after being held at about 4° C. Motility of each treated semen sample was examined again after 2 h at about 37° C. Cyclodextrin again proved to be spermicidal in this study, and therefore motility data is not shown for those treatments. In addition the berry sediment was spermicidal (likely due to pH related issues, not issues inherent to the berry sediment), and therefore motility data is not shown. Now referring to Tables 5 and 6, the results show that the treated semen samples containing adjuvants with juice extract at either 2.5% or 5% v/v (Extract 3 (24) at either 2.5% or 5% v/v) exhibited a reduced loss of motility of sperm cells as compared with the untreated control semen sample. EXAMPLE 3 In a third experiment (data set out to the right of Exp 2 in Tables 1-6) Semen was collected from one stallion via artificial vagina. The motility of the ejaculate was determined subjectively via light microscope prior to cooling. The semen was diluted to a final concentration of about 6×108motile cells/ml with E-Z Freezin LE (Animal Reproduction Systems, Chino, Calif.) containing about 20% egg yolk and about 0.1% orvus paste plus and each of seven 0.25 mL aliquots was combined with a corresponding one each of adjuvant as follows: (1) no addition-control, 2) emulsion of about 5% seed oil, 3) emulsion of about 2.5% seed oil, 4) emulsion of about 5% pulp oil, 5) emulsion of about 2.5% pulp oil, 6) about 5% juice, and 7) about 2.5% juice. Following dilution of each semen sample with E-Z Freezin LE plus the corresponding adjuvant (treated semen samples), the treated semen samples were cooled by reducing the temperature at a controlled rate to about 4° C. Motility was evaluated subjectively via light microscope immediately at about 24, 48, and 72 h post-cooling using the Hamilton-Thorn sperm analyzer five minutes after warming to about 37° C. and again after being held for about 2 hr at about 37° C. Now referring to Tables 1-6 below, the results show that in general the treated semen samples containing adjuvents with pulp oil extract, seed oil extract, or juice extract at either 2.5% or 5% v/v (Extracts 1, 2, or 3 (2)(15)(24) at either 2.5% or 5% v/v) exhibited a reduced loss of motility as compared with the untreated control semen sample. EXAMPLE 4 In a fourth through seventh experiment (the data set out to the right of Exp 4, Exp 5, Exp 6 & Exp 7 in Tables 1-14), semen was collected from two stallions (experiments 6 & 7; one stallion experiments 4 & 5) via artificial vagina. The motility of each ejaculate was determined objectively using a Hamilton-Thorn Sperm Analyzer prior to cooling. The semen was diluted to a final concentration of about 6×108motile cells/ml with E-Z Mixin CST (Animal Reproduction Systems, Chino, Calif.) containing about 20% egg yolk and about 0.1% orvus paste and to each of 15 0.25 mL aliquots was combined one adjuvant as follows: (1) no addition-control; 2) about 5% pulp oil; 3) about 2.5% pulp oil; 4) about 5% seed oil; 5) about 2.5% seed oil; 6) about 5% juice; 7) about 2.5% juice; 8) about 5% fruit alcohol extract; 9) about 2.5% fruit alcohol extract; 10) about 5% leaf alcohol extract; 11) about 2.5% leaf alcohol extract; 12) about 5% leaf glycerine extract; 13) about 2.5% leaf glycerine extract; 14) about 5% leaf paraffin; or 15) about 2.5% leaf paraffin. Following dilution of each semen sample with E-Z Mixin CST and containing the corresponding adjuvant (treated semen samples), the treated semen samples were cooled by reducing the temperature at a controlled rate to about 4° C. Motility was evaluated objectively using a Hamilton-Thorn Sperm Analyzer at about 24, 48 and 72 hours after cooling. Motility was evaluated after being warmed for about 5 minutes to about 37° C., and again after being held for about 120 minutes at about 37° C. For each evaluation a minimum of 500 sperm per treatment were analyzed. For experiment 6 and 7, motility data from two stallions is averaged for each treatment, and each time point. Now referring to Tables 1-14 below, the results show that embodiments of the invention which combine pulp oil extract (2), seed oil extract (15), juice extract (24), fruit tincture extract (23), leaf tincture extract (31), leaf paraffin extract (36), or leaf hydroglycerin extract41(and in particular embodiments of the invention which combine Extracts 1-3 and 6-9) with reproductive cells (and in particular sperm cells) can reduce loss of function (and in particular the loss of post warming motility in sperm cells at about 24, 48 and 72 hours after cooling), when compared to the control samples. With respect to sperm cells, the reduced loss of motility associated with the use of extracts can even be more pronounced after sperm cells are held for 2 hours. While there can be variability with respect to the reduced loss of motility in response to the use of extracts both between stallions as well as within ejaculates from the same stallion, the juice extract (24), leaf tincture extract (31), and fruit tincture extract (23) consistently reduce loss of motility at and between both time points (see Tables 5-8 and 13-14 column labeled ‘difference 2hr-2hr control for a reflection of the difference in motility for the extract treated sperm cells versus the control sperm cells). Only 2.5% leaf paraffin extract (36)(but effective at 5% leaf paraffin extract) does not appear to be effective by itself, for cooled sperm cells but might be effective in combination with other extracts, or for frozen sperm cells. Additionally, the average speed of the motile sperm cells treated with Extracts 1-3 and 6-9 can be increased as compared to the untreated control sperm cell samples, both immediately after warming to about 37° C., and after about 2 hours at room temperature. EXAMPLE 5 The antioxidant potential of Extracts 1-3 and 6-9 was analyzed using a kit by Oxford Biomedical Research, P.O. Box 522, Oxford MI 48371. This colorimetric microplate assay allows comparison of each Extract 1-3 and 6-9 to a standard to determine the total copper reducing equivalents. Generally the assay was performed by preparing the standards, and allowing dilution buffer, copper solution and stop solution to equilibrate to room temperature for about 30 minutes prior to running the assay. Both Extracts 1-3 and 6-9 samples and standards were diluted 1:40 in the provided dilution buffer (e.g. 15 mL serum+585 mL buffer). Next, 200 mL of diluted Extract samples or standards were placed in each well. The plate was read at 490 nanometers (nm) for a reference measurement. Then 50 mL of Cu++ solution was added to each well and incubated about 3 minutes at room temperature. 50 mL of stop solution was added and the plate read a second time at 490 nm. The data in Table 15 demonstrates the antioxidant potential of each of Extracts 1-3 and 6-9 at two different concentrations. The data further explains the effectiveness of extracts against damaging oxidant or ROS events (above discussed) generated during in vitro processing of reproductive cells. EXAMPLE 6 In a sixth experiment, avian semen (42) containing avian sperm cells (44) was obtained from sexually mature roosters using abdominal massage. Avian semen (42) containing avian sperm cells (43) was diluted 1 part avian semen to 2 parts Lake's extender (containing per liter 19.2 g glutamate, 1.28 g potassium citrate, 5 g sodium acetate, 0.6 g magnesium chloride, 10 g fructose adjusted to pH 7.2) admixed with 13.5% glycerol, 20% egg yolk and 0.1% orvus paste (the “control extender”) or the control extender further containing 3%Hippophaepulp oil extract (2) or seed oil extract (14). Pulp oil extract (2) and seed oil extract (14) ofHippophaewere added to the control extender at 3% (v:v) and homogenized using an ultrasonic homogenizer to produce a homogenous solution. An aliquot of diluted avian semen (42) was removed to determine pre-freeze membrane intactness or membrane quality. Diluted avian semen (42) was loaded into 0.5 mL straws, placed in a cooler held at about 4° C. Samples were incubated at about 4° C. for about one hour prior to freezing. Straws containing dilute avian semen (42) were frozen by exposure to liquid nitrogen vapor for about 12 minutes and then plunged into liquid nitrogen. Straws containing frozen diluted avian semen and avian sperm cells (44) were stored in liquid nitrogen until thawed to evaluate post-thaw membrane intactness or membrane quality. Straws containing frozen diluted avian semen (42) were thawed as described in Relationship Between Thirty Post-Thaw Spermatozoal Characteristics and the Field Fertility of 11 High-use Australian Dairy AI Sirs, Phillips et al., Animal Reproduction Science, 81 (2004), hereby incorporated by reference, to remove glycerol from the thawed diluted avian semen (42). In brief, straws containing frozen diluted avian semen (42) were transferred to a water bath at about 4° C. for about 5 minutes. The thawed diluted semen (42) from each straw was then transferred to a corresponding 15 mL tube. About 2 mL of Lake's extender containing 9% glycerol was then added to each tube containing thawed diluted avian semen (42). Lake's extender without glycerol was added to the thawed diluted avian semen (42) in each tube using the following method: 0.1 mL Lake's extender 10 times; 0.2 mL Lake's extender 10 times; 0.5 mL Lake's extender 10 times, 1 mL Lake's extender 10 times each aliquot added at about one minute intervals. The extended thawed avian semen (42) in each tube was centrifuged at about 300×g for about 25 minutes at about 4° C. The supernant was removed and the pellet of avian sperm cells (44) obtained was resuspended in 1 mL Lake's extender. Two microliters of propidium iodine (2.4 mM) were added to the resuspended avian sperm cells (44). The resuspended avian sperm cells (44) were allowed to incubate at room temperature for about 30 minutes. Aliquots of the incubated avian sperm cells (44) were viewed under a fluorescence microscope to determine avian sperm cell membrane integrity. A minimum of 100 avian sperm cells (44) were counted to assess percentage avian sperm cell membrane integrity. Now referring toFIG.2, the effect of treating avian sperm cells (44) with pulp oil extract (2) as above-described is shown. There was no apparent difference as to membrane integrity as between avian sperm cells (44) treated with the control extender and extender containing 3% pulp oil extract (2) on pre-freezing membrane integrity (P>0.10). In both cases, post-thaw membrane integrity was lower as compared to pre-freezing membrane integrity (P<0.05) but the addition of pulp oil extract (2) significantly improved (P<0.05) post-thaw membrane integrity of avian sperm cells (44). Therefore, the treatment of avian sperm cells (44) with pulp oil extract (2) prior to freezing confers a substantial advantage by increasing percentage membrane integrity (about 25%) of avian sperm cells (44) upon thawing. Now referring toFIG.3, the effect of treating avian sperm cells (44) with seed oil extract (15) as above-described is shown inFIG.3. There was no apparent difference in avian sperm cell (43) membrane integrity between avian sperm cells (44) treated with the control extender and control extender containing 3% seed oil extract (15) before freezing (P>0.05). As to both, cryopreservation significantly reduced avian sperm cell (44) membrane integrity (P<0.05). The reduction in avian sperm cell (44) membrane integrity was greater in avian sperm cells (44) frozen in control extender compared to sperm frozen in control extender containing seed oil extract (14). Therefore, the treatment of avian sperm cells (44) with seed oil extract (14) prior to freezing confers a substantial advantage by increasing the percentage membrane integrity (about 25%) of avian sperm cells (44) after thawing. Taken together these data demonstrate an improvement in avian sperm quality when Extracts (2) and (15) are added to diluted avian sperm cells (44). EXAMPLE 7 Bovine semen (45) containing bovine sperm cells (46) was collected from ten bulls using an artificial vagina and bovine sperm cell (46) concentration was determined in each ejaculate. Bovine semen (45) was diluted to 160×106motile bovine sperm cells/mL in a Tris diluent (200 mM Tris, 65 mM citric acid, and 55 mM glucose) and split into 12 treatments. Each split of the diluted bovine sperm cells (46) was diluted 1:1 (v:v) in Tris diluent with 20% egg yolk and slowly cooled to about 5° C. After reaching about 5° C., each split was further diluted 1:1 (v:v) in Tris diluent containing 10% egg yolk and 16% glycerol supplemented with 0% or about 2%, about 6%, about 10%, about 14%, or 18% (v:v) seed oil extract (15) or pulp oil extract (2) ofHippophaeresulting in a final concentration of 40×106sperm cells/mL in 0 about 1%, about 3%, about 5%, about 7%, or about 9% (v:v) seed oil extract (15) (Extract 2) or pulp oil extract (2) (Extract 1). Emulsions of seed oil extract (15) or pulp oil extract (2) were prepared as described by Nii and Ishii (2005), hereby incorporated by reference. Pulp oil extract (2) or seed oil extract (15) was added to the Tris diluent at the desired percentages (v:v). The Tris diluent containing the pulp oil extract (2) or the seed oil extract (15) is sonicated five times for about 1 min with intervals of 0.5 min on ice using ultrasonication (20 kHz). Diluted bovine sperm cells (46) treated with Tris diluent containing the pulp oil extract (2) or the seed oil extract (15) at the desired percentages were allowed to equilibrate for a minimum of 15 min and then transferred into 0.5 cc straws. Straws containing the treated bovine sperm cells (46) were exposed to liquid nitrogen vapor for 20 min and plunged into liquid nitrogen. Straws containing control and treated bovine sperm cells (46) were stored in liquid nitrogen until analyzed. Acrosome assay: An aliquot of thawed control or treated diluted bovine sperm cells (46) was washed three times in phosphate buffered saline (“PBS”) to remove particles of egg yolk and seed oil extract (15) or pulp oil extract (2). The diluted bovine sperm cells (46) were centrifuged at 300×g for about 5 min at room temperature to remove seed oil extract (15) or pulp oil extract (2) and harvest bovine sperm cells (46) between washings. After final wash, the pellet of bovine sperm cells (46) was resuspended in 1 mL PBS containing 10 μg/mL fluorescein isothiocyanate-peanut agglutinin (“FITC-PNA”). The resuspended pellets of bovine sperm cells (46) were incubated in the dark for about 60 min at room temperature. After incubation, the resuspended pellets of bovine sperm cells (46) were analyzed using flow cytometry. As a positive control, an aliquot of washed control bovine sperm cells (0% extract (2) or (15)) was treated with 1.3 mg of methyl-β-cyclodextrin for about 30 min at about 37° C. followed by 25 μg/ml lysophosphatidylcholine for about 30 min to induce acrosome reaction. Bovine sperm cells (46) were then be stained with FITC-PNA and analyzed as described above. Stained bovine sperm cells were analyzed immediately after staining (time0) and after about 3 hours at about 37C. As a negative control, FITC-PNA was omitted from the staining protocol. Results are shown in Table 16 and Table 17. Data represents the average of 1 or 2 replicates from each of 10 bulls. Bovine sperm cells (46) having acrosomes determined unreacted can fertilize an egg. Bovine sperm cells (46) having reacted/reacting acrosomes cannot fertilize an egg. Therefore, one reliable measure of bovine sperm cell (46) fertility can be a measure of whether the acrosome is intact. Now referring primarily to Table 16 and Table 17, it can be understood that over a 3 hour period the number of unreacted acrosomes in the control bovine sperm cells (46) decreased, while in the bovine sperm cells (46) treated with seed oil extract (15) (see Table 17) or pulp oil extract (2) (see Table 16) exhibited little decrease in unreacted acrosomes indicating that the seed oil extract (15) and the pulp oil extract (2) acted to protect the membranes of bovine sperm cells (46) treated as above-described. EXAMPLE 8 Bovine semen (45) containing bovine sperm cells (46) was obtained from ten bulls and extended to 20×106bovine sperm cells/mL in Tyrode's Albumin Lactate Pyruvate (“TALP”) alone (referred to as the “control”), or TALP containing 5% fruit alcohol (Extract 6)(23), or TALP containing 5% leaf alcohol extract (Extract 7)(31). Diluted bovine sperm cells (46) were held at about 37° C. for about 180 min. Terminal deoxynucleotidyl transferase dUTP nick end labeling (hereinafter referred as “TUNEL”) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL assays were performed according to the protocol provided by Invitrogen Inc., Eugene, Oregon with the TUNEL assay kit, hereby incorporated by reference, with modification for bovine sperm cells (46). In brief, bovine sperm cells (46) were washed twice with PBS to remove TALP or TALP containing 5% fruit alcohol (Extract 6)(23), or TALP containing 5% leaf alcohol extract (Extract 7)(31) as above described. Washed bovine sperm cells (46) were centrifuged at about 300×g for about 5 min at room temperature to harvest bovine sperm cells (46) between washings. After final wash, the pellets containing bovine sperm cells (46) were resuspended to approximately 1×105cells/mL in PBS containing 1% paraformaldehyde (w:v). Resuspended bovine sperm cells (46) were incubated for about 20 min at about 4° C. to fix the bovine sperm cells (46). Incubated bovine sperm cells (46) were washed two times in PBS as described to remove paraformaldehde. Following final wash, the pellets of bovine sperms cells (46) were resuspended in 1 ml 1% Triton X-100 ((C14H22O(C2H4O)n)(v:v) and incubated at room temperature for about 5 min to increase permeability of bovine sperm cells (46). Bovine sperm cells (46) were washed twice as described and the pellet of bovine sperm cells (46) resuspended in 70% ethanol and stored at −20° C. for a minimum of 30 min or until further analysis. DNA labeling and staining: Bovine sperm cell (46) suspensions were centrifuged at 300×g for about 5 min at room temperature to remove ethanol. Bovine sperm cells were washed twice with PBS and the pellet resuspended in 50 μl DNA-labeling solution after final wash (10 μl reaction buffer, 0.75 μl TdT enzyme, 8 μl of BrdUTP, and 31.25 μl water). Bovine sperm cells were incubated at about 37° C. for about 60 min. After incubation with DNA-labeling solution, bovine sperm cells (46) were centrifuged at 300×g for 5 min and the pellet was resuspended in 1 mL rinse buffer. Bovine sperm cells were washed twice with rinse buffer with centrifugation at about 300×g for about 5 min. After final wash, the pellets of bovine sperm cells (46) were resuspended in 100 μl antibody solution (5.0 μl Alexa Fluor 488 dye-labeled anti-BrdU antibody and 95 μl rinse buffer) and incubated at room temperature for about 30 min. At the end of incubation, 0.5 mL of propidium iodide/RNase A staining buffer were added to each of the tube containing bovine sperm cells and incubated for about 30 min at room temperature. As a positive control, an aliquot of control bovine sperm cells (46) (0% extract) were treated with 10 units of DNAase I for about 30 min at about 37° C. to induce DNA fragmentation. For a negative control, an aliquot of control bovine sperm cells (46) underwent the above described procedures but the TdT enzyme was omitted. All procedures were conducted in low lighting to prevent UV damage to DNA. Bovine sperm cells treated in accordance with the above procedure were analyzed using flow cytometry. DNA damage (positive TUNEL result) was determined immediately after cells were extended (Initial) and at 180 min post-stress at 37° C. using TUNEL assay. Now, referring primarily to Table 18 which evidences the percentage of TUNEL positive bovine sperm cells (46). The deoxyribonucleic acid (hereinafter referred to as “DNA”) of bovine sperm cells (46) only incorporate stain if it is damaged, or contains single or double stranded breaks. Therefore an increase in the percentage of bovine sperm cells (46) that are TUNEL positive indicates the bovine sperm cells (46) have correspondingly greater percentage of damaged DNA. The addition of Extract 6 (23) and Extract 7 (31) as above described in treatment of the bovine sperm cells (46) substantially decreased DNA damage. Treatments containing different numbers in column 3 (significance) are statistically different if labeled using a different letter. P<0.05 when compared to initial time point; pooled SEM=5.2. TABLE 12.5% Pulpoil (2)treatedcontroldifference0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen40223215772 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled4035602312Exp 4 - cooled3936421818Exp 5 - cooled54103321−11Exp 6 cooled3936421818Exp 7 cooled56103421−11 TABLE 25% PulpOil (2)treatedcontroldifference0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen462832151372 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled42156023−8Exp 4 - cooled212442186Exp 5 - cooled402133210Exp 6 cooled212442186Exp 7 cooled402134210 TABLE 32.5% SeedOil (15)treatedcontroldifference0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen453532152072 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled3636602313Exp 4 - cooled5238421820Exp 5 - cooled52123321−9Exp 6 cooled5138421820Exp 7 cooled522234211 TABLE 45% SeedOil (15)treatedcontroldifference0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen483032151572 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled43186023−5Exp 4 - cooled221942181Exp 5 - cooled482333212Exp 6 cooled241942181Exp 7 cooled4743342122 TABLE 5difference2.5% Juice(2 hr − 2 hr(24)treatedcontrolcontrol)0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen41283215130 hr24 hours0 hr24 hoursmotilitypost coolmotilitypost coolExp 2 - cooled77.55567.552.52.572 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled582860235Exp 4 - cooled4328421810Exp 5 - cooled632233211Exp 6 cooled5138421820Exp 7 cooled652434213 TABLE 6difference(2 hr − 2 hr5% Juice (24)treatedcontrolcontrol)0 hr2 hr @0 hr2 hr @motility37 C.motility37 C.Exp 1 - frozen3953215−100 hr24 hours0 hr24 hoursmotilitypost coolmotilitypost coolExp 2 - cooled8067.567.552.51572 hr2 hr @72 hr2 hr @motility37 C.motility37 C.Exp 3 - cooled6056602333Exp 4 - cooled5838421820Exp 5 - cooled622633215Exp 6 cooled5739421821Exp 7 cooled642634215 TABLE 7treatedcontrol2.5% Fruit72 hr2 hr @72 hr2 hr @Tincture (23)motility37 C.motility37 C.Exp 4 - cooled5833421815Exp 5 - cooled6342332121Exp 6 cooled5833421815Exp 7 cooled6443342122 TABLE 8treatedcontrol5% Fruit72 hr2 hr @72 hr2 hr @Tincture (23)motility37 C.motility37 C.Exp 4 - cooled5230421812Exp 5 - cooled652133210Exp 6 cooled5230421812Exp 7 cooled662234211 TABLE 9treatedcontrol2.5% Leaf72 hr2 hr @72 hr2 hr @Paraffin (36)motility37 C.motility37 C.Exp 442114218−7Exp 5 - cooled4053321−16Exp 6 cooled43124218−6Exp 7 cooled4073421−14 TABLE 10treatedcontrol5% Leaf72 hr2 hr @72 hr2 hr @Paraffin (36)motility37 C.motility37 C.Exp 45128421810Exp 5 - cooled53203321−1Exp 6 cooled5128421810Exp 7 cooled55203421−1 TABLE 112.5% LeaftreatedcontrolGlycerine72 hr2 hr @72 hr2 hr @(41)motility37 C.motility37 C.Exp 4 - cooled611942181Exp 5 - cooled552133210Exp 6 cooled621842180Exp 7 cooled52123421−9 TABLE 12treatedcontrol5% Leaf72 hr2 hr @72 hr2 hr @Glycerine (41)motility37 C.motility37 C.Exp 4 - cooled681842180Exp 5 - cooled732233211Exp 6 cooled68174218−1Exp 7 cooled72163421−5 TABLE 13treatedcontrol2.5% Leaf72 hr2 hr @72 hr2 hr @Tincture (31)motility37 C.motility37 C.Exp 4 - cooled582242184Exp 5 - cooled71113321−10Exp 6 cooled582342185Exp 7 cooled72123421−9 TABLE 14treatedcontrol5% Leaf72 hr2 hr @72 hr2 hr @Tincture (31)motility37 C.motility37 C.Exp 4 - cooled632142183Exp 5 - cooled7051332130Exp 6 cooled622142183Exp 7 cooled7041342120 TABLE 15μM CopperμM Copperreducingreducingequivalents atequivalents atExtract2.5% of Extract5% of ExtractExtract 1: pulp oil extract (2)330.51148.8Extract 2: Seed oil extract (15)149.5967.8Extract 3: juice extract (24)1290.41377Extract 6: fruit tincture extract (23)653.1613.7Extract 7: leaf tincture extract (31)1038.61565.8Extract 8: leaf paraffin extract (36)692.41219.6Extract 9: leaf hydroglycerine1691.71479.3extract (41) TABLE 16Extract% Unreacted1: pulpacrosomesoilTime 0Time 3 hr% decreasecontrol60.6052.7213%1%45.7848.29−5%3%48.0649.29−3%5%42.3142.89−1%7%54.1350.337% TABLE 17Extract% unreacted2: SeedacrosomeoilTime 0Time 3 hr% decreasecontrol60.6052.7213%1%45.6250.90−12%3%43.7848.24−10%5%53.9348.4910%7%51.2846.1010% TABLE 18Treatment% TUNEL positiveSignificancePre-cooling untreated3BPost-cooling, untreated28APost-cooling, 5% Extract:6BFruit alcoholPost-cooling, 5% Extract:4BLeaf alcohol Production of Embryos and Embryo Storage. The production of embryos via artificial means such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), zona drilling, zona dissection, and subzonal insertion of sperm (SUZI), involves much manipulation and potential exposure to oxygen, ROS and other potentially negative compounds. A particular embodiment of the present invention comprises the combination or addition of extracts to the media, diluent, or to the tools and containers used during such manipulation. In another embodiment of the invention, extracts can be combined or added to the solution used to buffer the sperm cells during micro-manipulation for such procedures as IVF, ICSI, GIFT, ZIFT and other procedures which utilize the in vitro addition of sperm cells to an oocyte. Alternatively, or in addition, extracts can be utilized to coat the interior surfaces of tools (e.g., petri dish, micromanipulating needles, and the like). After the addition of sperm cells to the oocyte, the resulting embryo must be washed to remove excess seminal plasma and additional sperm cells. In another embodiment of the present invention, this washing step via centrifugation, gravity column, or simple washing could utilize extracts, in the diluents or solutions, or attached by various methods, to the media used for separation. Additionally, oocytes can be stored for approximately 24 hours post-fertilization. Extracts could be present in the incubating media or diluent in order to protect the rapidly dividing cell from internally or externally generated free radicals. In another embodiment of the invention, extracts can be added to the solution utilized to preserve embryos via freezing. Such an addition would confer protection immediately, and post-thaw when membranes, negatively affected by freezing, would be susceptible to free radical attack. In yet another embodiment of the present invention, extracts would be added to the solution utilized to flush embryos from superovulated, inseminated females. The collected embryos can then be washed using extract containing media or diluents, and transferred to diluents or solutions, which may contain extracts in various concentrations, for freezing. Alternatively, the collected embryos may be transferred directly to a recipient. In still another embodiment of the present invention, the addition of extracts to the media would include, equipment or containers for embryos as they are manipulated for embryo splitting, embryo sexing or during production of clones from cell-banked tissue lines, or other tissue sources or production of ESCs. Such addition would confer protection during manipulation, during freezing, and during post-thaw implantation, and/or manipulation. In another embodiment of the invention, extracts could be utilized in solution, on containers or equipment used to produce, or achieve cloned offspring, transgenic animals or similar embryo-manipulating technologies. Reproductive Aids. In a particular embodiment of the present invention, extracts can be used to condition the in vivo reproductive tract for conception. Extracts would be added vaginally in the form of an insert, gel, foam, pessary or suppository. Such compositions may contain gelatinizing agents, lubricating agents and other additives compatible with Extracts and the reproductive environment. Vaginal administration may occur at a predetermined time prior to sexual intercourse or artificial insemination, or may occur immediately after sexual intercourse or artificial insemination. In an alternative or complimentary embodiment, Extracts may be administered as a cream or gel on the male genitalia prior to sexual intercourse. In yet another embodiment, extracts are administered directly to the uterine environment. The presence of extracts in the vaginal environment and/or uterus would decrease oxidative damage to reproductive cells, reduce inflammation and/or act as an antibiotic. In particular, the compositions and methods of the present invention will result in an increased number of sperm reaching the ooctye, thereby improving the odds of conceiving. Extracts as an Additive. This invention is directed to the medium additive extracts described above, media containing extracts, a method of making the medium supplement, a method of making media containing extracts and kits containing the medium supplement. Extracts can be added to any cell culture media, including but not limited to, a reproductive cell wash media, a reproductive cell culture media a reproductive cell cooling media, a reproductive cell culture media, a reproductive cell transfer media, a reproductive cell cryopreservation media, (to include both freezing and vitrification procedures), cell collection media and cell sorting, or sexing media. Any media that can support reproductive cell development can be used, which includes, bicarbonate buffered medium, Hepes-buffered or MOPES buffered medium, phosphate buffered saline and the like. Extracts may be prepared as a separate media supplement that is added to the media after media preparation, or it can be added directly to the media during media preparation or may be added to a component of the media prior to commutation. By way of example, extracts may be prepared on its own as follows. Extracts may be made into a stock solution by adding water, saline or media to make a concentrated stock solution. Alternatively, the solution may be obtained as a stock solution. Alternatively, extracts can be added directly to the media as either a powder or stock solution. Extracts may also be added to a buffer or a buffer containing limited components such as egg yolk citrate, or milk then added to the other components. Extracts may also be adhered to a container or equipment useful for producing, manipulating, culturing or processing reproductive cells. In a particular embodiment, Extracts may be adhered to beads useful for separation of reproductive cells via centrifugation, gravity sedimentation, column elution and the like. In another particular embodiment, Extracts may adhere to a container via antibodies, proteins and the like. In yet another particular embodiment, extracts are applied to a container to create a liquid coating of such container. In yet another particular embodiment, Extracts is applied to a container as part of a solid coating or film which dissolves or otherwise releases Extracts upon contact with liquid media. As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. The invention involves numerous and varied compositions or extracts obtained from plant sources including without limitation the genusHippophaeand methods of using such compositions or extracts to reduce loss of reproductive cell function. As such, the particular embodiments or elements of the invention disclosed by the description or shown in the figures or tables accompanying this application are not intended to be limiting, but rather exemplary of the numerous and varied embodiments generically encompassed by the invention or equivalents encompassed with respect to any particular element thereof. In addition, the specific description of a single embodiment or element of the invention may not explicitly describe all embodiments or elements possible; many alternatives are implicitly disclosed by the description and figures. It should be understood that each element of an apparatus or each step of a method may be described by an apparatus term or method term. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all steps of a method may be disclosed as an action, a means for taking that action, or as an element which causes that action. Similarly, each element of an apparatus may be disclosed as the physical element or the action which that physical element facilitates. As but one example, the disclosure of a “diluent” should be understood to encompass disclosure of the act of “diluting” —whether explicitly discussed or not—and, conversely, were there effectively disclosure of the act of “diluting”, such a disclosure should be understood to encompass disclosure of a “diluent” and even a “means for diluting.” Such alternative terms for each element or step are to be understood to be explicitly included in the description. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood to included in the description for each term as contained in the Random House Webster's Unabridged Dictionary, second edition, each definition hereby incorporated by reference. Thus, the applicant(s) should be understood to claim at least: i) each of the compositions or extracts herein disclosed and described, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods, iv) those alternative embodiments which accomplish each of the functions shown, disclosed, or described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the previous elements disclosed. All numeric values herein are assumed to be modified by the term “about”, whether or not explicitly indicated. For the purposes of the present invention, ranges may be expressed as from “about” one particular value to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value. The recitation of numerical ranges by endpoints includes all the numeric values subsumed within that range. A numerical range of one to five includes for example the numeric values 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. When a value is expressed as an approximation by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The background section of this patent application provides a statement of the field of endeavor to which the invention pertains. This section may also incorporate or contain paraphrasing of certain United States patents, patent applications, publications, or subject matter of the claimed invention useful in relating information, problems, or concerns about the state of technology to which the invention is drawn toward. It is not intended that any United States patent, patent application, publication, statement or other information cited or incorporated herein be interpreted, construed or deemed to be admitted as prior art with respect to the invention. The claims set forth in this specification, if any, are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to use all of or a portion of such incorporated content of such claims as additional description to support any of or all of the claims or any element or component thereof, and the applicant further expressly reserves the right to move any portion of or all of the incorporated content of such claims or any element or component thereof from the description into the claims or vice-versa as necessary to define the matter for which protection is sought by this application or by any subsequent application or continuation, division, or continuation-in-part application thereof, or to obtain any benefit of, reduction in fees pursuant to, or to comply with the patent laws, rules, or regulations of any country or treaty, and such content incorporated by reference shall survive during the entire pendency of this application including any subsequent continuation, division, or continuation-in-part application thereof or any reissue or extension thereon. The claims set forth below are intended to describe the metes and bounds of a limited number of the preferred embodiments of the invention, and are not to be construed as the broadest embodiment of the invention or a complete listing of embodiments of the invention that may be claimed. The applicant does not waive any right to develop further claims based upon the description set forth above as a part of any continuation, division, or continuation-in-part, or similar application. | 70,477 |
11857589 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates to a unique method of solubilizing cannabinoid extract/distillate for use in formulating pharmaceutical compositions containing dry powders, including tablets, capsules, dry powder inhalers, edibles, beverages and powdered mixes. The inventor has surprisingly discovered that extracts from cannabinoids can be effectively and inexpensively solubilized using a solvent and a combination of emulsifiers. The resulting composition is a water-soluble powder that may in turn be used in pharmaceuticals. The cannabis extracts of the invention are any that can be derived or extracted from cannabis plants, hemp or synthetically derived. Cannabis plants produce a unique family of terpeno-phenolic compounds called cannabinoids, which produce the “high” one experiences from consuming marijuana. There are 483 identifiable chemical constituents known to exist in the cannabis plant, and at least 185 different cannabinoids have been isolated from the plant. The two cannabinoids usually produced in greatest abundance are cannabidiol (CBD) and/or (−)-trans-Δ9-tetrahydrocannabinol (THC), but only THC is psychoactive. Thus, as used here, the term “cannabis” is intended to include not only cannabis, but hemp, synthetic cannabinoids, and terpenes. Cannabis plants are categorized by their chemical phenotype or “chemotype,” based on the overall amount of THC produced, and on the ratio of THC to CBD. Although overall cannabinoid production is influenced by environmental factors, the THC/CBD ratio is genetically determined and remains fixed throughout the life of a plant. Non-drug plants produce relatively low levels of THC and high levels of CBD, while drug plants produce high levels of THC and low levels of CBD. Besides CBD and THC, other cannabinoids include, but are not limited to, cannabichromene (CBC), cannabigerol (CBG) cannabinidiol (CBND), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), and all acidic forms, precursors, and derivatives thereof. Cannabinoids are derived from their respective 2-carboxylic acids (2-COOH) by decarboxylation (catalyzed by heat, light, or alkaline conditions). As a general rule, the carboxylic acid form of the cannabinoid have the function of a biosynthetic precursor. As noted, the present invention relates to the solubilization of use of any cannabis plant extract in any form. In addition to cannabinoids, cannabis plants produce terpenes, a diverse group of organic hydrocarbons that are the building blocks of the cannabinoids. Over 100 different terpenes have been identified in the cannabis plant, and every strain tends toward a unique terpene type and composition. The terpenes act synergistically with the cannabinoids to provide a therapeutic effect. The terpenes in the extract include, but are not limited to, myrcene, alpha-bisabolol, caryophyllene, limonene, eucalyptol, nerolidol, terpineol, camphene, valencene, geraniol, humulene, delta-3-carene, borneol, alpha-pinene and beta-pinene, and linalool. In various aspects the invention provides cannabis extracts with predefined ratios of cannabinoids. Standard conditions for cannabinoid assays, and methods of calculating cannabinoid content (as %) are well known in the art. The cannabinoid extract starting materials are typically mixtures of at least 90% total cannabinoids which include terpenes and/or flavonoids. Preferably the extracts contain a mixture of at least four cannabinoid such as tetrahydrocannabinolic acid (THCa), cannabidiolic acid (CBDa), cannabinolic acid (CBNa) cannabichromenic acid (CBCa), tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and cannabichromene (CBC). The terpene and/or flavonoids in the extract include, but are not limited to, myrcene, alpha-bisabolol, caryophyllene, limonene, eucalyptol, nerolidol, terpineol, camphene, valencene, geraniol, humulene, delta-3-carene, borneol, alpha-pinene and beta-pinene, and linalool. Therefore, in a further aspect the invention provides a method of making a water soluble powdered extract composition comprising, as an active agent, a substance which is an extract from at least one cannabis plant variety. Separate extracts may be prepared from single cannabis plant varieties having differing cannabinoid content (e.g. high THC and high CBD plants) and then mixed or blended together prior to formulation to produce the final pharmaceutical composition. This approach is preferred if, for example, it is desired to achieve a defined ratio by weight of individual cannabinoids in the final formulation. Alternatively, plant material from one or more cannabis plant varieties of defined cannabinoid content may be mixed together prior to extraction of a single botanical drug substance having the desired cannabinoid content, which may then be formulated into a final pharmaceutical composition. A preferred formulation includes a cannabinoid mixture where THC is greater than or equal to 90%; a CBD is less than 1%; CBN is less than 3%; and CBC is less than 1%. In some aspects the formulation further includes d-limonene, linalool, 1,8-cineole (eucalyptol), alpha-pinene, terpineol-4-ol, p-cymene, borneol, delta-3-carene, beta-sitosterol, cannflavin A, apigenin, and quercetin. Another preferred formulation includes a cannabinoid mixture where the THC is less than or equal to 30%; CBD is greater than or equal to 60%; CBN is less than 1%; and CBC is less than 1%. In some aspects the formulation further includes d-limonene, linalool, 1,8-cineole (eucalyptol), alpha-pinene, terpineol-4-ol, p-cymene, borneol, delta-3-carene, beta-sitosterol, cannflavin A, apigenin, and quercetin. In yet another preferred embodiment the formulation includes a cannabinoid mixture where the THC is greater than or equal to 45%; CBD is greater than or equal to 45%; CBN is less than 1%; and CBC is less than 1%. In some aspects the formulation further includes beta-myrcene, beta-caryophyllene, pulegone, alpha-terpineol, beta-sitosterol, cannflavin A, apigenin, and quercetin. In accordance with the methods of the invention, the constituents of the powdered extract may all be combined at once, or combined in stages. The following materials may be used to manufacture the powdered extract of the claimed invention:from about 0.1-85% by weight cannabinoid extract, terpene extract, or combinations thereof, with about 10-50% by weight being preferred, and about 20-30% by weight being most preferred;from about 0.1-75% by weight solvent, with about 20-60% by weight being preferred, and about 20-40% by weight being most preferred;from about 0.1-80% by weight carbohydrate substrate, with about 20-60% by weight being preferred, and about 30-50% by weight being most preferred. In one embodiment of the invention, cannabinoid extract is first combined with an emulsifier(s) and a pharmaceutically acceptable solvent to form a dissolved extract. The solvent is one that is capable of dissolving the extract but one that does not dissolve the substrate emulsifier used in the second step. The solvent also needs to be one that can be evaporated or otherwise incorporated into from the composition to form the resulting powder. Suitable class 1-3 solvents for this purpose include, but are not limited to, n-hexane, ethyl acetate, diethyl ether, 2-propanol, acetone, ethanol, ethanol/water, butane, propane, benzyl alcohol, 1,3-butylene glycol, citric acid esters of mono- and di-glycerols, glycerin, glyceryl triacetate, glyceryl tributyrate, isopropyl alcohol, monoglyceride citrate, propylene glycol, triethyl citrate, diethylene glycol and propylene glycol mono- and de-esters. In one embodiment of the invention, the solvent is 95% ethanol. The solvent is combined with the extract in an amount sufficient to dissolve the components of the extract. In one embodiment, the solvent is combined with equal parts extract. The solvent/extract mixture is preferably heated to a range of about 50-85° C. with about 50-60° C. being preferred and about 55° C. being most preferred. The mixture may optionally be stirred/agitated to more thoroughly combine the ingredients. The dissolved extract is next combined with an emulsifier and/or emulsifying system to form a water-soluble powder. The cannabinoids absorb onto and are coated by the substrate emulsifier. Thus, the emulsifier is used to absorb the oil and make the extract water soluble. The emulsifier is preferably a sugar or carbohydrate substrate that may include, but is not limited to, starch, maltodextrins, glucose syrup, crystalline glucose (dextrose, sucrose, fructose), poloxamers including 188 and 407, caramel, sorbitol, maltitol, mannitol, isomalt, beta/hydroxylpropyl cyclodextrins, lecithin, soy-derived glycerol phosphatides, acacia, gum arabic, xanthan gum, carrageenan, polyglycols, poloxamers, hydroxystearate polyoxyl 15 (Kolliphor HS15), locust bean gum, tapioca, carboxymethylcellulose, sodium tripolyphosphate (STPP), and combinations of the same. In one embodiment, the emulsifying system includes an oil soluble emulsifier combined with a water soluble emulsifier. In another embodiment, the emulsifying system is a combination of maltodextrin and gum arabic (acacia). In one embodiment, the emulsifying system includes STPP in a concentration of about 0.1-5% by weight. In one embodiment of the invention, the dissolved and/or emulsified extract may also include one or more other components or methods to improve the solubility and/or dissolution rate of the extract. For example, the cannabis and/or other active ingredients of the invention may be complexed with cyclodextrins. Cyclodextrins are molecular donut-shaped structures consisting of several glucose molecules. Depending on the denotation (alpha-, beta-, or gamma cyclodextrins), 6 to 8 glucopyranose units are cyclically joined to make a “truncated cone”. The interior diameter of the structure measures between 0.6 nm to 0.8 nm. Due to the orientation of the carbons of the sugars, the polarity of the cavity is comparable to the polarity of ethanol, which provides a local favorable environment for lipophilic drugs. However, solubility enhancement with cyclodextrins is restricted by their cavity size: the alpha form does not provide sufficient space for many drug molecules, and the larger gamma form is comparably expensive. The complexation of a drug with cyclodextrin furthermore leads to an enhanced dissolution rate. Mesoporous silica refers to any number of a variety of materials synthesized to produce a SiO2mesoporous structure. Mesoporous silica can be ordered or non-ordered. It has been widely reported that mesoporous silica can act as a solubility enhancer by adsorbing and stabilizing active pharmaceutical ingredients (APIs) in the amorphous form within the porous network. There are various methods of loading crystalline API onto mesoporous silica, which can be grouped into three broad categories: solvent-based, mechanical activation, and vapor-mediated. API is dissolved in organic solvent (thus removing any crystal lattice) and added to mesoporous silica. Adsorption of API onto the silica is then initiated through mechanical agitation or sonication of the slurry. Finally, solvent is removed, which can be achieved using a number of methods including vacuum drying, spray drying, lyophilization or rotary evaporation. The cannabis or other active ingredients of the invention may be loaded onto the silica through conventional methods well known to persons skilled in the art. Mesoporous silica has particular advantages in pre-clinical development due to the low capital investment requirements and relatively accessible loading method. The loading of mesoporous silica can be achieved using simple laboratory equipment and scaled to commercial batches using regular manufacturing equipment. Mesoporous silica materials (MSMs) are characterized by very large surface areas, uniform pore size, significant biocompatibility and easy surface functionalization. Generally, MSMs are prepared by hydrolysis and condensation of silica precursors such as tetraethoxysilane (TEOS) around the micelle templates (generated by supramolecular self-assemblies of surfactant molecules) followed by template removal by calcination or solvent extraction. There are two common mechanisms, i.e., cooperative self-assembly of micelle and silica source and liquid-crystal templating to describe the synthesis of MSMs. MSMs can be prepared with different sizes from nanoscale to microscale range, with large surface area (from 700 to 1000 m2/g) and pore volume (from 0.6 to 1 cm2/g). Also, depending on the reaction condition, diverse morphologies (e.g., spherical, rod, ellipsoid, and platelet) can also be made each suitable for a specific biological application. While typical pore diameters of MSMs are ˜2-5 nm, it is also possible to synthesize pore diameter up to 30 nm, thereby allowing accommodating not only small molecules but also larger chemicals such as proteins within the mesopores. Furthermore, owing to surface silanol groups, the surface properties of MSMs can range from hydrophobic to hydrophilic, thus providing the possibility of a proper drug loading parameters as well as drug release profile for different drug substances. The substrate is preferably selected according to the bulk density necessary for the pharmaceutical product in which the final powder product is incorporated, as well as to match formulation granulation. For instance, if the powder will be used to formulate a tablet, the substrate should have a higher bulk density, while a nasal inhalation pharmaceutical will have a lower bulk density. The types of pharmaceuticals and their desired bulk densities, and appropriate substrates to achieve such bulk densities are as follows (ratios of each can change):compressed tablet, bulk density of 0.4-1.5 g/cm3, appropriate substrates: spray dried lactose or direct compression lactose/acacia/STPP/poloxamer 188;immediate-release tablet, bulk density of 0.2-1.5 g/cm3, appropriate substrates: sieved lactose/acacia/STPP/poloxamer;powder, bulk density of 0.1-1.0 g/cm3, appropriate substrates: maltodextrin or milled lactose/acacia/STPP/poloxamer;dry-granulations, bulk density of 0.4-1.5 g/cm3, appropriate substrates: milled lactose/acacia/STPP/poloxamer;oral inhalation powder, bulk density of 0.1-0.5 g/cm3, appropriate substrates: fine milled and sieved lactose/acacia/STPP/poloxamer/CD The process can be applied to a range of core materials in numerous particles sizes and shapes and densities. No matter the shape, crystalline, spherical, irregular, amorphous, the process is capable of creating unique formulations to achieve the desired properties. Understanding the flow related properties, particle size and density is critical in the processing of these powders for their intended formulations. This invention eliminates most trial and error. If an emulsifying system is used, the system includes about 1:20-20:1 water soluble emulsifier to oil soluble emulsifier. In one embodiment, the system includes about 5% by weight oil soluble emulsifier to about 95% by weight water soluble emulsifier. A higher concentration of substrate, with or without emulsifier, will result in higher solubility of the final powder composition. The carbohydrate substrates themselves act as emulsifiers/colloids allowing the water insoluble colloids, cannabinoids or other drugs to become solubilized. The ratios of substrate alone or in combination with other emulsifiers allow for easy wetting and enhanced solubility, which is further enhanced as dilution continues. In one embodiment, the dissolved extract is sprayed onto the emulsifier while the emulsifier is under agitation and vacuum. In a preferred embodiment, a Ross vertical cone screw blender is utilized whereby the powdered solids are combined under vacuum then immediately subjected to high sheer mixing at a point in the blender where flow if most turbulent. In another embodiment, the dissolved extract is dripped into the emulsifier while mixing. If more than one emulsifier is used in this step it is preferred to mix the emulsifiers prior to combining with the dissolved extract. In one embodiment of the invention, the extract is ultrasonically homogenized or microfluidized to form microemulsions and nanoemulsions. When added to liquids, the resulting homogenized extracts are tasteless and translucent, as compared to most emulsifiers used in microemulsions which have a strong, bitter taste. In one embodiment, the globule sizes are less than about 100 nm, with an average globule size of about 50 nm. The key to the invention in this respect is the combination, ratio, and concentration of the carbohydrate components. The solvent/extract mixture is next dried to form a micronized powder by preferably heating the mixture for several hours to a range of about 50-85° C. with about 50-60° C. being preferred and about 55° C. being most preferred. The mixture may optionally be stirred/agitated during this step to more thoroughly combine the ingredients. In one embodiment, the mixture is agitated under vacuum. The solvent/extract mixture may also be dried using conventional methods including, but not limited to, air drying, spray drying, freeze drying, etc. The mixture is dried and/or placed under vacuum for a time period sufficient to provide a flowable powder free of aggregates, with a moisture content generally ranging from about 2-8% by weight, with about 4% moisture by weight being preferred. The powdered product may be further processed into consumer and pharmaceutical formulations and/or used for testing. In another embodiment, small batches of product may be prepared by hand on a benchtop. The resulting micronized powder has a particle size of no more than 5000 nm, with a resulting particles size of about 20-1000 nm to as small as about 20-50 nm depending upon the amount and extent of processing. Unlike other known preparation methods, the resulting product does not require encapsulation and/or microencapsulation. The product and its method of processing are further distinguished from prior methods in that the materials do not require sterilization during any of the processing steps. The preparation of the compositions of the invention may be easily scaled up with the use of equipment that is known in the art including, but not limited to, Wurster fluidizer, vertical blender with vacuum and spray dryer, and/or hand spraying/mixing. The compositions of the invention may be inexpensively manufactured on a commercial scale. A wet solution is sprayed into the fluid bed coater to agglomerate the primary powder together and create larger granules by suspending the particles inside the chamber through high velocity air. This material is then dried upon completion of the spraying by hot air to the unit. The particles exit the chamber in different shapes, sizes and densities based on the movement of the material in the chamber and through particle interaction. The powder is flowable and stable against oxidation and moisture. The water soluble powdered extract may be formulated with any convenient pharmaceutically acceptable diluents, carriers or excipients to produce a pharmaceutical composition. The choice of diluents, carriers or excipients will depend on the desired dosage form, which may in turn be dependent on the intended route of administration to a patient. Oral dosage forms include, but are not limited to, tablets, capsules, suspensions, granules, and solutions. The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example the pharmaceutical preparations may be made by means of conventional mixing, granulating, dissolving, lyophilizing processes and vibrational atomization. The processes to be used will depend ultimately on the physical properties of the active ingredient used. Suitable excipients are, in particular, fillers such as sugars for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulating agents and lubricants, for example, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate and/or polyethylene glycol. Oral dosage forms may be provided with suitable coatings which, if desired, may be resistant to gastric juices. For this purpose concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, dyestuffs and pigments may be added to the tablet coatings, for example, for identification or in order to characterize different combinations of compound doses. Other pharmaceutical preparations which can be used orally include capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids; such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition stabilizers may be added. The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example the pharmaceutical preparations may be made by means of conventional mixing, granulating, dissolving, lyophilizing processes. Such dosage forms may be prepared in accordance with standard principles of pharmaceutical formulation, known to those skilled in the art. The extract may be formulated for oral use (e.g. capsules) in dosage forms that provide, for example, less than 5 mg, 5 mg, 10 mg, 20 mg, 100 mg, or more than 100 mg of total cannabinoids per dose. The following examples are offered to illustrate but not limit the invention. Thus, it is presented with the understanding that various formulation modifications as well as method of delivery modifications may be made and still are within the spirit of the invention. Example 1 A pharmaceutical composition was prepared as described below. The following products were used in the amounts and concentrations specified:1. About 20 g cannabinoid distillate2. About 35 g Ethanol 95%3. About 40 g maltodextrin/gum acacia mixtureThe cannabinoid distillate was weighed in a glass beaker. Ethanol 95% was added to the same beaker. The contents of the beaker were allowed to dissolve on a hot plate set to 55° C. The above solution was combined with the maltodextrin/gum acacia in a planar mixer and was gently mixed until well incorporated. The above mixture was passed through a granulation screen into a second bowl. This bowl was placed into a vacuum oven at 55° C. for 12 hours. The powder was stirred at least one during this time frame. The formulation above was tested for potency and stability after 1 year of storage. After this period, no loss of potency was observed (as measured by HPLC), the formulation was visibly stable at room temperature and readily fluid when shaken. Example 2 A pharmaceutical composition was prepared as described below. The following products were used in the amounts and concentrations specified:1. The cannabinoid distillate (or terpenes or isolate or combinations thereof) is weighed into a glass beaker. The beaker was tared and then ethanol 95% was weighed into the same beaker. The contents of the beaker were allowed to completely dissolve on a hot plate set to 55° C.2. Maltodextrin/gum acacia powder was weighed into a glass mixing vessel with paddle attachment.3. The product of step 1 was slowly dripped into the maltodextrin/acacia powder while mixing with vertical blender using gentle shear.4. After the distillate/solvent has been added the powder is mixed for an additional 5 minutes.5. The wetted powder is passed through a 3.35 mm granulation screen and allowed to dry in a vacuum oven set to 55° C. for at least 12 hours.6. The powder was gently mixed at 2 hours and 6 hours. It should be appreciated that minor dosage and formulation modifications of the composition and the ranges expressed herein may be made and still come within the scope and spirit of the present invention. Having described the invention with reference to particular compositions, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary. The foregoing description has been presented for the purposes of illustration and description. It is not intended to be an exhaustive list or limit the invention to the precise forms disclosed. It is contemplated that other alternative processes and methods obvious to those skilled in the art are considered included in the invention. The description is merely examples of embodiments. It is understood that any other modifications, substitutions, and/or additions may be made, which are within the intended spirit and scope of the disclosure. From the foregoing, it can be seen that the exemplary aspects of the disclosure accomplish at least all of the intended objectives. | 26,809 |
11857590 | DETAILED DESCRIPTION OF THE INVENTION Various embodiments are described more fully hereinafter with reference to the accompanying drawings, which form a part hereof, and which show, by way of illustration, specific exemplary embodiments by which the innovations may be practiced. The embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art. Among other things, the various embodiments may be methods, formulations, compositions, and the like. The following detailed description is, therefore, not to be taken in a limiting sense. Definitions As used herein, the term “about” means plus or minus 5% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. As a non-limiting example, a temperature of about 150° C., means a range of 142.5° C. to 157.5° C., inclusive of the endpoints and all numbers therebetween. “Broad spectrum hemp extract (BSHE)” as used herein BSHE is a composition derived from theCannabisgenus of plants which has undergone at least some purification in order to refine the extract. Typically, BSHE comprises between 60 and 99.9% CBD and least one additional cannabinoid, selected from the group consisting of Δ9-THC, THCA, THCV, Δ8-THC, CBC, CBCA, CBG, CBGA, CBDA, CBDV, CBN, CBL, and combinations thereof the sum of which is between 0.1 and 40%. As used here, the term “cannabis extract” (CE) is a composition derived from theCannabisgenus of plants (including hemp). Typically, a cannabis extract contains cannabidiol, and more typically comprises both cannabidiol (CBD) and at least one additional cannabinoid, selected from the group consisting of Δ9-THC, THCA, THCV, Δ8-THC, CBC, CBCA, CBG, CBGA, CBDA, CBDV, CBN, CBL, and combinations thereof at between 0.1 and 40%. Cannabis extracts according to the present invention are typically enriched in cannabidiol, and may comprise between 1 and 99.9% CBD, preferably between 20 and 99.9% CBD, more preferably between 50 and 99.9% CBD, even more preferably between 70 and 99.9% CBD, and most preferably between 90 and 99.9% CBD. Full spectrum hemp extract, broad spectrum hemp extract, CBD isolate, and CBDA isolate are forms of cannabis extract utilized herein, as non-limiting examples of the CE. “Composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. “CBD/CBD isolate” as used herein encompasses a purified concentration of CBD molecules to a purity of greater than 98%. As used herein, the term “full spectrum hemp extract (FSHE)” is a composition derived from theCannabisgenus of plants which contains CBD, and quantities of THC above 0, preferably, between 0.01% and 5%, most preferably being between 0.01% and 0.3%. The FSHE may comprise additional cannabinoids, yielding a product that comprises at least 50-99% CBD, at least 0.01% to 10% THC (the sum of Δ9-THC, THCA, THCV, Δ8-THC), and total cannabinoids of between 50% and 99% of the weight of the CE. “Source material” as used herein encompasses the cannabis plants that are harvested to be used in an extraction process. “Cannabis-based green material” (GM) as used herein encompasses the parts of the source material producing the most cannabinoids including the flowers/inflorescence and some leaf material. Preferably, “cannabis-based green material” refers to inflorescence and leaf material of plants of thecannabisgenus. “Extract” as used herein encompasses an extraction solvent and the extracted materials dissolved in the extraction solvent. “Concentrate/Concentrated extract” as used herein encompasses extracted materials from the extract that remain after the extraction solvent has been removed. “Partitioned concentrate” as used herein encompasses the materials in the concentrate that are soluble in a nonpolar solvent and result from a partitioning of concentrate between an aqueous phase and the nonpolar solvent. “Crude oil” as used herein encompasses the resultant oil after hexane from a portioning step has been distilled off. “Residue” as used herein encompasses the material left behind as a result of distillation, and more particularly the material left behind as a result of a distillation step in a short pass still. “Distillate” as used herein encompasses the material removed (i.e., which is evaporated and recondensed) in a distillation step, and more particularly the material removed as a result of distillation in a short pass still. “At least a portion of” as used herein refers to at least 5%, preferably at least 10%, more preferably at least 20%, still more preferably at least 50%, and most preferably at least 75%, for example from 75% to 99%, from 75% to 95%, or from 75% to 90%. Thus, distilling at least a portion of the extraction solvent off of the extract typically means distilling at least 5%, preferably at least 10%, more preferably at least 20%, still more preferably at least 50%, and most preferably at least 75%, for example from 75% to 99%, from 75% to 95%, or from 75% to 90%, of the extraction solvent off of the extract. Similarly, removing at least a portion of water-soluble substances from the concentrate typically means removing at least 5%, preferably at least 10%, more preferably at least 20%, still more preferably at least 50%, and most preferably at least 75%, for example from 75% to 99%, from 75% to 95%, or from 75% to 90%, of the water-soluble substances from the concentrate. “Nonpolar solvent” as used herein refers to a solvent having a polarity index of 3.5 or less in accordance with the values calculated by Burdick and Jackson (list available at https://macro.lsu.edu/howto/solvents/polarity %20index.htm). Preferably, the nonpolar solvent is selected from pentane, hexane, heptane, toluene, the fraction of petroleum ether boiling between 30-40° C., the fraction of petroleum ether boiling between 40-60° C., toluene, xylene, diethyl ether and dichloromethane. Most preferably, the nonpolar solvent is hexane. “THC-free fraction” as used herein is a fraction obtained from centrifugal partition chromatography which contains less than 0.1% or less Δ-9-tetrahydrocannabinol (THC), preferably 0.008% or less, and more preferably less than 0.0027% THC. “Reclaim fraction” as used herein is a fraction obtained from centrifugal partition chromatography which contains more than 2% Δ-9-tetrahydrocannabinol (THC) and also more than 40% cannabidiol (CBD). Such a fraction can be recycled in the centrifugal partition step in order to obtain more of the desired BSHE product as described herein. “Waste fraction” as used herein is a fraction obtained from centrifugal partition chromatography which contains 10% or less cannabidiol (CBD) and is either recycled or discarded following the centrifugal partition chromatography step. Ralph Mechoulam coined the term ‘entourage effect’ to describe the inexplicable synergy that manifests when multiple naturally occurring compounds extracted fromCannabisplants are consumed in tandem. This effect is thought to be the result of multi-pathway activation and signaling from various nutrients in a hemp extract comprising more than just isolated CBD alone. The Full Spectrum Hemp Extracts (FSHE) resultant from processes described herein include multiple naturally occurring cannabinoids extracted from hemp plants. Such FSHE can also include, without limitation, additional naturally occurring phytonutrients such as essential fatty acids, flavonoids, terpenes, vitamins, and minerals such as, without limitation, omega-3 and omega-6 fatty acids, antioxidants, potassium, magnesium, iron, zinc, calcium, phosphorus, vitamin E, and other molecules. Broad Spectrum Hemp Extracts (BSHE) are produced by removing detectable amounts of THC from the FSHE while retaining other cannabinoids including CBD. BSHE also include some or all of the additional non-cannabinoid phytonutrients that are found in FSHE. Thus, to obtain the benefit of multiple types of cannabinoids plus other naturally occurring phytonutrients, one should use a FSHE or BSHE such as those produced by the processes described herein and not from a CBD isolate, which is only cannabidiol. Extracting cannabinoids and other phytochemicals from hemp plants is riddled with numerous challenges towards commercial viability of products. Firstly, phytocannabinoids are nearly insoluble in water; thus, they must be extracted using alcohols and/or nonpolar organic solvents. These solvents will also extract many impurities and other undesirables in addition to the desired molecules. The impurities, undesirable molecules, and solvents all need to be separated and removed from the desired cannabinoids and phytonutrients, which can be complicated, time consuming, and expensive. Some processes are insufficient in removal capabilities leaving impurities and sometimes harmful substances in their resultant extract. Conversely, in an effort to produce a quality product, other processes may remove many desired cannabinoids and phytonutrients in addition to the impurities etc. Either way, many of these processes were time consuming and suffered from inconsistent cannabinoid profiles between batches. Furthermore, many production processes ended with unsustainably low yields, leading to high prices for the generated extraction product oil. Applicant has discovered improved ways to extract cannabinoids and other phytonutrients from hemp plants and to purify such extractions via systems and processes that are more efficient, generate greater yields of desired key compounds, increase the diversity of desirous cannabinoids, terpenes, and other molecules found in the extractions, and providing greater consistency of the final product. The resultant high quality FSHE and BSHE are suitable for both personal and pharmaceutical use. Although our previous process worked well, it could be improved, especially with respect to throughput. Per the prior process, cannabinoids and other phytonutrients were extracted from frozen GM using ethanol as a solvent. Ethanol was removed from the extracted materials using a horizontal wiped evaporator. The result of ethanol removal was a black tarry substance, which upon distillation yielded hemp oils. The black tarry substance clung to glassware and other equipment, was difficult to work with, and removal oftentimes caused damage to glassware and equipment with physical removal. In fact, it could take up to two weeks to clean the substance from glassware and other equipment as compared to extraction/purification, which took about one day. Furthermore, yield was incomplete as cannabinoids, etc. were lost in the black tarry substance that was left behind on the glassware/equipment. Thus, the prior process could be improved at least because of the high running costs of materials and the time needed for cleaning between processing one batch of GM to then next, but also by dramatically increasing the yield. After much experimentation, we discovered a multi cannabinoid extraction and purification process that, at a minimum, decreased cost and waste while improving throughput, yield, consistency of results, and the variety of cannabinoids retained in the purified extract.FIG.1is an overview of an improved process (1) for producing a FSHE, andFIG.2is a block diagram of a system (200) on which the process (1) may be performed. Generally, the process (1) includes the following steps and associated system (200) components: preparing hemp GM for extraction (2,202), extracting soluble materials/substances from the prepared GM (3,206), distilling the extraction solvent to concentrate the extract (4,210), partitioning the concentrate into two phases based on solubility characteristics (5,212), distilling the nonpolar solvent from the upper phase of the partitioned concentrate to obtain a crude oil (6a,214), decarboxylating and degassing the crude oil (6b,216), and refining the crude oil to obtain a FSHE (7,218). The FSHE may undergo optional additional process (FIG.8) such as to produce a BSHE. The particulars of each of the forgoing steps are addressed in the ensuing paragraphs and associated figures. Preparing the GM begins with the grower. Specifically, it starts with the seeds planted and continues through harvest and packing. Plants used to produce the hemp extracts detailed herein are recognized by the United States Department of Agriculture (USDA) as “hemp” having less than 0.3% w/w THC. Moreover, the harvested source material should have less than 0.01 ppm pesticides and, after drying, a sustained water activity (a w) of less than 0.8, and preferably less than 0.7 to prevent mold growth. To assist growers in providing the best product possible, experiments were conducted emulating packing and/or weather conditions experienced by many growers. Generally, source material was packed at different rates and access to fresh air was controlled to mimic actual packed source material in barns. For the best results, it was found that source material should dry to have an a w below 0.8 before packing. Once the a w is below 0.8, mold growth is not observed when packed. Drying to have an a, below 0.8 took about 24 hours at room temperature if source material is loosely packed. Weather may also affect source material a w especially if hung in barns and threshed. From experiments designed to test hemp source material a w in wet weather, it was determined that one or two days of rain did not detrimentally affect the a w of the source material as it hung in a barn, but continuous rain over a few days raised the a w above acceptable limits, increasing susceptibility to mold growth. With the forgoing in mind, growers may plan for optimal growing, harvesting, and packing of source material to initially obtain an acceptable a w and to maintain that a w while the source material is in their hands. Overall yield depends not only on the quality of the source material, but also upon the degree to which the desired molecules are removed from green material (GM) and retained without loss. Green material prepared for extraction generally refers to the parts of the hemp plant that produce the most cannabinoids such as flowers/inflorescence and leaves. The steps of preparing hemp GM (2) and extracting substances (3) from the hemp GM are detailed in method (300), which is outlined inFIG.3. The method (300) may include inspecting the GM from the grower (302) before extraction such as by visual inspection for mold or other infestations and by testing, if not previously done. For example, the GM is tested to ensure that it contains less than 0.3% w/w THC and less than 0.01 ppm pesticides, and that is has an a w below 0.7. Dried GM is chopped/ground to about 0.25 inches and weighed to a predetermined weight. Also referring toFIG.2, the prepared GM is loaded (304) into an extractor drum (206) via a box dumper and auger conveyer (202). During loading, the prepared GM may need to occasionally be tamped down to fit the entire predetermined weight into the extractor drum (206). The extractor drum (206) is fitted with a mesh screen (208) to create a false bottom and to help keep particulate GM from the extraction solvent that collects at the bottom of the extractor drum (206). The extraction solvent is also prepared (306) before use by cooling it to a temperature at or below freezing. Generally, the extraction solvent is cooled in a solvent tank (204) having a jacket through which a chilled fluid is pumped. In this way, the extraction solvent is chilled to a temperature between about 0° C. and about −20° C. before use. When the extraction solvent is at a desired temperature, it is pumped into the extractor drum (206) until it reaches a desired volume, but at least as much as is needed to saturate the GM within the extractor drum (206). In a preferred embodiment, the extraction solvent is completely denatured alcohol formulation CDA-12A-1(completely denatured alcohol) from Greenfield Global, which comprises 5% heptane and 95%200proof ethanol, although embodiments are not limited thereto. When the extractor drum (206) is filled with the appropriate amounts of prepared GM and extraction solvent, the GM is run through a first extraction cycle (310). Generally, the extraction solvent flows through the GM and collects in the false bottom of the extractor drum (206). From there, the extraction solvent is pumped back to the top of the extractor drum (206) to percolate through the GM and collect in the false bottom where it is again pumped back to the top of the extractor drum (206). The cycling of the extraction solvent continues for about one hour at atmospheric pressure. Thereafter, the extract from the first extraction cycle is collected (312) by being pumped through a fine mesh filter to remove any particulate GM and into a holding tank. The GM in the extractor drum (206) may be subject to a second extraction cycle (314), although it is not required. If a second extraction cycle (314) is desired, the extraction drum (214) is filled with cold extraction solvent to a volume that is about ⅓ to ½ of the volume used for the first extraction cycle (310). The second extraction cycle (312) circulates the extraction solvent in the same way as the first extraction cycle for at least about an hour to extract any additional soluble material. If multiple extractions are performed, the extractions from each can be combined from each extraction step. Thereafter, the extract from the second extraction cycle (314) is pumped out to the holding tank and the GM is discarded. The extraction solvent including materials/substances from the GM dissolved therein is generally referred to as the extract. The extract contains all soluble substances extracted from the GM including, without limitation, waxes, sugars, cellulose, chlorophyll, dirt, cellular debris, and of course, cannabinoids and other phytonutrients. Since the purpose of the extraction step (3) is to extract hemp oils (comprising cannabinoids) from hemp GM, improvements at this stage will improve yield. Thus, the foregoing method for extracting substances from hemp GM was developed by changing certain parameters and examining yield. Since impurities in the black tarry substance seem to contribute to the difficulty in working with the tarry substance, different extraction solvents were examined to see if impurities could be left behind (i.e., not extracted) while maintaining, or preferably increasing, yield. Specifically, yield was examined when hexane, ethanol, and completely denatured ethanol were used as extraction solvents. Hexane produced suboptimal results. When extracting with ethanol, yield was better, and extraction with completely denatured alcohol yield was the best. Furthermore, chilled completely denatured alcohol (as described above) gave the best yield overall even though the black tarry substance remained an issue. Other parameters were tested to see if their effect on yield. Surprisingly, the size of prepared GM influenced outcome. This is especially true when the size of the GM was considered in combination with the false bottom of the extractor drum (206). Again, in an effort to keep as much debris, impurities, and other contaminants out of the extract as possible, filtering the extract through a natural material such as carbon, diatomaceous earth, or sand, was examined as was filtering through one or more synthetic filters. The synthetic filters, such as the one (208) creating the false bottom and or the ones through which the extract is pumped to the holding tanks, sufficiently kept particulate GM out of the extract. Furthermore, it was found that the optimal size of the GM in combination with the synthetic filters is about 0.25 inches. This size allows for complete extraction of desired cannabinoids and other phytonutrients without being so small to generate losses due to fine cutting or grinding, which would also necessitate a more expensive and cumbersome filtering of particulate GM from the extract. Another parameter examined during extraction redevelopment was the necessity of freezing the GM before extraction. Yields compared from extracts of frozen GM and room temperature GM showed that freezing the GM before extraction does not improve efficiency. The combination of the forgoing results decreased cost and time by omitting a freezing of the GM and filtering the extract through a natural material. It also increased our yield by allowing more quantities and types of cannabinoids to be extracted with completely denatured alcohol. The surprising result, however, was finding that the size of the prepared GM from which the cannabinoids are extracted greatly improved yield. The next step (4) of production process (1) begins the purification aspect by distilling the extraction solvent from the extract to form a concentrate. The concentrate contains all soluble extracted materials regardless of desirability. In other words, this step primarily removes just the extraction solvent. The removed extraction solvent is collected for reuse whereas the resultant concentrate undergoes additional processing. Referring theFIG.2, the holding tank (not shown) in which the extract from the previous step is stored connects to an evaporator (210). The evaporator (210) uses plate heat exchange technology to evaporate and condense the extraction solvent. As a result, the condensed extraction solvent is separated from the concentrated extract or concentrate. Referring to the method (400) detailed inFIG.4and more specifically, the extract is fed (402) from the holding tank to the evaporator (210), and particularly through a plate heat exchanger at a pressure of between about 5 and 10 psi. Steam is supplied to the plate heat exchanger at a pressure of between about 20 and 30 psi. The steam heats the extract to a temperature above about 160° F. and preferably at about 190° F., which forces the extraction solvent to vaporize (404). A temperature of between about 160° F. and 190° F. must remain fairly constant during extraction solvent vaporization and condensation. Thereafter, vaporized extraction solvent passes through a condenser set at about 70° F. to cause the vapors to condense (406). The condenser liquid can be any source, however, a 50:50 mix of propylene glycol:water is useful for this purpose. The condensed extraction solvent is collected for reuse (408). When little extraction solvent remains, the temperature of the concentrate circulating in the evaporator (210) is allowed to reach between about 240° F. to about 260° F. This temperature causes remaining extraction solvent to vaporize and condense and at least some of the remaining cannabinoids to decarboxylate. The warm concentrate is pumped (410) to a holding tank (not shown). Notably, the heat and condensing components of the evaporator (210) should be set before distillation begins and a backpressure of about 5-10 psi should be applied to the heated plate exchanger to prevent distillation inside the plates. The pressure of the holding tank in which the extract is contained is at atmospheric pressure. After the extraction solvent and concentrate are removed from the evaporator (210), additional ethanol or completely denatured alcohol can be used to remove accumulated concentrate from the evaporator (210). The next step in the process (1), the partitioning step (5), is detailed in the method (500) ofFIG.5. This step was newly created for the purification part of the process (1) and is designed to reduce the difficulty in working with the concentrate, which is the black tarry substance. As water soluble impurities in the concentrate are largely responsible for the difficulty in working with the concentrate, and the attempt to diminish extraction of water-soluble impurities with hexane (directly from GM) did not result in a sufficient yield, experiments were performed to see if water soluble impurities could be removed from the extract without a loss in yield. Initial experiments focused on liquid/liquid extraction using water and hexane on the extract, which includes the extraction solvent. The idea behind liquid/liquid extraction being that hydrophobic materials/substances will dissolve and separate from hydrophilic materials, which could be removed with an aqueous phase. After testing various ratios of hexane, extract, and water, it was found that the best ratio for separating components in 2 phases without loss of yield was 5:1:1 (hexane:extract:water). This ratio also made purification at a subsequent distillation step much easier and overall cleaning faster and easier. An optimal time need for phase separation was also determined. With continued experimentation, it was discovered that better results were achieved when the extraction solvent was removed (e.g., step4FIG.1;FIG.4,400) before the liquid-liquid extraction. Namely, it was found that when partitioning the extract, CBDA gravitated to the aqueous phase and was being removed with the water-soluble impurities. CBDA is the precursor to CBD, thus its retention is paramount to yield. Several unexpected advantages came with removing the extraction solvent before partitioning. One was that the extraction solvent could be recovered and reused instead of disposing of it as waste. As a consequence, the waste produced by partitioning was cleaner being primarily water, which also decreases cost of disposal. More importantly, however, was the realization that the heat from the evaporator (210) causes CBDA to decarboxylate to CBD. Since CBD dissolves more readily in hexane than in water, when more CBD is generated (due to the decarboxylation of CBDA) going into the partition step, yield dramatically increased. Thus, to ensure that CBDA is decarboxylated to CBD, the previous step (step4/FIG.4), was modified to force decarboxylation of CBDA (410). Specifically, circulating the concentrate in the evaporator (210) at temperatures at about 240° F. to about 260° F. will cause at least a portion of extracted CBDA to decarboxylate. Another advantage of removing the extraction solvent before partitioning is that more concentrate than extract can be added for each batch partitioned due to the loss of extraction solvent volume. In turn, less time and energy are spent on the partitioning step. Since the concentrate is more suitable to partitioning than the extract, solvent ratios and other parameters also had to change. For example, to get the best yields, it was discovered that there is a certain sequence in which the solvents and concentrate should be mixed, time needed for the phases to separate, and that the aqueous phase could be collected and repartitioned. Additionally, it was found that the connection between the evaporator (210) and the partition funnel (212) could be flushed with ethanol, which can be added to the partition funnel (212) for even more cannabinoid recovery. Referring toFIG.2andFIG.5together, potable water is added to the partition funnel (212) before anything else (502). There should be enough water so that the final ratio of water to concentrate to hexane optimally is 1 part water, 2 parts concentrate, and 2 parts hexane. There is flexibility, however, such that hexane may range from about 2 parts to 5 parts, concentrate may range from about 2 parts to 5 parts and water may range from about 1 part to 5 parts. Thereafter, concentrate is pumped from the evaporator (210) to the partition funnel (212,504). The concentrate and water should mix as the concentrate is pumped into the partition funnel (212). Thereafter food grade hexane (98% pure) is added to the partition funnel (506). The partition funnel is set to circulate the hexane, concentrate, and water at atmospheric pressure for between 1 and 20 minutes, and preferably between 5 and 10 minutes (508). A longer run time is acceptable, but not necessary. Circulating allows the phases to mix and for the extracted materials to dissolve in either the aqueous phase or the hexane phase. For example, water soluble impurities such as sugars, cellulose, chlorophyll, and the like will dissolve in water and hydrophobic materials such as oils (e.g., containing cannabinoids), waxes, and the like will dissolve in hexane. After mixing, the two phases (i.e., hexane/hydrophobic, and water/aqueous) should separate after about 60 minutes (510). A longer duration may be used but is not necessary. A shorter duration for separation may be effective, but risks losing some of the yield. After the phases have separated, the water (and water-soluble material) is drained into a holding container (512). The hexane layer with soluble material dissolved therein is pumped (514) to the falling film evaporator (214). Optionally, for example if the separation of the two phases was not clear, the collected water may be repartitioned by adding it back into the partition funnel (212) and reprocessing. For the second partition process, however, an equal amount of hexane and water are added to the partition funnel (212) before repeating the partitioning. Repartitioning the aqueous phase may increase cannabinoid yield, especially if CBDA was not fully converted to CBD via evaporator (210). The second partition may recover any CBDA that may have been lost in the aqueous phase. As mentioned, the connection from the evaporator (210) to the partition funnel (212) may be flushed with a small amount (e.g., 5 liters) of ethanol, which can be added to the contents of the partition funnel soon after the concentrate is added to the partition funnel (504,506). The ethanol will separate out with the aqueous layer without affecting yield. The addition of the partitioning step (5) to the purification process tremendously increased throughput by diminishing the extensive cleaning time from about two weeks to less than 3 hours. It also increased throughput by rendering a cleaned partitioned concentrate that did not need extensive cleaning thereafter. This step (5) also improved a capacity constant by improving the flow and reducing the cleaning time. Equipment was capable of more up-time thus improving capacity without added complexities and hours of operating. The most significant benefit obtained by including the partitioning step (5) into to the overall process was increase in yield; quantities of FSHE were greater than before and cannabinoids in the FSHE previously lost were now retained. That is, without this step (5) only about 50-70% of cannabinoids were recovered; with this step (5) there is a 30% increase of cannabinoid recovery. See, e.g., Table 2. The overall process (1) was further modified to remove hexane from the partitioned concentrate. Generally, hexane is removed via distillation (6a). In an embodiment, hexane is vaporized in an evaporator such as a falling film evaporator (214) and condensed via plate heat exchanger. Hexane removal from the partitioned concentrate results in a crude oil. The step (6a) of removing hexane from the partitioned concentrate is detailed in the method (600) ofFIG.6. To ensure hexane vapors condense properly, a chiller is turned on and set to about 50° F. before the partitioned concentrate is pumped (602) to the falling film evaporator (214). The heating element of the falling film evaporator (214) applies steam to heat the partitioned concentrate to a temperature of from about 160° F. to about 200° F., until the hexane vaporization occurs (604). The steam amount is adjusted to ensure that the chiller compressor operates at less than 100% and preferably at 60%. Hexane vapors condense (606) via the plate heat exchanger, which utilizes chilled water to reduce hexane vapor temperature. The partitioned concentrate cycles through this system until hexane is completely distilled based on visual assessment. While cycling, the evaporation temperature can reach as high as about 275° F. The chilled water, however, should not rise more than a few degrees about the 50° F. set point. Thus, the steam to the evaporator should be monitored and adjusted accordingly. Once the hexane has visually stopped distilling, a vacuum may be applied to the system (608). Generally, the vacuum will remove any remaining hexane that was not removed via distillation at atmospheric pressure. Steam flow to the evaporator (214) is decreased and a low pressure vacuum is applied (e.g., <10 torr). Pressure is gradually increased to between about 20 and 25 torr. Steam flow may also be increased if possible. Hexane distillation under vacuum continues for about an hour. Thereafter, the resultant crude oil is pumped out for degassing in a vacuum oven (610, step6b). The vacuum oven (216) reduces the pressure even more than that of the falling film evaporator (214), which evaporates residual solvent from the crude oil. The temperature in the vacuum oven is set to about 310° F. and the vacuum is set to less than 2 torr. At this temperature and pressure, trace solvents are evaporated off of the crude oil. Moreover, carboxylic acids, including cannabinoids in their carboxylic acid forms are decarboxylated, if not already decarboxylated at a prior step. Certain light terpenes may also evaporate in the vacuum oven (216). Crude oil is left in the vacuum oven until visual bubbling has stopped (612). Typically, this is from between 1 minute to 6 hours, and preferably, for less than about 3, 2, or 1 hour. Degassing in the vacuum oven is also a step newly developed for the purification process. Before adding this step (6b), degassing occurred during subsequent distillation. It was found that degassing before subsequent distillation improved yield. Moreover, degassing during subsequent distillation potentially allowed heavy metals to escape into the final product, which is a source of unacceptable contamination. Thus, degassing (step6b) in the vacuum oven (216) has at least a twofold benefit: increased yield and decreased contamination of the end product. After degassing (6b), the crude oil is ready for refining (7). Refining removes any remaining impurities such as chlorophyll, solvents, heavy metals, and the like. Refining is accomplished by at least one, preferably two, and optionally three passes through a wiped film short path still (218). The first pass separates volatile compounds such as terpenes from the crude oil and the second pass separates cannabinoids from less volatile impurities. If desired, impurities collected from the second pass may be subjected to a third pass to evaporate and separate any remaining cannabinoids from the impurities. Still parameters are adjusted for each pass to obtain optimal results. See Table 1, below. Generally, to refine a substance in the wiped film short path still (218), the substance may be heated before or as it enters a head of the still (218). The still wall is heated by either a heated jacket or electrically. Heating causes the substance to flow into the still (218) to run down the inside of the wall. As the substance runs down the wall, it spreads into a thin layer by rotating wipers. The still (218) is also kept under a vacuum. Thus, the heat and the vacuum cause volatile components to evaporate and condense against an internal condenser. The condensate at the internal condenser is a distillate. Whatever has not evaporated and condensed exits the still (218) as a residue. Both the distillate and the residue may be collected. In an embodiment, the still may be connected to an external condenser (not shown) which will condense vapors that have flowed upward and out of the top of the still. If vapors pass the external condenser, they may condense in a cold trap that lies between the external condenser and a vacuum pump. Parameters for each pass of the wiped film short path still (218) are as follows: TABLE 1WIPED FILM SHORT-PASS STILL PARAMETERSInternalExternalVacuumInfeedWiperHeaterCondenserCondensergas trapPressureTemperatureBlades(° C.)(° C.)(° C.)(° C.)(Torr)(° C.)(RPM)First150−15 to −5−15 to −5−95 to −85<260260-300PassSecond170-18570-90−15 to −5−95 to −85<50060260-300PassmTorrThird180-19070-90−15 to −5−95 to −85<50080260-300PassmTorr Referring toFIG.7, the first pass through the still (218) is called the “terpene pass” because certain terpenes and other highly volatile molecules are removed during this pass. When crude oil is fed (702) into the still (218) that set as indicated in Table 1, terpenes, some impurities, and the like evaporate and condense as the “first distillate” (704). What has not evaporated and condensed is the first residue, which is a more refined crude oil. The first residue is collected (706) and may optionally be dissolved in canola oil (10:1) before being run through a second distillation (708) on the same still or a sequential still. For the second pass distillation, the still is heated to a higher temperature and is set at a lower pressure (Table 1) since the higher pressure terpenes have already been removed in the first pass. The internal condenser is also at a higher temperature (Table 1). These changes to still parameters force cannabinoids to evaporate and condense inside the still leaving behind heavier, less volatile impurities. That is, within the still, cannabinoids evaporate from the first residue and condense on the internal condenser to flow out of the still as the second distillate (710). The heavier, less volatile materials do not evaporate and exit the still as a second residue (712). The second distillate is a refined oil comprising a FSHE, which is collected. Any cannabinoids remaining in the second residue may be extracted via an optional third pass through the same still or a sequential still. Before running the second residue through a third pass distillation, the second residue is mixed with canola oil (714) in a ratio of 10:1 (second residue: canola oil) to thin the second residue for easier distillation. On the third pass through the still, the feed is pre-warmed to a higher temperature and the still is heated at a slightly warmer temperature as comparted to the second pass through the still (see Table 1). The remaining settings are essentially the same as with the second pass. During this third pass (716), any remaining cannabinoids evaporate and condense against the internal condenser to be collected as the third distillate (718) which is an oil comprising FSHE and the third residue is collected to be disposed of as waste (720). The third distillate is added to the second distillate as they are both refined oil comprising FSHE extracted from the same starting materials (716). Generally, the second distillate and third distillate are passed to a heated tank (220) where they are mixed into a homogenous mixture and collected into glass jars for quality testing. An independent lab tests the FSHE for pesticides, heavy metals, microbes, residual solvents, and mycotoxins. The independent lab also provides a cannabinoid profile determined by liquid chromatography diode array detector (LC-DAD). An exemplary cannabinoid profile resultant from the forgoing extraction and purification process (1) is summarized in Table 2. TABLE 2EXEMPLARY CANNABINOID PROFILE OFFSHE AS DETECTED BY LC-DADCannabinoidmg/g%Δ8-THCNDNDΔ9-THC25.472.547Δ9-THCANDNDTHCVNDNDTHCVANDNDCBD799.4379.943CBDANDNDCBC18.891.889CBCANDNDCBDV7.790.779CBG16.701.670CBGANDNDCBN1.650.165Total THC25.472.547Total CBD799.4379.943Total Cannabinoids869.9486.994Sum of Cannabinoids869.9486.994where ND means not detected; total THC = Δ9-THC + (0.877 × Δ9-THCA); total CBD = CBD + (0.877 × CBDA); and total cannabinoids = Σ(neutral cannabinoids) + [0.877 × Σ(acidic cannabinoids)] Notably the cannabinoid profile shown in Table 2 is a profile from an actual FSHE production lot. Quantities and types of cannabinoids in a particular production lot may differ. Even so, most FSHE lots have a profile that is similar to that shown in Table 2 with quantities ranging from about ±25% of the quantities listed in Table 2 for minor elements (below 5% by weight, and ±10% for the major component, CBD. Moreover, it is not unusual to extract and recover small amounts of other cannabinoids such as THCV and CBL, which may be included at up to 2.5% each. In certain applications, the FSHE is desired; other applications, however, require a Δ9THC-free hemp extract. Thus, the FSHE produced by the extraction and purification method (1) may be further processed to produce a BSHE, which is Δ9 THC-free, meaning below the level of quantitation. Other aspects of a B SHE, such as the cannabinoid profile, are substantially similar to the FSHE from which it was produced. The primary steps for producing BSHE from FSHE include (i) separating THC from other cannabinoids using Centrifugal Partition Chromatography (CPC), and (ii) distilling solvent from THC-free fractions obtained via CPC. These steps are detailed inFIG.8as process (8). CPC is a type of chromatography that uses two liquids, one as a stationary phase, and the other as a mobile phase. The stationary phase is immobilized by a strong centrifugal force and the mobile phase moves through the stationary phase to separate different molecules. This technique typically requires a two-phase mixture of solvents. Different compounds can be separated based on the components of the particular solvent system and the partition coefficients of the molecules in the selected solvent system. Thus, by using two different solvents together with CPC, similar substances can be separated. Generally, using CPC, as one liquid is introduced into the machine, another exits from the CPC machine. Molecules having different partition coefficients will come off of the CPC machine at different times and/or in different fractions due to separation through the stationary phase in the CPC machine. The solvent system used to separate THC from other cannabinoids includes a nonpolar solvent (e.g., hexane) and two polar solvents (e.g., methanol and water). The ratio of these solvents is important as is the density of the non-polar solvent mixture. For freshly prepared (802) solvents, the ratio of hexane to methanol to water is 5:4:1. Per the BSHE production process (800) solvents can be reused (812,814to802). Nevertheless, the ratio of the solvents (5:4:1) should be maintained for proper THC separation. Generally, collected used solvents are allowed to separate in a container where they will form an upper layer that is primarily hexane, and a lower layer that is primarily water and methanol. After separation, the two layers are transferred to respective containers. The density of the lower phase is adjusted by adding water and/or methanol (as indicated by the particular instance) until the density is about 0.852 g/cm3at 70° F. To prepare FSHE for CPC separation, the desired amount of FSHE is warmed in an oven until it is fully dissolved. The warmed FSHE is added to the upper phase (e.g., hexane) such that the ratio of hexane to FSHE is about 2:1 (804). The FSHE should be completely dissolved in the hexane before being loaded into the CPC machine. At this point the solvents and sample are ready to load into the CPC machine. In brief, the CPC machine is filled with clean upper phase mixture of water and methanol and the centrifuge within the CPC machine starts rotating. Thereafter, the FSHE dissolved in hexane is loaded into the CPC machine and allowed to separate (806). Additional lower phase is added during FSHE separation. Thereafter upper phase is added to the CPC machine. Meanwhile, fractions coming off of the CPC machine are collected (808). Fractions can be monitored via a UV absorbance spectrum. At the beginning and the ending of a CPC run, where cannabinoids are not expected to come off the machine, the UV absorbance spectrum is low. In contrast, the presence of cannabinoids will cause the UV absorbance spectrum to show high points. This absorbance spectrum lets an operator of the CPC machine know when cannabinoids are coming through the system. Cannabinoids without THC have a different absorbance spectrum than those with THC, so it is possible to estimate which fractions are THC-Free and which are not. Thus, per the solvent system, sample loading, and CPC parameters, fractions can be divided into three categories: Waste, THC-free, and Reclaim. Simplified parameters of the CPC run method are outlined in Table 3. Since non-THC cannabinoids (e.g., CBD) dissolve better in the lower phase than THC, they come off the CPC machine first. Conversely, as THC dissolves better in the upper phase, it will come off the CPC machine later in the process. Reclaim is a mix of THC and CBD that can be separated in a second pass through the CPC. TABLE 3TIME, INPUT, OUTPUT, AND FLOW PARAMETERSFOR AN EXEMPLARY CPC RUNTimeInput SourceFraction/OutputFlow(min)(% Infeed)Type(mL/min)0100 Lower PhaseWaste2500.2100 SampleWaste1501.5100 Lower PhaseReclaim2506100 Lower PhaseTHC-free25012100 Lower PhaseReclaim25013100 Upper PhaseReclaim25016100 Upper PhaseWaste250 Before Reclaim fractions can be rerun on the CPC, the solvent is first removed by one or more distillation processes (810). As a nonlimiting example, Reclaim fractions may have solvent distilled off in the falling film evaporator (214), the wiped film short pass still (218), or both. These distillations are essentially the same as was described for FSHE purification. Thereafter, the resultant oil (e.g., Reclaim oil) may be rerun in the CPC machine. The run of Reclaim oil is similar to that of the initial run except that the ratio of hexane to Reclaim oil is 2.5 (hexane) to 1 (Reclaim oil) and only two fractions collected: THC-Free and Waste. Before continuing with removal of solvent, THC-Free fractions are internally verified to be truly THC-Free. If not, the fractions can be rerun through the CPC (e.g., as Reclaim fractions) and reverified as being THC-Free. The THC-Free fractions are combined for solvent removal via a horizontal wiped film evaporator. To remove methanol from the THC-free fractions using the horizontal wiped film evaporator (812), a heating jacket is set to heat the evaporator from about 60° C. 100° C., or higher, so long as below methanol's flash point, and at full vacuum, which is about 100 torr. As the THC-free fractions move into the evaporator, they are spread into a thin layer by the wipers, to enhance solvent (e.g., methanol) evaporation. The solvent vapors move to the condenser, which is set at 4° C. to condense the vapors back to liquid solvent. As was previously mentioned, the distilled solvent can be reused in the CPC. After about 4 hours, the undistilled portion containing concentrated THC-Free cannabinoids is collected. As solvent may still be present in the concentrated THC-Free portion, a second distillation is performed on a rotovap evaporator (814), with the bath set to at least about 100° C. under the strongest possible vacuum to distill off the remaining methanol/solvents. Vaporized solvent is condensed by a condenser set to about −20° C. After the remaining methanol is removed from the concentrated THC-free portion, the contents, which includes water, are allowed to cool for at least 1 hour, but a longer time will not affect yield. Once cooled, water and the THC-Free concentrate can be separated on the evaporator (816). To do so, the vacuum is turned on, but not the heat. The vacuum causes the oil to clump and separate from the water. After being under vacuum for at least 1 hour, water and the THC-Free product should be fully separated. Water is removed from the THC-Free product and the THC-Free product is returned to the evaporator to distill off the remainder of the water (818). For example, at a temperature of about 100° C. and a vacuum pressure of about less than 150 millitorr, water will evaporate away from the THC-Free product. When the THC-Free oil changes from cloudy to clear and there is little to no bubbling of the oil, the water has been distilled off and the THC-Free product, which is a BSHE (820), is sent to an independent lab for testing. TABLE 4EXEMPLARY CANNABINOID PROFILE OFBSHE AS DETECTED BY LC-DADCannabinoidmg/g%Δ8-THCNDNDΔ9-THCNDNDΔ9-THCANDNDTHCVNDNDTHCVANDNDCBD898.4989.849CBDANDNDCBCNDNDCBCANDNDCBDVNDNDCBG17.141.714CBGANDNDCBN1.650.165Total THCNDNDTotal CBD898.4989.849Total Cannabinoids915.6391.563Sum of Cannabinoids915.6391.563where ND means not detected; total THC = Δ9-THC + (0.877 × Δ9-THCA); total CBD = CBD + (0.877 × CBDA); and total cannabinoids = Σ(neutral cannabinoids) + [0.877 × Σ(acidic cannabinoids)] Notably, the cannabinoid profile shown in Table 4 is a profile from an actual BSHE production lot. Quantities and types of cannabinoids in a particular production lot may differ. Even so, most BSHE lots have a profile that is similar to that shown in Table 4 with quantities ranging from about ±10% of the quantities listed in Table 4. Moreover, it is not unusual to extract and recover small amounts of other cannabinoids such as CBC, CBG, and CBL. Table 5 below provides an acceptable variance for elements within the BSHE. TABLE 5BSHECannabinoidmg/g%Δ8-THCND0-1Δ9-THCND0-0.1Δ9-THCAND0-0.3THCVNDNDTHCVANDNDCBD90070-99CBDAND0-2.5CBCND0-3.5CBCAND0-5.0CBDVND0-2.5CBG150.1-3.5CBGAND0-3.5CBN2.00.01-0.5Total THCND0-1.5Total CBD898.4970-99Total Cannabinoids915.6371-99Sum of additional00-10Cannabinoids Table 6 below provides an acceptable variance for elements within the FSHE. TABLE 6FSHECannabinoidmg/g%Δ8-THCND0-3.0Δ9-THC250.01-5.0Δ9-THCAND0-1.0CBD80065-98CBC190-3.5CBDV80-2.5CBG170.1-3.5CBN1.650-0.5Total THC25.470.3-5.0Total CBD799.4365-98Total Cannabinoids869.9465-99.9Sum of additional00-10.0Cannabinoids A simplified approach to the formulations is that the BSHE includes between 60-95% of a CBD, Δ9-THC of 0-0.1%, but preferably not detectable, and additional cannabinoids between 0.1 and 20%. Whereas, the FSHE includes 0.01 to 5% THC, but preferably between and 0.3 as required in certain jurisdictions. Additional elements include between 0.1 and 20% of waxes and fatty acids. Either of the BSHE or FSHE once made, can now be utilized in a number of ways. Unfortunately, each of the BSHE and FSHE have a bitter flavor for oral mucosal or oral dosing, and so it is optimal to add the BSHE or FSHE to a suitable oral carrier for such dosage forms. Oils such as long chain triglyceride (LCT) oils or medium chain triglyceride oils (MCT) oils are readily available. In a preferred embodiment, the FSHE is added v/v into a mixture of cold pressed hemp seed oil, coconut oil, or combinations thereof. This makes the resulting oil palatable for oral administration. Typically, the FSHE is added at about 1-10% v/v with cold pressed hemp seed oil and the MCT oil (e.g., coconut oil) each at between 10 and 99% v/v. A flavoring can be added at about 0.1% to 5.0% v/v. Flavors include well known examples including but not limited to citrus flavors, fruit flavors, mint or wintergreen, and the like, which may be natural or synthetic. In certain embodiments, the FSHE or BSHE can be added to the carrier at up to 99% of the total volume or weight of the composition, inclusive of all ranges from 1-99%. A preferred embodiment is made by combining the BSHE or FSHE w/w into a mixture of cold pressed hemp seed oil and an MCT oil, and further comprising a terpene blend. In other embodiments, the BSHE or FSHE are added only to an MCT oil, or only to a cold pressed hemp seed oil. In preferred embodiments, a composition comprises between 40 and 70% cold pressed hemp seed oil, and between 30 and 50% MCT oil, and between 1 and 20% of a BSHE or FSHE. More preferably, the embodiment further comprises a terpene mix or other mixture to improve the profile of the composition. In certain embodiments, a terpene blend is included at between 0.1 and 2.0 v/v. A particular blend may comprise one, two, three, or more terpenes. A terpene blend may include β-myrcene, β-caryophyllene, linalool, α-pinene, citral, D-limonene, and/or eucalyptol. A particular terpene blend comprises a terpene profile including β-myrcene at 15-25%, β-caryophyllene at 15-25%, linalool at 5-15%, α-pinene at 5-15%, citral at 15-40%, D-limonene at 10-30%, and eucalyptol at 0.1-5%. In preferred embodiments, the terpene blend includes at least one, two, three, four, five, six or all seven of the recited seven terpenes. Preferably, the range of each of the terpenes is from between about 0.1% to about 50% of the total volume of the terpene blend. Most preferably, the terpene blend comprises at least 5 terpenes with each terpene representing a concentration of no more than 40% of the total volume of the terpene blend. A composition comprising the FSHE, cold pressed hemp seed oil, coconut oil, and a terpene blend is optimized for use as a tincture for oral administration. In preferred embodiments, a flavor is further added at between 0.1% to 2.0% v/v of the composition. Such application is provided for sublingual application wherein the FSHE and cannabinoids are intended for uptake through the oral mucosa. It is understood that the material is often eventually swallowed and that additional material is taken through the rear of the mouth, the esophagus as well as into the stomach and undergoes first pass metabolism. In certain embodiments, the FSHE is replaced by a refined BSHE. In further embodiments, the compositions may be formulated into a softgel, wherein the gel coating is produced to form as a dissolvable shell around a quantity of the composition. Such manufacture is well understood by one of ordinary skill in the art. In further embodiments, the composition is admixed into gelatin or another carrier, with the addition of one or more excipients, including but not limited to a flavorant, a sweetener, a color, for manufacture of a gummy product. For topical administration, the carrier may further include an emulsifying agent. In certain embodiments, a fat may be further included, such as shea butter, which can be utilized as the carrier in place of the MCT oil. In such embodiments, the composition comprises FSHE, cold pressed hemp seed oil, a second oil or fat, and optionally a terpene blend. In preferred embodiments, the second oil or fat is shea butter. In certain embodiments, the FSHE is replaced by a BSHE. Accordingly, a product may comprise between 1 to 50% of a BSHE or FSHE and between 99 and 50% of additional excipients, including the carrier and other components, which is suitable for topical administration. The oil, whether a BSHE or a FSHE is manufactured and bottled. In a subsequent processing step, the BSHE or FSHE may be encapsulated in a gelatin material for oral administration. Typical soft gelatin capsules comprise between 0.4 and 0.6 mL per capsule of the oils. The manufacture of the gelatin capsule may include further excipients necessary to make the gelatin shell. A further embodiment is directed towards a mucosal composition. In preferred embodiments, the mucosal composition may be intended for the oral mucosa, the nasal mucosa, the vaginal mucosa, or the rectal mucosa. In preferred embodiments, the mucosal composition is an intravaginal composition. The intravaginal compositions comprises a BSHE or a FSHE at between 1 and 99%, a carrier, preferably a fat or an oil. Additional excipients may be included for stabilizing the formulation and for modifying the flowability or character of the final product. A preferred fat is shea butter. The intravaginal composition may further comprise a terpene blend as detailed herein. In certain embodiments, the pH is modified, for example from a native pH of about 10.5 of the FSHE or BSHE to an acidic pH. An acidic buffer solution, comprising an appropriate conjugate acid and base can be utilized by one of ordinary skill in the art to make the pH acidic, such as between 3.5 and 6, and serve as the pH modifier. Osmolality modifiers may also be utilized, including the addition of salts, such as sodium chloride to the composition. In certain embodiments, a mucoadhesive agent is included, wherein the mucoadhesive agent is chitosan, hydroxyethyl cellulose, methyl cellulose, polyacrylic acid, poly vinyl pyrrolidone, poly vinyl alcohol, poly ethylene glycol, or combinations thereof. It will be appreciated that the embodiments and illustrations described herein are provided by way of example and that the present invention is not limited to what has been particularly disclosed. Rather, the scope of the present invention includes both combinations and sub combinations of the various features described above, as well as variations and modifications thereof that would occur to persons skilled in the art upon reading the forgoing description and that are not disclosed in the prior art. Therefore, the various methods, formulations, and compositions detailed herein may include one or all of the limitations of an embodiment, be performed in any order, or may combine limitations from different embodiments, as would be understood by those implementing the various methods and systems detailed herein. | 57,428 |
11857591 | DETAILED DESCRIPTION OF EMBODIMENTS Technical solutions of the present disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. Other embodiments made by those skilled in the art without sparing any creative effort should fall within the scope of the disclosure. Embodiment 1 20 parts by weight ofAndrographis herba,20 parts by weight ofIsatidis folium,10 parts by weight ofPolygonum hydropiper,10 parts by weight ofRheum palmatumL., 10 parts by weight ofGalla chinensis,10 parts by weight of taurine, and 20 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −20° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 2 Freshwater fish mainly rely on their non-specific immune system to protect themselves. The levels of lysozyme, immunoglobulin M, superoxide dismutase, malondialdehyde and total antioxidant capacity are directly related to the immune status of the body. The activities of aspartate aminotransferase and alanine aminotransferase can reflect the health status of liver. Intestinal health can be tested by the activities of digestive enzymes such as trypsin, lipase and amylase. Therefore, the embodiment evaluated the immunity and organism health of the crucian carp through the above indicators. Growth test: nine crucian carp ponds (identical in size, density of crucian carp stocking, and mode of management) were randomly selected and divided into groups in Huangshagang area of Sheyang County, Jiangsu Province, China. Control group A (fed with a basic commercial feed); Control group B (supplemented with 0.5%Andrographis herbain the basic commercial feed); Control group C (supplemented with 0.5%Isatidis foliumin the basic commercial feed); Control group D (supplemented with 0.5%Polygonum hydropiperin the basic commercial feed); Control group E (supplemented with 0.5%Rheum palmatumL. in the basic commercial feed); Control group F (supplemented with 0.5%Galla chinensisin the basic commercial feed); Control group G (supplemented with 0.15%Andrographis herba,0.1%Isatidis folium,0.05%Polygonum hydropiper,0.05%Rheum palmatumL., 0.05%Galla chinensisand 0.1% diatomite in the basic commercial feed); Control group H (supplemented 0.15%Andrographis herba,0.1%Isatidis folium,0.05%Polygonum hydropiper,0.05%Rheum palmatumL., 0.05%Galla chinensisand 0.1% taurine in the basic commercial feed); Test group (supplemented with 0.5% of the compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity prepared in embodiment 1 in the basic commercial feed). Each group was fed once a day in the morning, middle and evening (07:00, 12:00, 19:00). Each group was fed continuously for 10 days/month, and the rest of the time was fed with the basic commercial feed. After 4 months of domestication, the samples were collected and the indexes were tested in late July (10 fish were randomly taken from each pond). The activities of lysozyme (LZM), immunoglobulin M (IgM), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in liver, and trypsin (TPS), lipase (LPS) and amylase (AMS) in intestine were detected. The test results are shown in Table 1. TABLE 1GroupControlControlControlControlControlControlControlControlTestIndexgroup Agroup Bgroup Cgroup Dgroup Egroup Fgroup Ggroup HgroupLZM11.9418.1614.1716.1114.0315.0722.2227.7833.33(U/ml)IgM1.321.491.421.461.511.411.912.102.32(mg/ml)ALT4.874.324.414.54.464.623.143.162.83(U/L)AST27.6220.7623.7422.823.8823.519.3920.6218.02(U/L)SOD904.5967.15961.15948.58945.06932.96957.16977.231058.29(U/mgprot)MDA21.7114.2714.1712.6916.5216.1712.7213.2511.09(nmol/mgprot)T-AOC0.10.150.150.140.150.130.160.180.19(mmol/gprot)TPS30017.4334885.1436154.9338673.5235504.6835221.4547104.2945049.6249158.97(U/mgprot)LPS1.631.922.062.161.892.222.552.643.10(U/mgprot)AMS72.7186.1486.8189.5186.3289.5191.1595.5108.77(U/mgprot) The results showed that: compared with the control groups A-H, the levels of lysozyme (LZM) in serum in the test group were increased by 179.15%, 83.54%, 135.22%, 106.89%, 137.56%, 121.17%, 50.00% and 19.98%, respectively; the content of immunoglobulin M (IgM) in serum in the test group increased by 75.76%, 55.70%, 63.38%, 58.90%, 53.64%, 64.54%, 21.47% and 10.48% respectively; the activity of alanine aminotransferase (ALT) in serum in the test group decreased by 41.89%, 34.49%, 35.83%, 37.11%, 36.55%, 38.74%, 9.87% and 10.44%, respectively; the activity of aspartate aminotransferase (AST) in the test group decreased by 34.76%, 13.20%, 24.09%, 20.96%, 24.54%, 23.32%, 7.07% and 12.61%, respectively; the activity of superoxide dismutase (SOD) in the liver of fish in the test group increased by 17.00%, 9.42%, 10.11%, 10.57%, 11.98%, 13.43%, 10.57% and 8.29%, respectively; the content of malondialdehyde (MDA) in the test group decreased by 48.92%, 22.28%, 22.28%, 12.81%, 32.87%, 31.42%, 12.81% and 16.30%, respectively; the total antioxidant capacity (T-AOC) in the liver of fish in the test group increased by 90.00%, 26.67%, 26.67%, 35.71%, 26.67%, 46.15%, 18.75% and 5.56%, respectively; the activity of intestinal trypsin (TPS) in the test group increased by 63.77%, 40.92%, 35.97%, 27.11%, 38.46%, 39.57%, 4.36% and 9.12%, respectively; the activity of intestinal lipase (LPS) in the test group increased by 90.18%, 61.46%, 50.49%, 43.52%, 64.02%, 39.64%, 21.57% and 17.42%, respectively; the activity of intestinal amylase (AMS) in the test group increased by 49.59%, 26.27%, 25.30%, 21.52%, 26.01%, 21.52%, 19.33% and 13.90%, respectively. Therefore, compared with the control groups, the compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity with seven components can improve the immunity and antioxidant capacity of freshwater fish, and promote the health of liver and intestine. Embodiment 3 Challenge test: after the 4-month growth test in embodiment 2, all groups of crucian carp were fasted for 24 hours, and the crucian carp with the same specifications were selected from each pond forAeromonas hydrophilainfection test. The challenge test was carried out in a plastic box (1.0 m×0.4 m×0.4 m), with three replicates in each group and 30 fish in each replicate.Aeromonas hydrophilawas provided by the fish disease laboratory of Jiangsu Freshwater Fisheries Research Institute. The final concentration ofAeromonas hydrophilawas about 5×107 cells/ml diluted with sterile normal saline. The physiological activities of fish were observed after intraperitoneal injection of 1.0 ml bacterial solution per 100 g fish. The cumulative mortality of nine groups of fish at 0 h, 12 h, 24 h, 48 h and 96 h after challenge was counted, respectively. The results are shown in Table 2. TABLE 2Cumulative mortality (%)Group0 h12 h24 h48 h96 hControl group A0.0010.0021.1141.1161.11Control group B0.003.336.6717.7838.89Control group C0.006.6710.0023.3348.89Control group D0.003.337.7817.7844.44Control group E0.005.5613.3322.2246.67Control group F0.004.4412.2218.8945.56Control group G0.002.225.5614.4431.11Control group H0.002.223.3313.3327.78Test Group0.001.112.227.7818.89 The results showed that after the crucian carp was infected withAeromonas hydrophila, the viability began to decrease, and the abdomen and gills of the dead crucian carp had bleeding symptoms in various degrees (the comparison of symptoms is shown inFIG.1-4). Compared to the control groups A-H, the mortality of test fish 12 h after challenge was reduced by 88.90%, 66.70%, 83.35%, 66.70%, 80.02%, 75.03%, 50.05% and 50.05%, respectively; the mortality of test fish 24 h after challenge was reduced by 89.48%, 66.70%, 77.80%, 71.46%, 83.35%, 81.84%, 60.04% and 33.40%, respectively; the mortality of test fish 48 h after challenge was reduced by 81.08%, 56.24%, 6%, 6.66%, 56.24%, 64.99%, 58.81%, 46.14% and 41.65%, respectively; the mortality of test fish 96 h after challenge was reduced by 69.09%, 51.43%, 61.36%, 57.50%, 59.52%, 58.53%, 39.28% and 22.00%, respectively. Therefore, the compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity with seven components can improve freshwater fish's ability to resistAeromonas hydrophilainfection and reduce death caused by pathogenic bacteria. Embodiment 4 15 parts by weight ofAndrographis herba,18 parts by weight ofIsatidis folium,15 parts by weight ofPolygonum hydropiper,12 parts by weight ofRheum palmatumL., 12 parts by weight ofGalla chinensis,10 parts by weight of taurine, and 18 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −20° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 5 25 parts by weight ofAndrographis herba,20 parts by weight ofIsatidis folium,10 parts by weight ofPolygonum hydropiper,8 parts by weight ofRheum palmatumL., 10 parts by weight ofGalla chinensis,8 parts by weight of taurine, and 19 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −20° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 6 23 parts by weight ofAndrographis herba,22 parts by weight ofIsatidis folium,14 parts by weight ofPolygonum hydropiper,9 parts by weight ofRheum palmatumL., 8 parts by weight ofGalla chinensis,9 parts by weight of taurine, and 15 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −20° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 7 21 parts by weight ofAndrographis herba,21 parts by weight ofIsatidis folium,12 parts by weight ofPolygonum hydropiper,11 parts by weight ofRheum palmatumL., 11 parts by weight ofGalla chinensis,7 parts by weight of taurine, and 17 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −15° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 8 19 parts by weight ofAndrographis herba,19 parts by weight ofIsatidis folium,13 parts by weight ofPolygonum hydropiper,12 parts by weight ofRheum palmatumL., 12 parts by weight ofGalla chinensis,9 parts by weight of taurine, and 16 parts by weight of diatomite were weighed. Each of the raw materials was crushed coarsely. Each of the crushed raw materials was pulverized by an ultra-micro crusher with wall breaking at −15° C. Each of the pulverized raw materials was sieved with a sieve of 300 mesh, and all the raw materials were mixed well to obtain a compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity. Embodiment 9 Following the method of the growth test in embodiment 2 and the challenge test in embodiment 3, the physiological and biochemical indexes and the cumulative mortality (only 24 h and 48 h after challenge) of crucian carp fed with the compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity prepared in embodiments 4-9 were determined, respectively. The control group was fed with the basic commercial feed, and each test group was fed with the basic commercial feed added with 0.5% of the compound Chinese herbal medicine composition for protecting liver, protecting intestines and enhancing immunity prepared in embodiments 4-9, respectively (test group A, test group B, test group C, test group D, and test group E, respectively). The results are presented in Tables 3 and Tables 4. TABLES 3GroupControlTestTestTestTestTestIndexgroupgroup Agroup Bgroup Cgroup Dgroup ELZM11.9429.1737.5536.2132.7630.55(U/ml)IgM1.322.252.752.542.162.37(mg/ml)ALT4.872.852.562.742.952.93(U/L)AST27.6220.1019.4720.0219.2519.72(U/L)SOD904.50992.481104.781099.45988.451033.51(U/mgprot)MDA21.7113.4610.9512.4412.6013.68(nmol/mgprot)T-AOC0.100.180.190.190.180.17(nmol/gprot)TPS30017.4349186.0648457.1249020.4549798.3450280.21(U/mgprot)LPS1.633.052.842.782.943.35(U/mgprot)AMS72.71107.51100.2498.24100.84109.39(U/mgprot) TABLES 4Cumulative mortality (%)Group24 h48 hControl group21.1141.11Test group A6.678.89Test group B3.335.56Test group C5.567.78Test group D6.678.89Test group E5.567.78 The general principle as defined herein may be achieved in other embodiments without departing from the spirit or scope of the disclosure. The present disclosure will therefore not be restricted to these embodiments shown herein, but rather to comply with the broadest scope consistent with the principles and novel features disclosed herein. | 14,138 |
11857592 | DETAILED DESCRIPTION OF THE INVENTION All of the features disclosed in this specification may be combined in any combination. An alternative feature serving the same, equivalent, or similar purpose may replace each feature disclosed in this specification. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. Example 1 Preparation of Radix Polygalae (Polygala tenuifoliaWilld) Extract PDC-1421 Dry whole root ofP tenuifoliaWilld is cooked in water twice with a ratio of 100 kg to 800 kg under refluxing for one hour and a ratio of 100 kg to 700 kg under refluxing for another one hour. An aqueous extract is obtained after mixing the two cooked mixtures. The aqueous extract is concentrated to 400 kg by evaporation in vacuo and filtered by centrifugal filter. Then, the resulting concentrate is filtered by the resin column for chromatography. The specific filtering eluent is collected. The eluent is concentrated by evaporation in vacuo and dried by spray drier to yield a powder product PDC-1421. Example 2 In Vivo Tetrabenazine-Induced Hypothermia Assay The around 70% of PDC-1421 in Example 1 are composed of sucroester (34%), saponin (18%) and xanthone (17%). Some active compounds of in the powder product of Example 1, including polygalatenosides A/B/C (2nd fraction 1), ploygalaxanthone III, sibiricoses A5, and 7-O-methylmangiferin are also discovered through in vivo tetrabenazine-induced hypothermia test (one of rodent models for evaluating antidepressant-like activity in mice). Imipramine (as the positive control), PDC-1421, 1st Fraction 10-19, 1st Fraction 33, 2nd Fraction-1 (wherein the major components are polygalatenosides A/B/C), 2nd Fraction-3, TMCA, Polygalaxanthone III, Onjisaponin B, Sibiricose A5, 7-O-Methylmangiferin and Sibiricaxanthone B are dissolved in water for injection (WFI) and administered by oral gavage. The dosing volume is 10 mL/kg. Test substances and vehicle (WFI) are administered by oral gavage to groups of 8 ICR male mice weighing 23±3 g for 60 minutes, and then tetrabenazine (TBZ, 85 mg/kg) is injected intraperitoneally. Body temperature is recorded before (0 min), and 60, 90 and 120 min after TBZ challenge. The temperature recovery (%) of test substances is shown in theFIG.1. Therefore, drug-induced hypothermia (e.g., TBZ and reserpine) animal model is a typical experiment to evaluate the activity of NRI on depression. Temperature recovery of TBZ-induced hypothermic response is considered as indication of a anti-depressant activity.FIG.1shows that the PDC-1421 and its fractions of “1st Fraction 33”, “Polygalaxanthone III”, “Sibiricoses A5” and “7-O-Methylmangiferin” had a temperature recovery at 120 min after TBZ challenge. Example 3 In Vivo Attention Deficit/Hyperactivity Disorder Animal Model The male spontaneously hypertensive rat (SHR) is orally given PDC-1421 of Example 1 dissolved in saline at a dosage of 75, 225 or 675 mg/kg bodyweight the negative control groups (Saline) are orally administered once a day for four days. A local motor activity assay is performed 60 minutes after administration on all animals at the first and the fourth day by recording horizontal activity of SHR within one hour in Automated Locomotor Activity Analysis System Chamber. Total activity, phase analysis, central/peripheral activity, and rearing are measured in local motor activity assay. Total activity is the most meaningful index for ADHD. The results reveal that oral administration of PDC-1421 for 4 days significantly inhibits total activity and rearing of SHR rats at the concentration of 675 mg/kg, as shown inFIG.2. Example 4 Clinical Trial of Radix Polygalae (Polygala tenuifoliaWilld) Extract PDC-1421 Preclinical Study for Recommended Dosage and Route of Administration Based on the FDA's guidance documents, no observable adverse effect levels (NOAELs) from toxicity studies are used to estimate the starting dose. Table 2 summarizes the MRSD for 60 kg human/day calculated from the NOAEL of 28 day repeating dose studies in rats and in dogs using safety margin at 75-fold. TABLE 1Calculation of starting doseDose for60 kgStudySpeciesDosageHEDhuman/day28-day repeated dose subacuteRat3000 mg/kg483.9mg/kg387 mg/dayoral toxicity study in rats(NOAEL)(MRSD)28-day repeated dose subacuteDog3000 mg/kg1666.7mg/kg1333 mg/dayoral toxicity study in dogs(NOAEL)(MRSD)NOAEL: No observable adverse effect levelHED: Human equivalent doseMRSD: Maximum recommended starting dose*The calculation of HED and MRSD are referred to FDA document. Based on these calculations, the starting dose of PDC-1421 for human is 380 mg/day. In the proposed Phase I study, a single dose of PDC-1421 (oral administration) is used to evaluate the safety when taken by healthy subjects. The starting dose and dosage regimen are 380 mg PDC-1421 (one capsule of PDC-1421 Capsule) once a day after meal. Preparation of PDC-1421 Capsule A solid dosage form of a botanical extract of Radix Polygalae (Polygala tenuifoliaWilld), such as PDC-1421, can be prepared into a gelatin capsule by conventional techniques known to those skilled in the art. In a preferred embodiment of the invention, the matrix comprises two excipients. The first excipient is silicon dioxide served as a glidant which may constitutes from about 2 to 10%, more preferably 5% of filling, and the second excipient is magnesium stearate served as a lubricant which may constitutes from about 2 to 10%, more preferably 5% of filling. The practical composition of PDC-1421 capsule for clinical trial is shown in Table 2. TABLE 2Composition of PDC-1421 CapsuleName of IngredientMg per CapsuleFunctionPDC-1421380mgActive ingredientSilicon dioxide10mgExcipientMagnesium stearate10mgExcipient Phase I Tested in Healthy Subject for Screening for Safety In phase I trial, a total of 85 subjects are screened at study site and 30 subjects are enrolled. The 30 subjects are divided into four groups: 9 subjects in cohort A with 7 administered PDC-1421 (380 mg) and 2 administered placebo, 1 of 7 PDC-1421 subjects had no laboratory test data at baseline; 8 subjects in cohort B with 6 administered PDC-1421 (1140 mg) and 2 administered placebo; 4 subjects in cohort C with 3 administered PDC-1421 (2280 mg) and 1 administered placebo; and 9 subjects in cohort D with 7 administered PDC-1421 (3800 mg) and 2 administered placebo, 1 of 7 PDC-1421 subjects had abnormal laboratory data at screening visit. Physical examination is determined to be “normal” on each body system in each cohort and no subject had dose-limiting toxicity (DLT) and toxicity grade. All of the changes of vital signs from baseline of PDC-1421 and placebo group are mild and do not exceed the limit of normal range. Furthermore, all of the toxicity grades of vital signs are the lowest, systolic blood pressure in grade 1, increase >20 mm/Hg than baseline at 4 hours. No medical intervention/therapy is required. There is no correlation of changes from baseline or changes in the toxicity grade of vital signs between doses of PDC-1421. Only two grade 2 toxicity (at 24 hours in glucose in cohort A and at 4 hours in glucose in cohort B) occur in placebo group and no medical intervention/therapy were required for these cases Electrocardiogram (ECG) is determined to be “normal” in each time point and in each cohort. No subject had DLT and toxicity grade. Columbia-suicide severity rating scale (C-SSRS) are all 0 point on suicidal ideation, intensity of ideation, suicidal behavior in each cohort. No subject has serious adverse event, and no subject is discontinued due to adverse event, and no clinically significant finding in physical examinations, vital signs, electrocardiogram, laboratory measurements, or C-SSRS is observed throughout the treatment period. The oral administration of PDC-1421 in healthy volunteers is safe and well-tolerated for the dose from 380 mg to 3800 mg. During the treatment period, 5 subjects are reported to experience 8 mild adverse events shown as Table 3. The severity of these 5 adverse events is all mild and no medical action required. There is no correlation of number, severity, relationship and outcome of adverse events found between doses of PDC-1421 and placebo. Further, there is no deviation of electrolyte level and no significant gastrointestinal discomfort during monitoring in the clinical trial. There are two mild adverse events such as lower heart rate and higher systolic blood pressure. Lower heart rate is that coupled to the dog telemetry study but not higher systolic blood pressure. TABLE 3Frequencies of Adverse EventsFrequencyCohort ACohort BCohort CCohort DAdverse events(380 mg)(1140 mg)(2280 mg)(3800 mg)PlaceboBODY SystemN = 7N = 6N = 3N = 7N = 7Digest System3/70001/7FLATULENCE2/70001/7CONSTIPATION1/70000Nervous System002/302/7SOMNOLENCE001/302/7STOMATITIS ULCER001/300 In summary, no subject has serious adverse event, and no subject is discontinued due to adverse event. No clinically significant findings in physical examinations, vital signs, electrocardiogram, laboratory measurements, and C-SSRS are observed throughout the treatment period. The oral administration of PDC-1421 in healthy volunteers is safe and well-tolerated for the dose from 380 mg to 3800 mg. Phase II in Attention-Deficit Hyperactivity Disorder (ADHD) Patients The Phase II clinical trial is aimed to evaluate the safety and efficacy of PDC-1421 in adults with ADHD. The study is a single center, open label, dose escalation evaluation with two dosage levels in six subjects. Six subjects are initially evaluated for safety and efficacy assessments at low-dose (1 capsule of PDC-1421, three times a day (TID)) for 28 days. The subjects who pass the safety checkpoint are further evaluated for safety and efficacy assessments at high-dose (2 capsules of PDC-1421 TID) for next 28 days. The primary objective is to determine the efficacy profile of PDC-1421 capsule in ADHD with ADHD Rating Scale-IV (ADHD-RS-IV). The secondary objective is to evaluate the safety of PDC-1421 capsule in subjects receiving PDC-1421 at various dose levels. A total of 6 subjects were enrolled in the study. All enrolled subjects received the low-dose of PDC-1421 capsules for 4 weeks, and all of the subjects received the high-dose of PDC-1421 capsules for next 4 weeks after passing the safety checkpoint. The safety population included 6 subjects, the intention-to-treat (ITT) population included 6 subjects, and the per protocol (PP) population included 5 subjects in the study. One subject was excluded from PP population due to drug compliance less than 80%. Efficacy Results For the primary endpoints, the percentages of improvement of 40% or greater in ADHD-RS-IV score from baseline to 8 weeks treatment are 83.3% (N=5) in the ITT population and 80.0% (N=4) in the PP population shown, as shown in Table 4. The net mean changes of ADHD-RS-IV scores from baseline to 8 weeks treatment are −25.7 in the ITT population and −24.6 in the PP population, as shown in Table 5. TABLE 4Summary of improvement ≥40% in inattention subscale (IA), hyperactivity-impulsivity(HI) and total subscale of ADHD-RS-IV from baseline up to 8 weeks treatmentNo. of subject,ITT Population (N = 6)PP Population (N = 5)n (%)IAHITotal scoreIAHITotal scoreAt Week 11 (16.7%)001 (16.7%)00At Week 21 (16.7%)3(50.0%)1(16.7%)1 (20.0%)3(60.0%)1(20.0%)At Week 34 (66.7%)3(50.0%)3(50.0%)4 (80.0%)3(60.0%)3(60.0%)At Week 43 (50.0%)3(50.0%)3(50.0%)3 (60.0%)3(60.0%)3(60.0%)At Week 64 (66.7%)5(83.3%)5(83.3%)3 (60.0%)4(80.0%)4(80.0%)At Week 84 (66.7%)6(100.0%)5(83.3%)3 (60.0%)5(100.0%)4(80.0%) TABLE 5Summary of the inattention subscale, hyperactivity-impulsivitysubscale and total scale raw score of ADHD-RS-IVTotal scaleADHD-RS-IVMean (SD)Treatment PeriodITT population (N = 6)PP population (N = 5)Week 0 (baseline)41.8(6.6)40.2(5.9)Net change at Week 1−4.8(6.0)−5.2(6.6)Net change at Week 2−14.0(7.9)−15.2(8.3)Net change at Week 3−18.8(10.0)−20.2(10.6)Net change at Week 4−17.7(13.2)−20.4(12.7)Net change at Week 6−24.7(10.0)−22.8(10.0)Net change at Week 8−25.7(11.0)−24.6(12.0) For the secondary endpoints, a statistically significant improvement in the ADHD index subscale and Impulsivity subscale of CAARS-S:S at Week 8 compared to baseline is occurred. The mean changes of CAARS-S:S from baseline to 8 weeks treatment for the ADHD index subscale and impulsivity subscale are −10.8 (P=0.0313) and −15.2 (P=0.0313) in the ITT population, and 10.6 (P=0.0625) and −14.0 (P=0.0625) in the PP population, as shown in Table 6. In addition, a significant improvement in ADHD index subscale is observed after 3 week low-dose PDC-1421 treatment (mean change=−8.7, P=0.0313). The T-scores of CAARS-S:S for other subscales including inattention/memory subscale, hyperactivity subscale and self-concept subscale, also show improvement at Week 8 compared to baseline, but no statistical significance. TABLE 6Summary of CAARS-S:S subscale T-scoreCAARS-S:SInattention/ADHDmemoryHyperactivityImpulsivitySelf-conceptindexsubscalesubscalesubscalesubscalesubscaleTreatment PeriodMean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)ITT population (N = 6)Week 0 (baseline)74.2(7.9)65.2(6.3)63.7(10.2)65.0(14.8)73.5(8.3)Net change at Week 1−1.5(13.1)0.5(4.4)−4.5(5.0)−1.8(3.3)−2.3(5.2)Net change at Week 2−5.7(9.0)−0.2(3.7)−4.2(6.5)1.7(9.4)−0.3(5.9)Net change at Week 3−12.2(11.7)−3.2(8.4)−11.2(5.7)*−4.0(4.8)−8.7(6.1)*Net change at Week 4−10.0(10.2)−6.2(7.1)−10.8(7.1)−5.7(7.0)−8.2(7.4)*Net change at Week 6−7.8(12.7)−5.2(7.6)−10.3(10.9)−3.3(5.4)−8.0(5.5)*Net change at Week 8−10.5(11.0)−5.2(5.9)−15.2(7.9)*−4.7(6.2)−10.8(6.2)*PP population (N = 5)Week 0 (baseline)72.2(7.0)63.4(5.1)60.8(8.2)60.2(10.1)70.2(2.0)Net change at Week 1−1.2(14.7)1.2(4.5)−3.2(4.3)−2.2(3.5)−2.8(5.7)Net change at Week 2−6.2(10.0)−0.2(4.1)−2.8(6.2)2.0(10.5)−0.2(6.5)Net change at Week 3−11.8(13.1)−3.8(9.3)−11.2(6.4)−4.8(4.9)−9.8(6.1)Net change at Week 4−11.4(10.7)−7.4(7.2)−10.2(7.8)−6.8(7.2)−9.6(7.2)Net change at Week 6−6.6(13.8)−5.6(8.4)−9.6(12.0)−3.2(6.1)−7.8(6.2)Net change at Week 8−10.6(12.3)−5.6(6.5)−14.0(8.2)−4.8(6.9)−10.6(6.9)*p-value < 0.05 For the both ITT and PP populations, the percentages of CGI-ADHD-Improvement score less than or equal to 2 at Week 8 are 100% (N=6 for ITT, N=5 for PP), as shown in Table 7. For ITT population, the mean scores of CGI-ADHD-S at baseline and Week 8 are 5.3 (SD=0.5) and 2.7 (SD=1.0) respectively. The net mean changes of CGI-ADHD-Severity score from base line to Week 8 is 2.6 points. TABLE 7Summary of clinical global impression scale (CGI) score of 2 or LowerCGIITT Population (N = 6)PP Population (N = 5)CGI-ADHD-CGI-ADHD-CGI-ADHD-CGI-ADHD-Treatment PeriodSeverity of illnessImprovementSeverity of illnessImprovementCGI score, mean (SD)At Week 0 (baseline)5.3(0.5)—5.2(0.4)—At Week 15.0(0.9)3.7(0.5)4.8(0.8)3.6(0.5)At Week 24.3(0.5)2.8(0.4)4.2(0.4)2.8(0.4)At Week 33.7(0.8)2.7(0.5)3.4(0.5)2.6(0.5)At Week 44.0(0.9)3.0(0.6)3.8(0.8)3.0(0.7)At Week 63.3(0.5)2.2(0.8)3.4(0.5)2.2(0.8)At Week 82.7(1.0)1.5(0.5)2.4(0.9)1.4(0.5)No. of subject of CGI score of 2 or Lower, n (%)At Week 10(0.0%)0(0.0%)0(0.0%)0(0.0%)At Week 20(0.0%)1(16.7%)0(0.0%)1(20.0%)At Week 30(0.0%)2(33.3%)0(0.0%)2(4.0%)At Week 40(0.0%)1(16.7%)0(0.0%)1(20.0%)At Week 60(0.0%)4(66.7%)0(0.0%)3(60.0%)At Week 82(33.3%)6(100.0%)3(60.0%)5(100.0%) Safety Results The safety evaluation is based on the ITT population, who takes at least one dose of the IPs and has any of the post-baseline safety data collected. Regarding the incidence of adverse events (AEs), a total 4 subjects (66.7%) experience at least one AE and a total of 23 AEs are observed duration of the study. No subjects withdraw from the study due to AE. For more common AEs, the most frequently reported AEs in the study include “Gastrointestinal disorders” (Overall: N=3 [50.0%]; Unlikely: N=1; Possibly: N=2) and “Respiratory, thoracic and mediastinal disorders” (Overall: N=3 [50.0%]; Unrelated: N=3). There are no life-threatening AEs and deaths. For level of severity, the grade of most AEs is moderate (N=13). There are 1 case of severe AE and 9 cases of mild AEs. One event (“Dizziness”) rated as severe is judged as “Possibly” to study drug by the investigator. Regarding the laboratory values at each scheduled visit, few abnormal values without clinically significant are observed. The results suggest that treatment of PDC-1421 did not increase the abnormalities of laboratory data. For vital signs and electrocardiogram (ECG), no abnormal body temperature and ECG are observed and some abnormal blood pressure and heart rate without clinically significant are observed in the study. The results suggest that treatment of PDC-1421 does not increase the abnormalities of vital signs. For physical examination, no subjects are evaluated as abnormal for all the scheduled visits (Visit 1 and Visit 8) in the study. No physical evaluation becomes worse during treatment and follow-up period. For C-SSRS evaluation, no suicide-related treatment-emergent event occurs in the study. The results suggest that treatment of PDC-1421 does not increase the risk of either suicidal ideation or suicidal behavior. In summary, the present invention provides a method of treating ADHD in a safer and more effective way by administering a composition comprising a Radix Polygalae extract. In clinical trial, the subjects given the treatment of the composition have significant improvements in many ADHD related scale evaluations. And compared with the current medications for ADHD, the method of present shows no side effects on cardiovascular system or mental state. It is showed in all cases that the method of the present invention is a better treatment for ADHD and relatively safe. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims. | 18,185 |
11857593 | DETAILED DESCRIPTION In an exemplary embodiment of a gel-based nutraceutical food product, the gel comprises a solid polymeric matrix throughout which a liquid is dispersed. The solid polymeric matrix may comprise a galactomannan, a polysaccharide (e.g., xanthan, a fruit pectin, etc.), gelatin, or any other suitable gelling agent. In some embodiments, a plurality of gelling agents are used to provide desired properties, such as viscosity, consistency, and edibility. Various embodiments of nutraceutical food products that incorporate teachings of the present invention include a fruit component. The fruit component includes at least one fruit that naturally includes OPC or a juice or other extract of such a fruit. By way of nonlimiting example, the fruit component may include one or more of açai, elderberry, grape, and pomegranate, or an extract thereof. OPC is a known antioxidant and may, therefore, be useful in neutralizing or otherwise acting against free radicals and other oxidants, which may adversely affect cell membranes, cause accelerated cellular aging, and are known or believed to be at least indirectly responsible for a wide variety of disease states, as well as compromised immunity, in living beings. In embodiments of nutraceutical food products that include fruit components, the fruit component may be in solid form, in liquid form, or in some combination of solid and liquid forms. When the fruit component is included in solid form, it may be incorporated into the matrix (e.g., as pectin, such as pectin of an OPC-containing fruit, etc.) or it may be dispersed throughout the matrix (e.g., as chunks, bits, etc.). An immune modulator may be present within (e.g., dispersed throughout, dissolved in, etc.) the liquid component of various embodiments of nutraceutical food products of the present invention. When mixed with the liquid component, the immune modulator may retain substantially all of one or more of its activities (e.g., components of the liquid component may not interfere with one or more activities of the immune modulator), or one or more of the activities of the immune modulator may actually be enhanced by one or more components of the liquid component of the nutraceutical food product. The immune modulator may include transfer factor. The transfer factor may include any type of transfer factor, as well as a combination of two or more types of transfer factor. For example, avian transfer factor, bovine transfer factor, or any other type of transfer factor may be included in the transfer factor component. The transfer factor of the transfer component factor may be derived from any suitable, acceptable source. For example, avian transfer factor may be obtained from eggs, such as by a process disclosed in U.S. Pat. No. 6,468,534 to Hennen et al. (hereinafter “Hennen”), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. An example of the manner in which bovine transfer factor may be obtained is disclosed in U.S. Pat. No. 4,816,563 to Wilson et al. (hereinafter “Wilson”), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. Compositions that include two or more types of transfer factor, as well as processes for combining and processing two or more types of transfer factor, are disclosed in U.S. Pat. No. 6,866,868 to Lisonbee et al. (hereinafter “Lisonbee”), the disclosure of which is hereby incorporated herein, it its entirety, by this reference. Transfer factor is known or believed to improve the oxidative balance of a living being, as well as to enhance the effectiveness of antioxidants, as demonstrated by the disclosure of the international patent application filed pursuant to the Patent Cooperation Treaty and having International Publication Number WO 2004/041071 A2 (hereinafter “Dadali”), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. As an alternative to transfer factor, the immune modulator of various embodiments of a nutraceutical food product of the present invention may comprise a nanofraction immune modulator of the type disclosed by U.S. patent application Ser. No. 11/855,944. Other embodiments of nutraceutical food products of the present invention may include a plurality of different immune modulators, such as combinations of transfer factor and nanofraction immune modulators. Various examples of compositions that include transfer factor and nanofraction immune modulators are also disclosed by U.S. patent application Ser. No. 11/855,944. A nutraceutical food product of the present invention may also include one or more preservatives. Suitable preservatives, such as those accepted for use in foods and beverages, may be used. Any preservatives that are included in a nutraceutical food product of the present invention may be able to withstand pasteurization processes. Examples of preservatives that may be included in an edible preparation of the present invention include, but are not limited to, sodium benzoate and preservatives from the paraben family of chemicals. A particular embodiment of gel-based nutraceutical food product that incorporates teachings of the present invention is described in the following example: Example 1 TABLE 1% of% ofTotalTotalDensityJuicesIngredient(w/w)(g/mL)(v/v)Water75.4901.000RioVida Juice Blend15.965Apple Juice1.34619Purple Grape Juice1.33019Blueberry Juice1.31518Pomegranate JuiceElderberry Juice1.31515Açai Powder0.32514Transfer Factor Tri-Factor Blend1.880Glycerine (Vegetable)4.2101.249Grape color concentrate (e.g.,0.5101.306MEGANATURAL ™ purplefrom Canandaigua Concentrates& Colors, a Division ofCanandaigua Wine Company ofMadera, California)Vitamin C0.190Flavorings0.428Berry flavor (BE-01407)1.000Berry flavor (BE-01271)1.000Natural Vanilla (VA-01239)1.000Monolaurin (glycerol monolaurate)0.002Gums (Xanthan and Guar Gum, at a1.000ratio of 1:1 w/w) Transfer Factor Tri-Factor Blend includes transfer factor (including bovine transfer factor from cow colostrum and avian transfer factor from the yolk of a chicken's egg) and nanofraction immune modulators. The flavorings listed in TABLE 1 are available from Flavors Inc. Glycerol monolaurate is a surfactant. A surfactant may be useful for maintaining homogeneity (i.e., for keeping the components of the nutraceutical food product, including, but not limited to, any immune support component present in the nutraceutical food product, dispersed substantially homogeneously throughout the nutraceutical food product). A daily dosage of about one fluid ounce (about 30 ml) or more (e.g., about two fluid ounces, or 60 ml, etc.) of a composition with ingredients in the proportions listed in TABLE 1 may be administered to or consumed by a subject. In addition to the numerous known and believed benefits of antioxidants, including the benefits of OPC and OPC-containing fruits such as açai, administration or consumption of a nutraceutical food product that incorporates teachings of the present invention provides the subject with the additional and sometimes synergistic beneficial effects of transfer factor, which are known in the art, as evidenced by the disclosures of Dadali, Hennen, Lisonbee, and Wilson, and of nanofraction immune modulators. An edible preparation may be made by mixing components of a food base with transfer factor by processes that are known in the art. Suitable processes that may be used to manufacture edible preparations of a variety of different forms are well known and within the skill of those in the relevant art. Known techniques, such as those disclosed in “Principles and Practices of Small- and Medium-Scale Fruit Juice Processing,” Food and Agricultural Organization of the United Nations (FAO) Services Bulletin 146 (Rome, 2001), the entire disclosure of which is hereby incorporated herein by this reference, may be used in one or more parts of a process for manufacturing various embodiments of nutraceutical food products of the present invention. Processes that are used to manufacture nutraceutical food products according to the present invention may be effected at a low temperature (e.g., between about 0° C. and about 10° C., at about 4° C., etc.), such as in a refrigerated environment, then transported and stored at such temperatures to reduce the likelihood of microbial growth or proliferation therein. Alternatively, a nutraceutical food product that is in a form that is not completely dry may be pasteurized or sterilized. Pasteurization processes, which decrease the number of microorganisms present, but do not entirely eliminate the microorganisms, improve the stability of products that are to be stored at reduced temperatures (e.g., frozen or refrigerated, or “chilled”). When a nutraceutical food product is sterilized, all or substantially all microorganisms therein are killed or inactivated, facilitating prolonged storage of the nutraceutical food product at room temperature or even higher temperatures. As an example, a nutraceutical food product that includes transfer factor and/or nanofraction immune modulators may be sterilized by known superheated steam injection processes. The temperatures and durations of such processes depend, of course, upon the form and ingredients of the composition to be sterilized. When making a liquid preparation, the resulting nutraceutical food product may be “flash” heated to a particular temperature (e.g., 250° F.) for a corresponding duration (e.g., two seconds). Alternatively, a sterilization or pasteurization process of different duration and temperature may be used, so long as the duration and temperature of the process are in substantial accord with a practice that has been accepted in the art, such as use of the following equation: tp=5·1014·e−0.4353·Tmo, where tpis the minimum duration of the process, and Tmois the temperature at which the process is effected. Of course, processes that reduce microbial load on a nutraceutical food product of the present invention need not comprise heat-treatment techniques. Sterilization or other microbial load-reducing techniques that employ other means (e.g., filtration, antimicrobial ingredients, etc.) may also be used in manufacturing a nutraceutical food product. Examples of suitable processes are disclosed in Hughes, D. E., and Nyborg, W., “Minimally Processed Fruits and Vegetables: Reducing Microbial Load by Nonthermal Physical Treatments,” Food Technology 52(6): 66-71 (1997), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. It is desirable that, following pasteurization or sterilization, the transfer factor and/or nanofraction immune modulators retain some if not substantially all or all of their activity. A variety of pasteurization or sterilization processes may be employed, including pasteurization or sterilization processes that may be used to reduce microbial counts or completely eliminate microorganisms from foods. As many sterilization processes are known to significantly reduce the activity of certain proteins, including antibodies, a study was performed to determine whether transfer factor retains at least some of its activity following pasteurization. In the study, mouse footpad assay techniques, similar to those disclosed in J. Natl. Cancer Inst. 55(5):1089-95 (November 1975), were used to determine the affects of heat pasteurization or sterilization processes (specifically, superheated steam injection processes) on liquid nutraceutical food products including transfer factor. Two sterilized samples were compared with an unsterilized sample, as well as with a negative control and a positive control. Separate populations of six mice were tested for each of the five samples and controls. The tests were conducted in two phases, a first that immediately followed heat sterilization of the samples, and a second that was conducted after storing the two heat sterilized samples at a temperature of about 40° C. for about three months, which is well-accepted in the art to be the equivalent of about one year of storage at room temperature. Thirty different mice were used in each phase of the study. The following procedures were followed in each phase of the study. In the positive control (i.e., the “fifth group”), fourteen days prior to testing, the footpads of the right rear feet of six BALB/c mice having ages of about nine weeks to about ten weeks were anesthetized with isoflurane. Then 0.02 ml of an about 50/50 (wt/wt) mixture of Freund's adjuvant and bovine rhinotracheitis virus diarrhea vaccine was administered intramuscularly to each mouse by way of two injections at the base of each side of the mouse's tail. This early injection of antigen allows the mice of the positive control group to elicit their own primary immune response and secondary, or delayed-type hypersensitivity response to the antigen. The mice of the other five groups were not preexposed to the antigen in this manner. About twenty-four hours before evaluating the hind footpads of the mice, the six BALB/c mice of each group, which were of similar age to the mice of the positive control group, were anesthetized with isoflurane. About 0.5 ml of a sample solution or control solution was then administered by subcutaneous injection at the back of the neck of each mouse. In the first group (see EXAMPLE 2 below), which was the negative control group, the back of the neck of each mouse was injected with about 0.5 ml of sterile saline solution. In the second group (see EXAMPLE 3 below), the sample solution included 16% solids (w/v) of a reconstituted (in distilled, deionized water) lyophilized colostrum fraction that included transfer factor. The solution was set at a pH of 4.0, which was intended to estimate the pH of a fruit juice preparation (the actual pH of which is about 3.6 or about 3.7). Following reconstitution and pH adjustment, the solution was sterilized by heating the same to a temperature of 250° F. for about two seconds. In the third group (see EXAMPLE 4 below), the sample solution included 16% solids (w/v) of a reconstituted (in distilled, deionized water) lyophilized colostrum fraction that included transfer factor. The pH of the resulting solution was not adjusted and, thus, was neutral (i.e., 7.0) or slightly basic (i.e., greater than 7.0)). Following reconstitution, the solution was sterilized by heating the same to a temperature of 250° F. for about two seconds. In the fourth group (see EXAMPLE 5 below), the sample solution was a concentrate of a colostrum fraction that included transfer factor, which had been diluted to about 16% solids (w/v) in distilled, deionized water. This solution was not heat sterilized or pH adjusted. The mice of the fifth group (see EXAMPLE 6 below), which was the positive control groups, respectively, received sterile saline solution. At the start of the mouse footpad assay, the right hind footpad and the left hind footpad of each mouse were measured, such as with a Starrett gauge. The right hind footpad of each of the thirty mice during each phase of the study was then subcutaneously injected with an antigen-containing solution. The footpad on the left hind foot of each of the thirty mice in each phase, which was used as a control, was injected with about the same volume of a control solution, such as a sterile saline diluent, as the volume of antigen-containing solution that was injected into right hind footpad. After a sufficient amount of time (e.g., about twenty-four hours) for the secondary immune response components of the immune system of each mouse to respond, each mouse was again anesthetized and the distances across right and left hind footpads were again measured. A significant amount of swelling, determined by an increase in the distance across a right hind footpad of a mouse from the initial measurement to the second measurement, is indicative of the occurrence of a delayed-type hypersensitivity reaction in that footpad. The results of the mouse foot pad assays, and some accompanying analysis, are set forth in EXAMPLES 2 through 5 and 7: Example 2 In the first phase of the study, the footpads on the right hind feet of the six mice of the negative control, or first group, exhibited, on average, about 6.35 micrometers more swelling about twenty-four hours after they were injected with the antigen solution than the swelling measured in the footpads of the left hind feet of these mice, which were merely inoculated with sterile saline. The results for the negative control group during the second phase of the study are set forth in the following table: TABLE 2Foot PadFoot PadFoot Pad(difference)Foot(untreated)(final)(micro-Mouse(left/right)(micrometers)(micrometers)meters)1Left (control)1930.401955.8025.40Right (test)1905.001930.4025.402Left (control)1981.202006.6025.40Right (test)2006.602057.4050.803Left (control)2057.402057.400.00Right (test)2032.002057.4025.404Left (control)2006.602032.0025.40Right (test)2032.002057.4025.405Left (control)1955.802006.6050.80Right (test)1930.401955.8025.406Left (control)1905.001930.4025.40Right (test)1876.601955.8076.20 Similar to the results from the first phase, the footpads of the right hind feet of the mice of the negative control group exhibited, on average, only 12.70 micrometers more swelling about twenty-four hours after antigen injection than the footpads of the left hind feet of the same mice exhibited twenty-four hours after sterile saline injection. As twenty-four hours is not a sufficient period of time for a mouse to mount a primary (i.e., antibody-mediated) immune response to the antigen, these insignificant differences in swelling show that the mice did not exhibit a significant secondary immune response to the antigen. Example 3 In the first phase of the study, about twenty-four hours after they were injected with the antigen solution, the footpads on the right hind feet of the six mice of the second group of mice (which mice had previously been inoculated with a solution including 16% solids (w/v) colostrum at pH=4.0) swelled, on average, by 50.80 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice. These results indicate that there was a greater secondary, or delayed-type hypersensitivity, immune response in the footpads into which antigen was injected than in the footpads into which no antigen was injected, which were likely swollen merely because they were pierced by a needle. In the second phase of the study, similar results were obtained, as set forth in the following table: TABLE 3Foot PadFoot PadFoot Pad(difference)Foot(untreated)(final)(micro-Mouse(left/right)(micrometers)(micrometers)meters)1Left (control)1955.802006.6050.80Right (test)1981.202057.4076.202Left (control)1930.402006.6076.20Right (test)1955.802108.20152.403Left (control)1955.802006.6050.80Right (test)1981.202082.80101.604Left (control)2032.002057.4025.40Right (test)2057.402108.2050.805Left (control)1930.402006.6076.20Right (test)1955.802032.0076.206Left (control)2057.402108.2050.80Right (test)2032.002159.00127.00 More specifically, the footpads of the right hind feet of the six mice of the second group swelled so that they measured, on average, 42.33 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice before and after inoculation of their foot pads with the antigen solution. The similar results between the first and second phases of the study indicate that, once a liquid solution that includes transfer factor has been heat sterilized, there is little or no change in the activity of the transfer factor after prolonged storage of the solution. Example 4 The results for the third group of mice (which mice had previously been inoculated with a solution including 16% solids (w/v) colostrum at normal pH) were similar to the results for the second group in the first and second phases of the study. In the first phase of the study, about twenty-four hours after the footpad injections, the antigen solution-inoculated footpads on the right hind feet of the six mice of the third group of mice swelled, on average, by 35.98 micrometers more than the swelling that was measured in the sterile saline-inoculated footpads of the left hind feet of these mice. These results indicate that there was a greater secondary, or delayed-type hypersensitivity, immune response in the footpads into which antigen was injected than in the footpads into which no antigen was injected, which were likely swollen merely because they were pierced by a needle. In the second phase of the study, similar results were obtained, as set forth in the following table: TABLE 4Foot PadFoot PadFoot Pad(difference)Foot(untreated)(final)(micro-Mouse(left/right)(micrometers)(micrometers)meters)1Left (control)2006.602032.0025.40Right (test)2032.002082.8050.802Left (control)2057.402057.400.00Right (test)2006.602108.20101.603Left (control)1981.202006.6025.40Right (test)2057.402082.8025.404Left (control)2006.602057.4050.80Right (test)2032.002082.8050.805Left (control)2057.402082.8025.40Right (test)2082.802159.0076.206Left (control)2082.802108.2025.40Right (test)2108.202159.0050.80 These results show that the footpads of the right hind feet of the six mice of the third group swelled so that they measured, on average, 33.87 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice before and after inoculation of the foot pads with the antigen solution. The similar results between the first and second phases of the study indicate that, following prolonged storage, there was little or no change in the activity of the transfer factor in a heat-sterilized solution. Example 5 These results were confirmed by the results that were obtained from the fourth group of mice. In particular, during the first phase of the study, the footpads of the right hind feet of mice in the fourth group (which included mice that had been inoculated with a diluted liquid colostrum fraction that was not heat sterilized) exhibited, on average, about 35.98 micrometers more swelling than the foot pads of left hind feet of these mice about twenty-four hours after these footpads had been inoculated with antigen solution and sterile saline, respectively. Similar results were obtained during the second phase of the study, in which the average difference was 42.33 micrometers, as evidenced by the following data: TABLE 5Foot PadFoot PadFoot Pad(difference)Foot(untreated)(final)(micro-Mouse(left/right)(micrometers)(micrometers)meters)1Left (control)1955.802032.0076.20Right (test)1981.202082.80101.602Left (control)2006.602057.4050.80Right (test)2032.002108.2076.203Left (control)1955.802006.6050.80Right (test)1930.402057.40127.004Left (control)1955.802082.80127.00Right (test)1905.002032.00127.005Left (control)2032.002082.8050.80Right (test)2057.402184.40127.006Left (control)1955.801955.800.00Right (test)2006.602057.4050.80 As these results are comparable to (i.e., not significantly greater than) those obtained with heat-sterilized solutions (see the results from EXAMPLES 3 and 4), it is apparent that heat sterilization of a solution that includes transfer factor does not significantly diminish or reduce the activity of the transfer factor. Example 6 This conclusion was verified by data from another mouse footpad assay, in which six BALB/c mice were inoculated, behind the neck, with 0.5 ml of a solution including 16% solids (w/v) of a spray-dried colostrum fraction that had been reconstituted in distilled, deionized water. About twenty-four hours later, the mice were anesthetized with isoflurane, then footpads on their hind feet measured and inoculated in the manner described above (i.e., left footpad with sterile saline, right footpad with the antigen solution). After about another twenty-four hours, the footpads were again measured. The right footpads of these mice swelled, on average, about 42.33 micrometers more than the footpads on the left hind feet of these mice. This value is comparable to (i.e., not significantly different from) the differences noted above with respect to the second, third, and fourth groups of mice in both the first and second phases of the study detailed in EXAMPLES 2 through 5 and 7, further supporting the conclusion that heat sterilization of a solution that includes transfer factor, such as the solutions that were tested on the second and third groups of mice (EXAMPLES 3 and 4) does not have a significant adverse affect on the activity of the transfer factor. Example 7 The fact that the transfer factor with which the mice were inoculated was responsible for the increased secondary immune response is supported by the results from the fifth group, or positive control group, of mice during the second phase of the study, as set forth in the following table: TABLE 6Foot PadFoot PadFoot Pad(difference)Foot(untreated)(final)(micro-Mouse(left/right)(micrometers)(micrometers)meters)1Left (control)1981.202006.6025.40Right (test)2006.602082.8076.202Left (control)1828.801854.2025.40Right (test)1879.602082.80203.203Left (control)1905.001930.4025.40Right (test)1981.202082.80101.604Left (control)2006.602057.4050.80Right (test)2032.002184.40152.405Left (control)2032.002057.4025.40Right (test)2057.402184.40127.006Left (control)2108.202108.200.00Right (test)2082.802184.40101.60 These results, which show on average, 101.60 micrometers more swelling in the footpads that were inoculated with antigen solution over those that were inoculated with sterile saline, are similar to the 124.88 micrometer difference seen in the mice of the positive control group during the first phase of the mouse footpad study. The greater swelling in the antigen solution-inoculated footpads of the mice of the positive control group is indicative of a greater secondary immune response than that induced artificially by administration of transfer factor, as the mice of the positive control group had a sufficient period of time (i.e., two weeks) to generate their own transfer factor and, thus, to mount their own secondary immune response to the antigen. Once a nutraceutical food product of the present invention has been manufactured, it may be introduced into a clean or sterile container for subsequent transport and storage. Example 8 In another study, mouse footpad assays were conducted to determine the effectiveness of transfer factor in heat-treated samples of a liquid solution that included transfer factor that had been stored for one year. In total, four samples were prepared, two each having a pH of about 4 and two each having a pH of about 7. All of the samples had been flash sterilized at a temperature of about 250° F. for about two seconds to about four seconds. The samples were subsequently stored for one year, with one each of the pH=4 and pH=7 samples having been stored at room temperature (which varied from about 65° F. to about 74° F.) and one each of the pH=4 and pH=7 samples having been refrigerated (at temperatures of about 40° F.). After one year, the samples were lyophilized. Prior to testing, the lyophilized samples were reconstituted to desired concentrations, then administered in the manner described above. In a first sample, which included liquid having a pH of about 4 that was stored at room temperature, footpad swelling was, on average, 50.80 micrometers greater in footpads that had been injected with antigen versus footpads that had merely been injected with saline. These results were repeated in second (liquid of a pH of about 7 that was stored at room temperature), third (liquid of a pH of about 4 that was refrigerated), and fourth (liquid of a pH of about 7 that was refrigerated) samples, in which hind footpads that had been injected with antigen were, on average, respectively swollen 59.27, 67.73, and 63.50 micrometers more than hind footpads that were merely injected with saline. Additionally, positive and negative controls were prepared as discussed above. In the positive control, the average difference in swelling between antigen-injected footpads and saline-injected footpads was 114.30 micrometers. In the negative control, the average difference in swelling between antigen-injected footpads and saline-injected footpads was only 38.10 micrometers. Taken together, these data indicate that the increased swelling was due to the presence of transfer factor in the mice in the areas (hind footpads) into which antigen was introduced. Additionally, these data indicate that the transfer factor lost little or none of its effectiveness after heat-treatment and prolonged storage. The activity of transfer factor in refrigerated samples appears to have been slightly higher than the activity of transfer factor in the room temperature samples. Further, it appears from the foregoing that the pH at which the transfer factor is maintained (about 4 or about 7) has little or no effect on its long term viability. Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby. | 29,831 |
11857594 | DETAILED DESCRIPTION Some embodiments provide a composition comprising an amount of one or more curcumin extracts formulated as a curcumin composition. In certain embodiments, a curcumin composition can comprise an amount of diferuloylmethane, an amount of demethoxycurcumin (DMC), an amount of bis-demethoxycurcumin (BDMC), an amount of tetrahydrocurcumin, an amount of dihydrocurcumin, an amount of hexahydrocurcumin, an amount of octahydrocurcumin, or a combination of any of the foregoing. In some embodiments, a curcumin composition, as described herein, may comprise a pharmaceutically acceptable vehicle, carrier, or diluent. Diferuloylmethane is depicted below: DMC is depicted below: BDMC is depicted below: Tetrahydrocurcumin is depicted below: Dihydrocurcumin is depicted as follows: Hexahydrocurcumin is depicted as follows: Octahydrocurcumin is depicted as follows: In some embodiments, a curcumin composition, as described herein, may comprise an amount of at least two of diferuloylmethane, DMC, BDMC, tetrahydrocurcumin, dihydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. For example, a curcumin composition can comprise an amount of diferuloylmethane and an amount of DMC present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to DMC, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of diferuloylmethane and an amount of BDMC present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to BDMC, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of diferuloylmethane and an amount of tetrahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to tetrahydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of diferuloylmethane and an amount of dihydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to dihydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of diferuloylmethane and an amount of hexahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to hexahydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of diferuloylmethane and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of diferuloylmethane to octahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of DMC and an amount of BDMC present in a ratio of about 1:1, or about 20:1 to about 1:20 of DMC to BDMC, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of DMC and an amount of tetrahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of DMC to tetrahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of DMC and an amount of dihydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of DMC to dihydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of DMC and an amount of hexahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of DMC to hexahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of DMC and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of DMC to octahydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of BDMC and an amount of tetrahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of BDMC to tetrahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of BDMC and an amount of dihydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of BDMC to dihydrocurcumin. or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of BDMC and an amount of hexahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of BDMC to hexahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of BDMC and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of BDMC to octahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of tetrahydrocurcumin and an amount of dihydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of tetrahydrocurcumin to dihydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of tetrahydrocurcumin and an amount of hexahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of tetrahydrocurcumin to hexahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of tetrahydrocurcumin and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of tetrahydrocurcumin to octahydrocurcumin, or any range therebetween. In some embodiments, a curcumin composition can comprise an amount of dihydrocurcumin and an amount of hexahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of dihydrocurcumin to hexahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of dihydrocurcumin and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of dihydrocurcumin to octahydrocurcumin, or any range therebetween. In certain embodiments, a curcumin composition can comprise an amount of hexahydrocurcumin and an amount of octahydrocurcumin present in a ratio of about 1:1, or about 20:1 to about 1:20 of hexahydrocurcumin to octahydrocurcumin, or any range therebetween. As described further herein, any two of the aforementioned compounds can be present in a ratio of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, or any ratio in between. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a curcumin composition of the instant disclosure to achieve the results described herein. In certain embodiments, a curcumin composition, as described herein, may comprise an amount of three or more of diferuloylmethane, DMC, BDMC, tetrahydrocurcumin, dihydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. The three or more compounds may be present in a ratio. The ratio can be understood as comprising a “part” of any compound. For example, a curcumin composition may comprise a ratio of 4:1:10, diferuloylmethane to DMC to BDMC, corresponding to 4 parts diferuloylmethane, 1 part DMC, and 10 parts BDMC. The individual amount of any of the aforementioned compounds may be as low as 1 part, may be as high as 20 parts, or may any value therebetween. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a curcumin composition of the instant disclosure to achieve the results described herein. Some embodiments provide a composition comprising an amount of one or more green tea extracts formulated as a green tea extract composition. In certain embodiments, a green tea extract composition can comprise an amount of epigallocatechin gallate (EGCG), an amount of epicatechin (EC), an amount of epigallocatechin (EGC), an amount of epicatechin gallate (ECG), or a combination of any of the foregoing. In some embodiments, a green tea extract composition, as described herein, may comprise a pharmaceutically acceptable vehicle, carrier, or diluent. EGCG is depicted below: EC is depicted below: EGC is depicted below: ECG is depicted below: In some embodiments, a green tea extract composition, as described herein, may comprise an amount of at least two of EGCG, EC, EGC, and ECG. For example, a green tea extract composition can comprise an amount of EGCG and an amount of EC present in a ratio of about 1:1, or about 20:1 to about 1:20 of EGCG to EC, or any range therebetween. In some embodiments, a green tea extract composition can comprise an amount of EGCG and an amount of EGC present in a ratio of about 1:1, or about 20:1 to about 1:20 of EGCG to EGC, or any range therebetween. In certain embodiments, a green tea extract composition can comprise an amount of EGCG and an amount of ECG present in a ratio of about 1:1, or about 20:1 to about 1:20 of EGCG to ECG, or any range therebetween. In some embodiments, a green tea extract composition can comprise an amount of EC and an amount of EGC present in a ratio of about 1:1, or about 20:1 to about 1:20 of EC to EGC, or any range therebetween. In certain embodiments, a green tea extract composition can comprise an amount of EC and an amount of ECG present in a ratio of about 1:1, or about 20:1 to about 1:20 of EC to ECG, or any range therebetween. In some embodiments, a green tea extract composition can comprise an amount of EGC and an amount of ECG present in a ratio of about 1:1, or about 20:1 to about 1:20 of EGC to ECG, or any range therebetween. As described further herein, any two of the aforementioned compounds can be present in a ratio of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, or any ratio in between. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a green tea extract composition of the instant disclosure to achieve the results described herein. In certain embodiments, a green tea extract composition, as described herein, may comprise an amount of three or more of EGCG, EC, EGC, and ECG. The three or more compounds may be present in a ratio. The ratio can be understood as comprising a “part” of any compound. For example, a green tea extract composition may comprise a ratio of 4:1:10, EGCG to EC to EGC, corresponding to 4 parts EGCG, 1 part EC, and 10 parts EGC. The individual amount of any of the aforementioned compounds may be as low as 1 part, may be as high as 20 parts, or may any value therebetween. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a green tea extract composition of the instant disclosure to achieve the results described herein. Some embodiments provide a composition comprising an amount of one or more phycocyanin-related compounds formulated as a phycocyanin composition. As used herein, the term “phycocyanin-related compounds” refers to compounds that are pigment-proteins found inSpirulina, or compounds derived therefrom either by extraction, enzymatic degradation, or other known methods in the art. Exemplary examples of phycocyanin-related compounds include, but are not limited to phycocyanin, extracts of phycocyanin, phycocyanin peptides, and phycocyanin oligopeptides, and the like. In certain embodiments, a phycocyanin composition can comprise an amount of phycocyanin, an amount of an extract of phycocyanin, an amount of phycocyanin peptides, an amount of phycocyanin oligopeptides, or a combination of any of the foregoing. In some embodiments, a phycocyanin composition, as described herein, may comprise a pharmaceutically acceptable vehicle, carrier, or diluent. In some embodiments, a phycocyanin composition, as described herein, may comprise an amount of at least two of phycocyanin or an extract thereof, phycocyanin peptides, or phycocyanin oligopeptides. For example, a phycocyanin composition can comprise an amount of phycocyanin and an amount of an extract of phycocyanin present in a ratio of about 1:1, or about 20:1 to about 1:20 of phycocyanin to an extract of phycocyanin, or any range therebetween. In some embodiments, a phycocyanin composition can comprise an amount of phycocyanin and an amount of phycocyanin peptides present in a ratio of about 1:1, or about 20:1 to about 1:20 of phycocyanin to phycocyanin peptides, or any range therebetween. In certain embodiments, a green tea extract composition can comprise an amount of phycocyanin and an amount of phycocyanin oligopeptides present in a ratio of about 1:1, or about 20:1 to about 1:20 of phycocyanin to phycocyanin oligopeptides, or any range therebetween. In some embodiments, a phycocyanin composition can comprise an amount of an extract of phycocyanin and an amount of phycocyanin peptides present in a ratio of about 1:1, or about 20:1 to about 1:20 of an extract of phycocyanin to phycocyanin peptides, or any range therebetween. In certain embodiments, a green tea extract composition can comprise an amount of an extract of phycocyanin and an amount of phycocyanin oligopeptides present in a ratio of about 1:1, or about 20:1 to about 1:20 of an extract of phycocyanin to phycocyanin oligopeptides, or any range therebetween. In some embodiments, a green tea extract composition can comprise an amount of phycocyanin peptides and an amount of phycocyanin oligopeptides present in a ratio of about 1:1, or about 20:1 to about 1:20 of phycocyanin peptides to phycocyanin oligopeptides, or any range therebetween. As described further herein, any two of the aforementioned compounds can be present in a ratio of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, or any ratio in between. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a phycocyanin composition of the instant disclosure to achieve the results described herein. In certain embodiments, a phycocyanin composition, as described herein, may comprise an amount of three or more of phycocyanin, an extract of phycocyanin, phycocyanin peptides, and phycocyanin oligopeptides. The three or more compounds may be present in a ratio. The ratio can be understood as comprising a “part” of any compound. For example, a phycocyanin composition may comprise a ratio of 4:1:10, phycocyanin to phycocyanin peptides to phycocyanin oligopeptides, corresponding to 4 parts phycocyanin, 1 part phycocyanin peptides, and 10 parts phycocyanin oligopeptides. The individual amount of any of the aforementioned compounds may be as low as 1 part, may be as high as 20 parts, or may any value therebetween. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a phycocyanin composition of the instant disclosure to achieve the results described herein. Some embodiments provide a composition comprising an amount of a curcumin composition, as described herein, an amount of a green tea extract composition, as described herein, an amount of a phycocyanin composition, as described herein, or any combination of the foregoing formulated as a combination composition. In some embodiments, a combination composition, as described herein, may comprise an amount of at least two of a curcumin composition, as described herein, a green tea extract composition, as described herein, and a phycocyanin composition, as described herein. For example, a combination composition can comprise an amount of a curcumin composition, as described herein, and an amount of a green tea extract composition, as described herein, present in a ratio of about 1:1, or about 20:1 to about 1:20 of a curcumin composition, as described herein, to a green tea extract composition, as described herein, or any range therebetween. In some embodiments, a combination composition can comprise an amount of a curcumin composition, as described herein, and an amount of a phycocyanin composition, as described herein, present in a ratio of about 1:1, or about 20:1 to about 1:20 of a curcumin composition, as described herein, to a phycocyanin composition, as described herein, or any range therebetween. In certain embodiments, a combination composition can comprise an amount of a green tea extract composition, as described herein, and an amount of a phycocyanin composition, as described herein, present in a ratio of about 1:1, or about 20:1 to about 1:20 of a green tea extract composition, as described herein, to a phycocyanin composition, as described herein, or any range therebetween. As described further herein, any two of the aforementioned compositions comprising a combination composition can be present in a ratio of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, or any ratio in between. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a combination composition of the instant disclosure to achieve the results described herein. In certain embodiments, a combination composition, as described herein, may comprise an amount of a curcumin composition, as described herein, an amount of a green tea extract composition, as described herein, and an amount of a phycocyanin composition, as described herein, provided as a ratio. The ratio of curcumin composition to green tea extract composition to phycocyanin may be provided in a ratio of about 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, 1:2:10, 1:3:1, 1:3:2, 1:3:3, 1:3:4, 1:3:5, 1:3:6, 1:3:7, 1:3:8, 1:3:9, 1:3:10, 1:4:1, 1:4:2, 1:4:3, 1:4:4, 1:4:5, 1:4:6, 1:4:7, 1:4:8, 1:4:9, 1:4:10, 1:5:1, 1:5:2, 1:5:3, 1:5:4, 1:5:5, 1:5:6, 1:5:7, 1:5:8, 1:5:9, 1:5:10, 1:6:1, 1:6:2, 1:6:3, 1:6:4, 1:6:5, 1:6:6, 1:6:7, 1:6:8, 1:6:9, 1:6:10, 1:7:1, 1:7:2, 1:7:3, 1:7:4, 1:7:5, 1:7:6, 1:7:7, 1:7:8, 1:7:9, 1:7:10, 1:8:1, 1:8:2, 1:8:3, 1:8:4, 1:8:5, 1:8:6, 1:8:7, 1:8:8, 1:8:9, 1:8:10, 1:9:1, 1:9:2, 1:9:3, 1:9:4, 1:9:5, 1:9:6, 1:9:7, 1:9:8, 1:9:9, 1:9:10, 1:10:1, 1:10:2, 1:10:3, 1:10:4, 1:10:5, 1:10:6, 1:10:7, 1:10:8, 1:10:9, 1:10:10, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:2:1, 2:2:2, 2:2:3, 2:2:4, 2:2:5, 2:2:6, 2:2:7, 2:2:8, 2:2:9, 2:2:10, 2:3:1, 2:3:2, 2:3:3, 2:3:4, 2:3:5, 2:3:6, 2:3:7, 2:3:8, 2:3:9, 2:3:10, 2:4:1, 2:4:2, 2:4:3, 2:4:4, 2:4:5, 2:4:6, 2:4:7, 2:4:8, 2:4:9, 2:4:10, 2:5:1, 2:5:2, 2:5:3, 2:5:4, 2:5:5, 2:5:6, 2:5:7, 2:5:8, 2:5:9, 2:5:10, 2:6:1, 2:6:2, 2:6:3, 2:6:4, 2:6:5, 2:6:6, 2:6:7, 2:6:8, 2:6:9, 2:6:10, 2:7:1, 2:7:2, 2:7:3, 2:7:4, 2:7:5, 2:7:6, 2:7:7, 2:7:8, 2:7:9, 2:7:10, 2:8:1, 2:8:2, 2:8:3, 2:8:4, 2:8:5, 2:8:6, 2:8:7, 2:8:8, 2:8:9, 2:8:10, 2:9:1, 2:9:2, 2:9:3, 2:9:4, 2:9:5, 2:9:6, 2:9:7, 2:9:8, 2:9:9, 2:9:10, 2:10:1, 2:10:2, 2:10:3, 2:10:4, 2:10:5, 2:10:6, 2:10:7, 2:10:8, 2:10:9, 2:10:10, 3:1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:2:1, 3:2:2, 3:2:3, 3:2:4, 3:2:5, 3:2:6, 3:2:7, 3:2:8, 3:2:9, 3:2:10, 3:3:1, 3:3:2, 3:3:3, 3:3:4, 3:3:5, 3:3:6, 3:3:7, 3:3:8, 3:3:9, 3:3:10, 3:4:1, 3:4:2, 3:4:3, 3:4:4, 3:4:5, 3:4:6, 3:4:7, 3:4:8, 3:4:9, 3:4:10, 3:5:1, 3:5:2, 3:5:3, 3:5:4, 3:5:5, 3:5:6, 3:5:7, 3:5:8, 3:5:9, 3:5:10, 3:6:1, 3:6:2, 3:6:3, 3:6:4, 3:6:5, 3:6:6, 3:6:7, 3:6:8, 3:6:9, 3:6:10, 3:7:1, 3:7:2, 3:7:3, 3:7:4, 3:7:5, 3:7:6, 3:7:7, 3:7:8, 3:7:9, 3:7:10, 3:8:1, 3:8:2, 3:8:3, 3:8:4, 3:8:5, 3:8:6, 3:8:7, 3:8:8, 3:8:9, 3:8:10, 3:9:1, 3:9:2, 3:9:3, 3:9:4, 3:9:5, 3:9:6, 3:9:7, 3:9:8, 3:9:9, 3:9:10, 3:10:1, 3:10:2, 3:10:3, 3:10:4, 3:10:5, 3:10:6, 3:10:7, 3:10:8, 3:10:9, 3:10:10, 4:1:1, 4:1:2, 4:1:3, 4:1:4, 4:1:5, 4:1:6, 4:1:7, 4:1:8, 4:1:9, 4:1:10, 4:2:1, 4:2:2, 4:2:3, 4:2:4, 4:2:5, 4:2:6, 4:2:7, 4:2:8, 4:2:9, 4:2:10, 4:3:1, 4:3:2, 4:3:3, 4:3:4, 4:3:5, 4:3:6, 4:3:7, 4:3:8, 4:3:9, 4:3:10, 4:4:1, 4:4:2, 4:4:3, 4:4:4, 4:4:5, 4:4:6, 4:4:7, 4:4:8, 4:4:9, 4:4:10, 4:5:1, 4:5:2, 4:5:3, 4:5:4, 4:5:5, 4:5:6, 4:5:7, 4:5:8, 4:5:9, 4:5:10, 4:6:1, 4:6:2, 4:6:3, 4:6:4, 4:6:5, 4:6:6, 4:6:7, 4:6:8, 4:6:9, 4:6:10, 4:7:1, 4:7:2, 4:7:3, 4:7:4, 4:7:5, 4:7:6, 4:7:7, 4:7:8, 4:7:9, 4:7:10, 4:8:1, 4:8:2, 4:8:3, 4:8:4, 4:8:5, 4:8:6, 4:8:7, 4:8:8, 4:8:9, 4:8:10, 4:9:1, 4:9:2, 4:9:3, 4:9:4, 4:9:5, 4:9:6, 4:9:7, 4:9:8, 4:9:9, 4:9:10, 4:10:1, 4:10:2, 4:10:3, 4:10:4, 4:10:5, 4:10:6, 4:10:7, 4:10:8, 4:10:9, 4:10:10, 5:1:1, 5:1:2, 5:1:3, 5:1:4, 5:1:5, 5:1:6, 5:1:7, 5:1:8, 5:1:9, 5:1:10, 5:2:1, 5:2:2, 5:2:3, 5:2:4, 5:2:5, 5:2:6, 5:2:7, 5:2:8, 5:2:9, 5:2:10, 5:3:1, 5:3:2, 5:3:3, 5:3:4, 5:3:5, 5:3:6, 5:3:7, 5:3:8, 5:3:9, 5:3:10, 5:4:1, 5:4:2, 5:4:3, 5:4:4, 5:4:5, 5:4:6, 5:4:7, 5:4:8, 5:4:9, 5:4:10, 5:5:1, 5:5:2, 5:5:3, 5:5:4, 5:5:5, 5:5:6, 5:5:7, 5:5:8, 5:5:9, 5:5:10, 5:6:1, 5:6:2, 5:6:3, 5:6:4, 5:6:5, 5:6:6, 5:6:7, 5:6:8, 5:6:9, 5:6:10, 5:7:1, 5:7:2, 5:7:3, 5:7:4, 5:7:5, 5:7:6, 5:7:7, 5:7:8, 5:7:9, 5:7:10, 5:8:1, 5:8:2, 5:8:3, 5:8:4, 5:8:5, 5:8:6, 5:8:7, 5:8:8, 5:8:9, 5:8:10, 5:9:1, 5:9:2, 5:9:3, 5:9:4, 5:9:5, 5:9:6, 5:9:7, 5:9:8, 5:9:9, 5:9:10, 5:10:1, 5:10:2, 5:10:3, 5:10:4, 5:10:5, 5:10:6, 5:10:7, 5:10:8, 5:10:9, 5:10:10, 6:1:1, 6:1:2, 6:1:3, 6:1:4, 6:1:5, 6:1:6, 6:1:7, 6:1:8, 6:1:9, 6:1:10, 6:2:1, 6:2:2, 6:2:3, 6:2:4, 6:2:5, 6:2:6, 6:2:7, 6:2:8, 6:2:9, 6:2:10, 6:3:1, 6:3:2, 6:3:3, 6:3:4, 6:3:5, 6:3:6, 6:3:7, 6:3:8, 6:3:9, 6:3:10, 6:4:1, 6:4:2, 6:4:3, 6:4:4, 6:4:5, 6:4:6, 6:4:7, 6:4:8, 6:4:9, 6:4:10, 6:5:1, 6:5:2, 6:5:3, 6:5:4, 6:5:5, 6:5:6, 6:5:7, 6:5:8, 6:5:9, 6:5:10, 6:6:1, 6:6:2, 6:6:3, 6:6:4, 6:6:5, 6:6:6, 6:6:7, 6:6:8, 6:6:9, 6:6:10, 6:7:1, 6:7:2, 6:7:3, 6:7:4, 6:7:5, 6:7:6, 6:7:7, 6:7:8, 6:7:9, 6:7:10, 6:8:1, 6:8:2, 6:8:3, 6:8:4, 6:8:5, 6:8:6, 6:8:7, 6:8:8, 6:8:9, 6:8:10, 6:9:1, 6:9:2, 6:9:3, 6:9:4, 6:9:5, 6:9:6, 6:9:7, 6:9:8, 6:9:9, 6:9:10, 6:10:1, 6:10:2, 6:10:3, 6:10:4, 6:10:5, 6:10:6, 6:10:7, 6:10:8, 6:10:9, 6:10:10, 7:1:1, 7:1:2, 7:1:3, 7:1:4, 7:1:5, 7:1:6, 7:1:7, 7:1:8, 7:1:9, 7:1:10, 7:2:1, 7:2:2, 7:2:3, 7:2:4, 7:2:5, 7:2:6, 7:2:7, 7:2:8, 7:2:9, 7:2:10, 7:3:1, 7:3:2, 7:3:3, 7:3:4, 7:3:5, 7:3:6, 7:3:7, 7:3:8, 7:3:9, 7:3:10, 7:4:1, 7:4:2, 7:4:3, 7:4:4, 7:4:5, 7:4:6, 7:4:7, 7:4:8, 7:4:9, 7:4:10, 7:5:1, 7:5:2, 7:5:3, 7:5:4, 7:5:5, 7:5:6, 7:5:7, 7:5:8, 7:5:9, 7:5:10, 7:6:1, 7:6:2, 7:6:3, 7:6:4, 7:6:5, 7:6:6, 7:6:7, 7:6:8, 7:6:9, 7:6:10, 7:7:1, 7:7:2, 7:7:3, 7:7:4, 7:7:5, 7:7:6, 7:7:7, 7:7:8, 7:7:9, 7:7:10, 7:8:1, 7:8:2, 7:8:3, 7:8:4, 7:8:5, 7:8:6, 7:8:7, 7:8:8, 7:8:9, 7:8:10, 7:9:1, 7:9:2, 7:9:3, 7:9:4, 7:9:5, 7:9:6, 7:9:7, 7:9:8, 7:9:9, 7:9:10, 7:10:1, 7:10:2, 7:10:3, 7:10:4, 7:10:5, 7:10:6, 7:10:7, 7:10:8, 7:10:9, 7:10:10, 8:1:1, 8:1:2, 8:1:3, 8:1:4, 8:1:5, 8:1:6, 8:1:7, 8:1:8, 8:1:9, 8:1:10, 8:2:1, 8:2:2, 8:2:3, 8:2:4, 8:2:5, 8:2:6, 8:2:7, 8:2:8, 8:2:9, 8:2:10, 8:3:1, 8:3:2, 8:3:3, 8:3:4, 8:3:5, 8:3:6, 8:3:7, 8:3:8, 8:3:9, 8:3:10, 8:4:1, 8:4:2, 8:4:3, 8:4:4, 8:4:5, 8:4:6, 8:4:7, 8:4:8, 8:4:9, 8:4:10, 8:5:1, 8:5:2, 8:5:3, 8:5:4, 8:5:5, 8:5:6, 8:5:7, 8:5:8, 8:5:9, 8:5:10, 8:6:1, 8:6:2, 8:6:3, 8:6:4, 8:6:5, 8:6:6, 8:6:7, 8:6:8, 8:6:9, 8:6:10, 8:7:1, 8:7:2, 8:7:3, 8:7:4, 8:7:5, 8:7:6, 8:7:7, 8:7:8, 8:7:9, 8:7:10, 8:8:1, 8:8:2, 8:8:3, 8:8:4, 8:8:5, 8:8:6, 8:8:7, 8:8:8, 8:8:9, 8:8:10, 8:9:1, 8:9:2, 8:9:3, 8:9:4, 8:9:5, 8:9:6, 8:9:7, 8:9:8, 8:9:9, 8:9:10, 8:10:1, 8:10:2, 8:10:3, 8:10:4, 8:10:5, 8:10:6, 8:10:7, 8:10:8, 8:10:9, 8:10:10, 9:1:1, 9:1:2, 9:1:3, 9:1:4, 9:1:5, 9:1:6, 9:1:7, 9:1:8, 9:1:9, 9:1:10, 9:2:1, 9:2:2, 9:2:3, 9:2:4, 9:2:5, 9:2:6, 9:2:7, 9:2:8, 9:2:9, 9:2:10, 9:3:1, 9:3:2, 9:3:3, 9:3:4, 9:3:5, 9:3:6, 9:3:7, 9:3:8, 9:3:9, 9:3:10, 9:4:1, 9:4:2, 9:4:3, 9:4:4, 9:4:5, 9:4:6, 9:4:7, 9:4:8, 9:4:9, 9:4:10, 9:5:1, 9:5:2, 9:5:3, 9:5:4, 9:5:5, 9:5:6, 9:5:7, 9:5:8, 9:5:9, 9:5:10, 9:6:1, 9:6:2, 9:6:3, 9:6:4, 9:6:5, 9:6:6, 9:6:7, 9:6:8, 9:6:9, 9:6:10, 9:7:1, 9:7:2, 9:7:3, 9:7:4, 9:7:5, 9:7:6, 9:7:7, 9:7:8, 9:7:9, 9:7:10, 9:8:1, 9:8:2, 9:8:3, 9:8:4, 9:8:5, 9:8:6, 9:8:7, 9:8:8, 9:8:9, 9:8:10, 9:9:1, 9:9:2, 9:9:3, 9:9:4, 9:9:5, 9:9:6, 9:9:7, 9:9:8, 9:9:9, 9:9:10, 9:10:1, 9:10:2, 9:10:3, 9:10:4, 9:10:5, 9:10:6, 9:10:7, 9:10:8, 9:10:9, 9:10:10, 10:1:1, 10:1:2, 10:1:3, 10:1:4, 10:1:5, 10:1:6, 10:1:7, 10:1:8, 10:1:9, 10:1:10, 10:2:1, 10:2:2, 10:2:3, 10:2:4, 10:2:5, 10:2:6, 10:2:7, 10:2:8, 10:2:9, 10:2:10, 10:3:1, 10:3:2, 10:3:3, 10:3:4, 10:3:5, 10:3:6, 10:3:7, 10:3:8, 10:3:9, 10:3:10, 10:4:1, 10:4:2, 10:4:3, 10:4:4, 10:4:5, 10:4:6, 10:4:7, 10:4:8, 10:4:9, 10:4:10, 10:5:1, 10:5:2, 10:5:3, 10:5:4, 10:5:5, 10:5:6, 10:5:7, 10:5:8, 10:5:9, 10:5:10, 10:6:1, 10:6:2, 10:6:3, 10:6:4, 10:6:5, 10:6:6, 10:6:7, 10:6:8, 10:6:9, 10:6:10, 10:7:1, 10:7:2, 10:7:3, 10:7:4, 10:7:5, 10:7:6, 10:7:7, 10:7:8, 10:7:9, 10:7:10, 10:8:1, 10:8:2, 10:8:3, 10:8:4, 10:8:5, 10:8:6, 10:8:7, 10:8:8, 10:8:9, 10:8:10, 10:9:1, 10:9:2, 10:9:3, 10:9:4, 10:9:5, 10:9:6, 10:9:7, 10:9:8, 10:9:9, 10:9:10, 10:10:1, 10:10:2, 10:10:3, 10:10:4, 10:10:5, 10:10:6, 10:10:7, 10:10:8, 10:10:9, 10:10:10, or any ratio in between. In view of the results and discussion contained herein, one of skill in the art would understand how to formulate a combination composition comprising a curcumin composition, as described herein, a green tea extract, as described herein, and a phycocyanin composition, as described here, to achieve the results described herein. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of diferuloylmethane to EGCG to phycocyanin or an extract thereof, about 6:3:1 of diferuloylmethane to EGCG to phycocyanin or an extract thereof, about 7.2:1, 8:1 of diferuloylmethane to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of diferuloylmethane to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of diferuloylmethane to EC to phycocyanin or an extract thereof, about 6:3:1 of diferuloylmethane to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of diferuloylmethane to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of diferuloylmethane to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of diferuloylmethane to EGC to phycocyanin or an extract thereof, about 6:3:1 of diferuloylmethane to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of diferuloylmethane to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of diferuloylmethane to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of diferuloylmethane to ECG to phycocyanin or an extract thereof, about 6:3:1 of diferuloylmethane to ECG phycocyanin or an extract thereof, about 7, 2:1.8:1 of diferuloylmethane to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of diferuloylmethane to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of DMC to EGCG to phycocyanin or an extract thereof, about 6:3:1 of DMC to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of DMC to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of DMC to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of DMC to EC to phycocyanin or an extract thereof, about 6:3:1 of DMC to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of DMC to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of DMC to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of DMC to EGC to phycocyanin or an extract thereof, about 6:3:1 of DMC to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of DMC to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of DMC to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of DMC to ECG to phycocyanin or an extract thereof, about 6:3:1 of DMC to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of DMC to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of DMC to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of BDMC to EGCG to phycocyanin or an extract thereof, about 6:3:1 of BDMC to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of BDMC to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of BDMC to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of BDMC to EC to phycocyanin or an extract thereof, about 6:3:1 of BDMC to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of BDMC to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of BDMC to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of BDMC to EGC to phycocyanin or an extract thereof, about 6:3:1 of BDMC to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of BDMC to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of BDMC to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of BDMC to ECG to phycocyanin or an extract thereof, about 6:3:1 of BDMC to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of BDMC to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of BDMC to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of tetrahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 6:3:1 of tetrahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of tetrahydrocurcumin to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of tetrahydrocurcumin to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of tetrahydrocurcumin to EC to phycocyanin or an extract thereof, about 6:3:1 of tetrahydrocurcumin to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of tetrahydrocurcumin to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of tetrahydrocurcumin to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of tetrahydrocurcumin to EGC to phycocyanin or an extract thereof, about 6:3:1 of tetrahydrocurcumin to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of tetrahydrocurcumin to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of tetrahydrocurcumin to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of tetrahydrocurcumin to ECG to phycocyanin or an extract thereof, about 6:3:1 of tetrahydrocurcumin to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of tetrahydrocurcumin to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of tetrahydrocurcumin to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of dihydrocurcumin to EGCG to phycocyanin or an extract thereof, about 6:3:1 of dihydrocurcumin to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of dihydrocurcumin to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of dihydrocurcumin to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of dihydrocurcumin to EC to phycocyanin or an extract thereof, about 6:3:1 of dihydrocurcumin to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of dihydrocurcumin to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of dihydrocurcumin to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of dihydrocurcumin to EGC to phycocyanin or an extract thereof, about 6:3:1 of dihydrocurcumin to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of dihydrocurcumin to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of dihydrocurcumin to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of dihydrocurcumin to ECG to phycocyanin or an extract thereof, about 6:3:1 of dihydrocurcumin to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of dihydrocurcumin to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of dihydrocurcumin to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of hexahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 6:3:1 of hexahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of hexahydrocurcumin to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of hexahydrocurcumin to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of hexahydrocurcumin to EC to phycocyanin or an extract thereof, about 6:3:1 of hexahydrocurcumin to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of hexahydrocurcumin to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of hexahydrocurcumin to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of hexahydrocurcumin to EGC to phycocyanin or an extract thereof, about 6:3:1 of hexahydrocurcumin to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of hexahydrocurcumin to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of hexahydrocurcumin to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of hexahydrocurcumin to ECG to phycocyanin or an extract thereof, about 6:3:1 of hexahydrocurcumin to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of hexahydrocurcumin to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of hexahydrocurcumin to ECG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of octahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 6:3:1 of octahydrocurcumin to EGCG to phycocyanin or an extract thereof, about 7.2:1.8:1 of octahydrocurcumin to EGCG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of octahydrocurcumin to EGCG to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of octahydrocurcumin to EC to phycocyanin or an extract thereof, about 6:3:1 of octahydrocurcumin to EC to phycocyanin or an extract thereof, about 7.2:1.8:1 of octahydrocurcumin to EC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of octahydrocurcumin to EC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of octahydrocurcumin to EGC to phycocyanin or an extract thereof, about 6:3:1 of octahydrocurcumin to EGC to phycocyanin or an extract thereof, about 7.2:1.8:1 of octahydrocurcumin to EGC to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of octahydrocurcumin to EGC to phycocyanin or an extract thereof. In certain embodiments, a combination composition can comprise about 4.5:4.5:1 of octahydrocurcumin to ECG to phycocyanin or an extract thereof, about 6:31 of octahydrocurcumin to ECG to phycocyanin or an extract thereof, about 7.2:1.8:1 of octahydrocurcumin to ECG to phycocyanin or an extract thereof, or any ratio between about 8:1:1 to about 1:8:1 of octahydrocurcumin to ECG to phycocyanin or an extract thereof. In some embodiments, a combination composition, as described herein, may comprise between about 10 μg to about 10 g of one or more of a curcumin composition, as described herein, a green tea extract composition, as described herein, and a phycocyanin composition, as described herein. For example, some embodiments include a combination composition comprising one or more of a of a curcumin composition, as described herein, a green tea extract composition, as described herein, and a phycocyanin composition, as described herein present at an amount of about 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 725 μg, 750 μg, 775 μg, 800 μg, 825 μg, 850 μg, 875 μg, 900 μg, 925 μg, 950 μg, 975 μg, 1000 μg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, or any range or amount in between any two of the preceding values and any other ranges or amounts disclosed herein. In some embodiments, a curcumin composition, a green tea extract composition, a phycocyanin composition, or a combination composition, as described herein, can comprise one or more supplement ingredients. As used herein, the term supplement ingredient can refer to essential fatty acids such as linolenic acid and linoleic acid, and essential amino acids such as tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine, isoleucine, arginine, and histidine, and n-acetyl cysteine. Also included within the meaning of supplement ingredients are vitamins such as retinol (vitamin A), thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine, pyridoxamine, orpyridoxal (vitamin 86), biotin (vitamin B7) or pharmaceutically acceptable salts thereof, folic acid (vitamin B9) or pharmaceutically acceptable salts thereof, cobalamin (vitamin B12), choline, ascorbic acid (vitamin C) or pharmaceutically acceptable salts thereof, ergocalciferol (vitamin D2), calciferol (vitamin D3), 22-dihydroergocalciferol (vitamin D4), sitocalciferol (vitamin D5), tocopherol (vitamin E), phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), or any combination of the foregoing. Other vitamins not explicitly listed would readily be envisaged by those of skill in the art, in view of the disclosure contained herein. Supplement ingredients can further include dietary minerals such as, for example, chromium, bromine, cobalt, copper, fluorine, germanium, iodine, iron, magnesium, manganese, molybdenum, potassium, selenium, silicon, zinc, calcium, phosphorous, sodium, sulfur, and vanadium. Supplement ingredients can also comprise cranberry extract, turmeric, royal jelly, açai berry, beet root, coral calcium, oyster shell, gotu kola,Gingko biloba, lions mane mushroom, pomegranate, hibiscus flower, strawberry powder, dandelion root, celery powder, parsley powder, peppermint leaf, cinnamon bark powder, maca root, nicotinamide riboside, resveratrol, NAD+ precursors, Coenzyme Q10, omega-3-fatty acids, cabbage powder, pterostilbene, nicotinamide mononucleotide, and combinations thereof. Supplement ingredients can include nitrates such as citrulline nitrate, creatine nitrate, beta-alanine nitrate, and the like. Compositions described herein can include one or more of the foregoing supplement ingredients, as would be understood by one of skill in the art. In some embodiments, an amount of least one supplemental ingredient, as disclosed herein, can be about 10 μg to about 10 g. For example, the amount of the at least one supplemental ingredient in the composition can be about 10 μg, 15 μg, 20 μg, 25 μg, 30 sg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 725 μg, 750 μg, 775 μg, 800 μg, 825 μg, 850 μg, 875 μg, 900 μg, 925 μg, 950 μg, 975 μg, 1000 μg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, or any range or amount in between any two of the preceding values and any other ranges or amounts disclosed herein. In certain embodiments, a curcumin composition, a green tea extract composition, a phycocyanin composition, or a combination composition, as described herein, can be formulated as a dietary supplement or pharmaceutical agent. The compounds comprising curcumin compositions, green tea extract compositions, and phycocyanin compositions, as described herein, may be an active agent present in a therapeutically effective amount. By way of example, a “therapeutically effective amount” and/or an “effective amount” of the compound disclosed herein can be (on a dosage weight per subject weight basis), for example, 0.1 μg/kg, 0.5 μg/kg, 1 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 2.5 μg/kg, 3.0 μg/kg, 3.5 μg/kg, 4.0 μg/kg, 4.5 μg/kg, 5.0 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg, 250 μg/kg, 300 μg/kg, 350 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 80 μg/kg 0, 850 μg/kg, 900 μg/kg, 1 mg/kg, 1.5 mg·kg, 2.0 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, or more, or any fraction or integer in between any two of the preceding amounts of the compound. An effective amount may include any of the ranges and amounts discussed herein. Accordingly, in some embodiments, the dose of the compound in compositions disclosed herein (corresponding to the therapeutically effective amount), can be about 10 μg to about 10 g, preferably per day. For example, the amount of the composition can be 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 725 μg, 750 μg, 775 μg, 800 μg, 825 μg, 850 μg, 875 μg, 900 μg, 925 μg, 950 μg, 975 μg, 1000 μg, 1.25 g, 1.5 g, 1.75 g, 2.0 g, 2.25 g, 2.5 g, 2.75 g, 3.0 g, 3.25 g, 3.5 g, 3.5 g, 3.75 g, 4.0 g, 4.25 g, 4.5 g, 4.75 g, 5.0 g, 5.25 g, 5.5 g, 5.75 g, 6.0 g, 6.25 g, 6.5 g, 6.75 g, 7.0 g, 7.25 g, 7.5 g, 7.75 g, 8.0 g, 8.25 g, 8.5 g, 8.75 g, 9.0 g, 8.25 g, 9.5 g, 9.75 g, 10 g, or more, or any range or amount in between any two of the preceding values and any other ranges or amounts disclosed herein. In some embodiments, a curcumin composition, as described herein, a green tea extract composition, as described herein, and a phycocyanin composition, as described herein, are provided in a synergistic ratio in a combination composition, and optionally, the combination composition may be provided as a dietary supplement or pharmaceutical agent. The synergistic ratio is not particularly limited. In some embodiments the synergistic ratio can comprise 4.5:4.5:1 of curcumin composition to green tea extract composition to phycocyanin composition. In some embodiments the synergistic ratio can comprise 6:3:1 of curcumin composition to green tea extract composition to phycocyanin composition. In some embodiments the synergistic ratio can comprise 7.2:1.8:1 of curcumin composition to green tea extract composition to phycocyanin composition. In some embodiments the synergistic can comprise any ratio between about 8:1:1 to about 1:8:1 of curcumin composition to green tea extract composition phycocyanin composition. In some embodiments, compositions, as described herein, can be administered in the methods described elsewhere herein on an hourly basis, e.g., every one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three hours, or any interval in between, or on a daily basis, every two days, every three days, every four days, every five days, every six days, every week, every eight days, every nine days, every ten days, every two weeks, every month, or more or less frequently, as needed to achieve the desired therapeutic effect. In some embodiments, a ramping administration protocol where a subject is administered temporally increasing amounts of compositions described herein can be utilized. For example, a subject could be administered with 100 mg of a composition as described herein per day for 7 days, followed by 200 mg, for the next 7 days, followed by 300 mg for the next 7 days. Administration protocols can also follow a pattern whereby the dosage amount decreases over time. For example, 300 mg of a composition as described herein per day for 7 days, followed by 200 mg, for the next 7 days, followed by 100 mg for the next 7 days. In some embodiments, the methods as described herein can be utilized in combination with a calorie restriction protocol in a subject. In certain embodiments, the compositions described herein may be administered before, after, or during a meal. In addition, the appropriate dosage of the compositions can depend, for example, on the condition to be treated, the severity and course of the condition, whether the composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition, the type of composition used, and the discretion of the attending physician. The composition can be suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The composition may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question. The present disclosure discloses combination compositions comprising one or more of a curcumin composition, a green tea extract composition, and a phycocyanin composition, optionally formulated as a nutritional supplement or pharmaceutical agent, useful as an NK3 antagonist, and methods of using the same. Some embodiments provide solid dosage forms of the compositions disclosed herein. Certain embodiments provide aqueous solutions of compositions disclosed herein. Embodiments described herein comprising compositions disclosed herein as a nutritional supplement means that the composition disclosed herein is present in an unnatural form, i.e., is presented in a supplement (e.g., in a pill or powder) that is different from that which occurs naturally, or the nutritional or dietary supplement results in unnatural supplementation that is unachievable through a non-supplemented diet. Some embodiments can further comprise a matrix material such as a fatty acid, fatty acid ester, triglycerides, oils, lipid solvents, and the like. In some embodiments, a composition is a solid composition. In some embodiments, the composition comprises a sustained-release matrix. In some embodiments, the composition is enteric coated. Some embodiments provide physiologically compatible compositions, as disclosed herein, including hydrates, crystalline forms, polymorphic forms, solid forms having specific bulk densities or tap densities, and solid forms having specific particle sizes. Some embodiments provide compositions coated with pharmaceutically acceptable materials intended to modify its release and/or bioavailability, including, but not limited to Eudragit®, microcrystalline cellulose, hydroxypropylmethylcellulose phthalate, and the like. For oral administration, the compositions disclosed herein can be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup, elixir, or beverage. Solid dosage forms such as tablets and capsules may comprise an enteric coating. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutically acceptable compositions and such compositions may include one or more of the following agents: sweeteners, flavoring agents, coloring agents, coatings, and preservatives. The sweetening and flavoring agents will increase the palatability of the preparation. Tablets containing the complexes in admixture with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture are acceptable. Pharmaceutically acceptable vehicles such as excipients are compatible with the other ingredients of the formulation (as well as non-injurious to the patient). Such excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents such as starch, gelatin, or acacia; and lubricating agents such as magnesium stearate, stearic acid or tale. Tablets can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax can be employed. Formulations for oral use can also be presented as hard gelatin-containing or non-gelatinous capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Aqueous suspensions can contain the complex of the described herein admixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, dispersing, or wetting agents, one or more preservatives, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin. Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil, such asarachisoil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspension can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by an added antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water can provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Additional excipients, for example sweetening, flavoring, and coloring agents, can also be present. Syrups and elixirs can be formulated with sweetening agents, such as glycerol, sorbitol, or sucrose. Such formulations can also contain a demulcent, a preservative, a flavoring, or a coloring agent. The composition for parenteral administration can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butanediol. Suitable diluents include, for example, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectable preparations. It will be appreciated that the amount of the compound may be combined with a carrier material to produce a single dosage form. Such forms will vary depending upon the host treated and the particular mode of administration. In some aspects, compositions described herein may be administered via supplements or dosages designed for animals. In some animal applications, the compound or composition may be added to and/or comprise a pet treat or biscuit, for example, a dog biscuit or a cat treat. Aqueous suspensions may contain the compound disclosed herein in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, dispersing, or wetting agents, one or more preservatives, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin. Utilization of controlled release vehicles would readily be envisaged by those of skill in the pharmaceutical sciences in view of the disclosure contained herein, and these aspects can be applied to nutritional and dietary supplements. The technology and products in this art are variably referred to as controlled release, sustained release, prolonged action, depot, repository, delayed action, retarded release, and timed release; the words “controlled release” as used herein is intended to incorporate each of the foregoing technologies. Numerous controlled release vehicles can be used, including biodegradable or bioerodable polymers such as polylactic acid, polyglycolic acid, and regenerated collagen. Controlled release drug delivery devices can include creams, lotions, tablets, capsules, gels, microspheres, liposomes, ocular inserts, minipumps, and other infusion devices such as pumps and syringes. Implantable or injectable polymer matrices, and transdermal formulations, from which active ingredients are slowly released, and can be used in the disclosed methods. Controlled release preparations can be achieved by the use of polymers to form complexes with or absorb a curcumin composition. The controlled delivery can be exercised by selecting appropriate macromolecules such as polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamine sulfate, and the concentration of these macromolecule as well as the methods of incorporation are selected in order to control release of active complex. Controlled release of active complexes can be taken to mean any of the extended release dosage forms. The following terms may be considered to be substantially equivalent to controlled release, for the purposes of the present disclosure: continuous release, controlled release, delayed release, depot, gradual release, long term release, programmed release, prolonged release, programmed release, proportionate release, protracted release, repository, retard, slow release, spaced release, sustained release, time coat, time release, delayed action, extended action, layered time action, long acting, prolonged action, sustained action medications and extended release, release in terms of pH level in the gut and intestine, breakdown of the molecule and based on the absorption and bioavailability. Hydrogels, wherein a composition as disclosed herein is dissolved in an aqueous constituent to gradually release over time, can be prepared by copolymerization of hydrophilic mono-olefinic monomers such as ethylene glycol methacrylate. Matrix devices, wherein a curcumin composition is dispersed in a matrix of carrier material, can be used. The carrier can be porous, non-porous, solid, semi-solid, permeable, or impermeable. Alternatively, a device comprising a central reservoir of a composition disclosed herein surrounded by a rate controlling membrane can be used to control the release of the complex. Rate controlling membranes include ethylene-vinyl acetate copolymer or butylene terephthalate/polytetramethylene ether terephthalate. Use of silicon rubber or ethylene-vinyl alcohol depots are also contemplated. Controlled release oral formulations can also be used. In an embodiment, a composition as described herein is incorporated into a soluble or erodible matrix, such as a pill or a lozenge. In another example, the oral formulations can be a liquid used for sublingual administration. These liquid compositions can also be in the form a gel or a paste. Hydrophilic gums, such as hydroxymethylcellulose, are commonly used. A lubricating agent such as magnesium stearate, stearic acid, or calcium stearate can be used to aid in the tableting process. In some embodiments, dosing for oral administration may be with a regimen calling for single daily dose, or for a single dose every other day, or for a single dose within 72 hours of the first administered dose, or for multiple, spaced doses throughout the day. The active agents which make up the therapy may be administered simultaneously, either in a combined dosage form or in separate dosage forms intended for substantially simultaneous oral administration. The active agents which make up the therapy may also be administered sequentially, with either active component being administered by a regimen calling for two-step ingestion. Thus, a regimen may call for sequential administration of the active agents with spaced-apart ingestion of the separate, active agents. The time period between the multiple ingestion steps may range from a few minutes to as long as about 72 hours, depending upon the properties of each active agent such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the agent, as well as depending upon the age and condition of the patient. The active agents of the therapy whether administered simultaneously, substantially simultaneously, or sequentially, may involve a regimen calling for administration of one active agent by oral route and the other active agent by intravenous route. In one aspect, the embodiments described herein can achieve therapeutic and/or nutraceutical benefits not previously recognized or achievable, and thus, unexpectedly, and surprisingly achieve improved abilities for using the compositions. In some embodiments a composition is formulated for intravenous administration because a more concentrated solution can be produced. Whether the active agents of the therapy are administered by oral or intravenous route, separately or together, each such active agent will be contained in a suitable pharmaceutical formulation of pharmaceutically-acceptable excipients, diluents, or other formulations components. In certain embodiments, a combination composition according to the disclosure contained herein is administered to a subject as an NK3 antagonist. In certain embodiments, a composition, as described herein, is administered to a subject to treat, ameliorate, prevent, or reduce the effects of polycystic ovary syndrome. In certain embodiments, a composition as described herein is administered to a subject to support and/or maintain healthy levels of NK3 antagonism. In certain embodiments, a composition, as described herein, is administered to a subject to treat, ameliorate, prevent, or reduce the effects of infertility in women, hirsutism, acne, obesity, chronic anovulation, metabolic syndrome, type II diabetes mellitus, and combinations thereof. In certain embodiments, a composition, as described herein, is administered to support and/or maintain healthy fertility in women. In some embodiments, a composition, as described herein, is administered in combination with metabolic modulators such as metformin. In certain embodiments, a composition, as described herein, is administered to a subject to treat, ameliorate, prevent, or reduce the effects of cardiovascular disease and/or hypertension. In certain embodiments, a composition, as described herein, is administered to a subject to support and/or maintain healthy stress levels. In certain embodiments a woman with a body mass index exceeding 15 kg/m2and identified to have polycystic ovary syndrome is administered a composition, as described herein, to treat, ameliorate, prevent, or reduce the effects of polycystic ovary syndrome. In some embodiments, a composition, as described herein, is administered in combination with luteinizing hormone (LH) is administered to a subject to treat, ameliorate, prevent, or reduce the effects of one or more of the ailments described herein. In some embodiments, a composition, as described herein, is administered to a subject to treat, ameliorate, prevent, or reduce the effects of hot flashes. In some embodiments, a composition, as described herein, is administered to a menopausal woman to maintain a healthy level of hot flashes, i.e., to reduce the number, frequency, and/or severity of hot flashes. Without being bound by any particular theory, it is believed that the compositions and/or dietary supplements disclosed herein act as antagonists by targeting signaling pathways associated with menopause. It is believed that the symptoms associated with menopause are characterized by, and result from, increased secretion of LH and follicle-stimulating hormone (FSH) from the pituitary gland. The increase in LH and FSH secretion is believed to be mediated by increased secretion of gonadotropin hormone-releasing hormone (GnRH) and resulting from increased kisspeptin and NKB signaling. Accordingly, it is believed that by blocking and/or restricting the increased signaling of kisspeptin and NKB in their associated pathways, with antagonists, the increased secretion of GnRH can be reduced, and ultimately the secretion of LH and FSH. By reducing secretion of LH and FSH, it is believed that the symptoms associated with menopause can treated, prevented, ameliorated, and/or the effects associated therewith can be reduced. In some embodiments, a composition as described herein is administered to a subject to treat, ameliorate, prevent, or reduce the effects of mental, psychotic, and/or neuropsychiatric disorders such as schizophrenia, cognitive decline during aging, dementia, depression, Alzheimer's, and the like. In some embodiments, a composition as described herein is administered to support and/or maintain healthy cognitive function. In certain embodiments, a composition as described herein is administered to a subject to treat, ameliorate, prevent, or reduce the effects of neuroinflammation. In some embodiments, a composition as described herein is administered in combination with an antipsychotic drug, including but not limited to, aripiprazole, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, haloperidol, osanetant, and/or talnetant. In some embodiments, a composition as described herein is administered to a subject to treat, ameliorate, prevent, or reduce the effects of drug, nicotine, and/or alcohol addiction. In some embodiments, composition as described herein is administered to a subject to reduce drug-seeking behavior. In some embodiments, a composition as described herein is administered to a subject to treat, ameliorate, prevent, or reduce the effects of disorders associated with inflammation. For example, and without limitation, this includes central nervous system disorders (e.g., anxiety, psychosis, movement and convulsive disorders, and Parkinson's), respiratory and pulmonary inflammatory disorders, skin disorders and itch, gastrointestinal disorders, renal and bladder diseases, inflammatory bowel disease, eating disorders, and chronic pain. In certain embodiments, a curcumin composition is administered to treat hormonal variations. As used herein, “identifying,” refers to detecting or selecting a subject from a population of potential subjects, for example, to establish that a particular subject possesses certain properties or characteristics. “Identifying” may include, for example, self-identification, self-diagnosis, and diagnosis by a medical professional. As used herein, the terms “prophylactic treatment,” “prevent,” or “preventing,” can refer to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. A “disorder” is any condition that would benefit from treatment with the compositions described herein. As used herein, the terms “treating”, “treatment” and the like are used herein to generally refer to obtaining a desired pharmacological and physiological effect and can also refer to a nutritional or nutraceutical effect, the scopes, and meanings of which will be clear to the skilled artisan based upon the context in which these terms are used. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease. The term “treatment” as used herein encompasses any treatment of a disease in a mammal, particularly a human and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease or arresting its development; or (c) relieving the disease, causing regression of the disease and/or its symptoms, conditions, and co-morbidities. The terms “optimum” or “healthy” and the like may be used to refer to the physiological amounts of NK3 activity in a mammal, wherein administration of compositions as described herein may be administered to a mammal that may not have a disease or symptoms of a disease associated with NK3 activity but may be administered to antagonize NK3 along with the other physiological results described herein. As used in the claims below and throughout this disclosure, the phrase “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and can or cannot be present depending upon whether or not they affect the activity or action of the listed elements. For example, the use of a composition “consisting essentially of a composition” for the treatment of a particular disease or disorder, or the maintenance of a healthy condition, would exclude other ingredients that would materially alter the intended outcome of the curcumin composition. As used herein, the meaning of the term “hot flash” would immediately be envisaged by the skilled artisan. The etiology of hot flashes would be understood by the skilled artisan to refer to the sudden feeling of warmth, usually most intense over the face, neck, and chest, and profuse sweating, which is most commonly due to menopause. However, compositions described herein can also be administered to treat, ameliorate, prevent, or reduce the effects of hot flashes that are not caused by menopause. As used herein, a composition that “substantially” comprises a compound means that the composition contains more than about 80% by weight, more preferably more than about 90% by weight, even more preferably more than about 95% by weight, and most preferably more than about 98% by weight of the compound. The term “pharmaceutical formulation”, “formulation”, “composition” and the like can refer to preparations which are in such a form as to permit the biological activity of the active ingredients to be effective, and therefore may be administered to a subject for therapeutic use along with dietary and/or nutritional supplement use. The meaning of these terms will be clear to the skilled artisan based upon the context in which they are used. A “therapeutically effective amount” as used herein includes within its meaning a non-toxic but sufficient amount of a compound active ingredient or composition comprising the same for use in the embodiments disclosed herein to provide the desired therapeutic effect. Similarly, “an amount effective to” or “an effective amount” as used herein includes within its meaning a non-toxic but sufficient amount of a compound active ingredient or composition comprising the same to provide the desired effect. A “therapeutically effective amount” or an “effective amount” includes amounts of compounds that would not be achievable through a standard diet, but requires supplementation and dosing as described herein. The exact amount of the active ingredient disclosed herein required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the weight of the subject, and the mode of administration and so forth. Thus, it may not always be possible to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art in view of the disclosure contained herein. In some aspects, a therapeutically effective amount may include a dosing regimen. For example, a therapeutically effective amount may include about 100 mg of a curcumin composition orally consumed each day for fourteen consecutive days. In some aspects, a therapeutically effective amount may include about 100 mg of a composition orally consumed each day for thirty consecutive days. Compositions including a composition may include, for example, between 0.1-1000 grams of the composition. As used herein, the terms “synergy”, “synergistic”, “synergism” and the like are used herein to generally refer to the therapeutic efficacy of the composition being at least equal to the sum of the efficacy of the individual components in the composition administered independently. “Synergy”, “synergistic”, “synergism” and the like may also refer to the therapeutic efficacy of a composition being greater than the sum of the efficacy of the individual components in the composition administered independently. The scopes and meanings of which will be clear to the skilled artisan based upon the context in which these terms are used. As provided herein, the disclosure of a “ratio” of compounds and compositions corresponds to a ratio provided in terms of mass of the components present in the ratio. As used herein, the term “pharmaceutically acceptable solvent” can refer to water, water for injection, aqueous buffer solutions that are physiologically compatible, or aqueous solutions containing organic solvents that are physiologically compatible. A non-comprehensive list of pharmaceutically acceptable solvents is provided in U.S. Department of Health & Human Services, Food & Drug Administration, “Guidance for Industry: Q3C Impurities: Residual Solvents,” December 1997 or its current issue. As used herein, the term “bioavailability” refers to the amount of a substance that is absorbed in the intestines and ultimately available for biological activity in a subject's tissue and cells. As used herein, the term “enhancing the bioavailability” and the like are used herein to refer to obtaining a desired pharmacological and/or physiological effect of antagonizing NK3 that is absorbed from the intestine or is taken up by tissues and cells after administration of a composition to a mammal, which does not occur naturally. The effect may be prophylactic in terms of preventing or partially preventing the incidence, risk, or severity of an adverse symptom or condition caused by or related to the deficiency of a therapeutic agent. As used herein, the term “excipient material” refers to any compound that is part of a formulation that is not an active ingredient, i.e., one that has no relevant biological activity, and which is added to the formulation to provide specific characteristics to the dosage form, including by way of example, providing protection to the active ingredient from chemical degradation, facilitating release of a tablet or caplet from equipment in which it is formed, and so forth. For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom which includes but is not limited to mammals and birds. In certain embodiments described herein, a mammal may, for example but without limitation, be a horse, dog, or cat. The most preferred mammal of this application is a human. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about.” It is understood that whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations due to the experimental and/or measurement conditions for such given value. While the present invention has been described in some detail for purposes of clarity and understanding, one will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. EXAMPLES Example 1 Method The NK3 antagonistic activity of compositions disclosed herein were studied at the human NK3 receptor expressed in transfected CHO cells. The antagonistic activity was determined by measuring their effect on Neurokinin B (NKB, a potent NK3 agonist)-induced cytosolic Ca2′ ion mobilization using a fluorometric detection method. Several ingredients were administered to transfected CHO cells, the results of which are shown inFIGS.1A-1C, and described further below. Experimental Protocol The CHO cells were suspended in DMEM buffer (Invitrogen) complemented with 0.1% FCSd, then distributed in microplates at a density of 3×104cells/well. The fluorescent probe (Fluo4 Direct, Invitrogen) was mixed with probenecid in HBSS buffer (Invitrogen) complemented with 20 mM HEPES (invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 60 min at 37° C. then 15 min at 22° C. Thereafter, the assay plates were positioned in a microplate reader (CellLux, PerkinElmer) which was used for the addition of the test items, curcumin extract as defined herein, or HBSS buffer, followed by the addition (5 min later) of 1 nM [MePhe7]-NKB or HBSS buffer (basal control). Next, the changes in fluorescence intensity was determined which varies proportionally to the free cytosolic Ca2+ion concentration. The standard reference antagonist is SB 222200, which was tested at several concentrations to generate a concentration-response curve from which its IC50 value is calculated. The results are expressed as a percent inhibition of the control response to 1 nM [MePhe7]-NKB as shown inFIGS.1A-1Cupon administration of a curcumin composition, a green tea extract composition, and a phycocyanin composition, as described herein, respectively.FIGS.1A-1Cshow superior and unexpected NK3 inhibition achieved by administration of various compounds, including a curcumin composition (FIG.1A), a green tea extract composition (FIG.1B), and a phycocyanin composition (FIG.1C), expressed as the IC50 (μg/mL), and were 2.92 μg/ml, 16.9 μg/ml, and no IC50 value for the phycocyanin composition was obtained, respectively. Example 2 Method In vivo experiments were performed to evaluate the efficacy of a curcumin composition, a green tea extract composition, a phycocyanin composition, and the combination thereof as NK3 antagonists, by measuring hypothalamic levels of NKB. Experimental Procedure Female Wistar rats were purchased from Japan SLC Inc. (Shizuoka, Japan). At the age of 8 weeks, rats were ovariectomized or given a sham operation (control) under isoflurane anesthesia. All animals were individually housed under clean conditions with controlled temperature, humidity, and light (12-hr light-dark cycle) and provided a standard commercial diet and water ad libitum. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Tsukuba Research Center of Astellas Pharma Inc., which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals. Four weeks after surgery rats are randomly divided into 6 groups: sham-operated rats treated with a vehicle consisting of 0.5% methylcellulose solution (C); ovariectomized rats treated with a vehicle consisting of 0.5% methylcellulose solution (OVX); and ovariectomized rats treated with 600 mg HED of a curcumin composition, as described herein (OVX+CL); 300 mg HED of a green tea extract composition, as described herein (OVX+EGCG); 100 mg HED of a phycocyanin composition, as described herein (OVX+P); and a combination composition, as described herein, comprising 600 mg HED of a curcumin composition, as described herein, 300 mg HED of a green tea extract composition, as described herein, 100 mg HED of a phycocyanin composition, as described herein (OVX+CL+EGCG+P). Vehicles or active drugs were administered for 8 days. Body weight and food intake were measured twice: before the first administration and on the final day of administration day. At day 5 of repeated administration, one small temperature data logger (Thermochron SL, KN Laboratories Inc., Osaka, Japan) was surgically implanted into the abdominal cavity under isoflurane anesthesia and another was attached to the skin of the tail using surgical tape and a handmade aluminum protector to prevent detachment. At day 7 of repeated administration, core, skin, and room temperatures were measured every 30 min for 24 hr. Two to five hours after the final drug administration (10:00-14:00), blood samples were collected from the abdominal vena cava under isoflurane anesthesia and the uterus was isolated and weighed. Blood samples were centrifuged, and plasma was separated and stored at −80° C. until assay. Blood samples were analyzed for (i) serum levels of glucose, triglycerides, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, malondialdehyde (MDA), antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx), curcuminoids, EGCG, and phycocyanin extracts; (ii) calcium (Ca), inorganic phosphorus (P), and alkaline phosphatase (ALP); and (ii) plasma hormone levels such as estradiol, LH, FSH, progesterone and testosterone, according to known methods in the art. Brain tissue analysis was performed to evaluate levels of kisspeptin, NKB, GnRH, transient receptor potential cation channel subfamily V member 1 (TrpV1), and protein c-fos. Brain tissues were embedded in paraffin, and hypothalamic slices (3-μm thickness) containing the MnPO were prepared according to a rat brain atlas (Paxinos and Watson, 2007) and standard procedures. After deparaffinization, the sections were incubated with Immunosaver (Nisshin EM Co., Ltd., Tokyo, Japan) at 100° C. for 15 min for antigen retrieval followed by 3% hydrogen peroxide to block endogenous per-oxidases. The samples were incubated with 1% bovine serum albumin for 20 min at room temperature followed by anti-kisspeptin, NKB, GnRH, TrpV1, or c-fos monoclonal antibody (dilution 1:100) overnight at 4° C. The samples were then treated with the DAKO EnVision System (Agilent Technologies, Santa Clara, CA, USA). ImmPACT DAB (Vector Laboratories, Inc., Burlingame, CA, USA) was used for color development. The densitometric analysis of the relative intensity according to the control group of the western blot bands was performed with β-actin normalization to ensure equal protein loading. Blots were repeated at least three times (n=3) and a representative blot is shown. ANOVA and Tukey's post-hoc test were performed for statistical comparisons (p<0.05). Data are presented as mean±standard deviation. Different lowercase letters above data series (a-d) indicate a statistical difference between groups. Quantitative assessment of c-Fos-positive neurons (stained-reddish-brown) was performed using a computerized image analysis system (WinROOF Ver. 6.0; Mitani Corp., Fukui, Japan) to automatically (optimal color wavelength and approximate cell size were entered in advance) count the number of reddish-brown (c-Fos-positive) cells in the MnPO under light microscopy at a magnification of 100×. Results of hypothalamus levels of NKB following the treatment scheme of Example 2 are shown inFIG.2.FIG.2shows superior and unexpected results. As expected, rats receiving the ovariectomy treatment (OVX), demonstrated the highest levels of NKB hypothalamus levels. Surprisingly, individual compositions according to the disclosure, significantly reduced NKB levels, below that of the OVX rats. Unexpectedly, a combination composition, as described herein (OVX+CL+EGCG+P), returned NKB levels to those seen in non-ovariectomized mice receiving a saline treatment (C). Example 3 Method In vivo experiments were performed to evaluate the efficacy of combination compositions, of the disclosure, comprising a curcumin extract composition, a green tea extract composition, and a phycocyanin composition of the disclosure, as NK3 antagonists, by measuring hypothalamic levels of NKB. Experimental Procedure The experimental procedure of Example 3 is the same procedure and analysis as described in Example 2. In Example 3, various combination compositions, as disclosed herein, comprising varied amounts of a curcumin composition, a green tea extract composition, and a phycocyanin composition according to the disclosure. In Example 3, the experimental groups were: sham-operated rats treated with a vehicle consisting of 0.5% methylcellulose solution (C); ovariectomized rats treated with a vehicle consisting of 0.5% methylcellulose solution (OVX); ovariectomized rats treated with 600 mg HED of a curcumin composition, 300 mg HED of a green tea extract composition, 100 mg RED of a phycocyanin composition (OVX+CL1+EGCG1+P); 450 mg HED of a curcumin composition, 450 mg HED of a green tea extract composition, 100 mg HED of a phycocyanin composition (OVX+CL2+EGCG2+P); and 720 mg HED of a curcumin composition, 180 mg HED of a green tea extract composition, 100 mg HED of a phycocyanin composition (OVX+CL3+EGCG3+P). Vehicles or active drugs were administered for 8 days. Data are presented as mean±standard deviation. Different lowercase letters above data series (a-d) indicate a statistical difference between groups. Results of hypothalamus levels of NKB following the treatment scheme of Example 2 are shown inFIG.3.FIG.3shows superior and unexpected results. As expected, rats receiving the ovariectomy treatment (OVX), demonstrated the highest levels of NKB hypothalamus levels. Surprisingly, all of the combination compositions evaluated, significantly reduced NKB levels, below that of the OVX rats. | 85,534 |
11857595 | DETAILED DESCRIPTION OF THE INVENTION Treatment of Acromegaly Acromegaly is caused by a benign (non-cancerous) tumor (an adenoma) within the pituitary gland that secretes excess growth hormone (GH), leading to elevated levels of insulin-like growth factor-1 (IGF-1). This combined effect of elevated GH and IGF-1 levels causes the enlargement of body parts, including the hands, feet and facial features, along with serious morbidities such as cardiovascular, metabolic and respiratory diseases. If exposed to long-term elevated levels of GH and IGF-1, acromegaly patients face a two- to three-fold increased risk of death. The current treatment of acromegaly is summarized by Giustina et al 2014 (Ref 13) which is hereby incorporated by reference. Biochemical control of the disease, as measured by both GH and IGF-1 levels, is the primary goal of treatment. Other disease management objectives include tumor shrinkage and improvement in clinical signs and symptoms. Thus the main goals of treatment are to control GH and IGF-1 levels and to control acromegaly symptoms. Various forms of pharmaceutical therapy are used in the art for treatment of acromegaly: most are receptor-based, directed at the pituitary adenoma (the somatostatin receptor ligands—SRLs—octreotide, lanreotide and pasireotide which are all given by injection) and the dopamine agonist cabergoline given orally; and one is directed at decreasing and /or blocking GH effects in the periphery viz., the GH receptor antagonist pegvisomant given by injection. SRLs may be given in slow release formulation or in an immediate release formulation. Surgery is the primary treatment option if the tumor is resectable. SRLs (injectable octreotide or injectable lanreotide) are the primary first-line treatment after surgery and are the primary treatment option if surgery is not appropriate. Some physicians prescribe dopamine agonists as the primary first-line treatment after surgery. SRLs and dopamine agonists and pegvisomant may also be given before surgery or instead of surgery. The octreotide capsule described herein is an oral product indicated for long-term maintenance therapy in acromegaly patients; in certain embodiments the patients are those in whom prior treatment with somatostatin analogs (by injection) has been shown to be effective and tolerated. The goal of treatment in acromegaly is to control GH and IGF-1 levels and to lower the GH and IGF-1 levels to as close to normal as possible. The oral octreotide capsule should preferably be administered with a glass of water on an empty stomach (i.e., at least 1 hour prior to a meal or at least 2 hours after a meal). Patients currently receiving somatostatin analog therapy by injection can be switched to octreotide capsules with an initial dose of 20 mg BID given orally. Blood levels of IGF-1 and clinical symptoms should be monitored. If IGF-1 is normal and clinical symptoms are controlled or response level (biochemical and symptomatic response) is maintained, maintain oral octreotide capsule dosage at 20 mg BID (ie 40 mg daily). Dosage may be adjusted to 60 mg daily (40 mg morning+20 mg evening) if IGF-1 levels are increased, as determined by the treating physician, or in case of symptomatic exacerbation. Monitoring is continued, while applying the above algorithm for maintaining or increasing the dose up to 40 mg BID is 80 mg daily. The administering throughout occurs at least 2 hours after a meal, or at least 1 hour before a meal. In another embodiment of the invention, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from 60 mg daily to 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In another embodiment, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from 30 mg daily (only one capsule taken) to 60 mg daily to 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In a further embodiment of the invention, if a capsule containing less than 20 mg octreotide is administered e.g. 10 mg, then the above algorithm is adjusted concomitantly. For example in an embodiment of the invention, if a capsule containing about 10 mg octreotide is administered, then the above algorithm is used to adjust the dose from 20 mg daily to 30 mg daily and a maximum of 60 mg daily as needed; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. The invention may be used in the treatment of naive patients or patients already treated with parenteral injections. Patients who are not adequately controlled following dose titration can return to therapy by injections at any time. Proton pump inhibitors (PPIs), H2-receptor antagonists, and antacids may lead to a higher dosing requirement of oral octreotide to achieve therapeutic levels. One embodiment of the invention is a method of treating acromegaly in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 5 mg to about 35 mg (e.g. 5, 10, 15, 20, 25, 30 or 35 mg), and wherein the administering occurs at least 1 hour before a meal or at least 2 hours after a meal, to thereby treat the subject. Another embodiment of the invention is a method of treating acromegaly in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 18 mg to about 22 mg, and wherein the administering occurs at least 1 hour before a meal or at least 2 hours after a meal to thereby treat the subject. A dosage form is essentially a pharmaceutical product in the form in which it is marketed for use, typically involving a mixture of active drug components and nondrug components (excipients), along with other non-reusable material that may not be considered either ingredient or packaging (such as a capsule shell, for example). The oily suspension as used herein comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight such as 11%-15%, or 11%, 12%, 13%, 14%, 15% or more by weight. Oral formulations of octreotide, comprising the oily suspension, have been described and claimed, for example in co-assigned U.S. Pat. No. 8,329,198 which is hereby incorporated by reference; see for example claims1-26. In a particular embodiment of the method of the invention the oily suspension is formulated into a capsule, which may be enterically coated. In another embodiment of the method of the invention the capsule consists of an oily suspension. In another embodiment of the method of the invention the subject is dosed every 8-16 hours (e.g., every 12 hours). In another embodiment of the method of the invention one administration takes place at least 6, 8, 10 or 12 hours before a second administration. In a preferred embodiment the subject is a human. For clarity, the twice daily administration comprises a first administration and a second administration. In a further embodiment a first administration includes one or two dosage forms and a second administration includes one or two dosage forms, and more particularly the first administration includes one dosage form and the second administration includes one dosage form, or the first administration includes two dosage forms and the second administration includes one dosage form, or the first administration includes two dosage forms and the second administration includes two dosage forms. In embodiments of the invention the first administration is in the morning (normally 5 am to noon) and the second administration is in the evening (normally 5 pm to midnight). All the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. Particular embodiments of the invention are as follows: one dosage form is administered twice daily; two dosage forms are administered once a day and one dosage form is administered once a day; and two dosage forms are administered twice daily. Other embodiments of the invention are as follows: one dosage form is administered once a day; two dosage forms are administered once a day; three or more dosage forms are administered once a day; and two or more dosage forms (e.g. three dosage forms) are administered twice a day. All the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. In some embodiments of the invention, the administration may be self-administration; in other embodiments of the invention or a caregiver or health professional may administer the dosage form. In certain embodiments of the invention each dosage form comprises from about 19 to about 21 mg of octreotide and in a particular embodiment of the invention each dosage form comprises 20 mg of octreotide which is about 3% w/w octreotide or 3.3% w/w octreotide. In certain embodiments of the invention the total amount of octreotide administered per day is from about 36 to about 44 mg (e.g., from about 38 to about 42 mg, or 40 mg). In certain embodiments of the invention the total amount of octreotide administered per day is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In certain embodiments of the invention the total amount of octreotide administered per day is from about 72 to about 88 mg (e.g., from about 76 to about 84 mg, or 80 mg). In certain embodiments of the invention the total amount of octreotide administered per day is from about 90 to about 110 mg (e.g., from about 95 to about 105 mg, or 100 mg). All the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. In certain embodiments of the invention each dosage form comprises from about 27 to about 33 mg of octreotide and in a particular embodiment of the invention each dosage form comprises 30 mg of octreotide which is about 5% w/w octreotide or 4.96% w/w octreotide. This may be administered as one, two, three or four capsules per day, wherein administering occurs at least 1 hour before a meal or at least 2 hours after a meal. In another embodiments of the invention each dosage form comprises less than 20 mg octreotide and in a particular embodiment of the invention each dosage form comprises about 10 mg. This may be administered as one, two, three or four capsules per day, wherein administering occurs at least 1 hour before a meal or at least 2 hours after a meal. In further embodiments, the method of the invention occurs over a duration of at least 7 months, occurs over a duration of at least 13 months and over a duration of greater than 13 months. In a particular embodiment the method of treatment is for long-term maintenance therapy. Long-term maintenance therapy in a subject suffering from acromegaly continues as long as the subject is suffering from acromegaly and the IGF-1 levels are maintained at equal or less than 1.3 times the upper limit of the age-adjusted normal range (ULN). Thus the duration may be unlimited. In particular embodiments the long-term maintenance therapy may be for at least one, two, three, four or five years. In a particular embodiment upon administration of octreotide, an in vivo amount of growth hormone integrated over 2 hours is obtained which is equal or less than 2.5 ng/mL or equal or less than 1.0 ng/mL. In further embodiments, upon administration of octreotide, an in vivo concentration of IGF-I is obtained of equal or less than 1.3 times the upper limit of the age-adjusted normal range (ULN), or equal or less than 1.0 or 1.1 or 1.2 or 1.4 or 1.5 or 1.6 times the upper limit of the age-adjusted normal range (ULN). In certain embodiments, an in vivo mean peak plasma concentration upon administration of octreotide of about 3.5+/−0.5 ng/mL is achieved. In certain embodiments an in vivo mean area under the curve upon administration of octreotide is about 15+/−4 h×ng/mL is obtained. In particular embodiments of the method of the invention the subject has had prior treatment for acromegaly, and the prior treatment for acromegaly was surgical and/or medicinal; in certain embodiments the medicinal treatment was a somatostatin analog (=somatostatin receptor ligand) e.g. injectable octreotide or injectable lanreotide or injectable pasireotide and/or a dopamine agonist e.g. cabergoline and/or a GH receptor antagonist e.g. pegvisomant. In particular embodiments the prior treatment of the subject with a somatostatin analog has been shown to be effective and tolerated. In particular embodiments the prior treatment of the subject produced an IGF-1 level in the subject of equal or less than 1.3 times upper limit of normal (ULN), and/or prior treatment of the subject produced 2-hour integrated growth hormone (GH) of less than 2.5 ng/mL or less than 1.0 ng/mL Preferably the oral octreotide capsule should be administered on an empty stomach (i.e., at least 1 hour prior to a meal or at least 2 hours after a meal. In particular embodiments of all inventions describes herein, a meal comprises 100-1000 calories, or 300-600 calories which may be a high-fat meal or a high calorie meal and may comprise carbohydrates and/or fat and or protein e.g. 100, 200, 300, 400 calories or 500-1000 calories or 700-800 calories. The invention also contemplates titrating a patient suffering from acromegaly to determine the effective dose of octreotide. Such an embodiment of the invention relates to a method of titrating a patient having acromegaly, the method comprising orally administering to the subject at least once daily (e.g. twice daily) at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 18 mg to about 22 mg, wherein the total amount of octreotide administered per day is from about 36 to about 44 mg; and subsequent to the administration, evaluating an IGF-1 level (and/or a GH level) in a subject and comparing the level to a reference standard; wherein if the IGF-1 level (and/or the GH level) is above the reference standard, increasing the total amount of octreotide administered per day to from about 54 to about 66 mg; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. Another such embodiment of the invention relates to a method of titrating a patient having acromegaly, the method comprising orally administering to the subject at least once daily (e.g. twice daily) at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 18 mg to about 22 mg, wherein the total amount of octreotide administered per day is from about 54 to about 66 mg; and subsequent to the administration, evaluating an IGF-1 level (and /or a GH level) in a subject and comparing the level to a reference standard; wherein if the IGF-1 level (and/or the GH level) is above the reference standard, increasing the total amount of octreotide administered per day to from about 72 to about 88 mg; wherein the administering occurs at least 2 hours after a meal or at least 1 hour before a meal. In one embodiment of the invention, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from 60 mg daily to 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In another embodiment, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from 30 mg daily (only one capsule taken) to 60 mg daily (two capsules) to 90 mg daily (three capsules) and a maximum of 120 mg daily (four capsules); wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In a further embodiment of the invention, if a capsule containing less than 20 mg octreotide is administered e.g. 10 mg, then the above algorithm is adjusted concomitantly. In further embodiments of the titrating invention the oily suspension is formulated into a capsule; the capsule is enterically coated; the oral administration is twice daily comprising a first and second administration; the subject is dosed every 8-16 hours (e.g., every 12 hours); one administration takes place at least 6, 8, 10 or 12 hours before a second administration; and the subject is a human. In a further embodiment of the titrating invention the first administration prior to evaluation includes one or two dosage forms and the second administration includes one or two dosage forms. In a further embodiment of the titrating invention, the first daily administration prior to evaluation includes one dosage form and the second daily administration prior to evaluation includes one dosage form. In a further embodiment of the titrating invention the first daily administration prior to evaluation includes two dosage forms and the second daily administration prior to evaluation includes one dosage form. In a further embodiment of the titrating invention the first daily administration after evaluation includes two dosage forms and the second daily administration after evaluation includes two dosage forms. In a further embodiment of the invention one dosage form is administered once a day and two dosage forms are administered once a day, prior to evaluation. In a further embodiment of the invention two dosage forms are administered twice daily after evaluation. Administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In a further embodiment of the invention each dosage form comprises from about 19 to about 21 mg of octreotide, more particularly 20 mg of octreotide which is about 3% w/w octreotide. In a further embodiment of the invention the total amount of octreotide administered per day prior to evaluation is from about 36 to about 44 mg (e.g., from about 38 to about 42 mg, or 40 mg). In a further embodiment of the invention the total amount of octreotide administered per day prior to evaluation is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In a further embodiment of the invention the total amount of octreotide administered per day subsequent to evaluation is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In a further embodiment of the invention the total amount of octreotide administered per day subsequent to evaluation is from about 72 to about 88 mg (e.g., from about 76 to about 84 mg, or 80 mg). In a further embodiment of the invention the evaluation takes place at least two months from start of therapy (i.e. from start of administration of the dosage forms), 2-5 months from start of therapy or after 5 months from start of therapy (e.g. after 5, 6, 7 or 8 months or more from start of therapy). In a specific embodiment of the invention the blood levels of IGF-1 and clinical symptoms are monitored when oral octreotide capsule dosage at 40 mg (20 mg BID), and if IGF-1 is normal and clinical symptoms are controlled or response level (biochemical and symptomatic response) is maintained, then oral octreotide capsule dosage is continued at 40 mg (20 mg BID). In a further specific embodiment of the invention the blood levels of IGF-1 and clinical symptoms are further monitored when oral octreotide capsule dosage is at 40 mg, and if IGF-1 is not normal and clinical symptoms are not controlled or response level (biochemical and symptomatic response) is not maintained, then oral octreotide capsule dosage is increased to 60 mg daily (40 mg morning+20 mg evening). In a further specific embodiment of the invention the blood levels of IGF-1 and clinical symptoms are further monitored when oral octreotide capsule dosage is at 60 mg, and if IGF-1 is normal and clinical symptoms are controlled or response level (biochemical and symptomatic response) is maintained, then oral octreotide capsule dosage is continued at 60 mg daily. In a further specific embodiment of the invention the blood levels of IGF-1 and clinical symptoms are further monitored when oral octreotide capsule dosage is at 60 mg, and if IGF-1 is not normal and clinical symptoms are not controlled or response level (biochemical and symptomatic response) is not maintained, then oral octreotide capsule dosage is increased to 80 mg (40 mg morning+40 mg evening) In a further embodiment of the invention the reference standard is an in vivo amount of growth hormone integrated over 2 hours is obtained which is equal or less than 2.5 ng/mL (for example equal or less than 1.0 ng/mL). In a further embodiment of the invention the reference standard is an in vivo concentration of IGF-I is obtained of equal or less than 1.3 times the upper limit of the age-adjusted normal range (ULN). In a further embodiment of the invention an in vivo mean peak plasma concentration upon administration of octreotide after evaluation is about 3.5+/−0.5 ng/mL. In a further embodiment of the invention an in vivo mean area under the curve upon administration of octreotide after evaluation is about 15+/−4 h×ng/mL. In a further embodiment of the titrating invention the subject has had prior treatment for acromegaly which was surgical and/or pharmaceutical e.g. the pharmaceutical treatment was a somatostatin receptor ligand e.g. octreotide or lanreotide and was administered by injection. In a further embodiment of the titrating invention prior treatment of the subject with a somatostatin analog has been shown to be effective and tolerated. In a further embodiment of the invention the prior pharmaceutical treatment was pegvisomant or a dopamine agonist e.g. cabergoline. In a further embodiment of the invention, prior treatment of the subject produced an IGF-1 level in the subject of equal or less than 1.0 to 1.5 times upper limit of normal (ULN) e.g. equal or less than 1.3 times upper limit of normal (ULN). In a further embodiment of the invention prior treatment of the subject produced 2-hour integrated growth hormone (GH) of less than 2.5 ng/mL e.g. less than 1.0 ng/mL. A further embodiment of the invention is a method of predicting subsequent response to oral octreotide capsules in a patient receiving injectable treatment. Thus an embodiment of the invention is a method of predicting subsequent response to oral octreotide capsules comprising the oily suspension in a patient suffering from acromegaly, the method comprising measuring the degree of baseline control on injectable SRLs; and thereby determining if the patient is likely to respond to the oral octreotide capsules. In an embodiment of the invention the desired baseline control is IGF-I≤1 ULN and GH<2.5 ng/mL when the patient is maintained on low to mid doses of injectable SRLs (octreotide<30 mg or lanreotide<120 mg). Treatment of Idiopathic Intracranial Hypertension (IIH) Another embodiment of the invention is a method of treating idiopathic intracranial hypertension (IIH) in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 5 mg to about 35 mg (e.g. 5, 10, 15, 20, 25, 30 or 35 mg), and wherein the administering occurs at least 1 hour before a meal or at least 2 hours after a meal, to thereby treat the subject. In a particular embodiment the octreotide in each dosage form is from about 18 mg to about 22 mg. In another embodiment the octreotide in each dosage form is from about 27 mg to about 33 mg e.g. about 30 mg. The oily suspension as used herein comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight such as 11%-15%, or 11%, 12%, 13%, 14%, 15% or more by weight. The oily suspension of the invention is as described herein. In a particular embodiment of the method of the invention the oily suspension is formulated into a capsule, which may be enterically coated. In another embodiment of the method of the invention the capsule consists of an oily suspension. In another embodiment of the method of the invention the subject is dosed every 8-16 hours (e.g., every 12 hours). In another embodiment of the method of the invention one administration takes place at least 6, 8, 10 or 12 hours before a second administration. In a preferred embodiment the subject is a human. For clarity, the twice daily administration comprises a first administration and a second administration. In a further embodiment a first administration includes one or two dosage forms and a second administration includes one or two dosage forms, and more particularly the first administration includes one dosage form and the second administration includes one dosage form or the first administration includes two dosage forms and the second administration includes one dosage form or the first administration includes two dosage forms and the second administration includes two dosage forms. In embodiments of the invention the first administration is in the morning (normally 5 am to noon) and the second administration is in the evening (normally 5 pm to midnight). Particular embodiments of the invention are as follows: one dosage form is administered twice daily; two dosage forms are administered once a day and one dosage form is administered once a day; and two dosage forms are administered twice daily. Other embodiments of the invention are as follows: one dosage form is administered once a day; two dosage forms are administered once a day; three or more dosage forms are administered once a day; and two or more dosage forms (e.g. three dosage forms) are administered twice a day. In some embodiments of the invention, the administration may be self-administration; in other embodiments of the invention or a caregiver or health professional may administer the dosage form. In certain embodiments of the invention each dosage form comprises from about 19 to about 21 mg of octreotide and in a particular embodiment of the invention each dosage form comprises 20 mg of octreotide which is about 3% w/w octreotide or 3.3% w/w octreotide. In certain embodiments of the invention the total amount of octreotide administered per day is from about 36 to about 44 mg (e.g., from about 38 to about 42 mg, or 40 mg). In certain embodiments of the invention the total amount of octreotide administered per day is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In certain embodiments of the invention the total amount of octreotide administered per day is from about 72 to about 88 mg (e.g., from about 76 to about 84 mg, or 80 mg). In certain embodiments of the invention each dosage form comprises from about 5 to about 35 mg of octreotide and in a particular embodiment of the invention each dosage form comprises about 5 or 10 or 15 or 20 or 25 or 30 or 35 mg of octreotide. In further embodiments of the invention the method occurs over a duration of at least 7 months or more. In further embodiments of the invention the method can be tapered off after a few months e.g. over 2 months or more. In further embodiments of the invention the method can be tapered off after about 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or more. In further embodiments of the invention the capsule comprises 20 mg octreotide which is about 3% w/w octreotide. In further embodiments of the invention the capsule comprises about 10 mg octreotide or about 30 mg octreotide. In further embodiments of the invention the oily suspension comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight (e.g. at an amount of 11%-16% or more such as 11%, 12%, 13%,14%, 15%, 16% by weight). In particular embodiments of the invention upon administration of octreotide, headache is relieved. In particular embodiments of the invention upon administration of octreotide, visual disturbances are reduced. In particular embodiments of the invention upon administration of octreotide, papilledema subside. In particular embodiments of the invention upon administration of octreotide, the CSF opening pressure is reduced e.g. to to 8-23 cm H2O preferably 10-18 cm H2O. In another embodiment another SRL (e.g., lanreotide) may be used orally to treat IIH. Thus an embodiment of the invention is a method of treating idiopathic intracranial hypertension (IIH) in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form containing lanreotide to thereby treat the subject. In a specific embodiment the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. The invention also contemplates titrating a patient suffering from IIH to determine the effective dose of octreotide. This embodiment comprises titrating a patient having idiopathic intracranial hypertension (IIH), the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 18 mg to about 22 mg, wherein the total amount of octreotide administered per day is from about 36 to about 44 mg; and subsequent to the administration, evaluating an IIH symptom in a subject and comparing the level to a reference standard; wherein if the IIH symptom is above the reference standard, increasing the total amount of octreotide administered per day to from about 54 to about 66 mg; wherein the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. In one embodiment of the invention, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from 60 mg daily to 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In another embodiment, if a capsule containing about 30 mg octreotide is administered, then the above algorithm is used to adjust the dose from30 mg daily (only one capsule taken) to 60 mg daily to 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In a further embodiment of the invention, if a capsule containing less than 20 mg octreotide is administered e.g. 10 mg, then the above algorithm is adjusted concomitantly. This embodiment also comprises a method of titrating a patient having idiopathic intracranial hypertension (IIH), the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 18 mg to about 22 mg, wherein the total amount of octreotide administered per day is from about 54 to about 66 mg; and subsequent to the administration, evaluating an IIH symptom in a subject and comparing the level to a normal reference standard; wherein if symptom is above the reference standard, increasing the total amount of octreotide administered per day to from about 72 to about 88 mg; wherein the administering occurs at least 2 hours after a meal or at least 1 hour before a meal. In further embodiments of the titrating invention the oily suspension is formulated into a capsule; the capsule is enterically coated; the oral administration is twice daily comprising a first and second administration; the subject is dosed every 8-16 hours (e.g., every 12 hours); one administration takes place at least 6, 8, 10 or 12 hours before a second administration; and the subject is a human. In a further embodiment of the titrating invention the first administration prior to evaluation includes one or two dosage forms and the second administration includes one or two dosage forms. In a further embodiment of the titrating invention, the first daily administration prior to evaluation includes one dosage form and the second daily administration prior to evaluation includes one dosage form. In a further embodiment of the titrating invention the first daily administration prior to evaluation includes two dosage forms and the second daily administration prior to evaluation includes one dosage form. In a further embodiment of the titrating invention the first daily administration after evaluation includes two dosage forms and the second daily administration after evaluation includes two dosage forms. In a further embodiment of the invention one dosage form is administered once a day and two dosage forms are administered once a day, prior to evaluation. In a further embodiment of the invention two dosage forms are administered twice daily after evaluation. In a further embodiment of the invention each dosage form comprises from about 19 to about 21 mg of octreotide, more particularly 20 mg of octreotide which is about 3% w/w octreotide. In a further embodiment of the invention the total amount of octreotide administered per day prior to evaluation is from about 36 to about 44 mg (e.g., from about 38 to about 42 mg, or 40 mg). In a further embodiment of the invention the total amount of octreotide administered per day prior to evaluation is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In a further embodiment of the invention the total amount of octreotide administered per day subsequent to evaluation is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg). In a further embodiment of the invention the total amount of octreotide administered per day subsequent to evaluation is from about 72 to about 88 mg (e.g., from about 76 to about 84 mg, or 80 mg). In a further embodiment of the invention the evaluation takes place about one week or one month from start of therapy (i.e. from start of administration of the dosage forms), 2-5 months from start of therapy or after 5 months from start of therapy (e.g. after 5, 6, 7 or 8 months or more from start of therapy). In a further embodiment of the titrating invention, the oily suspension comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight (e.g. at an amount of 11%-16% or more such as 11%, 12%, 15%, 16% by weight). In a particular embodiment the capsule comprises 20 mg octreotide which is about 3% w/w octreotide. In another particular embodiment the capsule comprises about 10 mg octreotide or about 30 mg octreotide. In a further embodiment of the titrating invention the IIH symptom is one or more of headache, papilledema and visual disturbance. In a further embodiment of the titrating invention the reference standard is the normal for a healthy person not suffering from IIH e.g. no headache, no papilledema and no visual disturbance. Treatment of Vascular Headaches A further embodiment of the invention is a method of treating or prophylaxis of headaches in particular vascular headaches, which are thought to involve abnormal function of the brain's blood vessels or vascular system. The most common type of vascular headache is migraine headache. Other kinds of vascular headaches include cluster headaches and headaches caused by a rise in blood pressure. Migraines typically present with self-limited, recurrent severe headache associated with autonomic symptoms. Cluster headache is a neurological disorder characterized by recurrent, severe headaches on one side of the head, typically around the eye. There are often accompanying autonomic symptoms during the headache such as eye watering, nasal congestion and swelling around the eye, typically confined to the side of the head with the pain. The use of oral octreotide is envisaged to treat headaches in particular vascular headaches including migraines and cluster headaches. Thus an embodiment of the invention is a method of treating headaches in particular vascular headaches including migraines and cluster headaches in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form containing octreotide to thereby treat the subject. In a specific embodiment, the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. The treatment may comprise aborting a headache or prophylactic treatment wherein oral octreotide is taken on an ongoing prophylactic basis. In another embodiment another SRL (e.g., lanreotide) may be used orally to treat headaches in particular vascular headaches including migraines and cluster headaches. Thus an embodiment of the invention is a method of treating headaches in particular vascular headaches including migraines and cluster headaches in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form containing lanreotide to thereby treat the subject. In a specific embodiment the administering occurs at least 1 hour before a meal or at least 2 hours after a meal. The treatment may comprise aborting a headache or prophylactic treatment wherein oral lanreotide is taken on an ongoing prophylactic basis. A particular embodiment of the invention is a method of prophylactically treating or aborting headache in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form comprising an oily suspension comprising octreotide, wherein the octreotide in each dosage form is from about 5 mg to about 35 mg (e.g. 5, 10, 15, 20, 25, 30 or 35 mg), and wherein the administering occurs at least 1 hour before a meal or at least 2 hours after a meal, to thereby treat the subject. In particular embodiments of the invention the headache may be a vascular headache, which may be a migraine or a cluster headache or the headache may be caused by IIH. In a particular embodiment of the invention the oily suspension is formulated into a capsule, and the capsule may be enterically coated. In particular embodiments of the invention the oral administration is twice daily (e.g., administering one or two dosage forms at each administration), comprising a first and second administration; the subject is dosed every 8-16 hours (e.g., every 12 hours); one administration takes place at least 6, 8, 10 or 12 hours before a second administration; the subject is a human. In particular embodiments of the invention the first administration includes one or two dosage forms and the second administration includes one or two dosage forms. In further embodiments of the invention the first administration includes one dosage form and the second administration includes one dosage form or the first administration includes two dosage forms and the second administration includes one dosage form. or the first administration includes two dosage forms and the second administration includes two dosage forms. In further embodiments of the invention one dosage form is administered twice a day or two dosage forms are administered twice a day or one dosage form is administered once a day and two dosage forms are administered once a day. In particular embodiments of the invention each dosage form comprises from about 19 to about 21 mg of octreotide or each dosage form comprises 20 mg of octreotide. In another embodiment of the invention each dosage form comprises from about 27 to about 33 mg of octreotide or each dosage form comprises 30 mg of octreotide In particular embodiments of the invention the total amount of octreotide administered per day is from about 36 to about 44 mg (e.g., from about 38 to about 42 mg, or 40 mg); or the total amount of octreotide administered per day is from about 54 to about 66 mg (e.g., from about 57 to about 63 mg, or 60 mg); or the total amount of octreotide administered per day is from about 72 to about 88 mg (e.g., from about 76 to about 84 mg, or 80 mg). If a capsule containing about 30 mg octreotide is administered, then the dose is 30 mg daily or 60 mg daily or 90 mg daily and a maximum of 120 mg daily; wherein the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. In certain embodiments of the invention each dosage form comprises from about 5 to about 35 mg of octreotide and in a particular embodiment of the invention each dosage form comprises about 5 or 10 or 15 or 20 or 25 or 30 or 35 mg of octreotide. In particular embodiments of the invention the method occurs over a duration of at least 7 months, for prophylactic treatment and in particular embodiments of the invention the method can be tapered off after a few months. In particular embodiments of the invention the method the method occurs over a duration of about a day, or about one to two days or more for abortive treatment. In particular embodiments of the invention the capsule comprises 20 mg octreotide which is about 3% w/w octreotide. In other embodiments of the invention the capsule comprises 10 mg octreotide or 30 mg octreotide. In particular embodiments of the invention the oily suspension comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight (e.g. at an amount of 11%-16% or more such as 11%, 12%, 15%, 16% by weight). In particular embodiments of the invention, upon administration of octreotide, the headache is relieved or prophylactically prevented. Another embodiment of the invention is a method of prophylactically treating headache in a subject, the method comprising administering to the subject at least once daily at least one dosage form comprising octreotide, to thereby treat the subject. In particular embodiments of the invention the headache is a vascular headache; in further embodiments of the invention the vascular headache is a migraine or a cluster headache; in a further embodiment of the invention the headache is caused by IIH; in a further embodiment of the invention the octreotide is administered orally; in a further embodiment of the invention the administering occurs at least 2 hours after a meal, or at least 1 hour before a meal. Another embodiment of the invention is a method of aborting a headache in a subject, the method comprising orally administering to the subject at least once daily at least one dosage form comprising octreotide, to thereby treat the subject. In particular embodiments of the invention the headache is a vascular headache; in further embodiments of the invention the vascular headache is a migraine or a cluster headache; in a further embodiment of the invention the headache is caused by IIH; and in a further embodiment of the invention the administering occurs at least 2 hours after a meal or at least 1 hour before a meal. The Oily Suspension The oily suspension as used herein comprises an admixture of a hydrophobic medium (lipophilic fraction) and a solid form (hydrophilic fraction) wherein the solid form comprises a octreotide and at least one salt of a medium chain fatty acid, and wherein the medium chain fatty acid salt is present in the composition at an amount of 10% or more by weight or 11-20% or 11%, or 12% or 13% or 14% or 15% or 16% or 17%. In further embodiments of the methods of the invention, the medium chain fatty acid salt in the solid form has a chain length from about 6 to about 14 carbon atoms; the medium chain fatty acid salt is sodium hexanoate, sodium heptanoate, sodium octanoate, sodium nonanoate, sodium decanoate, sodium undecanoate, sodium dodecanoate, sodium tridecanoate or sodium tetradecanoate, or a corresponding potassium or lithium or ammonium salt or a combination thereof; the fatty acid salt is sodium octanoate (sodium caprylate); the medium chain fatty acid salt is present in the oily suspension at an amount of 11% to 40% by weight, or at an amount of 12% to 18% by weight, preferably 15% by weight. In a specific embodiment the oily suspension comprises 15% w/w sodium octanoate. In another specific embodiment the oily suspension comprises 10-20% e.g. 15% w/w sodium decanoate. In another embodiment the solid form in the oily suspension additionally comprises a matrix forming polymer, which can be for example dextran or polyvinylpyrrolidone (PVP). In another embodiment the polyvinylpyrrolidone is present in the oily suspension at an amount of about 2% to about 20% by weight, or about 5% to about 15% by weight or about 10% by weight. In a specific embodiment the polyvinylpyrrolidone is PVP-12 and has a molecular weight of about 2500-3000. In another embodiment the hydrophobic medium comprises glyceryl tricaprylate and in a specific embodiment herein the oily suspension comprises 50-70% w/w glyceryl tricaprylate. In another embodiment the hydrophobic medium comprises a mineral oil, paraffin, a fatty acid such as octanoic acid, a monoglyceride, a diglyceride, a triglyceride, an ether or an ester, or a combination thereof. In another embodiment the triglyceride is a long chain triglyceride, a medium chain triglyceride or a short chain triglyceride. In another embodiment the triglyceride is a short chain triglyceride or a medium chain triglyceride or a mixture thereof. In another embodiment the short chain triglyceride is glyceryl tributyrate and the medium chain triglyceride is glyceryl tricaprylate. In another embodiment the hydrophobic medium further comprises an ionic surfactant or a non-ionic surfactant. In further embodiments the surfactant is a monoglyceride, a cremophore, a polyethylene glycol fatty alcohol ether, a sorbitan fatty acid ester, a polyoxyethylene sorbitan fatty acid ester, Solutol HS15(polyoxyethylene esters of 12-hydroxystearic acid), or a poloxamer or a combination thereof. In further embodiments of the methods of the invention the monoglyceride is glyceryl monocaprylate, glyceryl monoocatnoate, glyceryl monodecanoate, glyceryl monolaurate, glyceryl monomyristate, glyceryl monopalmitate or glyceryl monooleate or glyceryl monostearate or a combination thereof. In further embodiments of the method, the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monooleate (also termed polysorbate 80 or Tween 80). In further embodiments the oily suspension comprises 3% w/w polyoxyethylene sorbitan monooleate. In further embodiments the hydrophobic medium additionally contains glyceryl monocaprylate and the oily suspension comprises 4% w/w glyceryl monocaprylate. In further embodiments the hydrophobic medium consists essentially of glyceryl tricaprylate and glyceryl monocaprylate. In further embodiments the hydrophobic medium comprises a triglyceride and a monoglyceride; in some embodiments the monoglyceride has the same fatty acid radical as the triglyceride; in some embodiments the triglyceride is glyceryl tricaprylate and the monoglyceride is glyceryl monocaprylate. In some embodiments of the method the medium chain fatty acid salt in the water-soluble composition has the same fatty acid radical as the medium chain monoglyceride or the medium chain triglyceride or a combination thereof. In some embodiments of the method the medium chain fatty acid salt is sodium caprylate (sodium octanoate) and the monoglyceride is glyceryl monocaprylate and the triglyceride is glyceryl tricaprylate. In some embodiments of the method the oily suspension comprises magnesium chloride. In one embodiment of the method, the oily suspension comprises about 3% octreotide, 5-15% PVP-12, 10-20% sodium caprylate (sodium octanoate), 2-10% surfactants, 50-70% lipid and stabilizer. In a particular embodiment the formulation consists essentially of an oily suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of octreotide and about 10-20% preferably 15% medium chain fatty acid salt preferably sodium octanoate, and about 5-10% preferably 10% PVP-12; and wherein the hydrophobic medium comprises about 20-80%, preferably 30-70% triglyceride preferably glyceryl tricaprylate or glyceryl tributyrate or castor oil or a mixture thereof, about 3-10% surfactants, preferably about 6%, preferably glyceryl monocaprylate and Tween 80; in particular embodiments the octreotide is present at an amount of less than 33%, or less than 25%, or less than 10%, or less than 5% or less than 1%. The solid form may be a particle (e.g., consist essentially of particles, or consists of particles). The particle may be produced by lyophilization or by granulation. In a particular embodiment the solid form may be a particle and may be produced by lyophilization or by granulation. In a further embodiment the formulation consists essentially of an oily suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of octreotide and about 10-20% preferably 15% medium chain fatty acid salt preferably sodium octanoate and about 5-10% preferably 10% PVP-12; and wherein the hydrophobic medium comprises about 20-80%, preferably 30-70% medium or short chain triglyceride preferably glyceryl tricaprylate or glyceryl tributyrate, about 0-50% preferably 0-30% castor oil, about 3-10% surfactants, preferably about 6%, preferably glyceryl monocaprylate and Tween 80; in particular embodiments the octreotide is present at an amount of less than 33%, or less than 25%, or less than 10%, or less than 5% or less than 1%. Oral Dosage Form In an embodiment, the oral octreotide is administered in a dosage form described herein. An exemplary oral dosage forms includes an enteric-coated oral dosage form comprising a composition comprising a suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of octreotide, at least one salt of a medium chain fatty acid and polyvinylpyrrolidone (PVP), wherein the polyvinylpyrrolidone is present in the composition at an amount of 3% or more by weight (e.g., about 3% to about 20% by weight or about 5% to about 15% by weight), and wherein the at least one salt of a medium chain fatty acid salt is present in the composition at an amount of at least 12% or more by weight (e.g., about 12% to 40% by weight or about 12% to 18% by weight). In an embodiment, the hydrophobic medium comprises glyceryl tricaprylate and the solid form consists of polyvinylpyrrolidone with a molecular weight of about 3000, and sodium octanoate. In an embodiment, the hydrophobic medium additionally comprises castor oil or glyceryl monocaprylate or a combination thereof and a surfactant. In an embodiment, the hydrophobic medium consists of glyceryl tricaprylate, glyceryl monocaprylate, and polyoxyethylene sorbitan monooleate. In an embodiment, the solid form consists essentially of octreotide, polyvinylpyrrolidone with a molecular weight of about 3000, and sodium octanoate. In an embodiment, the composition comprises about 41% of glyceryl tricaprylate, about 27% castor oil, about 4% glyceryl monocaprylate, about 2% polyoxyethylene sorbitan monooleate, about 15% sodium octanoate, about 10% polyvinylpyrrolidone with a molecular weight of about 3000, and about 1-3.5% by weight octreotide e.g. 1.5% or 2% or 2.5% or 3% or 3.3% octreotide. In an embodiment, the composition comprises about 65% glyceryl tricaprylate, about 4% glyceryl monocaprylate, about 2% polyoxyethylene sorbitan monooleate, about 15% sodium octanoate, about 10% polyvinylpyrrolidone with a molecular weight of about 3000 and about 1-5.5% by weight octreotide e.g. 1.5% or 2% or 2.5% or 3% or 3.3% or 4% or 5% or 5.5% octreotide. In an embodiment, the composition comprises a therapeutically effective amount of octreotide, about 12-21% of sodium octanoate, about 5-10% of polyvinylpyrrolidone with a molecular weight of about 3000, about 20-80% of glyceryl tricaprylate, about 0-50% castor oil, and about 3-10% surfactant. In an embodiment, the composition comprises a therapeutically effective amount of octreotide, about 12-21% of sodium octanoate, about 5-10% of polyvinylpyrrolidone with a molecular weight of about 3000, about 20-80% of glyceryl tricaprylate, and about 3-10% surfactant. In an embodiment, the octreotide is present at an amount of less than 33% (e.g., less than 25%, less than 10%, less than 5%, less than 1%). In an embodiment, the composition comprises about 15% of sodium octanoate, about 10% of polyvinylpyrrolidone with a molecular weight of about 3000, about 30-70% glyceryl tricaprylate and about 6% of surfactant. In an embodiment, the surfactant is glyceryl monocaprylate or polyoxyethylene sorbitan monooleate. In an embodiment, the solid form comprises a particle or a plurality of particles. In an embodiment, the solid form further comprises a stabilizer. In an embodiment, the polyvinylpyrrolidone has a molecular weight of about 3000. In an embodiment, the medium chain fatty acid salt has a chain length from about 6 to about 14 carbon atoms. In an embodiment, the medium chain fatty acid salt is sodium hexanoate, sodium heptanoate, sodium octanoate, sodium nonanoate, sodium decanoate, sodium undecanoate, sodium dodecanoate, sodium tridecanoate or sodium tetradecanoate, or a corresponding potassium or lithium or ammonium salt or a combination thereof. In an embodiment, the medium chain fatty acid salt is sodium octanoate. In another embodiment, the medium chain fatty acid salt is sodium decanoate. In an embodiment, the hydrophobic oily medium comprises a mineral oil, a paraffin, a fatty acid a monoglyceride, a diglyceride, a triglyceride, an ether or an ester, or a combination thereof. In an embodiment, the medium chain fatty acid salt is a lithium, potassium or ammonium salt. In an embodiment, the hydrophobic oily medium comprises glyceryl tricaprylate. In an embodiment, the composition further comprises a surfactant. The compositions described herein can be administered to a subject i.e., a human or an animal, in order to treat the subject with a pharmacologically or therapeutically effective amount of a therapeutic agent (octreotide) described herein. The animal may be a mammal e.g., a mouse, rat, pig, dog horse, cow or sheep. As used herein the terms “pharmacologically effective amount” or “therapeutically effective amount” or “effective amount” means that amount of a drug or pharmaceutical agent (the therapeutic agent) that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician and/or halts or reduces the progress of the condition being treated or which otherwise completely or partly cures or acts palliatively on the condition, or prevents development of the condition. As used herein, the term “treatment” as for example in “method of treatment” or “treat” or “treating” refers to therapeutic treatment, wherein the object is to reduce or reverse or prevent the symptoms of a disease or disorder. In some embodiments, the compounds or compositions disclosed herein are administered prior to onset of the disease or disorder. In some embodiments, the compounds or compositions disclosed herein are during or subsequent to the onset of the disease or disorder. The function and advantages of these and other embodiments will be more fully understood from the following example. This example is intended to be illustrative in nature and is not to be considered as limiting the scope of the systems and methods discussed herein. EXAMPLE A novel oral octreotide formulation was tested for efficacy and safety in a phase III multicenter open-label dose-titration baseline-controlled study for acromegaly. Methods: 155 complete or partially controlled patients were enrolled [IGF-I<1.3 upper limit of normal (ULN), and 2-hr integrated growth hormone (GH)<2.5 ng/mL] while receiving injectable somatostatin receptor ligand (SRL) for ≥3 months. Subjects were switched to 40 mg/day oral octreotide capsules (OOC), dose escalated to 60, and up to 80 mg/day, to control IGF-I. Subsequent fixed-doses were maintained for 7 month core treatment, followed by voluntary 6 month extension. Results: Of 151 evaluable subjects initiating OOC, 65% maintained response and achieved the primary endpoint of IGF-I<1.3 ULN and mean integrated GH<2.5 ng/mL at the end of the core treatment period and 62% at the end of treatment (up to 13 months). The effect was durable and 85% of subjects initially controlled on OOC, maintained this response up to 13 months. When controlled on OOC, GH levels were reduced compared to baseline and acromegaly-related symptoms improved. Of 102 subjects completing core treatment, 86% elected to enroll into 6-month extension. 26 subjects considered treatment failures (IGF-I≥1.3 ULN), terminated early and 23 withdrew for adverse events, consistent with those known for octreotide or disease-related. Conclusions: OOC, an oral therapeutic peptide achieves efficacy in controlling IGF-I and GH following switch from injectable SRLs, for up to 13 months, with a safety profile consistent with approved SRLs. OOC appears to be effective and safe as acromegaly monotherapy. Oral octreotide capsules (OOC) were employed which facilitate intestinal octreotide absorption by a novel transient permeability enhancer (TPE) formulation (28). The capsule containing 20 mg non-modified octreotide acetate formulated with TPE enables transient and reversible paracellular tight junction passage of molecules <70 kDa. The size limitation and limited permeability duration ensures that luminal pathogens and endobacterial toxins, are excluded (28). Ingestion of OOC by healthy volunteers achieved circulating octreotide levels and exposure comparable to those observed after subcutaneous octreotide injection (29). As a single 20 mg dose of OOC suppressed basal and GHRH-elicited GH levels in healthy volunteers (29), the drug was tested for efficacy and safety in a phase III, multicenter, open-label, dose-titration baseline-controlled study, in acromegaly. Objectives were to determine OOC effectiveness in maintaining baseline biochemical response for up to 13 months, in acromegaly patients in whom prior treatment with an injectable SRL had been effective i.e. to assess the proportion of subjects maintaining baseline response levels following a switch to OOC. This open-label, maintenance of response, baseline controlled, withdrawal study was conducted to evaluate OOC safety and efficacy in patients with acromegaly shown to tolerate and respond to injectable SRLs. This IRB-approved multicenter international study continued from March 2012 to November 2013 in 37 sites for ˜15 months and included screening, and baseline periods of ˜2 months, core treatment period of ≥7 months, voluntary 6-month extension for patients who completed the core study, and a follow-up period of 2 weeks. Patient Population Subjects had confirmed biochemical and clinical evidence for active acromegaly and were required to receive a stable dose of parenteral SRLs for at least 3 months prior to screening. At screening, patients had to demonstrate complete or partial response to SRLs, defined as IGF-I<1.3×ULN for age and integrated GH response over 2 hours <2.5 ng/mL. Patients were excluded if they received GH antagonists (within <3 months) or dopamine agonists (within <2 months), received radiotherapy within 10 years, or underwent pituitary surgery within 6 months prior to screening. Screening and Baseline Periods Screening and baseline periods (median 42 days) enabled assessment of subject eligibility and for establishing baseline disease control (IGF-1 and GH measurements), while receiving parenteral SRL injections. The first OOC dose was administered ≥4-weeks after the last SRL injection. On average, the last SRL dose was given approximately 2 weeks following Screening visit and 2 weeks prior to Baseline visit. Treatment Period The OOC treatment period lasted ≥13 months and comprised a dose escalation (2-5 months) followed by a fixed dose period (8-11 months). The fixed dose period included the time periods up to the completion of the core and extension treatment phases (at 7 and 13 months respectively). Enrollment into the extension phase was voluntary. OOC was administered in the morning and evening (≥1 hour prior to a meal and ≥2 hours after a meal). Dose-Escalation First OOC dose (20 mg+20 mg) was dispensed ≥4 weeks [mean (SD) 33.3 (12.62), median (P25,P75) 31.0 (29.0,35.0) days] after last SRL injection. OOC dose escalations (to 40+20 mg and if required to 40+40 mg), occurred after 2 successive visits if IGF-I was inadequately controlled on a stable dose i.e. >20% increase over prior levels, or emergence of acromegaly symptoms. Visits occurred every 14 days for IGF-I measurements, and results used to guide dosing decisions at the subsequent visit. Integrated GH levels (measured 2-4 hours following OOC administration) were measured with every dose escalation. Subjects could revert to parenteral SRL therapy at any time, for either safety or efficacy, at the discretion of the site. Fixed-Dose Subjects entered into the fixed-dose period when IGF-I levels were normalized or returned to baseline levels, during ≥2 successive visits. Per protocol, adequately controlled subjects completing the core treatment period were offered the option to continue a 6-month extension. At each monthly visit during the core treatment and bi-monthly during the extension, IGF-I was measured and acromegaly symptoms assessed. Integrated GH levels were measured at the beginning and end of the fixed-dose period (core and extension). The optimally effective OOC dose achieved during dose escalation was continued for the duration of the fixed-dose period, for up to 13 months. Endpoints and Statistical Analysis The primary efficacy endpoint was descriptive and defined as the proportion of responders at the end of the core treatment, with an exact 95% CI in the modified intent-to-treat (mITT) population (i.e. all subjects who had ≥1 post-first-dose efficacy assessment). Response was defined, similarly to the inclusion criteria as IGF-I<1.3 ULN for age and integrated GH<2.5 ng/mL (utilizing Last Observation Carried Forward imputation (LOCF)). At the end of extension, the primary endpoint was the proportion of responders, of all subjects who entered the extension (extension-ITT), and for those who entered the extension as responders, with an exact 95% CI. When continuous measures were reasonably symmetric, mean values and SD were used, otherwise both mean and median values are presented. Secondary and exploratory descriptive endpoints included the proportion of subjects who achieved categorical response levels at end of treatment, based on IGF-I and/or GH levels, and the proportion of subjects who maintained response i.e. who remained responders from the beginning of the fixed-dose to end of the treatment periods. Acromegaly symptoms (headache, asthenia, perspiration, swelling of extremities and joint pain), were scored by severity at each visit: absent=0, mild=1, moderate=2, severe=3. The proportion of subjects with improvement, no change or worsening in overall scores, as well as those with 1, 2 or 3 active symptoms from baseline to end of treatment was calculated. Assays IGF-I and GH were measured centrally by IDS-iSYS IGF-I(30) (IS-3900, Immunodiagnostic Systems, Boldon, UK) and IDS-iSYS hGH(31) (IS-3700, Immunodiagnostic Systems) assays, at the Endocrine Laboratory, Universitat Munchen, Germany, and Solstas Lab (Greensboro, NC, USA). Recombinant standards (98/574 for GH and 02/254 for IGF-I) yielded inter-assay variability of 4-8.7% (IGF-I) and 1.1-3.4% (GH), and sensitivity 8.8 ng/mL (IGF-I) and 0.04 ng/mL (GH) (30,31). Integrated GH levels were calculated from the mean of 5 samples collected every 30±5 minutes for 2 hours beginning 2 hours following drug dosing (or at time zero at screening and baseline visits) (31). IGF-I measurements were assayed from a single sample (time zero) and compared to age-related reference ranges (30). Routine laboratory safety assessments were performed centrally, and all samplings were after ≥8-hour fasting. During the fixed dose period 46 subjects at a subset of sites underwent pharmacokinetic (PK) evaluation. Results Baseline Characteristics Enrolled subjects had been receiving long-acting SRL injections for 3 months to >20 years at all dose ranges. Of the 155 subjects enrolled, 95 had IGF-1≤1 ULN and GH<2.5 ng/mL at baseline, of whom 67 (43%) had GH<1 ng/mL. 42 subjects entered the study with 1<IGF-1<1.3 and GH<2.5 ng/mL. While eligible patients had to meet criteria of complete or partial response to injectable SRLs at screening to enter the study, only 88.7% of these subjects were responding to injectable SRLs at baseline and 17 patients (11%) had IGF-1≥1.3 ULN and/or GH≥2.5 ng/mL. (See Table 1). 81% of subjects had active acromegaly symptoms despite treatment on injectables. TABLE 1Baseline Characteristics of All Subjects Enrolled [N = 155]Demographics [n (%)]Symptomatic & Biochemical ControlAgeAcromegaly symptoms [n (%)]Mean (SD)54.2 (11.54)Headache64 (41.3)GenderPerspiration65 (41.9)Female gender88 (56.8)Asthenia68 (43.9)Disease Characteristics [n (%)]Swelling of extremities58 (37.4)Duration of acromegalyJoint pain87 (56.1)<10 years74 (47.7)At least one symptom125 (80.6)10-<20 years53 (34.2)At least two symptoms91 (61.3)≥20 years28 (18.1)At least three symptoms67 (43.2)Pituitary tumor characteristicIGF-I (ULN)Microadenoma51 (32.9)Mean (SD)0.94 (0.250)Intrasellar macroadenoma53 (34.2)Median (P25, P75)0.89 (0.76, 1.07)Extrasellar macroadenoma46 (29.7)GH (mean ng/mL)Other5 (3.2)Mean (SD)0.93 (0.716)Medical Treatment [n (%)]Median (P25, P75)0.77 (0.44, 1.23)Previous treatments for acromegalyBiochemical control [n (%)]Surgery121 (78.1)IGF-I ≤1 ULN &95 (61)GH <2.5 ng/mLMedication, other than SRLs61 (39.4)IGF-1 ≤1 ULN and67 (43)GH <1 ng/mLRadiation13 (8.4)IGF-1 ≤1 ULN and28 (18)1 ≤ GH <2.5 ng/mLSurgery followed by radiation8 (5.2)1 < IGF-I <1.3 &42 (27)GH <2.5 ng/mLRadiation followed by surgery1 (0.6)IGF-I ≥1.3 and/or18 (12)GH ≥2.5 ng/mLPrevious SRLs treatment [n (%)]Octreotide LAR1(mg)97 (62.6)10, 2064 (66% ofpts onoctreotide)30, 40, 6033 (34% ofpts onoctreotide)Lanreotide2(mg)58 (37.4)60, 9027 (47% ofpts onlanreotide)12031 (53% ofpts onlanreotide)Time receiving parenteral SRLs [n (%)]<1 year21 (13.5)1-<5 years63 (40.6)5-<10 years37 (23.9)>=10 years34 (21.9)Subjects on Combo18 (11.6)cabergoline/pegvisomant3[n (%)]1Sandostatin LAR,2Somatuline Autogel3Subjects on combination therapy with cabergoline/pegvisomant within the last 6 months prior to screening. Subject Disposition 235 patients were screened and most of those failing to meet inclusion criteria had IGF-I≥1.3 ULN. 155 subjects (67 males, 88 females) were enrolled, 151 underwent at least one biochemical assessment after first OOC dose, (mITT), 110 (71%) entered the fixed dose period, 88 elected to continue into the 6 months extension and 82 subjects completed 13 months treatment. 59 subjects discontinued treatment during the course of the study, most (n=45; 76%), during the dose-escalation period. Early terminations were due to treatment failure (IGF-I>1.3 ULN; n=26; 16. 8%), adverse events (n=23; 14.8%), patient choice (n=7; 4.5%), lost to follow-up (n=2; 1.3%) and sponsor request (n=1; 0.6%). Efficacy Overall, 65% of all enrolled subjects (mITT population, N=151, 95% CI 58.4-74.2), were responders up to 7 months, and 62% were responders up to 13 months (95% CI 54.9-71.7), as compared to 88.7% at the baseline visit while on injectable SRLs. Sensitivity analysis (Markov Chain Monte Carlo multiple imputation), showed 65.6% response, consistent with primary LOCF analysis. The effect was durable as 85% and 89% of subjects who entered the fixed dose and extension periods respectively as responders, maintained response for up to 13 months treatment. 78.4% [95% CI 68.4, 86.5] of subjects who entered the extension were responders at end of treatment (up to 13 months). At the beginning of the fixed dose phase 51/110 (46%) were treated on 40 mg, 25/110 (23%) on 60 mg and 34/110 (31%) on 80 mg. The response up to 13 months, for those patients that entered the fixed dose, was 88% (95% CI 76.1-95.6), 84% (95% CI 63.9-95.5) and 47% (95% CI 29.8-64.9), for 40 mg, 60 mg and 80 mg respectively. Table 2 depicts biochemical response categories at baseline and end of treatment for all evaluable patients. Integrated GH levels <2.5 ng/mL were achieved in 93% of mITT subjects at the end of treatment versus 96% at baseline, while GH levels <1 ng/mL were achieved in 78% of subjects versus 66% at baseline. GH levels were decreased from 0.77 at baseline to 0.48 ng/mL at the end of treatment. While GH was maintained or reduced in 93% of subjects enrolled, 64% achieved IGF-I<1.3×ULN at the end of treatment versus 91% at baseline. 65 subjects (43% of mITT) entered the study with IGF-1≤1 ULN and GH<1 ng/mL, and 49 (32.5%) subjects exhibited this control at end of treatment. TABLE 2IGF-I and Mean Integrated GH Suppression at Baseline andEnd of TreatmentBaselineEnd of Treatmentn (%)n (%)mITT populationN = 151N = 151IGF-I <1.3 ULN134 (88.7)93 (61.6)and GH <2.5 ng/mLIGF-1 ≤1 ULN and65 (43.0)49 (32.5)GH <1 ng/mLIGF-I ≥1.3 ULN AND/OR17 (11.3)58 (38.4)GH ≥2.5 ng/mLIGF-I <1.3 ULN138 (91.4)97 (64.2)IGF-I ≤1.0 ULN96 (63.6)57 (37.7)GH <2.5 ng/mL145 (96.0)140 (92.7)GH <1.0 ng/mL100 (66.2)117 (77.5)Median IGF-1 levels (Q1-Q3)0.90 (0.76-1.07)1.120 (0.870-1.440)Median GH levels (Q1-Q3)0.77 (0.44-1.23)0.488 (0.244-0.870) Table 2 shows IGF-I and GH categories at Baseline and end of treatment (core+extension), for all enrolled subjects, with at least one efficacy measure on post first OOC dose (mITT population). This analysis also includes the 59 subjects who terminated early during the course of the study. For this analysis the last concentrations of IGF-I and GH on treatment were carried forward. mITT, modified Intent to Treat; IGF-1, Insulin Growth Factor−1; GH, Growth Hormone; ULN-Upper Limit of Normal. Q1-Q3, interquartile range Table 3 depicts biochemical response categories at the beginning of the fixed dose and end of 13 months treatment for those 110 subjects stabilized on OOC, who entered the fixed dose phase. Of these subjects, 91 (83%) were responders at the beginning of the fixed dose phase and 82 (75%) were responders at the end of treatment (LOCF imputation). During the fixed dose phase, both GH and IGF1 responses were largely maintained. TABLE 3IGF-I and Mean Integrated GH Suppression at the Beginningof Fixed-dose Period and at the End of 13-Month Treatment.BeginningEnd ofof Fixed DoseTreatmentn (%)n (%)Fixed dose populationN = 110N = 110IGF-I <1.3 ULN and GH <2.5 ng/mL91 (82.7)82 (74.5)IGF-I ≥1.3 ULN OR GH ≥2.5 ng/mL19 (17.3)28 (25.5)IGF-I <1.3 ULN91 (82.7)84 (76.4)IGF-I ≤1.0 ULN59 (53.6)52 (47.3)GH <2.5 ng/mL109 (99.1)105 (95.5)GH <1.0 ng/mL97 (88.2)90 (81.8)Median IGF-1 levels (Q1-Q3)0.98 (0.79-1.19)1.04 (0.83-1.26)Median GH levels (Q1-Q3)0.40 (0.23-0.66)0.43 (0.23-0.76) Table 3 shows IGF-I and GH categories at Baseline and end of treatment (core+extension), for all subjects controlled on OOC and entering the fixed dose phase. For this analysis the last on treatment concentrations of IGF-I and GH were carried forward. IGF-1, Insulin Growth Factor−1; GH, Growth Hormone; ULN, Upper Limit of Normal. Q1-Q3, interquartile range. Exploratory analysis showed that the degree of baseline control on injectable SRLs predicted subsequent response to OOC. The combination of IGF-I≤<1 ULN/GH<2.5 ng/mL and low to mid doses of injectable SRLs (octreotide<30 mg or lanreotide<120 mg), at screening, yielded an OOC response rate of 84.5% (49 of 58 subjects). FIGS.1A-1Dshow that mean IGF-I levels were stably maintained between the beginning to the end of the fixed-dose period, up to 13 months in both the mITT and fixed dose population. The slight increase in mean values from baseline towards the end of the dose-escalation period in the mITT population reflects those subjects failing to be controlled on OOC and discontinuing the study early, all of whom were included in the mITT analysis. Median GH levels at Baseline (0.77 ng/mL), were attenuated within 2 hours of the first OOC dose to 0.40 ng/mL and remained suppressed by the end the extension (0.49 ng/mL). In the fixed dose population median GH levels were 0.77 at baseline, and 0.43 ng/mL at the end of treatment. 80% of subjects entering the fixed dose improved or maintained acromegaly symptoms (26% maintained, 54% improved). Proportion of subjects with at least 1, 2 or 3 acromegaly symptoms decreased from 79%, 63% and 45% respectively at baseline to 68%, 48% and 31% at end of treatment. Acromegaly symptoms improved as demonstrated by the decline from baseline (on injectables) to end of treatment (OOC), in the proportion of subjects with active acromegaly symptoms. Compliance Over 94% of subjects fully complied with study drug administration in both the core treatment period and the extension, based on capsule counts, daily diaries, and a general drug administration and food habits questionnaire. Pharmacokinetics In 46 subjects studied during the fixed dose phase, mean plasma octreotide concentrations increased dose-dependently (seeFIG.2), and mean plasma octreotide trough values (at time zero), were comparable for the 40 and 60 mg regimens, each of which represent a prior 20 mg overnight dose, with a higher mean trough for the 80 mg regimen, which represents a 40 mg prior overnight dose. Steady-state mean apparent elimination half-life (t½) ranged from 3.19±1.07 (mean±SD, on 40 mg) to 4.47±2.02 hrs (on 80 mg) Safety Of 155 subjects in the safety population, 138 (89%) experienced an AE. Ninety two percent of events were mild to moderate (see below). Most commonly reported organ systems included gastrointestinal, neurologic and musculoskeletal, consistent with the known octreotide safety profile (1,20). Common gastrointestinal AEs (occurring in ≥5%), were nausea, diarrhea, dyspepsia, abdominal pain and distention, flatulence and vomiting, which mostly occurred within the first two months of treatment, and mostly resolved with treatment continuation (median AE duration=13 days). Common neurologic AEs were headache and dizziness and in the musculoskeletal system, arthralgia and back pain. Infections related to the gastrointestinal system included a single case of viral gastroenteritis. Hypoglycemia or hyperglycemia were reported in 7 and 11 subjects respectively (4.5% and 7%), neither of which led to early discontinuation. Hepatobiliary disorders were reported in 18 (11.6%); with cholelithiasis in 12 (7.7%). Clinically meaningful alterations were not observed in laboratory safety parameters, vital signs, ECG or physical examinations. Forty seven percent of AEs occurred within the first 3 months of treatment and the incidence significantly decreased with time from the dose escalation to the fixed dose phase. Twenty one subjects (13.5%) experienced 39 serious AEs. Two were considered possibly related to OOC-elevated hepatic transaminases and jaundice occurred in a subject with severe dehydration and a subject with suspected bile duct obstruction. Four malignancies were reported, none of which were considered study drug-related. Serious gastrointestinal infections were not reported. Twenty-three patients discontinued because of an AE, 19 of which were study-drug related, mostly in the first 3 months of treatment; ten earlier terminations were due to gastrointestinal symptoms, including nausea, diarrhea and abdominal pain. Two deaths were reported, neither of which were considered OOC-related. (See below). Overall, OOC safety was consistent with the known octreotide safety profile and acromegaly disease burden, with no new emerging safety signals related to the novel formulation and route of administration. Discussion In healthy volunteers 20 mg oral OOA yielded systemic drug exposure (AUC) comparable to 0.1 mg SC dose of octreotide (29). We now show clinical utility and unique mode of action of TPE, whereby a therapeutic peptide is effectively and safely delivered orally. OOC is shown to exhibit efficacy in controlling and maintaining IGF-I and integrated GH levels, for ≥13 months in biochemically controlled acromegaly subjects after switching from injectable SRLs. The primary efficacy endpoint was achieved by 65% of subjects at the end of the core treatment and by 62% at the end of 13 months, compared to 89% on injectable SRLs at baseline. The effect was durable and 85% of 91 subjects who entered the fixed-dose period as responders maintained this response for up to 13 months. These results are comparable to those reported for 41 acromegaly patients responding to injectable octreotide LAR (IGF-1≤1.2 and GH <2.5 ng/mL). 84% of these maintained baseline IGF-I/GH control at 6 months (32). Predictors of the degree of OOC responsiveness included good baseline control on injectable SRLs, (IGF-I≤1 ULN/GH<2.5 ng/mL), and low to mid doses of injectable SRLs. OOC also showed efficacy in maintaining clinical response; improved acromegaly symptom severity was noted in subjects who entered the fixed dose phase. As activity and safety of octreotide are well characterized, the primary goal was to assess safety and efficacy of an oral octreotide formulation. Parenteral treatment, shown to be effective, was withdrawn and replaced with OOC. As long-term maintenance of response to parenteral octreotide therapy is well established (33) and octreotide tachyphylaxis does not occur in acromegaly, a baseline-control of SRL responders shown here reflects an appropriate study design. This design also anticipates clinical practice whereby patients eligible to receive OOC would be those responding to and tolerating parenteral SRLs and then switched to an oral formulation. The enrolled patient population is representative of acromegaly patients suitable for OOC therapy. Despite being biochemically controlled by receiving SRL injections as the standard of care, 81% of subjects still exhibited persistent acromegaly symptoms at baseline. The duration of residual IGF-I suppression after long-acting SRL withdrawal is not known, but is not expected beyond 8-12 weeks from withdrawal in a patient with active disease (34). In fact, GH levels may revert between 4-6 weeks after octreotide LAR withdrawal (35). Accordingly, SRL was withdrawn 4 weeks prior to the first OOC test dose and clinical and biochemical response measured for ≥13 subsequent months. Several additional factors highlight disease activity of the enrolled subjects. Thirty-nine percent had IGF-I>1 ULN at baseline. Of the patients enrolled, 41% were being treated with the highest doses of parenteral octreotide and lanreotide for disease control. Ninety patients (58%) required >40 mg OOC doses to maintain response. Furthermore, dose up-titration against rising IGF-I levels, as well as the observed sustained IGF-I normalization achieved with OOC over the 13-month duration of the study, allayed the concern of parenteral SRL carryover effect. OOC doses selected for dose titration to enable optimal IGF-I control were based on PK modeling to achieve effective therapeutic exposure to octreotide (21,36). Distribution of the fixed dose population by OOC dose requirements were similar to the experience with injectable SRLs where higher doses are not usually required for adequate control (37,38). PK analyses demonstrated dose proportional exposure to oral octreotide. Octreotide levels measured prior to the morning dose are reflective of trough levels of the previous night dose, and were within the range shown to effectively inhibit GH secretion (21,36). The results show that under fasting conditions, OOC suppressed GH levels in nearly all subjects. However, in contrast to GH inhibition, the proportion of subjects maintaining IGF-1<1.3 ULN was lower. This suggests that OOC bioavailability was not a cause of non-response. Hepatic IGF-I generation is log-linear with GH levels (39). Octreotide acts primarily on the pituitary to suppress GH secretion, but also directly inhibits hepatic IGF-I (24,25), and the observed mild discordant GH and IGF-I responses are commonly observed with SRL injections. The enhanced response of GH to OOC may also reflect that fasting GH levels were measured within 2-4 hours following the morning OOC dose, hence may not reflect trough levels. These results underscore that the somatotroph SSTR2 receptor is a primary target for the oral ligand and point to central control of GH hypersecretion by OOC, similar to the primary action of injectables. The short GH half-life and the pulsatile nature of GH secretion (40,41) confound the accuracy of assessing GH levels based on a single blood test. The cutoff value of <2.5 ng/mL (integrated) for GH was chosen to distinguish excess from normal mortality in acromegaly. IGF-I<1.3 ULN was chosen because of the wide variances of IGF-I values and the challenge of reproducing a rigorous IGF-I<1 ULN even within individual patients (30,42). OOC side effects are largely consistent with underlying acromegaly, as well as known to be associated with SRLs (16,20) only with no injection site reactions. Most adverse events occurred within the first 60 days and mostly resolved on treatment. Fluctuations in circulating octreotide levels (e.g. after withdrawal of injectable SRLs and followed by OOC initiation) are known to result in transient AEs (Sandostatin LAR label). Gastrointestinal symptoms, associated with octreotide, were also largely transient and reported early in the study and resolved on continued treatment. Adverse events were not dose-related. No route-of-administration-related safety signals or formulation-related AEs were encountered. As OOC exhibits GH/IGF-I control, responders to parenteral SRL injection could be switched to OOC and avoid the burden of injections. Although compliance with food restrictions might be perceived as challenging for some, the advantages of an oral vs parenteral SRL preparation include convenience with ease of administration, precluding painful injections, and obviating monthly clinic visits and dependence on health care providers and/or family members for injection. Moreover, dose titration and symptomatic control could be achieved more efficiently with an oral SRL than with a 30-day preparation. This novel TPE technology safely and successfully allowed oral delivery of a therapeutic peptide that achieved systemic endocrine effects. Twice daily OOC appears to offer a safe option for acromegaly monotherapy. SeeFIG.4, which provides a flowchart of the study. Pharmacokinetic Sampling During the second monthly visit of the fixed dose phase, and after receiving the therapeutic regimen for at least 2 months, 46 subjects at a subset of sites underwent pharmacokinetic (PK) evaluation. Octreotide plasma concentrations were determined at 0 (pre-dose, up to 60 minutes before the morning drug administration)), and thereafter at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, and 12 hours post-dosing. Plasma concentrations of octreotide were measured using a validated LC/MS/MS method by PPD (Richmond, VA). The limit of quantitation (LOQ) for plasma octreotide concentrations was 0.0227 ng/mL. Pharmacokinetics Analysis Actual blood sampling times were used for pharmacokinetic (PK) analyses and per protocol times were used to calculate concentrations for graphical displays. Values below LLOQ up to the time at which the first quantifiable concentration or at last time were set to zero. Values below LLOQ that are embedded between two quantifiable values were set to missing. PK calculations were done using SAS®. PK parameters were derived from the plasma concentration actual time data, calculated using non compartmental analysis. Concentrations that were missing or not reportable were treated as missing values. PK parameters C0, Cmax, Tmax, and Tlagwere taken directly from the concentration time data. The elimination rate constant, λz, was calculated as the negative of the slope of the terminal log-linear segment of the plasma concentration-time curve. The slope was determined from a linear regression of the natural logarithm of the terminal plasma concentrations against time; at least 3 terminal plasma concentration time points, beginning with the final concentration≥LOQ, were selected for the determination of λz and the regression had to have a coefficient of determination (r2)≥0.9000. The range of data used for each subject was determined by visual inspection of a semi-logarithmic plot of concentration vs. time. Elimination half-life (t½) was calculated according to the following equation: t1/2=0.693λz Area under the curve to the final sample with a concentration≥LOQ [AUC(0−t)] was calculated using the linear trapezoidal method. Safety Two deaths were reported, neither of which were reported as study drug related. One was a 37-year-old male with a 10-year history of multiple surgeries for extrasellar pituitary macroadenoma. Six months after OOC initiation he had a suspected biliary obstruction, and subsequently also developed sepsis and multiple organ failure. At autopsy, no evidence for biliary obstruction was observed. The second was a 60-year-old male with cardiovascular risk factors, diagnosed with pancreatic cancer after six months into the study, and suffered a fatal myocardial infarction. TABLE 4Incidence of Most Common (≥5%) Adverse Events by System OrganClass and Preferred Term in all enrolled patients (n = 155),up to 13 months treatment.Number ofAdverse Event by System Organ Classsubjectsand Preferred term(%)Gastrointestinal disordersNausea46 (29.7)Diarrhea31 (20)Abdominal pain upper15 (9.7)Dyspepsia14 (9)Abdominal pain12 (7.7)Flatulence10 (6.5)Abdominal distension10 (6.5)Vomiting10 (6.5)Nervous system disordersHeadache56 (36.1)Dizziness9 (5.8)Musculoskeletal and connective tissue disorderArthralgia46 (29.7)Back Pain9 (5.8)General disorders and administration site conditionsAsthenia38 (24.5)Peripheral edema26 (16.8)Fatigue8 (5.2)Infections and infestationsNasopharyngitis12 (7.7)Influenza11 (7.1)Upper respiratory tract infection11 (7.1)Skin and subcutaneous tissue disordersHyperhidrosis36 (23.2)Hepatobiliary disordersCholelithiasis12 (7.7)Vascular disordersHypertension11 (7.1) REFERENCES 1. Melmed S. Medical progress: Acromegaly. N Engl J Med 2006; 355:2558-25732. 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Wass J A. Octreotide treatment of acromegaly. Horm Res 1990; 33 Suppl 1:1-5; discussion 637. Fleseriu M. Clinical efficacy and safety results for dose escalation of somatostatin receptor ligands in patients with acromegaly: a literature review. Pituitary 2011; 14:184-19338. Turner H E, Thornton-Jones V A, Wass J A. Systematic dose-extension of octreotide LAR: the importance of individual tailoring of treatment in patients with acromegaly. Clin Endocrinol (Oxf) 2004; 61:224-23139. Barkan A L, Beitins I Z, Kelch R P. Plasma insulin-like growth factor-I/somatomedin-C in acromegaly: correlation with the degree of growth hormone hypersecretion. J Clin Endocrinol Metab 1988; 67:69-73.40. Giustina A, Veldhuis J D. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocr Rev 1998; 19:717-79741. Reutens A T, Hoffman D M, Leung K C, Ho K K. Evaluation and application of a highly sensitive assay for serum growth hormone (GH) in the study of adult GH deficiency. J Clin Endocrinol Metab 1995; 80:480-48542. Clemmons D R. Consensus statement on the standardization and evaluation of growth hormone and insulin-like growth factor assays. Clin Chem 2011; 57:555-559 Having thus described several aspects of at least one embodiment, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure and are intended to be within the scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and the scope of the invention should be determined from proper construction of the appended claims, and their equivalents. | 97,422 |
11857596 | DESCRIPTION OF EMBODIMENTS [Core] When the present inventors comprehensively investigated physiologically active substances, it was found that at least one selected from the group consisting of bacteriocins, polyphenols, amino acids or derivatives thereof, organic acids or derivatives thereof, heat shock protein (HSP) inducers, antioxidants, and polysaccharides was observed to have an excellent membrane-strengthening ability. Bacteriocins are classified into two classes, Class I and Class II, according to the classification of Paul D. Cotter (Nat. Rev. Microbiol. 2005 Volume 3 (10), pp 777-88). Bacteriocins belonging to Class I are also called lantibiotics, and their structure has an abnormal amino acid generated by post-translational modification. Specific examples include nisin A, nisin Z, nisin Q, subtilin, duramycin, mersacidin, and lactisin-481. Class II are peptides containing no abnormal amino acids in their structure. Class II is further classified into three subclasses a to c. Specific examples of bacteriocins belonging to Class IIa include pediocin PA-1 and enterocin A. It is known that bacteriocins belonging to Class IIb are two peptides produced at the same time, and they synergistically enhance the effects of each other. Specific examples include plantaricin (PlnE, PlnF), enterocin X (Xalpha, Xbeta), and lactococcin Q (Qalpha, Qbeta). Bacteriocins belonging to Class IIc have a cyclic structure in which the N-terminal and C-terminal are linked via a peptide bond. Specific examples include gassericin A, circularin A, and lactocyclicin Q. The bacteriocin used in the present invention is preferably bacteriocin belonging to Class I, Class IIb, and Class IIc. The bacteriocin used in the present invention is more preferably nisin, gassericin, plantaricin, and subtilin. Note that, when no alphabetical letter is attached after the name of each bacteriocin, it means the generic name of that bacteriocin (for example, the term “nisin” is a concept including nisin A, nisin Z, and the like). The experiments of the present inventors observed a membrane-strengthening ability in nisin, so that the index for screening bacteriocin having a membrane-strengthening ability was examined, and it was found that the membrane-strengthening ability was not necessarily correlated with the antibacterial activity or antibacterial spectrum (see Experimental Example B described later). It is known that the antibiotics used as AGP are not decomposed by digestive enzymes, so that the activity continues even in the body and that they have antibacterial activity even after excreted as feces. On the other hand, bacteriocin, which is a protein or peptide, is easily decomposed by digestive enzymes, so that its activity is lost in the digestive tract. When used in feed applications, it is desirable to protect (coat) it so that it will not be inactivated by digestive enzymes. If protected, 10 to 100 ppm in the small intestine will exhibit sufficient membrane-strengthening ability. After that, the bacteriocin transferred to the feces is safe because it is inactivated by the enzymes in the feces. Therefore, even when a feed additive or feed containing bacteriocin instead of AGP is administered to livestock, there is an advantage that the problem of antibiotic resistant bacteria does not occur. Note that, in the present specification, “ppm” means “ppm by mass.” The polyphenol used in the present invention is quercetin, curcumin, and the like, and quercetin is preferable. The amino acid used in the present invention is glutamine (Gin), tryptophan (Trp), valine (Val), tyrosine (Tyr), phenylalanine (Phe), and the like. The amino acid derivative is phenyl lactatic acid (PLA) and the like. The organic acid used in the present invention is butyric acid and the like. The organic acid has a total of about 2 to 5 carbon atoms. The organic acid derivative is, for example, an ester with an alcohol having 1 to 5 carbon atoms. The HSP inducer (heat shock protein (HSP) inducer) used in the present invention is polyphosphate and a sporulation factor (competence and sporulation factor (CSF)) derived fromBacillus subtilis. The antioxidant used in the present invention is astaxanthin and the like. The polysaccharide used in the present invention is gum arabic, pullulan, galactoglucomannan (GGM), xanthan gum (XG), and the like. In the present invention, the physiologically active substances may be used alone or in combination of two or more kinds. The physiologically active substance used in the present invention is preferably a bacteriocin, a polysaccharide, an amino acid, or a combination thereof, and more preferably nisin, Tyr, or a combination thereof. Note here that it is known polyphosphoric acid strengthens the barrier function by increasing the productivity of heat shock protein (HSP inducer) (Shuichi Segawa et. al., PLoS ONE, 2011, 6(8): e23278). Induction of stress response proteins typified by heat shock proteins is believed to strengthen the intercellular barrier function. Meanwhile, the tight junction is a layer which is formed by the cell membranes of adjacent cells adhering to each other with their outer membrane fused to each other, and is located at the boundary between the basolateral membrane and the apical membrane of epithelial cells, and is considered to prevent the membrane proteins and lipids from intermingling between them (Iwanami “Seibutugaku Jiten,” Fourth Edition, CD-ROM Version, 1998). Without wishing to be bound by any theory, it is presumed that the physiologically active substance used in the present invention has a membrane-strengthening ability due to one or both of the function of preventing substance diffusion by the tight junction and the intercellular barrier function by the stress response protein. It was considered that strengthening the tight junction would lead to health, but there was no feed that could allow a substance capable of strengthen the tight junction to reach the intestines. In addition, it was not known that an antibacterial agent capable of strengthening the tight junction in vitro exhibited a body weight gain effect when administered to livestock. The purity of the physiologically active substance used in the present invention is not limited as long as the desired effect is obtained. For example, a physiologically active substance obtained by being produced by a microorganism can be used together with the culture. The solid content in the fermentation broth can be obtained by freeze-drying, or the fermentation broth can be obtained as a solid content by spray granulation. Alternatively, as the physiologically active substance used in the present invention, a secreted product or extracted product from plants, algae, crustaceans, or fish can be used as it is. As the physiologically active substance used in the present invention, a commercially available product can be used as it is. Among the physiologically active substances, plants containing polyphenols include grapes, wine, tea, apples, blueberries, persimmons, bananas, turmeric, cinnamon, coffee beans, citrus fruits, onions, and the like. As a microorganism that produces polysaccharides among physiologically active substances, for example, galactoglucomannan (GGM) produced byLipomyces starkeyican be obtained by the methods described in Japanese Patent Application Publication No. Hei 7-298873 and Japanese Patent Application Publication No. Hei 9-131199. Microorganisms that produce polysaccharides among physiologically active substances includeXanthomonas campestris, Aureobasidium pullulans, Lipomyces Starkeyi, Phaeophyta as brown algae, and Eucheuma as algae. In addition, the cases of plants include gum arabic (Acacia senegal), guar beans (Cyamopsis tetragonoloba), ibaranori (Hypnea musciformis), cod (Tara spinosa), locust beans (Ceratonia siliqua), and the like. Among the physiologically active substances, microorganisms that produce amino acids or derivatives thereof includeBrevibacterium flavum, Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and the like. Among the physiologically active substances, microorganisms that produce organic acids or derivatives thereof includeLactobacillus, Bifidobacterium, Clostridium, and the like. Among the physiologically active substances, microorganisms that produce HSP inducers includeBacillus, Lactococcus lactis, Lactobacillus, Leuconostoc, andPediococcus. Among physiologically active substances, microorganisms that produce antioxidants includePhaffia rhodozymaandPseudomonas thiazolinophilum, andHaematococcus pluvialisas algae. Also, bacteriocin, which is one of the physiologically active substances used in the present invention, is preferably a culture product of at least one microorganism selected from the group consisting ofBacillus, Lactococcus, Lactobacillus, Leuconostoc, andPediococcus. In particular, the microorganism that produces bacteriocin is preferably a culture product ofBacillus subtilis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri(cattle),Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis(swine),Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus lactis, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus zeae, Lactococcus lactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Pediococcus acidilactici, Pediococcus cerevisiae/damnosus, Pediococcus dextrinicus, andPediococcus pentosaceus, which are registered with the Association of American Feed Control Officials (AAFCO) or the European Food Safety Authority (EFSA). In particular, culture products ofLactococcus lactis, Bacillus subtilis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus gasseri, andClostridium beijerinckiiare preferable. Among these, a culture product ofLactococcus lactisis preferable. Particularly, culture products ofLactococcus lactisFERM BP-8552 (nisin Z-producing bacterium),Bacillus subtilisATCC 6633 (subtilin-producing bacterium),Lactococcus lactisNCIMB 702054 (nisin Z-producing bacterium),Lactobacillus plantarumJCM 1057 (plantaricin-producing bacterium),Lactobacillus gasseriLA39 JCM 11657 (gassericin A-producing bacterium),Clostridium beijerinckiiJCM 1390 (circularin A-producing bacterium) are preferable.Lactococcus lactisFERM BP-8552 (nisin Z-producing bacterium) andLactococcus lactisNCIMB 702054 (nisin Z-producing bacterium) are most preferable. Note thatLactococcus lactisFERM BP-8552 was deposited on Nov. 19, 2003 with the International Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology (postal code 305-8566, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, Japan, currently the National Institute of Technology and Evaluation, postal code 292-0818, 2-5-8 Kazusakamatari, Kisarazu, Chiba Prefecture, Japan). In the present specification, this strain may be referred to as AJ110212. Bacillus subtilisATCC 6633 has been deposited with the American Type Culture Collection, Manassas, VA, USA. Lactococcus lactisNCIMB 702054 has been deposited with the National Collection of Industrial, Food and Marine Bacteria, NCIMB Ltd., Aberdeen, Scotland, UK. Lactobacillus plantarumJCM 1057,Lactobacillus gasseriLA39 JCM 11657, andClostridium beijerinckiiJCM 1390 have been deposited with Japan Collection of Microorganisms, RIKEN BioResource Research Center (postal code 305-0074, 3-1-1 Koyadai, Tsukuba City, Ibaraki Prefecture). The core may contain a protective agent. The protective agent includes skim milk, amino acid salts such as sodium glutamate, sugar alcohols such as sorbitol, and disaccharides such as trehalose and sucrose. The core may contain an excipient. The excipient is not particularly limited as long as it is one commonly used for improving shape formation, and examples thereof include calcium carbonate, silicon dioxide, calcium silicate, zeolite, sorbitol, corn starch, talc, yeast bentonite, rice husk, liquid paraffin, polysaccharides other than polysaccharides having a property of aggregating gram-negative bacteria, monosaccharides, and disaccharides. When the core contains an excipient, the amount of the excipient is usually preferably 0.1 to 100 parts by mass based on 100 parts by mass of the core. The core may also contain any additives that may be included in conventional feed. When the core contains an optional additive, the amount of the optional additive is usually preferably 0.1 to 100 parts by mass based on 100 parts by mass of the core. [Coating Agent] The coating agent is a substance capable of forming an enteric coating and can be used without particular limitation as long as it is a substance safe for livestock to ingest. The coating agent may be used alone or in combination of two or more kinds. From the viewpoint of easy handling and economical efficiency, the coating agent is preferably hydrogenated vegetable oil, or shellacs, zein, hydroxypropyl methylcellulose, maltitol, and the like which are substances commonly used as tablet coating agents. The hydrogenated vegetable oil includes hydrogenated oils of rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice oil, olive oil, palm oil, palm kernel oil, or coconut oil. The coating agent is preferably hydrogenated rapeseed oil and shellac. The layer of hydrogenated rapeseed oil is preferable because it can dissolve the core in a short period of time. The layer of shellac is preferable because it can dissolve the core with neutral to alkaline (after passing the stomach). Alternatively, the coating agent may be a microorganism itself that produces such a physiologically active substance. The coating agent is in an amount of preferably 5 to 90% by mass, and more preferably 20 to 30% by mass, based on the total mass of the coated-type feed additive of the present invention. The coating agent may also contain any additives that may be included in conventional feed. The coating may be a single layer or multiple layers of two or more layers. The multi-layer coating is preferable because it is easier to control the dissolution rate in the body. In particular, it is preferable that the outermost layer is a layer of hydrogenated rapeseed oil and the innermost layer in contact with the core is a layer of shellac, because the coating agent dissolves in the intestine rather than in the stomach. The dissolution rate of the coated-type feed additive of the present invention in gastric juice is desirably less than 50%, and the difference between the dissolution rate in intestinal juice and the dissolution rate in artificial gastric juice is desirably 10% or more. For the purpose of achieving such a dissolution rate, it can be adjusted by forming a two-layer membrane or a multi-layer membrane, or by controlling the type of coating agent and the membrane thickness for each layer. [Coating Method] The method of coating the core is not particularly limited, and for example, it is possible to obtain a coated-type feed additive by spraying a coating agent in a liquid state, heated to a temperature higher than the melting point, while allowing the powdered or granular core to flow with a commercially available fluidized bed spray granulator. The coated-type feed additive obtained from powdered or granular polysaccharides has a size of preferably about 0.05 to 5 mm because handling is easy. In addition, the temperature for heating the coating agent is not particularly limited as long as it is equal to or higher than the melting point of the coating agent, but it is preferably higher than the melting point of the coating agent by about 5° C. to 15° C. [Feed] The coated-type feed additive of the present invention can be given to livestock as it is, or can be used as a feed together with an excipient or diluent such as corn, soybean flour, rice bran, fish meal, or brewer's yeast. The feed of the present invention may also contain any additives that may be included in feed. The feed of the present invention is suitable for continuous daily intake. The feed intake varies depending on the size of livestock. For example, in the case of chickens, it is desirable that the physiologically active substance is fed at a daily intake of about 1 to 200 ppm and preferably 10 to 100 ppm, based on the feed other than the coated-type feed additive. In the present specification, the term “livestock” refers to creatures that are bred by humans Specific examples include ruminants such as cows, sheep, and goats, and monogastric animals such as horses, pigs, chickens, dogs, and fish. It is particularly preferable to give the feed of the present invention to monogastric animals. The method of feeding the coated-type feed additive of the present invention is not particularly limited. EXAMPLES Experimental Example A: Membrane Strengthening Experiment The membrane-strengthening ability of the test substance was evaluated according to the description in J. Nutr. (2009) volume 139(5), pp 965-974. Caco-2 cells (human intestinal epithelial cells (ECACC, Code 86010202)) were seeded in a double-layered Transwell system, and cultured in DMEM medium at 37° C. On the 12th day of culture, TNF-α, was added to reduce the barrier function of Tight Junction. On the 14th day of culture, the test substance was added, the mixture was cultured at the same temperature for 24 hours, and then Millicell ERS-2 (manufactured by Millipore) was used to measure the transepithelial electrical resistance value TER (Ω*cm2), thereby evaluating the restoration (recovery ratio %) of the barrier function.FIG.1AtoFIG.1Hpresent the results. As illustrated inFIG.1A, it was confirmed that AGP, such as avilamycin and colistin, had a function of strengthening the barrier function even at a low concentration. With the group added with TNF-α alone used as a control, a recovery ratio/control of 1.1 or more was determined to have the membrane-strengthening ability. Table 1 presents the concentration of the material itself when the recovery ratio/control of each physiologically active substance is around 1.1. Quercetin, grape seed extract (manufactured by Ajinomoto Co., Inc.), nisin A, tyrosine (manufactured by Ajinomoto Co., Inc.), astaxanthin (manufactured by DSM), and the like were also observed to have the function of strengthening the barrier function. TABLE 1RecoveryRecoveryMaterialMaterialconc.ratio/ctr*1ratio (%)Antibiotic GrowthAvilamycin10uM1.10127Promoter (AGP)Tylosin10uM1.17135Colistin1uM1.13130Zinc Bacitracin10uM1.10124Salinomycin1uM1.10124Monensin1uM1.13126Enramycin100uM1.28144BacteriocinNisin (Class I)10uM1.20139Duramycin (Class I)1uM1.04120Gassericin (sup)*2 (Class IIc)1unit1.44162Plantaricin (sup)*3 (Class IIb)1unit1.52171PolyphenolQuercetin100uM1.23138Curcumin200uM1.14128Grape Seed Extract*4200uM1.19157Amino acidGlutamine20mM1.18136Tryptophan4mM1.11128Valine0.2mM1.13130Phenyl-lactate0.2mM1.12129Tyrosin20uM1.19138Organic acidButylate2.0mM1.13131chelate /HSP inducerPoly-Phosphate (750)10ppm1.12129Anti-oxidantAstaxanthin10mg/L1.14132Poly SaccharideArabic Gum1ppm1.20139Pullulan100ppm1.11124GGM1ppm1.08122XG1ppm1.06119*1: Comparison value with Ctr (group added with TNFα alone)*2: Gassericin (sup): Culture supernatant of the production bacteria (Lactobacillus gasseriLA39 JCM 11657) concentrated three times with an ultrafiltration membrane (MW: 3,000)*3: Plantaricin (sup): Culture supernatant of the production bacteria (Lactobacillus plantarumJCM 1057) concentrated three times with an ultrafiltration membrane (MW: 3,000)*4: “OmniVin ™ 10R” manufactured by Ajinomoto Example 1: Coated-Quercetin 1-1: Preparation of Coated-Quercetin Quercetin (reagent manufactured by Tokyo Chemical Industry Co., Ltd. (purity 95%)) was used as the core, and hydrogenated rapeseed oil (melting point 67° C.) and natural resin shellac were used as the coating agents. The powdered or granular core was sprayed with a predetermined amount of coating agent, liquefied by heating to a temperature higher than the melting point, to obtain a coated-type feed additive. The coating was carried out by spraying 5 parts by mass of shellac as the first layer (inner layer) and 17 parts by mass of hydrogenated rapeseed oil as the second layer (outer layer), based on 77 parts by mass of the core. 1-2: Acid Resistance and Enteric Test (Artificial Gastric Juice Treatment) To pure water produced using a pure water production device manufactured by Merck Millipore, 0.2 mass % NaCl and 0.2 mass % pepsin (from Porcine stomach Mucosa, 1:5,000, 2,500 unit/mg) were added to adjust the pH to 2, and then the coated-type feed additive prepared in 1-1 above was charged therein, followed by enzyme treatment at 37° C. for 2 hours. The dissolution rate was measured by automatically and continuously measuring the optical density during this period. Note that the “2 hours” assumes the time from when the feed reaches the stomach of the chicken until it passes. (Artificial Intestinal Juice Treatment) After the artificial gastric juice treatment, 0.2% trypsin (from Porcine Pancreas, 1:5,000; 4,500 unit/mg) was added to adjust the pH to 6, followed by enzyme treatment at 37° C. for 2 hours. The dissolution rate was measured by automatically and continuously measuring the optical density during this period. Note that the “2 hours” assumes the time from when the feed reaches the intestine of the chicken until it passes. For pH adjustment, hydrochloric acid and sodium hydroxide were used. The optical density was measured at OD 660 nm using Biophoto-recorder TVS062CA manufactured by ADVANTEC. FIG.2presents the results. FromFIG.2, in the two-layer coating, the dissolution rate within 2 hours from the start of gastric juice treatment was suppressed to 25%, while in the intestinal juice treatment, 65% was dissolved. From these results, it was found that the feed additive having a hydrogenated rapeseed oil layer as the outer layer and a shellac layer as the inner layer had acid resistance and enteric properties, and was excellent in release control. 1-3: Chicken Growth Test The coated-quercetin prepared by the two-layer coating method of 1-1 above was added to the feed matrix having the composition presented in Table 2 so that the amount of the core agent was 20 ppm and 200 ppm to obtain a feed composition. In a flat poultry house, 25 neonate broiler chickens in 1 section were used, fed with the feed composition, and bred for 22 days at 0 to 21 days of age in triplicate to evaluate the chicken body weight gain (BWG) and feed conversion ratio (FCR=Feed/BWG). Note that a conventional antimicrobial growth promoter (AGP), avilamycin, was used as a positive control. As avilamycin, a commercially available product (Surmax 200 (registered trademark) manufactured by ELANCO, uncoated) was used as it was. The results are presented with a negative control of 100. Table 3 presents the results. Although it is known that uncoated quercetin has a negative effect on the intestinal microbiota (J. Nutr. 139: 965-974, 2009.), coated-quercetin exhibited a body weight gain effect and feed conversion ratio improvement effect. Note that, since uncoated quercetin has a negative effect on the intestinal microbiota, the chicken growth test using uncoated quercetin was not carried out. TABLE 2Raw MaterialMixing Ratio, Mass %Corn45.4Grain Sorghum10.0Soybean Meal30.0Corn Gluten Meal4.00Fishmeal (CP 65%)3.00L-Lysine Hydrochloride0.31DL-Methionine0.35L-Threonine0.12L-Arginine0.16Animal Oil and/or Fat3.49Dibasic Calcium Phosphate1.45Calcium Carbonate1.06Salt0.30Vitamin and Mineral Premix0.25Choline Chloride0.02L-Valine0.07Total100*Feed Composition TABLE 3Addition0-3 weeksCategoryppmBWGFCRAGPPC1/Avilamycin10110%96%MembraneCoated Quercetin20106%98%StrengtheningCoated Quercetin200104%98% Example 2: Coated-Phe and Coated-Tyr 2-1: Preparation of Coated-Phe and Coated-Tyr Coated-Phe and coated-Tyr were prepared according to the description in 1-1 of Example 1. 2-2: Acid Resistance and Enteric Test The coated-Tyr obtained above was used to carry out acid resistance and enteric test in the same manner as in 1-2 of Example 1.FIG.3presents the results. 2-3: Chicken Growth Test The coated-Phe and coated-Tyr obtained above were added to the feed matrix having the composition presented in Table 2 so that the amount of the core material was 200 ppm, to thereby evaluate the body weight gain effect and feed conversion ratio of chickens according to the method described in 1-3 of Example 1. Table 4 presents the results. A certain degree of body weight gain effect was confirmed even when Phe and Tyr were not coated, but Phe or Tyr was not detected in the intestinal tract contents or blood as a result of metabolome analysis. It is thus considered that coated-Phe and coated-Tyr are assimilated by intestinal bacteria in feces and rapidly metabolized in blood. Therefore, it is considered that the coated amino acids more stably exhibit the effect than the uncoated amino acids. TABLE 4Addition0-3 weeksCategoryppmBWGFCRPC1/Avilamycin10107%95%Phe200106%98%Coated Phe200103%97%Tyr200104%98%Coated Tyr200105%99% 2-4: Evaluation of L-Tyr and Coated-L-Tyr bySalmonellaInfection Test in Chickens The coated-Tyr and Tyr prepared in the same manner as in Example 2-1 were added to the feed matrix having the composition presented in Table 2 so that the amount of the core material was 200 ppm, to thereby prepare a feed. Broilers at 1 day of age were introduced into a breeding facility for infection test (6 broilers/repeat and 2 repeats/test group), thenSalmonella enterica(SE) was orally administered to the broilers at 2 days of age, and the test feed was fed for 21 days to evaluate the body weight gain effect and feed conversion ratio of chickens. Note that a conventional antimicrobial growth promoter, enramycin, was used as a positive control. As enramycin, a commercially available product (“Enramycin F-80” manufactured by Scientific Feed Laboratory Co., Ltd., uncoated) was used as it was. The results are presented with a negative control of 100. Table 5 presents the results. TABLE 5Addition,0-3 weekTest GroupppmBWGFCRControl Group—100%100%Enramycin (AGP)10107%98%L-Tyr20096%100%Coated-L-Tyr200109%97% As a result, there was no effect in uncoated tyrosine (“L-Tyr”), but coated-Tyr exhibited the same body weight gain effect and feed conversion ratio improvement effect as those of enramycin. Example 3: Coated-Nisin A 3-1: Preparation of Coated-Nisin A Nisin A (reagent manufactured by Sigma-Aldrich (nisin content 2.5% by mass, balance sodium chloride and denatured milk solids) was used as the core, and hydrogenated rapeseed oil (melting point 67° C.) and natural resin shellac were used as the coating agents. A coated-type feed additive with a two-layer coating was obtained according to the description in 1-1 of Example 1. 3-2: Acid Resistance and Enteric Test The coated-nisin A prepared above was used to carry out acid resistance and enteric test in the same manner as in 1-2 of Example 1.FIG.4presents the results. 3-3: Chicken Growth Test The coated-nisin A prepared in 3-1 was added to the feed matrix having the composition presented in Table 2 so that the amount of the core material was 1 ppm and 10 ppm, to thereby evaluate the body weight gain effect and feed conversion ratio of chickens. In a flat poultry house, 25 neonate broiler chickens in 1 section were used, and bred at 0 to 21 days of age in triplicate. Table 6 presents the results. Although nisin decreased in activity by gastric acid and was completely inactivated by the digestive enzyme trypsin, coated-nisin A was observed to have a body weight gain effect and feed conversion ratio improvement effect. TABLE 6Addition0-3 weeksCategory(ppm)BWGFCRNC100%100%PC1 (W/Avilamycin)10104%97%PC2 (W/Avilamycin)10107%94%Coated Nisin A10103%97%Coated Nisin A1101%98% 3-4: Evaluation of Coated-Nisin A and Coated-Pullulan bySalmonellaInfection Test in Chickens The coated-nisin A prepared in 3-1 was used alone or in combination with coated-pullulan, and added to the feed matrix having the composition presented in Table 2 so that the amount of each core agent added was 10 ppm, to thereby prepare a feed. Broilers at 0 days of age were introduced into a breeding facility for infection test (2 repeats/test group and 6 broilers/repeat), and thenSalmonella enterica(SE) were administered at 107 counts/broiler with a probe into the crop of the broilers at 2 days of age, and the test feed was fed for 21 days to evaluate the body weight gain effect and feed conversion ratio. Table 7 presents the results. TABLE 7Amount Added0-3 wTest Group[ppm]BWGFCRControl Group—100%199%Coated-Nisin A Group10102%92%Coated-Nisin A +10, 10105%98%Coated-Pullulan Group As a result, both the coated-nisin group and the coated-nisin+coated-pullulan group exhibited a body weight gain effect and feed conversion ratio improvement effect. 3-5: Evaluation of Nisin A and Coated-Nisin A bySalmonellaInfection Test in Chickens The coated-nisin A and nisin A prepared in the same manner as in Example 3-1 were added to the feed as presented in Table 2 to prepare a feed. Broilers at 0 days of age were introduced into a breeding facility for infection test (6 broilers/repeat and 2 repeats/test group), thenSalmonella enterica(SE) was orally administered to the broilers at 2 days of age, and the test feed was fed for 21 days to evaluate the body weight gain effect and feed conversion ratio. Note that a conventional antimicrobial growth promoter, enramycin, was used as a positive control. As enramycin, a commercially available product (“Enramycin F-80” manufactured by Scientific Feed Laboratory Co., Ltd., uncoated) was used as it was. Table 8 presents the results. TABLE 8Volume,0-3 weekTest GroupppmBWGFCRControl Group—100%100%Enramycin (AGP)10107%98%Nisin A10101%100%Coated-Nisin A10107%97% As a result, there was no effect in uncoated nisin A, but coated-nisin A exhibited the same body weight gain effect and feed conversion ratio improvement effect as those of enramycin. Experimental Example B: Evaluation of Antibacterial Activity and Evaluation of Antibacterial Spectrum for Bacteriocin B-1: Measurement of Minimum Inhibitory Concentration The minimum inhibitory concentrations of AGP and nisin were measured and compared. Lactococcuslactic AJ110212 (FERM BP-8552) was used as the nisin Z-producing bacterium. The nisin Z-producing bacteria were cultured at 100 rpm at 30° C. in a medium (1 L of Lactobacilli MRS Broth manufactured by BD Difco) in a 5 L Sakaguchi flask. The culture was carried out for 20 hours as a standard, and the culture solution was prepared by measuring the optical density at a wavelength of 610 nm with a spectrophotometer (Biophoto-recorder TVS062CA manufactured by ADVANTEC) to be 0.1 or more when diluted 26 times. The obtained culture solution was centrifuged (6,000 G×10 min, 4° C.) to separate a cell fraction (wet cells). The following strains were used as test bacteria. The medium and culture temperature are written in parentheses at the end of the strain. The MRS medium used was Lactobacilli MRS Broth manufactured by Difco, the GAM medium and LB medium used were manufactured by Nissui Pharmaceutical Co., Ltd., and the NB medium used was manufactured by Difco. Gram-Positive Bacteria Lactobacillus acidophilusAJ13778 (MRS, 37° C., corresponding to that deposited at accession number JCM 1132)Lactobacillus salivariusAJ110152 (MRS, 37° C., corresponding to that deposited at accession number JCM 1231)Bifidobacterium thermophilumAJ110569 (GAM, 37° C., corresponding to that deposited at accession number JCM 1207)Bacteroides fragilisJCM 11019 (GAM, 37° C.)Escherichia coliMG1655 (LB, 37° C., corresponding to that deposited at accession number ATCC 700926)Clostridium perfringensAJ3350 (GAM, 37° C., corresponding to that deposited at accession number ATCC 10873) Gram-Negative BacteriaEnterococcus faecalisAJ110149 (MRS, 30° C., corresponding to that deposited at accession number JCM 5803)Salmonella entericaAJ2785 (NB, 37° C., corresponding to that deposited at accession number IAM 1648) Note that the depositary institution for bacteria identified by an accession number starting with JCM is Japan Collection of Microorganisms, RIKEN BioResource Research Center (postal code 305-0074, 3-1-1 Koyadai, Tsukuba City, Ibaraki Prefecture). The depositary institution for bacteria identified by an accession number starting with ATCC is the American Type Culture Collection, Manassas, VA, USA. The depositary institution for bacteria identified by an accession number starting with IAM is IAM Culture Collection, Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan (collection transferred to JCM). The minimum inhibitory concentration was calculated by measuring the antibacterial activity by the spot-on-lawn method described in Mayr-Harting, A. et al., Methods Microbiol. 1972, 7A, pp 315-422. In the case of using a culture solution, a qualitative judgment was made based on the size of the inhibition circle. Table 9 presents the results. TABLE 9Comparative data on the minimum inhibitory concentrations of AGP and nisinMinimum inhibitory concentration (mg/mL)Antibiotic Growth Promotor (AGP)Class IClass IIIClass IVPolyether-Based,Macrolide-OthersPolypeptide-AlternativeIonophorePolypeptide-BasedBasedChlortetra-Avila-Basedto AGPIndicator strainSalinomycinMonensinBacitracinEnramycinTylosincyclinemycinColistinNisinLactobacillus22320.51416>1003.1acidophilusAJ13778Lactobacillus11320.5138>1001.6salivariusAJ110152Clostridium11150140.0621>1001.6perfringensAJ3350Enterococcus0.5212844ND2>1003.1faecalisAJ110149Escherichia coli>100>100>100>100>10050>1004>25MG1665Bacteroides64128>10064448>1003.1fragilisJCM11019Bifidobacterium2240.50.0116161281.6thermophilumAJ110569Salmonella>100>100>100>100>10050>100>100>25entericaAJ2785“ND”: indicates no effect detected (not detected). B-2: Measurement of Antibacterial Spectrum For compounds difficult to obtain with reagents, the following producing bacteria were cultured in Lactobacilli MRS Broth medium manufactured by Difco at 30° C. to prepare a bacteriocin-containing culture solution.As bacteriocin/class I: nisin, nisin A used in 3-1 of Example 3 and nisin Z (producing bacteria wereLactococcus lactisNCIMB 702054) were used. As the subtilin-producing bacteria,Bacillus subtilisATCC 6633 was used. As duramycin, a reagent manufactured by Sigma-Aldrich (1 mg/ml) was used.As bacteriocin/class IIb: plantaricin-producing bacteria,Lactobacillus plantarumJCM 1057 was used.As bacteriocin/class IIc: gassericin A-producing bacteria,Lactobacillus gasseriLA39 JCM 11657 was used, and circularin A-producing bacteria used wasClostridiumbeijerinckii JCM 1390. The antibacterial spectrum was evaluated by the spot-on lawn method using a 10-fold concentrated culture solution supernatant. Table 10 presents the results of the investigation. TABLE 10Antibacterial Spectrum of BacteriocinClass IClass IIbClass IIcIndicator StrainNisin A*Nisin ZSubtilinDuramycin*Planta-ricinGassericin ACircularin AClostridium perfringens++++++++++++++++NDNDLactobacillus acidophilus++++++++++++++Lactobacillus salivarius++++++++++ND+NDNDAJ110152Bifidobacterium thermophilum+++++++++++++++AJ110569Bacteroides fragilis+++++ND++++JCM11019Escherichia coli++NDNDNDNDNDNDMG1655Enterococcus faecalis+++++++++ND++NDNDAJ110149Nisin A (1 mg/ml), Duramycin (1 mg/ml), Others: Bacteriocin producers“+” to “+++++”: indicates the strength of antibacterial effect. The more “+”, the stronger the effect.“ND”: indicates no effect detected (not detected). From the comparison between Table 9 and Table 10 and Table 1, it was found that the membrane-strengthening function was unrelated to the presence or absence of antibacterial activity and its strength. Example 4: Coated-Bacteriocin-Producing Bacteria Probiotics 4-1: Culture of Bacteriocin-Producing Bacteria While Example 3 used the nisin A reagent, Example 4 usedLactococcus lactisFERM BP-8552 as the nisin Z-producing bacteria. The nisin Z-producing bacteria were cultured at 100 rpm at 30° C. in a medium (1 L of Lactobacilli MRS Broth manufactured by BD Difco) in a 5 L Sakaguchi flask. The culture was carried out for 20 hours as a standard, and the culture solution was prepared by measuring the optical density at a wavelength of 610 nm with a spectrophotometer (Biophoto-recorder TVS062CA manufactured by ADVANTEC) to be 0.1 or more when diluted 26 times. In the same manner, subtilin and plantaricin cell fractions were obtained. Note thatBacillus subtilisATCC 6633 was used as the subtilin-producing bacterium.Lactobacillus plantarumJCM 1057 was used as the plantaricin-producing bacterium. 4-2: Preparation of Bacteriocin-Producing Bacteria Powder The cell fraction of nisin Z obtained in 4-1 was added to 120 ml of the protective agent, and dried by a spray dryer (inlet temperature 80° C. and outlet temperature 50° C.) or depressurized freeze-drying. The protective agent is as follows. (A) skim milk 10% by mass (manufactured by BD)+sodium glutamate 3% by mass (MSG, AJICO) (B) MSG 3% by mass, sorbitol 10% by mass, trehalose 10% by mass, and sucrose 10% by mass ((MSG was manufactured by AJICO, and the others except for MSG were manufactured by Wako Pure Chemical Industries, Ltd.) The viable cell count in the obtained nisin Z-producing bacteria powder was measured. The viable cell count was measured as follows. The powder sample in an amount of 0.01 g was suspended in 1 ml of physiological saline, and the physiological saline was diluted 10 times in sequence, 0.1 ml of which was smeared on an MRS agar plate, and cultured at 30° C. for 24 hours, and the number of colonies formed was used to measure the colony formed unit (cfu)/g. Similarly, subtilin- and plantaricin-producing bacteria powder was obtained, and the producing bacteria were measured. 4-3: Preparation of Coated-Nisin Z-Producing Bacteria Powder, Coated-Subtilin-Producing Bacteria Powder, and Coated-Plantaricin-Producing Bacteria Powder The nisin Z-producing bacteria powder prepared in 4-2 was coated in the same manner as described in 1-1 of Example 1 to obtain coated-nisin Z-producing bacteria powder. Similarly, coated-subtilin-producing bacteria powder and coated-plantaricin-producing bacteria powder were prepared. 4-4: Acid Resistance and Enteric Test The coated bacteriocin-producing bacteria powder obtained in 4-3 was subjected to the artificial gastric juice treatment and artificial intestinal juice treatment described in 1-2 of Examples 1. The acid resistance was evaluated by measuring the viable cell count and antibacterial activity of each bacteriocin after artificial gastric juice treatment and artificial intestinal juice treatment. The viable cell count was measured according to the description in 4-2. The antibacterial activity was measured according to the description in B-2 of Experimental Example B. Table 11 presents the results. Note that the meanings of “+” and “ND” in the table are the same as those described in Table 10. TABLE 11Ctr 0 (hr)Gastric Juice Treatment 2 (hr)Intestinal Juice Treatment 4 (hr)AntibacterialAntibacterialAntibacterialBacteriocinGrowth, cfuActivityGrowth, cfuActivityGrowth, cfuActivityNisin Z2.1*10{circumflex over ( )}10++++1.4*10{circumflex over ( )}2ND1.0*10{circumflex over ( )}2NDSubtilin2.1*10{circumflex over ( )}8++++1.1*10{circumflex over ( )}8++1.0*10{circumflex over ( )}8++Plantaricin7.1*10{circumflex over ( )}10++2.0*10{circumflex over ( )}5ND2.0*10{circumflex over ( )}5ND 4-5: Chicken Growth Test Using Coated-Nisin Z-Producing Bacteria Powder The coated-nisin Z-producing bacteria powder prepared in 4-3 was added to the feed matrix presented in Table 2 so that the viable cell count was as in the table below, and the method described in 1-3 of Example 1 was followed to evaluate the body weight gain effect and feed conversion ratio of chickens. TABLE 120-3 weeksCategoryAmount Added (cfu)BWGFCRNon Treatment—100100Nisin Producer1010103100 Example 5: Chicken Growth Test Three conditions were prepared, a AGP-containing feed (PC) obtained by adding antibiotics (lasalocid 0.05% by mass and avilamycin 0.01% by mass) to a standard feed, a PRB-supplemented feed (nisin (Lc)) supplemented with 2% of nisin A culture solution obtained by culturingLactococcus lactisNCIMB 8780 in the same manner as in Example 4-1, and a AGP-free feed (standard feed only) (NC), and were administered to newborn chicks. Note that, for one condition, ten Cobb Broiler male newborn chicks were used, and the experiment was repeated three times to evaluate the body weight gain effect and feed conversion ratio of chickens. For the drug-free group (NC), a standard feed (ME 3160 kcal and CP 22% by mass without antibiotics used) was used. For the PC and nisin addition group, 2% by mass of the antibiotics (lasalocid and avilamycin) or nisin Z-containing liquid was added to the standard feed (ME 3160 kcal and CP 22% by mass), respectively. TABLE 13BWGFCRCategory1 w2 w1 W2 WNC106.2 ± 3.0330.3 ± 7.81.19 ± 11.34 ± 0.02PC*110.4 ± 4.7379.9 ± 10.01.08 ± 01.23 ± 0.02Nisin (Lc)**111.1 ± 5.4352.4 ± 27.91.23 ± 21.35 ± 0.03*Antibiotics: lasalocid 0.05% and avilamycin 0.01% added.**For nisin, 2%Lactococcuslactisculture solution was added. | 42,138 |
11857597 | DETAILED DESCRIPTION OF THE INVENTION The high frequency of multidrug resistant bacteria, and in particular, Gram-positive bacteria, both in the healthcare setting and the community present a significant challenge for the management of infections (Krause et al. (2008).Antimicrobial Agents and Chemotherapy52(7), pp. 2647-2652, incorporated by reference herein in its entirety for all purposes). Moreover, methicillin resistantS. aureus(MRSA) infections in cystic fibrosis (CF) patients is a concern, and there is a lack of clinical data regarding approaches to eradicate such infections (Goss and Muhlebach (2011).Journal of Cystic Fibrosis10, pp. 298-306, incorporated by reference herein in its entirety for all purposes). Due to the high frequency of resistant pathogens, novel compounds and methods are needed to treat infections due to such pathogens. Moreover, it has been found that semi synthetic glycopeptides containing primary amino conjugated lipophilic moieties can accumulate in tissue and can exhibit long half-lives at the site of administration following administration (e.g., administration via inhalation). As such, glycopeptides that promote clearance from the site of administration are needed. The present invention addresses the need for new antibiotics and treatment methods by providing certain glycopeptides containing a primary amino conjugated lipophilic moiety that is cleavable by enzymatic hydrolysis, and methods for using the same. The lipophilic moiety is conjugated to the primary amino group via a functional group that is capable of undergoing enzymatic hydrolysis. The functional group that undergoes enzymatic hydrolysis, in one embodiment, in conjugated to the primary amino group via a straight chain or branched alkyl group, e.g., a methyl, ethyl, propyl or butyl group. In another embodiment, the functional group is an amide that comprises the nitrogen atom from the primary amino group of the glycopeptide. Glycopeptides of the present invention are referred to herein in various embodiments, as lipo-glycopeptide cleavable (LGPC) derivatives. Without being bound by any particular theory or mechanism, it is believed that the cleavage of the lipophilic moiety promotes clearance of the glycopeptide from the site of administration. In one embodiment, the LGPC derivative clears more rapidly from the site of administration (e.g., the lung) as compared to a structurally similar glycopeptide having a non-cleavable lipophilic moiety conjugated to the counterpart primary amino group. As an example, in one embodiment, a glycopeptide containing a cleavable lipophilic group attached to a primary amino group of the glycopeptide clears from the site of administration at a faster rate than a glycopeptide having a non-cleavable lipophilic group attached to the same primary amino group. In another embodiment, the LGPC has a half-life (T1/2) at the site of administration that is shorter than the T1/2of a glycopeptide having a non-cleavable lipophilic group attached to the primary amino group. The comparison of clearance is made in one embodiment, between glycopeptides having the same core structure, but for the different primary amino group conjugated moieties. One embodiment of an appropriate comparison is shown in Table A. TABLE ASemi-synthetic glycopeptide primaryLGPC derivative primary aminoamino conjugated non-cleavableconjugated cleavable moietymoiety-(alkyl)n1-Y1-lipophilic groupvs.-(alkyl)n1-Y2-lipophilic groupEach n1 is the same for each comparison, or differs by 1, 2 or 3 carbon atoms.Y1is a functional group that can undergo enzymatic hydrolysis, e.g., —O—C(O)—; —NH—C(O)—; —C(O)—O—; —C(O)—NH—; —O—C(O)—NH; NH—C(O)—O; O—C(O)—OY2is a functional group that cannot undergo enzymatic hydrolysis, e.g., —O—; —NH—; —S—S—; —SO2—;Alkyl is either substituted or unsubstituted.Each lipophilic group is the same, or differs in length by one carbon or two carbon atoms.The lipophilic group, in one embodiment, is an alkyl group, and can be straight chain or branched. In a further embodiment, the alkyl group is substituted at one, two or three carbon atoms. As such, in one embodiment, the LGPCs provided herein are intended to promote glycopeptide clearance from tissue, for example, increased clearance from the lung after local administration via inhalation. As cleavage of the delivered LGPC derivative occurs over a time T1, an effective amount of LGPC derivative can remain at the site of action during T1, or a portion thereof. Cleavage, in one embodiment, is via an esterase. In another embodiment, cleavage occurs in vivo via an amidase. In another embodiment, cleavage occurs in vivo via a protease such as a peptidase. Importantly, the compounds provided herein would not be considered prodrugs, even though they each contain a labile moiety. Rather, the uncleaved LGPCs provided herein are more active than their cleaved metabolite. In one embodiment, the LGPC derivative provided herein has a shorter T1/2than a counterpart uncleavable lipophilic derivatized glycopeptide. In one embodiment, the T1/2of the LGPC is about 5-75% of the T1/2of the uncleavable lipophilic derivatized glycopeptide, including about 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 15-20%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, or 70-75% of the T1/2of the uncleavable lipophilic derivatized glycopeptide. In one embodiment, the LGPC derivative provided herein has a faster clearance rate from the site of administration than a counterpart uncleavable lipophilic derivatized glycopeptide. In one embodiment, the clearance rate of the LGPC is about 5-75% of the clearance rate of the uncleavable lipophilic derivatized glycopeptide, including about 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 15-20%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, or 70-75% of the clearance rate of the uncleavable lipophilic derivatized glycopeptide. In one embodiment, the LGPC derivative provided herein has a minimum inhibitory concentration (MIC) against a particular bacterium that is lower than its cleaved metabolite. In particular embodiments the MIC of the LGPC is about 5-75% of the MIC of the cleaved metabolite, including about 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 15-20%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, or 70-75% of the MIC of the cleaved glycopeptide. In certain embodiments, the bacterium is a Gram-positive bacterium. In a further embodiment, the bacterium is methicillin-resistant Staphylococcus aureus (MRSA). In the methods provided herein, the bacterial infection can comprise planktonic bacteria, bacterial biofilm, or a combination thereof. One or more compounds provided herein, e.g., a LGPC of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, is delivered to a patient in need of treatment of the bacterial infection. In one embodiment, the bacterial infection is a pulmonary bacterial infection and the composition is administered via the pulmonary route (e.g., inhalation). “Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable addition salt refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (e.g., as lactate), lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, acetic acid (e.g., as acetate), tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid (TFA), undecylenic acid, and the like. In one embodiment, the pharmaceutically acceptable salt is HCl, TFA, lactate or acetate. A pharmaceutically acceptable base addition salt retains the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Inorganic salts include the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Organic bases that can be used to form a pharmaceutically acceptable salt include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. In one aspect, the present invention relates to methods for treating bacterial infections, for example, Gram-positive bacterial infections, and diseases associated with the same. In one embodiment, the Gram-positive bacterial infection is a pulmonary infection. In one embodiment, the infection is a bacterial biofilm infection. The method, in one embodiment, comprises administering to a patient in need thereof, a composition comprising an effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof. The composition can be administered by any route. In the case of a pulmonary infection, in one embodiment, the composition is administered via a nebulizer, dry powder inhaler or a metered dose inhaler. In one aspect of the present invention, an LGPC derivative of Formula (I) or (II), or a pharmaceutically acceptable salt, is provided. The LGPC derivatives of the present invention include a biologically-labile moiety (e.g., amide, ester) that is conjugated to a glycopeptide via an amine group, e.g., a primary amine, on the glycopeptide. Upon administration, the biologically-labile moiety undergoes cleavage (e.g., via hydrolysis or enzymatic cleavage), providing one or more glycopeptide metabolites. In some embodiments, the metabolite provides a decreased residence time in the lungs compared to the unmetabolized compounds, thereby assisting in elimination of the therapeutic agent from the organ (e.g., lung in the case of pulmonary administration). The compounds and formula described herein are set forth graphically without depicting stereochemistry. However, one of ordinary skill in the art will understand that the LGPC derivatives described herein each have a stereochemical configuration. In some embodiments, a stereoisomer (e.g., enantiomer, diastereomer) or a combination of stereoisomers of the respective LGPC derivative are provided. In one embodiment, the present invention is directed to a compound of Formula (I), or a pharmaceutically acceptable salt thereof: Glycopeptide-R1(I) R1is conjugated to the Glycopeptide at a primary amine group of the Glycopeptide; R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3; —(CH2)n1—C(O)—NH—(CH2)n2—CH3; —C(O)—(CH2)n2—CH3; —(CH2)n1—NH—C(O)—(CH2)n2—CH3; —(CH2)n1—O—C(O)—(CH2)n2—CH3; —(CH2)n1—O—C(O)—NH—(CH2)n2—CH3; —(CH2)n1—O—(CO)—O—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—O—(CH2)n2—CH3or n1 is 1, 2, 3,4 or 5; and n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, the Glycopeptide is vancomycin, telavancin, chloroeremomycin or decaplanin. In a further embodiment, the Glycopeptide is telavancin, chloroeremomycin or decaplanin. The structures of hundreds of natural and semisynthetic glycopeptides have been determined. These structures are highly related and fall within five structural subtypes, I-V, and the present invention is not limited to a particular subtype, so long as the glycopeptide includes a primary amine group to conjugate the R1group. Of the varying structural subtypes, type I structures contain aliphatic chains, whereas types II, III, and IV include aromatic side chains within these amino acids. Unlike types I and II, types III and IV contain an extra F—O—G ring system. Type IV compounds have, in addition, a long fatty-acid chain attached to the sugar moiety. Structures of type V, such as complestatin, chloropeptin I, and kistamincin A and B, contain the characteristic tryptophan moiety linked to the central amino acid. In one embodiment, one of the glycopeptides described in PCT publication no. WO 2014/085526, the disclosure of which is incorporated by reference herein for all purposes, can be used as the glycopeptide set forth in Formula (I). In one embodiment of Formula (I), the Glycopeptide is A477, A35512, A40926, A41030, A42867, A47934, A80407, A82846, A83850, A84575, AB-65, actaplanin, actinoidin, ardacin, avoparcin, azureomycin, chloroorienticin chloropolysporin, chloroeremomycin, decaplanin, N-demethylvancomycin, eremomycin, galacardin, helvecardin A, helvecardin B, izupeptin, kibdelin, LL-AM374, mannopeptin, MM45289, MM47761 , MM47766. MM55266, MM55270, OA-7653, orienticin, parvodicin, ristocetin, ristomycin, synmonicin, teicoplanin, telavancin, UK-68597, UK-69542, UK- 72051, vancomycin, or a pharmaceutically acceptable salt of one of the foregoing. In one embodiment of Formula (I), the Glycopeptide is vancomycin. In one embodiment of Formula (I), the Glycopeptide is telavancin. In one embodiment of Formula (I), the Glycopeptide is chloroeremomycin. In one embodiment of Formula (I), the Glycopeptide is decaplanin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n1 is 2 and n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, n1 is 1, 2 or 3; and n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n1 is 2 and n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n1 is 2 and n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, n1 is 1, 2 or 3; and n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n1 is 2 and n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n1 is 2 and n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)n2—CH3. In a further embodiment, n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n2 is 10. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In even a further embodiment, n1 is 2 and n2 is 10 or 11. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In even a further embodiment, n1 is 2 and n2 is 10 or 11. a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, n1 is 2 or 3; and n2 is 10, 11, 12 or 13. In even a further embodiment, n1 is 1, 2 or 3 and n2 is 10 or 11. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In even a further embodiment, n1 is 2 and n2 is 10 or 11. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In even a further embodiment, n1 is 2 and n2 is 10 or 11. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin In one embodiment of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)n2—CH3. In a further embodiment, n2 is 10, 11, 12 or 13. In even a further embodiment, n2 is 10 or 11. In a further embodiment, the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In even a further embodiment, the Glycopeptide is vancomycin. In another embodiment, a compound of Formula (II), or a pharmaceutically acceptable salt thereof, is provided: wherein, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3; —(CH2)n1—C(O)—NH—(CH2)n2—CH3; —C(O)—(CH2)n2—CH3; —(CH2)n1—NH—C(O)—(CH2)n2—CH3; —(CH2)n1—O—C(O)—(CH2)n2—CH3; —(CH2)O—O—C(O)—NH—(CH2)n2—CH3; —(CH2)n1—O—(CO)—O—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—O—(CH2)n2—CH3; n1 is 1, 2, 3,4 or 5; n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; R2is OH or NH—(CH2)q—R5; q is 1, 2, 3, 4, or 5; R3is H or R4is H or CH2—NH—CH2—PO3H2; and R5is —N(CH3)2, —N+(CH3)3, —N+(CH3)2(n-C14H29), or In some embodiments of Formula (II), R2is OH. In a further embodiment, R4is H. In some embodiments of Formula (II), R2is OH. In a further embodiment, R4is CH2—NH—CH2—PO3H2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R2is —NH—(CH2)3—R3. In a further embodiment, R3and R4are H. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R2is —NH—(CH2)3—R3. In a further embodiment, R4is CH2—NH—CH2—PO3H2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R2is —NH—(CH2)q—R5. In a further embodiment, R2is —NH—(CH2)3—N(CH3)2. In another embodiment, R2is —NH—(CH2)3—N+(CH3)3. In yet other embodiments, R2is —NH—(CH2)3—N+(CH3)2(n-C14H29). In a further embodiment, R2is In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R2is —NH—(CH2)q—N(CH3)2. In another embodiment, R2is —NH—(CH2)q—N+(CH3)3. In another embodiment, R2is —NH—(CH2)q—R5and R5is —N+(CH3)2(n-C14H29). In yet another embodiment, R2is —NH—(CH2)q—R5and R5is In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)2—CH3. In a further embodiment, R2is OH and R3and R4are H. In a further embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is and R4is H. In even a further embodiment, n1 is 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)2—CH3. In a further embodiment, R2is OH, R3is an R4is H. In even a further embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, R1is —(CH2)n1—O—C(O)—(CH2)2—CH3. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)2—CH3. In a further embodiment, R2is OH, R3is H and R4is CH2—NH—CH2—PO3H2. In even a further embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n2 is 10. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3or —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, R1is ˜(CH2)n1—O—C(O)—(CH2)n2—CH3. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3and R4are H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3and R4are H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—O—(CH2)n2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3and R4are H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3is H and R4is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In one embodiment of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, R1is —C(O)—(CH2)2—CH3. In a further embodiment, R2is —NH—(CH2)q—R5, R3is H and R4is H. In even a further embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n2 is 10. In yet even a further embodiment, q is 2 or 3 and R5is N(CH3)2. In yet another embodiment, a compound of Formula (I) or (II) is provided, wherein one or more hydrogen atoms is replaced with a deuterium atom. For example, in one embodiment of a compound of Formula (II), R3or R4is deuterium. The compounds of present disclosure, i.e., the compounds of Formulae (I) and (II) can be prepared according to methods and steps known to those of ordinary skill in the art. For example, the compounds of the present may be prepared according to methods described in U.S. Pat. No. 6,392,012;U.S. Patent Application Publication No. 2017/0152291;U.S. Patent Application Publication No. 2016/0272682, each of which is hereby incorporated by reference in their entirety for all purposes. Methods described in International Publication No. WO 2018/08197, the disclosure of which is incorporated by reference in its entirety, can also be employed. Synthesis schemes are also provided at the Example section, herein. Compositions provided herein can be in the form of a solution, suspension or dry powder. Compositions can be administered by techniques known in the art, including, but not limited to intramuscular, intravenous, intratracheal, intranasal, intraocular, intraperitoneal, subcutaneous, and transdermal routes. In addition, as discussed throughout, the compositions can also be administered via the pulmonary route, e.g., via inhalation with a nebulizer, a metered dose inhaler or a dry powder inhaler. In one embodiment, the composition provided herein comprises a plurality of nanoparticles of the antibiotic of any of Formula (I)-(II) in association with a polymer. The mean diameter of the plurality of nanoparticles, in one embodiment, is from about 50 nm to about 900 nm, for example from about 10 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm or from about 100 nm to about 500 nm. In one embodiment, the plurality of nanoparticles comprise a biodegradable polymer and the glycopeptide antibiotic of Formulae (I)-(II). In a further embodiment, the biodegradable polymer is poly(D,L-lactide), poly(lactic acid) (PLA), poly(D,L-glycolide) (PLG), poly(lactide-co-glycolide) (PLGA), poly-(cyanoacrylate) (PCA), or a combination thereof. In even a further embodiment, the biodegradable polymer is poly(lactic-co-glycolitic acid) (PLGA). Nanoparticle compositions can be prepared according to methods known to those of ordinary skill in the art. For example, coacervation, solvent evaporation, emulsification, in situ polymerization, or a combination thereof can be employed (see, e.g., Soppimath et al. (2001). Journal of Controlled Release 70, pp. 1-20, incorporated by reference herein in its entirety). The amount of polymer in the composition can be adjusted, for example, to adjust the release profile of the compound of Formula (I) or (II) from the composition. In one embodiment, a dry powder composition disclosed in one of U.S. Pat. Nos. 5,874,064, 5,855,913 and/or U.S. Patent Application Publication No. 2008/0160092 is used to formulate one of the glycopeptides of Formula (I) or (II), or a pharmaceutically acceptable salt thereof. The disclosures of U.S. Pat. Nos. 5,874,064; 5,855,913 and U.S. Patent Application Publication No. 2008/0160092 are each incorporated by reference herein in their entireties. In one embodiment, the composition delivered via the methods provided herein are spray dried, hollow and porous particulate compositions. For example, the hollow and porous particulate compositions as disclosed in WO 1999/16419, hereby incorporated in its entirety by reference for all purposes, can be employed. Such particulate compositions comprise particles having a relatively thin porous wall defining a large internal void, although, other void containing or perforated structures are contemplated as well. Compositions delivered via the methods provided herein, in one embodiment, yield powders with bulk densities less than 0.5 g/cm3or 0.3 g/cm3, for example, less 0.1 g/ cm3, or less than 0.05 g/cm3. By providing particles with very low bulk density, the minimum powder mass that can be filled into a unit dose container is reduced, which eliminates the need for carrier particles. Moreover, the elimination of carrier particles, without wishing to be bound by theory, can minimize throat deposition and any “gag” effect, since the large lactose particles can impact the throat and upper airways due to their size. In some embodiments, the particulate compositions delivered via the methods provided herein comprise a structural matrix that exhibits, defines or comprises voids, pores, defects, hollows, spaces, interstitial spaces, apertures, perforations or holes. The particulate compositions in one embodiment, are provided in a “dry” state. That is, the particulate composition possesses a moisture content that allows the powder to remain chemically and physically stable during storage at ambient temperature and easily dispersible. As such, the moisture content of the microparticles is typically less than 6% by weight, and for example, less 3% by weight. In some embodiments, the moisture content is as low as 1% by weight. The moisture content is, at least in part, dictated by the formulation and is controlled by the process conditions employed, e.g., inlet temperature, feed concentration, pump rate, and blowing agent type, concentration and post drying. Reduction in bound water can lead to improvements in the dispersibility and flowability of phospholipid based powders, leading to the potential for highly efficient delivery of powdered lung surfactants or particulate composition comprising active agent dispersed in the phospholipid. The composition administered via the methods provided herein, in one embodiment, is a particulate composition comprising a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, a phospholipid and a polyvalent cation. In particular, the compositions of the present invention can employ polyvalent cations in phospholipid-containing, dispersible particulate compositions for pulmonary administration to the respiratory tract for local or systemic therapy via aerosolization. Without wishing to be bound by theory, it is thought that the use of a polyvalent cation in the form of a hygroscopic salt such as calcium chloride stabilizes a dry powder prone to moisture induced stabilization. Without wishing to be bound by theory, it is thought that such cations intercalate the phospholipid membrane, thereby interacting directly with the negatively charged portion of the zwitterionic headgroup. The result of this interaction is increased dehydration of the headgroup area and condensation of the acyl-chain packing, all of which leads to increased thermodynamic stability of the phospholipids. Other benefits of such dry powder compositions are provided in U.S. Pat. No. 7,442,388, the disclosure of which is incorporated herein in its entirety for all purposes. The polyvalent cation for use in the present invention in one embodiment, is a divalent cation. In a further embodiment, the divalent cation is calcium, magnesium, zinc or iron. The polyvalent cation is present in one embodiment, to increase the Tm of the phospholipid such that the particulate composition exhibits a Tm which is greater than its storage temperature Ts by at least 20° C. The molar ratio of polyvalent cation to phospholipid in one embodiment, is 0.05, e.g., from about 0.05 to about 2.0, or from about 0.25 to about 1.0. In one embodiment, the molar ratio of polyvalent cation to phospholipid is about 0.50. In one embodiment, the polyvalent cation is calcium and is provided as calcium chloride. According to one embodiment, the phospholipid is a saturated phospholipid. In a further embodiment, the saturated phospholipid is a saturated phosphatidylcholine. Acyl chain lengths that can be employed range from about C16to C22. For example, in one embodiment an acyl chain length of 16:0 or 18:0 (i.e., palmitoyl and stearoyl) is employed. In one phospholipid embodiment, a natural or synthetic lung surfactant is provided as the phospholipid component. In this embodiment, the phospholipid can make up to 90 to 99.9% w/w of the lung surfactant. Suitable phospholipids according to this aspect of the invention include natural or synthetic lung surfactants such as those commercially available under the trademarks ExoSurf, InfaSurf® (Ony, Inc.), Survanta, CuroSurf, and ALEC. The Tm of the phospholipid-glycopeptide particles, in one embodiment, is manipulated by varying the amount of polyvalent cations in the composition. Phospholipids from both natural and synthetic sources are compatible with the compositions administered by the methods provided herein, and may be used in varying concentrations to form the structural matrix. Generally compatible phospholipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. The incorporated phospholipids in one embodiment, are relatively long chain (i.e., C16-C22) saturated lipids and in a further embodiment, comprise saturated phospholipids. In even a further embodiment, the saturated phospholipid is a saturated phosphatidylcholine. In even a further embodiment, the saturated phosphatidylcholine has an acyl chain lengths of 16:0 or 18:0 (palmitoyl or stearoyl). Exemplary phospholipids useful in the disclosed stabilized preparations comprise, phosphoglycerides such as dipalmitoylphosphatidylcholine (DPPC), disteroylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine dibehenoylphosphatidylcholine, diphosphatidyl glycerol, short-chain phosphatidylcholines, long-chain saturated phosphatidylethanolamines, long-chain saturated phosphatidylserines, long-chain saturated phosphatidylglycerols, long-chain saturated phosphatidylinositols. In addition to the phospholipid, a co-surfactant or combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the particulate compositions can be used in the compositions delivered via the methods provided herein. By “associated with or comprise” it is meant that the particulate compositions may incorporate, adsorb, absorb, be coated with or be formed by the surfactant. Surfactants include fluorinated and nonfluorinated compounds and can include saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants and combinations thereof. In one embodiment comprising stabilized dispersions, nonfluorinated surfactants are relatively insoluble in the suspension medium. Compatible nonionic detergents suitable as co-surfactants in the compositions provided herein include sorbitan esters including sorbitan trioleate (Span™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) (Brij® S20), sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic® F-68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. The phospholipid-glycopeptide particulate compositions can include additional lipids such as a glycolipid, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; a lipid bearing a polymer chain such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; a lipid bearing sulfonated mono-, di-, and polysaccharides; a fatty acid such as palmitic acid, stearic acid, and/or oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. In addition to the phospholipid and polyvalent cation, the particulate composition delivered via the methods provided herein can also include a biocompatible, and in some embodiments, biodegradable polymer, copolymer, or blend or other combination thereof. The polymer in one embodiment is a polylactide, polylactide-glycolide, cyclodextrin, polyacrylate, methylcellulose, carboxymethylcellulose, polyvinyl alcohol, polyanhydride, polylactam, polyvinyl pyrrolidone, polysaccharide (e.g., dextran, starch, chitin, chitosan), hyaluronic acid, protein (e.g., albumin, collagen, gelatin, etc.). Besides the aforementioned polymer materials and surfactants, other excipients can be added to a particulate composition, for example, to improve particle rigidity, production yield, emitted dose and deposition, shelf-life and/or patient acceptance. Such optional excipients include, but are not limited to: coloring agents, taste masking agents, buffers, hygroscopic agents, antioxidants, and chemical stabilizers. Other excipients may include, but are not limited to, carbohydrates including monosaccharides, disaccharides and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and the like; trisaccharides such as raffinose and the like; and other carbohydrates such as starches (hydroxyethylstarch), cyclodextrins and maltodextrins. Mixtures of carbohydrates and amino acids are further held to be within the scope of the present invention. The inclusion of both inorganic (e.g., sodium chloride), organic acids and their salts (e.g., carboxylic acids and their salts such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffers can also be undertaken. Salts and/or organic solids such as ammonium carbonate, ammonium acetate, ammonium chloride or camphor can also be employed. According to one embodiment, the particulate compositions may be used in the form of dry powders or in the form of stabilized dispersions comprising a non-aqueous phase. The dispersions or powders of the present invention may be used in conjunction with metered dose inhalers (MDIs), dry powder inhalers (DPIs), atomizers, or nebulizers to provide for pulmonary delivery. While several procedures are generally compatible with making certain dry powder compositions described herein, spray drying is a particularly useful method. As is well known, spray drying is a one-step process that converts a liquid feed to a dried particulate form. With respect to pharmaceutical applications, it will be appreciated that spray drying has been used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996, which is incorporated herein by reference in its entirety for all purposes. In general, spray drying consists of bringing together a highly dispersed liquid, and a sufficient volume of hot air to produce evaporation and drying of the liquid droplets. The preparation to be spray dried or feed (or feed stock) can be any solution, suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. In one embodiment, the feed stock comprises a colloidal system such as an emulsion, reverse emulsion, microemulsion, multiple emulsion, particulate dispersion, or slurry. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. It will further be appreciated that spray dryers, and specifically their atomizers, may be modified or customized for specialized applications, e.g., the simultaneous spraying of two solutions using a double nozzle technique. More specifically, a water-in-oil emulsion can be atomized from one nozzle and a solution containing an anti-adherent such as mannitol can be co-atomized from a second nozzle. In one embodiment, it may be desirable to push the feed solution though a custom designed nozzle using a high pressure liquid chromatography (HPLC) pump. Examples of spray drying methods and systems suitable for making the dry powders of the present invention are disclosed in U.S. Pat. Nos. 6,077,543, 6,051,256, 6,001,336, 5,985,248, and 5,976,574, each of which is incorporated in their entirety by reference. While the resulting spray-dried powdered particles typically are approximately spherical in shape, nearly uniform in size and frequently are hollow, there may be some degree of irregularity in shape depending upon the incorporated glycopeptide of Formulae (I)-(II) and the spray drying conditions. In one embodiment, an inflating agent (or blowing agent) is used in the spray-dried powder production, e.g., as disclosed in WO 99/16419, incorporated by reference herein in its entirety for all purposes. Additionally, an emulsion can be included with the inflating agent as the disperse or continuous phase. The inflating agent can be dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 PSI. This process forms an emulsion, and in some embodiments, an emulsion stabilized by an incorporated surfactant, and can comprise submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The blowing agent in one embodiment, is a fluorinated compound (e.g., perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. Other suitable liquid blowing agents include nonfluorinated oils, chloroform, Freons, ethyl acetate, alcohols and hydrocarbons. Nitrogen and carbon dioxide gases are also contemplated as a suitable blowing agent. Perfluorooctyl ethane is the blowing agent, in one embodiment. Whatever components are selected, the first step in particulate production in one embodiment, comprises feed stock preparation. The selected glycopeptide is dissolved in a solvent, for example water, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, ethanol, methanol, or combinations thereof, to produce a concentrated solution. The polyvalent cation may be added to the glycopeptide solution or may be added to the phospholipid emulsion as discussed below. The glycopeptide may also be dispersed directly in the emulsion, particularly in the case of water insoluble agents. Alternatively, the glycopeptide is incorporated in the form of a solid particulate dispersion. The concentration of the glycopeptide used is dependent on the amount of glycopeptide required in the final powder and the performance of the delivery device employed (e.g., the fine particle dose for a MDI or DPI). As needed, cosurfactants such as poloxamer 188 or span 80 may be dispersed into this annex solution. Additionally, excipients such as sugars and starches can also be added. In one embodiment, a polyvalent cation-containing oil-in-water emulsion is then formed in a separate vessel. The oil employed in one embodiment, is a fluorocarbon (e.g., perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin) which is emulsified with a phospholipid. For example, polyvalent cation and phospholipid may be homogenized in hot distilled water (e.g., 60° C.) using a suitable high shear mechanical mixer (e.g., Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5 minutes. In one embodiment, 5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting polyvalent cation-containing perfluorocarbon in water emulsion is then processed using a high pressure homogenizer to reduce the particle size. In one embodiment, the emulsion is processed at 12,000 to 18,000 PSI, 5 discrete passes and kept at 50 to 80° C. The glycopeptide solution (or suspension) and perfluorocarbon emulsion are then combined and fed into the spray dryer. In one embodiment, the two preparations are miscible. While the glycopeptide is solubilized separately for the purposes of the instant discussion it will be appreciated that, in other embodiments, the glycopeptide may be solubilized (or dispersed) directly in the emulsion. In such cases, the glycopeptide emulsion is simply spray dried without combining a separate glycopeptide preparation. Operating conditions such as inlet and outlet temperature, feed rate, atomization pressure, flow rate of the drying air, and nozzle configuration can be adjusted in accordance with the manufacturer's guidelines in order to produce the desired particle size, and production yield of the resulting dry particles. The selection of appropriate apparatus and processing conditions are well within the purview of a skilled artisan. In one embodiment, the particulate composition comprises hollow, porous spray dried micro- or nano-particles. Along with spray drying, particulate compositions useful in the present invention may be formed by lyophilization. Those skilled in the art will appreciate that lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. Methods for providing lyophilized particulates are known to those of skill in the art. The lyophilized cake containing a fine foam-like structure can be micronized using techniques known in the art. Besides the aforementioned techniques, the glycopeptide particulate compositions or glycopeptide particles provided herein may also be formed using a method where a feed solution (either emulsion or aqueous) containing wall forming agents is rapidly added to a reservoir of heated oil (e.g., perflubron or other high boiling FCs) under reduced pressure. The water and volatile solvents of the feed solution rapidly boils and are evaporated. This process provides a perforated structure from the wall forming agents similar to puffed rice or popcorn. In one embodiment, the wall forming agents are insoluble in the heated oil. The resulting particles can then be separated from the heated oil using a filtering technique and subsequently dried under vacuum. In another embodiment, the particulate compositions of the present invention may also be formed using a double emulsion method. In the double emulsion method, the medicament is first dispersed in a polymer dissolved in an organic solvent (e.g., methylene chloride, ethyl acetate) by sonication or homogenization. This primary emulsion is then stabilized by forming a multiple emulsion in a continuous aqueous phase containing an emulsifier such as polyvinylalcohol. Evaporation or extraction using conventional techniques and apparatus then removes the organic solvent. The resulting particles are washed, filtered and dried prior to combining them with an appropriate suspension medium. In order to maximize dispersibility, dispersion stability and optimize distribution upon administration, the mean geometric particle size of the particulate compositions in one embodiment, is from about 0.5-50 μm, for example from about 0.5 μm to about 10 μm or from about 0.5 to about 5μm. In one embodiment, the mean geometric particle size (or diameter) of the particulate compositions is less than 20 μm or less than 10 μm. In a further embodiment, the mean geometric diameter is ≤about 7 μm or ≤5 μm. In even a further embodiment, the mass geometric diameter is ≤about 2.5 μm. In one embodiment, the particulate composition comprises a powder of dry, hollow, porous spherical shells of from about 0.1 to about 10 μm, e.g., from about 0.5 to about 5 μm in diameter, with shell thicknesses of approximately 0.1 μm to approximately 0.5 μm. Methods for treating infectious diseases, especially those caused by Gram-positive microorganisms, are provided. The method comprises, in one embodiment, administering to a patient in need of treatment, a composition comprising an effective amount of an LGPC derivative, or a pharmaceutically acceptable salt thereof. The LGPC derivative, contains a primary amino conjugated lipophilic moiety that is cleavable by enzymatic hydrolysis. The lipophilic moiety is conjugated to the primary amino group via a functional group that is capable of undergoing enzymatic hydrolysis. The functional group that undergoes enzymatic hydrolysis, in one embodiment, in conjugated to the primary amino group via a straight chain or branched alkyl group, e.g., a methyl, ethyl, propyl or butyl group. In another embodiment, the functional group is an amide that comprises the nitrogen atom from the primary amino group of the glycopeptide. The method comprises, in one embodiment, administering the composition comprising the LGPC derivative to the patient in need of treatment via inhalation. In one embodiment of the methods provided herein, a composition comprising an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt of one of the foregoing, is administered to a patient in need of treatment. Without wishing to be bound by a particular theory, it is believed that the R1groups conjugated to the glycopeptides provided herein facilitate cellular uptake of the glycopeptide at the site of infection, for example, macrophage uptake. An “effective amount” of a compound of Formula (I) or (II) or a pharmaceutically acceptable salt of Formula (I) or (II), is an amount that can provide the desired therapeutic response. The effective amount can refer to a single dose as part of multiple doses during an administration period, or as the total dosage of the LGPC given during an administration period. A treatment regimen can include substantially the same dose for each LGPC administration, or can comprise at least one, at least two or at least three different dosages. According to one embodiment, a method is provided to treat an infection due to a Gram-positive bacterium, including, but not limited to, generaStaphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebaclerium, Nocardia, Clostridium,andListeria.In one embodiment, the infection is due to a Gram-positive cocci bacterium. In a further embodiment, the Gram-positive cocci infection is aStaphylococcus, EnterococcusorStreptococcusinfection. The bacterial infection treated by the methods provided herein may be present as planktonic free-floating bacteria, a biofilm, or a combination thereof. In one embodiment, the infection treated with the methods provided herein is a pulmonary infection. In one embodiment, the bacterial infection is a Gram-positive bacterial infection. In a further embodiment, the bacterial infection is a pulmonary Gram-positive bacterial infection. In one embodiment, the Gram-positive bacterial infection is a Gram-positive cocci infection. In a further embodiment, the Gram-positive cocci infection is aStreptococccus, Enterococcusor aStaphylococcusinfection. Over the past few decades, there has been a decrease in the susceptibility of Gram-positive cocci to antibacterials for the treatment of infection. See, e.g., Alvarez-Lerma et al. (2006) Drugs 66, pp. 751-768, incorporated by reference herein in its entirety for all purposes. As such, in one aspect, the present invention addresses this need by providing a composition comprising an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, in a method for treating a patient in need thereof for a Gram-positive cocci infection that is resistant to a different antibacterial. For example, in one embodiment, the Gram-positive cocci infection is a penicillin resistant or a vancomycin resistant bacterial infection. In a further embodiment, the resistant bacterial infection is a methicillin-resistantStaphylococcusinfection, e.g., methicillin-resistantS. aureusor a methicillin-resistantStaphylococcus epidermidisinfection. In another embodiment, the resistant bacterial infection is an oxacillin-resistantStaphylococcus(e.g.,S. aureus) infection, a vancomycin-resistantEnterococcusinfection or a penicillin-resistantStreptococcus(e.g.,S. pneumoniae) infection. In yet another embodiment, the Gram-positive cocci infection is a vancomycin-resistant enterococci (VRE), methicillin-resistantStaphylococcus aureus(MRSA), methicillin-resistantStaphylococcus epidermidis(MRSE), vancomycin resistantEnterococcus faeciumalso resistant to teicoplanin (VRE Fm Van A), vancomycin resistantEnterococcus faeciumsensitive to teicoplanin (VRE Fm Van B), vancomycin resistantEnterococcus faecalisalso resistant to teicoplanin (VRE Fs Van A), vancomycin resistantEnterococcus faecalissensitive to teicoplanin (VRE Fs Van B), or penicillin-resistantStreptococcus pneumoniae(PSRP). According to one embodiment, a method is provided for treating a bacterial infection comprising administering a composition comprising an effective amount of a compound of Formula (I) or (II), or a pharmaceutically-acceptable salt thereof, to the patient. For example, the composition can be administered to the patient via pulmonary administration or via parenteral administration (e.g., intravenous). As provided herein, LGPC derivatives of Formulae (I) and (II) are provided. Such compounds are useful in the treatment of bacterial infections, including, but not limited to, pulmonary infections, and specifically, pulmonary infections caused by Gram-positive bacteria. The LGPC derivatives provided herein possess a biologically-labile moiety (e.g., amide, ester) connected via an amine group of the glycopeptide, e.g., a primary amine. Subsequent to administration, the biologically-labile moiety undergoes cleavage by any available mechanism (e.g., hydrolysis or enzymatic cleavage), providing one or more glycopeptide metabolites. In some embodiments, the glycopeptide metabolite provides a decreased residence time in the lungs compared to the unmetabolized glycopeptide compound, thereby assisting in elimination of the therapeutic agent from this organ. In one embodiment, the compound of Formula (I) or (II), and its respective metabolite, provide a synergistic effect against the bacterial infection being treated. Metabolites of LGPC derivatives of Formula (I) (or a pharmaceutically acceptable salt thereof), in one embodiment, have the following structures (Glycopeptide, R1, n1 and n2 as defined above).Glycopeptide-(CH2)n1—OH (a metabolite of a compound of Formula (I), where R1is —(CH2)n1—O—C(O)—(CH2)n2—CH3; —(CH2)n1—O—C(O)—O—(CH2)n2—CH3; or —(CH2)n1—O—C(O)—NH—(CH2)n2—CH3)Glycopeptide-(CH2)n1—NH2(metabolite of a compound of Formula (I), where R1is —(CH2)n1—NH—C(O)—(CH2)n2—CH3; or —(CH2)n1—NH—C(O)—O—(CH2)2—CH3)Glycopeptide—(CH2)n1—C(O)OH (a metabolite of a compound of Formula (I), where R1is —(CH2)n1—C(O)—NH—(CH2)n2—CH3or —(CH2)n1—C(O)—O—(CH2)n2—CH3) Metabolites of LGPC derivatives of Formula (II) (or a pharmaceutically acceptable salt thereof), have the following structures (R1, R2, R3, R4, n1 and n2 defined above): In one embodiment, a Gram-positive cocci infection is treated with one of the methods provided herein. In a further embodiment, the Gram-positive cocci infection is aStaphylococcusinfection.Staphylococcusis Gram-positive non-motile bacteria that colonizes skin and mucus membranes.Staphylococciare spherical and occur in microscopic clusters resembling grapes. The natural habitat ofStaphylococcusis nose; it can be isolated in 50% of normal individuals. 20% of people are skin carriers and 10% of people harborStaphylococcusin their intestines. Examples ofStaphylococciinfections treatable with the methods and compositions provided herein, includeS. aureus, S. epidermidis, S. auricularis, S. carnosus, S. haemolyticus, S. hyicus, S. intermedius, S. lugdunensis, S. saprophytics, S. sciuri, S. simulans,andS. warneri.In one embodiment, theStaphylococcusinfection is aStaphylococcus aureus(S. aureus) infection. While there have been about 20 species ofStaphylococcusreported, onlyStaphylococcus aureusandStaphylococcus epidermisare known to be significant in their interactions with humans. In one embodiment, theStaphylococcusinfection is aStaphylococcus haemolyticus(S. haemolyticus) infection. In another embodiment, theStaphylococcusinfection is aStaphylococcus epidermis(S. epidermis) infection. AStaphylococcusinfection, e.g.,S. aureusis treated in one embodiment, in a patient that has been diagnosed with mechanical ventilation-associated pneumonia. In one embodiment, theS. aureusinfection is a methicillin-resistantStaphylococcus aureus(MRSA) infection. In another embodiment, theS. aureusinfection is a methicillin-sensitiveS. aureus(MSSA) infection. In another embodiment, theS. aureusinfection is aS. aureus(VISA) infection, or a vancomycin-resistantS. aureus(VRSA) infection. In one embodiment, theStaphylococcusspecies is resistant to a penicillin such as methicillin. In a further embodiment, theStaphylococcusspecies is methicillin-resistantStaphylococcus aureus(MRSA) or methicillin-resistantStaphylococcusepidermidis (MRSE). TheStaphylococcusspecies, in another embodiment, is methicillin-sensitiveS. aureus(MSSA), vancomycin-intermediateS. aureus(VISA), or vancomycin-resistantS. aureus(VRSA). S. aureuscolonizes mainly the nasal passages, but it may be found regularly in most anatomical locales, including skin oral cavity, and gastrointestinal tract. In one embodiment, aS. aureusinfection is treated with one of the methods and/or compositions provided herein. TheS. aureusinfection can be a healthcare associated, i.e., acquired in a hospital or other healthcare setting, or community-acquired. In one embodiment, theStaphylococcalinfection treated with one of the methods and /or compositions provided herein, causes endocarditis or septicemia (sepsis). As such, the patient in need of treatment with one of the methods and/or compositions provided herein, in one embodiment, is an endocarditis patient. In another embodiment, the patient is a septicemia (sepsis) patient. In one embodiment, the bacterial infection is erythromycin-resistant (ermR), vancomycin-intermediateS. aureus(VISA) heterogeneous vancomycin-intermediateS. aureus(hVISA),S. epidermidiscoagulase-negative staphylococci (CoNS), penicillin-intermediateS. pneumoniae(PISP), or penicillin-resistantS. pneumoniae(PRSP). In even a further embodiment, the administering comprises administering via inhalation. In one embodiment, the Gram-positive cocci infection is aStreptococcusinfection.Streptococciare Gram-positive, non-motile cocci that divide in one plane, producing chains of cells. The primary pathogens includeS. pyrogenesandS. pneumoniaebut other species can be opportunistic.S. pyrogenesis the leading cause of bacterial pharyngitis and tonsillitis. It can also produce sinusitis, otitis, arthritis, and bone infections. Some strains prefer skin, producing either superficial (impetigo) or deep (cellulitis) infections.Streptoccocus pnemoniaeis treated, in one embodiment, in a patient that has been diagnosed with community-acquired pneumonia or purulent meningitis. S. pneumoniaeis the major cause of bacterial pneumonia in adults, and in one embodiment, an infection due toS. pneumoniaeis treated via one of the methods and/or compositions provided herein. Its virulence is dictated by its capsule. Toxins produced by streptococci include: streptolysins (S & O), NADase, hyaluronidase, streptokinase, DNAses, erythrogenic toxin (which causes scarlet fever rash by producing damage to blood vessels; requires that bacterial cells are lysogenized by phage that encodes toxin). Examples of Streptococcus infections treatable with the compositions and methods provided herein include,S. agalactiae, S. anginosus, S. bovis, S. canis, S. constellatus, S. dysgalactiae, S. equi, S. equinus, S. Mae, S. intermedins, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. peroris, S. pneumoniae, S. pyogenes, S. ratti, S. salivarius, S. salivariusssp.thermophilics, S. sanguinis, S. sobrinus, S. suis, S. uteris, S. vestibularis, S. viridans,andS. zooepidemicus. In one embodiment, the Streptococcus infection is aS. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. suis, S. mitis, S. pneumoniae,or aS. mutansinfection. In another embodiment, theStreptococcusinfection is aS. mutansinfection. In still another embodiment, theStreptococcusinfection is aS. pneumoniaeinfection. In yet another embodiment, the theStreptococcusinfection is aS. dysgalactiaeinfection. In a further embodiment, theStreptococcusinfection is aS. pyogenesinfection. In one embodiment, the Gram-positive cocci infection is anEnterococcusinfection. In another embodiment, theEnterococcusinfection is a vancomycin resistant infection (VRE). In a further embodiment, theEnterococcusinfection is a vancomycin sensitive infection (VSE). The genusEnterococciconsists of Gram-positive, facultatively anaerobic organisms that are ovoid in shape and appear on smear in short chains, in pairs, or as single cells.Enterococciare important human pathogens that are increasingly resistant to antimicrobial agents. Examples ofEnterococcitreatable with the methods and compositions provided herein areE. avium, E. durans, E. faecalis, E. faecium, E. gallinarum,andE. solitarius.AnEnterococcusspecies is treated, in one embodiment, in a patient that has been diagnosed with a urinary-catheter related infection. In one embodiment of the methods provided herein, a patient in need thereof is treated for anEnterococcus faecalis(E. faecalis) infection. In a further embodiment, the infection is a pulmonary infection. In another embodiment, a patient in need thereof is treated for anEnterococcus faecium(E. faecium) infection. In a further embodiment, the infection is a pulmonary infection. In one embodiment, a patient in need thereof is treated for anEnterococcusinfection that is resistant or sensitive to vancomycin or resistant or sensitive to penicillin. In a further embodiment, theEnterococcusinfection is anE. faecalisorE. faeciuminfection. In a specific embodiment, theEnterococcusinfection is anEnterococcus faecalis(E. faecalis) infection. In one embodiment, theE. faecalisinfection is a vancomycin-sensitiveE. faecalisinfection. In another embodiment, theE. faecalisinfection is a vancomycin-resistantE. faecalisinfection. In yet another embodiment, theE. faecalisinfection is an ampicillin-resistantE. faecalisinfection. In another embodiment, theEnterococcusinfection is anEnterococcus faecium(E. faecium) infection. In still another embodiment, theE. faeciuminfection is a vancomycin-resistantE. faeciuminfection. In a further embodiment, theE. faeciuminfection is an ampicillin-resistantE. faeciuminfection. In yet a further embodiment, theE. faeciuminfection is a vancomycin-sensitiveE. faeciuminfection. Bacteria of the genusBacillusare aerobic, endospore-forming, Gram-positive rods, and infections due to such bacteria are treatable via the methods and compositions provided herein.Bacillusspecies can be found in soil, air, and water where they are involved in a range of chemical transformations. In one embodiment, a method is provided herein to treat aBacillus anthracis(B. anthracis) infection with a glycopeptide composition.Bacillus anthracis,the infection that causes Anthrax, is acquired via direct contact with infected herbivores or indirectly via their products. The clinical forms include cutaneous anthrax, from handling infected material, intestinal anthrax, from eating infected meat, and pulmonary anthrax from inhaling spore-laden dust. The route of administration of the glycopeptide will vary depending on how the patient acquires the B. anthracis infection. For example, in the case of pulmonary anthrax, the patient, in one embodiment, is treated via a dry powder inhaler, nebulizer or metered dose inhaler. Several otherBacillusspecies, in particular,B. cereus, B. subtilisandB. licheniformis,are associated periodically with bacteremia/septicemia, endocarditis, meningitis, and infections of wounds, the ears, eyes, respiratory tract, urinary tract, and gastrointestinal tract, and are therefore treatable with the methods and compositions provided herein. Examples of pathogenicBacillusspecies whose infection is treatable with the methods and compositions provided herein, include, but are not limited to,B. anthracis, B. cereusandB. coagulans. Corynebacteriaare small, generally non-motile, Gram-positive, non sporalating, pleomorphic bacilli and infections due to these bacteria are treatable via the methods provided herein. Corybacterium diphtheria is the etiological agent of diphtheria, an upper respiratory disease mainly affecting children, and is treatable via the methods and compositions provided herein. Examples of otherCorynebacteriaspecies treatable with the methods and compositions provided herein include Corynebacterium diphtheria, Corynebacterium pseudotuberculosis, Corynebacterium tenuis, Corynebacterium striatum, and Corynebacterium minutissimum. The bacteria of the genusNocardiaare Gram-positive, partially acid-fast rods, which grow slowly in branching chains resembling fungal hyphae. Three species cause nearly all human infections:N. asteroides, N. brasiliensis,andN. caviae,and patients with such infections can be treated with the compositions and methods provided herein. Infection is by inhalation of airborne bacilli from an environmental source (soil or organic material). Other Nocardial species treatable with the methods and compositions provided herein includeN. aerocolonigenes, N. africana, N. argentinensis, N. asteroides, N. blackwellu, N. brasiliensis, N. brevicalena, N. cornea, N. caviae, N. cerradoensis, N. corallina, N. cyriacigeorgica, N. dassonvillei, N. elegans, N. farcinica, N. nigiitansis, N. nova, N. opaca, N. otitidis-cavarium, N. paucivorans, N. pseudobrasiliensis, N. rubra, N. transvelencesis, N. uniformis, N. vaccinii,andN. veterana. Clostridia are spore-forming, Gram-positive anaerobes, and infections due to such bacteria are treatable via the methods and compositions provided herein. In one embodiment, one of the methods provided herein are used to treat aClostridium tetani(C. tetani) infection, the etiological agent of tetanus. In another embodiment, one of the methods provided herein is used to treat aClostridium botidinum(C. botidinum) infection, the etiological agent of botulism. In yet another embodiment, one of the methods provided herein is used to treat aC. perfringensinfection, one of the etiological agents of gas gangrene. OtherClostridiumspecies treatable with the methods and compositions of the present invention, include,C. difficile, C. perfringens,and/orC. sordellii.In one embodiment, the infection to be treated is aC. difficileinfection. Listeria are non-spore-forming, nonbranching Gram-positive rods that occur individually or form short chains. Listeria monocytogenes (L. monocytogenes) is the causative agent of listeriosis, and in one embodiment, a patient infected withL. monocytogenesis treated with one of the methods and compositions provided herein. Examples of Listeria species treatable with the methods and compositions provided herein, includeL. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, L. murrayi,andL. welshimeri. In some embodiments, the methods disclosed herein are useful in treating Gram-negative infections. In one embodiment, the bacterial infection is aBurkholderiainfection. In some embodiments, theBurkholderiainfection is aBurkholderia pseudomallei(B. pseudomallei),B. dolosa, B. fungorum, B. gladioli, B. multivorans, B. vietnamiensis, B. ambifaria, B. andropogonis, B. anthina, B. brasilensis, B. calcdonica, B. caribensisor aB. caryophylliinfection. Burkholderiais a genus of Proteobacteria whose pathogenic members include among other theBurkholderia cepaciacomplex which attacks humans;Burkholderia pseudomallei,causative agent of melioidosis; andBurkholderia cepacia,an important pathogen of pulmonary infections in people with cystic fibrosis. TheBurkholderia(previously part ofPseudomonas) genus name refers to a group of virtually ubiquitous Gram-negative, obligately aerobic, rod-shaped bacteria that are motile by means of single or multiple polar flagella, with the exception ofBurkholderia malleiwhich is nonmotile. In other embodiment, the bacterial infection is aYersinia pestis(Y. pestis) infection. Yersinia pestis(formerlyPasteurella pestis) is a Gram-negative, rod-shaped coccobacillus, non-mobile with no spores. It is a facultative anaerobic organism that can infect humans via the oriental rat flea. It causes the disease plague, which takes three main forms: pneumonic, septicemic, and bubonic plagues. In yet another embodiment, the bacterial infection is aFrancisella tularensis(F. tularensis) infection.Francisella tularensisis a pathogenic species of Gram-negative, rod-shaped coccobacillus, an aerobe bacterium. It is non-spore forming, non-motile and the causative agent of tularemia, the pneumonic form of which is often lethal without treatment. It is a fastidious, facultative intracellular bacterium which requires cysteine for growth. The bacterial infection in one embodiment, is a respiratory tract infection. In a further embodiment, the infection is a resistant bacterial infection, for example, one of the infections provided above. The patient treatable by the methods and compositions provided herein, in one embodiment, has been diagnosed with a community-acquired respiratory tract infection, for example, pneumonia. In one embodiment, the bacterial infection treated in the pneumonia patient is aS. pneumoniaeinfection. In another embodiment, the bacterial infection treated in the pneumonia patient isMycoplasma pneumoniaor aLegionellaspecies. In another embodiment, the bacterial infection in the pneumonia patient is penicillin resistant, e.g., penicillin-resistantS. pneumoniae. The bacterial infection, in one embodiment, is a hospital acquired infection (HAI), or acquired in another health care facility, e.g., a nursing home, rehabilitation facility, outpatient clinic, etc. Such infections are also referred to as nosocomial infections. In a further embodiment, the bacterial infection is a respiratory tract infection or a skin infection. In one embodiment, the HAI is pneumonia. In a further embodiment, the pneumonia is due toS. aureus,e.g., MRSA. Respiratory infections and in particular pulmonary infections are quite problematic for patients afflicted with cystic fibrosis (CF). In fact, such infections are the main cause of pulmonary deterioration in this population of patients. The lungs of CF patients are colonized and infected by bacteria from an early age. These bacteria thrive in the altered mucus, which collects in the small airways of the lungs. The formation of biofilms makes infections of this origin difficult to treat. Consequently, more robust treatments options are needed. Thus, in one embodiment, the methods disclosed herein are useful in treating a patient with cystic fibrosis having a bacterial infection. In some embodiments, the bacterial infection is a pulmonary infection. In other embodiments, the pulmonary infection is comprised of a biofilm. With respect to pulmonary infections, the compounds and compositions provided herein can be delivered to a patient in need of treated via an inhalation delivery device that provides local administration to the site of infection. The inhalation delivery device employed in embodiments of the methods provided herein can be a nebulizer, dry powder inhaler (DPI), or a metered dose inhaler (MDI), or any other suitable inhalation delivery device known to one of ordinary skill in the art. The device can contain and be used to deliver a single dose of the composition or the device can contain and be used to deliver multi-doses of the composition of the present invention. According to one embodiment, a dry powder particulate composition is delivered to a patient in need thereof via a metered dose inhaler (MDI), dry powder inhaler (DPI), atomizer, nebulizer or liquid dose instillation (LDI) technique to provide for glycopeptide delivery. With respect to inhalation therapies, those skilled in the art will appreciate that where a hollow and porous microparticle composition is employed, the composition is particularly amenable for delivery via a DPI. Conventional DPIs comprise powdered formulations and devices where a predetermined dose of medicament, either alone or in a blend with lactose carrier particles, is delivered as an aerosol of dry powder for inhalation. The medicament is formulated in a way such that it readily disperses into discrete particles with an MMD between 0.5 to 20 μm, for example from 0.5-5 μm, and are further characterized by an aerosol particle size distribution less than about 10 μm mass median aerodynamic diameter (MMAD), and in some embodiments, less than 5.0 μm. The MMAD of the powders will characteristically range from about 0.5-10 μm, from about 0.5-5.0 μm, or from about 0.5 -4.0 μm. The powder is actuated either by inspiration or by some external delivery force, such as pressurized air. Examples of DPIs suitable for administration of the particulate compositions of the present invention are disclosed in U.S. Pat. Nos. 5,740,794, 5,785,049, 5,673,686, and 4,995,385 and PCT application Nos. 00/72904, 00/21594, and 01/00263, the disclosure of each of which is incorporated by reference in their entireties for all purposes. DPI formulations are typically packaged in single dose units such as those disclosed in the aforementioned patents or they employ reservoir systems capable of metering multiple doses with manual transfer of the dose to the device. The compositions disclosed herein may also be administered to the nasal or pulmonary air passages of a patient via aerosolization, such as with a metered dose inhaler (MDI). Breath activated MDIs are also compatible with the methods provided herein. Along with the aforementioned embodiments, the compositions disclosed herein may be delivered to a patient in need thereof via a nebulizer, e.g., a nebulizer disclosed in PCT WO 99/16420, the disclosure of which is hereby incorporated in its entirety by reference, in order to provide an aerosolized medicament that may be administered to the pulmonary air passages of the patient. A nebulizer type inhalation delivery device can contain the compositions of the present invention as a solution, usually aqueous, or a suspension. For example, the prostacyclin compound or composition can be suspended in saline and loaded into the inhalation delivery device. In generating the nebulized spray of the compositions for inhalation, the nebulizer delivery device may be driven ultrasonically, by compressed air, by other gases, electronically or mechanically (e.g., vibrating mesh or aperture plate). Vibrating mesh nebulizers generate fine particle, low velocity aerosol, and nebulize therapeutic solutions and suspensions at a faster rate than conventional jet or ultrasonic nebulizers. Accordingly, the duration of treatment can be shortened with a vibrating mesh nebulizer, as compared to a jet or ultrasonic nebulizer. Vibrating mesh nebulizers amenable for use with the methods described herein include the Philips Respironics I-Neb®, the Omron MicroAir, the Nektar Aeroneb®, and the Pari eFlow®. The nebulizer may be portable and hand held in design, and may be equipped with a self contained electrical unit. The nebulizer device may comprise a nozzle that has two coincident outlet channels of defined aperture size through which the liquid formulation can be accelerated. This results in impaction of the two streams and atomization of the formulation. The nebulizer may use a mechanical actuator to force the liquid formulation through a multiorifice nozzle of defined aperture size(s) to produce an aerosol of the formulation for inhalation. In the design of single dose nebulizers, blister packs containing single doses of the formulation may be employed. In the present invention, the nebulizer may be employed to ensure the sizing of particles is optimal for positioning of the particle within, for example, the pulmonary membrane. Upon nebulization, the nebulized composition (also referred to as “aerosolized composition”) is in the form of aerosolized particles. The aerosolized composition can be characterized by the particle size of the aerosol, for example, by measuring the “mass median aerodynamic diameter” or “fine particle fraction” associated with the aerosolized composition. “Mass median aerodynamic diameter” or “MMAD” is normalized regarding the aerodynamic separation of aqua aerosol droplets and is determined by impactor measurements, e.g., the Andersen Cascade Impactor (ACI) or the Next Generation Impactor (NGI). The gas flow rate, in one embodiment, is 2example8 Liter per minute for the ACI and 15 liters per minute for the NGI. “Geometric standard deviation” or “GSD” is a measure of the spread of an aerodynamic particle size distribution. Low GSDs characterize a narrow droplet size distribution (homogeneously sized droplets), which is advantageous for targeting aerosol to the respiratory system. The average droplet size of the nebulized composition provided herein, in one embodiment is less than 5 μm or about 1 μm to about 5 μm, and has a GSD in a range of 1.0 to 2.2, or about 1.0 to about 2.2, or 1.5 to 2.2, or about 1.5 to about 2.2. “Fine particle fraction” or “FPF,” as used herein, refers to the fraction of the aerosol having a particle size less than 5 μm in diameter, as measured by cascade impaction. FPF is usually expressed as a percentage. In one embodiment, the mass median aerodynamic diameter (MMAD) of the nebulized composition is about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm or about 1 μm to about 2 μm, as measured by the Anderson Cascade Impactor (ACI) or Next Generation Impactor (NGI). In another embodiment, the MMAD of the nebulized composition is about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less, as measured by cascade impaction, for example, by the ACI or NGI. In one embodiment, the MMAD of the aerosol of the pharmaceutical composition is less than about 4.9 μm, less than about 4.5 μm, less than about 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less than about 4.0 μm or less than about 3.5 μm, as measured by cascade impaction. In one embodiment, the MMAD of the aerosol of the pharmaceutical composition is about 1.0 μm to about 5.0 μm, about 2.0 μm to about 4.5 μm, about 2.5 μm to about 4.0 μm, about 3.0 p.m to about 4.0 μm or about 3.5 μm to about 4.5 μm, as measured by cascade impaction (e.g., by the ACI or NGI). In one embodiment, the FPF of the aerosolized composition is greater than or equal to about 50%, as measured by the ACI or NGI, greater than or equal to about 60%, as measured by the ACI or NGI or greater than or equal to about 70%, as measured by the ACI or NGI. In another embodiment, the FPF of the aerosolized composition is about 50% to about 80%, or about 50% to about 70% or about 50% to about 60%, as measured by the NGI or ACI. In one embodiment, a metered dose inhalator (MDI) is employed as the inhalation delivery device for the compositions of the present invention. In a further embodiment, the prostacyclin compound is suspended in a propellant (e.g., hydroflourocarbon) prior to loading into the MDI. The basic structure of the MDI comprises a metering valve, an actuator and a container. A propellant is used to discharge the formulation from the device. The composition may consist of particles of a defined size suspended in the pressurized propellant(s) liquid, or the composition can be in a solution or suspension of pressurized liquid propellant(s). The propellants used are primarily atmospheric friendly hydroflourocarbons (HFCs) such as 134a and 227. The device of the inhalation system may deliver a single dose via, e.g., a blister pack, or it may be multi dose in design. The pressurized metered dose inhalator of the inhalation system can be breath actuated to deliver an accurate dose of the lipid-containing formulation. To insure accuracy of dosing, the delivery of the formulation may be programmed via a microprocessor to occur at a certain point in the inhalation cycle. The MDI may be portable and hand held. In one embodiment, a dry powder inhaler (DPI) is employed as the inhalation delivery device for the compositions of the present invention. In one embodiment, the DPI generates particles having an MMAD of from about 1μm to about 10 μm, or about 1 μm to about 9 μm, or about 1 μm to about 8 μm, or about 1 μm to about 7 μm, or about 1 μm to about 6 μm, or about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm, or about 1 μm to about 2 μm in diameter, as measured by the NGI or ACI. In another embodiment, the DPI generates particles having an MMAD of from about 1 μm to about 10 μm, or about 2 μm to about 10 μm, or about 3 μm to about 10 μm, or about 4 μm to about 10 μm, or about 5 μm to about 10 μm, or about 6 μm to about 10 μm, or about 7 μm to about 10 μm, or about 8μm to about 10 μm, or about 9μm to about 10 μm, as measured by the NGI or ACI. In one embodiment, the MMAD of the particles generated by the DPI is about 1 μm or less, about 9 μm or less, about 8 μm or less, about 7 μm or less, 6 μm or less, 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less, as measured by the NGI or ACI. In one embodiment, each administration comprises 1 to 5 doses (puffs) from a DPI, for example, 1 dose (1 puff), 2 dose (2 puffs), 3 doses (3 puffs), 4 doses (4 puffs) or 5 doses (5 puffs). The DPI, in one embodiment, is small and transportable by the patient. In one embodiment, the MMAD of the particles generated by the DPI is less than about 9.9 μm, less than about 9.5 μm, less than about 9.3 μm, less than about 9.2 μm, less than about 9.1 μm, less than about 9.0 μm, less than about 8.5 μm, less than about 8.3 μm, less than about 8.2 μm, less than about 8.1 μm, less than about 8.0 μm, less than about 7.5 μm, less than about 7.3 μm, less than about 7.2 μm, less than about 7.1 μm, less than about 7.0 μm, less than about 6.5 μm, less than about 6.3 μm, less than about 6.2 μm, less than about 6.1 μm, less than about 6.0 μm, less than about 5.5 μm, less than about 5.3 μm, less than about 5.2 μm, less than about 5.1 μm, less than about 5.0 μm, less than about 4.5 μm, less than about 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less than about 4.0 μm or less than about 3.5 μm, as measured by the NGI or ACI. In one embodiment, the MMAD of the particles generated by the DPI is about 1.0 μm to about 10.0 μm, about 2.0 μm to about 9.5 μm, about 2.5 μm to about 9.0 μm, about 3.0 μm to about 9.0 μm, about 3.5 μm to about 8.5 μm or about 4.0 μm to about 8.0 μm. In one embodiment, the FPF of the prostacyclin particulate composition generated by the DPI is greater than or equal to about 40%, as measured by the ACI or NGI, greater than or equal to about 50%, as measured by the ACI or NGI, greater than or equal to about 60%, as measured by the ACI or NGI, or greater than or equal to about 70%, as measured by the ACI or NGI. In another embodiment, the FPF of the aerosolized composition is about 40% to about 70%, or about 50% to about 70% or about 40% to about 60%, as measured by the NGI or ACI. EXAMPLES The present invention is further illustrated by reference to the following Examples. However, it is noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way. Example 1 Synthesis of LGPC Derivatives Lipo glycopeptide cleavable (LGPC) derivatives were prepared as follows. Reductive Amination To a reactor vessel equipped with temperature control and agitation was added anhydrous DMF and DIPEA. The resulting solution was heated to 65° C. with agitation and vancomycin HCl was added slowly in portions. Heating was continued until all of vancomycin HCl had dissolved (5-10 min). The beige colored solution was allowed to cool to room temperature after which a solution of the desired aldehyde dissolved in DMF was added over 5-10 min. The resulting solution was allowed to stir overnight, typically producing a clear red-yellow solution. MeOH and TFA were introduced and stirring was further continued for at least 2 h. At the end of the stirring period, the imine forming reaction mixture was analyzed by HPLC which was characteristically typical. Borane tert-butylamine complex was added in portions and the reaction mixture was stirred at ambient temperature for an additional 2 h after which an in-process HPLC analysis of the reaction mixture indicated a near quantitative reduction of the intermediate imine group. After the reaction was over, the reaction mixture was purified using reverse phase C18 column chromatography (Phenomenex Luna 10 μM PREP C18(2) 250×21.2 mm column) using gradients of water and acetonitrile, each containing 0.1% (v/v) of TFA. Fractions were evaluated using HPLC and then pertinent fractions containing the target product were pooled together for the isolation of the product via lyophilization. Typical products were isolated as fluffy white solids. The procedure is shown atFIG.1. Aldehyde Preparation Aldehydes used in the reductive amination reaction to form the LGPC can be prepared as set forth below and in Scheme 2. To a reaction equipped with a stir bar was added an alcohol reagent containing an ester or amide bond and a suitable organic solvent (typically DCM or THF). The reaction mixture was stirred for approximately 5 min. to fully dissolve the starting material, at which point sodium bicarbonate and dess-martin periodinane were added to the reaction mixture. The reaction mixture was allowed to stir for 2 hours at which point TLC analysis was used to assess progress. In the instance that a large amount of unreacted starting material was present, an additional aliquot of dess-martin periodinane was added to the reaction mixture and progress was re-assessed after an additional 2 h of stirring. Once the reaction was complete, the reaction mixture was treated with DCM and a solution of 10% sodium thiosulfate saturated with NaHCO3 for 90 min. The reaction mixture was then extracted with the sodium thiosulfate solutions (3×100 mL) and brine (2×100 mL) while retaining the organic layer. The organic layer was dried over Na2SO4, filtered, and solvent was removed under reduced pressure to yield the target aldehyde. The final material was typically used without further purification. However, in some instances, the aldehyde may be purified by either silica gel flash column chromatography or preparatory HPLC. Cleavable Bond Formation (Ester and Amide Coupling Reactions) Depending on the type of LGPC desired, one of the following coupling reactions is chosen to make the alcohol reactant for the aldehyde synthesis reaction. Glycol+Acid chloride (Scheme 1). To a reaction vessel was added the appropriate glycol such as ethylene glycol and a suitable organic solvent such as THF or DCM. Temperature was adjusted to be 0° C. and stirring was initiated. Once the temperature stabilized, triethylamine was added in a single aliquot. Separately, a solution of the appropriate acid chloride such as decanoyl chloride and suitable organic solvent such as THF or DCM was prepared and charged into a dosing apparatus. The acid chloride solution was added drop wise over the course of few hours while stirring at 0° C. The reaction mixture was warmed to 25° C. over a 2 h period and the reaction mixture was allowed to stir for approximately 18 h at which point stirring was stopped. The reaction mixture was filtered to remove a white precipitate that had formed. Solvent was removed under reduced pressure to yield a thick, colorless oil. The crude material was dissolved in EtOAc and washed with saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to yield crude product, typically as a white solid. The crude material was purified using prep-HPLC with a CN column and an isocratic method with 10% isopropyl alcohol as the mobile phase. Pure fractions were combined and solvent was removed to yield the target compound, typically as a white solid. Glycol+Carboxylic Acid+Coupling Reagent (Scheme 2). To a clean vessel was added a suitable organic solvent (typically N,N-Dimethylformamide), DIPEA, the appropriate carboxylic acid such as decanoic acid, an coupling reagent such as HATU or PyBOP, and the appropriate glycol such as ethylene glycol. The vial was vortexed for 30 seconds to help dissolve the compounds. The reaction was allowed to shake overnight at 40° C. and ˜125 rpm. Solvent was removed under reduced pressure and the crude reaction mixture was purified using silica gel flash column chromatography with a gradient method using hexanes, EtOAc, and IPA as the mobile phases. Pure fractions were combined and solvent was removed to yield the target compound, typically as a white solid. Hydroxy Alkyl Halide+Carboxylic Acid (Scheme 3). To a vial was added a suitable organic solvent such as N,N-Dimethylformamide, the appropriate acid chloride such as decanoyl chloride, and a hydroxyl alkyl halide such as 2-iodoethanol. The reaction mixture was then placed in an incubated shaker set at 40° C. and ˜125 rpm where it was left to shake overnight. Solvent was removed under reduced pressure and the residue was subjected to liquid-liquid extraction using H2O (40 mL) and hexanes (3×75 ml). Organic layers were combined and solvent was removed under reduced pressure. The crude material was purified via silica gel flash column chromatography using a gradient method with hexanes and ethylacetate as the mobile phases. Fractions of interest were combined and solvent was removed under reduced pressure to produce the target compound, typically as a thick oil. Alkyl Halide+Hydroxy Acid coupling reaction (Scheme 4). To a vial was added a suitable organic solvent such as N,N-Dimethylformamide, an appropriate hydroxyl acid such as glycolic acid, and an alkyl halide such as 1-lododecane. The reaction mixture was then placed in an incubated shaker set at 40° C. and ˜125 rpm where it was left to shake overnight. Solvent was removed under reduced pressure and the residue was subjected to liquid-liquid extraction using H2O (40 mL) and hexanes (3×75 ml). Organic layers were combined and solvent was removed under reduced pressure. The crude material was purified via silica gel flash column chromatography using a gradient method with hexanes and ethylacetate as the mobile phases. Fractions of interest were combined and solvent was removed under reduced pressure to produce the target compound, typically as a thick oil. Amino alcohol+Acid Chloride (Scheme 5). To a reactor vessel was added the appropriate amino alcohol such as ethanolamine and a suitable organic solvent such as THF or DCM. Temperature was adjusted to be 0° C. and stirring was initiated. Once the temperature stabilized triethylamine was added in a single aliquot. Separately, a solution of the appropriate acid chloride such as decanoyl chloride and suitable organic solvent such as THF or DCM was prepared and charged into a dosing apparatus. The acid chloride solution was added drop wise over the course of few hours while stirring at 0° C. The reaction mixture was warmed to 25° C. over a 2 h period and the reaction mixture was allowed to stir for approximately 18 h at which point stirring was stopped. The reaction mixture was filtered to remove a white precipitate that had formed. Solvent was removed under reduced pressure to yield a thick, colorless oil. The crude material was dissolved in EtOAc and washed with 0.1M HCl, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to yield crude product, typically as a white solid. The crude material was purified using prep-HPLC with a CN column and an isocratic method with 10% isopropyl alcohol as the mobile phase. Pure fractions were combined and solvent was removed to yield the target compound, typically as a white solid. Amino alcohol+Carboxylic Acid+Coupling Reagent coupling reaction (Scheme 6). To a clean vessel was added a suitable organic solvent such as N,N-Dimethylformamide), DIPEA, the appropriate carboxylic acid such as decanoic acid, a coupling reagent such as HATU or PyBOP, and the appropriate amino alcohol such as ethanolamine. The vial was vortexed for 30 seconds to help dissolve the compounds. The reaction was allowed to shake overnight at 40° C. and ˜125 rpm. Solvent was removed under reduced pressure and the crude reaction mixture was purified using silica gel flash column chromatography with a gradient method using hexanes, EtOAc, and IPA as the mobile phases. Pure fractions were combined and solvent was removed to yield the target compound, typically as a white solid. Alkyl amine+Hydroxy Acid+Coupling Reagent coupling reaction (Scheme 7). To a clean vessel was added a suitable organic solvent such as N,N-Dimethylformamide), DIPEA, the appropriate hydroxy acid such as glycolic acid, a coupling reagent such as HATU or PyBOP, and the appropriate alkyl amine such 1-aminodecane. The vial was vortexed for 30 s to help dissolve the compounds. The reaction was allowed to shake overnight at 40° C. and ˜125 rpm. Solvent was removed under reduced pressure and the crude reaction mixture was purified using silica gel flash column chromatography with a gradient method using hexanes, EtOAc, and IPA as the mobile phases. Pure fractions were combined and solvent was removed to yield the target compound, typically as a white solid. Example 2 Synthesis of LGPC Derivative RV65 Ester Bond Coupling (Scheme 8). To a clean 20 mL scintillation vial was added N,N-Dimethylformamide (5 mL, Potassium Carbonate (0.862 g, 6.24 mmol), Lauric acid (0.5 g, 2.5 mmol), and 2-iodo-ethanol (0.43 g, 0.20 mL, 2.5 mmol). The reaction mixture was then placed in an incubated shaker set at 40° C. and ˜125 rpm where it was left to shake overnight. Solvent was removed under reduced pressure and the residue was subjected to liquid-liquid extraction using H2O (40 mL) and hexanes (3×75 ml). Organic layers were combined and solvent was removed under reduced pressure. The crude material was purified via silica gel flash column chromatography using a gradient method with hexanes and ethyl acetate as the mobile phases. Fractions of interest were combined and solvent was removed under reduced pressure to produce the target compound (91.9 mg, 0.38 mmol) as a thick, slightly yellow-tinged oil. Oxidation to Aldehyde To a 20 mL scintillation vial was added 2-hydroxyethyl dodecanoate (0.184 g, 0.753 mmol), dess-martin periodinane (0.639 g, 1.506 mmol), and (S1) Dichloromethane (3.68 mL). The mixture was allowed to stir overnight and reaction progress was monitored via TLC. To the reaction mixture was added 2 mL of sodium thiosulfate (10% in water) and 2 mL of saturated sodium bicarbonate at the same time; at which point a white precipitate formed, the solution turned pink, and a small amount of bubbles were formed. The aqueous layer was washed with DCM (3×25 mL) at which point organic layers were combined, washed with brined, dried over Na2SO4, and filtered. The crude sample was evaporated to dryness under reduced pressure to produce 2-oxoethyl dodecanoate (0.26 g, 1.08 mmol) as a slightly pink-tinged solid. The final material was analyzed by TLC using a 2,4-DNP stain to reveal the presence of an aldehyde. Reductive Amination To a 40 mL vial equipped a stir bar was added anhydrous DMF (20 mL) and DIPEA (0.24 mL). The resulting solution was heated to 65° C. on an incubated shaker and vancomycin HCl (1.0 g, 0.7 mmol) was added slowly in portions. Heating was continued until all of vancomycin HCl had dissolved (5-10 min). The beige colored solution was allowed to cool to room temperature after which a solution of 2-oxoethyl dodecanoate (250 mg, 1.03 mmol) and DMF (5 mL) was added over 5-10 min. The resulting solution was allowed to stir overnight to give a clear red-yellow solution. MeOH (10 mL) and TFA (0.21 mL, 2.8 mmol) were introduced to the reaction mixture producing a small amount of white smoke; the reaction mixture also turned yellow. Stirring was further continued for at least 2 h. At the end of the stirring period, the imine forming reaction mixture was analyzed by HPLC which was characteristically typical. Borane tert-butylamine complex (60 mg, 0.7 mmol) was added in portions and the reaction mixture was stirred at ambient temperature for an additional 2 h after which an in-process HPLC analysis of the reaction mixture indicated a near quantitative reduction of the intermediate imine group. After the reaction was over the reaction mixture is purified using reverse phase C18 column chromatography (Phenomenex Luna 10 uM PREP C18(2) 250×21.2 mm column) using gradients of water and acetonitrile, each containing 0.1% (v/v) of TFA. Fractions were evaluated using HPLC and then pertinent fractions containing RV65 were pooled together for the isolation of the product via lyophilization. The target compound, RV65 (150 mg, 0.09 mmol, 13% overall yield), was obtained as a white solid in >97% purity (by HPLC). Example 3 Synthesis of LGPC Derivative RV62 Coupling (Scheme 11). To a 400 mL reactor vessel equipped with pH monitoring, stirring, temperature control, inert gas, and a dosing apparatus was set up. To the reactor was added ethanolamine (3.461 g, 3.42 mL, 56.66 mmol, 2.1 equiv.) and THF (150 mL, 0.18 M, 25.412 Vols). The temperature was adjusted to be 0° C., stirring was initiated at 500 rpm, and pH monitoring was initiated. Once the temperature stabilized Triethylamine (4.095 g, 5.641 mL, 40.472 mmol, 1.5 equiv.) was added in a single aliquot. Separately, a solution of dodecanoyl chloride (5.903 g, 6.423 mL, 26.981 mmol, 1 equiv.) and THF (50 mL, 0.54 M, 8.471 Vols) was prepared and used to fill the dosing apparatus. The dodecanoyl chloride solution was added drop wise over the course of 5 h while controlling the temperature at 0° C. and the pH to basic conditions. The reaction mixture temperature was warmed to 25° C. over a 2 h period and the reaction mixture was allowed to stir for approximately 18 h at which point stirring was stopped. The reaction mixture was filtered to remove a white precipitate that had formed. Solvent was removed under reduced pressure to yield a thick, colorless oil. The crude material was dissolved in EtOAc (300 mL) and was washed with 0.1M HCl (3×100 mL), saturated NaHCO3(3×100 mL), and brine (3×100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to yield 4.45 g of crude product as a white solid. The crude material was purified using prep-HPLC with a CN column and an isocratic method with 10% isopropyl alcohol as the mobile phase. Pure fractions were combined and solvent was removed to yield the target compound as a white solid (3.15g, 12.94 mmol, 48% yield). Oxidation to Aldehyde To a 40 mL vial equipped with a stir bar was added N-(2-hydroxyethyl)decanamide (1 g, 4.109 mmol, 1 equiv.), dichloromethane (20 mL, 0.205 M, 20 Vols), and THF (10 mL, 0.411 M, 10 Vols). The reaction mixture was stirred for approximately 5 min. to fully dissolve the starting material at which point NaHCO3(0.69 g, 8.217 mmol, 2 equiv.) and dess-martin periodinane (2.178 g, 5.136 mmol, 1.25 equiv.) were added to the reaction mixture. The reaction mixture was allowed to stir for 2 h at which point TLC analysis indicated the reaction had reached completion. The reaction mixture was then treated with and a solution of 10% sodium thiosulfate saturated with NaHCO3 for 90 min. The reaction mixture was then extracted with the sodium thiosulfate solutions (3×100 mL) and brine (2×100 mL) while retaining the organic layer. The organic layer (DCM) was dried over Na2SO4, filtered, and solvent was removed under reduced pressure to yield 673.1 mg (2.79 mmol, 68.9% yield) of the target compound a white solid that was used without further purification. Reductive Amination To a 400 mL reactor vessel equipped with pH monitoring, overhead stirring, temperature control, inert gas, and a dosing apparatus was prepared. To the reactor was added anhydrous DMF (50 mL) and DIPEA (0.694 mL). The resulting solution was heated to 65° C. with stirring and vancomycin HCl (2.9 g, 2.0 mmol) was added slowly in portions. Heating was continued until all of vancomycin HCl had dissolved (5-10 min). The beige colored solution was allowed to cool to 30° C. after which a solution of N-(2-oxoethyl)dodecanamide (673 mg, 2.8 mmol) and DMF was added over 5-10 min. The resulting solution was allowed to stir overnight to give a clear red-yellow solution. MeOH (25 mL) and TFA (0.61 mL, 8 mmol) were introduced and stirring was further continued for at least 2 h. At the end of the stirring period, the imine forming reaction mixture was analyzed by HPLC which was characteristically typical. Borane tert-butylamine complex (173 mg, 2.0 mmol) was added in portions and the reaction mixture was stirred at ambient temperature for an additional 2 h after which an in-process HPLC analysis of the reaction mixture indicated a near quantitative reduction of the intermediate imine group. After the reaction was over the reaction mixture is purified using reverse phase C18 column chromatography (Phenomenex Luna 10 uM PREP C18(2) 250×21.2 mm column) using gradients of water and acetonitrile, each containing 0.1% (v/v) of TFA. Fractions were evaluated using HPLC and then pertinent fractions containing RV62 were pooled together for the isolation of the product via lyophilization. The target compound, RV62 (600 mg, 0.35 mmol, 18% overall yield), was obtained as a white solid in >97% purity (by HPLC). Example 4 Synthesis of LGPC Chloroeremomycin Derivative To a 20 mL scintillation vial equipped with a stir bar was added chloroeremomycin and a solution of copper (II) acetate in MeOH. The reaction mixture was stirred at room temperature until the chloroeremomycin had dissolved. To the reaction mixture was then added the appropriate aldehyde and sodium cyanoborohydride as a 1M solution in THF. The reaction mixture was transferred to an incubated shaker set to 45° C. and reaction progress was monitored by HPLC. In some instances, it was necessary to add an additional aliquot of aldehyde reagent. The reaction mixture was allowed to shake overnight at 45° C. The reaction mixture was cooled to RT and sodium borohydride was added to convert residual aldehyde reagent to the corresponding alcohol. The pH was adjusted to between 7-8 using either acetic acid or 0.1M NaOH and volatile solvents were removed by blowing N2(g) with gentle heat. To the reaction mixture was added acetontrile to precipitate the crude product as an off-white solid. The reaction mixture was centrifuged and the liquid was decanted. The solid was dissolved in 10% MeCN/H2O containing 0.1% phosphoric acid to decomplex the copper at which point the solution briefly turned purple and then took on a yellow tinge. Preparatory HPLC was used to purify final product and LCMS was used to confirm compound identity and purity. A diagram of the reaction is provided below as scheme 14. Example 5 C-terminus Modification of LGPC Derivative To a round bottom flask equipped with a stir bar was added a LPGC derivative, a 1:1 solution of DMF:DMSO, and DIPEA. To the reaction mixture was then added HBTU and the appropriate amine (e.g., 3-(dimethylamino)- 1-propylamine). Reaction progress was monitored by HPLC. Once complete, the reaction was quenched upon addition of 1:1H2O: MeOH. The crude material was then purified using reverse phase C18 preparatory HPLC. Purified fractions were lyophilized to yield the target products, typically as a white fluffy powder in modest yield and high purity. Example 6 Resorcinol-Like Modification of LGPC Derivative. To a round bottom flask equipped with a stir bar was added (Aminomethyl)phosphoic acid, water, and DIPEA. The reaction mixture was allowed to stir for 15 minutes at room temperature. To the reaction mixture was then added acetonitrile and formaldehyde, 37% solution in H2O. The reaction mixture was allowed to stir for an additional 15 min. at which point a LGPC derivative and additional DIPEA were added. Reaction progress was closely monitored using HPLC. Once complete the reaction mixture was purified using reverse phase C18 preparative HPLC. Purified fractions were lyophilized to yield the target product as a white fluffy powder. Example 7 Minimum Inhibitory Concentration (MIC) of Compounds of Formula (II) Compounds of the invention were evaluated for their ability to inhibit bacterial growth in two MRSA strains—MRSA 1556 and MRSA 29213. The minimal inhibitory concentrations MICs are summarized in Table 1. Table 2 provides MIC concentrations for metabolites of the ester and amide compounds, RV80 (metabolite of RV65 ester) and RV82 (metabolite of RV62 amide). MIC values for vancomycin and telavancin are also provided. MIC Testing: Glycopeptide compounds were dissolved in 100% DMSO. In vitro activities were determined using CLSI-guided broth susceptibility testing to measure drug minimum inhibitory concentrations (MICs) of the compounds against the quality control strain ATCC 29213 (MSSA) and the MRSA isolate ATCC BAA-1556. TABLE 1MIC Values,μg/mLMRSAMSSACompoundClass155629213R1R2R3R4RV90Ester0.2500.250—(CH2)2—O—C(O)—(CH2)6—CH3OHHHRV67Ester0.1570.094—(CH2)2—O—C(O)—(CH2)7—CH3OHHHRV54Ester0.1670.146—(CH2)2—O—C(O)—(CH2)8—CH3OHHHRV66Ester0.1250.125—(CH2)2—O—C(O)—(CH2)9—CH3OHHHRV65Ester0.0940.063—(CH2)2—O—C(O)—(CH2)10—CH3OHHHRV88Ester0.1250.125—(CH2)2—O—C(O)—(CH2)12—CH3OHHHRV89Ester1.0000.500—(CH2)2—O—C(O)—(CH2)14—CH3OHHHRV55Ester0.2500.250—(CH2)3—O—C(O)—(CH2)9—CH3OHHHRV93Amide0.1250.125—(CH2)2—NH—C(O)—(CH2)6—CH3OHHHRV60Amide0.0630.063—(CH2)2—NH—C(O)—(CH2)7—CH3OHHHRV56Amide0.0630.063—(CH2)2—NH—C(O)—(CH2)8—CH3OHHHRV61Amide0.0310.031—(CH2)2—NH—C(O)—(CH2)9—CH3OHHHRV62Amide0.0230.023—(CH2)2—NH—C(O)—(CH2)10—CH3OHHHRV92Amide0.0310.023—(CH2)2—NH—C(O)—(CH2)12—CH3OHHHRV91Amide0.2500.188—(CH2)2—NH—C(O)—(CH2)14—CH3OHHHRV94Amide0.0310.031—CH2—C(O)—NH—(CH2)9—CH3OHHHRV95Amide0.0310.031—CH2—C(O)—NH—(CH2)11—CH3OHHHRV72Amide0.50.5—(CH2)2—NH—C(O)—(CH2)9—CH3NH—(CH2)3—N(CH3)2HHRV73Amide0.50.5—(CH2)2—NH—C(O)—(CH2)9—CH3OHHCH2—NH—CH2—PO3H2 TABLE 2MIC Values, μg/mLMRSAMSSACompound155629213Vancomycin11Telavancin0.0630.063RV8011RV8233 MIC values for amide derivatives were lower than the ester derivatives (Table 1). RV62 was found to be about 3x more efficacious than RV65, the ester with the lowest measured MIC. Example 8 Degradation of RV62 and RV65 RV62 and RV65 degradation was determined according to the following procedures. Compounds were dissolved and diluted with 1 mM Tris buffer (pH 6.99) to achieve a concentration of 54 μg/mL (stock solution). 0.5 mL of stock solution was further diluted with acetonitrile to a total volume of 10 mL. The stock solution was incubated at 40° C., with samples withdrawn at 3, 6, 24, and 72 h and tested by HPLC. HPLC method: Samples were injected onto a 100 x 2.1 mm Waters Cortecs HILIC with a particle size of 1.6 μm. The mobile phase consisted of water (0.1% formic acid) and acetonitrile (0.1% formic acid). The analytic method utilized a gradient from 10% water (0.1% formic acid) /90% acetonitrile (0.1% formic acid) to 70% water (0.1% formic acid)/30% acetonitrile (0.1% formic acid). The HPLC instrument was equipped with a UV detector (280 nm). Compounds were identified by mass. FIG.3shows the extent of hydrolysis of RV62 and RV65 at 3, 6, 24, and 72 h. The amount of cleaved glycopeptide was found to increase steadily up to the 24 h time point for RV65. For RV65, between 24 and 72 h, the rate of cleavage appears to plateau, such that at 72 h, the peak area for the cleaved glycopeptide was determined to be about 42% of the total. Example 9 Enzyme Mediated Hydrolysis of LGPC Glycopeptide Ester Derivatives The respective LGPC was dissolved in propanol:TBA:H2O (1:1:1) at ˜3mg/mL, with DSPE-PEG2000 (˜1.5mg/mL), and lactose:leucine (7:3 at ˜20 mg/mL). The solution was flash frozen and lyophilized. The lyophilized cake was suspended in PBS (pH=8.0) at 2 mg/mL LGPC. The LGPC was suspended at 0.5-1 mg/mL in PBS (pH adjusted to 8.0 with NaOH) and placed at 37° C. in the presence and absence of esterase (0.2 U/mL). Aliquots were removed at preselected time intervals of 0, 15, 30, 45, 60, 90 and 120 min. Aliquots (125 μL) were diluted in 500 μL 1:1 acetonitrile (ACN):H2O with 0.1% formic acid to stop enzymatic degradation. Diluted samples were analyzed by HPLC to determine the relative peak area for the parent and the metabolite for each LGPC tested. FIG.4is a graph of percent LGPC degradation as a function of time. Esterase mediated hydrolysis of ester LGPC derivatives is chain length dependent. Example 10 Metabolism of RV62 and RV65 in Rat Plasma RV62 and RV65 were dissolved in 100% DMSO. Stock solutions were diluted using rat plasma to contain less than 1% organic solvent with a final drug concentration of 50 μg/mL. Samples were briefly vortexed and then incubated in a shaker set to 37° C. and 300 rpm. Aliquots were removed at specified time points and store at −80° C. until extraction and analysis. Samples were extracted using a solution of 10% TCA and analyzed using LCMS. The hydrolysis of amide RV62 and ester RV65 incubated in rat plasma was determined (FIG.5). That data shows that RV65 (ester) was metabolized faster in plasma compared to RV62 (amide). For RV65, ˜90% of degradant (RV80) was detected in plasma after only 6 h incubation. In contrast, only 6% of degradant of RV62 (RV82) was detected in plasma even after 24 h incubation. As such, under the test conditions, the ester moiety was found to be more labile than the corresponding amide moiety. Example 11 Pharmacokinetics (PK) of RV62 and its Hydrolysis Product RV82, Given by Nose Only Inhalation in Rats The structures are RV62 and RV82 are provided below. Male Sprague-Dawley rats from Charles River Laboratories weighing between 250 g and 300 g at the start of dosing were used in the study. RV62 solution 5 mg/mL in Bicine buffer 0.8 mg/mL at pH 9.5 was prepared prior to animal dosing. RV62 was administered using Aeroneb nebulizer (Aerogen) which delivers a mass mean aerosol diameter between 2.5 to 4 μm and a range of 0.2-0.4 mL/min of nebulization rate. The volume of material to be nebulized was 6 mL, and the total administration time was ˜20 min. On the day of dosing, the eleven rats were placed into the nose-cone restraint chambers which are connected to a 12-port nose-only inhalation chamber (CH Technologies). The test article was delivered from the nebulizer to the chamber with an airflow of 6 L/min. At the end of the compound exposure, the rats were either returned to their cage or sacrificed at 0.5 h after the end of nebulization which was defined as the immediately post dose (IPD) collection. For the terminal time points, rats were anesthetized with 2% isoflurane inhaled with pure oxygen and blood samples of 2.0 mL were obtained by heart puncture and transferred into a 2.0 mL K2-EDTA tube. The tubes were centrifuged at 4° C. to separate the plasma and aliquoted into three conical tubes and stored at −50° C. Lungs were extracted, weighed, and stored at −50° C. for subsequent analysis of lung drug concentrations. RV62 and RV82 were measured in both blood plasma and the lung by LC-MS/MS method. Results of the study are provided atFIG.6(lung) andFIG.7(blood plasma). All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes. The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. | 120,535 |
11857598 | DETAILED DESCRIPTION Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature. Definitions As used herein, “control” is an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable. As used herein, “biocompatible” or “biocompatibility” refers to the ability of a material to be used by a patient without eliciting an adverse or otherwise inappropriate host response in the patient to the material or a derivative thereof, such as a metabolite, as compared to the host response in a normal or control patient. As used herein, “biodegradable” refers to the ability of a material or compound to be decomposed by bacteria or other living organisms or organic processes. As used herein, “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within±10% of the indicated value, whichever is greater. As used herein, “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. As used herein, “administering” can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. For example a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. As used herein, “preventative” refers to hindering or stopping a disease or condition before it occurs or while the disease or condition is still in the sub-clinical phase. As used herein, “therapeutic” can refer to treating or curing a disease or condition. As used interchangeably herein, “subject,” “individual,” or “patient,” refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term “pet” includes a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term farm animal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like. As used herein, the terms “cancer,” “cancer cells,” “neoplastic cells,” “neoplasia,” “tumor,” and “tumor cells” (used interchangeably) refer to cells which exhibit relatively autonomous growth so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign. A metastatic cell or tissue means that the cell can invade and destroy neighboring body structures. The cancer can be selected from astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brain stem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, ductal cancer, endometrial cancer, ependymoma, Ewing sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer and Wlms tumor. In some embodiments, the cancer is prostate cancer. The terms “guide polynucleotide,” “guide sequence,” or “guide RNA” as used herein refers to any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The degree of complementarity between a guide polynucleotide and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). A guide polynucleotide (also referred to herein as a guide sequence and includes single guide sequences (sgRNA)) can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 90, 100, 110, 112, 115, 120, 130, 140, or more nucleotides in length. The guide polynucleotide can include a nucleotide sequence that is complementary to a target DNA sequence. This portion of the guide sequence can be referred to as the complementary region of the guide RNA. In some contexts, the two are distinguished from one another by calling one the complementary region or target region and the rest of the polynucleotide the guide sequence or trans-activating crRNAln (tracrRNA). The guide sequence can also include one or more miRNA target sequences coupled to the 3′ end of the guide sequence. The guide sequence can include one or more MS2 RNA aptamers incorporated within the portion of the guide strand that is not the complementary portion. As used herein the term guide sequence can include any specially modified guide sequences, including but not limited to those configured for use in synergistic activation mediator (SAM) implemented CRISPR (Nature517, 583-588 (29 Jan. 2015). A guide polynucleotide can be less than about 150, 125, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. In some ebodiments, a guide polynucleotide can be 150 nucleotides or more, including, but not limited to 175, 200, 250, 300, 450, 500 or more. It will also be appreciated that the exact number of nucleotides may be any integer bweteen any of the specific numbers given, for example 1, 4, 123, 36, etc. and are all within the spirit and scope of this disclosure. The ability of a guide polynucleotide to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide polynucleotide to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing binding or rate of cleavage at the target sequence between the test and control guide polynucleotide reactions. Other assays are possible, and will occur to those skilled in the art. A complementary region of the gRNA can be configured to target any DNA region of interest. The complementary region of the gRNA and the gRNA can be designed using a suitable gRNA design tool. Suitable tools are known in the art and are available to the skilled artisan. As such, the constructs described herein are enabled for any desired target DNA so long as it is CRISPR compatible according to the known requirements for CRISPR activation. A guide polynucleotide can be selected to reduce the degree of secondary structure within the guide polynucleotide. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker & Stiegler ((1981)Nucleic Acids Res.9, 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. Gruber et al., (2008)Cell106: 23-24; and Carr & Church (2009)Nature Biotechnol.27: 1151-1162). Homology-directed repair (HDR) refers to a mechanism in cells to repair double-stranded and single stranded DNA breaks. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. (2010)Annu. Rev. Biochem.79: 181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirements between the donor and acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR. Homology-directed repair at nicks (single-stranded breaks) can occur via a mechanism distinct from HDR at double-strand breaks. Error-prone DNA repair refers to mechanisms that can produce mutations at double-strand break sites. The Non-Homologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et al., (2006)DNA Repair5: 1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible. The two ends of one double-strand break are the most prevalent substrates of NHEJ (Kirik et al., (2000)EMBO J.19: 5562-5566), however if two different double-strand breaks occur, the free ends from different breaks can be ligated and result in chromosomal deletions (Siebert & Puchta, (2002)Plant Cell14:1121-1131), or chromosomal translocations between different chromosomes (Pacher et al., (2007)Genetics175: 21-29). It will also be appreciated that CRISPR can also be used to activate specific genes through CRISPR/synergistic activation mediator procedures. These procedures can utilize a guide polynucleotide that incorporates 2 MS2 RNA aptamers at the tetraloop and the stem-loop of the guide RNA such as that described in, but not limited to (Nature517, 583-588 (29 Jan. 2015). The term “operatively linked” as used herein can refer to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operatively linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operatively linked to regulatory sequences in a sense or antisense orientation. In one example, the complementary RNA regions can be operatively linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA. The term “operatively linked” as used herein can also refer to the direct or indirect linkage of any two nucleic acid sequences on a singly nucleic acid fragment such that they are indirectly or directly physically connected on the same nucleic acid fragment. The term “operatively linked” as used herein can also refer to the insertion of a nucleic acid within the 5′ and 3′ end of another nucleic or the direct coupling of a nucleic acid to the 5′ or 3′ end of another nucleic acid. As used herein, “specific binding” can refer to binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferably binds to a single epitope and to no other epitope within the family of proteins. As another non-limiting example, a miRNA can specifically bind preferably to a miRNA target and not to a non-specific nucleic acid sequence or if binding to a non-specific nucleic acid sequence occurs that no change in the expression or function of the non-specific nucleic acid can be observed or detected. As used herein, “differentially expressed,” refers to the differential production of RNA, including but not limited to mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA transcribed from a gene or regulatory region of a genome or the protein product encoded by a gene as compared to the level of production of RNA or protein by the same gene or regulator region in a normal or a control cell. In another context, “differentially expressed,” also refers to nucleotide sequences or proteins in a cell or tissue which have different temporal and/or spatial expression profiles as compared to a normal or control cell. As used herein, “polypeptides” or “proteins” are amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). As used herein, “gene” can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. “Gene” also refers to the specific sequence of DNA that is transcribed into an RNA transcript that can be translated into a polypeptide or be a catalytic RNA molecule including but not limited to tRNA, siRNA, piRNA, miRNA, long-non-coding RNA and shRNA. As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. RNA can be in the form of non-coding RNA such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), microRNA (miRNA), or ribozymes, aptamers or coding mRNA (messenger RNA). As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined elsewhere herein. As used herein, “DNA molecule” includes nucleic acids/polynucleotides that are made of DNA. As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions can include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein. As used herein, “microRNA” can refer to a small non-coding RNA molecule containing about 21 to about 23 nucleotides found in organisms, which functions in transcriptional and post-transcriptional regulation of transcription and translation of RNA. “MicroRNA” can exist as part of a larger nucleic acid molecule such as a stem-loop structure that can be processed by a cell and yield a microRNA of about 21-23 nucleotides. As used herein, “pharmaceutically acceptable carrier, diluent, binders, lubricants, glidant, preservative, flavoring agent, coloring agent, and excipient” refers to a carrier, diluent, binder, lubricant, glidant, preservative, flavoring agent, coloring agent, or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. The term “treating”, as used herein, can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. As used herein, “overexpressed” or “overexpression” refers to an increased expression level of an RNA (coding or non-coding RNA) or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell. As used herein, “underexpressed” or “underexpression” refers to decreased expression level of an RNA (coding or non-coding RNA) or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell. As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins. As used herein “gene editing”, “genome editing,” “genome modification” can refer to the non-natural manipulation of genomic DNA such that the genomic DNA contains one or more additional nucleotides or has one or more nucleotides removed from the genomic sequence. Such genome editing can be achieved by techniques such as viral integration of a transgene, homologous recombination insertion of a transgene, and CRISPR related methods and techniques, including but not limited to, the self-replicating RNA and self-replicating cell-selective CRISPR methods described herein. “Gene editing” and “genome editing” or “genome modification” can refer to the deletion and/or addition of nucleotides into the genomic DNA. As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, and protein/peptides, “corresponding to” can refer to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively “corresponding to” can direct them to determine the possible underlying and/or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined. As used herein, “promoter” can refer to all nucleotide sequences capable of driving or initiating transcription of a coding or a non-coding DNA sequence. The term “promoter” as used herein can refer to a DNA sequence generally described as the 5′ regulator region of a gene, located proximal to the start codon. The transcription of an adjacent coding sequence(s) is initiated at the promoter region. The term “promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. As used herein, “selectable marker” can refer to a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene. For instance, a recombinant nucleic acid may include a selectable marker operatively linked to a gene of interest and a promoter, such that expression of the selectable marker indicates the successful transformation of the cell with the gene of interest. As used herein, “constitutive promoter” can refer to a promoter that allows for continual or ubiquitous transcription of its associated gene or polynucleotide. Constitutive promoters are generally are unregulated by cell or tissue type, time, or environment. As used herein, “inducible promoter” can refer to a promoter that allows transcription of its associated gene or polynucleotide in response to a substance or compound (e.g. an antibiotic, or metal), an environmental condition (e.g.temperature), developmental stage, or tissue type. As used herein, “electroporation” is a transformation method in which a high concentration of plasmid DNA (containing exogenous DNA) or RNA is added to a suspension of host cell protoplasts, and the mixture shocked with an electrical field of about 200 to 600 V/cm. As used herein, “plasmid” can refer to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell. As used herein, the term “vector” can refer to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector can include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments can include promoter and terminator sequences, internal ribosome entry site, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, microRNA target sequences etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or can contain elements of both. The term “vector” can also include RNA or circular RNA vectors linked to additional segments that provide for its translation upon introduction into a host cell or host cell organelles. Such additional segments can include 5′Cap, one or more selectable markers, an enhancer, a polyadenylation signal, polyA tail, microRNA target sequences etc. As used herein, “identity,” can refer to a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptides or polynucleotides as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Neede/man and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure. As used herein, the term “transfection” can refer to the introduction of an exogenous and/or recombinant nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus, or chloroplast. The nucleic acid may be in the form of naked DNA or RNA (unmodified or modified), it may be associated with various proteins or regulatory elements (e.g., a promoter and/or signal element, miRNA target sequences as described herein), or the nucleic acid may be incorporated into a vector or a chromosome. As used herein, “transformation” or “transformed” can refer to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding or non-coding portions of the introduced nucleic acid. As used herein a “transformed cell” can refer to a cell transfected with a nucleic acid sequence. As used herein, a “transgene” can refer to an artificial gene which is used to transform a cell of an organism, such as a bacterium or a plant. As used herein, “transgenic” can refer to a cell, tissue, or organism that contains a transgene. As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids can include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a “fusion protein” (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g, a nucleic acid and a constitutive promoter etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man, including but not limited to miRNA target sequences described herein. As used herein, the term “exogenous DNA” or “exogenous RNA” or exogenous nucleic acid sequence” or “exogenous polynucleotide” can refer to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection. Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and/or it may be synthetic and/or recombinant. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g., an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid). Typically, the introduced exogenous sequence is a recombinant sequence. As used herein “miRNA target” or “miRNA target sequence” can refer to the nucleic acid sequence, typically RNA, that a miRNA specifically binds to. The miRNA target can be or include a sequence that is complementary to the miRNA. As an example, microRNA 126 (miR-126) can specifically bind a miR-126 target. Binding of a miRNA to a miRNA target can result in transcription and/or translation inhibition of the nucleic acid sequence, such as through degradation of the nucleic acid sequence (typically mRNA or other type of RNA), that the miRNA target is part of). A microRNA does not have to have perfect complementarity to a miRNA target for specific binding or transcription inhibition to occur. As used herein “seed sequence” or “seed region” can refer to the conserved heptametrical sequence of a microRNA that has perfect complementarity to the miRNA target. The seed sequence can be at about positions 2-7 from the miRNA 5′-end. As used herein, “nonstructural viral protein” and similar phrases can refer to proteins encoded by a virus, but are not incorporated into the viron particle. As used herein “differentially expressed”, “differential expression,” and the like can refer to the difference in spatial, temporal, and/or amount of expression of a gene, transcript, and/or protein that can be observed between the same or different genes, transcripts, and/or proteins. Discussion Despite the remarkable advance in therapeutic strategies over the past decades, the lack of cell selective therapies remains a major hurdle in clinical medicine. Side effects and reduced efficacy due to poor or no selectivity remains the Achilles' heel in the treatment of many diseases and contributes to the significant morbidity and mortality rates associated with many diseases. Gene therapy holds significant promise to treat, if not cure, many diseases for which there currently is no effective treatment or cure. However, the benefits of gene therapy still remain illusive. Traditional gene therapy approaches rely on pseudo-viral packaging and delivery of the gene to cells. Off-target effects due to ubiquitous overexpression and oncogenesis due to insertional mutagenesis as a result of poor cell selectivity and insertion control remain issues with virus-based gene therapy. Further, size limitations of currently viral-based systems prevent the use of many viral-based systems for the delivery of many genes of interest. With that said, described herein are cell-selective RNA molecules that can include a gene of interest or guide sequence RNA (gRNA) operatively coupled to one or more miRNA target sites. The cell-selective RNA molecules can be linear or circular, self-replicating, and/or contain one or more chemical modifications that can reduce immunogenecity of the cell-selective RNA molecules. The compositions described herein can be used for the treatment of diseases or symptoms thereof and/or cell selective gene editing. Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure. Cell Selective RNA Molecules As shown inFIG.1, the cell-selective RNA molecules can contain one or more RNA molecules of interest. The RNA molecule(s) can be operatively coupled to one or more miRNA target sequences. When introduced to cells that have miRNA that can specifically bind to the miRNA target(s), the miRNA can specifically bind the miRNA target(s) of the cell-selective RNA molecules. This can result in degradation and/or prevent translation of the cell-selective RNA molecules through endogenous pathways (e.g. RISC complex-mediated degradation). Thus, there is no observable expression of the RNA of interest. When introduced into cells that do not express miRNA that can specifically bind to the miRNA target(s) of the cell-selective RNA molecule, the cell-selective RNA molecule can be translated by the cell. It will be appreciated that the same outcome can be realized if a cell expresses the miRNA that can specifically bind the miRNA target(s) at such a low level that binding to the cell-selective RNA does not reduce translation (through degradation or otherwise) of enough cell-selective RNA molecules to inhibit or ablate effective expression of the RNA of interest. In this way expression and/or translation of the RNA of interest can be selective to only cells that do not express miRNA that can bind to the miRNA target(s) in the cell-selective RNA molecule. By way of non-limiting example,FIGS.2-3show cell-selective expression of p27, Cas9, and/or gRNA that can be used as part of a CRISPR/Cas9 genome modification system. In some embodiments, the cell-selective RNA molecule can be a RNA molecule. In other embodiments, the cell-selective molecule can be a DNA molecule that corresponds to (or encodes) cell-selective RNA molecule. The cell-selective RNA molecule or its corresponding DNA molecule can exist as naked RNA or DNA molecule (i.e. not contained in a vector) or contained within a vector. RNA Molecules of Interest The cell-selective RNA molecules can contain one or more RNA molecules of interest (ROI). The RNA molecule can correspond to a gene of interest. The ROI can correspond to an untranslated RNA molecule. The ROI can be any RNA molecule, linear or circular. The ROI can contain one or more ROI separated by a self-cleaving 2a peptide sequence. The ROI can contain a sequence corresponding to B18R to mitigate the innate immune response. The B18R can have a sequence that can be 90-100% identical to a sequence that corresponds to SEQ ID NO. 1. ROI can correspond to p27 that can be 90-100% identical to a sequence that corresponds to SEQ ID NO. 2, or any other tumor suppressor gene or suicide gene. The ROI can be a RNA corresponding to a Cas9 protein. The Casp can have a polypeptide sequence that is identical to or that corresponds to SEQ NO. 3, Cas9n (D10A nickase version of the Cas9 enzyme generates a single-strand DNA break) that correspond to SEQ NO. 4, dCas9 (A catalytically inactive Cas9 or dCas9-repressor peptide fusion can be used to knock-down gene expression by interfering with transcription of the gene) that correspond to SEQ NO. 5 or dCAS9-VP64 activator that corresponds to SEQ NO. 6. The ROI can have or include a sequence 100% identical to any one of SEQ ID NOs: 2-6. The ROI can have or include a sequence that is 90-100% identical to a sequence corresponding to any one of SEQ ID NOs: 2-6. The ROI can be a guide RNA (gRNA). The ROI can have or include a sequence that is 90-100% identical to SEQ ID NOs: 7. The ROI can be gRNA incorporating two MS2 RNA aptamers gRNA(MS2) cloning backbone include a sequence that is 90-100% identical to SEQ ID NO: 8. The ROI can encode MS2-P65-HSF1 activation helper proteins separated by 2a peptide sequence that have or include a sequence that is 90-100% identical to SEQ ID NO: 9 that can be used as part of a CRISPR/Cas9 genome modification system. The ROI can be self-replicating cell-selective RNA molecules (described below). miRNA Targets The cell-selective RNA molecules can contain one or more miRNA targets. In some embodiments, the number of miRNA targets can range from 1 to 20 or more. For example, in some embodiments, the cell-selective RNA molecules can contain 1, 2, 3, 4, or 5 miRNA targets. The miRNA target(s) can be operatively linked to the 5′ and/or 3′ end of the ROI. The miRNA target(s) can be operatively linked to a 5′ untranslated region (UTR) of the ROI and/or a 3′ UTR of the ROI. In embodiments having one or more miRNA targets, the miRNA targets can be the same miRNA target, they can each be a different miRNA target. In some embodiments at least two of the miRNA targets are the same. In some embodiments, at least two of the miRNA targets are different. The miRNA targets can be operatively linked to each other and/or the ROI of interest. The miRNA target(s) are nucleotide sequences that can specifically bind one or more miRNAs. The miRNA(s) can have differential spatial and temporal expression. As such, effective expression of the ROI can be controlled both spatially and temporally depending on the miRNA target(s) included in the cell-selective RNA molecule as previously described. Some exemplary constructs incorporating various ROls are demonstrated inFIGS.4,5,17,18,19,20,22, and31-42. Suitable miRNA targets include, but are not limited to, targets for miR-126, miR-145, miR-296, miR-21, miR-22, miR-15a, miR-16, miR-19b, miR-92, miR-93, miR-96, miR-130, miR-130b, miR-128, miR-9, miR-125b, miR-131, miR-178, miR-124a, miR-266, miR-103, miR-9*, miR-125a, miR-132, miR-137, miR-139, miR-7, miR-124b, miR-135, miR-153, miR-149, miR-183, miR-190, miR-219, miR-18, miR-19a, miR-24, miR-32, miR-213, miR-20, miR-141, miR-193, miR-200b, miR99a, miR127, miR142-a, miR-142-s, miR-151, miR-189b, miR-223, miR-142, miR-122a, miR-152, miR-194, miR-199, miR-215, miR-1b, miR-1d, miR-133, miR-206, miR-208, miR-143, miR-30b, miR-30c, miR-26a, miR-27a, let-7a, and miR-7b. In some embodiments, an miRNA target can have a sequence or include a sequence that is about 20-100% identical to the complement of any one of SEQ ID NOs: 10-135. In some embodiments, an miRNA target can have a sequence or include a sequence that is about 30-100% identical to the complement of a portion of any one of SEQ ID NOs: 10-135, where the portion is at least 5 consecutive nucleotides that corresponds to the seed sequence. The miRNAs that can specifically bind to the miRNA target(s) included in the cell-selective RNA molecule can have or include a sequence or portion thereof that is about 20-100% identical to any of SEQ ID NOs: 10-135, where a portion is at least 6 consecutive nucleotides that corresponds to the seed sequence. Where a stem-loop sequence is provided, those of skill in the art will appreciate, which portions correspond to the mature miRNA sequences that can be produced from the stem-loop sequences. Self-Replicating Cell-Selective RNA Molecules The cell-selective RNA molecules can be configured such that they are self-replicating. In other words, the cell-selective RNA molecules can be RNA replicons. The cell-selective RNA molecules can also include one or more viral RNA sequences that confer self-replication functionality once the cell-selective RNA molecule is introduced to a cell. The viral RNA sequence(s) can be alphavirus RNA sequences. The viral RNA sequence(s) can be Venezuelan Equine Encephalitis, Sindbis, and/or Semliki Forest virus sequence(s). Such sequences will be appreciated and determined by those of skill in the art. The sequences can encode one or more nonstrucutrual proteins and/or an RNA replicase. The viral sequences can be operatively linked to the ROI and/or miRNA target(s). Noninfectious self-replicating viral RNA that lacks the genes encoding the viral structural proteins that encodes four nonstructural replication complex proteins (NSPs) as a single open reading frame (ORF) in addition to the ROI. In some embodiments, the cell-selective RNA molecules do not include an RNA replicase. Replicases are generally known in the art. Non-limiting examples include, but are not limited to, those set forth in Geall et al., (2012) PNAS 109(36) 14604-14609 and Yoshioka et al. (2013) Stem Cell. 13(2):246-254, which are incorporated by reference as if expressed in their entirety. Promoter Sequences and Other Transcription Elements The cell-selective RNA molecules can contain one or more promoters. The promoter(s) can be operatively linked to the 5′ end of the cell selective RNA molecule. The promoter(s) can be operatively linked at any position between the 5′ and 3′ end of the cell-selective RNA molecules. The promoter(s) can be operatively linked to the 5′ end of an ROI. The promoter(s) can be operatively linked to the 5′ end of a miRNA target. The promoter(s) can drive in vitro and/or in vivo transcription of the cell-selective RNA molecule or corresponding DNA molecule. The promoter can be a eukaryotic promoter. The promoter can be a prokaryotic promoter. The promoter can be a Pol I promoter. The promoter can be a Pol II promoter. The promoter can be a Pol III promoter. The promoter can be a T7, Sp6 or T3 polymerase promoter. Example promoters include, but are not limited to 26S subgenomic promoter, CMV, CAG, SV40, EF1a, PGK1, Unc, beta actin promoter, TRE, UAS, Ac5, Polyhedrin, CaMKlla, GAL1, GAL10, TEF1, GDS, GAPDH promoter, ADH1, cAMV355, Ubi, H1, 7SK, U6, T7, SP6, T7lac. araBAD, trp, lac, Ptac, and pL. The sequences and variants thereof of these promoters as well as other promoters that would be suitable to one of ordinary skill in the art in view of this description are generally known in the art. See also Yoshioka et al. (2013) Stem Cell. 13(2):246-254 and Addgene Plasmid No.: 58976, which are incorporated by reference as if expressed in their entirety. Markers The cell-selective RNA molecule can include one or more markers or reporter molecules. Example markers and reporter molecules include, but are not limited to, Examples of selectable markers include, but are not limited to, DNA and/or RNA segments that contain restriction enzyme sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and/or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and/or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. FLAG- and His-tags), and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art. The terms “plasmid”, “vector” and “cassette” as used herein can refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA. Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell. Vectors Also provided herein are nucleic acid vectors containing a nucleic acid molecule corresponding to a cell-selective RNA molecule described herein. All or part of the vectors can be capable of being transcribed in vitro without a host cell or in a host cell. The vectors can be capable of being replicated by a host cell. All or part of the vector or a RNA molecule produced from the vector template can be capable of being integrated directly or indirectly into a host cell genome. The vectors can be viral vectors, i.e. vectors that are virus based or incorporate viral proteins or nucleic acids corresponding to a viral protein. Suitable viral vectors can include adenoviral, lentiviral, retroviral, and alpha viral vectors. Synthesis of the Cell-Selective RNA Molecules The cell-selective RNA molecules can be synthesized using de novo chemical synthesis. The cell-selective RNA molecules can be synthesized using in vitro transcription from a DNA molecule template. In vitro transcription can occur within a host cell or without a host cell. The cell-selective RNA molecules can also be transcribed in vivo after delivery to a subject. The DNA molecule template can be a DNA molecule or DNA vector containing a DNA sequence corresponding to the cell-selective RNA molecules. These DNA molecules and vectors are described elsewhere herein. The cell-selective RNA molecules can be polyadenylated at the 3′ end. The cell-selective RNA molecules can be 5′ capped in vitro. The cell-selective RNA molecules can be synthesized with an ARCA Cap at the 5′ end. The RNA molecule can be expressed in a bacterial, viral, yeast, plant, insect, or mammalian expression system. Suitable systems will be appreciated by those skilled in the art. The RNA molecule produced from transcription can be purified from a solution and/or other cellular components. Methods of RNA purification will be appreciated by those of skill in the art. In some embodiments, the cell-selective RNA molecules are modified. The modification can occur during or after synthesis (transcription of the RNA molecule). The modification can be a nucleotide modification. As such, the cell-selective RNA molecules can be synthesized with one or more modified nucleotides. Suitable nucleotide modifications include, but are not limited to, pseudouridine (ΨU), N-1-methylpseudouridine, 5-methoxyuridine, and 5-hydroxymethylcytidine. In some embodiments 0-100% of the nucleotides are substituted. The modifications can reduce immunogenicity of the cell-selective RNA molecules. The modifications can increase transcription and/or translation of the cell-selective RNA molecules. Delivery of the Cell-Selective RNA Molecules The cell-selective RNA molecules, corresponding DNA molecule, vectors, virons, or pseudoviral particles described herein can be delivered to a subject as part of a pharmaceutical formulation as described herein. As described elsewhere herein, the cell-selective RNA molecules can be delivered to a cell by transfection, transduction, or by other suitable method. In other embodiments, a DNA based vector or polynucleotide that encodes a cell-selective RNA molecule as described herein can be configured to be delivered to a cell as being incorporated in a virus, pseudovirus, or virus particle. In these embodiments, the cells can be transduced or infected with a virus, pseudovirus, or other virus particle that can deliver the corresponding DNA molecule to the cell. In other embodiments, the cell-selective RNA molecules and/or corresponding DNA molecule can be delivered to a cell via chemical transfection of a cell. Chemical transfection methods include encapsulating the cell-selective RNA molecules and/or corresponding DNA molecules in a liposome or micelle (e.g. cationic liposome), which can then be taken in by the cell via endocytosis. Suitable transfection reagents will be appreciated by those of skill in the art. In further embodiments, the cell-selective RNA molecules and/or corresponding DNA molecules can be incorporated with mesoporous silica nanoparticles that can include a polycation adjunct or large pore mesoporous silica nanoparticles. The mesoporous silica nanoparticles that include the cell-selective RNA molecules and/or corresponding DNA molecules can be taken up by the cell via endocytosis. In other embodiments, the cell-selective RNA molecules and/or corresponding DNA molecules can couple to organic/inorganic silica hybrid nanoparticleswhich can be taken up by a cell via endocytosis. Other delivery methods include electroporation or chitosan polymers. Other delivery methods will be appreciated by those of ordinary skill in the art. Pharmaceutical Formulations Also provided herein are pharmaceutical formulations containing an amount of a cell-selective RNA molecule, corresponding DNA molecule (including vectors), and/or viron particle as described herein. The amount can be an effective amount. Pharmaceutical formulations can be formulated for delivery via a variety of routes and can contain a pharmaceutically acceptable carrier. Techniques and formulations generally can be found inRemmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20th Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use. Suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxyl methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition. The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition. The pharmaceutical formulations can be administered to a subject in need thereof. The subject in need thereof can have a disease, disorder, or a symptom thereof. Example disease or disorder can include, but are not limited to, a cardiovascular disease, a pulmonary disease, a brain disease, a renal disease, a liver disease, a blood disease, a nervous system disease, an intestinal disease, an ocular disease, and cancer. The pharmaceutical formulations can be disposed on or otherwise coupled to or integrated with a medical device, such as, but not limited to, catheters or stents, such that the pharmaceutical formulation is eluted from the medical device over a time period. The pharmaceutical formulation can therefore be delivered to a subject in need thereof during and/or after a procedure such as an angioplasty, vein draft or organ transplant. Other procedures where such a medical device would be useful will be appreciated by those of skill in the art. A pharmaceutical formulation can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The construct, biologic molecules and pharmaceutical formulations thereof described herein can be disposed on or otherwise integrated with or coupled to a medical device such as, but not limited to, a catheter or stent, such that the construct, biological molecule can be released to the surrounding local area or systemically over a period of time after insertion or implantation into a subject in need thereof. These can also be referred to as drug eluting medical devices. Pharmaceutical formulations suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers can include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Injectable pharmaceutical formulations can be sterile and can be fluid to the extent that easy syringability exists. Injectable pharmaceutical formulations can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by incorporating an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating any of the cell-selective RNA molecules, corresponding DNA molecules, or viron particles as described herein in an amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the nucleic acid vectors into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fluidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the cell-selective RNA molecules, corresponding DNA molecules, and/or viron particles can be formulated into ointments, salves, gels, or creams as generally known in the art. In some embodiments, the cell-selective RNA molecules, corresponding DNA molecules, and/or viron particles can be applied via transdermal delivery systems, which can slowly release the cell-selective RNA, corresponding DNA molecule, and/or viron particles for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media. Transdermal patches are described in for example, U.S. Pat. Nos. 5,407,713; 5,352,456; 5,332,213; 5,336,168; 5,290,561; 5,254,346; 5,164,189; 5,163,899; 5,088,977; 5,087,240; 5,008,110; and 4,921,475. Administration of the cell-selective RNA molecules, corresponding DNA molecules, and/or viron particles is not restricted to a single route, but may encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan. The pharmaceutical formulations can be administered to a subject by any suitable method that allows the agent to exert its effect on the subject in vivo. For example, the formulations or other compositions described herein can be administered to the subject by known procedures including, but not limited to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation, via nasal delivery, vaginally, rectally, and intramuscularly. The formulations or other compositions described herein can be administered parenterally, by epifascial, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymatous, and/or sublingual delivery. Delivery can be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray. In some embodiments, the nucleic acid vectors of the invention are administered to the subject by way of delivery directly to the heart tissue, such as by way of a catheter inserted into, or in the proximity of the subject's heart, or by using delivery vehicles capable of targeting the drug to the heart. For example, the cell-selective RNA molecules, corresponding DNA molecules, and/or viron particles described herein can be conjugated to or administered in conjunction with an agent that is targeted to the heart, such as an aptamer, antibody or antibody fragment. The cell-selective RNA molecules, corresponding DNA molecules, and/or viron particles can be administered to the subject by way of delivery directly to the tissue of interest, such as by way of a catheter inserted into, or in the proximity of the subject's tissue of interest, or by using delivery vehicles capable of targeting the nucleic acid vectors to the muscle, such as an antibody or antibody fragment. For oral administration, a formulation as described herein can be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation can contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, cornstarch, gelatins, disintegrators, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants, and/or or magnesium stearate. For parenteral administration (i.e., administration by through a route other than the alimentary canal), the formulations described herein can be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation can be prepared by dissolving the active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering the solution sterile. The formulation can be presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation can be delivered by injection, infusion, or other means known in the art. For transdermal administration, the formulation described herein can be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the nucleic acid vectors of the invention and permit the nucleic acid vectors to penetrate through the skin and into the bloodstream. The formulations and/or compositions described herein can be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which can be dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch. Dosage Forms The pharmaceutical formulations or compositions described herein can be provided in unit dose form such as a tablet, capsule or single-dose injection or infusion vial. Where appropriate, the dosage forms described herein can be microencapsulated. The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the complexed active agent can be the ingredient whose release is delayed. In other embodiments, the release of an auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington—The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more. Coatings may be formed with a different ratio of water soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form. Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides. In some embodiments, such as for treatments of plants, the topical formulation of a composition or pharmaceutical formulation described herein can be further formulated as a spray and can include a suitable surfactant, wetting agent, adjuvants/surfactant (stickers, extender, plant penetrant, compatibility agents, buffers, drift control additives, and defoaming agents), or any combination thereof so as to formulated as a spray. The compounds, any optional auxiliary active ingredient, suitable surfactant, wetting agent, adjuvants, or any combination thereof can be formulated as a solution, suspension, or emulsion. The spray dosage from can be administered through a spraying device. In some embodiments, the spraying device can be configured to generate the sprayable formulation as a liquid solution is contacted with the complexed active agent compound or formulation thereof. In other embodiments, the sprayable dosage form is pre-made prior to spraying. As such, the spraying device can act solely as an applicator for these embodiments. In further embodiments, such as for treatments of plants (e.g. such as a herbicide), the dosage form of composition or pharmaceutical formulation described herein thereof can be further formulated as a dust and can include a suitable dry inert carrier (e.g. talc chalk, clay, nut hull, volcanic ash, or any combination thereof so as to be formulated as a dust. The dust can contain dust particles of varying sizes. In some embodiments, the particle size can be substantially homogenous. In other embodiments, the particle size can be heterogeneous. Dosage forms adapted as a dust can contain one or more adjuvants/surfactants (stickers, extender, plant penetrant, compatibility agents, buffers, drift control additives, and defoaming agents). In some embodiments, the dosage form can be formulated as a bait. In these embodiments, the complexed active agent compound or other formulation thereof can be further formulated to include a food or other attractive substance that can attract one or more insect or other pest. The bait dosage form can be formulated as a dust, paste, gel, or granule. Dosage forms adapted as baits can contain one or more adjuvants/surfactants (stickers, extender, plant penetrant, compatibility agents, buffers, drift control additives, and defoaming agents). In additional embodiments, the dosage form can be formulated as granules or pellets that can be applied to the environment. These dosage formulations are similar to dust formulations, but the particles are larger and heavier. The granules can be applied to soil or other environmental area. Dosage forms adapted as granules or pellets can contain one or more adjuvants/surfactants (stickers, extender, plant penetrant, compatibility agents, buffers, drift control additives, and defoaming agents). The dusts, granules, and pellets described herein can be formulated as wetable dusts, granules, and pellets, soluble dusts granules, and pellets, and/or water-dispersible granules, and/or dry flowables. The dosage form can be adapted for impregnating (saturating) an object or device, which then can be carried by, worn, or otherwise coupled to an organism in need thereof. In some embodiments, the dosage form can be impregnated onto a collar, bracelet, patch, adhesive tape, livestock ear tags, clothing, blankets, plastics, nets, and paints. The composition or pharmaceutical formulation thereof can be formulated and impregnated in the object or device such that the composition or pharmaceutical formulation evaporates over time, which releases the composition and/or pharmaceutical formulation into the air and/or environment surrounding the organism and/or onto the organism. The dosage form can be adapted as a fumigant, which is a formulation that forms a gas when utilized or applied. In some embodiments, the composition and/or pharmaceutical formulation thereof can be supplied as a liquid when packaged under pressure and change to a gas when they are released. In other embodiments, the composition and/or pharmaceutical formulation thereof can be supplied as a volatile liquid when enclosed in a container (not under pressure). Others can be formulated as solids that release gases when applied under conditions of high humidity or in the presence of high water vapor. Dosage forms adapted as fumigants can contain one or more adjuvants/surfactants (stickers, extender, plant penetrant, compatibility agents, buffers, drift control additives, and defoaming agents). Effective Amounts The pharmaceutical formulations can contain an effective amount of a composition described herein and/or an effective amount of an auxiliary agent. In some embodiments, the effective amount ranges from about 0.001 pg to about 1,000 g or more of the composition described herein. In some embodiments, the effective amount of the composition described herein can range from about 0.001 mg/kg body weight to about 1,000 mg/kg body weight. In yet other embodiments, the effective amount of the composition can range from about 1% w/w to about 99% or more w/w, w/v, or v/v of the total pharmaceutical formulation. Combination Therapy The pharmaceutical formulations or other compositions described herein can be administered to a subject either as a single agent, or in combination with one or more other agents. Additional agents include but are not limited to DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, and chemotherapeutics. Multiple cell-selective ROI can be administered simultaneously in a combination treatment. Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g. choline salicylate, magnesium salicylae, and sodium salicaylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine. Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g. alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotenergic antidepressants (e.g. selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, afobazole, selank, bromantane, emoxypine, azapirones, barbituates, hyxdroxyzine, pregabalin, validol, and beta blockers. Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzaprine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine. Suitable analgesics include, but are not limited to, paracetamol/acetaminophen, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylate, and sodium salicylate). Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g. submandibular gland peptide-T and its derivatives). Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g. cimetidine, famotidine, lafutidine, nizatidine, rafitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and β2-adrenergic agonists. Suitable anti-infectives include, but are not limited to, amebicides (e.g. nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine, and iodoquinol), aminoglycosides (e.g. paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel, abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g. azole antifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g. caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g. nystatin, and amphotericin b), antimalarial agents (e.g. pyrimethamine/sulfadoxine, artemether/lumefantrine, atovaquone/proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g. aminosalicylic acid), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethanmbutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine, emtricitabine/tenofovir, cobicistat/elvitegravir/emtricitabine/tenofovir, efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine, lamivudine/zidovudine, emtricitabine/tenofovir, emtricitabine/opinavir/ritonavir/tenofovir, interferon alfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpiviirine, delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem, and cilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erthromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procaine penicillin, oxaxillin, dicloxacillin, and nafcillin), quinolones (e.g. lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline, demeclocycline, minocycline, doxycycline/salicyclic acid, doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue). Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, aspargainase erwinia chyrsanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid. Devices Containing the Cell Selective RNA Molecules Also described herein are medical devices that can contain a composition or pharmaceutical formulation as described herein. In some embodiments, the composition or pharmaceutical formulation described herein can be encapsulated in one or more polymers. In some embodiments, the polymers are biocompatible polymers. The polymers can be FDA approved polymers. Suitable polymers can include, but are not limited to phosphorylcholine-based polymers, poly lactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), chitosan, cationic nanoemulsion, cationic electrodeposition coating or lipid nanoparticles. The composition or pharmaceutical formulations can be encapsulated in the polymer and the polymer can be applied to the medical device. In use, the compositions and/or pharmaceutical formulations described herein can be released from the polymer into the surrounding environment and be utilized by cells in the surrounding environment. Suitable medical devices include, but are not limited to, stents, spinal plates, pins, screws, replacement joints and components thereof, catheters, coated balloons, cannulas, bone plates, spacers, replacement discs, stabilization rods, and surgical mesh. Uses of the Cell Selective RNA Molecules and Devices The compositions and formulations described herein can be used to selectively deliver a translated RNA (e.g. a gene) or untranslated RNA (gRNA) to a cell. In this way, the compositions and formulations described herein can be used to treat and/or prevent a disease, disorder, or symptom thereof by delivering the ROI to a cell. Selective expression of the ROI can increase treatment or preventive efficacy by decreasing side effects. When the ROI includes a gRNA or sgRNA(MS2), dCas9, dCas9-VP64 fusion or MS2-P65-HSF1 the compositions can be also used for genome editing using the CRISPR/Cas9 system, which includes the addition or deletion of one or more nucleotides to the genome or exon skipping. This can result in gene knock down, knock out, gene repair, gene suppression or gene activation. Thus, in this way genome editing can be cell-selective because the gRNA and/or sgRNA(MS2), Cas9, dCas9, dCas9-VP64 fusion or MS2-P65-HSF1 will only be present in cells lacking the miRNA that can bind the target(s) in the cell-selective RNA molecule. EXAMPLES Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. Example 1 Despite the remarkable advances in therapeutic strategies over the past decades the lack of cell selective therapies remain a major overarching problem in clinical medicine. One clear example for this challenge is the deadly consequences of the non-selective agent used on the drug-eluting stents (DES) that were developed to inhibit neointimal overgrowth of vascular smooth muscle cells (VSMCs) following percutaneous intervention. While DES significantly reduced the occurrence of restenosis compared with a bare-metal stent (BMS), they do not completely eliminate this challenging problem. Moreover, stent thrombosis (ST), reinfarction, and neoatherosclerosis within stent segments have emerged as major safety concerns with DES, all predominantly attributed to the lack of reendothelialization of diseased vessel walls with competent endothelial cells (ECs). DES deployment inevitably traumatizes the normal competent endothelium structure. Compounding this insult, the drugs eluted from the stents, while not delivered systemically, are still universally deleterious to all cell types exposed to the eluted drug, proving toxic to ECs and drastically reducing the quality of regenerating endothelium. This incompetent endothelium, with poorly formed cell-to-cell junctions reduced expression of antithrombotic molecules and endothelial nitric oxide synthase (eNOS), can no longer function normally to maintain vascular tone and fluid-tissue homeostasis. Thus, this requires patients to comply with at least 12 months of dual anti-platelet therapy. Newer generations of DES, with more sophisticated platforms, thinner struts, and biocompatible polymers have been developed to combat DES-associated risks. Yet, they still deploy the same non-selective drugs (paclitaxel, sirolimus or its analogs, everolimus, zotarolimus and biolimus, with improved pharmacokinetics). Current DES devices with low rates of restenosis and ST translate into a significant number of patients that will suffer from myocardial infarction and death due to the large number of percutaneous coronary intervention (PCI) procedures performed worldwide every year. In fact, percutaneous interventions are among the most performed procedures in Medicine. In the US alone, nearly 1 million patients undergo PCI for symptomatic coronary artery disease (CAD) every year, and non-selective DES are deployed in at least 75% of these cases. In 2010, the DES segment contributed 55%-60% of the global coronary stent market and is expected to reach USD 5.3 billion in 2016 due to the growing aging population and lifestyle changes leading to obesity. The market for DES is growing at 9.0% in the United States, 3.1% in Europe, 10% in Asia-Pacific and 3.1% in the rest of the world. These numbers will potentially expand as DES deployment is used in percutaneous interventions to treat peripheral artery disease (PAD) that affects more than 10 million people in the US, with symptomatic lower-extremity PAD, renal artery stenosis and carotid artery disease. Although percutaneous transluminal angioplasty (PTA) and stenting achieve a greater lumen diameter, vessel remodeling and restenosis remain its Achilles' heel contributing to significant morbidity and mortality rates in these patients. To date, cell-selective drugs that can discriminate between proliferating VSMCs, inflammatory cells and ECs are not available. Since vascular ECs provide crucial protection against thrombosis, lipid uptake and inflammation, there is a need to develop a cell-selective therapy that can inhibit VSMC proliferation and inhibit infiltration of inflammatory cells, yet spare ECs to carry on their vital functions. A viral vector approach has been previously developed that contained an EC specific miRNA target sequence (target for miR-126). In that work an adenoviral vector (Ad-p27-126TS) containing target sequences complementary to the mature miR-126-3p strand at the 3′-end of the cyclin-dependent kinase inhibitor p27Kip1 (p27). This approach allowed for exogenous p27 to be selectively overexpressed in VSMCs and infiltrate inflammatory cells, yet preserving the ECs. This therapy achieved results in an established rat model of balloon angioplasty, selectively inhibiting neointimal hyperplasia and inflammation while simultaneously promoting vessel reendothelialization, reducing hypercoagulability and restoring the endothelium-dependent vasodilatory response to levels indistinguishable from uninjured controls. (FIGS.21-29). Due to complications with viral gene delivery, this Example describes a messenger RNA based, cell-selective nanotherapy utilizing the mRNA or self-replicating RNA that can be based on an alphavirus genome. This strategy can match the potency of viral vectors yet avoid the serious safety concerns associated with recombinant virus-based therapeutics. This approach can reduce the need for repeated revascularization, reduce the need for prolonged dual anti-platelet drug regimens, and ultimately reduce the morbidity and mortality of CAD and PAD patients. This mRNA based cell-selective nanotherapy could not only replace the use of stents, but potentially treat lesions where DES cannot be deployed or DES is contraindicated, such as in stent restenosis, bifurcations, torturous vessels, small vessels or long calcifications. In addition to its potential benefit in CAD and PAD, this approach can be applied to benefit multiple other stenotic conditions, including transplant vasculopathy, arteriovenous fistulae, and vein graft failure, which all result from VSMC hyperplasia and EC dysfunction. Over 200,000 cardiovascular surgical procedures utilizing venous grafts fail each year in the US, primarily due to restenosis caused by VSMC hyperplasia. This mRNA based cell-selective approach may also inhibit vascular remodeling in deadly diseases such as pulmonary hypertension and pulmonary fibrosis, which still has no cure. The versatile concept of mRNA based, cell-selective nanotherapy can be broadly applied across disciplines and for treating any disease, including but not limited to, liver cirhosis and cancer. This tailored mRNA-based, cell-selective nanotherapy represents an exciting translational and potentially transformative approach in modern clinical medicine. mRNA-based therapeutics represent a game-changing technology and hold great promise for the treatment of human diseases including genetic disorders, infections, degenerative diseases and cancer. Many are the advantages of these versatile mRNA-based therapies. They can be produced very quickly, cost effectively and in a cell-free system at good manufacturing practice quality. Furthermore, any nucleotide sequence needed can be synthetically produced and stored at room temperature. Importantly, chemically modified mRNA that does not change the amino acid sequence of the corresponding protein not only decreases activation of the innate immune pathway but also improves the stability and enhances the translation levels. mRNA is non-replicative and therefore considered a very safe biomolecule that allows transient protein expression of every protein in virtually all cell types including non-dividing cells. Moreover, no nuclear localization, promoter elements or transcription is required and unlike recombinant virus-vectors the probability of genomic integration is nearly nonexistent. Lastly, chemically modified mRNAs that elude the body's innate immune response make therapeutic gene products and protein replacement therapies possible. However, there still exists the challenge of cell- or tissue-specific delivery that hinders virtually any type of therapeutic agent. To overcome the deficiencies stated above the miRNA-based cell selective approach can be utilized in a chemically modified self-replicating RNA platform to achieve long lasting cell-selective targeting that can inhibit proliferating VSMCs and infiltration of inflammatory cells while allowing ECs to reendothelialize vessel walls and maintain their crucial function. miR-126 is enriched in ECs and is a pivotal regulator of vascular integrity and angiogenesis. Moreover, miR-126 was observed to be up-regulated following arterial injury and in atherosclerotic plaques. The approach described herein utilizes EC-specific expression of miR-126 to drive cell-specific expression of therapeutic genes of interest. As proof of principle, an adenoviral vector (Ad-p27-126TS) containing target sequences complementary to the mature miR-126-3p strand at the 3′-end of the p27 to selectively avoid p27 overexpression in ECs, but not in VSMCs was designedFIG.22-23. Employing this single comprehensive nanotherapy in a rat carotid balloon injury model we were able to inhibit neointimal hyperplasia, inhibit infiltration of inflammatory cells to the injury site, and at the same time complete reendothelialization of the vessels was achieved as soon as 2 weeks post injury restoring the endothelium-dependent vasodilatory response to levels indistinguishable from uninjured controls and reducing the plasma D-Dimer levels of injured vessels to levels observed in uninjured controlsFIG.25-29. Incomplete and incompetent endothelial coverage in the vessel when deployed with current DES in PCI procedures greatly increased the risk of potentially catastrophic events such as late ST, primarily caused by the lack of drug specificity. Therefore, a therapy that would selectively inhibit VSMC proliferation, migration and inflammatory cell infiltration without affecting reendothelialization and EC function in the treated vessels would be advantageous. To establish proof of principle for cell-selective inhibition we chose to overexpress p27 in a cells selective manner due to the proven role it plays in the pathophysiology of vascular remodeling. As one of the most potent members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, p27 binds and modulates cyclin D-, E- and A-dependent kinases, resulting in G1/S transition failure and cell cycle arrest. In healthy arteries, p27 is constitutively expressed in quiescent VSMCs. Upon vascular injury, however, multiple response mechanisms are initiated to conclude with a rapid downregulation of p27, activating and enabling VSMCs to resume cell division. This reentry into the cell cycle triggers intimal hyperplasia, leading to vascular restenosis. p27 knockout mice display a significant increase in VSMC proliferation and develop extensive arterial lesions. In contrast, overexpression of exogenous p27 in VSMCs instigates G1 phase arrest, resulting in VSMC growth inhibition and a significant reduction of neointimal lesion formation in both a porcine femoral arterial injury model as well as a rat carotid model of balloon angioplasty. Moreover, p27 has been shown to play a key role in atherosclerosis. Indeed, p27 deficiency leads to increased atherosclerotic plaque formation in Apoe-/-mice. Lastly, previous reports show that p27 directly regulates the proliferation and migration of bone marrow-derived cells (hematopoietic and nonhematopoeitic) to the damaged vessels to reconstitute vascular lesions. Therefore, p27 is a particularly ideal candidate for cell-selective regulation. This approach also takes advantage of the established fact that miR-126 is robustly enriched in EC and is a pivotal regulator of vascular integrity and angiogenesis. Moreover, miR-126 was shown to be up-regulated following arterial injury and in atherosclerotic plaques. An adenoviral (Ad) vector encoding p27 a known cell-cycle inhibitor, and incorporating 4 complementary target sequences for the mature miR-126-3p strand at its 3′ end (Ad-p27-126TS)FIGS.22-23. Our aim was to overexpress exogenous p27, yet effectively regulate its overexpression in a cell-specific manner with the incorporated EC-specific miR-126-3p target sequencesFIG.24. Employing this single comprehensive nanotherapy (Ad-p27-126TS) in a rat carotid balloon injury model the following was achieved: 1) inhibition of neointimal hyperplasia (FIG.25); 2) inhibition of infiltration of inflammatory cells to the injury site (FIG.26); 3) reendothelialize vessels rapidly and extensively (FIGS.27A-B) 4) reduce the plasma D-Dimer levels of injured vessels to levels observed in uninjured controls (FIG.28); and 5) restore the endothelium-dependent vasodilatory response to levels indistinguishable from uninjured controls (FIG.29). These data demonstrate that the simple incorporation of miR-126 target sequences within the Ad-p27-126TS vector provided robust EC protection and at the same time achieved significant inhibition of the neointimal hyperplasia and infiltration of inflammatory cells to the injury site. Although viral delivery systems are very efficient for in vivo transduction and delivery of nucleic acids, they suffer major drawbacks including their possible toxicity, immunogenicity, insertional mutagenicity and oncogenicity. Additionally, viral vectors are difficult and expensive to produce in large quantities. As such the design and engineering of alternative nonviral systems for delivery of therapeutic agents is needed. Presented here are mRNA-based, cell-selective nanotherapies that can self-replicate and can consistently express high levels of exogenous p27 and at the same time retain its cell-selective degradation in a miRNA-controlled fashion, in some cases over multiple cellular divisions. To provide proof of concept and to show the feasibility of our cell-selective mRNA approach we constructed the following GFP-encoding plasmids: A) GFP coding sequence placed under the control of the bacteriophage T7 RNA polymerase promoter. To increase the stability of the mRNA we flanked GFP coding sequence with β-globin 5′- and 3′-UTR (FIG.4); B) GFP-2x126TS, a GFP-coding sequence placed under the control of the bacteriophage T7 RNA polymerase promoter, flanked with β-globin 5′- and 3′-UTR containing 2 or 3 tandem copies of a 22-bp target sequence perfectly complementary to the mature miR-126-3p strand at its 3′ end (FIG.4). In vitro transcription reaction from the linear plasmid templates described inFIG.4was performed using T7 RNA polymerase to produce unmodified (regular nucleotides, NTPs) or modified RNA in which we substituted 100% of the uridine with pseudouridine. After removal of free NTPs, 5′ capping and poly(A) tail addition was performed resulting in a high-yield RNA transcript. HEK cells were transfected with miR-126, miR-143 or control mimic and after 24 hr cells were transfected with either unmodified or modified GFP, GFP-2x126TS or GFP-3x126TS mRNAs. The effect of over-expression miR-126, miR-143 or control on GFP protein levels was assessed after 24 hr (FIGS.6-14). First, a significant increase in GFP expression was observed when uridine was 100% substituted with pseudouridine (FIGS.7and13. In cells transfected with the control unmodified or modified GFP mRNA neither over-expression of miR-126-3p or miR-143 had an effect on GFP expression (FIGS.6-10and13-14). HEK cells transfected with GFP-2xmiR-126TS or GFP-3x126TS mRNA, over-expression of miR-126 dramatically inhibited GFP expression of the unmodified or the modified mRNA (FIGS.6-8and11-14). These data demonstrate that pseudouridine modified GFP-2x126TS mRNA is susceptible to argonaute-dependent gene silencing by miR-126. This effect was miR-126 specific since over-expression of miR-143 had no effect on GFP expression in cells transfected with either unmodified or modified GFP-2xmiR-126TS or GFP-3x126TS (FIGS.6-8and11-14). The data show that pseudouridine modified mRNA is susceptible to the endogenous miRNA machinery. Adding target sites in the 3′UTR of chemically modified mRNA can affect its expression only when this specific miRNA is expressed. Example 2 A Flag tagged p27 encoding plasmids can be engineered to facilitate in vitro transcription of p27 encoding mRNA (FIG.17): A) Flag-tagged p27); B) Flag-tagged p27 followed by two 2 fully complementary target sequences for the mature miR-126-3p strand at its 3′-UTR (p27-2x126TS). A Flag-tag can be incorporated to distinguish between endogenous and exogenous p27 expression. To reduce innate immune responses and toxicity and at the same time maximize the efficiency and duration of expression of the mRNA encoding p27 described inFIG.17, the following modified nucleotide substitutions or combinations thereof can be used: 1) Pseudouridine; 2) N-1-methylpseudouridine; 3) 5-methoxy-U; 4) 5-hydroxymethyl-C; 5) 5-methyl-C and 6) combination of Pseudouridine and 5-methyl-C. mRNAs can be in vitro transcribed using T7 RNA polymerase followed by 5′ capping and poly(A) tail addition using a Vaccinia Capping Enzyme andE. coliPoly(A)Polymerase (New England BioLabs Inc.), respectively. Example 3 Example 1 demonstrates that substitution of uridine with pseudouridine and addition of miR-126 target sequences at the 3′-UTR of GFP mRNA increased the translational efficiently in a cell-selective manner. However, to increase its therapeutic potential, it is desirable that sustained cell selective inhibition matches the potency and the durability of the drugs eluted from the DES. To develop a long-lasting cell-selective mRNA-based therapy, we focused our efforts on an approach that (1) utilizes a single RNA species capable of self-replicating for a limited number of cell divisions; (2) is capable of encoding our gene of interest; (3) consistently expresses the protein at high threshold levels over multiple cellular divisions; and/or (4) can be susceptible to endogenous miRNA degradation in a cell-selective fashion. In this Example, an approach utilizing a noninfectious, self-replicating Venezuelan equine encephalitis (VEE) virus (lacks the genes encoding the viral structural proteins) and mimics cellular mRNA with a 5′-cap and poly(A) tail and does not utilize a DNA intermediate is described. This approach lacks the potential for genomic integration. VEE virus is a positive-stranded RNA that encodes four nonstructural replication complex proteins (NSPs) as a single open reading frame (ORF). Design of self-replicating, cell-selective mRNA vectors: To ectopically express p27 in a cell-selective manner, a p27-coding sequence can be placed at the 5′ end of VEE-structural protein genes. The following VEE10 GFP or Flag tagged p27 encoding plasmids will be engineered: A) VEE-p27, 3′-ORF replaced with flag-tag p27 coding sequence followed by β-globin 3′-UTR (FIG.19); (B) VEE-p27-2x126TS, 3′-ORF replaced with flag-tag p27-coding sequence followed by two tandem copies of a 22-bp target sequence perfectly complementary to the mature miR-126-3p at its 3′ end (FIG.19). A Flag-tag can be included to distinguish between endogenous and exogenous p27 expression. More than two miR-126 targets can be incorporated (FIG.18). Self-replicating mRNA depicted inFIGS.18-19can be in vitro transcribed using T7 RNA polymerase in the presence of the unmodified and modified nucleotides (Pseudouridine, N-1-methylpseudouridine, 5-methoxy-U, 5-hydroxymethyl-C, 5-methyl-C or combination of Pseudouridine and 5-methyl-C). mRNAs can be in vitro transcribed using T7 RNA polymerase followed by 5′ capping and poly(A) tailing. The self-replicating mRNA can also be designed to include RNA encoding the B18R protein. This can reduce immunogenic reactions (FIG.20). The self-replicating mRNA can also incorporate RNA encoding a marker protein such as GFP.FIG.18. Example 4 Cell-selective alternatives to the current DES used in percutaneous interventions are needed to inhibit restenosis while promoting reendothelialization. The advantage of such a treatment is the local non-invasive administration of drug in conjunction with balloon angioplasty limiting systemic toxicity. In this Example, an approach using FDA approved polymers to encapsulate the cell-selective mRNA or cell-selective self-replicating mRNA transcripts to facilitate efficient gene delivery in vivo is presented. The cell-selective RNA molecules can be encapsulated with a polymer to form RNA polymer nanoparticles. The RNA polymer nanoparticles can be attached to the stent or other medical device using a suitable method such as surface by dip- or by spray-coating. The following FDA approved polymers can be used: Phosphorylcholine-based polymers, Poly lactic-co-glycolic acid (PLGA), chitosan, cationic nanoemulsion, cationic electrodeposition coating or lipid nanoparticles. Example 5 Manipulation of gene expression via the RNAi system is a powerful new tool for the treatment of many diseases, including Hepatitis C and heart failure. The current trend in miRNA therapies focuses on the use of antagomirs, short RNA fragments that block the activity of a specific miRNA. RNAs with numerous target sites for miRNAs, referred to as miRNA sponges, are also able to effectively restore the repressed targeted mRNAs by sequestering the endogenous miRNA. Cell-specific miRNAs can be used to confer cell-protectivity to a gene therapy by incorporating miRNA target sites into the 3′ UTR. This strategy prevents expression of the inserted gene in a specific cell type based on the miRNA target sequences. To demonstrate that miRNA can be used to specifically regulate gene of interest a GFP-expressing lentiviral vector was used in which four target sequences for miR-126 were inserted, a vascular endothelial cell (VEC)-specific miRNA, in the GFP 3′UTR (GFP-4xmiR-126TS). To control for miRNA specificity, four target sequences for miR-143 (GFP-4xmiR-143TS) were inserted, which is vascular smooth muscle cell (VSMC)-specific, or four scramble sequences (GFP) in the GFP-3′UTR region HEK cells were transduced with the different GFP expressing lentiviruses, and the effect of over-expression or inhibition of miR-143, miR-126 or control on GFP expression was assessed. In cells transduced with the control GFP virus, neither over-expression nor inhibition of miR-143 or miR-126 had an effect on GFP expression (FIG.15, left). However, in HEK cells transduced with GFP-4xmiR-143TS virus, only over-expression of miR-143 dramatically inhibited GFP expression (FIG.15, middle). Similarly, over-expression of miR-143 had no effect on GFP expression in cells transduced with GFP-4xmiR-126TS, while over-expression of miR-126 did inhibit GFP expression in these cells (FIG.15, right). This strategy was used in VEC to ensure that the endogenous miR-126 targets the viral constructs as expected. VEC were transduced with the same lentiviruses. In control GFP virus transduced cells, neither over-expression nor inhibition of miR-143 or miR-126 had an effect on GFP expression (FIG.16, left). Similarly, over-expression of miR-143 inhibited GFP expression only in GFP-4xmiR-143TS transduced cells, while over-expression of miR-126 had no effect (FIG.16, middle). Interestingly, because miR-126 is highly expressed in VEC, it abolished the expression of GFP in the GFP-4xmir-126TS infected cells. Only inhibition of the endogenous miR-126 with a miR-126 antagomir rescued the expression of GFP (FIG.16, right). These results demonstrate the sensitivity and specificity of endogenous miRNA for target sites in the 3′ UTR of unmodified mRNAs. Predictable patterns of expression can be observed when using cell-specific miRNA target to regulate expression of the inserted gene. Example 6 Modified nucleotides can increase the translational efficiency and reduce cellular toxicity caused by the immunogenic response to exogenous mRNA. Here, 4 different representative modified nucleotide compositions (FIG.30) can be included in the cell-selective mRNA. Each of the modified nucleotides can be used as a complete substitute for the unmodified nucleotides to achieve the maximum effect. Modified and unmodified mRNA can be synthesized with a 5′ cap using an ARCA cap analog (TriLink) and PolyA tail using reagents to increase the stability of the mRNAs (TriLink). To determine if modified nucleic acids affect target site recognition, modified and unmodified mRNA encoding green fluorescent protein (GFP) or luciferase (Luc) followed by 4 target sequences (TS) for miR-126, miR-143 or control scrambled sequences will be transcribed in vitro by T7 RNA polymerase (FIG.5). Example 7 The number of miRNA silencing can be dependent on the number of miRNA target sites present in the construct. As demonstrated inFIGS.6and7, an increase from 0 (GFP) to 2 (2X126TS) to 3 (3x126TS) miRNA target sites within the construct can result in a positively correlated increase in silencing. The data ofFIGS.6and7further demonstrates the specificity of the miRNA target sequences. Example 8 To test whether modified nucleic acids affect the efficiency of miRNA-target site recognition and miRNA-dependent gene silencing, GFP or GFP4x126TS mRNA was in vitro transcribed with substitutions of uridine with pseudouridine (0%, 25%, 50% or 100%). Since ad-HEK293 cells do not express miR-126 or miR-143, we transfected with miR-126 mimic or miR-143 mimic 24 hr prior to transfection with GFP or GFP4x126TS mRNAs to ensure for miRNA/RISC assembly and GFP expression levels were assessed after 24 hr. In the control, unmodified (0% Pseudouridine) GFP transfected cells, the over-expression of miR-126-3p or miR-143 did not have any effect on GFP expression (FIGS.43A-43C). This was also observed in cells transfected with GFP-mRNA containing increasing percentage of Pseudouridine (FIGS.43A-43C). However, in cells transfected with GFP4x126TS mRNA, the over-expression of miR-126 and not miR-143 dramatically reduced the percentage of GFP positive cells and inhibited GFP expression. The miR-126-specific inhibition was not affected by increasing the percentage of pseudouridine substitution (FIGS.43A-43C). Example 9 It was also tested whether 100% substitution of both uridine and cytosine with pseudouridine and 5-methylcystidine nucleotides would affect miRNA-dependent gene silencing. To that end, GFP or GFP-4x126TS mRNA were in vitro transcribed with 100% pseudouridine and 100% 5-methylcystidine and transfected into Ad-HEK293 cells that were priory (24 hr) transfected with miR-126 or miR-143 mimics. The inhibitory effect of miR-126 on the expression of the double-modified GFP-4x126TS mRNA were compared to the unmodified mRNA after 24 hr. Cells transfected with pseudouridine and 5-methylcystidine modified mRNA, miR-126 and not miR-143 reduced the percentage of GFP positive cells and inhibited GFP expression to the same extent as the unmodified mRNA (FIGS.44A-44C). Thus, our data show that complete substitution of pseudouridine, or combination of pseudouridine and 5-methylcystidine modified mRNA, can still be targeted by microRNA-dependent silencing. Example 10 To test whether this miRNA dependent silencing of modified mRNAs is not limited to miR-126, GFP coding mRNA containing target sites for miR-21 or miR-145 was designed. Ad-HEH293 cells were transfected with miR-21, miR-145 or miR-143 mimics 24 hr prior to transfection with unmodified or pseudouridine and 5-methylcystidine modified GFP, GFP4x21TS or GFP4x145TS mRNAs to ensure for RISC assembly and the GFP expression levels were assessed after 24 hr. Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x21TS mRNA, the over-expression of miR-21 and not miR-145, miR-21 or moR-143 reduced the expression GFP to the same extent as cells transfected with unmodified GFP4x21TS mRNA (FIGS.45A-45C). Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x154TS mRNA, showed the same reduction in GFP expression as unmodified mRNA when miR-145 mimic was added and not when miR-21, miR-126 or miR-143 mimics were added (FIGS.45A-45C). In the control transfected cells with unmofdified or 100% substitution with Pseudouridine and 5-methylcystidine GFP mRNA (with no miRNA target sites), the over-expression of miR-21, miR-145 or miR-143 did not have an effect on GFP expression (FIGS.45A-45C). Example 11 Cell-Selective Gene Activation Using CRISPR/CAS9 Synergistic Activation Mediator (SAM) System As proof of principle the modified CRISPR/dCas9 Synergistic Activation Mediator (SAM) system (Konermann et al. 2015. Nature, 517:583-588) was employed that included the sgRNA2.0, MS2-p65-HSF1 and NLS-dCas9-VP64. To activate LIN28A in a cell selective manner, we added four tandem copies of a 22-bp target sequence perfectly complementary to the mature miR-126-3p strand at dCas9 3′UTR (dCas9-4x126TS). Ad-HEK293 cells were transfected with miR-126 mimic 24 hours prior to transfection with CRISPR-SAM components containing dCas9 or dCas9-4x126TS and the expression levels of LIN28A was assessed after 72 hours after. Transient transfected adHEK293 cells transfected with CRISPR-SAM components with dCas9 activated the transcription of LIN28A by >5000 fold while adHEK293 cells transfected with CRISPR-SAM and dCas9-4x126TS showed only 600-fold in LIN28A (FIG.46). | 116,075 |
11857599 | DETAILED DESCRIPTION OF THE INVENTION 1. Overview In part, the disclosure provides heteromultimers that comprise an ALK4 polypeptide and an ActRIIB polypeptide for use in treating spinal muscular atrophy (SMA). Preferably such ALK4 polypeptides comprise a ligand-binding domain of an ALK4 receptor and such ActRIIB polypeptides comprise a ligand-binding domain of an ActRIIB receptor. In certain preferred embodiments, ALK4:ActRIIB heteromultimers of the disclosure have an altered TGFβ superfamily ligand binding profile/specificity compared to a corresponding sample of a homomultimer (e.g., an ALK4:ActRIIB heterodimer compared to an ActRIIB:ActRIIB homodimer or an ALK4:ALK4 homodimer). The TGF-β superfamily is comprised of over 30 secreted factors including TGF-betas, activins, nodals, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and anti-Mullerian hormone (AMH) [Weiss et al. (2013) Developmental Biology, 2(1): 47-63]. Members of the superfamily, which are found in both vertebrates and invertebrates, are ubiquitously expressed in diverse tissues and function during the earliest stages of development throughout the lifetime of an animal. Indeed, TGF-β superfamily proteins are key mediators of stem cell self-renewal, gastrulation, differentiation, organ morphogenesis, and adult tissue homeostasis. Ligands of the TGF-beta superfamily share the same dimeric structure in which the central 3½ turn helix of one monomer packs against the concave surface formed by the beta-strands of the other monomer. The majority of TGF-beta family members are further stabilized by an intermolecular disulfide bond. This disulfide bonds traverses through a ring formed by two other disulfide bonds generating what has been termed a ‘cysteine knot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck et al. (2012) FEBS Letters 586: 1860-1870]. TGF-beta superfamily signaling is mediated by heteromeric complexes of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) upon ligand stimulation [Massagué (2000) Nat. Rev. Mol. Cell Biol. 1:169-178]. These type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase specificity. In general, type I receptors mediate intracellular signaling while the type II receptors are required for binding TGF-beta superfamily ligands. Type I and II receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors. The TGF-beta family can be divided into two phylogenetic branches based on the type I receptors they bind and the Smad proteins they activate. One is the more recently evolved branch, which includes, e.g., the TGF-betas, activins, GDF8, GDF9, GDF11, BMP3 and nodal, which signal through type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBS Letters 586:1860-1870]. The other branch comprises the more distantly related proteins of the superfamily and includes, e.g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7, which signal through Smads 1, 5, and 8. Activins are members of the TGF-beta superfamily and were initially discovered as regulators of secretion of follicle-stimulating hormone, but subsequently various reproductive and non-reproductive roles have been characterized. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related β subunits (βAβA, βBβB, and βAβB, respectively). The human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing βCor βEare also known. In the TGF-beta superfamily, activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos [DePaolo et al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997) Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963]. In several tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, in the regulation of follicle-stimulating hormone (FSH) secretion from the pituitary, activin promotes FSH synthesis and secretion, while inhibin reduces FSH synthesis and secretion. Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP, also known as FLRG or FSTL3), and α2-macroglobulin. As described herein, agents that bind to “activin A” are agents that specifically bind to the PA subunit, whether in the context of an isolated PA subunit or as a dimeric complex (e.g., a βAβAhomodimer or a βAβBheterodimer). In the case of a heterodimer complex (e.g., a βAβBheterodimer), agents that bind to “activin A” are specific for epitopes present within the PA subunit, but do not bind to epitopes present within the non-βAsubunit of the complex (e.g., the βBsubunit of the complex). Similarly, agents disclosed herein that antagonize (inhibit) “activin A” are agents that inhibit one or more activities as mediated by a PA subunit, whether in the context of an isolated βAsubunit or as a dimeric complex (e.g., a βAβAhomodimer or a βAβBheterodimer). In the case of βAβBheterodimers, agents that inhibit “activin A” are agents that specifically inhibit one or more activities of the PA subunit, but do not inhibit the activity of the non-βAsubunit of the complex (e.g., the βBsubunit of the complex). This principle applies also to agents that bind to and/or inhibit “activin B”, “activin C”, and “activin E”. Agents disclosed herein that antagonize “activin AB” are agents that inhibit one or more activities as mediated by the PA subunit and one or more activities as mediated by the βBsubunit. The same principle also applies to agent that bind to and/or inhibit “activin AC”, “activin BC”, “activin AE”, and “activin BE”. The BMPs and GDFs together form a family of cysteine-knot cytokines sharing the characteristic fold of the TGF-beta superfamily [Rider et al. (2010) Biochem J., 429(1):1-12]. This family includes, for example, BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4, BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known as GDF2), BMP10, BMP11 (also known as GDF11), BMP12 (also known as GDF7), BMP13 (also known as GDF6), BMP14 (also known as GDF5), BMP15, GDF1, GDF3 (also known as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, and decapentaplegic. Besides the ability to induce bone formation, which gave the BMPs their name, the BMP/GDFs display morphogenetic activities in the development of a wide range of tissues. BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signaling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists resemble TGF-beta superfamily ligands. As demonstrated herein, a soluble ALK4:ActRIIB heterodimer, which binds to various ligands including activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10, is effective in reducing the severity of muscle and bone loss in an SMA model. Moreover, ALK4:ActRIIB treatment increased muscle strength in SMA patients. While not wishing to be bound to any particular mechanism, it is expected that the effects of ALK4:ActRIIB heterodimers are caused primarily by a ALK4-ActRIIB signaling antagonist effect, particularly as mediated by one or more of activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10. Regardless of the mechanism, it is apparent from the data presented herein that ALK4:ActRIIB signaling antagonists do reduce the severity of muscle and bone loss as well as having other positivity effects in treating SMA. It should be noted that muscle and bone growth/loss are dynamic, with changes depending on a balance of factors that increase muscle/bone and factors that decrease muscle/bone. Muscle mass and bone density can be increased by increasing factors that increase muscle and bone growth; decreasing factors that inhibit muscle and bone growth; or both. The terms increasing muscle mass and bone density refer to the observable physical changes in bone and muscle tissues and are intended to be neutral as to the mechanism by which the changes occur. The animal models for SMA that was used in the studies described herein are considered to be predicative of efficacy in humans, and therefore, this disclosure provides methods for using ALK4:ActRIIB heterodimer and other ALK4:ActRIIB antagonists to treat SMA, particularly preventing or delaying onset and/or reducing the severity or duration of one or more complications of SMA, in humans. As disclosed herein, the term ALK4:ActRIIB antagonist refers a variety of agents that may be used to antagonize ALK4-ActRIIB signaling including, for example, antagonists that inhibit one or more ligands [e.g., activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10]; antagonists that inhibit one or more type I- or type II-(e.g., ALK4 and ActRIIB); and antagonists that inhibit one or more downstream signaling components (e.g., Smad proteins such as Smads 2 and 3). ALK4:ActRIIB antagonists to be used in accordance with the methods and uses of the disclosure include a variety of forms, for example, ligand traps (e.g., ALK4:ActRIIB heteromultimers), antibody antagonists (e.g., antibodies that inhibit one or more of ALK4, ActRIIB, activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10), small molecule antagonists [e.g., small molecules that inhibit one or more of ALK4, ActRIIB, activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10 and one or more Smad proteins (e.g., Smads 2 and 3)], and nucleotide antagonists [e.g., nucleotide sequences that inhibit one or more of ALK4, ActRIIB, activin A, activin B, GDF11, GDF8, BMP6, GDF3, and BMP10 and one or more Smad proteins (e.g., Smads 2 and 3)]. The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which it is used. The terms “heteromer” or “heteromultimer” refer to a protein complex comprising at least a first polypeptide chain and a second polypeptide chain, wherein the second polypeptide chain differs in amino acid sequence from the first polypeptide chain by at least one amino acid residue. The heteromer can comprise a “heterodimer” formed by the first and second polypeptide chains or can form higher order structures where one or more polypeptide chains in addition to the first and second polypeptide chains are present. Exemplary structures for the heteromultimer include heterodimers, heterotrimers, heterotetramers and further oligomeric structures. Heterodimers are designated herein as X:Y or equivalently as X-Y, where X represents a first polypeptide chain and Y represents a second polypeptide chain. Higher-order heteromers and oligomeric structures are designated herein in a corresponding manner. In certain embodiments a heteromultimer is recombinant (e.g., one or more polypeptide components may be a recombinant protein), isolated and/or purified. “Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin. The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin. “Percent (%) sequence identity” with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid (nucleic acid) sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. “Agonize”, in all its grammatical forms, refers to the process of activating a protein and/or gene (e.g., by activating or amplifying that protein's gene expression or by inducing an inactive protein to enter an active state) or increasing a protein's and/or gene's activity. “Antagonize”, in all its grammatical forms, refers to the process of inhibiting a protein and/or gene (e.g., by inhibiting or decreasing that protein's gene expression or by inducing an active protein to enter an inactive state) or decreasing a protein's and/or gene's activity. The terms “about” and “approximately” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is ±10%. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably <5-fold and more preferably <2-fold of a given value. Numeric ranges disclosed herein are inclusive of the numbers defining the ranges. The terms “a” and “an” include plural referents unless the context in which the term is used clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two or more specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 2. ALK4:ActRIIB Heteromultimers In certain aspects, the present disclosure relates to heteromultimers comprising one or more ALK4 receptor polypeptides (e.g., SEQ ID NOs: 9, 10, 19, 20, 42, 44, 47, 48, 74, and 76) and one or more ActRIIB receptor polypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, 46, 70, 72, 78, and 80) which are generally referred to herein as “ALK4:ActRIIB heteromultimer complexes” or “ALK4:ActRIIB heteromultimers”. Preferably, ALK4:ActRIIB heteromultimers of the disclosure are soluble, for example, a heteromultimer may comprises a soluble portion (domain) of an ALK4 receptor and a soluble portion (domain) of an ActRIIB receptor. In general, the extracellular domains of ALK4 and ActRIIB correspond to a soluble portion of these receptors. Therefore, in some embodiments, heteromultimers of the disclosure comprise an extracellular domain of an ALK4 receptor and an extracellular domain of an ActRIIB receptor. Example extracellular domains ALK4 and ActRIIB receptors are disclosed herein and such sequences, as well as fragments, functional variants, and modified forms thereof, may be used in accordance with the inventions of the disclosure (e.g., ALK4:ActRIIB heteromultimer compositions and uses thereof). ALK4:ActRIIB heteromultimers of the disclosure include, e.g., heterodimers, heterotrimers, heterotetramers and higher order oligomeric structures. See, e.g.,FIG.6. In certain preferred embodiments, heteromultimers of the disclosure are ALK4:ActRIIB heterodimers. Preferably, ALK4:ActRIIB heteromultimers of the disclosure bind to one or more TGF-beta superfamily ligands. In some embodiments, ALK4:ActRIIB heteromultimers may bind to one or more of activin (e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8, GDF11, BMP6, GDF3, and BMP10. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin A. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin B. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin C. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin E. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin AB. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin AC. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin AE. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin BC. In some embodiments, ALK4:ActRIIB heteromultimers bind to activin BE. In some embodiments, ALK4:ActRIIB heteromultimers bind to GDF11. In some embodiments, ALK4:ActRIIB heteromultimers bind to GDF8. In some embodiments, ALK4:ActRIIB heteromultimers bind to BMP6. In some embodiments, ALK4:ActRIIB heteromultimers bind to GDF3. In some embodiments, ALK4:ActRIIB heteromultimers bind to BMP10. In some embodiments, ALK4:ActRIIB heteromultimers do not bind to, or no not substantially bind to BMP9 (e.g., have indeterminate Kaor Kddue to the transient nature of the interaction between BMP9 and an ALK4:ActRIIB heteromultimer). In some embodiments, ALK4:ActRIIB heteromultimers binds with stronger affinity to activin B compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers binds with weaker affinity to GDF3 compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers binds with weaker affinity to BMP9 compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers binds with weaker affinity to BMP10 compared to a corresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIB heteromultimers may further bind to one or more of BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7, GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty. In some embodiments, ALK4:ActRIIB heteromultimers may be used to inhibit (antagonize) signaling (e.g., Smad 2/3 and/or Smad 1/5/8 signaling) mediated by one or more TGFβ superfamily ligands. In particular, ALK4:ActRIIB heteromultimers of the disclosure may be used to inhibit intracellular signaling by one or more TGFβ superfamily ligands in, for example, a cell-based assay such as those described herein. For example, ALK4:ActRIIB heteromultimers may inhibit signaling mediated by one or more of activin (e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8, GDF11, BMP6, GDF3, and BMP10 in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin A signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin B signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin C signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin D signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin E signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin AB signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin AC signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin BC signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin AE signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit activin BE signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit GDF11 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit GDF8 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit BMP6 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit GDF3 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibit BMP9 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers do not inhibit, or do not substantially inhibit BMP9 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors of activin B signaling in a cell-based assay compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers are weaker inhibitors of BMP10 signaling in a cell-based assay compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors of GDF3 signaling in a cell-based assay compared to a corresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors of BMP9 signaling in a cell-based assay compared to a corresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIB heteromultimers may further inhibit intracellular signaling by one or more of BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7, GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty in a cell-based assay. As used herein, the term “ActRIIB” refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification. Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity. The term “ActRIIB polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIB polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication Nos. WO 2006/012627, WO 2008/097541, and WO 2010/151426, which are incorporated herein by reference in their entirety. Numbering of amino acids for all ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence provided below (SEQ ID NO: 1), unless specifically designated otherwise. The human ActRIIB precursor protein sequence is as follows: (SEQ ID NO: 1)1MTAPWVALAL LWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCE51GEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVY101FCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLA YSLLPIGGLS151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV501TNVDLPPKES SI The signal peptide is indicated with asingle underline; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated with adouble underline. The processed extracellular ActRIIB polypeptide sequence is as follows: (SEQ ID NO: 2)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT In some embodiments, the protein may be produced with an “SGR . . . ” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated by asingle underline. The sequence with the “tail” deleted (a Δ15 sequence) is as follows: (SEQ ID NO: 3)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64) is also reported in the literature See, e.g., Hilden et al. (1994) Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fc fusion protein comprising an extracellular domain of ActRIIB with the A64 substitution has a relatively low affinity for activin and GDF11. By contrast, the same ActRIIB-Fc fusion protein with an arginine at position 64 (R64) has an affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, sequences with an R64 are used as the “wild-type” reference sequence for human ActRIIB in this disclosure. The form of ActRIIB with an alanine at position 64 is as follows: (SEQ ID NO: 4)1MTAPWVALAL LWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCE51GEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVY101FCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLA YSLLPIGGLS151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV501TNVDLPPKES SI The signal peptide is indicated bysingle underlineand the extracellular domain is indicated by bold font. The processed extracellular ActRIIB polypeptide sequence of the alternative A64 form is as follows: (SEQ ID NO: 5)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT In some embodiments, the protein may be produced with an “SGR . . . ” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated bysingle underline. The sequence with the “tail” deleted (a Δ15 sequence) is as follows: (SEQ ID NO: 6)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA A nucleic acid sequence encoding the human ActRIIB precursor protein is shown below (SEQ ID NO: 7), representing nucleotides 25-1560 of Genbank Reference Sequence NM_001106.3, which encode amino acids 1-513 of the ActRIIB precursor. The sequence as shown provides an arginine at position 64 and may be modified to provide an alanine instead. The signal sequence is underlined. (SEQ ID NO: 7)1ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC51CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG101CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA151GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC201TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT251GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC301TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC351AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA401CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC451CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA501CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC551TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC601TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA651GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT701TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC751GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT801CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT851GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC901CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT951TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA1001CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA1051CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC1101TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA1151TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC1201AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA1251GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA1301AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG1351GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC1401TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT1451CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC1501ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC A nucleic acid sequence encoding processed extracellular human ActRIIB polypeptide is as follows (SEQ ID NO: 8). The sequence as shown provides an arginine at position 64, and may be modified to provide an alanine instead. (SEQ ID NO: 8)1GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT251GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC An alignment of the amino acid sequences of human ActRIIB extracellular domain and human ActRIIA extracellular domain are illustrated inFIG.1. This alignment indicates amino acid residues within both receptors that are believed to directly contact ActRII ligands. For example, the composite ActRII structures indicated that the ActRIIB-ligand binding pocket is defined, in part, by residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through F101. At these positions, it is expected that conservative mutations will be tolerated. In addition, ActRIIB is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. For example,FIG.2depicts a multi-sequence alignment of a human ActRIIB extracellular domain compared to various ActRIIB orthologs. Many of the ligands that bind to ActRIIB are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIB-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant of substitution without significantly altering normal ActRIIB-ligand binding activities. Therefore, an active, human ActRIIB variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIB, or may include a residue that is similar to that in the human or other vertebrate sequences. Without meaning to be limiting, the following examples illustrate this approach to defining an active ActRIIB variant. L46 in the human extracellular domain (SEQ ID NO: 53) is a valine inXenopusActRIIB (SEQ ID NO: 55), and so this position may be altered, and optionally may be altered to another hydrophobic residue, such as V, I or F, or a non-polar residue such as A. E52 in the human extracellular domain is a K inXenopus, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y and probably A. T93 in the human extracellular domain is a K inXenopus, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, K, R, E, D, H, G, P, G and Y. F108 in the human extracellular domain is a Y inXenopus, and therefore Y or other hydrophobic group, such as I, V or L should be tolerated. E111 in the human extracellular domain is K inXenopus, indicating that charged residues will be tolerated at this position, including D, R, K and H, as well as Q and N. R112 in the human extracellular domain is K inXenopus, indicating that basic residues are tolerated at this position, including R and H. A at position 119 in the human extracellular domain is relatively poorly conserved, and appears as P in rodents and V inXenopus, thus essentially any amino acid should be tolerated at this position. Moreover, ActRII proteins have been characterized in the art in terms of structural and functional characteristics, particularly with respect to ligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and 7,842,663]. In addition to the teachings herein, these references provide amply guidance for how to generate ActRIIB variants that retain one or more normal activities (e.g., ligand-binding activity). For example, a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Accordingly, the core ligand-binding domains of human ActRIIB, as demarcated by the outermost of these conserved cysteines, corresponds to positions 29-109 of SEQ ID NO: 1 (ActRIIB precursor). Thus, the structurally less-ordered amino acids flanking these cysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 residues at the N-terminus and/or by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary ActRIIB extracellular domains for N-terminal and/or C-terminal truncation include SEQ ID NOs: 2, 3, 5, and 6. Attisano et al. showed that a deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduced the affinity of the receptor for activin. An ActRIIB-Fc fusion protein containing amino acids 20-119 of present SEQ ID NO: 1, “ActRIIB(20-119)-Fc”, has reduced binding to GDF11 and activin relative to an ActRIIB(20-134)-Fc, which includes the proline knot region and the complete juxtamembrane domain (see, e.g., U.S. Pat. No. 7,842,663). However, an ActRIIB(20-129)-Fc protein retains similar, but somewhat reduced activity, relative to the wild-type, even though the proline knot region is disrupted. Thus, ActRIIB extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 (with respect to SEQ ID NO: 1) are all expected to be active, but constructs stopping at 134 or 133 may be most active. Similarly, mutations at any of residues 129-134 (with respect to SEQ ID NO: 1) are not expected to alter ligand-binding affinity by large margins. In support of this, it is known in the art that mutations of P129 and P130 (with respect to SEQ ID NO: 1) do not substantially decrease ligand binding. Therefore, an ActRIIB polypeptide of the present disclosure may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected to have reduced ligand binding. Amino acid 119 (with respect to present SEQ ID NO:1) is poorly conserved and so is readily altered or truncated. ActRIIB polypeptides ending at 128 (with respect to SEQ ID NO: 1) or later should retain ligand-binding activity. ActRIIB polypeptides ending at or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127), with respect to SEQ ID NO: 1, will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting. At the N-terminus of ActRIIB, it is expected that a protein beginning at amino acid 29 or before (with respect to SEQ ID NO: 1) will retain ligand-binding activity. Amino acid 29 represents the initial cysteine. An alanine-to-asparagine mutation at position 24 (with respect to SEQ ID NO: 1) introduces an N-linked glycosylation sequence without substantially affecting ligand binding [U.S. Pat. No. 7,842,663]. This confirms that mutations in the region between the signal cleavage peptide and the cysteine cross-linked region, corresponding to amino acids 20-29, are well tolerated. In particular, ActRIIB polypeptides beginning at position 20, 21, 22, 23, and 24 (with respect to SEQ ID NO: 1) should retain general ligand-biding activity, and ActRIIB polypeptides beginning at positions 25, 26, 27, 28, and 29 (with respect to SEQ ID NO: 1) are also expected to retain ligand-biding activity. It has been demonstrated, e.g., U.S. Pat. No. 7,842,663, that, surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 will have the most activity. Taken together, a general formula for an active portion (e.g., ligand-binding portion) of ActRIIB comprises amino acids 29-109 of SEQ ID NO: 1. Therefore ActRIIB polypeptides may, for example, comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at a residue corresponding to any one of amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding to any one amino acids 109-134 (e.g., ending at any one of amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1. Other examples include polypeptides that begin at a position from 20-29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or 21-29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and end at a position from 119-134 (e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-133 (e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133), 129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134), or 129-133 (e.g., any one of positions 129, 130, 131, 132, or 133) of SEQ ID NO: 1. Other examples include constructs that begin at a position from 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24 (e.g., any one of positions 21, 22, 23, or 24), or 22-25 (e.g., any one of positions 22, 22, 23, or 25) of SEQ ID NO: 1 and end at a position from 109-134 (e.g., any one of positions 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-134 (e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) or 129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1. Variants within these ranges are also contemplated, particularly those having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO: 1. The variations described herein may be combined in various ways. In some embodiments, ActRIIB variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10 or 15 conservative amino acid changes in the ligand-binding pocket, and zero, one, or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket. Sites outside the binding pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO: 1). An asparagine-to-alanine alteration at position 65 (N65A) actually improves ligand binding in the A64 background, and is thus expected to have no detrimental effect on ligand binding in the R64 background [U.S. Pat. No. 7,842,663]. This change probably eliminates glycosylation at N65 in the A64 background, thus demonstrating that a significant change in this region is likely to be tolerated. While an R64A change is poorly tolerated, R64K is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64 [U.S. Pat. No. 7,842,663]. Additionally, the results of the mutagenesis program described in the art indicate that there are amino acid positions in ActRIIB that are often beneficial to conserve. With respect to SEQ ID NO: 1, these include position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, and particularly tryptophan), position 37 (acidic, and particularly aspartic or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus, the disclosure provides a framework of amino acids that may be conserved in ActRIIB polypeptides. Other positions that may be desirable to conserve are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), 98 (polar or charged, particularly E, D, R or K), all with respect to SEQ ID NO: 1. In certain embodiments, the disclosure relates to heteromultimers that comprise at least one ActRIIB polypeptide, which includes fragments, functional variants, and modified forms thereof. Preferably, ActRIIB polypeptides for use in accordance with inventions of the disclosure are soluble (e.g., an extracellular domain of ActRIIB) In other preferred embodiments, ActRIIB polypeptides for use in accordance with the disclosure bind to one or more TGF-beta superfamily ligands. Therefore, in some embodiments, ActRIIB polypeptides for use in accordance with the disclosure inhibit (antagonize) activity (e.g., inhibition of Smad signaling) of one or more TGF-beta superfamily ligands. In some embodiments, heteromultimers of the disclosure comprise at least one ActRIIB polypeptide that comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at a residue corresponding to amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding to amino acids 109-134 (e.g., ending at any one of amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1. In certain preferred embodiments, heteromultimers of the disclosure comprise at least one ActRIIB polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 29-109 of SEQ ID NO: 1 In other preferred embodiments, heteromultimer complexes of the disclosure comprise at least one ActRIIB polypeptide that comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 25-131 of SEQ ID NO: 1. In some embodiments, heteromultimers of the disclosure comprise at least one ActRIIB polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, or 46. In certain preferred embodiments, heteromultimers of the disclosure comprise do not comprise an ActRIIB polypeptide wherein the position corresponding to L79 of SEQ ID NO: 1 is an acidic amino acid (i.e., is not a naturally occurring D or E amino acid residue or artificial acidic amino acid). In certain aspects, the present disclosure relates to protein complexes that comprise an ALK4 polypeptide. As used herein, the term “ALK4” refers to a family of activin receptor-like kinase-4 proteins from any species and variants derived from such ALK4 proteins by mutagenesis or other modification. Reference to ALK4 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK4 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity. The term “ALK4 polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ALK4 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Numbering of amino acids for all ALK4-related polypeptides described herein is based on the numbering of the human ALK4 precursor protein sequence below (SEQ ID NO: 9), unless specifically designated otherwise. A human ALK4 precursor protein sequence (NCBI Ref Seq NP_004293) is as follows: (SEQ ID NO: 9)1MAESAGASSF FPLVVLLLAG SGGSGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLD61GMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPS121MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ RLDMEDPSCE MCLSKDKTLQ181DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE VWRGRWRGGD VAVKIFSSRE241ERSWFREAEI YQTVMLRHEN ILGFIAADNK DNGTWTQLWL VSDYHEHGSL FDYLNRYTVT301IEGMIKLALS AASGLAHLHM EIVGTQGKPG IAHRDLKSKN ILVKKNGMCA IADLGLAVRH361DAVTDTIDIA PNQRVGTKRY MAPEVLDETI NMKHFDSFKC ADIYALGLVY WEIARRCNSG421GVHEEYQLPY YDLVPSDPSI EEMRKVVCDQ KLRPNIPNWW QSYEALRVMG KMMRECWYAN481GAARLTALRI KKTLSQLSVQ EDVKI The signal peptide is indicated by asingle underlineand the extracellular domain is indicated in bold font. A processed extracellular human ALK4 polypeptide sequence is as follows: (SEQ ID NO: 10)SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWGPVE A nucleic acid sequence encoding the ALK4 precursor protein is shown below (SEQ ID NO: 11), corresponding to nucleotides 78-1592 of Genbank Reference Sequence NM_004302.4. The signal sequence isunderlinedand the extracellular domain is indicated in bold font. (SEQ ID NO: 11)ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGGTCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAGGAAGACGTGAAGATC A nucleic acid sequence encoding an extracellular ALK4 polypeptide is as follows: (SEQ ID NO: 12)TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAG An alternative isoform of human ALK4 precursor protein sequence, isoform C (NCBI Ref Seq NP_064733.3), is as follows: (SEQ ID NO: 19)1MAESAGASSF FPLVVLLLAG SGGSGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLD61GMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPS121MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ RLDMEDPSCE MCLSKDKTLQ181DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE VWRGRWRGGD VAVKIFSSRE241ERSWFREAEI YQTVMLRHEN ILGFIAADNK ADCSFLTLPW EVVMVSAAPK LRSLRLQYKG301GRGRARFLFP LNNGTWTQLW LVSDYHEHGS LFDYLNRYTV TIEGMIKLAL SAASGLAHLH361MEIVGTQGKP GIAHRDLKSK NILVKKNGMC AIADLGLAVR HDAVTDTIDI APNQRVGTKR421YMAPEVLDET INMKHFDSFK CADIYALGLV YWEIARRCNS GGVHEEYQLP YYDLVPSDPS481IEEMRKVVCD QKLRPNIPNW WQSYEALRVM GKMMRECWYA NGAARLTALR IKKTLSQLSV541QEDVKI The signal peptide is indicated by asingle underlineand the extracellular domain is indicated in bold font. A processed extracellular ALK4 polypeptide sequence (isoform C) is as follows: (SEQ ID NO: 20)SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWGPVE A nucleic acid sequence encoding the ALK4 precursor protein (isoform C) is shown below (SEQ ID NO: 21), corresponding to nucleotides 78-1715 of Genbank Reference Sequence NM_020328.3. The signal sequence isunderlinedand the extracellular domain is indicated in bold font. (SEQ ID NO: 21)ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGGTCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGCAGACTGCTCATTCCTCACATTGCCATGGGAAGTTGTAATGGTCTCTGCTGCCCCCAAGCTGAGGAGCCTTAGACTCCAATACAAGGGAGGAAGGGGAAGAGCAAGATTTTTATTCCCACTGAATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAGGAAGACGTGAAGATC A nucleic acid sequence encoding an extracellular ALK4 polypeptide (isoform C) is as follows: (SEQ ID NO: 22)TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAG In certain embodiments, the disclosure relates to heteromultimers that comprise at least one ALK4 polypeptide, which includes fragments, functional variants, and modified forms thereof. Preferably, ALK4 polypeptides for use in accordance with inventions of the disclosure (e.g., heteromultimers comprising an ALK4 polypeptide and uses thereof) are soluble (e.g., an extracellular domain of ALK4). In other preferred embodiments, ALK4 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., Smad signaling) of one or more TGF-beta superfamily ligands. In some embodiments, heteromultimers of the disclosure comprise at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9, 10, 19, 20, 42, 44, 47, 48, 74, and 76. In some embodiments, heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9, 10, 19, 20, 42, 44, 47, 48, 74, and 76. ALK4 is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. For example,FIG.5depicts a multi-sequence alignment of a human ALK4 extracellular domain compared to various ALK4 orthologs. Many of the ligands that bind to ALK4 are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ALK4-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ALK4-ligand binding activities. Therefore, an active, human ALK4 variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ALK4, or may include a residue that is similar to that in the human or other vertebrate sequences. Without meaning to be limiting, the following examples illustrate this approach to defining an active ALK4 variant. V6 in the human ALK4 extracellular domain (SEQ ID NO: 59) is isoleucine inMus muculusALK4 (SEQ ID NO: 63), and so the position may be altered, and optionally may be altered to another hydrophobic residue such as L, I, or F, or a non-polar residue such as A, as is observed inGallus gallusALK4 (SEQ ID NO: 62). E40 in the human extracellular domain is K inGallus gallusALK4, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y, and probably a non-polar residue such as A. S15 in the human extracellular domain is D inGallus gallusALK4, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, T, R, E, K, H, G, P, G and Y. E40 in the human extracellular domain is K inGallus gallusALK4, indicating that charged residues will be tolerated at this position, including D, R, K, H, as well as Q and N. R80 in the human extracellular domain is K inCondylura cristataALK4 (SEQ ID NO: 60), indicating that basic residues are tolerated at this position, including R, K, and H. Y77 in the human extracellular domain is F inSus scrofaALK4 (SEQ ID NO: 64), indicating that aromatic residues are tolerated at this position, including F, W, and Y. P93 in the human extracellular domain is relatively poorly conserved, appearing as S inErinaceus europaeusALK4 (SEQ ID NO: 61) and N inGallus gallusALK4, thus essentially any amino acid should be tolerated at this position. Moreover, ALK4 proteins have been characterized in the art in terms of structural and functional characteristics, particularly with respect to ligand binding [e.g., Harrison et al. (2003) J Biol Chem 278(23):21129-21135; Romano et al. (2012) J Mol Model 18(8):3617-3625; and Calvanese et al. (2009) 15(3):175-183]. In addition to the teachings herein, these references provide amply guidance for how to generate ALK4 variants that retain one or more normal activities (e.g., ligand-binding activity). For example, a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Accordingly, the core ligand-binding domains of human ALK4, as demarcated by the outermost of these conserved cysteines, corresponds to positions 34-101 of SEQ ID NO: 9 (ALK4 precursor). Thus, the structurally less-ordered amino acids flanking these cysteine-demarcated core sequences can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, residues at the N-terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 residues at the C-terminus without necessarily altering ligand binding. Exemplary ALK4 extracellular domains for N-terminal and/or C-terminal truncation include SEQ ID NOs: 10 and 20. Accordingly, a general formula for an active portion (e.g., a ligand-binding portion) of ALK4 comprises amino acids 34-101. Therefore ALK4 polypeptides may, for example, comprise, consists essentially of, or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ALK4 beginning at a residue corresponding to any one of amino acids 24-34 (e.g., beginning at any one of amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO: 1 and ending at a position corresponding to any one amino acids 101-126 (e.g., ending at any one of amino acids 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126) of SEQ ID NO: 9. Other examples include constructs that begin at a position from 24-34 (e.g., any one of positions 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), 25-34 (e.g., any one of positions 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), or 26-34 (e.g., any one of positions 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO: 9 and end at a position from 101-126 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126), 102-126 (e.g., any one of positions 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126), 101-125 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125), 101-124 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124), 101-121 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121), 111-126 (e.g., any one of positions 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126), 111-125 (e.g., any one of positions 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125), 111-124 (e.g., any one of positions 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124), 121-126 (e.g., any one of positions 121, 122, 123, 124, 125, or 126), 121-125 (e.g., any one of positions 121, 122, 123, 124, or 125), 121-124 (e.g., any one of positions 121, 122, 123, or 124), or 124-126 (e.g., any one of positions 124, 125, or 126) of SEQ ID NO: 9. Variants within these ranges are also contemplated, particularly those having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO: 9. The variations described herein may be combined in various ways. In some embodiments, ALK4 variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10 or 15 conservative amino acid changes in the ligand-binding pocket. Sites outside the binding pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above), In some embodiments, the present disclosure contemplates making functional variants by modifying the structure of an ALK4 polypeptide and/or an ActRIIB polypeptide. Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the disclosure results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide, or to bind to one or more TGF-beta superfamily ligands including, for example, BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, activin BE, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty. In some embodiments, the present disclosure contemplates making functional variants by modifying the structure of an ALK4 and/or ActRIIB polypeptide for such purposes as enhancing therapeutic efficacy or stability (e.g., increase shelf-life and/or resistance to proteolytic degradation). In some embodiments, the present disclosure contemplates specific mutations of an ALK4 polypeptide and/or an ActRIIB polypeptide so as to alter the glycosylation of the polypeptide. Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where “X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the polypeptide (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on a polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylation may involve, for example, exposure of a polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. The sequence of a polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect, and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, heteromeric complexes of the present disclosure for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well. The present disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an ALK4 and/or an ActRIIB polypeptide as well as truncation mutants. Pools of combinatorial mutants are especially useful for identifying functionally active (e.g., TGF-beta superfamily ligand binding) ALK4 and/or ActRIIB sequences. The purpose of screening such combinatorial libraries may be to generate, for example, polypeptides variants which have altered properties, such as altered pharmacokinetic or altered ligand binding. A variety of screening assays are provided below, and such assays may be used to evaluate variants. For example, ALK4:ActRIIB complex variants may be screened for ability to bind to one or more TGF-beta superfamily ligands to prevent binding of a TGF-beta superfamily ligand to a TGF-beta superfamily receptor, and/or to interfere with signaling caused by an TGF-beta superfamily ligand. The activity of a ALK4:ActRIIB heteromultimer may be tested, for example, in a cell-based or in vivo assay. For example, the effect of an ALK4:ActRIIB heteromultimer on the expression of genes or activity of proteins involved in muscle production in a muscle cell may be assessed. This may, as needed, be performed in the presence of one or more TGF-beta superfamily ligands, and cells may be transfected so as to produce an ALK4:ActRIIB heteromultimer, and optionally, a TGF-beta superfamily ligand. Likewise, an ALK4:ActRIIB heteromultimer may be administered to a mouse or other animal, and one or more measurements, such as muscle formation and strength may be assessed using art-recognized methods. Similarly, the activity of an ALK4:ActRIIB heteromultimer, or variants thereof, may be tested, for example, in osteoblasts, adipocytes, and/or neuronal cells for any effect on growth of these cells, for example, by the assays as described herein and those of common knowledge in the art. A SMAD-responsive reporter gene may be used in such cell lines to monitor effects on downstream signaling. Combinatorial-derived variants can be generated which have increased selectivity or generally increased potency relative to a reference ALK4:ActRIIB heteromultimer. Such variants, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding unmodified ALK4:ActRIIB heteromultimer. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction, or otherwise inactivation, of an unmodified polypeptide. Such variants, and the genes which encode them, can be utilized to alter polypeptide complex levels by modulating the half-life of the polypeptide. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant polypeptide complex levels within the cell. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fc portion to alter one or more activities of the ALK4:ActRIIB heteromultimer including, for example, immunogenicity, half-life, and solubility. A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ALK4 and/or ActRIIB sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ALK4 and/or ActRIIB encoding nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art [Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res. 11:477]. Such techniques have been employed in the directed evolution of other proteins [Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815]. Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, ALK4:ActRIIB heteromultimers can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], by linker scanning mutagenesis [Gustin et al. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316], by saturation mutagenesis [Meyers et al., (1986) Science 232:613]; by PCR mutagenesis [Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by random mutagenesis, including chemical mutagenesis [Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of ALK4 and/or ActRIIB polypeptides. A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ALK4:ActRIIB heteromultimers. The most widely used techniques for screening large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Preferred assays include TGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, activin BE, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty) binding assays and/or TGF-beta ligand-mediated cell signaling assays. In certain embodiments, ALK4:ActRIIB heteromultimers may further comprise post-translational modifications in addition to any that are naturally present in the ALK4 and/or ActRIIB polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, ALK4:ActRIIB heteromultimers may comprise non-amino acid elements, such as polyethylene glycols, lipids, polysaccharide or monosaccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a heteromultimer complex may be tested as described herein for other heteromultimer variants. When a polypeptide of the disclosure is produced in cells by cleaving a nascent form of the polypeptide, post-translational processing may also be important for correct folding and/or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ALK4 and/or ActRIIB polypeptide as well as heteromultimers comprising the same. In certain preferred embodiments, heteromultimers described herein comprise at least one ALK4 polypeptide associated, covalently or non-covalently, with at least one ActRIIB polypeptide. Preferably, polypeptides disclosed herein form heterodimeric complexes, although higher order heteromultimeric complexes are also included such as, but not limited to, heterotrimers, heterotetramers, and further oligomeric structures (see, e.g.,FIG.6). In some embodiments, ALK4 and/or ActRIIB polypeptides comprise at least one multimerization domain. As disclosed herein, the term “multimerization domain” refers to an amino acid or sequence of amino acids that promote covalent or non-covalent interaction between at least a first polypeptide and at least a second polypeptide. Polypeptides disclosed herein may be joined covalently or non-covalently to a multimerization domain. Preferably, a multimerization domain promotes interaction between a first polypeptide (e.g., an ALK4 polypeptide) and a second polypeptide (e.g., an ActRIIB polypeptide) to promote heteromultimer formation (e.g., heterodimer formation), and optionally hinders or otherwise disfavors homomultimer formation (e.g., homodimer formation), thereby increasing the yield of desired heteromultimer (see, e.g.,FIG.6). Many methods known in the art can be used to generate ALK4:ActRIIB heteromultimers. For example, non-naturally occurring disulfide bonds may be constructed by replacing on a first polypeptide (e.g., an ALK4 polypeptide) a naturally occurring amino acid with a free thiol-containing residue, such as cysteine, such that the free thiol interacts with another free thiol-containing residue on a second polypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond is formed between the first and second polypeptides. Additional examples of interactions to promote heteromultimer formation include, but are not limited to, ionic interactions such as described in Kjaergaard et al., WO2007147901; electrostatic steering effects such as described in Kannan et al., U.S. Pat. No. 8,592,562; coiled-coil interactions such as described in Christensen et al., U.S.20120302737; leucine zippers such as described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584; and helix-turn-helix motifs such as described in Pack et al., (1993) Bio/Technology 11: 1271-1277. Linkage of the various segments may be obtained via, e.g., covalent binding such as by chemical cross-linking, peptide linkers, disulfide bridges, etc., or affinity interactions such as by avidin-biotin or leucine zipper technology. In certain aspects, a multimerization domain may comprise one component of an interaction pair. In some embodiments, the polypeptides disclosed herein may form protein complexes comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of an ALK4 polypeptide and the amino acid sequence of a first member of an interaction pair; and the second polypeptide comprises the amino acid sequence of an ActRIIB polypeptide and the amino acid sequence of a second member of an interaction pair. The interaction pair may be any two polypeptide sequences that interact to form a complex, particularly a heterodimeric complex although operative embodiments may also employ an interaction pair that can form a homodimeric complex. One member of the interaction pair may be fused to an ALK4 or ActRIIB polypeptide as described herein, including for example, a polypeptide sequence comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 2, 3, 5, 6, 10, and 20. An interaction pair may be selected to confer an improved property/activity such as increased serum half-life, or to act as an adaptor on to which another moiety is attached to provide an improved property/activity. For example, a polyethylene glycol moiety may be attached to one or both components of an interaction pair to provide an improved property/activity such as improved serum half-life. The first and second members of the interaction pair may be an asymmetric pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. Accordingly, first and second members of an asymmetric interaction pair may associate to form a heterodimeric complex (see, e.g.,FIG.6). Alternatively, the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference and thus may have the same or different amino acid sequences. Accordingly, first and second members of an unguided interaction pair may associate to form a homodimer complex or a heterodimeric complex. Optionally, the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates covalently with the second member of the interaction pair. Optionally, the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates non-covalently with the second member of the interaction pair. As specific examples, the present disclosure provides fusion proteins comprising ALK4 or ActRIIB fused to a polypeptide comprising a constant domain of an immunoglobulin, such as a CH1, CH2, or CH3 domain of an immunoglobulin or an Fc domain. Fc domains derived from human IgG1, IgG2, IgG3, and IgG4 are provided herein. Other mutations are known that decrease either CDC or ADCC activity, and collectively, any of these variants are included in the disclosure and may be used as advantageous components of a heteromultimeric complex of the disclosure. Optionally, the IgG1 Fc domain of SEQ ID NO: 31 has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered in accordance with the corresponding full-length IgG1). In certain cases, the mutant Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of binding to the Fcγ receptor relative to a wildtype Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., Asn-434 mutation) has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc domain. An example of a native amino acid sequence that may be used for the Fc portion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 31). Dotted underline indicates the hinge region, and solid underline indicates positions with naturally occurring variants. In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31. Naturally occurring variants in G1Fc would include E134D and M136L according to the numbering system used in SEQ ID NO: 31 (see Uniprot P01857). (SEQ ID NO: 31)151VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK An example of a native amino acid sequence that may be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 32). Dotted underline indicates the hinge region and double underline indicates positions where there are data base conflicts in the sequence (according to UniProt P01859). In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32. (SEQ ID NO: 32)151FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS201CSVMHEALHN HYTQKSLSLS PGK Two examples of amino acid sequences that may be used for the Fc portion of human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be up to four times as long as in other Fc chains and contains three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 33) contains a short hinge region consisting of a single 15-residue segment, whereas the second G3Fc sequence (SEQ ID NO: 34) contains a full-length hinge region. In each case, dotted underline indicates the hinge region, and solid underline indicates positions with naturally occurring variants according to UniProt P01859. In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 33 and 34. (SEQ ID NO: 33)151VSHEDPEVQF KWYVDGVEVH NAKTKPREEQYNSTFRVVSV LTVLHQDWLN101GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL151TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS201RWQQGNIFSC SVMHEALHNRFTQKSLSLSP GK(SEQ ID NO: 34)151101EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV LHQDWLNGKE151YKCKVSNKAL PAPIEKTISKTKGQPREPQV YTLPPSREEM TKNQVSLTCL201VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ251QGNIFSCSVM HEALHNRFTQ KSLSLSPGK Naturally occurring variants in G3Fc (for example, see Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, F221Y when converted to the numbering system used in SEQ ID NO: 33, and the present disclosure provides fusion proteins comprising G3Fc domains containing one or more of these variations. In addition, the human immunoglobulin IgG3 gene (IGHG3) shows a structural polymorphism characterized by different hinge lengths [see Uniprot P01859]. Specifically, variant WIS is lacking most of the V region and all of the CH1 region. It has an extra interchain disulfide bond at position 7 in addition to the 11 normally present in the hinge region. Variant ZUC lacks most of the V region, all of the CH1 region, and part of the hinge. Variant OMM may represent an allelic form or another gamma chain subclass. The present disclosure provides additional fusion proteins comprising G3Fc domains containing one or more of these variants. An example of a native amino acid sequence that may be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 35). Dotted underline indicates the hinge region. In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35. (SEQ ID NO: 35)151EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE101YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL151VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ201EGNVFSCSVM HEALHNHYTQ KSLSLSLGK A variety of engineered mutations in the Fc domain are presented herein with respect to the G1Fc sequence (SEQ ID NO: 31), and analogous mutations in G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc inFIG.3. Due to unequal hinge lengths, analogous Fc positions based on isotype alignment (FIG.3) possess different amino acid numbers in SEQ ID NOs: 31, 32, 33, 34, and 35. It can also be appreciated that a given amino acid position in an immunoglobulin sequence consisting of hinge, CH2, and CH3 regions (e.g., SEQ ID NOs: 31, 32, 33, 34, and 35) will be identified by a different number than the same position when numbering encompasses the entire IgG1 heavy-chain constant domain (consisting of the CHL hinge, CH2, and CH3 regions) as in the Uniprot database. For example, correspondence between selected CH3 positions in a human G1Fc sequence (SEQ ID NO: 31), the human IgG1 heavy chain constant domain (Uniprot P01857), and the human IgG1 heavy chain is as follows. Correspondence of CH3 Positions in Different Numbering SystemsG1FcIgG1 heavy chainIgG1 heavy chain(Numbering beginsconstant domain(EU numberingat first threonine(Numbering beginsscheme ofin hinge region)at CH1)Kabat et al., 1991*)Y127Y232Y349S132S237S354E134E239E356K138K243K360T144T249T366L146L251L368N162N267N384K170K275K392D177D282D399D179D284D401Y185Y290Y407K187K292K409H213H318H435K217K322K439*Kabat et al. (eds) 1991; pp. 688-696 inSequences of Proteins of Immunological Interest, 5thed., Vol. 1, NIH, Bethesda, MD. A problem that arises in large-scale production of asymmetric immunoglobulin-based proteins from a single cell line is known as the “chain association issue”. As confronted prominently in the production of bispecific antibodies, the chain association issue concerns the challenge of efficiently producing a desired multichain protein from among the multiple combinations that inherently result when different heavy chains and/or light chains are produced in a single cell line [Klein et al (2012) mAbs 4:653-663]. This problem is most acute when two different heavy chains and two different light chains are produced in the same cell, in which case there are a total of 16 possible chain combinations (although some of these are identical) when only one is typically desired. Nevertheless, the same principle accounts for diminished yield of a desired multichain fusion protein that incorporates only two different (asymmetric) heavy chains. Various methods are known in the art that increase desired pairing of Fc-containing fusion polypeptide chains in a single cell line to produce a preferred asymmetric fusion protein at acceptable yields [Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. Methods to obtain desired pairing of Fc-containing chains include, but are not limited to, charge-based pairing (electrostatic steering), “knobs-into-holes” steric pairing, SEEDbody pairing, and leucine zipper-based pairing [Ridgway et al (1996) Protein Eng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al (2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010); 285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S. Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605]. As described herein, these methods may be used to generate ALK4-Fc:ActRIIB-Fc heteromultimer complexes. SeeFIG.6. For example, one means by which interaction between specific polypeptides may be promoted is by engineering protuberance-into-cavity (knob-into-holes) complementary regions such as described in Arathoon et al., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g., tyrosine or tryptophan). Complementary “cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Where a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface. At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged and lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation and at the same time hinder homodimer formation. Attractive interactions take place between opposite charges and repulsive interactions occur between like charges. In part, protein complexes disclosed herein make use of the attractive interactions for promoting heteromultimer formation (e.g., heterodimer formation), and optionally repulsive interactions for hindering homodimer formation (e.g., homodimer formation) by carrying out site directed mutagenesis of charged interface residues. For example, the IgG1 CH3 domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439′, Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering in the second chain is indicated by (′)]. It should be noted that the numbering scheme used here to designate residues in the IgG1 CH3 domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction will represented twice in the structure (e.g., Asp-399-Lys409′ and Lys409-Asp399′). In the wild-type sequence, K409-D399′ favors both heterodimer and homodimer formation. A single mutation switching the charge polarity (e.g., K409E; positive to negative charge) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399′ and D399-K409E′). A similar mutation switching the charge polarity (D399K′; negative to positive) in the second chain leads to unfavorable interactions (K409′-D399K′ and D399K-K409′) for the second chain homodimer formation. But, at the same time, these two mutations (K409E and D399K′) lead to favorable interactions (K409E-D399K′ and D399-K409′) for the heterodimer formation. The electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360. The table below lists possible charge change mutations that can be used, alone or in combination, to enhance ALK4:ActRIIB heteromultimer formation. Examples of Pair-Wise Charged Residue Mutations to EnhanceHeterodimer FormationInteractingCorrespondingPosition inMutation inposition inmutation infirst chainfirst chainsecond chainsecond chainLys409Asp or GluAsp399′Lys, Arg, or HisLys392Asp or GluAsp399′Lys, Arg, or HisLys439Asp or GluAsp356′Lys, Arg, or HisLys370Asp or GluGlu357′Lys, Arg, or HisAsp399Lys, Arg, or HisLys409′Asp or GluAsp399Lys, Arg, or HisLys392′Asp or GluAsp356Lys, Arg, or HisLys439′Asp or GluGlu357Lys, Arg, or HisLys370′Asp or Glu In some embodiments, one or more residues that make up the CH3-CH3 interface in a fusion protein of the instant application are replaced with a charged amino acid such that the interaction becomes electrostatically unfavorable. For example, a positive-charged amino acid in the interface (e.g., a lysine, arginine, or histidine) is replaced with a negatively charged amino acid (e.g., aspartic acid or glutamic acid). Alternatively, or in combination with the forgoing substitution, a negative-charged amino acid in the interface is replaced with a positive-charged amino acid. In certain embodiments, the amino acid is replaced with a non-naturally occurring amino acid having the desired charge characteristic. It should be noted that mutating negatively charged residues (Asp or Glu) to His will lead to increase in side chain volume, which may cause steric issues. Furthermore, His proton donor- and acceptor-form depends on the localized environment. These issues should be taken into consideration with the design strategy. Because the interface residues are highly conserved in human and mouse IgG subclasses, electrostatic steering effects disclosed herein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4. This strategy can also be extended to modifying uncharged residues to charged residues at the CH3 domain interface. In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to be complementary on the basis of charge pairing (electrostatic steering). One of a pair of Fc sequences with electrostatic complementarity can be arbitrarily fused to the ALK4 or ActRIIB polypeptide of the construct, with or without an optional linker, to generate an ALK4:ActRIIB heteromultimer. This single chain can be coexpressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multichain construct (e.g., ALK4:ActRIIB heteromultimer). In this example based on electrostatic steering, SEQ ID NO: 23 [human G1Fc(E134K/D177K)] and SEQ ID NO: 24 [human G1Fc(K170D/K187D)] are examples of complementary Fc sequences in which the engineered amino acid substitutions are double underlined, and the TGF-beta superfamily type I or type II receptor polypeptide of the construct can be fused to either SEQ ID NO: 23 or SEQ ID NO: 24, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 23 and 24). (SEQ ID NO: 23)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLKSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK(SEQ ID NO: 24)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYDTTPPVLDSDG SFFLYSDLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered for steric complementarity. In part, the disclosure provides knobs-into-holes pairing as an example of steric complementarity. One of a pair of Fc sequences with steric complementarity can be arbitrarily fused to the ALK4 or ActRIIB polypeptide of the construct, with or without an optional linker, to generate an ALK4:ActRIIB heteromultimer. This single chain can be co-expressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multi-chain construct. In this example based on knobs-into-holes pairing, SEQ ID NO: 25 [human G1Fc(T144Y)] and SEQ ID NO: 26 [human G1Fc(Y185T)] are examples of complementary Fc sequences in which the engineered amino acid substitutions are double underlined, and the ALK4 or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 25 or SEQ ID NO: 26, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 25 and 26). (SEQ ID NO: 25)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK(SEQ ID NO: 26)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLTSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK An example of Fc complementarity based on knobs-into-holes pairing combined with an engineered disulfide bond is disclosed in SEQ ID NO: 27 [hG1Fc(S132C/T144W)] and SEQ ID NO: 28 [hG1Fc(Y127C/T144S/L146A/Y185V)]. The engineered amino acid substitutions in these sequences are double underlined, and the TGF-beta superfamily type I or type II polypeptide of the construct can be fused to either SEQ ID NO: 27 or SEQ ID NO: 28, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 27 and 28). (SEQ ID NO: 27)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK(SEQ ID NO: 28)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to generate interdigitating β-strand segments of human IgG and IgA CH3 domains. Such methods include the use of strand-exchange engineered domain (SEED) CH3 heterodimers allowing the formation of SEEDbody fusion proteins [Davis et al. (2010) Protein Eng Design Sel 23:195-202]. One of a pair of Fc sequences with SEEDbody complementarity can be arbitrarily fused to the ALK4 or ActIIB of the construct, with or without an optional linker, to generate an ALK4 or ActRIIB fusion polypeptide. This single chain can be co-expressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multi-chain construct. In this example based on SEEDbody (Sb) pairing, SEQ ID NO: 29 [hG1Fc(SbAG)] and SEQ ID NO: 30 [hG1Fc(SbGA)] are examples of complementary IgG Fc sequences in which the engineered amino acid substitutions from IgA Fc are double underlined, and the ALK4 or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 29 or SEQ ID NO: 30, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG1Fc, hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate an Fc monomer which may be used in the complementary IgG-IgA pair below (SEQ ID NOs: 29 and 30). (SEQ ID NO: 29)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PFRPEVHLLP PSREEMTKNQ VSLTCLARGF151YPKDIAVEWE SNGQPENNYK TTPSRQEPSQGTTTFAVTSK LTVDKSRWQQ201GNVFSCSVMH EALHNHYTQKTISLSPGK(SEQ ID NO: 30)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PPSEELALNELVTLTCLVKG151FYPSDIAVEWESNGQELPREKYLTWAPVLD SDGSFFLYSILRVAAEDWKK201GDTFSCSVMH EALHNHYTQK SLDRSPGK In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains with a cleavable leucine zipper domain attached at the C-terminus of the Fc CH3 domains. Attachment of a leucine zipper is sufficient to cause preferential assembly of heterodimeric antibody heavy chains [Wranik et al (2012) J Biol Chem 287:43331-43339]. As disclosed herein, one of a pair of Fc sequences attached to a leucine zipper-forming strand can be arbitrarily fused to the ALK4 or ActRIIB polypeptide of the construct, with or without an optional linker, to generate an ALK4 or ActRIIB fusion polypeptide. This single chain can be co-expressed in a cell of choice along with the Fc sequence attached to a complementary leucine zipper-forming strand to favor generation of the desired multi-chain construct. Proteolytic digestion of the construct with the bacterial endoproteinase Lys-C post purification can release the leucine zipper domain, resulting in an Fc construct whose structure is identical to that of native Fc. In this example based on leucine zipper pairing, SEQ ID NO: 36 [hG1Fc-Ap1 (acidic)] and SEQ ID NO: 37 [hG1Fc-Bp1 (basic)] are examples of complementary IgG Fc sequences in which the engineered complimentary leucine zipper sequences are underlined, and the ALK4 or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 36 or SEQ ID NO: 37, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that leucine zipper-forming sequences attached, with or without an optional linker, to hG1Fc, hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate an Fc monomer which may be used in the complementary leucine zipper-forming pair below (SEQ ID NOs: 36 and 37). (SEQ ID NO: 36)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LEKELQALEK ENAQLEWELQ251ALEKELAQGA T(SEQ ID NO: 37)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LKKKLQALKK KNAQLKWKLQ251ALKKKLAQGA T In certain aspects, the disclosure relates to ALK4 polypeptides (e.g., ALK4-Fc fusion proteins) comprising one or more amino acid modifications that alter the isoelectric point (pI) of the ALK4 polypeptide and/or ActRIIB polypeptides (e.g., ActRIIB-Fc fusion proteins) comprising one or more amino acid modifications that alter the isoelectric point of the ActRIIB polypeptide. In some embodiments, one or more candidate domains that have a pI value higher than about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 are selected for construction of the full multidomain protein. In other embodiments, one or more candidate domains that have a pI value less than about 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, or 5.0 are selected for construction of the full multidomain protein. It will be understood by one skilled in the art that a single protein will have multiple charge forms. Without wishing to be bound by any particular theory, the charge of a protein can be modified by a number of different mechanisms including but not limited to, amino acid substitution, cationization, deamination, carboxyl-terminal amino acid heterogeneity, phosphorylation and glycosylation. The pI of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see for example Bjellqvist et al., 1993, Electrophoresis 14:1023). In one embodiment, pI is determined using a Pharmacia Biotech Multiphor 2 electrophoresis system with a multi temp refrigerated bath recirculation unit and an EPS 3501 XL power supply. Pre-cast ampholine gels (e.g., Amersham Biosciences, pI range 2.5-10) are loaded with protein samples. Broad range pI marker standards (e.g., Amersham, pI range 3-10, 8 .mu.L) are used to determine relative pI for the proteins. Electrophoresis is performed, for example, at 1500 V, 50 mA for 105 minutes. The gel is fixed using, for example, a Sigma fixing solution (5×) diluted with purified water to 1× Staining is performed, for example, overnight at room temperature using Simply Blue stain (Invitrogen). Destaining is carried out, for example, with a solution that consisted of 25% ethanol, 8% acetic acid and 67% purified water. Isoelectric points are determined using, for example, a Bio-Rad Densitometer relative to calibration curves of the standards. The one or more metrics may further include metrics characterizing stability of the domain under one or more different conditions selected from different pH values, different temperatures, different shear stresses, and different freeze/thaw cycles. In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains by methods described above in combination with additional mutations in the Fc domain which facilitate purification of the desired heteromeric species. An example is complementarity of Fc domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 27-28, plus additional substitution of two negatively charged amino acids (aspartic acid or glutamic acid) in one Fc-containing polypeptide chain and two positively charged amino acids (e.g., arginine) in the complementary Fc-containing polypeptide chain (SEQ ID NOs: 66-67). These four amino acid substitutions facilitate selective purification of the desired heteromeric fusion protein from a heterogeneous polypeptide mixture based on differences in isoelectric point or net molecular charge. The engineered amino acid substitutions in these sequences are double underlined below, and the ALK4 or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 66 or SEQ ID NO: 67, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 66-67). (SEQ ID NO: 66)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTENQ VSLWCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQDSLS LSPGK(SEQ ID NO: 67)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF151YPSDIAVEWE SRGQPENNYK TTPPVLDSRG SFFLVSKLTV DKSRWQQGNV201FSCSVMHEAL HNHYTQKSLS LSPGK Another example involves complementarity of Fc domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 27-28, plus a histidine-to-arginine substitution at position 213 in one Fc-containing polypeptide chain (SEQ ID NO: 68). This substitution (denoted H435R in the numbering system of Kabat et al.) facilitates separation of desired heteromer from undesirable homodimer based on differences in affinity for protein A. The engineered amino acid substitution is indicated by double underline, and the ALK4 or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 68 or SEQ ID NO: 28, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (seeFIG.3) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair of SEQ ID NO: 68 (below) and SEQ ID NO: 28. (SEQ ID NO: 68)1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201FSCSVMHEAL HNRYTQKSLS LSPGK A variety of engineered mutations in the Fc domain are presented above with respect to the G1Fc sequence (SEQ ID NO: 31). Analogous mutations in G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc inFIG.3. Due to unequal hinge lengths, analogous Fc positions based on isotype alignment (FIG.3) possess different amino acid numbers in SEQ ID NOs: 31, 32, 33, 34, and 35 as summarized in the following table. Correspondence between CH3 Positions for Human Fc Isotypes*IgG1IgG4IgG2IgG3SEQ ID NO: 31SEQ ID NO: 35SEQ ID NO: 32SEQ ID NO: 33NumberingNumberingNumberingNumberingbegins atbegins atbegins atbegins atTHT . . .ESK . . .VEC . . .EPK . . .Y127Y131Y125Y134S132S136S130S139E134E138E132E141K138K142K136K145T144T148T142T151L146L150L144L153N162N166N160S169K170K174K168N177D177D181D175D184D179D183D177D186Y185Y189Y183Y192K187R191K185K194H213H217H211R220K217K221K215K224*Numbering based on multiple sequence alignment shown in FIG. 3 It is understood that different elements of the fusion proteins (e.g., immunoglobulin Fc fusion proteins) may be arranged in any manner that is consistent with desired functionality. For example, an ALK4 and/or ActRIIB polypeptide domain may be placed C-terminal to a heterologous domain, or alternatively, a heterologous domain may be placed C-terminal to an ALK4 and/or ActRIIB polypeptide domain. The ALK4 and/or ActRIIB polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains. For example, an ALK4 and/or ActRIIB receptor fusin protein may comprise an amino acid sequence as set forth in the formula A-B-C. The B portion corresponds to an ALK4 or ActRIIB polypeptide domain. The A and C portions may be independently zero, one, or more than one amino acid, and both the A and C portions when present are heterologous to B. The A and/or C portions may be attached to the B portion via a linker sequence. A linker may be rich in glycine (e.g., 2-10 (SEQ ID NO: 84), 2-5 (SEQ ID NO: 85), 2-4 (SEQ ID NO: 86), 2-3 glycine residues) or glycine and proline residues and may, for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO: 13), GGGG (SEQ ID NO: 14), TGGGG (SEQ ID NO: 15), SGGGG (SEQ ID NO: 16), TGGG (SEQ ID NO: 17), SGGG (SEQ ID NO: 18), or GGGGS (SEQ ID NO: 58) singlets, or repeats. In certain embodiments, an ALK4 and/or ActRIIB fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a leader (signal) sequence, B consists of an ALK4 and/or ActRIIB polypeptide domain, and C is a polypeptide portion that enhances one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of protein complexes, and/or purification. In certain embodiments, an ALK4 and/or ActRIIB fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a TPA leader sequence, B consists of a ALK4 or ActRIIB receptor polypeptide domain, and C is an immunoglobulin Fc domain. Preferred fusion proteins comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39, 41, 42, 44, 45, 46, 47, 48, 70, 72, 74, 76, 78, and 80. In some embodiments, ALK4:ActRIIB heteromultimers further comprise one or none heterologous portions (domains) so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy-chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen) useful with (HIS6(SEQ ID NO: 87)) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ligand trip polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation. In certain embodiments, ALK4 and/or ActRIIB polypeptides may contain one or more modifications that are capable of stabilizing the polypeptides. For example, such modifications enhance the in vitro half-life of the polypeptides, enhance circulatory half-life of the polypeptides, and/or reduce proteolytic degradation of the polypeptides. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ALK4 and/or ActRIIB polypeptide domain and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to a polypeptide of the disclosure), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from a polypeptide of the disclosure). As used herein, the term “stabilizer domain” not only refers to a fusion domain (e.g., an immunoglobulin Fc domain) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol. In preferred embodiments, ALK4:ActRIIB heteromultimers to be used in accordance with the methods described herein are isolated complexes. As used herein, an isolated protein (or protein complex) or polypeptide (or polypeptide complex) is one which has been separated from a component of its natural environment. In some embodiments, a heteromultimer of the disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). Methods for assessment of antibody purity are well known in the art [Flatman et al., (2007) J. Chromatogr. B 848:79-87]. In some embodiments, ALK4:ActRIIB heteromultimer preparations are substantially free of ALK4 and/or ActRIIB homomultimers. For example, in some embodiments, ALK4:ActRIIB heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers. In some embodiments, ALK4:ActRIIB heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIB homomultimers. In some embodiments, ALK4:ActRIIB heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers and less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIB homomultimers. In certain embodiments, ALK4 and/or ActRIIB polypeptides, as well as heteromultimers comprising the same, of the disclosure can be produced by a variety of art-known techniques. For example, polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, the polypeptides, including fragments or variants thereof, may be recombinantly produced using various expression systems [E. coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as is well known in the art. In a further embodiment, the modified or unmodified polypeptides may be produced by digestion of recombinantly produced full-length ALK4 and/or ActRIIB polypeptides by using, for example, a protease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. 3. Nucleic Acids Encoding ALK4 and/or ActRIIB Polypeptides In certain embodiments, the present disclosure provides isolated and/or recombinant nucleic acids encoding ALK4 and/or ActRIIB polypeptides (including fragments, functional variants, and fusion proteins thereof) disclosed herein. For example, SEQ ID NO: 11 encodes a naturally occurring human ALK4 precursor polypeptide, while SEQ ID NO: 12 encodes a processed extracellular domain of ALK4. The subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ALK4:ActRIIB heteromultimers as described herein. As used herein, isolated nucleic acid(s) refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. In certain embodiments, nucleic acids encoding ALK4 and/or ActRIIB polypeptides of the present disclosure are understood to include any one of SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83, as well as variants thereof. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions including allelic variants, and therefore, will include coding sequences that differ from the nucleotide sequence designated in any one of SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83. In certain embodiments, ALK4 and/or ActRIIB polypeptides of the present disclosure are encoded by isolated or recombinant nucleic acid sequences that comprise, consist essentially of, or consists of a sequence that is least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83. One of ordinary skill in the art will appreciate that nucleic acid sequences that comprise, consist essentially of, or consists of a sequence complementary to a sequence that is least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83 also within the scope of the present disclosure. In further embodiments, the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence or in a DNA library. In other embodiments, nucleic acids of the present disclosure also include nucleotide sequences that hybridize under stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83, the complement sequence of SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83, or fragments thereof. One of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the disclosure provides nucleic acids which hybridize under low stringency conditions of 6×SSC at room temperature followed by a wash at 2×SSC at room temperature. Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83 to degeneracy in the genetic code are also within the scope of the disclosure. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure. In certain embodiments, the recombinant nucleic acids of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In some embodiments, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. In certain aspects, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ALK4 and/or ActRIIB polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of ALK4 and/or ActRIIB polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a ALK4 and/or ActRIIB polypeptides. Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. A recombinant nucleic acid of the present disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant ALK4 and/or ActRIIB polypeptides include plasmids and other vectors. For instance, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such asE. coli. Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures [Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis Cold Spring Harbor Laboratory Press, 2001]. In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III). In a preferred embodiment, a vector will be designed for production of the subject ALK4 and/or ActRIIB polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will be apparent, the subject gene constructs can be used to cause expression of the subject ALK4 and/or ActRIIB polypeptide in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification. This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more of the subject ALK4 and/or ActRIIB polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, an ALK4 and/or ActRIIB polypeptide may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells [e.g. a Chinese hamster ovary (CHO) cell line]. Other suitable host cells are known to those skilled in the art. Accordingly, the present disclosure further pertains to methods of producing the subject ALK4 and/or ActRIIB polypeptides. For example, a host cell transfected with an expression vector encoding an ALK4 and/or ActRIIB polypeptide can be cultured under appropriate conditions to allow expression of the ALK4 and/or ActRIIB polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, ALK4 and/or ActRIIB polypeptide may be isolated from a cytoplasmic or membrane fraction obtained from harvested and lysed cells. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The subject polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of ALK4 and/or ActRIIB polypeptides and affinity purification with an agent that binds to a domain fused to ALK4 and/or ActRIIB polypeptide (e.g., a protein A column may be used to purify ALK4-Fc and/or ActRIIB-Fc fusion proteins). In some embodiments, the ALK4 and/or ActRIIB polypeptide is a fusion protein containing a domain which facilitates its purification. In some embodiments, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. An ALK4 and/or ActRIIB polypeptides, as well as fusion proteins thereof, may be purified to a purity of >90%, >95%, >96%, >98%, or >99% as determined by size exclusion chromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDS PAGE. The target level of purity should be one that is sufficient to achieve desirable results in mammalian systems, particularly non-human primates, rodents (mice), and humans. In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ALK4 and/or ActRIIB polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ALK4 and/or ActRIIB polypeptide, as well as heteromultimers thereof [Hochuli et al. (1987)J. Chromatography411:177; and Janknecht et al. (1991)PNAS USA88:8972]. Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence. See, e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992. 4. ALK4:ActRIIB Antibody Antagonists In certain aspects, the present disclosure relates to an ALK4:ActRIIB antagonist (inhibitor) that is antibody, or combination of antibodies. ALK4:ActRIIB antagonist antibody, or combination of antibodies, may bind to one or more ALK4:ActRIIB-associated ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] or one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In particular, the disclosure provides methods of using an ALK4:ActRIIB antagonist antibody, or combination of ALK4:ActRIIB antagonist antibodies, alone or in combination with one or more additional supportive therapies and/or active agents, to achieve a desired effect in a subject in need thereof (e.g., treating SMA, particularly preventing or reducing the severity of one or more complications of SMA). In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least GDF11. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least GDF11. As used herein, a GDF11 antibody (anti-GDF11 antibody) generally refers to an antibody that binds to GDF11 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF11. In certain embodiments, the extent of binding of an anti-GDF11 antibody to an unrelated, non-GDF11 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to GDF11 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-GDF11 antibody binds to an epitope of GDF11 that is conserved among GDF11 from different species. In certain preferred embodiments, an anti-GDF11 antibody binds to human GDF11. In other preferred embodiments, an anti-GDF11 antibody may inhibit GDF11 from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit GDF11-mediated signaling (e.g., Smad signaling) via these receptors. It should be noted that GDF11 has high sequence homology to GDF8 and therefore antibodies that bind to GDF11, in some cases, may also bind to and/or inhibit GDF8. In some embodiments, an anti-GDF11 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to an antibody that binds to GDF11 and GDF8. In some embodiments, the disclosure relates to an antibody that binds GDF11 and activin A. In some embodiments, the disclosure relates to an antibody that binds GDF11 and activin B. In some embodiments, the disclosure relates to an antibody that binds GDF11, GDF8, and activin A. In some embodiments, the disclosure relates to an antibody that binds GDF11, GDF8, and activin B. In some embodiments, the disclosure relates to an antibody that binds GDF11, GDF8, activin A, and activin B. In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-GDF11 antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11 and an antibody that binds to GDF8. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11 and an antibody that binds to activin A. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11 and an antibody that binds to activin B. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11, an antibody that binds to GDF8, and an antibody that binds to activin A. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11, an antibody that binds to GDF8, and an antibody that binds to activin B. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF11, an antibody that binds to GDF8, an antibody that binds to activin A, and an antibody that binds to activin B. In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least GDF8. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least GDF8. As used herein, a GDF8 antibody (anti-GDF8 antibody) generally refers to an antibody that binds to GDF8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF8. In certain embodiments, the extent of binding of an anti-GDF8 antibody to an unrelated, non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to GDF8 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-GDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8 from different species. In certain preferred embodiments, an anti-GDF8 antibody binds to human GDF8. In other preferred embodiments, an anti-GDF8 antibody may inhibit GDF8 from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit GDF8-mediated signaling (e.g., Smad signaling) via these receptors. It should be noted that GDF8 has high sequence homology to GDF11 and therefore antibodies that bind to GDF8, in some cases, may also bind to and/or inhibit GDF11. In some embodiments, an anti-GDF8 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4]. In some embodiments, the disclosure relates to an antibody that binds GDF8 and activin A. In some embodiments, the disclosure relates to an antibody that binds GDF8 and activin B. In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-GDF8 antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF8 and an antibody that binds to activin A. In some embodiments, the disclosure relates to a combination of antibodies comprising an antibody that binds to GDF8 and an antibody that binds to activin B. In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC). Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least activin. As used herein, an activin antibody (anti-activin antibody) generally refers to an antibody that binds to activin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting activin. In certain embodiments, the extent of binding of an anti-activin antibody to an unrelated, non-activin protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to activin as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-activin antibody binds to an epitope of activin that is conserved among activin from different species. In certain preferred embodiments, an anti-activin antibody binds to human activin. In other preferred embodiments, an anti-activin antibody may inhibit activin from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit activin-mediated signaling (e.g., Smad signaling) via these receptors. It should be noted that activins share sequence homology and therefore antibodies that bind to one activin (e.g., activin A) may bind to one or more additional activins (e.g., activin B, activin AB, activin C, activin E, activin AC). In some embodiments, an anti-activin antibody binds to at least activin A and activin B. In some embodiments, an anti-activin antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF11, GDF8, GDF3, BMP6, and BMP10] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-activin antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF8, GDF11, GDF3, BMP6, and BMP10] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least GDF3. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least GDF3. As used herein, a GDF3 antibody (anti-GDF3 antibody) generally refers to an antibody that binds to GDF3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF3. In certain embodiments, the extent of binding of an anti-GDF3 antibody to an unrelated, non-GDF3 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to GDF3 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-GDF3 antibody binds to an epitope of GDF3 that is conserved among GDF3 from different species. In certain preferred embodiments, an anti-GDF3 antibody binds to human GDF3. In other preferred embodiments, an anti-GDF3 antibody may inhibit GDF3 from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit GDF3-mediated signaling (e.g., Smad signaling) via these receptors. In some embodiments, an anti-GDF3 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), BMP6, and BMP10] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-GDF3 antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) BMP6, and BMP10] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least BMP6. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least BMP6. As used herein, a BMP6 antibody (anti-BMP6 antibody) generally refers to an antibody that binds to BMP6 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting BMP6. In certain embodiments, the extent of binding of an anti-BMP6 antibody to an unrelated, non-BMP6 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to BMP6 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-BMP6 antibody binds to an epitope of BMP6 that is conserved among BMP6 from different species. In certain preferred embodiments, an anti-BMP6 antibody binds to human BMP6. In other preferred embodiments, an anti-BMP6 antibody may inhibit BMP6 from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit BMP6-mediated signaling (e.g., Smad signaling) via these receptors. In some embodiments, an anti-BMP6 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and BMP10] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-BMP6 antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, and BMP10] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least BMP10. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least BMP10. As used herein, a BMP10 antibody (anti-BMP10 antibody) generally refers to an antibody that binds to BMP10 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting BMP10. In certain embodiments, the extent of binding of an anti-BMP10 antibody to an unrelated, non-BMP10 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to BMP10 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-BMP10 antibody binds to an epitope of BMP10 that is conserved among BMP10 from different species. In certain preferred embodiments, an anti-BMP10 antibody binds to human BMP10. In other preferred embodiments, an anti-BMP10 antibody may inhibit BMP10 from binding to a cognate type I and/or type II receptor (e.g., ActRIIB and ALK4) and thus inhibit BMP10-mediated signaling (e.g., Smad signaling) via these receptors. In some embodiments, an anti-BMP10 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to one or more additional ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and BMP6] and/or binds to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-BMP10 antibody and one or more additional antibodies that bind to, for example, different ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, and BMP6] and/or bind to one or more type I and/or type II receptors (e.g., ActRIIB and ALK4). In other aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least ActRIIB Therefore, in some embodiments, an ActRII antagonist antibody, or combination of antibodies, binds to at least ActRIIB As used herein, an ActRIIB antibody (anti-ActRIIB antibody) generally refers to an antibody that binds to ActRIIB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIB In certain embodiments, the extent of binding of an anti-ActRIIB antibody to an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to ActRIIB as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-ActRIIB antibody binds to an epitope of ActRIIB that is conserved among ActRIIB from different species. In certain preferred embodiments, an anti-ActRIIB antibody binds to human ActRIIB In other preferred embodiments, an anti-ActRIIB antibody may inhibit one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] from binding to ActRIIB It should be noted that ActRIIB has sequence homology to ActRIIA and therefore antibodies that bind to ActRIIB, in some cases, may also bind to and/or inhibit ActRIIA. In some embodiments, an anti-ActRIIB antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ActRIIB and one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10]. In some embodiments, an anti-ActRIIB antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ActRIIB and ALK4. In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises at least an anti-ActRIIB antibody and at least an ALK4 antibody. In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-ActRIIB antibody and one or more additional antibodies that bind to, for example, one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and ALK4. In certain aspects, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, of the disclosure is an antibody that inhibits at least ALK4. Therefore, in some embodiments, an ALK4:ActRIIB antagonist antibody, or combination of antibodies, binds to at least ALK4. As used herein, an ALK4 antibody (anti-ALK4 antibody) generally refers to an antibody that binds to ALK4 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ALK4. In certain embodiments, the extent of binding of an anti-ALK4 antibody to an unrelated, non-ALK4 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK4 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-ALK4 antibody binds to an epitope of ALK4 that is conserved among ALK4 from different species. In certain preferred embodiments, an anti-ALK4 antibody binds to human ALK4. In other preferred embodiments, an anti-ALK4 antibody may inhibit one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, BMP10, and BMP9] from binding to ALK4. In some embodiments, an anti-ALK4 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ALK4 and one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or ActRIIB In some embodiments, the disclosure relates to combinations of antibodies, as well as uses thereof, wherein the combination of antibodies comprises an anti-ALK4 antibody and one or more additional antibodies that bind to, for example, one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] and/or ALK4. The term antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894, 5,587,458, and 5,869,046. Antibodies disclosed herein may be polyclonal antibodies or monoclonal antibodies. In certain embodiments, the antibodies of the present disclosure comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme, or enzyme co-factor). In preferred embodiments, the antibodies of the present disclosure are isolated antibodies. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129-134 (2003); and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy-chain variable domain or all or a portion of the light-chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. See, e.g., U.S. Pat. No. 6,248,516. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g.,E. colior phage), as described herein. The antibodies herein may be of any class. The class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu. In general, an antibody for use in the methods disclosed herein specifically binds to its target antigen, preferably with high binding affinity. Affinity may be expressed as a KDvalue and reflects the intrinsic binding affinity (e.g., with minimized avidity effects). Typically, binding affinity is measured in vitro, whether in a cell-free or cell-associated setting. Any of a number of assays known in the art, including those disclosed herein, can be used to obtain binding affinity measurements including, for example, surface plasmon resonance (Biacore™ assay), radiolabeled antigen binding assay (RIA), and ELISA. In some embodiments, antibodies of the present disclosure bind to their target antigens [e.g., ActRIIB ALK4, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] with at least a KDof 1×10−7or stronger, 1×10−8or stronger, 1×10−9or stronger, 1×10−1° or stronger, 1×10−11or stronger, 1×10−12or stronger, 1×10−13or stronger, or 1×10−14or stronger. In certain embodiments, KDis measured by RIA performed with the Fab version of an antibody of interest and its target antigen as described by the following assay. Solution binding affinity of Fabs for the antigen is measured by equilibrating Fab with a minimal concentration of radiolabeled antigen (e.g.,125I-labeled) in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol. Biol. 293:865-881]. To establish conditions for the assay, multi-well plates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g., overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs) and subsequently blocked with bovine serum albumin, preferably at room temperature (e.g., approximately 23° C.). In a non-adsorbent plate, radiolabeled antigen are mixed with serial dilutions of a Fab of interest [e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab of interest is then incubated, preferably overnight but the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation, preferably at room temperature for about one hour. The solution is then removed and the plate is washed times several times, preferably with polysorbate 20 and PBS mixture. When the plates have dried, scintillant (e.g., MICROSCINT® from Packard) is added, and the plates are counted on a gamma counter (e.g., TOPCOUNT® from Packard). According to another embodiment, KDis measured using surface plasmon resonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000 (Biacore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips at about 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, Biacore, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. For example, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (about 0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate of approximately 25 μl/min. Association rates (kon) and dissociation rates (koff) are calculated using, for example, a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. [see, e.g., Chen et al., (1999) J. Mol. Biol. 293:865-881]. If the on-rate exceeds, for example, 106M−1s−1by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (e.g., excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nM anti-antigen antibody (Fab form) in PBS in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette. The nucleic acid and amino acid sequences of human ActRIIB ALK4, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10 are well known in the art and thus antibody antagonists for use in accordance with this disclosure may be routinely made by the skilled artisan based on the knowledge in the art and teachings provided herein. In certain embodiments, an antibody provided herein is a chimeric antibody. A chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. In general, chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody provided herein is a humanized antibody. A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR (a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing “resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing “FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; and Klimka et al. Br. J. Cancer (2000) 83:252-260 (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296]; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light-chain or heavy-chain variable regions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623]; human mature (somatically mutated) framework regions or human germline framework regions [see, e.g., Almagro and Fransson (2008) Front. Biosci. 13:1619-1633]; and framework regions derived from screening FR libraries [see, e.g., Baca et cd., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et cd., (1996) J. Biol. Chem. 271:22611-22618]. In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel (2001) Curr. Opin. Pharmacol. 5: 368-74 and Lonberg (2008) Curr. Opin. Immunol. 20:450-459. Human antibodies may be prepared by administering an immunogen [e.g., ActRIIB, ALK4, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see, for example, Lonberg (2005) Nat. Biotechnol. 23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab® technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE® technology); and U.S. Patent Application Publication No. 2007/0061900 (describing VelociMouse® technology). Human variable regions from intact antibodies generated by such animals may be further modified, for example, by combining with a different human constant region. Human antibodies provided herein can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described [see, e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol., 147: 86]. Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3562. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue (2006) 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein (2005) Histol. Histopathol., 20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp. Clin. Pharmacol., 27(3):185-91. Human antibodies provided herein may also be generated by isolating Fv clone variable-domain sequences selected from human-derived phage display libraries. Such variable-domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described herein. For example, antibodies of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. A variety of methods are known in the art for generating phage-display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J. and further described, for example, in the McCafferty et al. (1991) Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Marks et al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) in Methods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa, N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al. (2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad. Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods 284(1-2): 119-132. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. (1994) Ann. Rev. Immunol., 12: 433-455. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen [e.g., ActRIIB ALK4, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10] without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies directed against a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. (1993) EMBO J, 12: 725-734. Finally, naive libraries can also be made synthetically by cloning un-rearranged V-gene segments from stem cells and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. In certain embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies (typically monoclonal antibodies) have binding specificities for at least two different epitopes (e.g., two, three, four, five, or six or more) on one or more (e.g., two, three, four, five, six or more) antigens. Engineered antibodies with three or more functional antigen binding sites, including “octopus antibodies,” are also included herein (see, e.g., US 2006/0025576A1). In certain embodiments, the antibodies disclosed herein are monoclonal antibodies. Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. For example, by using immunogens derived from GDF11, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols [see, e.g., Antibodies: A Laboratory Manual (1988) ed. by Harlow and Lane, Cold Spring Harbor Press]. A mammal, such as a mouse, hamster, or rabbit can be immunized with an immunogenic form of the GDF11 polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a GDF11 polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody production and/or level of binding affinity. Following immunization of an animal with an antigenic preparation of GDF11, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256: 495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al. (1983) Immunology Today, 4:72], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a GDF11 polypeptide, and monoclonal antibodies isolated from a culture comprising such hybridoma cells. In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein thereby generating an Fc-region variant. The Fc-region variant may comprise a human Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution, deletion, and/or addition) at one or more amino acid positions. For example, the present disclosure contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet for which certain effector functions [e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)] are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII FcR expression on hematopoietic cells is summarized in, for example, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom, I. et al. (1986) Proc. Nat'l Acad. Sci. USA 83:7059-7063; Hellstrom, I et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1499-1502; U.S. Pat. No. 5,821,337; and Bruggemann, M. et al. (1987) J. Exp. Med. 166:1351-1361. Alternatively, non-radioactive assay methods may be employed (e.g., ACTI™, non-radioactive cytotoxicity assay for flow cytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656. C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity [see, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402]. To assess complement activation, a CDC assay may be performed [see, e.g., Gazzano-Santoro et al. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood 101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood 103:2738-2743]. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art [see, e.g., Petkova, S. B. et al. (2006) Int. Immunol. 18(12):1759-1769]. Antibodies of the present disclosure with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). In certain embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy-chain Fc region. Cysteine engineered antibodies may be generated as described, for example, in U.S. Pat. No. 7,521,541. In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md.), western blots, immunoprecipitation assays, and immunohistochemistry. In certain embodiments, amino acid sequence variants of the antibodies and/or the binding polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody and/or binding polypeptide. Amino acid sequence variants of an antibody and/or binding polypeptides may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody and/or binding polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within, the amino acid sequences of the antibody and/or binding polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target-binding (ActRIIB, ALK4, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10 binding). Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described in the art [see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J., (2001)]. In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted. In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two, or three amino acid substitutions. A useful method for identification of residues or regions of the antibody and/or the binding polypeptide that may be targeted for mutagenesis is called “alanine scanning mutagenesis”, as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody or binding polypeptide with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties. Amino-acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. In certain embodiments, an antibody and/or binding polypeptide provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody and/or binding polypeptide include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody and/or binding polypeptide may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody and/or binding polypeptide to be improved, whether the antibody derivative and/or binding polypeptide derivative will be used in a therapy under defined conditions. 5. Small Molecule Antagonists In other aspects, the present disclosure relates to an ALK4:ActRIIB antagonist (inhibitor) that is small molecule, or combination of small molecules. ALK4:ActRIIB antagonist small molecules may inhibit to one or more ALK4:ActRIIB-associated ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10], one or more type I and/or type II receptors (e.g., ActRIIB and ALK4), or one or more downstream signaling components (e.g., Smads 2 and/or 3). In particular, the disclosure provides methods of using an ALK4:ActRIIB antagonist small molecules, or combination of ALK4:ActRIIB antagonist small molecules, alone or in combination with one or more additional supportive therapies and/or active agents, to achieve a desired effect in a subject in need thereof (e.g., treating SMA, particularly preventing or reducing the severity of one or more complication of SMA). In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least GDF11. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits GDF11 further inhibits one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10], ALK4 and/or ActRIIB In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least GDF8. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits GDF8 further inhibits one or more ligands [e.g., GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10], ALK4 and/or ActRIIB In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least activin (e.g., activin A, activin B, activin C, activin E, activin AB, and activin AE). In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits activin further inhibits one or more ligands [e.g., GDF11, GDF8, GDF3, BMP6, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least GDF3. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits GDF3 further inhibits one or more ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), BMP6, and/or BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least BMP6. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits BMP6 further inhibits one or more ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and/or BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least BMP10. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits BMP10 further inhibits one or more ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and BMP6], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least ActRIIB In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits ActRIIB further inhibits one or more ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and/or BMP10] and/or ALK4. In some embodiments, an ActRII antagonist is a small molecule antagonist, or combination of small molecule antagonists, that inhibits at least ALK4. In some embodiments, a small molecule antagonist, or combination of small molecule antagonists, that inhibits ALK4 further inhibits one or more ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10] and/or ActRIIB. Small molecule antagonists can be direct or indirect inhibitors. For example, an indirect small molecule antagonist, or combination of small molecule antagonists, may inhibit the expression (e.g., transcription, translation, cellular secretion, or combinations thereof) of at least one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and/or BMP10], one or more type I and/or type II receptors (e.g., ActRIIB and ALK4), or one or more ActRII downstream signaling components (e.g., Smads 2 and/or 3). Alternatively, a direct small molecule ALK4:ActRIIB antagonist, or combination of small molecule antagonists, may directly bind to, for example, one or more of one or more ligands [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10], one or more type I and/or type II receptors (e.g., ActRIIB and ALK4), or one or more ActRII downstream signaling components (e.g., Smads 2 and/or 3). Combinations of one or more indirect and one or more direct small molecule antagonists may be used in accordance with the methods disclosed herein. Binding organic small molecule antagonists of the present disclosure may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general, small molecule antagonists of the disclosure are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules that are capable of binding, preferably specifically, to a polypeptide as described herein. Such small molecule antagonists may be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening organic small molecule libraries for molecules that are capable of binding to a polypeptide target are well-known in the art (see, e.g., international patent publication Nos. WO00/00823 and WO00/39585). Binding organic small molecules of the present disclosure may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, and acid chlorides. 6. Nucleotide Antagonists In other aspects, the present disclosure relates to an ALK4:ActRIIB antagonist (inhibitor) that is a polynucleotide, or combination of polynucleotides. ALK4:ActRIIB antagonist polynucleotides may inhibit to one or more ALK4:ActRIIB-associated ligands [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10], one or more type I and/or type II receptors (e.g., ActRIIB and ALK4), or one or more downstream signaling components (e.g., Smads 2 and/or 3). In particular, the disclosure provides methods of using an ALK4:ActRIIB antagonist polynucleotide, or combination of ALK4:ActRIIB antagonist polynucleotides, alone or in combination with one or more additional supportive therapies and/or active agents, to achieve a desired effect in a subject in need thereof (e.g., treating SMA, particularly preventing or reducing the severity of one or more complications of SMA). In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least GDF11. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits GDF11 further inhibits one or more ligand [e.g., GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least GDF8. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits GDF8 further inhibits one or more ligand [e.g., GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least activin (e.g., activin A, activin B, activin C, activin E, activin AB, and activin AE). In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits activin further inhibits one or more ligand [e.g., GDF11, GDF8, GDF3, BMP6, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB polynucleotide is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least GDF3. In some embodiments, a polynucleotide, or combination of polynucleotide antagonists, that inhibits GDF3 further inhibits one or more ligand [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), BMP6, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least BMP6. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits BMP6 further inhibits one or more ligand [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and BMP10], ActRIIB, and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least BMP10. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits BMP10 further inhibits one or more ligand [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, and BMP6], ActRIIB, and/or ALK4. In some embodiments, an ActRII antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least ActRIIB In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits ActRIIB further inhibits one or more ligand [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10] and/or ALK4. In some embodiments, an ALK4:ActRIIB antagonist is a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits at least ALK4. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, that inhibits ALK4 further inhibits one or more ligand [e.g., GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC), GDF3, BMP6, and BMP10] and/or ActRIIB The polynucleotide antagonists of the present disclosure may be an antisense nucleic acid, an RNAi molecule [e.g., small interfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)], an aptamer and/or a ribozyme. The nucleic acid and amino acid sequences of human ALK4, ActRIIB, GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and BMP10 are known in the art and thus polynucleotide antagonists for use in accordance with methods of the present disclosure may be routinely made by the skilled artisan based on the knowledge in the art and teachings provided herein. For example, antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed, for example, in Okano (1991) J. Neurochem. 56:560; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Cooney et al. (1988) Science 241:456; and Dervan et al., (1991) Science 251:1300. The methods are based on binding of a polynucleotide to a complementary DNA or RNA. In some embodiments, the antisense nucleic acids comprise a single-stranded RNA or DNA sequence that is complementary to at least a portion of an RNA transcript of a desired gene. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids of a gene disclosed herein, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Polynucleotides that are complementary to the 5′ end of the message, for example, the 5′-untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′-untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature 372:333-335]. Thus, oligonucleotides complementary to either the 5′- or 3′-untranslated, noncoding regions of a gene of the disclosure, could be used in an antisense approach to inhibit translation of an endogenous mRNA. Polynucleotides complementary to the 5′-untranslated region of the mRNA should include the complement of the AUG start codon. Antisense polynucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the methods of the present disclosure. Whether designed to hybridize to the 5′-untranslated, 3′-untranslated, or coding regions of an mRNA of the disclosure, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides. In one embodiment, the antisense nucleic acid of the present disclosure is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of a gene of the disclosure. Such a vector would contain a sequence encoding the desired antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding desired genes of the instant disclosure, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region [see, e.g., Benoist and Chambon (1981) Nature 29:304-310], the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al. (1980) Cell 22:787-797], the herpes thymidine promoter [see, e.g., Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], and the regulatory sequences of the metallothionein gene [see, e.g., Brinster, et al. (1982) Nature 296:39-42]. In some embodiments, the polynucleotide antagonists are interfering RNA or RNAi molecules that target the expression of one or more genes. RNAi refers to the expression of an RNA which interferes with the expression of the targeted mRNA. Specifically, RNAi silences a targeted gene via interacting with the specific mRNA through a siRNA (small interfering RNA). The ds RNA complex is then targeted for degradation by the cell. An siRNA molecule is a double-stranded RNA duplex of 10 to 50 nucleotides in length, which interferes with the expression of a target gene which is sufficiently complementary (e.g. at least 80% identity to the gene). In some embodiments, the siRNA molecule comprises a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the nucleotide sequence of the target gene. Additional RNAi molecules include short-hairpin RNA (shRNA); also short-interfering hairpin and microRNA (miRNA). The shRNA molecule contains sense and antisense sequences from a target gene connected by a loop. The shRNA is transported from the nucleus into the cytoplasm, and it is degraded along with the mRNA. Pol III or U6 promoters can be used to express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002) 16:948-958, 2002] have used small RNA molecules folded into hairpins as a means to effect RNAi. Accordingly, such short hairpin RNA (shRNA) molecules are also advantageously used in the methods described herein. The length of the stem and loop of functional shRNAs varies; stem lengths can range anywhere from about 25 to about 30 nt, and loop size can range between 4 to about 25 nt without affecting silencing activity. While not wishing to be bound by any particular theory, it is believed that these shRNAs resemble the double-stranded RNA (dsRNA) products of the DICER RNase and, in any event, have the same capacity for inhibiting expression of a specific gene. The shRNA can be expressed from a lentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70 nucleotides in length that are initially transcribed as pre-miRNA characterized by a “stem-loop” structure and which are subsequently processed into mature miRNA after further processing through the RISC. Molecules that mediate RNAi, including without limitation siRNA, can be produced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199, 2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase (Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-stranded RNA using a nuclease such asE. coliRNase III (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002). According to another aspect, the disclosure provides polynucleotide antagonists including but not limited to, a decoy DNA, a double-stranded DNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA, a double-stranded RNA, a molecule capable of generating RNA interference, or combinations thereof. In some embodiments, the polynucleotide antagonists of the disclosure are aptamers. Aptamers are nucleic acid molecules, including double-stranded DNA and single-stranded RNA molecules, which bind to and form tertiary structures that specifically bind to a target molecule, such as a ALK4, ActRIIB, GDF11, GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC) GDF3, BMP6, and/or BMP10 polypeptide. The generation and therapeutic use of aptamers are well established in the art. See, e.g., U.S. Pat. No. 5,475,096. Additional information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. Nucleic acid aptamers are selected using methods known in the art, for example via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules as described in, e.g., U.S. Pat. Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796, 5,763,177, 6,011,577, and 6,699,843. Another screening method to identify aptamers is described in U.S. Pat. No. 5,270,163. The SELEX process is based on the capacity of nucleic acids for forming a variety of two- and three-dimensional structures, as well as the chemical versatility available within the nucleotide monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric, including other nucleic acid molecules and polypeptides. Molecules of any size or composition can serve as targets. The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve desired binding affinity and selectivity. Starting from a mixture of nucleic acids, which can comprise a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; dissociating the nucleic acid-target complexes; amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids. The steps of binding, partitioning, dissociating and amplifying are repeated through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule. Typically, such binding molecules are separately administered to the animal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but such binding molecules can also be expressed in vivo from polynucleotides taken up by a host cell and expressed in vivo [see, e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)]. 7. Screening Assays In certain aspects, the present disclosure relates to the use of ALK4:ActRIIB heteromultimers to identify compounds (agents) which are ALK4:ActRIIB antagonists. Compounds identified through this screening can be tested to assess their ability to modulate tissues such as bone and muscle, to assess their ability to modulate tissue growth in vivo or in vitro. These compounds can be tested, for example, in animal models (SMA models of disease). There are numerous approaches to screening for therapeutic agents for modulating tissue growth by targeting TGFβ superfamily ligand signaling (e.g., SMAD signaling). In certain embodiments, high-throughput screening of compounds can be carried out to identify agents that perturb TGFβ superfamily receptor-mediated effects on a selected cell line. In certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ALK4:ActRIIB heteromultimer to a binding partner including for example, BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGβ31, TGβ32, TGβ33, activin A, activin B, activin AB, activin AC, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty. Alternatively, the assay can be used to identify compounds that enhance binding of an AALK4:ActRIIB heteromultimer to a binding partner such as a ligand. In a further embodiment, the compounds can be identified by their ability to interact with an ALK4:ActRIIB heteromultimer. A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. As described herein, the test compounds (agents) of the invention may be created by any combinatorial chemical method. Alternatively, the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In certain embodiments, the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons. The test compounds of the disclosure can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. Optionally, the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivatible crosslinkers or any combinations thereof. In many drug-screening programs which test libraries of compounds and natural extracts, high-throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity between an ALK4:ActRIIB heteromultimer to a binding partner including for example, BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGβ31, TGβ32, TGβ33, activin A, activin B, activin AB, activin AC, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty. Merely to illustrate, in an exemplary screening assay of the present disclosure, the compound of interest is contacted with an isolated and purified ALK4:ActRIIB heteromultimer which is ordinarily capable of binding to a TGFβ superfamily ligand, as appropriate for the intention of the assay. To the mixture of the compound and ALK4:ActRIIB heteromultimer is then added to a composition containing the appropriate ligand (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGβ31, TGβ32, TGβ33, activin A, activin B, activin C, activin E, activin AB, activin AC, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and Lefty). Detection and quantification of heteromultimer-superfamily ligand complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ALK4:ActRIIB heteromultimer and its binding protein. The efficacy of the compound can be assessed by generating dose-response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified ligand is added to a composition containing the ALK4:ActRIIB heteromultimer, and the formation of heteromultimer-ligand complex is quantitated in the absence of the test compound. It will be understood that, in general, the order in which the reactants may be admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysates may be used to render a suitable cell-free assay system. Binding of an ALK4:ActRIIB heteromultimer to another protein may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g.,32P,35S,14C or3H), fluorescently labeled (e.g., FITC), or enzymatically labeled ALK4:ActRIIB heteromultimer and/or a binding protein, by immunoassay, or by chromatographic detection. In certain embodiments, the present disclosure contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an ALK4:ActRIIB heteromultimer and a binding protein. Further, other modes of detection, such as those based on optical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the disclosure. Moreover, the present disclosure contemplates the use of an interaction trap assay, also known as the “two-hybrid assay,” for identifying agents that disrupt or potentiate interaction between ALK4:ActRIIB heteromultimer and a binding partner. See, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment, the present disclosure contemplates the use of reverse two-hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate interactions between an ALK4:ActRIIB heteromultimer and a binding protein [Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368]. In certain embodiments, the subject compounds are identified by their ability to interact with an ALK4:ActRIIB heteromultimer. The interaction between the compound and the ALK4:ActRIIB heteromultimer may be covalent or non-covalent. For example, such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography [Jakoby W B et al. (1974) Methods in Enzymology 46:1]. In certain cases, the compounds may be screened in a mechanism-based assay, such as an assay to detect compounds which bind to an ALK4:ActRIIB heteromultimer. This may include a solid-phase or fluid-phase binding event. Alternatively, the gene encoding an ALK4:ActRIIB heteromultimer can be transfected with a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library preferably by high-throughput screening or with individual members of the library. Other mechanism-based binding assays may be used; for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric endpoints or fluorescence or surface plasmon resonance. 8. Exemplary Therapeutic Uses As demonstrated by the examples, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer) can be used to increase muscle mass and strength as well as increase bone mineral density in a spinal muscular atrophy (SMA) model. These data support the use of ALK4:ActRIIB antagonist therapy prolong ambulation and slow bone loss in patients with SMA. Therefore, in some embodiments, the present disclosure relates to ALK4:ActRIIB antagonists (e.g., ALK4:ActRIIB heterodimers) that may be used to treat patients with SMA, particularly preventing or reducing the severity of one or more complications of SMA. The terms “subject,” an “individual,” or a “patient” are interchangeable throughout the specification. Any of the ALK4:ActRIIB antagonists of the disclosure can potentially be employed individually or in combination for therapeutic uses disclosed herein. For example, an ALK4:ActRIIB antagonist may be used in combination with one or more additional active agent or supportive therapy for treating SMA. The methods described herein are particularly aimed at therapeutic and prophylactic treatments of mammals including, for example, rodents, primates, and humans. As used herein, a therapeutic that “prevents” (“preventing”) a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. The term “treating” (“treat”) as used herein includes amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent. In general, treatment or prevention of a disease or condition as described in the present disclosure is achieved by administering an ALK4:ActRIIB antagonist of the present disclosure in an “effective amount”. An effective amount of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A “therapeutically effective amount” of an agent of the present disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Spinal muscular atrophy is linked to a genetic mutation in the SMN1 gene [Brzustowicz, L. M. et al. (1990) Nature 344 (6266): 540-541.] Human chromosome 5 contains two nearly identical genes at location 5q13: a telomeric copy SMN1 and a centromeric copy SMN2. In healthy individuals, the SMN1 gene codes the survival of motor neuron protein (SMN) which, as its name suggests, plays a crucial role in survival of motor neurons. The SMN2 gene, on the other hand undergoes alternative splicing at the junction of intron 6 to exon 8, with only 10-20% of SMN2 transcripts coding a fully functional SMN protein and 80-90% of transcripts resulting in a truncated protein compound (SMNA7) which is rapidly degraded in the cell. In individuals affected by SMA, the SMN1 gene is mutated in such a way that it is unable to correctly code the SMN protein—due to either a deletion occurring at exon 7 or to other point mutations (frequently resulting in the functional conversion of the SMN1 sequence into SMN2). Fortunately, almost all people have at least one functional copy of the SMN2 gene, which still codes small amounts of SMN protein—around 10-20% of the normal level—allowing some neurons to survive. However, reduced availability of the SMN protein results in gradual death of motor neuron cells, particularly in the anterior horn of spinal cord and the brain. Muscles that depend on these motor neurons for regulation have decreased innervation (also called denervation) and therefore have decreased central nervous system control. Decreased impulse transmission through the motor neurons leads to decreased contractile activity of the denervated muscle. Consequently, denervated muscles undergo progressive atrophy. Muscles of lower extremities are usually affected first, followed by muscles of upper extremities, spine and neck and, in more severe cases, pulmonary and mastication muscles. Proximal muscles are generally affected earlier and to a greater degree than distal. SMA manifests over a wide range of severity, affecting infants through adults. The disease spectrum is divided into several types based on the age of onset of symptoms or with the highest attained milestone of motor development. SMA1 (also known as Werdnig-Hoffmann disease) is a severe form of the disease that manifests in the first months of life, usually with a quick and unexpected onset (“floppy baby syndrome”). Rapid motor neuron death causes inefficiency of the major bodily organs, particularly especially of the respiratory system often leading to pneumonia-induced respiratory failure. Babies diagnosed with SMA type 1 do not generally live past two years of age. In some patients, death may occur as early as within weeks of birth (sometimes these most severe cases are termed SMA type 0). With proper respiratory support, those with milder SMA type I phenotypes, which account for around 10% of SMA1 cases, are known to live into adolescence and adulthood. In some embodiments, the disclosure relates to ALK4:ActRIIB antagonists for treating SMA1, particularly preventing or delaying onset and/or decreasing severity of one or more complications of SMA1. SMA2 (also known as Dubowitx disease) is an intermediate form of the disease that affects children. Most SMA2 patients cannot stand or walk but are able to maintain a sitting position at least some period of time in their life. The onset of weakness is usually noticed some time between 6 and 18 months. The progress is known to vary greatly, some people gradually grow weaker over time while others through careful maintenance avoid any progression. Scoliosis may be present in SMA2 children, and correction with a brace may help improve respiration. Resipiatory complications are common amount SMA2 patients and are a significant cause of mortality. Life expectancy is somewhat reduced but most people with SMA2 live well into adulthood. In some embodiments, the disclosure relates to ALK4:ActRIIB antagonists for treating SMA2, particularly preventing or delaying onset and/or decreasing severity of one or more complications of SMA2. SMA3 (also known as Kugelberg-Welander disease) is the juvenile form of the disease, usually manifests after 12 months of age. In general, people with SMA3 are able to walk without support at some period time in their life, although many later lose this ability. Respiratory involvement is less noticeable in SMA3, and life expectancy is normal or near normal. In some embodiments, the disclosure relates to treating SMA3, particularly preventing or delaying onset and/or decreasing severity of one or more complications of SMA3. SMA4 is the adult-onset form of the disease (sometimes classified as a late-onset SMA type 3), usually manifesting after the third decade of life. In general, SMA4 patients experience gradual weakening of muscles, mainly in proximal muscles of the extremities. Frequently, SMA4 disease progression results in patients requiring wheelchair for mobility. Other complications are rare in SMA4 patients, and life expectancy is generally unaffected. In some embodiments, the disclosure relates to ALK4:ActRIIB antagonists for treating SMA4, particularly preventing or delaying onset and/or decreasing severity of one or more complications of SMA4. The severity of SMA complications generally depends to how well the remaining SMN2 genes can make up for the loss of function of SMN1. This is partly related to the number of SMN2 gene copies present on the chromosome. While healthy individuals often carry two SMN2 gene copies, people with SMA may have between 1 and 4 (or even more) copies. Normally greater number of SMN2 copies in a patient is correlated with milder disease severity. Thus, most SMA type I babies have one or two SMN2 copies; people with SMA II and III usually have at least three SMN2 copies; and people with SMA IV normally have at least four copies. SMA-associated complications vary greatly depending on the type of SMA and stage of disease. SMA complications include, for example: areflexia, particularly in the extremities; overall muscle weakness; respiratory complications; respiratory failure; respiratory insufficiency; poor muscle tone; limpness or a tendency to flop; difficulty in achieving developmental milestones; difficulty sitting, standing, and/walking; loss of strength of respiratory muscles, often resulting in weak cough, weak cry, accumulation of secretions in lungs or throat, and/or respiratory distress; fasciculations of the tongue; bone loss (e.g., low bone mineral density) and/or weakness; and difficulty sucking or swallowing, often resulting in poor feeding. In some embodiments, the disclosure relates to ALK4:ActRIIB antagonists for treating one or more complication of SMA, particularly preventing or delaying onset and/or decreasing severity of one or more complications of SMA. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) is a validated, 16-item, 64-point scale shown to be reliable in SMA Type I subjects. CHOP INTEND was derived in part from the Test of Infant Motor Performance (TIMP, see below) and was designed to measure motor function in weak infants with neuromuscular disease. It includes both active movements, spontaneous or goal-directed, and elicited reflexive movements, and assesses head, neck, trunk, and proximal and distal limb strength. CHOP INTEND does not include respiratory or feeding assessments, but it has been structured to move from easiest to hardest. The grading includes lower scores (gravity eliminated) and higher scores (antigravity movements). The typical symptomatic SMA Type I infant has a CHOP INTEND score of approximately 20-22 points on the 0-64 point scale. In the studies from both the PNCR68 and the NeuroNEXT SMA study, no infants with 2 copies of SMN2 had a baseline value over 40 points. In the recently conducted NeuroNEXT SMA infant biomarker study, a total of 23 SMA infants and 14 control infants were assessed. The average CHOP INTEND score for SMA infants who had 2 copies of SMN2 was 20.2 (SD=7.4, n=16, range=10-33) and the maximum score in this subgroup was 33, a finding consistent with the results of the PNCR type I SMA natural history study. In some embodiments, ALK4:ActRIIB antagonists of the disclosure may be used to treat SMA patients, wherein the treatment results in at least a 4-point in motor function in accordance with CHOP INTEND. For example, treatment may result in at least a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 point increase in motor function in accordance with CHOP INTEND. The Hammersmith Infant Neurological Examination (HINE) is a neurological examination for infants between 2 and 24 months of age and consists of 37 items divided into 3 sections. Section I: Neurological Examination, includes assessments of cranial nerve function, posture, movements, tone, reflexes, and reactions. Section II assesses developmental milestones: head control, sitting, voluntary grasp, ability to kick, rolling, crawling, standing, and walking. Section III is a behavioural scale assessing the state of consciousness, emotional state, and social orientation. The HINE was utilized as an exploratory outcome measure in the Ionis CS3A open label infant SMA type I study of nusinersen, and because it was found to be informative, it evolved into a primary outcome measure in the randomized sham-controlled ENDEAR (CS3B) study. Each physical examination finding or milestone can be scored using a numerical scale, and therefore a global score can be created. In some embodiments, ALK4:ActRIIB antagonists of the disclosure may be used to treat SMA patients, wherein the treatment results in at least a 2-point in motor function in accordance with HIND. For example, treatment may result in at least a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 point increase in motor function in accordance with HIND. The Test of Infant Motor Performance Screening Items (TIMPSI) is a 29-item, 99-point scale that has been shown to be valid and reproducible in infants with type I SMA. It tests rolling and crawling but does not test sitting. It includes many items administered in a prone position, which is not tolerated well by type I infants. It was utilized in the recent NeuroNEXT SMA infant biomarker study as a measure to screen the participating SMA and healthy infants' motor performance. Subjects who scored less than 41 on the TIMPSI were then evaluated using CHOP INTEND, and subjects who scored 41 or greater on the TIMPSI were evaluated using the Alberta Infant Motor Scale (AIMS, see below). The average TIMPSI score for the SMA cohort was 34.9 points, significantly lower than the average score for healthy controls (66.1, range=50-96). SMA infants with 2 copies of SMN2 had an average TIMPSI score of 27.2 (SD=8.0, n=16, range=15-49). In some embodiments, ALK4:ActRIIB antagonists of the disclosure may be used to treat SMA patients, wherein the treatment results in at least a 2-point in motor function in accordance with TIMPSI. For example, treatment may result in at least a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 point increase in motor function in accordance with TIMPSI. The Bayley Scales of Infant Development (Bayley-III) includes assessment of motor function (gross and fine) as well as assessment of cognition and language (receptive and expressive) in infants and young children from 0-42 months. The Bayley-III language assessment is divided into expressive and receptive subtests; when these two subtests are combined, a composite score is determined. Similarly, the Bayley-III motor assessment includes scale scores for fine motor and gross motor development as well as a composite score. It has normative data, but in older/stronger children it takes a long time to administer. The main advantage of the Bayley scale is that it can also assess fine motor function, cognition and language; further, it can measure not only the acquisition of a milestone but also define when it was acquired. It has been used in the AveXis gene therapy type 1 trial. In some embodiments, ALK4:ActRIIB antagonists of the disclosure may be used to treat SMA patients, wherein the treatment results in at least a 2-point in motor function in accordance with Bayley-III. For example, treatment may result in at least a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 point increase in motor function in accordance with Bayley-III. In some embodiments, the disclosure relates to methods of treating SMA, particularly preventing or delaying onset of and/or reducing the severity of one or more complications of SMA, comprising administering an ALK4:ActRIIB antagonist in combination with one or more additional active agents or supportive therapies for treating SMA. As used herein, “in combination with”, “combinations of”, or “conjoint administration” refers to any form of administration such that additional therapies (e.g., second, third, fourth, etc.) are still effective in the body (e.g., multiple compounds are simultaneously effective in the patient, which may include synergistic effects of those compounds). Effectiveness may not correlate to measurable concentration of the agent in blood, serum, or plasma. For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially, and on different schedules. Thus, an individual who receives such treatment can benefit from a combined effect of different therapies. One or more ALK4:ActRIIB antagonist of the disclosure can be administered concurrently with, prior to, or subsequent to, one or more other additional agents or supportive therapies. In general, each therapeutic agent will be administered at a dose and/or on a time schedule determined for that particular agent. The particular combination to employ in a regimen will take into account compatibility of the antagonist of the present disclosure with the therapy and/or the desired. Since the underlying genetic cause of SMA was identified [Lefebvre et al. (1995) Cell. 80 (1): 155-165], several therapeutic approaches have been proposed and investigated that primarily focus on increasing the availability of SMN protein in motor neurons. The main research directions include: SMN1 gene replacement, SMN2 alternative splicing modulation, SMN2 gene activation, SMN stabilization, neuroprotection, and muscle restoration. In some embodiments, the disclosure relates to methods of treating SMA, particularly preventing or delaying onset of and/or reducing the severity of one or more complications of SMA, comprising administering an ALK4:ActRIIB antagonist in combination with one or more additional therapeutic approaches selected from: SMN1 gene replacement, SMN2 alternative splicing modulation, SMN2 gene activation, SMN stabilization, neuroprotection, and muscle restoration. Nusinersen is the only approved drug to treat spinal muscular atrophy. It is an antisense drug that administered directly to the central nervous system using an intrathecal injection. Like other antisense drugs, there is a risk of abnormalities in blood clotting and a reduction in platelets as well as a risk of kidney damage. In clinical trials, people treated with nusinersen had an increased risk of upper and lower respiratory infections and congestion, ear infections, constipation, aspiration, teething, and scoliosis. In some embodiments, the disclosure relates to methods of treating SMA, particularly preventing or delaying onset of and/or reducing the severity of one or more complications of SMA, comprising administering an ALK4:ActRIIB antagonist in combination with nusinersen. In general, gene therapy in SMA aims at restoring the SMN1 gene function through inserting specially crafted nucleotide sequence (a SMN1 transgene) into the cell nucleus using a viral vector; scAAV-9 and scAAV-10 are the primary viral vectors under investigation. For example, AVXS-101 uses self-complementary scAAV-9 as a vector to deliver the SMN1 transgene. Early results from an AVXS-101 clinical study show improvement in treated infants compared to the natural course of the disorder. In general, SMN2 alternative splicing modulation approaches aim at modifying the alternative splicing of the SMN2 to force it to code for higher percentage of full-length SMN protein. Sometimes it is also called gene conversion, because it attempts to convert the SMN2 gene functionally into SMN1 gene. Splicing modulators have reached clinical stage development include, for example, branaplam (LMI070), RG7916, RG3039 (Quinazoline495), and RG7800. Basic research has also identified other compounds which modified SMN2 splicing in vitro including, for example, sodium orthovanadate and aclarubicin. Other Morpholino-type antisense oligonucleotides with the same cellular target as nusinersen remain a subject of intense research. In general, SMN2 gene activation approaches aim at increasing expression (activity) of the SMN2 gene, thus increasing the amount of full-length SMN protein available. Oral salbutamol (albuterol), a popular asthma medicine, showed therapeutic potential in SMA both in vitro and in three small-scale clinical trials involving patients with SMA types 2 and 3, besides offering respiratory benefits. Other compounds that have shown promise in SMN2 gene activation include, for example, butyrates (sodium butyrate and sodium phenylbutyrate), valproic, hydroxycarbamide (hydroxyurea), histone deacetylase inhibitors, benzamide M344, hydroxamic acids (e.g., CBHA, SBHA, entinostat, panobinostat trichostatin A, and vorinostat), prolactin[as well as natural polyphenol compounds (e.g., resveratrol and curcumin), and p38 pathway activators (e.g., celecoxib). In general, SMN stabilization aims at stabilizing the SMNA7 protein, the short-lived defective protein coded by the SMN2 gene, so that it is able to sustain neuronal cells. SMN stabilization agents that have been considered include, for example, aminoglycosides and indoprofen. In general, neuroprotective drugs aim at enabling the survival of motor neurons even with low levels of SMN protein. For example, olesoxime is a proprietary neuroprotective compound which showed stabilizing effects in a phase II-III clinical trial involving people with SMA types 2 and 3. Other neuroprotective drugs that have been considered include, for example, thyrotropin-releasing hormone, riluzole, and β-lactam antibiotics (e.g., ceftriaxone). In general, muscle restoration approaches aim to counter the effect of SMA by targeting the muscle tissue instead of neurons. For example, CK-2127107 (CK-107) is a skeletal troponin activator that is used to increasing muscle reactivity despite lowered neural signaling. There are several supportive therapies for managing the various complications associated with SMA. For example, weak spine muscles may lead to development of kyphosis, scoliosis and other orthopaedic problems. Spine fusion is sometimes performed in people with SMA I/II once they reach the age of 8-10 to relieve the pressure of a deformed spine on the lungs. People with SMA might also benefit greatly from various forms of physiotherapy and occupational therapy. Orthotic devices can be used to support the body and to aid walking. For example, orthotics such as AFO's (ankle foot orthosis) are used to stabilize the foot and to aid gait, TLSO's (thoracic lumbar sacral orthosis) are used to stabilize the torso. Assistive technologies may help in managing movement and daily activity and greatly increase the quality of life. Respiratory system requires utmost attention in SMA as once weakened it never fully recovers. Weakened pulmonary muscles in people with SMA type I/II can make breathing more difficult and pose a risk of hypoxiation, especially in sleep when muscles are more relaxed. Impaired cough reflex poses a constant risk of respiratory infection and pneumonia. Non-invasive ventilation (BiPAP) is frequently used and tracheostomy may be sometimes performed in more severe cases. Booth of these methods of ventilation prolong survival in a comparable degree, although tracheostomy prevents speech development. Difficulties in jaw opening, chewing and swallowing food might put people with SMA at risk of malnutrition. A feeding tube or gastrostomy can be necessary in SMA type I and people with more severe type II. Additionally, metabolic abnormalities resulting from SMA impair β-oxidation of fatty acids in muscles and can lead to organic acidemia and consequent muscle damage, especially when fasting. It is suggested that people with SMA, especially those with more severe forms of the disease, reduce intake of fat and avoid prolonged fasting (i.e., eat more frequently than healthy people). An ALK4:ActRIIB antagonist of the disclosure may be conjointly administered with other bone-active pharmaceutical agents. ALK4:ActRIIB antagonists may be particularly advantageous if administered with other bone-active agents. A patient may benefit from conjointly receiving an ALK4:ActRIIB antagonist and taking calcium supplements, vitamin D, appropriate exercise and/or, in some cases, other medication. Examples of other medications include, bisphosphonates (alendronate, ibandronate and risedronate), calcitonin, estrogens, parathyroid hormone and raloxifene. The bisphosphonates (alendronate, ibandronate and risedronate), calcitonin, estrogens and raloxifene affect the bone remodeling cycle and are classified as anti-resorptive medications. Bone remodeling consists of two distinct stages: bone resorption and bone formation. Anti-resorptive medications slow or stop the bone-resorbing portion of the bone-remodeling cycle but do not slow the bone-forming portion of the cycle. As a result, new formation continues at a greater rate than bone resorption, and bone density may increase over time. Teriparatide, a form of parathyroid hormone, increases the rate of bone formation in the bone remodeling cycle. Alendronate is approved for both the prevention (5 mg per day or 35 mg once a week) and treatment (10 mg per day or 70 mg once a week) of postmenopausal osteoporosis. Alendronate reduces bone loss, increases bone density and reduces the risk of spine, wrist and hip fractures. Alendronate also is approved for treatment of glucocorticoid-induced osteoporosis in men and women as a result of long-term use of these medications (i.e., prednisone and cortisone) and for the treatment of osteoporosis in men. Alendronate plus vitamin D is approved for the treatment of osteoporosis in postmenopausal women (70 mg once a week plus vitamin D), and for treatment to improve bone mass in men with osteoporosis. Ibandronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken as a once-a-month pill (150 mg), ibandronate should be taken on the same day each month. Ibandronate reduces bone loss, increases bone density and reduces the risk of spine fractures. Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mg dose or 35 mg dose with calcium), risedronate slows bone loss, increases bone density and reduces the risk of spine and non-spine fractures. Risedronate also is approved for use by men and women to prevent and/or treat glucocorticoid-induced osteoporosis that results from long-term use of these medications (i.e., prednisone or cortisone). Calcitonin is a naturally occurring hormone involved in calcium regulation and bone metabolism. In women who are more than 5 years beyond menopause, calcitonin slows bone loss, increases spinal bone density, and may relieve the pain associated with bone fractures. Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an injection (50-100 IU daily) or nasal spray (200 IU daily). A patient may also benefit from conjointly receiving an ALK4:ActRIIB antagonist and additional bone-active medications. Estrogen therapy (ET)/hormone therapy (HT) is approved for the prevention of osteoporosis. ET has been shown to reduce bone loss, increase bone density in both the spine and hip, and reduce the risk of hip and spinal fractures in postmenopausal women. ET is administered most commonly in the form of a pill or skin patch that delivers a low dose of approximately 0.3 mg daily or a standard dose of approximately 0.625 mg daily and is effective even when started after age 70. When estrogen is taken alone, it can increase a woman's risk of developing cancer of the uterine lining (endometrial cancer). To eliminate this risk, healthcare providers prescribe the hormone progestin in combination with estrogen (hormone replacement therapy or HT) for those women who have an intact uterus. ET/HT relieves menopause symptoms and has been shown to have a beneficial effect on bone health. Side effects may include vaginal bleeding, breast tenderness, mood disturbances and gallbladder disease. Raloxifene, 60 mg a day, is approved for the prevention and treatment of postmenopausal osteoporosis. It is from a class of drugs called Selective Estrogen Receptor Modulators (SERMs) that have been developed to provide the beneficial effects of estrogens without their potential disadvantages. Raloxifene increases bone mass and reduces the risk of spine fractures. Data are not yet available to demonstrate that raloxifene can reduce the risk of hip and other non-spine fractures. Teriparatide, a form of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and men who are at high risk for a fracture. This medication stimulates new bone formation and significantly increases bone mineral density. In postmenopausal women, fracture reduction was noted in the spine, hip, foot, ribs and wrist. In men, fracture reduction was noted in the spine, but there were insufficient data to evaluate fracture reduction at other sites. Teriparatide is self-administered as a daily injection for up to 24 months. 9. Pharmaceutical Compositions In certain aspects, ALK4:ActRIIB antagonists of the present disclosure can be administered alone or as a component of a pharmaceutical formulation (also referred to as a therapeutic composition or pharmaceutical composition). A pharmaceutical formation refers to a preparation which is in such form as to permit the biological activity of an active ingredient (e.g., an agent of the present disclosure) contained therein to be effective and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. For example, one or more agents of the present disclosure may be formulated with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is generally nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, and/or preservative. In general, pharmaceutical formulations for use in the present disclosure are in a pyrogen-free, physiologically-acceptable form when administered to a subject. Therapeutically useful agents other than those described herein, which may optionally be included in the formulation as described above, may be administered in combination with the subject agents in the methods of the present disclosure. In certain embodiments, compositions will be administered parenterally [e.g., by intravenous (I.V.) injection, intraarterial injection, intraosseous injection, intramuscular injection, intrathecal injection, subcutaneous injection, or intradermal injection]. Pharmaceutical compositions suitable for parenteral administration may comprise one or more agents of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use. Injectable solutions or dispersions may contain antioxidants, buffers, bacteriostats, suspending agents, thickening agents, or solutes which render the formulation isotonic with the blood of the intended recipient. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical formulations of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, a therapeutic method of the present disclosure includes administering the pharmaceutical composition systemically, or locally, from an implant or device. Further, the pharmaceutical composition may be encapsulated or injected in a form for delivery to a target tissue site (e.g., bone marrow or muscle). In certain embodiments, compositions of the present disclosure may include a matrix capable of delivering one or more of the agents of the present disclosure to a target tissue site (e.g., bone marrow or muscle), providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of one or more agents of the present disclosure. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material may be based on one or more of: biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties. The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other potential materials are biodegradable and biologically well-defined including, for example, bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined including, for example, sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material including, for example, polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition (e.g., calcium-aluminate-phosphate) and processing to alter one or more of pore size, particle size, particle shape, and biodegradability. In certain embodiments, pharmaceutical compositions of present disclosure can be administered topically. “Topical application” or “topically” means contact of the pharmaceutical composition with body surfaces including, for example, the skin, wound sites, and mucous membranes. The topical pharmaceutical compositions can have various application forms and typically comprises a drug-containing layer, which is adapted to be placed near to or in direct contact with the tissue upon topically administering the composition. Pharmaceutical compositions suitable for topical administration may comprise one or more one or more ALK4:ActRIIB antagonists of the disclosure in combination formulated as a liquid, a gel, a cream, a lotion, an ointment, a foam, a paste, a putty, a semi-solid, or a solid. Compositions in the liquid, gel, cream, lotion, ointment, foam, paste, or putty form can be applied by spreading, spraying, smearing, dabbing or rolling the composition on the target tissue. The compositions also may be impregnated into sterile dressings, transdermal patches, plasters, and bandages. Compositions of the putty, semi-solid or solid forms may be deformable. They may be elastic or non-elastic (e.g., flexible or rigid). In certain aspects, the composition forms part of a composite and can include fibers, particulates, or multiple layers with the same or different compositions. Topical compositions in the liquid form may include pharmaceutically acceptable solutions, emulsions, microemulsions, and suspensions. In addition to the active ingredient(s), the liquid dosage form may contain an inert diluent commonly used in the art including, for example, water or other solvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acid ester of sorbitan, and mixtures thereof]. Topical gel, cream, lotion, ointment, semi-solid or solid compositions may include one or more thickening agents, such as a polysaccharide, synthetic polymer or protein-based polymer. In one embodiment of the invention, the gelling agent herein is one that is suitably nontoxic and gives the desired viscosity. The thickening agents may include polymers, copolymers, and monomers of: vinylpyrrolidones, methacrylamides, acrylamides N-vinylimidazoles, carboxy vinyls, vinyl esters, vinyl ethers, silicones, polyethyleneoxides, polyethyleneglycols, vinylalcohols, sodium acrylates, acrylates, maleic acids, NN-dimethylacrylamides, diacetone acrylamides, acrylamides, acryloyl morpholine, pluronic, collagens, polyacrylamides, polyacrylates, polyvinyl alcohols, polyvinylenes, polyvinyl silicates, polyacrylates substituted with a sugar (e.g., sucrose, glucose, glucosamines, galactose, trehalose, mannose, or lactose), acylamidopropane sulfonic acids, tetramethoxyorthosilicates, methyltrimethoxyorthosilicates, tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propylene glycol, glycerine, polysaccharides, alginates, dextrans, cyclodextrin, celluloses, modified celluloses, oxidized celluloses, chitosans, chitins, guars, carrageenans, hyaluronic acids, inulin, starches, modified starches, agarose, methylcelluloses, plant gums, hylaronans, hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses, hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins, low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graft copolymers, starch sodium polyacrylate, hydroxyethyl methacrylates, hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethyl methacrylates, polystyrenes, polyurethanes, polyalkanoates, polylactic acids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid), SEM (sulfoethylmethacrylate), SPM (sulfopropyl methacrylate), SPA (sulfopropyl acrylate), N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine, methacryllic acid amidopropyl-dimethyl ammonium sulfobetaine, SPI (itaconic acid-bis(1-propyl sulfonizacid-3) ester di-potassium salt), itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid), beta-carboxyethyl acrylate (acrylic acid dimers), and maleic anhydride-methylvinyl ether polymers, derivatives thereof, salts thereof, acids thereof, and combinations thereof. In certain embodiments, pharmaceutical compositions of present disclosure can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis such as sucrose and acacia or tragacanth), powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, or an elixir or syrup, or pastille (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and/or a mouth wash, each containing a predetermined amount of a compound of the present disclosure and optionally one or more other active ingredients. A compound of the present disclosure and optionally one or more other active ingredients may also be administered as a bolus, electuary, or paste. In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, and granules), one or more compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers including, for example, sodium citrate, dicalcium phosphate, a filler or extender (e.g., a starch, lactose, sucrose, glucose, mannitol, and silicic acid), a binder (e.g. carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone, sucrose, and acacia), a humectant (e.g., glycerol), a disintegrating agent (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, a silicate, and sodium carbonate), a solution retarding agent (e.g. paraffin), an absorption accelerator (e.g. a quaternary ammonium compound), a wetting agent (e.g., cetyl alcohol and glycerol monostearate), an absorbent (e.g., kaolin and bentonite clay), a lubricant (e.g., a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), a coloring agent, and mixtures thereof. In the case of capsules, tablets, and pills, the pharmaceutical formulation (composition) may also comprise a buffering agent. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using one or more excipients including, e.g., lactose or a milk sugar as well as a high molecular-weight polyethylene glycol. Liquid dosage forms for oral administration of the pharmaceutical composition may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient(s), the liquid dosage form may contain an inert diluent commonly used in the art including, for example, water or other solvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acid ester of sorbitan, and mixtures thereof]. Besides inert diluents, the oral formulation can also include an adjuvant including, for example, a wetting agent, an emulsifying and suspending agent, a sweetening agent, a flavoring agent, a coloring agent, a perfuming agent, a preservative agent, and combinations thereof. Suspensions, in addition to the active compounds, may contain suspending agents including, for example, an ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and combinations thereof. Prevention of the action and/or growth of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents including, for example, paraben, chlorobutanol, and phenol sorbic acid. In certain embodiments, it may be desirable to include an isotonic agent including, for example, a sugar or sodium chloride into the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of an agent that delay absorption including, for example, aluminum monostearate and gelatin. It is understood that the dosage regimen will be determined by the attending physician considering various factors which modify the action of the one or more of the agents of the present disclosure. In the case of a ALK4:ActRIIB antagonist that promotes red blood cell formation, various factors may include, but are not limited to, the patient's red blood cell count, hemoglobin level, the desired target red blood cell count, the patient's age, the patient's sex, the patient's diet, the severity of any disease that may be contributing to a depressed red blood cell level, the time of administration, and other clinical factors. The addition of other known active agents to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of one or more of red blood cell levels, hemoglobin levels, reticulocyte levels, and other indicators of the hematopoietic process. In certain embodiments, the present disclosure also provides gene therapy for the in vivo production of one or more of the agents of the present disclosure. Such therapy would achieve its therapeutic effect by introduction of the agent sequences into cells or tissues having one or more of the disorders as listed above. Delivery of the agent sequences can be achieved, for example, by using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred therapeutic delivery of one or more of agent sequences of the disclosure is the use of targeted liposomes. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or an RNA virus (e.g., a retrovirus). The retroviral vector may be a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody. Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing one or more of the agents of the present disclosure. Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes (gag, pol, and env), by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium. Another targeted delivery system for one or more of the agents of the present disclosure is a colloidal dispersion system. Colloidal dispersion systems include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In certain embodiments, the preferred colloidal system of this disclosure is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form [Fraley, et al. (1981) Trends Biochem. Sci., 6:77]. Methods for efficient gene transfer using a liposome vehicle are known in the art [Mannino, et al. (1988) Biotechniques, 6:682, 1988]. The composition of the liposome is usually a combination of phospholipids, which may include a steroid (e.g. cholesterol). The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Other phospholipids or other lipids may also be used including, for example a phosphatidyl compound (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, a sphingolipid, a cerebroside, and a ganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art. INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention, and are not intended to limit the invention. Example 1. Generation of an ALK4:ActRIIB Heterodimer Soluble ALK4-Fc:ActRIIB-Fc heteromeric complexes comprising the extracellular domains of human ActRIIB and human ALK4, which are each separately fused to an Fc domain with a linker positioned between the extracellular domain and the Fc domain, were constructed. The individual constructs are referred to as ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide, respectively, and the sequences for each are provided below. A methodology for promoting formation of ALK4-Fc:ActRIIB-Fc heteromeric complexes, as opposed to ActRIIB-Fc or ALK4-Fc homodimeric complexes, is to introduce alterations in the amino acid sequence of the Fc domains to guide the formation of asymmetric heteromeric complexes. Many different approaches to making asymmetric interaction pairs using Fc domains are described in this disclosure. In one approach, illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 39-41 and 42-44, respectively, one Fc domain is altered to introduce cationic amino acids at the interaction face, while the other Fc domain is altered to introduce anionic amino acids at the interaction face. ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader: (SEQ ID NO: 38)MDAMKRGLCCVLLLCGAVFVSP. The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 39) is shown below: (SEQ ID NO: 39)1MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV301EWESNGQPEN NYKTTPPVLKSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351EALHNHYTQK SLSLSPGK The leader (signal) sequence and linker areunderlined. To promote formation of ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing acidic amino acids with lysine) can be introduced into the Fc domain of the ActRIIB fusion protein as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 39 may optionally be provided with lysine (K) removed from the C-terminus. This ActRIIB-Fc fusion protein is encoded by the following nucleic acid sequence (SEQ ID NO: 40): (SEQ ID NO: 40)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC801ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG851TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC951CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT1051GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG1101TAAA The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 41) is as follows, and may optionally be provided with lysine (K) removed from the C-terminus. (SEQ ID NO: 41)1GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS251RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 42) is as follows: (SEQ ID NO: 42)1MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF151LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP201REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG251QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY301DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL351SLSPG The leader sequence and linker areunderlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 39 and 41 above, two amino acid substitutions (replacing lysines with aspartic acids) can be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 42 may optionally be provided with lysine (K) added at the C-terminus. This ALK4-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 43): (SEQ ID NO: 43)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT151GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT201GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT251ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC301TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC351TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA501GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT551TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG601CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT651CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA701ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG751CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT801GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA851GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC901GACACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTATAG951CGACCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT1001GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC1051TCCCTGTCTC CGGGT The mature ALK4-Fc fusion protein sequence (SEQ ID NO: 44) is as follows and may optionally be provided with lysine (K) added at the C-terminus. (SEQ ID NO: 44)1SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD151VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN201GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL251TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFF LYSDLTVDKS301RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G The ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 41 and SEQ ID NO: 44, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc. In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins the Fc domains are altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond as illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 45-46 and 47-48, respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader: (SEQ ID NO: 38)MDAMKRGLCCVLLLCGAVFVSP. The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 45) is shown below: (SEQ ID NO: 45)1MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351EALHNHYTQK SLSLSPGK The leader (signal) sequence and linker areunderlined. To promote formation of the ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a trytophan) can be introduced into the Fc domain of the fusion protein as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 45 may optionally be provided with lysine (K) removed from the C-terminus. The mature ActRIIB-Fc fusion polypeptide is as follows: (SEQ ID NO: 46)1GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC251REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 47) is as follows and may optionally be provided with lysine (K) removed from the C-terminus. (SEQ ID NO: 47)1MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF151LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP201REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG251QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESNGQPENNY301KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL351SLSPGK The leader sequence and the linker areunderlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 45 and 46 above, four amino acid substitutions can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 47 may optionally be provided with lysine (K) removed from the C-terminus. The mature ALK4-Fc fusion protein sequence is as follows and may optionally be provided with lysine (K) removed from the C-terminus. (SEQ ID NO: 48)1SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD151VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN201GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL251SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS301RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 46 and SEQ ID NO: 48, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc. Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, cation exchange chromatography, and epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope on ALK4 or ActRIIB) The purification could be completed with viral filtration and buffer exchange. In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins, the Fc domains are altered to introduce complementary hydrophobic interactions, an additional intermolecular disulfide bond, and electrostatic differences between the two Fc domains for facilitating purification based on net molecular charge, as illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 70-73 and 74-77, respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 38). The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 70) is shown below: (SEQ ID NO: 70)1MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPTGGGTHTCPPC151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251APIEKTISKA KGQPREPQVY TLPPCREEMTENQVSLWCLV KGFYPSDIAV301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351EALHNHYTQDSLSLSPG The leader sequence and linker areunderlined. To promote formation of the ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a trytophan) can be introduced into the Fc domain of the fusion protein as indicated bydouble underlineabove. To facilitate purification of the ALK4-Fc:ActRIIB-Fc heterodimer, two amino acid substitutions (replacing lysines with acidic amino acids) can also be introduced into the Fc domain of the fusion protein as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 70 may optionally be provided with a lysine added at the C-terminus. This ActRIIB-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 71): (SEQ ID NO: 71)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC801ACAGGTGTAC ACCCTGCCCC CATGCCGGGA GGAGATGACC GAGAACCAGG851TCAGCCTGTG GTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC951CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT1051GAGGCTCTGC ACAACCACTA CACGCAGGAC AGCCTCTCCC TGTCTCCGGG1101T The mature ActRIIB-Fc fusion polypeptide is as follows (SEQ ID NO: 72) and may optionally be provided with a lysine added to the C-terminus. (SEQ ID NO: 72)1GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC251REEMTENQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQDSLSLS PG This ActRIIB-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 73): (SEQ ID NO: 73)1GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT251GCTGTGAAGG CAACTTCTGC AACGAGCGCT TCACTCATTT GCCAGAGGCT301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACCGGTGG351TGGAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGGGGAC401CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC451CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC501TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA551AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGC601GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG651CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA701AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATGC751CGGGAGGAGA TGACCGAGAA CCAGGTCAGC CTGTGGTGCC TGGTCAAAGG801CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG851AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA CGGCTCCTTC901TTCCTCTATA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA951CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC1001AGGACAGCCT CTCCCTGTCT CCGGGT The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 74) is as follows and may optionally be provided with lysine removed from the C-terminus. (SEQ ID NO: 74)1MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF151LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP201REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG251QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESRGQPENNY301KTTPPVLDSRGSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL351SLSPGK The leader sequence and the linker areunderlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 70 and 72 above, four amino acid substitutions (replacing a tyrosine with a cysteine, a threonine with a serine, a leucine with an alanine, and a tyrosine with a valine) can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated bydouble underlineabove. To facilitate purification of the ALK4-Fc:ActRIIB-Fc heterodimer, two amino acid substitutions (replacing an asparagine with an arginine and an aspartate with an arginine) can also be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated bydouble underlineabove. The amino acid sequence of SEQ ID NO: 74 may optionally be provided with lysine removed from the C-terminus. This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 75): (SEQ ID NO: 75)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT151GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT201GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT251ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC301TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC351TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA501GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT551TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG601CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT651CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA701ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG751CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT801GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA851GCGACATCGC CGTGGAGTGG GAGAGCCGCG GGCAGCCGGA GAACAACTAC901AAGACCACGC CTCCCGTGCT GGACTCCCGC GGCTCCTTCT TCCTCGTGAG951CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT1001GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC1051TCCCTGTCTC CGGGTAAA The mature ALK4-Fc fusion polypeptide sequence is as follows (SEQ ID NO: 76) and may optionally be provided with lysine removed from the C-terminus. (SEQ ID NO: 76)1SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD151VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN201GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL251SCAVKGFYPS DIAVEWESRG QPENNYKTTP PVLDSRGSFF LVSKLTVDKS301RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 77): (SEQ ID NO: 77)1TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT51CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT101TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG151GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT201GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA251GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC301CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC351TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG401ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC451GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT501GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA551CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT601GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT651CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT701GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG751TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA801GAGCCGCGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG851ACTCCCGCGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC901AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT951GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 72 and SEQ ID NO: 76, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc. Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, cation exchange chromatography, epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope on ALK4 or ActRIIB), and multimodal chromatography (e.g., with resin containing both electrostatic and hydrophobic ligands). The purification could be completed with viral filtration and buffer exchange. In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins, the Fc domains are altered to introduce complementary hydrophobic interactions, an additional intermolecular disulfide bond, and a histidine-to-arginine substitution specifically in the ActRIIB-Fc polypeptide chain for facilitating purification based on protein A affinity, as illustrated in the ActRIIB-Fc polypeptide sequences of SEQ ID NOs: 78-81 and the ALK4-Fc polypeptide sequences of SEQ ID NOs: 47, 48, 82, and 83. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the TPA leader: (SEQ ID NO: 38)MDAMKRGLCCVLLLCGAVFVSP. The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 78) is shown below: (SEQ ID NO: 78)1MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPTGGGTHTCPPC151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351EALHNRYTQK SLSLSPGK The leader sequence and linker areunderlined. To promote formation of the ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a trytophan) can be introduced into the Fc domain of the ActRIIB-Fc fusion polypeptide as indicated bydouble underlineabove. Another amino acid substitution (replacing histidine with arginine) can also be introduced into the Fc domain of the fusion protein as indicated bydouble underlineabove to facilitate purification of the ALK4-Fc:ActRIIB-Fc heterodimer. The amino acid sequence of SEQ ID NO: 78 may optionally be provided with lysine removed from the C-terminus. This ActRIIB-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 79): (SEQ ID NO: 79)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC801ACAGGTGTAC ACCCTGCCCC CATGCCGGGA GGAGATGACC AAGAACCAGG851TCAGCCTGTG GTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC951CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT1051GAGGCTCTGC ACAACCGCTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG1101TAAA The mature ActRIIB-Fc fusion polypeptide is as follows (SEQ ID NO: 80) and may optionally be provided with lysine removed from the C-terminus. (SEQ ID NO: 80)1GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC251REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN RYTQKSLSLS PGK This ActRIIB-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 81): (SEQ ID NO: 81)1GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT251GCTGTGAAGG CAACTTCTGC AACGAGCGCT TCACTCATTT GCCAGAGGCT301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACCGGTGG351TGGAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGGGGAC401CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC451CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC501TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA551AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGC601GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG651CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA701AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATGC751CGGGAGGAGA TGACCAAGAA CCAGGTCAGC CTGTGGTGCC TGGTCAAAGG801CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG851AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA CGGCTCCTTC901TTCCTCTATA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA951CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CGCTACACGC1001AGAAGAGCCT CTCCCTGTCT CCGGGTAAA The complementary form of ALK4-Fc fusion polypeptide is SEQ ID NO: 47 (shown above), which contains four amino acid substitutions to guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 78 and 80 and may optionally be provided with lysine removed from the C-terminus. This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 82): (SEQ ID NO: 82)1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT151GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT201GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT251ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC301TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC351TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA501GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT551TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG601CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT651CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA701ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG751CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT801GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA851GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC901AAGACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCGTGAG951CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT1001GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC1051TCCCTGTCTC CGGGTAAA The mature ALK4-Fc fusion polypeptide sequence is SEQ ID NO: 48 (shown above) and may optionally be provided with lysine removed from the C-terminus. This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 83): (SEQ ID NO: 83)1TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT51CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT101TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG151GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT201GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA251GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC301CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC351TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG401ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC451GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT501GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA551CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT601GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT651CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT701GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG751TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA801GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG851ACTCCGACGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC901AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT951GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 80 and SEQ ID NO: 48, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc. Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography and epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope on ALK4 or ActRIIB), and multimodal chromatography (e.g., with resin containing both electrostatic and hydrophobic ligands). The purification could be completed with viral filtration and buffer exchange. Example 2. Ligand Binding Profile of ALK4-Fc:ActRIIB-Fc Heterodimer Compared to ActRIIB-Fc Homodimer and ALK4-Fc Homodimer A Biacore™-based binding assay was used to compare ligand binding selectivity of the ALK4-Fc:ActRIIB-Fc heterodimeric complex described above with that of ActRIIB-Fc and ALK4-Fc homodimer complexes. The ALK4-Fc:ActRIIB-Fc heterodimer, ActRIIB-Fc homodimer, and ALK4-Fc homodimer were independently captured onto the system using an anti-Fc antibody. Ligands were injected and allowed to flow over the captured receptor protein. Results are summarized in the table below, in which ligand off-rates (kd) most indicative of effective ligand traps are denoted by gray shading. Ligand binding profile of ALK4-Fc: ActRIIB-Fc heterodimer compared toActRIIB-Fc homodimer and ALK4-Fc homodimerActRIIB-FcALK4-FcALK4-Fc: ActRIIB-FchomodimerhomodimerheterodimerkakdKDkakdKDkakdKDLigand(1/Ms)(1/s)(pM)(1/Ms)(1/s)(pM)(1/Ms)(1/s)(pM)Activin A1.2 × 1072.3 × 10−4195.8 × 1051.2 × 10−2200001.3 × 1071.5 × 10−412Activin B5.1 × 1061.0 × 10−420No binding7.1 × 1064.0 × 10−56BMP63.2 × 1076.8 × 10−3190—2.0 × 1065.5 × 10−32700BMP91.4 × 1071.1 × 10−377—Transient*3400BMP102.3 × 1072.6 × 10−411—5.6 × 1074.1 × 10−374GDF31.4 × 1062.2 × 10−31500—3.4 × 1061.7 × 10−24900GDF88.3 × 1052.3 × 10−42801.3 × 1051.9 × 10−315000†3.9 × 1052.1 × 10−4550GDF115.0 × 1071.1 × 10−425.0 × 1064.8 × 10−3270†3.8 × 1071.1 × 10−43*Indeterminate due to transient nature of interaction†Very low signal—Not tested These comparative binding data demonstrate that ALK4-Fc:ActRIIB-Fc heterodimer has an altered binding profile/selectivity relative to either ActRIIB-Fc or ALK4-Fc homodimers. ALK4-Fc:ActRIIB-Fc heterodimer displays enhanced binding to activin B compared with either homodimer, retains strong binding to activin A, GDF8, and GDF11 as observed with ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. In particular, BMP9 displays low or no observable affinity for ALK4-Fc:ActRIIB-Fc heterodimer, whereas this ligand binds strongly to ALK4-Fc:ActRIIB-Fc heterodimer. Like the ActRIIB-Fc homodimer, the heterodimer retains intermediate-level binding to BMP6. SeeFIG.4. In addition, an A-204 Reporter Gene Assay was used to evaluate the effects of ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer on signaling by activin A, activin B, GDF11, GDF8, BMP10, and BMP9. Cell line: Human Rhabdomyosarcoma (derived from muscle). Reporter vector: pGL3(CAGA)12 (as described in Dennler et al, 1998, EMBO 17: 3091-3100). The CAGA12 motif is present in TGF-beta responsive genes (PAI-1 gene), so this vector is of general use for factors signaling through Smad2 and 3. An exemplary A-204 Reporter Gene Assay is outlined below. Day 1: Split A-204 cells into 48-well plate. Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or pGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene. Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to be pre-incubated with Factors for about one hr before adding to cells. About six hrs later, cells are rinsed with PBS and then lysed. Following the above steps, applicant performed a Luciferase assay. Both the ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer were determined to be potent inhibitors of activin A, activin B, GDF11, and GDF8 in this assay. In particular, as can be seen in the comparative homodimer/heterodimer IC50data illustrated inFIG.7, ALK4-Fc:ActRIIB-Fc heterodimer inhibits activin A, activin B, GDF8, and GDF11 signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fc homodimer. However, ALK4-Fc:ActRIIB-Fc heterodimer inhibition of BMP9 and BMP10 signaling pathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fc homodimer. This data is consistent with the above-discussed binding data in which it was observed that both the ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer display strong binding to activin A, activin B, GDF8, and GDF11, but BMP10 and BMP9 have significantly reduced affinity for the ALK4-Fc:ActRIIB-Fc heterodimer compared to the ActRIIB-Fc:ActRIIB-Fc homodimer. To reduce the risk of potential adverse immune-related events in mouse studies described herein, ALK4-Fc:ActRIIB-Fc heterodimers were generated using the human ALK4 and human ActRIIB extracellular domains and linker domains as described above but were instead fused to mouse Fc fusion domains. The mouse Fc fusion domains were modified as described above to promote heteromultimer formation. This construct is designated as ALK4-mFc:ActRIIB-mFc. Together, these data therefore demonstrate that ALK4-Fc:ActRIIB-Fc heterodimer is a more selective antagonist of activin B, activin A, GDF8, and GDF11 compared to ActRIIB-Fc homodimer. Accordingly, an ALK4-Fc:ActRIIB-Fc heterodimer will be more useful than an ActRIIB-Fc homodimer in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of one or more of activin A, activin B, activin AB, GDF8, and GDF11 but minimize antagonism of one or more of BMP9, BMP10, GDF3, and BMP6. Example 3. ALK4:ActRIIB Heteromultimer Treatment Improves Muscle Strength and Bone Mass in a Spinal Muscular Atrophy Model Motor neuron diseases (MND) such as spinal muscular atrophy (SMA) are characterized by loss of motor neurons resulting in muscular atrophy and weakness. Muscle weakness and bone loss are primary outcomes in patients with MND and result in decreased ambulation and increased morbidity. Therefore, therapeutic interventions are necessary that could increase muscle strength, reduce bone loss and prolong ambulation. The effects of the ALK4-mFc:ActRIIB-mFc was assessed in the SMA C/C mouse model [Min Liu et al. (2016) PLOS ONE. DOI:10.1371/journal.pone.0166803]. SMA C/C mice (21-week old, n=16) were randomized to receive either vehicle (PBS) or ALK4-mFc:ActRIIB-mFc. Dorsiflexion and tibialis anterior (TA) muscle contractility during isometric contraction was assessed by both in vivo and in situ methods. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry. In ALK4-mFc:ActRIIB-mFc treated SMA animals, TA muscle mass was increased by 62% (p<0.001) and its physiological cross-sectional area (pCSA) was increased by 50% (p<0.001) compared to control mice. The increase in muscle mass correlated with an increase in strength, with maximum tetanic force improving by 25% (p<0.01) compared to control mice. The maximum rate of contraction and relaxation were accelerated by 28% and 38% respectively (p<0.001) in the ALK4-mFc:ActRIIB-mFc treated mice compared to vehicle group. The longitudinal alteration of dorsiflexion force after 6 weeks treatment compared to baseline was a −24% reduction in the vehicle group, compared to a 78% increase with ALK4-mFc:ActRIIB-mFc treatment. BMD of ALK4-mFc:ActRIIB-mFc treated SMA mice was improved by 11% compared with vehicle group (p<0.001). Taken together, the data demonstrate that ALK4-mFc:ActRIIB-mFc therapy sustained muscle and bone parameters in SMA C/C mice. Therefore, the data indicate that ALK4-mFc:ActRIIB-mFc, and potentially other ALK4:ActRIIB antagonists, may be used as a therapy to treat SMA, particularly prolong ambulation and slow bone loss in SMA patients. | 306,891 |
11857600 | ABBREVIATIONS Unless indicated otherwise, the following includes abbreviations for terms disclosed herein: acute myeloid leukemia (AML), Adenosine A3 Receptor (ADORA3), Adenosine Receptor A2b (ADORA2B), adenovirus high-throughput system (AdHTS), Adenylate Cyclase Activating Polypeptide 1 (Pituitary) Receptor Type I (ADCYAP1R1), Adrenoceptor Alpha 1A (ADRA1A), Adrenoceptor Beta 2 (ADRB2), Apelin Receptor (APLNR), Atypical chemokine receptor 3 (ACKR3), bimolecular fluorescence complementation (BiFC), Bioluminescence Resonance Energy Transfer (BRET), bovine serum albumin (BSA), Calcitonin Receptor (CALCR), Cancer stem cells (CSCs), C—C chemokine receptor type 2 (CCR2), chemerin chemokine-like receptor 1 (CMKLR1), Cholinergic Receptor Muscarinic 1 (CHRM1), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic obstructive pulmonary disease (COPD), Complement C5a Receptor 1 (C5AR1), C-terminal fragment of Venus (VC), C—X—C Motif Chemokine ligand 12 (CXCL12), CXC receptor 4 (CXCR4), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), δ-opioid receptor (OPRD), Endothelin Receptor Type B (EDNRB), enzyme-linked immunosorbent assay (ELISA), formalin-fixed paraffin-embedded (FFPE), fluorescence resonance energy transfer (FRET), G protein-coupled receptor (GPCR), Galanin Receptor 1 (GALR1), glioblastoma multiforme (GBM), Glucagon receptor (GCGR), GPCR heteromer identification technology (GPCR-HIT), Granulocyte-colony stimulating factor (G-CSF), hematopoietic stem cells (HSCs), hepatocellular carcinoma (HCC), Histamine Receptor H1 (HRH1), human immunodeficiency virus (HIV), International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), μ-opioid receptor (MOR), Motilin Receptor (MLNR), Multiple myeloma (MM), multiplicity of infection (MOI), Myelodysplastic Syndromes (MDS), Neurotensin Receptor 1 (NTSR1), non-Hodgkin lymphoma (NHL), non-small-cell lung cancer (NSCLC), N-terminal fragments of Venus (VN), patient derived cell (PDC), Patient-Derived Xenograft (PDX), positron emission tomography (PET), Computed Tomography (CT), programmed cell death ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), Prostaglandin E Receptor 2 (PTGER2), Prostaglandin E Receptor 3 (PTGER3), proximity ligation assay (PLA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Single-photon emission computed tomography (SPECT), small lymphocytic lymphoma (SLL), small-cell lung cancer (SCLC), Somatostatin Receptor 2 (SSTR2), Stromal cell-derived factor 1 (SDF-1), systemic lupus erythematosus (SLE), Tachykinin Receptor 3 (TACR3), Threshold cycles (Ct), time-resolved FRET (TR-FRET), tumor microenvironment (TME), Vascular endothelial growth factor (VEGF), vascular smooth muscle cells (VSMC), WHIM syndrome (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis), green fluorescence protein (GFP), and yellow fluorescence protein (YFP). DETAILED DESCRIPTION OF THE INVENTION CXCR4 plays important roles in tumor formation and progression, but the development of CXCR4 antagonists as anticancer drugs has not been successful possibly due to side effects and lack of efficacy within acceptable dose ranges. Recently, various CXCR4-GPCR heteromers with distinct physiological and pharmacological properties were reported, but their roles in cancer biology or possibilities for developing anti-cancer therapeutics targeting CXCR4-GPCR heteromers have not been clearly understood. Traditionally GPCRs were believed to function as monomers that interact with heterotrimeric G proteins upon ligand binding, and drugs were developed based on monomeric or homomeric GPCRs (Milligan 2008). This view has changed drastically by the discoveries that GPCRs can form heteromers, and heteromerization is obligatory for some GPCRs. GPCR heteromerization is known to alter GPCR maturation and cell surface delivery, ligand binding affinity, signaling intensity and pathways, as well as receptor desensitization and recycling (Terrillon and Bouvier 2004; Ferre et al., 2010; Rozenfeld and Devi 2010; Gomes et al., 2016; Farran 2017). Different GPCR heteromers display distinct functional and pharmacological properties, and GPCR heteromerization can vary depending on cell types, tissues, and diseases or pathological conditions (Terrillon and Bouvier 2004; Ferre et al., 2010; Rozenfeld and Devi 2010; Gomes et al., 2016; Farran 2017). Now GPCR heteromerization is regarded as a general phenomenon, and deciphering GPCR heteromerization opens new avenues for understanding receptor function, physiology, roles in diseases and pathological conditions. Accordingly, identification of GPCR heteromers and their functional properties offers new opportunity for developing new pharmaceuticals or finding new use of old drugs with fewer side effects, higher efficacy, and increased tissue selectivity (Ferre et al., 2010; Rozenfeld and Devi 2010; Farran 2017). The identification of bona fide GPCR heteromer requires intensive and critical evaluation. To distinguish GPCR heteromers from simple association of GPCRs, researchers in this field and the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) have declared GPCR heteromer as “macromolecular complex composed of at least two (functional) receptor units [protomers] with biochemical properties that are demonstrably different from those of its individual components” and these heteromers exist in native tissue (Ferre et al., 2009; Gomes et al., 2016; Pin et al., 2007). They proposed three criteria to demonstrate GPCR heteromers: (1) Heteromers should exhibit appropriate co-localization and interaction to enable allosterism using co-immunoprecipitation, in situ hybridization, or proximity-based techniques including proximity ligation assays in cells/tissues that express both receptors and not in cells/tissues that lack one of the receptors; (2) Heteromers should exhibit distinct properties such as changes in signaling, ligand binding, and/or trafficking, only in cells/tissues expressing both receptors but not in cells/tissues that lack one of the receptors; and (3) Heteromer-selective reagents should alter heteromer-specific properties. Heteromer-selective reagents include heteromer-selective antibodies, membrane-permeable peptides, and bivalent/bifunctional ligands (Gomes et al., 2016; Pin et al., 2007). Although many GPCR heteromers have been identified in vitro using recombinant receptors expressed in heterologous cells, only a few have demonstrated novel properties, and very few have shown evidence for GPCR heteromerization in native tissue due to technical problems (Gomes et al., 2016). NC-IUPHAR announced that one should provide evidence that satisfies at least two of the above three criteria for approval of new GPCR heteromers (Pin et al., 2007). As disclosed herein, to establish whether criterion 1 of 3 is satisfied (relating to whether heteromer components colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism) in determining the presence/existence of a CXCR4-GPCRx heteromer, one or more of the following methods may be utilized to including, but not limited to: co-internalization assays; co-localization assays (determining co-localization of the receptor portomers within a cellular compartment) such as in situ hybridization, immunohistochemistry, or immunoelectron microscopy; proximity-based assays, such as proximity-based biophysical techniques, including resonance energy transfer (RET), bioluminescence RET (BRET), fluorescence RET (FRET), time-resolved fluorescence RET (TR-FRET), antibody-aided FRET, ligand-aided FRET, bimolecular fluorescence complementation (BiFC), and proximity ligation assays (PLA); co-immunoprecipitation assays; or fluorescent animals. For example, BiFC, co-internalization assay, or PLA, were utilized to evaluate whether a CXCR4-GPCRx heteromer satisfied criterion 1 of 3. As disclosed herein, to establish whether criterion 2 of 3 is satisfied (relating to whether a heteromer exhibits properties distinct from those of the individual protomers), such as a CXCR4-GPCRx heteromer that results in an enhanced downstream signaling, for example an enhanced calcium mobilization (such as determined by a calcium mobilization assay), a two-tiered approach was utilized on those CXCR4-GPCRx heteromers that satisfied criterion 1 of 3 discussed above: (1) determine the presence/absence of an enhanced downstream signaling, for example an enhanced calcium mobilization (synergism) in the individual protomer context—comparing calcium mobilization in cells co-expressing HA-VC and one of the protomers (either CXCR4 or GPCRx) upon (a) co-stimulation with CXCL12 and the respective selective agonist, relative to (b) stimulation with either CXCL12 alone or the respective selective agonist alone; and (2) determine the presence/absence of an enhanced downstream signaling, for example an enhanced calcium mobilization (synergism) in the CXCR4-GPCRx heteromer context—comparing calcium mobilization in cells co-expressing CXCR4 and GPCRx upon (a) co-stimulation with CXCL12 and the respective selective agonist, relative to (b) the sum of stimulation with either CXCL12 alone or the respective selective agonist alone. As disclosed herein, to satisfy criterion 2 of 3 and be considered a CXCR4-GPCRx heteromer that results in an enhanced downstream signaling, for example an enhanced calcium mobilization, there must be (1) an absence of an enhanced downstream signaling, for example an enhanced calcium mobilization in either protomer context (i.e., the CXCR4 and HA-VC context or the GPCRx and HA-VC context), and (2) a presence of an enhanced downstream signaling, for example an enhanced calcium mobilization in the CXCR4-GPCRx heteromer context. In both the protomer context and the CXCR4-GPCRx heteromer context, the concentration of CXCL12 utilized to stimulate the cells (either as a single agent or in combination with the respective selective GPCRx agonist) and the concentration of the selective GPCRx agonist (either an endogenous agonist or a known selective agonist for the respective GPCRx) utilized to stimulate the cells (either as a single agent or in combination with CXCL12) are independently at a concentration of 100× the EC50 concentration or lower. For example, the concentration of CXCL12 utilized to stimulate the cells (either as a single agent or in combination with the respective selective GPCRx agonist) was at a concentration of 15 nM (which is approximately the EC50 concentration against CXCR4). As disclosed herein, to establish whether criterion 3 of 3 is satisfied (relating to whether heteromer-selective reagents should alter heteromer-specific properties) in determining the presence/existence of a CXCR4-GPCRx heteromer, patient derived cells, having satisfied criterion 1 of 3 and 2 of 3, are effected in the presence of an antagonist (a CXCR4 antagonist, a GPCRx antagonist, or a CXCR4-GPCRx heteromer antagonist), such as effecting cell proliferation of the patient derived cells containing a CXCR4-GPCRx heteromer. In some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies at least two of the following criteria or characteristics to be considered a CXCR4-GPCRx heteromer, comprising: 1) the CXCR4-GPCRx heteromer components in a cell colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism, as determined via one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay; 2) an enhanced amount of calcium mobilization, such that: a) either CXCR4 or the respective GPCRx in an individual protomer context in a cell, upon co-stimulation with CXCL12 and a respective selective GPCRx agonist, results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; and b) the CXCR4-GPCRx heteromer exhibits an enhanced calcium mobilization upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; as determined via a calcium mobilization assay; or 3) a CXCR4-GPCRx heteromer-selective reagent: i) alters heteromer-specific properties of the CXCR4-GPCRx heteromer in a patient derived cell; ii) alters heteromer-specific function of the CXCR4-GPCRx heteromer in a patient derived cell; iii) alters heteromer-specific properties of a patient derived cell containing the CXCR4-GPCRx heteromer; or iv) decreases cell proliferation of a patient derived cell(s) containing the CXCR4-GPCRx heteromer. In some embodiments, the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of the CXCR4-GPCRx heteromer in a patient derived cell, as determined by at least one of the following methods: PLA, radioligand binding assays, [35S]GTP-γS Binding assays, Calcuim assay, cAMP assay, GTPase assay, PKA activation, ERK1/2 and/or Akt/PKB Phosphorylation assays, Src and STAT3 phosphorylation assays, CRE-reporter assay, NFAT-RE-reporter assay, SRE-reporter assay, SRF-RE reporter assay, NF-kB-RE reporter assay, Secreted alkaline phosphatase Assay, Inositol 1-Phosphate Production assay, Adenylyl Cyclase Activity assay, analysis of target gene expression by RT-PCR, RT-qPCR, RNAseq, next generation sequencing (NGS), or microarray. In some embodiments, the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific function of the CXCR4-GPCRx heteromer in a patient derived cell, as determined by at least one of the following methods: PLA, radioligand binding assays, [35S]GTP-γS Binding assays, Calcuim assay, cAMP assay, GTPase assay, PKA activation, ERK1/2 and/or Akt/PKB Phosphorylation assays, Src and STAT3 phosphorylation assays, CRE-reporter assay, NFAT-RE-reporter assay, SRE-reporter assay, SRF-RE reporter assay, NF-kB-RE reporter assay, Secreted alkaline phosphatase Assay, Inositol 1-Phosphate Production assay, Adenylyl Cyclase Activity assay, analysis of target gene expression by RT-PCR, RT-qPCR, RNAseq, or microarray. In some embodiments, the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of a patient derived cell containing the CXCR4-GPCRx heteromer, as determined by at least one of the following methods: assays on proliferation, migration, invasion, and drug resistance (survival) of cancer cells, modulation of immune cell function, angiogenesis, vasculogenesis, metastasis, drug resistance, tissue microarray (TMA), and cancer cell-tumor microenvironment (TME) interaction. For example, in some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies at least two of the following criteria or characteristics to be considered a CXCR4-GPCRx heteromer, comprising: 1) the CXCR4-GPCRx heteromer components in a cell colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism, as determined via one or more of the following: a co-internalization assay, bimolecular fluorescence complementation (BiFC), or a proximity ligation assay (PLA); 2) an enhanced amount of calcium mobilization, such that: a) either CXCR4 or the respective GPCRx in an individual protomer context in a cell, upon co-stimulation with CXCL12 and a respective selective GPCRx agonist, results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; and b) the CXCR4-GPCRx heteromer exhibits an enhanced calcium mobilization upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; as determined via a calcium mobilization assay; or 3) a CXCR4-GPCRx heteromer-selective reagent: i) alters heteromer-specific properties of the CXCR4-GPCRx heteromer in a patient derived cell; ii) alters heteromer-specific function of the CXCR4-GPCRx heteromer in a patient derived cell; iii) alters heteromer-specific properties of a patient derived cell containing the CXCR4-GPCRx heteromer; or iv) decreases cell proliferation of a patient derived cell(s) containing the CXCR4-GPCRx heteromer. In some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies criteria 1 and 2 to be considered a CXCR4-GPCRx heteromer. In some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies criteria 1 and 3 to be considered a CXCR4-GPCRx heteromer. In some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies criteria 2 and 3 to be considered a CXCR4-GPCRx heteromer. In some embodiments, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, include or rely upon establishing that an association of CXCR4 and a GPCRx in a cell satisfies criteria 1, 2, and 3, to be considered a CXCR4-GPCRx heteromer. As disclosed herein, the CXCR4-GPCRx heteromer satisfying at least two of the three criteria may have, cause, or produce an enhanced downstream signaling. The enhanced downstream signaling may be the result from the CXCR4-GPCRx heteromer, such as from agonism of the CXCR4-GPCRx heteromer, from CXCR4 agonism of the CXCR4-GPCRx heteromer, from GPCRx agonism of the CXCR4-GPCRx heteromer, and/or from CXCR4 agonism and GPCRx agonism of the CXCR4-GPCRx heteromer. In some embodiments, the enhanced downstream signaling may be downstream of the CXCR4, the respective GPCRx, or the CXCR4-GPCRx heteromer. In some embodiments, the enhanced downstream signaling may be from the CXCR4-GPCRx heteromer, relative to downstream signaling from a CXCR4 protomer or a respective GPCRx protomer in their respective individual protomer context. In some embodiments, the enhanced downstream signaling may be from the CXCR4-GPCRx heteromer, relative to downstream signaling from a CXCR4 protomer in an individual protomer context. In some embodiments, the enhanced downstream signaling may be from the CXCR4-GPCRx heteromer, relative to downstream signaling from a respective GPCRx protomer in an individual protomer context. In some embodiments, the enhanced downstream signaling may be from the CXCR4-GPCRx heteromer, relative to downstream signaling from a CXCR4 protomer and a respective GPCRx protomer in their respective individual protomer context. The enhanced downstream signaling from said CXCR4-GPCRx heteromer, may in some embodiments, be suppressed in the cancer patient, such as suppressed in the patient's cancer cells. In some embodiments, the enhanced downstream signaling from said CXCR4-GPCRx heteromer, may be an enhanced amount of calcium mobilization (or synergistic amount of calcium mobilization), which may be determined by an intracellular Ca2+ assay, such as a calcium mobilization assay. As disclosed herein, the CXCR4-GPCRx heteromer satisfying at least two of the three criteria may have, cause, or produce an enhanced downstream signaling, wherein the enhanced downstream signaling is an enhanced amount of calcium mobilization. The enhanced amount of calcium mobilization from the CXCR4-GPCRx heteromer may be a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is at least 10% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In some embodiments, the enhanced amount of calcium mobilization from the CXCR4-GPCRx heteromer may be a synergistic amount of calcium mobilization that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is at least 10% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. For example, the enhanced amount of calcium mobilization (or synergistic amount of calcium mobilization) from the CXCR4-GPCRx heteromer may be a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 90% greater, at least 100% greater, at least 150% greater, or at least 200% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In some embodiments, the enhanced amount of calcium mobilization (or synergistic amount of calcium mobilization) from the CXCR4-GPCRx heteromer may be a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, may be between 10-100% greater than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay, for example, may be a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, may be between 25-100% greater, 50-100% greater, 75-100% greater, or 100-200% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, as disclosed herein, the CXCR4-GPCRx heteromer satisfies at least two of the three criteria, thereby having, causing, or producing, an enhanced downstream signaling, wherein the enhanced downstream signaling is an enhanced amount of calcium mobilization, such that: a) either the CXCR4 or the respective GPCRx in an individual protomer context in the cell upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; and b) the CXCR4-GPCRx heteromer exhibits an enhanced calcium mobilization upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; as determined via a calcium mobilization assay. For example, in some embodiments, the enhanced downstream signaling from the CXCR4-GPCRx heteromer is an enhanced amount of calcium mobilization, such that: i) the calcium mobilization from the protomer CXCR4 or GPCRx, in the individual protomer context in the cell, is non-synergistic, as determined via calcium mobilization assay; and ii) the calcium mobilization from the CXCR4-GPCRx heteromer in the cell is synergistic, as determined via a calcium mobilization assay. In some embodiments, the individual protomer context may be: a) the individual protomer CXCR4 in the cell, in the absence of the respective individual protomer GPCRx; or b) the respective individual protomer GPCRx in the cell, in the absence of the individual protomer CXCR4; upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In some embodiments, the individual protomer context may be, independently: a) the individual protomer CXCR4 in the cell, in the absence of the respective individual protomer GPCRx; and b) the respective individual protomer GPCRx in the cell, in the absence of the individual protomer CXCR4; upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. For example, in some embodiments, the CXCR4-GPCRx heteromer, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, may result in a calcium mobilization amount that is greater than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. The term “CXCR4” as used herein refers to C—X—C Motif Chemokine Receptor 4, also identified by unique database identifiers (IDs) and alternate names as shown in Table 1 (Chatterjee et al., 2014; Debnath et al., 2013; Domanska et al., 2013; Guo et al., 2016; Peled et al., 2012; Roccaro et al., 2014; Walenkamp et al., 2017). The terms “GPCRx” as used herein refers to GPCRs that were used in this study to investigate if these GPCRs interact with CXCR4 and show properties distinct from those of individual protomers, including ADCYAP1R1 (ADCYAP Receptor Type I), ADORA2B (Adenosine A2b Receptor), ADORA3 (Adenosine A3 Receptor), ADRB2 (Adrenoceptor Beta 2), APLNR (Apelin Receptor), C5AR1 (Complement C5a Receptor 1), CALCR (Calcitonin Receptor), CCR5 (Chemokine (C—C Motif) Receptor 5), CHRM1 (Cholinergic Receptor Muscarinic 1), GALR1 (Galanin Receptor 1), EDNRB (Endothelin Receptor Type B), HRH1 (Histamine Receptor H1), MLNR (Motilin Receptor), NTSR1 (Neurotensin Receptor 1), PTGER2 (Prostaglandin E Receptor 2), PTGER3 (Prostaglandin E Receptor 3), SSTR2 (Somatostatin Receptor 2), and TACR3 (Tachykinin Receptor 3), also identified by unique database identifiers (IDs) and alternate names as shown in Table 1 and Table 2. Table 1 shown below provides the nomenclature of GPCRx that form heteromers with CXCR4 and synergistically enhance Ca2+ response upon co-stimulation with both agonists, and Table 2 shown below provides the nomenclature of GPCRx that form heteromers with CXCR4, but do not synergistically enhance Ca2+ response upon co-stimulation with both agonists. TABLE 1Gene nameFull nameOther namesIDsCXCR4C-X-C MotifLeukocyte-Derived SevenGCID: GC02M136114ChemokineTransmembrane Domain Receptor;HGNC: 2561Receptor 4Lipopolysaccharide-AssociatedEntrez Gene: 7852Protein 3; Stromal Cell-DerivedEnsembl: ENSG00000121966Factor 1 Receptor; Chemokine (C-OMIM: 162643X-C Motif) Receptor 4; LPS-UniProtKB: P61073Associated Protein 3 SevenTransmembrane Helix Receptor; C-X-C Chemokine Receptor Type 4;Neuropeptide Y Receptor Y3;Neuropeptide Y3 Receptor;Chemokine Receptor; Seven-Transmembrane-Segment Receptor,Spleen; Chemokine (C-X-C Motif),Receptor 4 (Fusin); SDF-1Receptor; CD184 Antigen; Fusin;LAP-3; LESTR; NPYRL; FB22;HM89; LCR1; D2S201E;HSY3RR; NPYY3R;CXC-R4; CXCR-4; CD184;NPY3R; WHIMS; LAP3; NPYR;WHIMADCYAP1R1ADCYAP ReceptorAdenylate Cyclase ActivatingGCID: GC07P031058Type IPolypeptide 1 (Pituitary) ReceptorHGNC: 242Type I; Pituitary AdenylateEntrez Gene: 117Cyclase-Activating PolypeptideEnsembl: ENSG00000078549Type 1 Receptor; PACAP Type IOMIM: 102981Receptor; PACAP Receptor 1;UniProtKB: P41586PACAP-R1; Pituitary AdenylateCyclase Activating Polypeptide 1Receptor Type I Hiphop; PituitaryAdenylate Cyclase-ActivatingPolypeptide Type I Receptor;PACAP-R-1; PACAPRI;PACAPR; PAC1R; PAC1ADORA2BAdenosine A2bAdenosine Receptor A2b;GCID: GC17P015927ReceptorADORA2HGNC: 264Entrez Gene: 136Ensembl: ENSG00000170425OMIM: 600446UniProtKB: P29275ADORA3Adenosine A3Adenosine Receptor A3; A3ARGCID: GC01M111499ReceptorHGNC: 268Entrez Gene: 140Ensembl: ENSG00000282608OMIM: 600445UniProtKB: P0DMS8ADRB2Adrenoceptor BetaAdrenergic, Beta-2-, Receptor,GCID: GC05P1488252Surface; Beta-2 Adrenoreceptor;HGNC: 286Beta-2 Adrenoceptor; ADRB2R;Entrez Gene: 154B2AR; Adrenoceptor Beta 2,Ensembl: ENSG00000169252Surface; Adrenoceptor Beta 2OMIM: 109690Surface; Beta-2 AdrenergicUniProtKB: P07550Receptor; Catecholamine Receptor;BETA2AR; ADRBR; BARC5AR1Complement C5aComplement Component 5aGCID: GC19P047290Receptor 1Receptor 1; ComplementHGNC: 1338Component 5 Receptor 1 (C5aEntrez Gene: 728Ligand);Ensembl: ENSG00000197405C5a Anaphylatoxin ChemotacticOMIM: 113995Receptor 1; C5a AnaphylatoxinUniProtKB: P21730Chemotactic Receptor;Complement Component 5Receptor 1; C5a AnaphylatoxinReceptor; C5a-R;C5R1; CSAR; CD88 Antigen; C5aLigand; CD88; C5aCALCRCalcitonin ReceptorCT-R; CTR1; CRT; CTRGCID: GC07M093424HGNC: 1440Entrez Gene: 799Ensembl: ENSG00000004948OMIM: 114131UniProtKB: P30988CHRM1CholinergicAcetylcholine Receptor,GCID: GC11M062927ReceptorMuscarinic 1;HGNC: 1950Muscarinic 1Muscarinic Acetylcholine ReceptorEntrez Gene: 1128M1; HM1; M1R; M1Ensembl: ENSG00000168539OMIM: 118510UniProtKB: P11229EDNRBEndothelin ReceptorEndothelin Receptor Non-SelectiveGCID: GC13M077895Type BType; ET-BR; ET-B;HGNC: 3180ETRB;Entrez Gene: 1910Endothelin Receptor Subtype B1;Ensembl: ENSG00000136160ABCDS; HSCR2;OMIM: 131244ETB1; ETBR; WS4A; HSCR; ETBUniProtKB: P24530HRH1Histamine ReceptorHH1R; H1R; Histamine Receptor,GCID: GC03P011113H1Subclass H1; Histamine H1HGNC: 5182Receptor; HisH1; H1-REntrez Gene: 3269Ensembl: ENSG00000196639OMIM: 600167UniProtKB: P35367MLNRMotilin ReceptorG Protein-Coupled Receptor 38;GCID: GC13P049220GPR38; MTLR1; G-ProteinHGNC: 4495Coupled Receptor 38; MTLREntrez Gene: 2862Ensembl: ENSG00000102539OMIM: 602885UniProtKB: O43193NTSR1NeurotensinHigh-Affinity Levocabastine-GCID: GC20P062708Receptor 1Insensitive Neurotensin Receptor;HGNC: 8039Neurotensin Receptor 1 (HighEntrez Gene: 4923Affinity); NT-R-1; NTR1; NTRH;Ensembl: ENSG00000101188Neurotensin Receptor Type 1;OMIM: 162651NTRR; NTRUniProtKB: P30989TACR3TachykininNeurokinin Beta Receptor;GCID: GC04M103586Receptor 3Neurokinin B Receptor; NK-3HGNC: 11528Receptor; NK-3R; NK3REntrez Gene: 6870NKR; Neuromedin-K Receptor;Ensembl: ENSG00000169836TAC3RL; TAC3R; HH11OMIM: 162332UniProtKB: P29371*GCID: Genecards identificationHGNC: HUGO Gene Nomenclature Committee TABLE 2Gene nameFull nameOther namesIDsAPLNRApelin ReceptorG-Protein Coupled Receptor HG11;GCID: GC11M057233Angiotensin II Receptor-Like 1;HGNC: 339Angiotensin Receptor-Like 1; APJEntrez Gene: 187(Apelin) Receptor; G Protein-CoupledEnsembl:Receptor APJ;ENSG00000134817G-Protein Coupled Receptor APJ;OMIM: 600052HG11 Orphan Receptor;UniProtKB: P35414APJ Receptor; AGTRL1; APJ; APJR;HG11CCR5Chemokine (C—CC—C Chemokine Receptor Type 5;GCID: GC03P046382Motif) Receptor 5Chemokine Receptor CCR5;HGNC: 1606Chemokine (C—C Motif) Receptor 5Entrez Gene: 1234(Gene/Pseudogene);Ensembl:C—C Motif Chemokine Receptor 5ENSG00000160791A159A; Chemokine Recptor CCR5OMIM: 601373Delta32; CD195 Antigen; HIV-1UniProtKB: P51681Fusion Coreceptor; CC-CKR-5;ChemR13; CMKBR5; CCR-5;C—C CKR-5; CCCKR5; IDDM22;CD195; CKR-5; CKR5GALR1Galanin Receptor 1GALNR1; GAL1-R; GALR-1;GCID: GC18P077250GALNR; Galanin Receptor Type 1HGNC: 4132Entrez Gene: 2587Ensembl:ENSG00000166573OMIM: 600377UniProtKB: P47211PTGER2Prostaglandin EProstaglandin E Receptor 2 (SubtypeGCID: GC14P052314Receptor 2EP2), 53 kDa; Prostaglandin E ReceptorHGNC: 95942 (Subtype EP2), 53 kD; PGE2Entrez Gene: 5732Receptor EP2 Subtype; PGE ReceptorEnsembl:EP2 Subtype; Prostanoid EP2ENSG00000125384Receptor; Prostaglandin E2 ReceptorOMIM: 176804EP2 Subtype; EP2UniProtKB: P43116PTGER3Prostaglandin EProstaglandin E Receptor 3 (SubtypeGCID: GC01M070852Receptor 3EP3); PGE2 Receptor EP3 Subtype;HGNC: 9595Prostanoid EP3 Receptor;Entrez Gene: 5733PGE2-R; Prostaglandin E ReceotorEnsembl:EP3 Subtype 3 Isoform; ProstaglandinENSG00000050628E2 Receptor EP3 Subtype;OMIM: 176806Prostaglandin Receptor (PGE-2); PGEUniProtKB: P43115Receptor, EP3 Subtype; PGE ReceptorEP3 Subtype; EP3-III; EP3-II; EP3-IV;EP3-VI; EP3-I; EP3e; EP3SSTR2SomatostatinSRIF-1; SS2R; Somatostatin ReceptorGCID: GC17P073165Receptor 2Type 2; SS-2-R; SS2-RHGNC: 11331Entrez Gene: 6752Ensembl:ENSG00000180616OMIM: 182452UniProtKB: P30874*GCID: Genecards identificationHGNC: HUGO Gene Nomenclature Committee Further information regarding the GPCRs evaluated herein as heteromers with CXCR4 are detailed below:(1) ADCYAP1R1—Pituitary adenylate cyclase-activating polypeptide type I receptor also known as PAC1, is a protein that in humans is encoded by the ADCYAP1R1 gene. This receptor binds pituitary adenylate cyclase activating peptide. ADCYAP1R1 is a membrane-associated protein and shares significant homology with members of the G-protein coupled class B glucagon/secretin receptor family. This receptor mediates diverse biological actions of adenylate cyclase activating polypeptide 1 and is positively coupled to adenylate cyclase (Vaudry et al., 2000). This is a receptor for PACAP-27 and PACAP-38. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. ADCYAP1R1 may regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, prolactin, epinephrine, and catecholamine. ADCYAP1R1 may play a role in spermatogenesis and sperm motility. ADCYAP1R1 causes smooth muscle relaxation and secretion in the gastrointestinal tract (Ogi et al., 1993). ADCYAP1R1 is expressed in the adrenal medulla, pancreatic acini, uterus, myenteric plexus and brain (Reubi, 2000; Reubi et al., 2000). It is also expressed in the trigeminal, otic and superior cervical ganglia (prejunctional) and cerebral arteries (postjunctional) (Knutsson and Edvinsson, 2002). Diseases associated with ADCYAP1R1 include post-traumatic stress disorder (Lowe et al., 2015) and anxiety disorder. Among its related pathways are G alpha(s) signalling events and RET signaling (Cooper et al., 2013).(2) ADORA2B—The adenosine A2B receptor, also known as ADORA2B, is a G-protein coupled adenosine receptor, and also denotes the human adenosine A2b receptor gene which encodes it. Adenosine functions as a signaling molecule through the activation of four distinct adenosine receptors—ADORA1, ADORA2A, ADORA2B, and ADORA3, also called as A1, A2A, A2B, and A3, respectively. This integral membrane protein stimulates adenylate cyclase activity in the presence of adenosine. This protein also interacts with netrin-1, which is involved in axon elongation. These receptors are widely expressed and have been implicated in several biological functions, both physiological and pathological. Both ADORA2A and ADORA2B receptors are important in cancer immunology. The tumor microenvironment is hypoxic, which promotes the expression of CD39 and CD73 by immune cells. CD39 and CD73 are ectonucleotidases that convert ATP to adenosine, elevating concentrations of adenosine locally. Adenosine is a crucial mediator of altered immune function in cancer as it binds ADORA2A and ADORA2B receptors on lymphocytes silencing the antitumor immune response (Chen et al., 2013). ADORA2B has a role in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation (Yang et al., 2008) and promotes progression of human oral cancer (Kasama et al., 2015). Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis (Desmet et al., 2013). ADORA2B receptors display high expression levels in the cecum, colon and bladder, with lower levels in the lung, blood vessels and eye (Fagerberg et al., 2014).(3) ADORA3—The adenosine A3 receptor, also known as ADORA3, is an adenosine receptor, but also denotes the human gene encoding it. ADORA3 receptors are G-protein coupled receptors that couple to Gi/Gq and are involved in a variety of intracellular signaling pathways and physiological functions. It mediates a sustained cardioprotective function during cardiac ischemia, it is involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury, it has been implicated in both neuroprotective and neurodegenerative effects, and it may also mediate both cell proliferation and cell death (Gao et al., 2008; Miwatashi et al., 2008). Cordycepin induces apoptosis in human bladder cancer cells via activation of ADORA3 receptors (Cao et al., 2017). Jafari et al. showed that ADORA3 receptor agonist inhibited survival of breast cancer stem cells via GLI-1 and ERK1/2 pathway (Jafari et al., 2017). ADORA3 is broadly expressed in adrenal (RPKM 4.4), small intestine (RPKM 2.9) and 20 other tissues containing brain, bladder, lymph node and colon (Fagerberg et al., 2014).(4) ADRB2—The beta-2 adrenergic receptor (β2 adrenoreceptor), also known as ADRB2, is a cell membrane-spanning beta-adrenergic receptor that interacts with epinephrine, a hormone and neurotransmitter (ligand synonym, adrenaline) whose signaling, via a downstream L-type calcium channel interaction, mediates physiologic responses such as smooth muscle relaxation and bronchodilation (Gregorio et al., 2017). ADRB2 functions in muscular system such as smooth muscle relaxation, motor nerve terminals, glycogenolysis and in circulatory system such as heart muscle contraction, cardiac output increase. In the normal eye, beta-2 stimulation by salbutamol increases intraocular pressure via net. In digestive system, the ADRB2 induces glycogenolysis and gluconeogenesis in liver and insulin secretion from pancreas (Fitzpatrick, 2004). ADRB2 signaling in the cardiac myocyte is modulated by interactions with CXCR4 (LaRocca et al., 2010). Norepinephrine attenuates CXCR4 expression and the corresponding invasion of MDA-MB-231 breast cancer cells via ADRB2 (Wang et al., 2015a). ADRB2 is expressed in several cancers such as pancreatic, prostate (Braadland et al., 2014; Xu et al., 2017), renal and breast cancer (Choy et al., 2016).(5) C5AR1—The C5a receptor also known as complement component 5a receptor 1 (C5AR1) or CD88 (Cluster of Differentiation 88) is a G protein-coupled receptor for C5a. It functions as a complement receptor. C5AR1 modulates inflammatory responses, obesity, development and cancers. (Gerard and Gerard, 1994). The C5AR1 is expressed on granulocytes, monocytes, dendritic cells, hepatoma-derived cell line HepG2, astrocytes, microglia (Klos et al., 2013). C5AR1 is related in several diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, experimental allergic encephalomyelitis, multiple sclerosis, meningitis, and brain trauma (Lee et al., 2008).(6) CALCR—The calcitonin receptor (CT) is a G protein-coupled receptor that binds the peptide hormone calcitonin and is involved in maintenance of calcium homeostasis, particularly with respect to bone formation and metabolism (Dacquin et al., 2004; Davey et al., 2008). CT works by activating the G-proteins Gs and Gq often found on osteoclasts, on cells in the kidney, and on cells in a number of regions of the brain. It may also affect the ovaries in women and the testes in men. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. The calcitonin receptor is thought to couple to the heterotrimeric guanosine triphosphate-binding protein that is sensitive to cholera toxin. The physiological effects of CT such as inhibition of osteoclast mediated bone resorption or increased Ca2+ excretion by the kidney are mediated by high affinity CTs (Albrandt et al., 1995). Loss of function mutation in CT identifies highly aggressive glioblastoma with poor outcome (Pal et al., 2018). Expression of CTRs in mammary gland, cartilage, nose and ear has been reported. The developmental regulation of CALCR gene expression in a variety of the above tissues suggests a role for CTRs in the morphogenesis of these tissues (Pondel, 2000).(7) CHRM1—The muscarinic cholinergic receptors belong to a larger family of G protein-coupled receptors. The functional diversity of these receptors is defined by the binding of acetylcholine and includes cellular responses such as adenylate cyclase inhibition, phosphoinositide degeneration, and potassium channel mediation. Muscarinic receptors influence many effects of acetylcholine in the central and peripheral nervous system (Rang, 2003). The muscarinic cholinergic receptor 1 is involved in mediation of vagally-induced bronchoconstriction and in the acid secretion of the gastrointestinal tract. It is predominantly found bound to G proteins of class Gq that use upregulation of phospholipase C and, therefore, inositol trisphosphate and intracellular calcium as a signaling pathway (Qin et al., 2011). Muscarinic receptors are widely distributed throughout the body and control distinct functions according to location and subtype (CHRM1-CHRM5). They are predominantly expressed in the parasympathetic nervous system where they exert both inhibitory and excitatory effects. This receptor is found mediating slow excitatory postsynaptic potential at the ganglion in the postganglionic nerve, is common in exocrine glands and in the CNS (Johnson, 2002). Diseases associated with CHRM1 include asthma and heart block, congenital and Pelizaeus-Merzbacher disease. Activators of CHRM1 muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease (Marlo et al., 2009).(8) EDNRB—Endothelin receptor type B (ETB) receptor was cloned shortly after the ETA receptor (EDBRA). Similarly to the EDNRA, it belongs to class A of G protein-coupled heptahelical receptors with an external amino terminus and internal carboxy terminus and binding sites intrinsic to the heptahelical portions of the receptor. Similar to the EDNRA, the endogenous agonist ET-1 has a high affinity for the EDNRB. The pharmacology of the EDNRB receptor is somewhat richer than that of the EDNRA in that multiple agonists of the EDNRB are recognized, including sarafotoxin 6c (S6c) and IRL1620. Selective antagonists of the EDNRB include BQ788, A192621, RES7011, and IRL2500. The EDNRB has been localized to cardiovascular tissues, the pulmonary system, neurons, bone, pancreas, and kidney (Watts, 2010). EDNRB is a G protein-coupled receptor which activates a phosphatidylinositol-calcium second messenger system. Its ligand, endothelin, consists of a family of three potent vasoactive peptides: ET1, ET2, and ET3. In melanocytic cells the EDNRB gene is regulated by the microphthalmia-associated transcription factor. Mutations in either gene are links to Waardenburg syndrome (Sato-Jin et al., 2008). The multigenic disorder, Hirschsprung disease type 2, is due to mutation in EDNRB gene (Tanaka et al., 1998). Diseases associated with EDNRB include Waardenburg syndrome, Type 4A and Abcd syndrome. Among its related pathways are calcium signaling pathway and prostaglandin synthesis and regulation of Waardenburg Syndrome (Sato-Jin et al., 2008).(9) HRH1—The H1 receptor is a histamine receptor belonging to the family of rhodopsin-like G-protein-coupled receptors. This receptor is activated by the biogenic amine histamine. The HRH1 is linked to an intracellular G-protein (Gq) that activates phospholipase C and the inositol triphosphate (IP3) signaling pathway. Antihistamines, which act on this receptor, are used as anti-allergy drugs. The crystal structure of the receptor has been determined and used to discover new HRH1 ligands in structure-based virtual screening studies (de Graaf et al., 2011). A subunit of the G-protein subsequently dissociates and affects intracellular messaging including downstream signaling accomplished through various intermediaries such as cyclic AMP, cyclic GMP, calcium, and nuclear factor kappa B. It functions in immune-cell chemotaxis, pro-inflammatory cytokine production, expression of cell adhesion molecules, and other allergic and inflammatory conditions (Canonica and Blaiss, 2011). In peripheral tissues, the HRH1 mediates the contraction of smooth muscles, increase in capillary permeability due to contraction of terminal venules, and catecholamine release from adrenal medulla, as well as mediating neurotransmission in the central nervous system. It is also known to contribute to the pathophysiology of allergic diseases such as atopic dermatitis, asthma, anaphylaxis and allergic rhinitis (Xie and He, 2005). It is expressed in smooth muscles, on vascular endothelial cells, in the heart, and in the central nervous system (de Graaf et al., 2011).(10) MLNR—Motilin receptor is a G protein-coupled receptor that binds motilin. Motilin in turn is an intestinal peptide that stimulates contraction of gut smooth muscle. Also, the MLNR mediates progastrokinetic effects. MLNR is an important therapeutic target for the treatment of hypomotility disorders (Depoortere, 2001). It is found at its highest concentrations in the nerves of the antral wall of the stomach and is also found at significant levels throughout the smooth muscle of the upper gastrointestinal tract (Kitazawa et al., 1995). Diseases associated with MLNR include gastroparesis and diabetic autonomic neuropathy (Kitazawa et al., 1997).(11) NTSR1—Neurotensin receptor 1 belongs to the large superfamily of G-protein coupled receptors. Neurotensin is a 13-amino acid peptide originally isolated from hypothalamic and later from intestines of bovine. In the brain, neurotensin is exclusively found in nerve cells, fibers, and terminals, whereas the majority of peripheral neurotensin is found in the endocrine N-cells located in the intestinal. Central or peripheral injections of neurotensin produce completely different pharmacological effects indicating that the peptide does not cross the blood-brain barrier. NTSR1 mediates the multiple functions of neurotensin, such as hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion (Vincent, 1995). Signaling is effected via G proteins that activate a phosphatidylinositol-calcium second messenger system. Signaling leads to the activation of downstream MAP kinases and protects cells against apoptosis (Heakal et al., 2011). Swift et al. showed altered expression of neurotensin receptors is associated with the differentiation state of prostate cancer (Swift et al., 2010).(12) PTGER2—Prostaglandin E2 receptor 2, also known as EP2, is a prostaglandin receptor for prostaglandin E2 (PGE2) encoded by the human gene PTGER2. PTGER2 is classified as a relaxant type of prostanoid receptor based on its ability, upon activation, to relax certain types of smooth muscle. When initially bound to PGE2 or any other of its agonists, it mobilizes G proteins containing the Gαs-Gβγ complex. The Gαs-Gβγ complexes dissociate into their Gαs and Gβγ subunits which in turn regulate cell signaling pathways. In particular, Gαs stimulates adenyl cyclase to raise cellular levels of cAMP thereby activating PKA; PKA activates various types of signaling molecules such as the transcription factor CREB which lead to different types of functional responses depending on cell type (Markovic et al., 2017; Woodward et al., 2011). PTGER2 also activates the GSK-3 pathway which regulates cell migratory responses and innate immune responses including pro-inflammatory cytokine and interleukin production and Beta-catenin pathway which regulates not only cell-cell adhesion but also activates the Wnt signaling pathway which, in turn, stimulates the transcription of genes responsible for regulating cell migration and proliferation (Woodward et al., 2011). The PTGER2 receptor can act as a tumor promoter. PTGER2 gene knockout mice have less lung, breast, skin, and colon cancers following exposure to carcinogens. Knockout of this gene in mice with the adenomatous polyposis coli mutation also causes a decrease in the size and number of pre-cancerous intestinal polyps that the animals develop. These effects are commonly ascribed to the loss of PTGER2-mediated: vascular endothelial growth factor production and thereby of tumor vascularization; regulation of endothelial cell motility and survival; interference with transforming growth factor-β's anti-cell proliferation activity; and, more recently, regulation of host anti-tumor immune responses (O'Callaghan and Houston, 2015). PTGER2 is widely distributed in humans. Its protein is expressed in human small intestine, lung, media of arteries and arterioles of the kidney, thymus, uterus, brain cerebral cortex, brain striatum, brain hippocampus, corneal epithelium, corneal choriocapillaries, Myometriuml cells, eosinophiles, sclera of the eye, articular cartilage, the corpus cavernosum of the penis, and airway smooth muscle cells; its mRNA is expressed in gingival fibroblasts, monocyte-derived dendritic cells, aorta, corpus cavernosum of the penis, articular cartilage, airway smooth muscle, and airway epithelial cells. In rats, the receptor protein and/or mRNA has been found in lung, spleen, intestine, skin, kidney, liver, long bones, and rather extensively throughout the brain and other parts of the central nervous system (Yagami et al., 2016). Pre-clinical studies indicate that PTGER2 may be a target for treating and/or preventing particular human disorders involving: allergic diseases such as asthma (particular aspirin and nonsteroidal inflammatory drug-induced asthma syndromes) and rhinitis (Machado-Carvalho et al., 2014); glaucoma (Doucette and Walter, 2017); various diseases of the nervous system (Yagami et al., 2016); fractures, osteoporosis, and other bone abnormalities (Li et al., 2007); pulmonary fibrosis; certain forms of malignant disease such as colon cancer including those that arise from Adenomatous polyposis coli mutations (O'Callaghan and Houston, 2015); and salt-sensitive forms of hypertension (Yang and Du, 2012); This receptor has also been suggested to be a target for contraception (Sugimoto et al., 2015).(13) TACR3—Tachykinin receptor 3, also known as TACR3, function as receptors for tachykinins. The tachykinins are a family of peptides that comprise substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the species divergent endokinins including endokinin B (EKB) in humans. These tachykinins are encoded on three different genes, preprotachykinin 1 (TAC1) encoding SP and NKA, TAC3 encoding NKB and TAC4 encoding EKB. Three tachykinin receptors have been identified, which interact with these tachykinins: TACR1, TACR2, and TACR3, also called as NK1, NK2 and NK3, respectively, whereby SP and EKB show the greatest potency for TACR1, NKA for TACR2 and NKB for TACR3. Receptor affinities are specified by variations in the 5′-end of the sequence. Tachykinin receptor-3 (TACR3) is the mediator of biologic actions encoded by the C-terminal sequence of tachykinins, for which neurokinin B is a more potent agonist than neurokinin A or substance P (Page et al., 2003). It is reported that four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R) (Topaloglu et al., 2009). NKB, a member of the substance P-related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin (Goodman et al., 2007), a recently identified regulator of gonadotropin-releasing hormone secretion (Gianetti and Seminara, 2008).(14) APLNR—The apelin receptor (also known as the APJ receptor) is a G protein-coupled receptor which binds apelin and Apela/ELABELA/Toddler (Medhurst et al., 2003). Receptor for apelin receptor early endogenous ligand (APELA) and apelin (APLN) hormones coupled to G proteins that inhibit adenylate cyclase activity. APLNR plays a key role in early development such as gastrulation and heart morphogenesis by acting as a receptor for APELA hormone. APLNR plays also a role in various processes in adults such as regulation of blood vessel formation, blood pressure, heart contractility, and heart failure by acting as a receptor for APLN hormone. A decade of investigations has shown that the apelinergic system has a broad range of biological functions, playing an important role particularly in maintaining homeostasis of the cardiovascular system and fluid metabolism. The activation of APLNR causes a broad spectrum of biochemical changes including cAMP suppression, phosphorylation of protein kinase B (Akt), ERK1/25 and p70S6K, calcium mobilization7 and nitric oxide synthase (NOS). However, the specificity linking these biochemical activities to a certain biological function is yet to be determined (Wang et al., 2015c). Also, It functions in embryonic and tumor angiogenesis (Wu et al., 2017) and as a human immunodeficiency virus (HIV-1) coreceptor (Zhou et al., 2003). Both APLNR and apelin are expressed in many tissues including heart, lung, endothelium, kidney and brain (Wang et al., 2015c).(15) CCR5—C—C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines. This is the process by which T cells are attracted to specific tissue and organ targets. Many forms of HIV, the virus that causes AIDS, initially use CCR5 to enter and infect host cells. Certain individuals carry a mutation known as CCR5-A32 in the CCR5 gene, protecting them against these strains of HIV. Certain populations have inherited the Delta 32 mutation resulting in the genetic deletion of a portion of the CCR5 gene. Homozygous carriers of this mutation are resistant to M-tropic strains of HIV-1 infection (Hutter et al., 2009). CCR5's cognate ligands include CCL3, CCL4 (also known as MIP 1α and 1β, respectively), and CCL3L1. CCR5 furthermore interacts with CCL5 (Struyf et al., 2001). It is likely that CCR5 plays a role in inflammatory responses to infection, though its exact role in normal immune function is unclear. Regions of this protein are also crucial for chemokine ligand binding, functional response of the receptor, and HIV co-receptor activity (Barmania and Pepper, 2013). CCR5 is predominantly expressed on T cells, macrophages, dendritic cells, eosinophils, microglia and a subpopulation of ether breast or prostate cancer cells (Sicoli et al., 2014; Velasco-Velazquez et al., 2012).(16) GALR1—Galanin receptor 1 (GAL1) is a G-protein coupled receptor encoded by the GALR1 gene. The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gi/Go as transducing elements (Habert-Ortoli et al., 1994). GALR1 has anti-proliferative effects in oral squamous cell carcinoma. GALR1 protein and mRNA expression and GAL secretion were detected at variable levels in immortalized human oral keratinocytes and human oropharyngeal squamous cell carcinoma cell lines. Upon competitive inhibition of GALR1, proliferation was up-regulated in immortalized and malignant keratinocytes (Henson et al., 2005) Stevenson et al. report the stem cell marker and regulator, galanin and GALR1 as key determinants of drug resistance and potential therapeutic targets for combating drug resistance. Mechanistically, they identify a novel role for the GALR1-galanin receptor-ligand axis as an important upstream regulator of expression of the anti-apoptotic protein FLIPL. Clinically, galanin mRNA was found to be overexpressed in colorectal tumours, and notably, high galanin mRNA expression correlated with poor disease-free survival in early stage disease (Stevenson et al., 2012). GALR1 is widely expressed in the brain and spinal cord, as well as in peripheral sites such as the small intestine and heart (Fagerberg et al., 2014)(17) PTGER3—Prostaglandin EP3 receptor (53 kDa), also known as EP3, is a prostaglandin receptor for prostaglandin E2 (PGE2) encoded by the human gene PTGER3; it is one of four identified EP receptors, the others being PTGER1, PTGER2, and PTGER4, also called as EP1, EP2, and EP4, respectively, all of which bind with and mediate cellular responses to PGE2 and also, but generally with lesser affinity and responsiveness, certain other prostanoids. PTGER3 activation promotes duodenal secretion in mice; this function is mediated by PTGER3 activation in humans. EP receptor functions can vary with species and most of the functional studies cited here have not translated their animal and tissue models to humans (Moreno, 2017). Studies of the direct effects of PTGER3 activation on cancer in animal and tissue models give contradictory results suggesting that this receptor does not play an important role in carcinogenesis. However, some studies suggest an indirect pro-carcinogenic function for the PTGER3: The growth and metastasis of implanted Lewis lung carcinoma cells, a mouse lung cancer cell line, is suppressed in PTGER3-deficient mice. This effect was associated with a reduction in the levels of Vascular endothelial growth factor and matrix metalloproteinase-9 expression in the tumor's stroma; expression of the pro-lymphangiogenic growth factor, VEGF-C and its receptor, VEGFR3; and a tumor-associated angiogenesis and lymphangiogenesis (O'Callaghan and Houston, 2015). PTGER3 is widely distributed in humans. Its protein and/or mRNA is expressed in kidney (i.e. glomeruli, Tamm-Horsfall protein negative late distal convoluted tubules, connecting segments, cortical and medullary collecting ducts, media and endothelial cells of arteries and arterioles); stomach (vascular smooth muscle and gastric fundus mucosal cells); thalamus (anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei); intestinal mucosal epithelia at the apex of crypts; myometrium (stromal cells, endothelial cells, and, in pregnancy, placenta, chorion, and amnion); mouth gingival fibroblasts; and eye (corneal endothelium and keratocytes, trabecular cells, ciliary epithelium, and conjunctival and iridal stroma cells, and retinal Müller cells) (Norel, 2016).(18) SSTR2—Somatostatin receptor type 2 is a protein that in humans is encoded by the SSTR2 gene. Somatostatin acts at many sites to inhibit the release of many hormones and other secretory proteins. The biologic effects of somatostatin are probably mediated by a family of G protein-coupled receptors that are expressed in a tissue-specific manner. SSTR2 has been shown to interact with shank2 (Zitzer et al., 1999). SSTR2 encodes somatostatin receptor that can inhibit the cell proliferation of solid tumors. SSTR2 promoter hypermethylation is associated with the risk and progression of laryngeal squamous cell carcinoma in males (Shen et al., 2016). Most pituitary adenomas express SSTR2, but other somatostatin receptors are also found (Miller et al., 1995). Somatostatin analogs (i.e. Octreotide, Lanreotide) are used to stimulate this receptors, and thus to inhibit further tumor proliferation (Zatelli et al., 2007). SSTR2 is expressed in highest levels in cerebrum and kidney (Fagerberg et al., 2014). The term “inhibitor” as used herein refers to molecule that inhibits or suppresses the enhanced function of CXCR4-GPCRx heteromer. Non-limiting examples of the inhibitor of the invention that can be used for treatment, amelioration, or prevention of a cancer or related symptoms include GPCRx antagonist, GPCRx inverse agonist, GPCRx positive and negative allosteric modulator, CXCR4-GPCRx heteromer-specific antibody or its antigen biding portions including single-domain antibody-like scaffolds, bivalent ligands which have a pharmacophore selective for CXCR4 joined by a spacer arm to a pharmacophore selective for GPCRx, bispecific antibody against CXCR4 and GPCRx, radiolabeled CXCR4 ligand linked with GPCRx ligand, and small molecule ligands that inhibit heteromer-selective signaling. Certain examples of inhibitors against GPCRx that form heteromers with CXCR4 and enhance Ca2+ response upon co-stimulation with both agonists are listed in Table 3. The term “antagonist” as used herein refers to a type of receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor, also called blockers. Antagonists have affinity but no efficacy for their cognate receptors, and their binding disrupts the interaction and inhibit the function of an agonist or inverse agonist at the cognate receptors. Certain examples of antagonists against GPCRx that form heteromers with CXCR4 and enhance Ca2+ response upon co-stimulation with both agonists are listed in Table 3. TABLE 3Examples of inhibitors against GPCRx that form heteromers with CXCR4 and enhancesCa2+ response upon co-stimulation with both agonists.Antibodies/nanobodies/Gene nameAntagonists/Inverse agonistsi-bodies/othersCXCR4ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor),AD-114, AD-114-AMD3465, ATI 2341, BKT140 (BL-8040; TF14016; 4F-Benzoyl-6H, AD-114-Im7-TN14003), CTCE-9908, CX549, D-[Lys3] GHRP-6, FC122, FC131, GMI-FH, AD-114-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t),PA600-6H, ALX-KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]Methyl-AMD3465,0651, LY2624587,POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012,PF-06747143,TG-0054 (Burixafor), USL311, viral macrophage inflammatory protein-IIulocuplumab(vMIP-II), WZ811, [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga]pentixafor,(MDX1338/BMS-[90Y]bentixather, [99mTc]O2-AMD3100, [177Lu]pentixather, and 508MCl936564), 12G5,(Compound 26).238D2, and 238D4ADCYAP1R1M65, Max.d.4, MK-0893, N-stearyl-[Nle17] neurotensin-(6-11)/VIP-(7-28),PACAP-(6-38), and PG 97-269ADORA2B3-isobutyl-8-pyrrolidinoxanthine, alloxazine, AS16, AS70, AS74, AS94,AS95, AS96, AS99, AS100, AS101, ATL802, BW-A1433, caffeine, CGS15943, CPX, CSC, CVT-6883, DAX, DEPX, derenofylline, DPCPX, FK-453, I-ABOPX, istradefylline, KF26777, LAS38096, LUF5981, MRE2029F20, MRE 3008F20, MRS1191, MRS1220, MRS1523, MRS1706,MRS1754, MSX-2, OSIP339391, pentoxifylline, preladenant, PSB-10, PSB-11, PSB36, PSB603, PSB-0788, PSB1115, rolofylline, SCH 58261,SCH442416, ST-1535, theophylline, tonapofylline, vipadenant, xanthineamine congener, XCC, and ZM-241385.ADORA3ATL802, BW-A1433, caffeine, CGS 15943, CSC, CVT-6883,derenofylline, dexniguldipine, DPCPX, FK-453, flavanone, flavone,galangin, I-ABOPX, istradefylline, KF26777, LAS38096, LUF5981, MRE2029F20, MRE 3008F20, MRE 3010F20, MRS1041, MRS1042, MRS1067,MRS1088, MRS1093, MRS1097, MRS1177, MRS1186, MRS1191,MRS1191, MRS1220, MRS1476, MRS1486, MRS1505, MRS1523,MRS1754, MRS928, MSX-2, nicardipine, preladenant, PSB-10, PSB-11,PSB36, PSB603, PSB1115, rolofylline, sakuranetin, SCH 58261,SCH442416, ST-1535, theophylline, tonapofylline, vipadenant, visnagin,VUF5574, VUF8504, VUF8507, xanthine amine congener, and ZM-241385ADRB2Alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol,carvedilol, CGP 12177, cicloprolol, ICI 118551, ICYP, labetalol,levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP,propafenone, propranolol, sotalol, SR59230A, and timolol.C5AR1A8Δ71-73, AcPhe-Orn-Pro-D-Cha-Trp-Arg, avacopan, C089, CHIPS,DF2593A, JPE1375, L-156,602, NDT9520492, N-methyl-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H, PMX205, PMX53, RPR121154, and W54011.CALCRα-CGRP-(8-37) (human), AC187, CT-(8-32) (salmon), and olcegepant.CHRM13-Quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB,AFDX384, amitriptyline, AQ-RA 741, atropine, benzatropine, biperiden,darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate,guanylpirenzepine, hexahydrodifenidol, hexahydrosiladifenidol,hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine,ML381, muscarinic toxin 1, muscarinic toxin 2, muscarinic toxin 3, N-methyl scopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine,propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine,silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine,trihexyphenidyl, tripitramine, UH-AH 37, umeclidinium, and VU0255035.EDNRBA192621, ambrisentan, atrasentan, bosentan (RO 470203, Tracleer);,BQ788, IRL 2500, K-8794, macitentan, RES7011, Ro 46-8443, SB209670,SB217242 (enrasentan), TAK 044, and tezosentan (RO610612).HRH1(−)-chlorpheniramine, (+)-chlorpheniramine, (−)-trans-H2-PAT, (+)-cis-H2-PAT, (+)-trans-H2-PAT, (±)-cis-H2-PAT, (±)-trans-H2-PAT, (R)-cetirizine,(S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239,alimemazine, amitriptyline, aripiprazole, arpromidine, asenapine,astemizole, AZD3778, azelastine, BU-E 47, cetirizine, chlorpheniramine,chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine,cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin,doxepin, epinastine, fexofenadine, fluphenazine, fluspirilene, haloperidol,hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen,loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine,pimozide, pipamperone, pitolisant, promethazine, pyrilamine, quetiapine,risperidone, sertindole, terfenadine, thioridazine, thiothixene,trifluoperazine, tripelennamine, triprolidine, ziprasidone, and zotepine.MLNRGM-109, MA-2029, and OHM-11526.NTSR1Meclinertant, SR48527, SR48692, and SR142948ATACR3[Trp7, β-Ala8] neurokinin A-(4-10), AZD2624, FK 224, GR138676, GSK172981, GSK 256471, N′,2-diphenylquinoline-4-carbohydrazide 8m, N′,2-diphenylquinoline-4-carbohydrazide, osanetant, PD 154740, PD 161182,PD157672, saredutant, SB 218795, SB 222200, SB 235375, SCH 206272,SSR 146977, and talnetant. In some embodiments, the method for treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, or the method of suppressing enhanced downstream signaling from a CXCR4-GPCRx heteromer in a cell of a patient suffering from cancer, as disclosed herein, may comprise administering, or the pharmaceutical kit or pharmaceutical kit, comprise, administering to the patient an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein: i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; and ii) the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In some embodiments, the pharmaceutical kit or pharmaceutical composition for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, as disclosed herein, may comprise an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. For example, in some embodiments, the CXCR4 inhibitor is an antagonist of CXCR4, an inverse agonist of CXCR4, a partial antagonist of CXCR4, an allosteric modulator of CXCR4, an antibody of CXCR4, an antibody fragment of CXCR4, a ligand of CXCR4, or an antibody-drug conjugate of CXCR4. For example, in some embodiments, the GPCRx inhibitor is an antagonist of GPCRx, an inverse agonist of GPCRx, a partial antagonist of GPCRx, an allosteric modulator of GPCRx, an antibody of GPCRx, an antibody fragment of GPCRx, a ligand of GPCRx, or an antibody-drug conjugate of GPCRx. For example, in some embodiments, the inhibitor of the CXCR4-GPCRx heteromer is an antagonist of the CXCR4-GPCRx heteromer, an inverse agonist of the CXCR4-GPCRx heteromer, a partial antagonist of the CXCR4-GPCRx heteromer, an allosteric modulator of the CXCR4-GPCRx heteromer, an antibody of the CXCR4-GPCRx heteromer, an antibody fragment of the CXCR4-GPCRx heteromer, a ligand of the CXCR4-GPCRx heteromer, a protein-protein interaction (PPI) inhibitor of the CXCR4-GPCRx heteromer, or an antibody-drug conjugate of the CXCR4-GPCRx heteromer. In some embodiments, the method for treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, as disclosed herein, includes a combination of inhibitors selected from the group consisting of: the CXCR4 inhibitor, the GPCRx inhibitor, and the inhibitor of the CXCR4-GPCRx heteromer, wherein the combination may be in a single pharmaceutical composition or a plurality of separate pharmaceutical compositions for each respective inhibitor. In some embodiments, the combination of inhibitors comprises the CXCR4 inhibitor and the GPCRx inhibitor. In some embodiments, the combination of inhibitors comprises the CXCR4 inhibitor and the inhibitor of the CXCR4-GPCRx heteromer. In some embodiments, the combination of inhibitors comprises the GPCRx inhibitor and the inhibitor of the CXCR4-GPCRx heteromer. In some embodiments, the combination of inhibitors are administered sequentially, concurrently, or simultaneously. In some embodiments, the combination of inhibitors are administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the combination of inhibitors are administered as separate pharmaceutical compositions, wherein the separate pharmaceutical compositions independently further comprise a pharmaceutically acceptable carrier. In some embodiments, the method for treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, as disclosed herein, comprises a therapeutically effective amount or a sub-therapeutically effective amount of the CXCR4 inhibitor, such as administering a therapeutically effective amount of the CXCR4 inhibitor to the patient. In some embodiments, the method for treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, as disclosed herein, comprises a therapeutically effective amount or a sub-therapeutically effective amount of the GPCRx inhibitor, such as administering a therapeutically effective amount of the GPCRx inhibitor to the patient. In some embodiments, the method for treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, as disclosed herein, comprises a therapeutically effective amount or a sub-therapeutically effective amount of the inhibitor of the CXCR4-GPCRx heteromer, such as administering a therapeutically effective amount of the inhibitor of the CXCR4-GPCRx heteromer to the patient. In some embodiments, the method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, as disclosed herein, may comprise: 1) determining whether the patient has the cancer cell containing the CXCR4-GPCRx heteromer having enhanced downstream signaling by: obtaining or having obtained a biological sample from the patient; and performing or having performed an assay on the biological sample to determine if: i) the patient's cancer cell containing said CXCR4-GPCRx heteromer; or ii) a CXCR4-GPCRx heteromer-selective reagent: alters heteromer-specific properties or function of said CXCR4-GPCRx heteromer in a patient derived cell(s); alters heteromer-specific properties of a patient derived cell(s) containing the CXCR4-GPCRx heteromer; or decreases cell proliferation of a patient derived cell(s) containing the CXCR4-GPCRx heteromer; and 2) if the patient has a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer to the cancer patient. In some embodiments, the method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, as disclosed herein, may comprise: 1) determining whether the patient's cancer cell contains the CXCR4-GPCRx heteromer by: obtaining or having obtained a biological sample from the patient and performing or having performed an assay on the biological sample to determine if said CXCR4-GPCRx heteromer is present in the patient's cancer cell; wherein: a) the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; and b) the assay performed on the biological sample is or comprises one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay, such as via a co-internalization assay, bimolecular fluorescence complementation (BiFC), or a proximity ligation assay (PLA); and 2) if the patient's cancer cell contains said CXCR4-GPCRx heteromer, then internally administering an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer to the patient. In some embodiments, the patient's biological sample is a biological fluid sample or is a biological tissue sample. In some embodiments, a liquid biopsy is performed on the biological fluid sample or performed on the biological tissue sample. In some embodiments, the biological fluid sample is a blood sample, a plasma sample, a saliva sample, a cerebral fluid sample, an eye fluid sample, or a urine sample. In certain embodiments, the biological fluid sample includes circulating tumor cells (CTCs), tumor-derived cell-free DNA (cfDNA), circulating small RNAs, and extracellular vesicles including exosomes, from bodily fluids as disclosed, for example, in Campos C D M et al., “Molecular Profiling of Liquid Biopsy Samples for Precision Medicine,” Cancer J. 2018 March/April; 24(2):93-103, which is incorporated hereby in its entirety. In some embodiments, the biological tissue sample is an organ tissue sample, a bone tissue sample, or a tumor tissue sample. The term “heteromer” as used herein refers to macromolecular complex composed of at least two GPCR units [protomers] with biochemical properties that are demonstrably different from those of its individual components. Heteromerization can be evaluated by in situ hybridization, immunohistochemistry, RNAseq, Reverse transcription-quantitative PCR (RT-qPCR, realtime PCR), microarray, proximity ligation assay (PLA), time-resolved FRET (TR-FRET), whole-body Single-photon emission computed tomography (SPECT) or Positron Emission Tomography/Computed Tomography (PET/CT). The phrase “effective amount” as used herein refers to an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc. The phrase “therapeutically effective amount” as used herein refers to the amount of a therapeutic agent (e.g., an inhibitor, an antagonist, or any other therapeutic agent provided herein) which is sufficient to reduce, ameliorate, and/or prevent the severity and/or duration of a cancer and/or a symptom related thereto. A therapeutically effective amount of a therapeutic agent can be an amount necessary for the reduction, amelioration, or prevention of the advancement or progression of a cancer, reduction, amelioration, or prevention of the recurrence, development or onset of a cancer, and/or to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of a inhibitor, an antagonist, or any other therapeutic agent provided herein). The phrase “therapeutic agent” refers to any agent that can be used in the treatment, amelioration, prevention, or management of a cancer and/or a symptom related thereto. In certain embodiments, a therapeutic agent refers to an inhibitor of CXCR4-GPCRx heteromer of the invention. A therapeutic agent can be an agent which is well known to be useful for, or has been or is currently being used for the treatment, amelioration, prevention, or management of a cancer and/or a symptom related thereto. The phrase “intracellular Ca2+ assay,” “calcium mobilization assay,” or their variants as used herein refer to cell-based assay to measure the calcium flux associated with GPCR activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. The dye binds the calcium released from intracellular store and its fluorescence increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. The phrase “proximity-based assay” as used herein refers to biophysical and biochemical techniques that are able to monitor proximity and/or binding of two protein molecules in vitro (in cell lysates) and in live cells, including bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), Proximity ligation assay (PLA), cysteine crosslinking, and co-immunoprecipitation (Ferre et al., 2009; Gomes et al., 2016). Alternative methods for detecting heteromer formation include, but are not limited to: immunostaing (Bushlin et al., 2012; Decaillot et al., 2008); immunoelectron microscopy (Fernandez-Duenas et al., 2015); BRET (Pfleger and Eidne, 2006); Time-resolved FRET assays (Fernandez-Duenas et al., 2015); In Situ Hybridization (He et al., 2011); FRET (Lohse et al., 2012); β-arrestin recruitment assay using GPCR heteromer identification technology (GPCR-HIT, Dimerix Bioscience) (Mustafa and Pfleger, 2011) using BRET, FRET, BiFC, Bimolecular Luminescence Complementation, enzyme fragmentation assay, and Tango Tango GPCR assay system (Thermo Fisher Scientific) (Mustafa, 2010); PRESTO-Tango system (Kroeze et al., 2015); regulated secretion/aggregation technology (ARIAD Pharmaceuticals) (Hansen et al., 2009); Receptor Selection and Amplification Technology (ACADIA Pharmaceuticals) (Hansen et al., 2009); DimerScreen (Cara Therapeutics) (Mustafa, 2010); Dimer/interacting protein translocation assay (Patobios) (Mustafa, 2010); Co-immunoprecipitation (Abd Alla et al., 2009); GPCR internalization assays using surface enzyme-linked immunosorbent assay (ELISA) (Decaillot et al., 2008) or Flow Cytometry (Law et al., 2005); Whole Cell Phosphorylation Assays (Pfeiffer et al., 2002); and Proximity-ligation assay (PLA) (Frederick et al., 2015). Alternative methods for detecting changes in pharmacological properties, signaling properties, and/or trafficking properties, in cells expressing both CXCR4 and GPCRx include, but are not limited to: Radioligand Binding Assays (Bushlin et al., 2012; Pfeiffer et al., 2002); Cell Surface Biotinylation and Immunoblotting (He et al., 2011); immunostaing (Bushlin et al., 2012; Decaillot et al., 2008); immunoelectron microscopy (Fernandez-Duenas et al., 2015); [35S]GTPQ□S Binding assays (Bushlin et al., 2012); Calcuim imaging or assays using dyes such as Fura 2-acetomethoxy ester (Molecular Probes), Fluo-4 NW calcium dye (Thermo Fisher Scientific), or FLIPR5 dye (Molecular Devices); cAMP assays using radioimmunoassay kit (Amersham Biosciences); AlphaScreen (PerkinElmer Life Sciences); Parameter Cyclic AMP Assay (R&D Systems); femto cAMP kit (Cisbio); cAMP Direct Immunoassay Kit (Calbiochem) or GloSensor cAMP assay (Promega); GTPase assay (Pello et al., 2008); PKA activation (Stefan et al., 2007); ERK1/2 and/or Akt/PKB Phosphorylation Assays (Callen et al., 2012); Src and STAT3 phosphorylation assays (Rios et al., 2006); reporter assays such as cAMP response element (CRE); nuclear factor of activated T-cells response element (NFAT-RE); serum response element (SRE); serum response factor response element (SRF-RE); and NF-κB-response element luciferase reporter assays; Secreted alkaline phosphatase Assay (Decaillot et al., 2011); Measurement of Inositol 1-Phosphate Production Using TR-FRET or [3H]myo-Inositol (Mustafa et al., 2012); RT-qPCR for measuring downstream target gene expression (Mustafa et al., 2012); and Adenylyl Cyclase Activity (George et al., 2000); next generation sequencing (NGS); and any other assay that can detect a change in receptor function as a result of receptor heterodimerization. The phrase “protein-protein interaction inhibitor,” “PPI inhibitor,” or their variants as used herein refer to any molecules that can interfere with protein-protein interactions. Protein-protein interaction, unlike enzyme-substrate interaction involving well-defined binding pockets, is a transient interaction or association between proteins over relatively large areas and is often driven by electrostatic interactions, hydrophobic interactions, hydrogen bonds, and/or Van der Waals forces. PPI inhibitors may include, but not limited to, membrane-permeable peptides or lipid fused to a peptide sequence that disrupts the GPCR heteromeric interface, for example, transmembrane helix, intracellular loop, or C-terminal tail of GPCRx. The PPI inhibitor of the CXCR4-GPCRx heteromer, for example, may be a membrane-permeable peptide or cell-penetrating peptide (CPP) conjugated with peptide that targets the CXCR4-GPCRx heteromeric interface(s), or may be a cell-penetrating lipidated peptide targeting the CXCR4-GPCRx heteromeric interface(s). For example, the membrane-permeable peptide or cell-penetrating peptide includes: HIV-1 TAT peptides, such as TAT48-60and TAT49-57; Penetratins, such as pAntp (43-58); Polyarginines (Rn such as R5 to R12); Diatos peptide vector 1047 (DPV1047, Vectocell®); MPG (HIV gp41 fused to the nuclear localization signal (NLS) of the SV40 large T antigen); Pep-1 (tryptophan-rich cluster fused to the NLS of SV40 large T antigen); pVEC peptide (vascular endothelial cadherin); p14 alternative reading frame (ARF) protein-based ARF(1-22); N-terminus of the unprocessed bovine prion protein BPrPr(1-28); Model amphipathic peptide (MAP); Transportans; Azurin-derived p28 peptide; amphipathic β-sheet peptides, such as VT5; proline-rich CPPs, such as Bac 7 (Bacl-24); hydrophobic CPPs, such as C105Y derived from al-Antitrypsin; PFVYLI derived from synthetic C105Y; Pep-7 peptide (CHL8 peptide phage clone); and modified hydrophobic CPPs, such as stapled peptides and prenylated peptides (Guidotti et al., 2017; Kristensen et al., 2016). The membrane-permeable peptide or cell-penetrating peptide can further include, for example, TAT-derived cell-penetrating peptides, signal sequence-based (e.g., NLS) cell-penetrating peptides, hydrophobic membrane translocating sequence (MTS) peptides, and arginine-rich molecular transporters. The cell-penetrating lapidated peptide includes, for example, pepducins, such as ICL1/2/3, C-tail-short palmitoylated peptides (Covic et al., 2002; O'Callaghan et al., 2012). The peptide(s) that target the CXCR4-GPCRx heteromeric interface may be, for example, a transmembrane domain of CXCR4, transmembrane domain of GPCRx, intracellular loop of CXCR4, intracellular loop of GPCRx, C-terminal domain of CXCR4, or C-terminal domain of GPCRx, extracellular loop of CXCR4, extracellular loop of GPCRx, N-terminal region of CXCR4, or N-terminal region of GPCRx. In some embodiments, the invention provides a pharmaceutical composition (sometimes referred to herein as “pharmaceutical formulations”) having an inhibitor of a CXCR4-GPCRx heteromer of the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier that can be used in the pharmaceutical compositions of the invention include any of the standard pharmaceutical carriers known in the art, such as physiologically acceptable carriers, excipients or stabilizers, for example, phosphate buffered saline solution, water and emulsions such as an oil and water emulsion, and various types of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dose forms or any other dosing form that is sufficient for delivery of the inhibitor of a CXCR4-GPCRx heteromer of the invention to the target area of the subject in need of treatment. For example, the pharmaceutical compositions can be prepared in any manner appropriate for the chosen mode of administration, e.g., intravascular, intramuscular, subcutaneous, intradermal, intrathecal, etc. Other optional components, e.g., pharmaceutical grade stabilizers, buffers, preservatives, excipients and the like can be readily selected by one of skill in the art. The preparation of a pharmaceutically composition, having due regard to pH, isotonicity, stability and the like, is within the level of skill in the art. Pharmaceutical formulations containing one or more inhibitors of a CXCR4-GPCRx heteromer of the invention provided herein can be prepared for storage by mixing the inhibitors having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight polypeptides of less than about 10 residues; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes, e.g., Zn-protein complexes; and/or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Thus, in some embodiments, the invention provides a method for treatment, amelioration, or prevention of a disease in a subject in need thereof. The methods of the invention can include administering a therapeutically effective amount of a pharmaceutical composition provided herein to the subject. For example, the pharmaceutical composition can include one or more inhibitors of a CXCR4-GPCRx heteromer provided herein. Diseases that can be treated or prevented using the methods of the invention include cancer, tumor, metastasis, and/or angiogenesis. In particular, the methods of the invention are useful for treating cancers or related symptoms wherein the cells of cancer, tumor, and/or microenvironment expresses the CXCR4-GPCRx heteromer. Non-limiting examples of cancers or tumors that can be treated, ameliorated, or prevented using the methods of the invention include tumors of the gastrointestinal tract, for example, breast cancer, lung cancer, small cell carcinoma of the lung, hepatocellular carcinoma, brain cancer, kidney cancer, pancreatic cancer or pancreatic adenocarcinoma, ovarian cancer, prostate cancer, melanoma, lymphoma, leukemia, multiple myeloma, renal cell carcinoma, soft tissue sarcoma, gastrointestinal cancer, stomach cancer, colon cancer, colorectal cancer, colorectal adenocarcinoma, bladder adenocarcinoma, esophageal cancer, and adenocarcinoma of the stomach, esophagus, throat, and urogenital tract. In some embodiments, the invention provides a method for treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, or provides a method of suppressing enhanced downstream signaling from a CXCR4-GPCRx heteromer in a cell of a patient suffering from cancer, the method comprising: administering to the patient an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein: i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; and ii) the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In some embodiments, the invention provides a pharmaceutical kit for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, the pharmaceutical kit comprising: an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. In some embodiments, the invention provides a pharmaceutical composition for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, the pharmaceutical composition comprising: i) an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; and ii) a pharmaceutically acceptable carrier; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the progression of the cancer in the patient having said cancer cell containing the CXCR4-GPCRx heteromer is decreased in the range of 5-100% more upon administration of the combination of inhibitors, relative to administering the CXCR4 inhibitor or GPCRx inhibitor as the single inhibitor to said patient, such as decreased in the range of 5-100% more, 10-100% more, 20-100% more, 30-100% more, 40-100% more, 50-100% more, 60-100% more, 75-100% more, 5-75% more, 5-50% more, or 5-25% more, upon administration of the combination of inhibitors, relative to administering the CXCR4 inhibitor or GPCRx inhibitor as the single inhibitor to said patient. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HIRH1; or selected from the group consisting of: ADRB2, HIRH, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the efficacy of a CXCR4 inhibitor is increased in the range of 5-2000% when administered in combination with the GPCRx inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the CXCR4 inhibitor when administered as a single inhibitor, such as increased in the range of 5-1750%, 5-1500%, 5-1250%, 5-1000%, 5-900%, 5-800%, 5-700%, 5-500%, 5-400%, 5-250%, 5-200%, 5-100%, 5-75%, 5-50%, 5-40%, 5-30%, 5-25%, 100-2000%, 200-2000%, 300-2000%, 500-2000%, 750-2000%, 1000-2000%, 1250-2000%, 1500-2000%, 5-1500%, 25-1500%, 50-1500%, 75-1500%, 100-1500%, 200-1500%, 300-1500%, 500-1500%, 750-1500%, 1000-1500%, or 1250-1500%, when administered in combination with the GPCRx inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the CXCR4 inhibitor when administered as a single inhibitor. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HRH1; or selected from the group consisting of: ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the efficacy of a GPCRx inhibitor is increased in the range of 5-2000% when administered in combination with the CXCR4 inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the GPCRx inhibitor when administered as a single inhibitor, such as increased in the range of 5-1750%, 5-1500%, 5-1250%, 5-1000%, 5-900%, 5-800%, 5-700%, 5-500%, 5-400%, 5-250%, 5-200%, 5-100%, 5-75%, 5-50%, 5-40%, 5-30%, 5-25%, 100-2000%, 200-2000%, 300-2000%, 500-2000%, 750-2000%, 1000-2000%, 1250-2000%, 1500-2000%, 5-1500%, 25-1500%, 50-1500%, 75-1500%, 100-1500%, 200-1500%, 300-1500%, 500-1500%, 750-1500%, 1000-1500%, or 1250-1500%, when administered in combination with the CXCR4 inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the GPCRx inhibitor when administered as a single inhibitor. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HRH1; or selected from the group consisting of: ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the method administers a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; or the pharmaceutical kit or pharmaceutical composition comprises a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer. In some embodiments, the combination of inhibitors is a combination of two inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the method administers a combination of a CXCR4 inhibitor and a GPCRx inhibitor; or the pharmaceutical kit or pharmaceutical composition comprises a combination of a CXCR4 inhibitor and a GPCRx inhibitor. In some embodiments, according to the method of treating, method of suppressing, pharmaceutical kit, or pharmaceutical composition, provided herein, the method administers a CXCR4-GPCRx heteromer inhibitor; or the pharmaceutical kit or pharmaceutical composition comprises a CXCR4-GPCRx heteromer inhibitor. In some embodiments, according to the method of treating or method of suppressing provided herein, or according to the use of the pharmaceutical kit or pharmaceutical composition provided herein, the administering of the combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-2000 fold, relative to single inhibitor administration, such as suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-1750 fold, 5-1500 fold, 5-1250 fold, 5-1000 fold, 5-900 fold, 5-800 fold, 5-700 fold, 5-500 fold, 5-400 fold, 5-250 fold, 5-200 fold, 5-100 fold, 5-75 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 100-2000 fold, 200-2000 fold, 300-2000 fold, 500-2000 fold, 750-2000 fold, 1000-2000 fold, 1250-2000 fold, 1500-2000 fold, 5-1500 fold, 25-1500 fold, 50-1500 fold, 75-1500 fold, 100-1500 fold, 200-1500 fold, 300-1500 fold, 500-1500 fold, 750-1500 fold, 1000-1500 fold, or 1250-1500 fold, relative to single inhibitor administration. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HRH1; or selected from the group consisting of: ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. In some embodiments, according to the method of treating or method of suppressing provided herein, or according to the use of the pharmaceutical kit or pharmaceutical composition provided herein, the administering of the inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-2000 fold, relative to suppression of downstream signaling from either a CXCR4 protomer or a GPCRx protomer in their respective individual protomer context, such as suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-1750 fold, 5-1500 fold, 5-1250 fold, 5-1000 fold, 5-900 fold, 5-800 fold, 5-700 fold, 5-500 fold, 5-400 fold, 5-250 fold, 5-200 fold, 5-100 fold, 5-75 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 100-2000 fold, 200-2000 fold, 300-2000 fold, 500-2000 fold, 750-2000 fold, 1000-2000 fold, 1250-2000 fold, 1500-2000 fold, 5-1500 fold, 25-1500 fold, 50-1500 fold, 75-1500 fold, 100-1500 fold, 200-1500 fold, 300-1500 fold, 500-1500 fold, 750-1500 fold, 1000-1500 fold, or 1250-1500 fold, relative to suppression of downstream signaling from either a CXCR4 protomer or a GPCRx protomer in their respective individual protomer context. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HRH1; or selected from the group consisting of: ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. Initial testing and evaluation of an inhibitor, or combination of inhibitors, regarding whether effective, or therapeutically effective, in suppressing an enhanced downstream signaling from a CXCR4-GPCRx heteromer, according to the methods disclosed herein, and/or in determining an IC50 value according to the assays disclosed herein, may utilize a concentration of the inhibitor in the range of between 1-10 μM (or each of the inhibitors of the combination at concentrations in the range of between 1-10 μM), such as at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. If suppression of the signal by the inhibitor is not appreciable enough for a determinable measurement, then a greater concentration of the inhibitor may be used to better evaluate a determinable measurement, such as an IC50 value. If suppression of the signal by the inhibitor is very strong, then a lower concentration of the inhibitor may be used to better evaluate a determinable measurement, such as an IC50 value. It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the invention disclosed herein. EXAMPLES Example 1. Evaluation of CXCR4-GPCRx Heteromer Formation by BiFC Assay To identify novel CXCR4-GPCRx heteromers, we made recombinant adenoviruses encoding 143 GPCRs fused with N-terminal fragments of yellow fluorescent protein Venus (VN) and 147 GPCRs fused with C-terminal fragment of Venus (VC) as described in Song et al. (Song et al., 2014; SNU patent; Song, thesis). CXCR4-GPCR heteromers were identified using bimolecular fluorescence complementation (BiFC) assay (FIG.1), in which two complementary VN and VC fragments of Venus reconstitute a fluorescent signal only when both fragments are close enough through interaction between two different proteins to which they are fused (Hu et al., 2002). U-2 OS cells were plated in 96-well plates, were co-transduced with 30 MOI each of adenoviruses encoding CXCR4-VN and GPCRx-VC or CXCR4-VC and GPCRx-VN, and were allowed to express GPCRs for 2 days. After staining the cells with Hoechst 33342, BiFC and nuclear images were obtained from three fields per well using IN Cell Analyzer 1000. Images of about 200 cells from each well were analyzed with Multi-target analysis software in IN Cell Developer ToolBox (GE Healthcare, Waukesha, WI). Cell boundary was marked based on Hoechst signal, and fluorescence intensity per cell was measured. Cells with fluorescence intensities higher than background level were considered as BiFC positive cells. Dead cells that showed extremely high intensities were excluded from the cell count. Positive cells were determined, and positive cell count ratio (“BiFC score”) was calculated as (positive cells/total cells)×100 (see Table 4 below). When CXCR4-VN was co-expressed with HA-VC (FIG.2A) or GCGR-VC, a GPCR encoding glucagon receptor (FIG.2C), no yellow fluorescence protein (YFP) signal (BiFC signal) was observed. In contrast, when CXCR4-VN was co-expressed with CXCR4-VC (FIG.2B), BiFC signal was observed in the plasma membrane and in the cytoplasm. Strong BiFC signal was observed in the plasma membrane and in the cytoplasm when CXCR4-VN was co-transduced with ADCYAP1R1-VC (FIG.2D), ADORA3-VC (FIG.2F), ADRB2-VC (FIG.2G), APLNR-VC (FIG.2H), C5AR1-VC (FIG.2I), CALCR-VC (FIG.2J), CCR5-VC (FIG.2K), CHRM1-VC (FIG.2L), GALR1-VC (FIG.2M), EDNRB-VC (FIG.2N), HRH1-VC (FIG.2O), MLNR-VC (FIG.2P), NTSR1-VC (FIG.2Q), PTGER2-VC (FIG.2R), SSTR2-VC (FIG.2T), and TACR3-VC (FIG.2U). Robust BiFC signal was also observed when cells were co-transduced with CXCR4-VC and ADORA2B-VN (FIG.2E) or PTGER3-VN (FIG.2S). Cells that showed BiFC fluorescence signal higher than background level was counted as BiFC positive cells, and BiFC score was calculated. Protein-protein interaction can be affected by fusion tags such as fluorescence protein fragments in BiFC or renilla luciferase in BRET through interfering with the expression, folding, or localization of the partner protein. Partner protein can also impair the expression or folding of the fusion tags, and affect proximity-based assay result. Thus, the absence of signal between two proteins in specific combination does not necessarily imply that the proteins do not interact, but simply that the attached donor and acceptor molecules are in particular conformation that does not allow interaction to occur (Eidne et al., 2002; Kerppola, 2006). Therefore, CXCR4 and GPCRx that gave BiFC signal in either CXCR4-VN and GPCRx-VC or CXCR4-VC and GPCRx-VN combination were considered as interacting proteins. The BiFC score of CXCR4-VN and CXCR4-VC pair, a well-known homomer, was 9.9. Thus, CXCR4-GPCRx pairs that gave BiFC score equal or higher than 10 were selected as candidates for CXCR-GPCRx heteromer as shown in Table 4. TABLE 4GPCRs that exhibited BiFC score equal or higher than10 when co-expressed with CXCR4.BiFC scoreCXCR4-VN&GPCRxGPCRx-VC1ADCYAP1R1232ADORA2B11*3ADORA3434ADRB2245APLNR636C5AR1427CALCR538CCR5449CHRM12410GALR16011EDNRB4112HRH14513MLNR1014NTSR14815PTGER25816PTGER313*17SSTR23018TACR333*CXCR4-VC and GPCRx-VN were used. Sixteen GPCRs, ADCYAP1R1, ADORA3, ADRB2, APLNR, C5AR1, CALCR, CCR5, CHRM1, GALR1, EDNRB, HRH1, MLNR, NTSR1, PTGER2, SSTR2, and TACR3, were identified as CXCR4-interacting GPCRs in CXCR4-VN and GPCRx-VC combination and two GPCRs, ADORA2B and PTGER3, gave BiFC score higher than 10 in CXCR4-VC and GPCRx-VN combination as shown in Table 4. Among those, ADRB2 and CCR5 have been reported to form heteromers with CXCR4 (LaRocca 2010, Nakai 2014, (Agrawal et al., 2004; LaRocca et al., 2010; Rodriguez-Frade et al., 2004; Sohy et al., 2007; Sohy et al., 2009; Martinez-Munoz et al., 2014)), and the remaining 16 GPCRs were found to be novel CXCR4-GPCRx heteromers that have not been reported to the best of our knowledge. Among GPCRs that have been known to interact with CXCR4, ADRA1A and CXCR3 were also included in our BiFC assay. These GPCRs were excluded from further investigation since they gave BiFC signal of less than 10 with CXCR4: ADRA1A (CXCR4-VN-ADRAA-VC: BiFC score 7.85) and CXCR3 (BiFC score 1.16). Cannabinoid Receptor 2 (CB2, sometimes referred to as CNR2) was identified as a CXCR4-interacting GPCR in our BiFC assay (CNR2-VN-CXCR4-VC configuration: BiFC score 4.25; CNR2-VC-CXCR4-VN configuration: BiFC score 38.1), and co-internalization assay as described inFIGS.3A-3B and4A-4Q. But CNR2 was not selected as the final candiadate because it failed to exhibit an enhanced downstream signaling in the calcium mobilization assay (see Example 3 below). Example 2. Evaluation of CXCR4-GPCRx Heteromer Formation by Co-Internalization Assay Some of the GPCR heteromers are known to exhibit, as a result of the heteromerization, altered trafficking properties such as maturation of the partner GPCR (GABA(B) receptor) (White, 1998), agonist-mediated internalization of the partner GPCR from the cell surface (DOR-GRPR, A2A-D2R) (Hillion et al., 2002; Liu et al., 2011; Torvinen et al., 2005), and changes in the localization of the partner GPCR from an intracellular compartment to the cell surface (DOR-CB1) (Rozenfeld et al., 2012). Co-internalization of pairs of co-expressed GPCRs in response to agonists selective for only one of the pair has been used to confirm GPCR heteromerization (Milligan, 2008). To examine if GPCRx modulates the trafficking of CXCR4 when co-expressed and forms CXCR4-GPCRx heteromer and also to confirm the physical interaction between CXCR4 and GPCRx identified using BiFC assay, cells were co-transduced with adenoviruses encoding CXCR4-GFP and GPCRx and GFP images were obtained before and 30 min after stimulation with GPCRx agonist. Loss of GFP expression on the cell surface or appearance of GFP granules inside the cells were considered as CXCR4-GFP co-internalization with GPCRx (FIGS.3A-3B). InFIGS.4A-4Q, cells were stimulated with GPCRx agonists specific for CXCR4 (FIG.4A), ADCYAP1R1 (FIG.4B), ADORA2B (FIG.4C), ADORA3 (FIG.4D), ADRB2 (FIG.4E), APLNR (FIG.4F), C5AR1 (FIG.4G), CCR5 (FIG.4H), CHRM1 (FIG.4I), GALR1 (FIG.4J), EDNRB (FIG.4K), HRH1 (FIG.4L), MLNR (FIG.4M), NTSR1 (FIG.4N), PTGER3 (FIG.4O), SSTR2 (FIG.4P), and TACR3 (FIG.4Q) as follows: 10 nM of CXCL12 (FIG.4A), 1 μM of vasoactive intestinal peptide (VIP) (FIG.4B), 1 μM of BAY 60-6583 (FIG.4C), 1 μM of CGS21680 (FIG.4D), 100 nM of formoterol (FIG.4E), 1 μM of apelin-13 (FIG.4F), 100 nM of C5a (FIG.4G), 100 nM of CCL2 (FIG.4H), 1 μM of acetylcholine (FIG.4I), 100 nM of galanin (FIG.4J), 1 μM of endothelin 1 (FIG.4K), 1 μM of histamine (FIG.4L), 100 nM of motilin (FIG.4M), 1 μM of neurotensin (FIG.4N), 1 μM of PGE2 (FIG.4O), 1 μM of SRIF-14 (FIG.4P), and 1 μM of senktide (FIG.4Q), respectively. Images were obtained before and 30 min after agonist stimulation, and analyzed using IN Cell Analyzer 2000. The selection of the concentration of these GPCRx agonists was initially set at the EC50 concentration (or alternatively, the Ki or Kd concentration) for the respective specific GPCRx, and if the resulting signal was too intense, then the concertation was lowered below the EC50, and if the resulting signal was too weak, then the concertation was increased to no more than 10,000× the EC50 concentration. Stimulation of cells expressing CXCR4-GFP with CXCL12 resulted in re-location of GFP from the plasma membrane to distinct intracellular granules, showing internalization of surface CXCR4-GFP into the cytoplasm (FIG.4A). Among 18 CXCR4-GPCRx heteromers identified by BiFC assay, 16 heteromers containing the following GPCRx induced internalization of CXCR4 as revealed by increased intracellular GFP granules and reduced GFP signal in the plasma membrane in cells co-expressing CXCR4-GFP and GPCRx, confirming heteromer co-internalization: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, APLNR, C5AR1, CCR5, CHRM1, GALR1, EDNRB, HRH1, MLNR, NTSR1, PTGER3, SSTR2, and TACR3 (FIG.4B-Q). CXCR4-GPCRx heteromers containing CALCR and PTGER2, on the other hand, did not show co-internalization of the heteromer upon GPCRx agonist treatment. These results demonstrate that certain GPCRx partners identified in the BiFC assay do form CXCR4-GPCRx heteromers in cells co-expressing both GPCRs, as evidenced by altered cellular trafficking properties of the heteromer. Example 3. Evaluation by Ca2+ Mobilization Assay of Enhanced CXCR4 Downstream Signaling Upon CXCR4-GPCRx Heteromer Formation and Inhibition of the Enhanced Signaling To further examine if these CXCR4-GPCRx heteromers exhibit properties distinct from those of the individual protomers (in cells lacking one of the receptors), we investigated calcium signaling in the presence of either or both agonists in cells expressing either GPCR or both GPCRs together. In this example, as noted in Example 2, the selection of the concentration of these GPCRx agonists was initially set at the EC50 concentration (or alternatively, the Ki or Kd concentration) for the respective specific GPCRx, and if the resulting Ca2+ signal was too intense, then the concertation was lowered below the EC50, and if the resulting Ca2+ signal was too weak, then the concertation was increased to no more than 100× the EC50 concentration. When MDA-MB-231 human breast cancer cells were transduced with adenoviruses encoding CXCR4, treatment of CXCL12 evoked intracellular calcium mobilization (FIG.5A). Stimulation of the cells with salmeterol, an ADRB2-selective agonist, did not induce calcium response, demonstrating the calcium response evoked by CXCL12 was mediated by CXCR4. Co-treatment of the cells with CXCL12 and salmeterol induced similar calcium response compared to the one induced by CXCL12 alone. In cells overexpressing ADRB2 alone, CXCL12 did not induced calcium response while salmeterol induced calcium response, showing that salmeterol induces calcium response through ADRB2 (FIG.5B). Co-treatment of both agonists induced similar calcium response to the one stimulated by salmeterol alone. In cells overexpressing both CXCR4 and ADRB2, stimulation with each agonist induced calcium responses similar to the ones shown in cells expressing CXCR4 or ADRB2 alone (FIGS.5Cvs5A and5B). In contrast, co-treatment of both agonists together significantly increased the calcium response compared to the ones evoked by individual agonists (FIGS.5C &5D). The enhanced calcium signaling was observed only in cells expressing both CXCR4 and ADRB2, but not in cells expressing either CXCR4 or ADRB2 alone. These results clearly demonstrate that CXCR4-ADRB2 heteromer exhibit properties distinct from those of the individual GPCRs. To examine if the other GPCRs identified to interact with CXCR4 in BiFC and co-internalization assay also show distinct properties in calcium signaling, experiments shown inFIGS.5A-5Dwere performed for other identified GPCRx partners. In cells co-expressing CXCR4 and either ADCYAP1R1 (FIG.6A), ADORA2B (FIG.6B), ADORA3 (FIG.6C), C5AR1 (FIG.6D), CALCR (FIG.6E), CHRM1 (FIG.6F), EDNRB (FIG.6G), HRH1 (FIG.6H), MLNR (FIG.6I), NTSR1 (FIG.6J), PTGER2 (FIG.6K), or TACR3 (FIG.6L), co-treatment of CXCL12 and respective GPCRx agonist significantly increased calcium responses compared with the sum of calcium responses that were induced by individual agonists as shown inFIGS.5C and5D, and as shown inFIGS.6A-6L. The cells co-expressing the CXCR4 and the GPCRx (either ADCYAP1R1 (FIG.6A), ADORA2B (FIG.6B), ADORA3 (FIG.6C), C5AR1 (FIG.6D), CALCR (FIG.6E), CHRM1 (FIG.6F), EDNRB (FIG.6G), HRH1 (FIG.6H), MLNR (FIG.6I), NTSR1 (FIG.6J), PTGER2 (FIG.6K), or TACR3 (FIG.6L)) exhibited an enhanced calcium mobilization upon co-stimulation (or co-treatment) with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined by the calcium mobilization assay. Notably, in cells co-expressing CXCR4 and CNR2, CXCL12-induced calcium response was significantly reduced, rather than enhanced, upon addition of CXCL12 alone or together with JWH-133, a selective CNR2 agonist, compared with the CXCL12-mediated calcium response observed in cells expressing CXCR4 alone (data not shown). Within the above set of co-expressed systems, it was also noted that an enhanced amount of calcium mobilization was observed in the individual protomer context in cells involving CXCR4 and PTGER2 (FIG.6K) (an individual protomer context, e.g., either CXCR4 expressed in the absence of the respective GPCRx, or the respective GPCRx expressed in the absence of the CXCR4), upon co-stimulation with CXCL12 and the respective selective GPCRx agonist. Specifically, an enhanced amount of calcium mobilization was observed for PTGER2 (FIG.6K), in the individual protomer context, in both situations where CXCR4 was expressed in the absence of the respective GPCRx, and where the respective GPCRx (PTGER2) was expressed in the absence of the CXCR4, upon co-stimulation with CXCL12 and the respective selective GPCRx agonist (5 μM of PGE2), the calcium mobilization amount resulting from the individual protomer context systems were greater than the sum of calcium mobilization amounts resulting from single agonist stimulations with either the CXCL12 or the respective selective GPCRx agonist (5 μM of PGE2). Accordingly, from these observations, those systems co-expressing CXCR4 and the GPCRx selected from the group consisting of ADCYAP1R1 (FIG.6A), ADORA2B (FIG.6B), ADORA3 (FIG.6C), C5AR1 (FIG.6D), CALCR (FIG.6E), CHRM1 (FIG.6F), EDNRB (FIG.6G), HRH1 (FIG.6H), MLNR (FIG.6I), NTSR1 (FIG.6J), and TACR3 (FIG.6L), are considered to have: (i) exhibited an enhanced calcium mobilization upon co-stimulation (or co-treatment) with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined by the calcium mobilization assay; (ii) exhibited a calcium mobilization amount equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist in an individual protomer context; and thus (iii) constituted CXCR4-GPCRx heteromers exhibiting properties distinct from those of the individual GPCR. The enhanced calcium responses were not observed in cells expressing individual GPCRs, thereby demonstrating that these CXCR4-GPCRx heteromers exhibit properties distinct from those of the individual GPCR. The enhanced calcium signaling induced by small amounts of individual agonists together clearly indicated that CXCR4-GPCRx heteromers would increase the sensitivity and dynamic range of these receptors at limiting ligand concentrations in vivo, and may be associated with poor prognosis. The enhanced calcium signaling by the CXCR4-GPCRx heteromers also indicates that the current diagnosis based on the level of CXCR4 expression alone needs to be changed to consider the expression levels of CXCR4 and GPCRx. In contrast, GPCRs such as APLNR (FIG.7A), CCR5 (FIG.7B), GALR1 (FIG.7C), PTGER3 (FIG.7D), and SSTR2 (FIG.7E) did not show enhanced calcium responses when cells expressing both receptors were co-treated with both agonists, although these GPCRs were shown to interact with CXCR4 in BiFC (Table 5) and CXCR4-GFP co-internalization assay. These results clearly demonstrate that the synergistic increase in calcium response shown inFIGS.5A-5D and6A-6Lis a unique feature of CXCR4-GPCRx heteromers, that is not shared by other CXCR4-GPCRx heteromers such as CXCR4-APLNR, CXCR4-CCR5, CXCR4-GALR1, CXCR4-PTGER3, and CXCR4-SSTR2. TABLE 5GPCRs that exhibited BiFC score equalor higher than 10 when co-expressed withCXCR4 but did not show enhanced Ca2+signaling upon co-treatment of both agonists.CXCR4-GPCRx heteromers that do not showenhanced Ca2+signaling upon co-treatment of bothagonistsBiFC scoreCXCR4-VN&GPCRxGPCRx-VC1APLNR632CCR5443GALR1604PTGER3*135SSTR230*CXCR4-VC and GPCRx-VN were used. It was further examined if enhanced calcium responses in cells co-expressing CXCR4 and GPCRx upon co-stimulation with CXCL12 and GPCRx ligands are inhibited by GPCRx antagonists. In cells co-expressing CXCR4 and either ADRB2 (FIG.8A), CHRM1 (FIG.8B&FIG.8C), HRH1 (FIGS.8F-8I), MLNR (FIG.8J), or NTSR1 (FIG.8K), treatment of 10 μM of ADRB2 antagonist carvedilol (FIG.8A), 1 μM of CHRM1-selective antagonist VU0255035 (FIG.8B), 10 μM of CHRM1 antagonist oxybutynin (FIG.8C), 1 μM of HRH1-selective antagonist cetirizine (FIG.8F), 1 μM of HRH1-selective antagonist pyrilamine (FIG.8G), 10 μM of HRH1 antagonist hydroxyzine (FIG.8H), 10 μM of HRH1-selective antagonist loratadine (FIG.8I), 1 μM of MLNR-selective antagonist MA-2029 (FIG.8J) and 1 μM of NTSR1-selective antagonist meclinertant (FIG.8K), respectively, suppressed the enhanced calcium signaling significantly. And co-treatment of both antagonists together resulted in a more complete suppression (FIGS.8A-8C, and8F-8K). These results demonstrated that GPCRx antagonists could be used as an efficient therapeutics against CXCR4-GPCRx heteromers where GPCRx represents ADRB2, CHRM1, HRH1, MLNR, and NTSR1. In cells co-expressing CXCR4 and CHRM1, 10 μM of muscarinic acetylcholine receptor antagonist umeclidinium reduced the enhanced calcium signaling, although not statistically significant (FIG.8D). In these cells, co-treatment of AMD3100 and umeclidinium almost completely inhibited calcium response induced by simultaneous addition of CXCL12 and bethanechol. In cells co-expressing CXCR4 and either EDNRB (FIG.8E) or TACR3 (FIG.8L), treatment of either 1 μM of AMD3100 alone (FIG.8EandFIG.8L), or 1 μM of endothelin receptor antagonist bosentan (FIG.8E) or 1 μM of TACR3-selective antagonist SSR 146977 alone (FIG.8L) failed to inhibit enhanced calcium response significantly. But when the cells were co-treated with AMD3100 and bosentan (FIG.8E) or AMD3100 and SSR 146977 together (FIG.8L), the enhanced calcium responses were significantly inhibited. In cells co-expressing CXCR4 and either CHRM1 (FIG.8B), EDNRB (FIG.8E), HRH1 (FIGS.8F and8G), or MLNR (FIG.8J), although 1 μM of AMD3100 alone failed to inhibit enhanced calcium signaling, co-treatment of 1 μM of both antagonists together significantly suppressed the enhanced calcium signaling. These results clearly demonstrate that co-treatment of small doses of antagonists targeting each protomer provides novel therapeutic tools to efficiently suppress CXCR4-GPCRx heteromer response while avoiding side effects associated with high doses of individual antagonists. Example 4. Inhibition of Internalization by GPCRx Antagonists To further study if co-internalization of CXCR4 heterodimer was blocked by partner GPCRx antagonists, internalization inhibition assay was performed. As shown inFIGS.4B-4Q, CXCR4-GFP expressing U-2 OS cells were co-internalized by partner GPCRx specific agonist when CXCR4 and GPCRx were simultaneously transduced to cells (control: CXCR4-GFP (FIG.4A)). If CXCR4 formed heterodimer with GPCRx and co-internalized by partner GPCRx, it can be blocked by GPCRx specific antagonist. U-2 OS cells stably expressing CXCR4-GFP were transduced with Adenoviruses encoding GPCRx (ADRB2, CHRM1, HRH1). After 2 days, images were obtained before and 20 minutes after stimulating cells with CXCR4-specific agonist, CXCL12 (SDF-1) (20 nM), and/or GPCRx-specific antagonist (10 μM). Internalization of CXCR4-GFP was observed as GFP granules using IN Cell Analyzer 2500. Loss of GFP expression on the cell surface or appearance of GFP granules inside the cells were considered as CXCR4-GFP co-internalization. CXCR4 agonist, CXCL12 induced internalization of CXCR4-GFP with GPCRx (FIGS.10A-C, first columns). GPCRx antagonists, treated as follows, had no effect on the internalization of the heteromer (FIGS.10A-C, second columns): Carvedilol, an ADRB2 antagonist (FIG.10A); Oxybutynin and Umeclidinium, CHRM1 antagonists (FIG.10B); Prometazine, Hydroxyzine, and Loratadine, HRH1 antagonists (FIG.10C). Internalization of CXCL12 stimulated CXCR4-GFP with GPCRx were inhibited by GPCRx specific antagonists (FIGS.10A-C, third columns). These data demonstrate that CXCR4-GPCRx co-internalization was heteromer specific event and CXCR4 and GPCRx formed heterodimer. These data further demonstrate that, by inhibition of internalization of CXCR4 heteromer, abnormal downstream signal in CXCR4-GPCRx heteromer overexpressed cells, such as cancers, can be blocked for therapeutics purposes. Example 5. Evaluation by Cell Proliferation Assay of Phenotypic Effects of Inhibitor of CXCR4-GPCRx Heteromer Signaling on Tumor Growth Several CXCR4 antagonists have been developed, but none have been approved as anticancer drugs until now. To overcome the limitation of CXCR4 inhibitors and to develop CXCR4 heteromer-based therapeutic agent, we tested the effect of GPCRx antagonist on cell proliferation. We prepared single cell suspensions of resected and dissociated glioblastoma tissues from patients. (provided by Samsung Seoul hospital in Seoul, Korea) These cells were cultured under conditions optimal for propagation and non-differentiation of normal neuronal stem cell. Media were composed serum free Neurobasal media supplemented with basic FGF and EGF. The effect of GPCRx antagonist on the survival of patient derived cells (PDCs) were assessed using ATPlite (PerkinElmer, Cat. No. 6016739 reagent. ATPlite is an Adenosine Triphospate monitoring system based on firefly luciferase. This luminescence assay is the alternative to colorimetric, fluorometric and radioisotopic assays for the quantitative evaluation of proliferation and cytotoxicity of cultured mammalian cells. Cells were seeded in 384-well plate at 500 cells/well in 40 μl culture media. After overnight growth, the cells were cultured for 7 days in the presence of several dose of GPCRx antagonist or DMSO alone. After the 7-day incubation, 15 μl ATPlite was added into the each well and the plate were shaken for 5 minutes in an orbital shaker at 700 rpm. The luminescent signal was detected within 30 minutes at PerkinElmer TopCount detection instrument. The cell viability was calculated using the equation: Cell viability(%)=(OD of antagonist treatment/OD of DMSO only treatment)×100%. As shown inFIGS.11A-C, when Carvedilol, ADRB2 specific antagonist, was treated in PDC expressing CXCR4 and ADRB2, growth of cells was inhibited significantly. (IC50=11.69 μM,FIG.11A). Oxybutynin and Umeclidinium, CHRM1 antagonists also inhibited on the survival of PDc. Oxybutynin or Umeclidinium each showed significantly decreased survival of cells at IC50=3.04 μM or 4.03 μM, respectively. (FIG.11B). Promethazine, Hydroxyzine, and or Loratadine as a HRH1 antagonist each showed decreased survival of PDc at IC50=18.39 uM, 12.79 μM or 5.29 μM, respectively. (FIG.11C). These results demonstrate that CXCR4 heteromer induced abnormal cell proliferation can be blocked by partner GPCRx specific antagonist in CXCR4-GPCRx heteromer expressing cells, indicating that inhibition of cancer cell growth using partner GPCRx antagonist in CXCR4-GPCRx heteromer bearing patients can overcome the limitations of CXCR4 inhibitors alone as cancer therapeutics. Example 6. Evaluation of CXCR4-GPCRx Heteromer Formation in Patient Derived Cells (PDC) Using Proximity Ligation Assay (PLA) To investigate the existence of GPCR complexes in native tissues, various approaches such as atomic force microscopy (Fotiadis et al., 2006), co-immunoprecipitation (Gomes et al., 2004) and binding or functional assays (Wreggett and Wells, 1995) have been used. The most convenient methods to monitor interactions are based on resonance energy transfer performed with labeled proteins. The labeling can be performed by selective probes such as antibodies or fluorescent ligands (Roess et al., 2000; Patel et al., 2002). Bazin et al. employed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based approach that offers a much higher signal-to-noise ratio (Bazin et al., 2002). FRET is based on the transfer of energy between two fluorophores, a donor and an acceptor, when in close proximity. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and by detecting the level of energy transfer. Introducing a time delay of approximately 50 to 150 μseconds between the system excitation and fluorescence measurement allows the signal to be cleared of all non-specific short-lived emissions. Proximity ligation assay (PLA) is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions and modifications with high specificity and sensitivity (Gullberg et al., 2004). Two primary antibodies raised in different species recognize the target antigen on the proteins of interest. Secondary antibodies directed against the constant regions of the different primary antibodies, called PLA probes, bind to the primary antibodies. Each of the PLA probes has a unique short DNA strand attached to it, If the PLA probes are in close proximity (that is, if the two original proteins of interest are in close proximity, or part of a protein complex, as shown in the figures), the DNA strands can participate in rolling circle DNA synthesis when appropriate substrates and enzymes are added. The DNA synthesis reaction results in several-hundred fold amplification of the DNA circle. Next, fluorescent-labeled complementary oligonucleotide probes are added, and they bind to the amplified DNA. The resulting high concentration of fluorescence is easily visible as a distinct bright spot when viewed with a fluorescence microscope (Gustafsdottir et al., 2005). CXCR4 overexpressing cell line, U2OS-CXCR4, was infected with ADRB2 expressing adenovirus, Ad-ADRB2 at the dose of 0, 2.5, 10, 40 MOIs for 2 days. PLA was performed as described previously (Brueggemann et al., 2014; Tripathi et al., 2014). To perform PLA, infected cells were fixed with 4% paraformaldehyde (PFA) on sixteen-well tissue culture slides. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santacruz, Sc-53534), Rabbit anti-ADRB2 (1:200, Thermoscientific, PA5-33333), Rabbit anti-CHRM1 (1:200, Ls bio, Ls-C313301) at 37° C. for 1 h in a humidifying chamber. Slides were then washed and incubated (1 h at 37° C.) with secondary anti-rabbit and anti-mouse antibodies conjugated with plus and minus Duolink II PLA probes. Slides were washed again and then incubated with ligation-ligase solution (30 min at 37° C.) followed by incubation with amplification-polymerase solution (2 h at 37° C.). Slides were then mounted with minimal volume of Duolink II mounting medium with 4′,6-diamidino-2phenylindole (DAPI) for 15-30 min, and PLA signals [Duolink In Situ Detection Reagents Green (λ excitation/emission 495/527 nm) or Red (λ excitation/emission 575/623 nm)] were identified as fluorescent spots under a IN Cell analyzer 2500. As shown in theFIGS.12A-12B, the PLA signal increases in a dose dependent manner as the expression level of ADRB2.FIG.12A: Images of PLA signal from U2OS cells expressing CXCR4-ADRB2 heteromer over a series of MOIs (multiplicity of infection).FIG.12B: The red signal spots were counted and calculated by normalization against negative control. The PLA signal increased proportionate to the expression level of ADRB2 in a dose dependent manner.FIG.12C: To investigate endogenous ADRB2 expression, qRT-PCR was performed with ADRB2 specific primers. As the results show, endogenous ADRB2 expression level of U2OS cell was quite high, indicating that the PLA signal is detected even in the section without virus infection (at 0 MOI for ADRB2). Traditionally Glioblastoma (GBM) is the most common and lethal primary brain tumor. Preclinical cancer biology has largely relied on the use of human cancer cell lines in vitro and the xenograft process of established these cell lines. However, the process of establishing conventional cell lines results in irreversible loss of important biological properties and, as a result, the xenograft tumor models do not maintain genomic and phenotypic characteristics present in the original tumor. Patient derived cell (PDC) derived directly from glioblastoma harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, and in vivo biology of human glioblastomas. To perform PLA with PDC samples, the patient derived cells were plated and fixed with 4% PFA on sixteen-well tissue culture slides. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santa Cruz, Sc-53534), rabbit anti-ADRB2 (1:200, Thermo Scientific, PA5-33333), rabbit anti-CHRM1 (1:200, Lsbio, Ls-C313301) at 37° C. for 1 h in a humidifying chamber. Slides were then washed and incubated (1 h at 37° C.) with secondary anti-rabbit and anti-mouse antibodies conjugated with plus and minus Duolink II PLA probes. Slides were washed again and then incubated with ligation-ligase solution (30 min at 37° C.) followed by incubation with amplification-polymerase solution (2 h at 37° C.). Slides were then mounted with minimal volume of Duolink II mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI) for 15-30 min, and PLA signals [Duolink In Situ Detection Reagents Green (λ excitation/emission 495/527 nm) or Red (λ excitation/emission 575/623 nm)] were identified as fluorescent spots under the IN Cell analyzer 2500. As shown inFIGS.13A-13BandFIGS.14A-14B, PLA ratio refers to the CXCR4 GPCRx heteromer, and the frequency of the heteromer formation varies depending on the patient. PLA ratio was calculated as: number of fluorescent spots in PDC sample/number of fluorescent spots in negative control. Negative control (NC) represents background fluorescence signal, indicated by the number of spots when only the secondary antibody conjugated with plus and minus Duolink II PLA probes is treated without primary antibody (mouse anti-CXCR4, rabbit anti-ADRB2, rabbit anti-CHRM1) treatment in PLA processing. These data demonstrate quantitative analysis of CXCR4-ADRB2 heterodimer in cancer patient samples. Example 7. Evaluation of CXCR4-GPCRx Heteromer Formation In Vivo Using PDX Model To perform PLA with PDX samples, the glioblastoma patient derived FFPE samples were used (provided by Samsung Seoul hospital in Seoul, Korea). After FFPE sample were de-paraffinized and performed heat induced antigen retrieval for 15 minutes at 100° C. Slides were blocked with blocking solution provided by Duolink and incubated with rabbit anti-CXCR4 (1:200, Thermoscientific, PA3305), mouse anti-ADRB2 (1:200, Santacruz, Sc-271322), at 37° C. for 1 h in a humidifying chamber. The other process was same as described above (PLA with PDC). In theFIG.15A, nuclei were visualized with DAPI staining, and CXCR4-ADRB4 heteromers were stained with PLA as small dots. As shown inFIG.15B, PLA ratio is different according to the patient and based on this result, indicating that it is possible to perform personalized medicine by the companion diagnostics. Example 8. Evaluation by Ca2+ Mobilization Assay of Enhanced CXCR4 Downstream Signaling Upon CXCR4-GPCRx Heteromer Formation In cells overexpressing both CXCR4 and ADRB2, stimulation with salmeterol alone did not elicit calcium mobilization dose-dependently (FIG.16A). But when the cells were co-stimulated with salmeterol in the presence of CXCL12, calcium signaling was greatly enhanced in a broad range of salmeterol concentrations, such as concentrations in the range of between 10 nM to 300 nM. Similarly, co-treatment with histamine and CXCL12 also significantly enhanced calcium responses in cells overexpressing both CXCR4 and HRH1 over a broad range of histamine concentrations (0.3 nM to 300 nM), even at histamine concentrations that did not evoke any calcium responses when treated alone (0.3 nM and 1 nM) (FIG.16B). Considering that the concentration of histamine in human plasma and glomeruli is below 10 nM and 2 μM, respectively (Sedor and Abboud, 1984), this result indicates that enhanced calcium signaling by CXCR4-HRH1 heteromer can occur at physiological levels of histamine concentration in vivo. In cells expressing CXCR4-APLNR (FIG.17A), CXCR4-PTGER3 (FIG.17B), or CXCR4-SSTR2 (FIG.17C), stimulation with GPCRx agonist (Apelin-13, PGE2, or octreotide, respectively) alone increased calcium signal dose-dependently. However, unlike in cells expressing CXCR4-ADRB2 or CXCR4-HRH1, calcium signal was not enhanced any further upon co-stimulation with both agonists (CXCL12 and GPCRx agonist) in any dose ranges tested in cells expressing CXCR4-APLNR, CXCR4-PTGER3, or CXCR4-SSTR2. These results are consistent with the results shown inFIGS.7A,7D,7E, respectively, and show that the lack of signal enhancement in cells expressing CXCR4-APLNR or CXCR4-SSTR2 upon co-stimulation was not due to the use of a specific dose of GPCRx agonist, but due to the intrinsic nature of these heteromers. These results also clearly demonstrate that the enhanced calcium responses shown inFIG.6A-6LandFIGS.16A-16Bare unique features of CXCR4-GPCRx heteromers such as CXCR4-ADRB2 or CXCR4-HRH1 described inFIGS.5C-5DandFIG.6H, respectively. Example 9. Evaluation of Enhanced Ca2+ Mobilization Upon Co-Stimulation of Endogenous CXCR4-HRH1 Heteromer in MDA-MB-231 Cells Using RT-qPCR, the endogenous expression levels of CXCR4 and HRH1 in MDA-MB-231 cells were measured. The threshold cycles (Ct) of CXCR4 and HRH1 were 28.5 and 27.7, respectively, when 12.5 ng of total RNA was analyzed. Stimulation of MDA-MB-231 cells with either CXCL12 (100 nM), or histamine (10 nM) alone, elicit weak calcium responses (FIG.18A). But when the cells were co-stimulated with CXCL12 and histamine together, greatly enhanced calcium response was observed similar to the enhancement observed in cells overexpressing CXCR4 and HRH1 together (FIGS.18A and18B, compared withFIG.16B). The result clearly indicates that the enhanced calcium response upon co-stimulation with both agonists could occur in native cells expressing both CXCR4 and HRH1 together. Example 10. Evaluation of Enhanced Migration of Cancer Cells by CXCR4-HRH1 Heteromer Upon Co-Stimulation with Both Ligands Since MDA-MB-231 cells express about twice more HRH1 mRNA compared with CXCR4 mRNA when measured by RT-qPCR, the MDA-MB-231 cells were transduced with small amount of lentivirus encoding CXCR4 (1 MOI) and chemotactic migration of the cells toward CXCL12 and HRH1 was measured (FIGS.19A and19B). Histamine (50 nM) alone, sufficient to produce calcium signaling as shown inFIG.27A(see below), did not induce cell migration. On the other hand, when treated together with CXCL12, histamine significantly enhanced the migration of MDA-MB-231 cells towards CXCL12. However, in the presence of pyrilamine, a RH-selective inverse agonist, co-stimulation of the cells with CXCL12 and histamine failed to enhance the cell migration elicited by CXCL12. The results clearly demonstrate that the enhancement of observed cancer cell migration was specifically induced by HRH1, and not by other histamine receptor subtypes. Example 11. Evaluation by Ca2+ Mobilization Assay of Enhanced CXCR4 Downstream Signaling Upon CXCR4-GPCRx Heteromer Formation and Inhibition of the Enhanced Signaling In cells overexpressing both CXCR4 and ADRB2, co-treatment of both agonists CXCL12, a CXCR4 agonist and salmeterol, an ADRB2-selective agonist significantly increased the calcium response compared to the ones evoked by individual agonists (FIG.20). These results clearly demonstrate that CXCR4-ADRB2 heteromer exhibit properties distinct from those of the individual GPCRs. It was examined if enhanced calcium responses in cells co-expressing CXCR4 and ADRB2 upon co-stimulation with CXCL12 and ADRB2 ligands are inhibited by anti-CXCR4 antibody as a CXCR4 antagonist. In cells co-expressing CXCR4 and ADRB2, treatment of 2 μg of anti-CXCR4 antibody, 12G5, suppressed the enhanced calcium signaling as shown inFIG.20. And co-treatment of both antagonists (Carvedilol, ADRB2 antagonist and 12G5, CXCR4 antagonist together) resulted in a more significant suppression. These results demonstrated that anti-CXCR4 antibody and ADRB2 antagonist could be used as an efficient therapeutics against CXCR4-ADRB2 heteromer. Utilizing the Calcium mobilization assay, MDA-MB-231 human breast cancer cells were seeded at 20,000 cells per well in a black clear bottom 96-well plate (Corning Costar, #3340) in 100 μL of RPMI 1640 supplemented with 10% FBS. The next day, cells were co-transduced with 10 MOI of CXCR4 and 30 MOI of GPCRx. After 2 days, cells were treated with ADRB2 antagonist, Carvedilol(Tocris), anti-CXCR4 antibody, 12G5 (Thermo Scientific, 35-8800) with indicated amounts and incubated with Cal 6 (FLIPR® Calcium 6 Assay Kit by Molecular Devices, Cat. R8191) for 2 hr. And then, cells were stimulated with indicated amounts of CXCL12, ADRB2 agonist, or CXCL12 and ADRB2 agonist. Calcium mobilization was measured using FlexStation 3 Multi-Mode Microplate Reader. The results were normalized for base-line activity. Calcium mobilization was quantified by calculating the area-under-the-curve (AUC) of each graph. Data were normalized to CXCL12-stimulated calcium response in cells expressing CXCR4 alone. Data represent three independent experiments (mean±SEM). *P<0.05; Student's t test. Example 12. Evaluation of CXCR4-GPCRx Heteromer Formation in CXCR4-GPCRx Expressing Cell Using PLA A process for screening a CXCR4-GPCRx heteromer is essential for the treatment of CXCR4-GPCRx targeted anticancer drug. First, for the quantitative detection of CXCR4 and GPCRx heteromer, PLA was performed in CXCR4-GPCRx expressing cells. CXCR4 overexpressing cell line, the U2OS-CXCR4 was infected with GPCRx expressing adenovirus, Ad-GPCRx at the dose of 0, 2.5, 10, 40 MOIs for 2 days. And then, the transduced cells were fixed with 4% paraformaldehyde and performed PLA was performed. The number of PLA signals means quantitatively demonstrating the formation of CXCR4-GPCRx heteromerization. As shown in theFIGS.21A-21B, the PLA signal increased proportionate to the expression level of CHRM1 (FIG.21A) and HRH1 (FIG.21B) in a dose dependent manner. These results demonstrate detection of different types of CXCR-GPCRx heteromer and quantitative analysis of CXCR4-GPCRx heteromer in cancer patient samples. Utilizing the PLA in cells, CXCR4 overexpression cell lines, the U2OS-CXCR4 was infected with GPCRx expressing adenovirus, Ad-GPCRx at the dose of 0, 2.5, 10, 40 MOIs for 2 days. PLA was performed as described previously (Brueggemann et al., 2014; Tripathi et al., 2014). To perform PLA, infected cells were fixed with 4% PFA on sixteen-well tissue culture slides. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (Santacruz, Sc-53534), rabbit anti-CHRM1 (LS Bio, LS-C313301), or rabbit anti-HRH1 (Thermoscientific, PA5-27817) at 37° C. for 1 h in a humidifying chamber. Slides were then washed and incubated (1 h at 37° C.) with secondary anti-rabbit and anti-mouse antibodies conjugated with plus and minus Duolink II PLA probes. Slides were washed again and then incubated with ligation-ligase solution (30 min at 37° C.) followed by incubation with amplification-polymerase solution (2 h at 37° C.). Slides were then mounted with minimal volume of Duolink II mounting medium with 4′,6-diamidino-2phenylindole (DAPI) for 15-30 min, and PLA signals [Duolink In Situ Detection Reagents Green (λ excitation/emission 495/527 nm) or Red (λ excitation/emission 575/623 nm)] were identified as fluorescent spots under a IN Cell analyzer 2500. Example 13. Ca2+ Mobilization Inhibition of the Enhanced CXCR4 Downstream Signaling Upon CXCR4-GPCRx Heteromer Formation—Comparison of Single Inhibitor Treatment to Combination Inhibitor Treatment The data shown above (see Example 3,FIG.8A) demonstrated that an increased Ca2+ signal due to CXCR4-ADRB2 heteromer formation was significantly decreased when treated simultaneously with a CXCR4 inhibitor and an ADRB2 inhibitor. The degree of inhibition (measured as IC50 values for Ca2+response) was compared for a series of CXCR4 inhibitors, evaluated as single treatment in cells expressing CXCR4 alone (individual protomer context), as single treatment in CXCR4-ADRB2 heteromer-expressing cells, and as co-treatment with an ADRB2 inhibitor (Carvedilol; 10 μM) in CXCR4-ADRB2 heteromer-expressing cells. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 only, or CXCR4 and ADRB. The cells were cultured for 2 days and were treated CXCR4 inhibitor alone or co-treated with a CXCR4 inhibitor and an ADRB2 inhibitor (Carvedilol; 10 uM). The cells were then stained with Cal-6 for 2 hours and stimulated with CXCR4 agonsit (CXCL12; 20 nM) and ADRB2 agonist (Salmeterol; 1 μM). Calcium mobilization was measured using FlexStation3, the results of which are shown in Table 6, showing IC50 of Ca2+response in: (1) MDA-MB-231 cells expressing CXCR4 alone (individual protomer context), treated with CXCR4 inhibitor only (2ndcolumn); (2) MDA-MB-231 cells expressing CXCR4-ADRB2 heteromer, treated simultaneously with CXCR4 inhibitor and ADRB2 inhibitor Carvedilol (3rdcolumn); and (3) MDA-MB-231 cells expressing CXCR4-ADRB2 heteromer, treated only with CXCR4 inhibitor (4thcolumn). TABLE 6CXCR4 + ADRB2CXCR4 + ADRB2CXCR4CXCR4(+Carvedilol)(−Carvedilol)inhibitorIC50[nM]IC50[nM]IC50[nM]AMD310057.80 ± 20.360.053 ± 0.0728.65 ± 13.23AMD07043.37 ± 0.571.40 ± 2.224.69 ± 8.8812G580.19 ± 48.52.784 ± 0.8613.95 ± 10.04Ulocuplumab0.42 ± 0.090.0008 ± 0.00021.12 ± 0.56BKT140434 ± 4.380.81 ± 0.41130.40 ± 49.78TZ1401123.93 ± 3.580.02 ± 0.0217.78 ± 7.84 As shown in Table 6, the IC50 value of Ca2+response was CXCR4 inhibitor dependent in the context of single treatment of MDA-MB-231 cells expressing CXCR4 alone, and in the single treatment context and the co-treatment context with an ADRB2 inhibitor of MDA-MB-231 cells expressing CXCR4-ADRB2 heteromer. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca2+response in the CXCR4-ADRB2 heteromer context decreased by 1400 times or more when treated in combination with an ADRB2 inhibitor (3rdcolumn), relative to single treatment with the CXCR4 inhibitor alone (4thcolumn). For instance, the IC50 value of Ca2+response in the CXCR4-ADRB2 heteromer context decreased about 540 fold (from 28.65 nM to 0.053 nM), about 1400 fold (from 1.12 nM to 0.0008 nM), or about 890 fold (from 17.78 nM to 0.02 nM), upon co-treatment of Carvedilol with AMD3100, Ulocuplumab, or TZ14011, relative to single treatment with AMD3100, Ulocuplumab, or TZ14011, respectively. These results suggest that co-treating with a CXCR4 inhibitor and ADRB2 inhibitor more effectively inhibits increased Ca2+response than single treatment with a CXCR4 inhibitor alone in CXCR4-ADRB2 heteromer expressing cells. To determine the possibility that CXCR4-ADRB2 heteromer formation induces conformation change and/or changes the binding affinity for the CXCR4 inhibitor, the IC50 values of Ca2+response were compared between single treatment with a CXCR4 inhibitor of cells expressing CXCR4 alone and cells expressing CXCR4-ADRB2 heteromer. The results showed that single treatment with a CXCR4 inhibitor altered the IC50 value only by about 0.4-5 fold in cells expressing CXCR4-ADRB2 heteromer (4thcolumn), relative to cells expressing CXCR4 alone (2ndcolumn). These results suggest that co-treatment with CXCR4 inhibitor and ADRB2 inhibitor can increase, to a dramatic extent for certain CXCR4 inhibitors, the therapeutic efficacy towards CXCR4-ADRB2 heteromer expressing patients and/or patient cells/tissues, compared with single treatment with CXCR4 inhibitor. Example 14. Effect of CXCR4-ADRB2 Heteromer on Tumor Growth To investigate the effect of CXCR4-ADRB2 heteromer on tumor growth, cell lines stably overexpressing CXCR4 alone or CXCR4-ADRB2 heteromer were prepared in A549 lung cancer cell, and the same amount of cells (1×107cell/head) were injected subcutaneously in nude mice to compare tumor growth rates. As shown inFIGS.22A-22B, the tumor size at 28 days after transplantation was 351.4±214.7 mm3for A549, 726.9±259.6 mm3for A549-CXCR4, and 1012.2±556.1 mm3for A549-CXCR4-ADRB2. The tumor growth rate of mice transplanted with the A549-CXCR4 overexpressing CXCR4 was faster than that of mice bearing only parental A549, and the fastest tumor growth was observed in mice transplanted with cells overexpressing the CXCR4-ADRB2 heteromer. These results suggest that the formation of CXCR4-ADRB2 heteromer synergistically increases the Ca2+response and thus promotes tumor growth. Images of three mice which were transplanted with either parental cell A549, A549-CXCR4 stably overexpressing CXCR4, or A549-CXCR4-ADRB2 stably overexpressing CXCR4-ADRB2 heteromer, at 28 days after transplantation, are shown inFIG.22A. These images show that the tumor size of the mouse bearing the cell expressing CXCR4-ADRB2 heteromer is the largest (accelerated the most) among them. The tumor growth rates of these three different mice over time are graphically illustrated inFIG.22B. Tumor growth was monitored every third or fourth day by measuring the length (L) and width (W) of the tumor and calculating tumor volume on the basis of the following formula: Volume=0.5 LW2. The mice bearing CXCR4-expressing cell show relatively fast tumor growth as compared to the mice bearing parental cell A549, and the tumor growth is the fastest in mice transplanted with cells overexpressing the CXCR4-ADRB2 heteromer. Example 15. Evaluation by Ca2+ Mobilization Assay of Enhanced CXCR4 Downstream Signaling Upon Co-Stimulation of CXCR4-GPCRx Heteromer with Endogenous Ligands The enhancement of the calcium response in MDA-MB-231 cells co-expressing CXCR4 and GPCRx upon co-stimulation with CXCL12 and endogenous ligand for GPCRx, are shown inFIGS.23A-23G. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and HA-VC, GPCRx and HA-VC, or CXCR4 and GPCRx where GPCRx represents ADCYAP1R1 (FIG.23A), ADORA2B (FIG.23B), ADORA3 (FIG.23C), CHRM1 (FIG.23D), EDNRB (FIG.23E), MLNR (FIG.23F), and TACR3 (FIG.23G). Cells were treated with CXCL12 alone, GPCRx endogenous ligand alone, or CXCL12 and GPCRx ligand together. Calcium mobilization was calculated as described inFIG.5D. Statistically significant difference between the sum (square with dots) and co-treatment (filled square) was determined by Student's t test. *P<0.05; **P<0.01; ***P<0.001; Mean±SEM (n=3). InFIG.6A, enhanced Ca2+signaling was observed in cells expressing CXCR4 and ADCYAP1R1 together, but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and VIP, a non-selective endogenous ADCYAP1R1 ligand. When the cells were co-treated with CXCL12 and PACAP-38, an ADCYAP1R1-selective endogenous ligand, enhanced Ca2+signaling was also observed (FIG.23A). The result demonstrates that enhanced signaling of CXCR4-ADCYAP1R1 heteromer upon co-stimulation with CXCL12 is not only limited to VIP, but also selective ADCYAP1R1 natural ligand. It further suggests this enhancement could happen in vivo in cells expressing CXCR4 and ADCYAP1R1 together by their natural ligands. Enhanced Ca2+signaling was observed in cells expressing CXCR4 and ADORA2B (FIG.6B) or CXCR4 and ADORA3 together (FIG.6C), but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and BAY 60-6583 (ADORA2B-selective agonist), or CXCL12 and Cl-IB-MECA (ADORA3-selective agonist), respectively. When these cells were co-treated with CXCL12 and adenosine, endogenous ligand for all adenosine receptors, enhanced Ca2+signaling was also observed (FIGS.23B and23C). The result demonstrates that natural endogenous ligand, not only synthetic agonists, enhances downstream signaling of CXCR4-ADORA2B and CXCR4-ADORA3 heteromers upon co-stimulation with CXCL12. It further suggests that synergistic enhancement of downstream signaling could happen in vivo in cells expressing CXCR4 and ADORA2B or CXCR4 and ADORA3 together by their natural ligands. InFIG.6F, enhanced Ca2+signaling was observed in cells expressing CXCR4 and CHRM1 together, but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and bethanechol, a synthetic CHRM1 agonist. When the cells were co-treated with CXCL12 and acetylcholine, endogenous ligand for all acetylcholine receptors, enhanced Ca2+signaling was also observed (FIG.23D). The result demonstrates that natural endogenous ligand, not only synthetic agonist, enhances signaling of CXCR4-CHRM1 heteromer upon co-stimulation with CXCL12. It further suggests that synergistic enhancement of downstream signaling could happen in vivo in cells expressing CXCR4 and CHRM1 together by their natural ligands. InFIG.6G, enhanced Ca2+signaling was observed in cells expressing CXCR4 and EDNRB together, but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and BQ3020, an EDNRB-selective agonist. When the cells were co-treated with CXCL12 and endothelin-1, endogenous ligand for endothelin receptors, enhanced Ca2+signaling was also observed (FIG.23E). The result demonstrates that natural endogenous ligand, not only synthetic agonist, enhances signaling of CXCR4-EDNRB heteromer upon co-stimulation with CXCL12. It further suggests that synergistic enhancement of downstream signaling could happen in vivo in cells expressing CXCR4 and EDNRB together by their natural ligands. InFIG.6I, enhanced Ca2+signaling was observed in cells expressing CXCR4 and MLNR together, but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and roxithromycin, an antibiotic as well as full MLNR agonist. When the cells were co-treated with CXCL12 and motilin, a selective endogenous ligand for MLNR, enhanced Ca2+signaling was also observed (FIG.23F). The result demonstrates that natural endogenous ligand, not only synthetic agonist, enhances signaling of CXCR4-MLNR heteromer upon co-stimulation with CXCL12. It further suggests that synergistic enhancement of downstream signaling could happen in vivo in cells expressing CXCR4 and MLNR together by their natural ligands. InFIG.6L, enhanced Ca2+signaling was observed in cells expressing CXCR4 and TACR3 together, but not in cells expressing individual GPCR alone, upon co-treatment with CXCL12 and senktide, a TACR3-selective agonist. When the cells were co-treated with CXCL12 and neurokinin B, selective endogenous ligand for TACR3, enhanced Ca2+signaling was also observed (FIG.23G). The result demonstrates that natural endogenous ligand, not only synthetic agonist, enhances signaling of CXCR4-TACR3 heteromer upon co-stimulation with CXCL12. It further suggests that synergistic enhancement of downstream signaling could happen in vivo in cells expressing CXCR4 and EDNRB together by their natural ligands. Example 16. Evaluation by Ca2+ Mobilization Assay of Enhanced CXCR4 Downstream Signaling Upon Co-Stimulation of CXCR4-GPCRx Heteromer In MDA-MB-231 cells co-expressing CXCR4 and ADCYAP1R1, stimulation with selective endogenous ligands CXCL12 alone or PACAP38 alone, respectively, produced calcium signaling dose-dependently (FIGS.24A-24B). MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and ADCYAP1R1. As shown inFIG.24A, addition of small amount of ADCYAP1R1 selective endogenous ligand (PACAP38; 1 nM) significantly enhanced calcium response in a broad range of CXCL12 concentrations compared with the sum values calculated by adding the responses obtained by PACAP38 alone and CXCL12 alone at indicated doses (FIG.24A). Maximal Ca2+response evoked by CXCL12 alone was taken as 100%. Sum represents the calculated additive value of the responses evoked by 1 nM of PACAP38 and CXCL12 alone at indicated doses. Similarly, as shown inFIG.24B, addition of CXCR4 selective endogenous ligand (CXCL12; 15 nM) significantly enhanced downstream response in a broad range of PACAP38 concentrations compared with the sum values calculated by adding the responses obtained by CXCL12 alone and PACAP38 alone at indicated doses, even at concentrations that did not evoke any response when treated alone (0.03˜0.3 nM) (FIG.24B). Maximal Ca2+response evoked by PACAP38 alone was taken as 100%. Sum represents the calculated additive value of the responses evoked by 15 nM of CXCL12 alone and PACAP38 alone at indicated doses. Statistically significant differences between the sum and co-treatment at each point were determined by Student's t test. *P<0.05; **P<0.01; ***P<0.001; Mean±SD (n=3). These results suggest an enhanced response in cells co-expressing CXCR4 and ADCYAP1R1 in vivo in the presence of small amounts of CXCL12 and ADCYAP1R1 ligands together. The enhancement of calcium response in a broad range of ligand concentrations in cells co-expressing CXCR4 and TACR3 is shown inFIGS.25A-25B. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and TACR3. Stimulation of the cells with CXCL12 alone or Neurokinin B alone, respectively, produced calcium signaling dose-dependently (FIGS.25A-25B). As shown inFIG.25A, addition of small amount of Neurokinin B (0.4 nM, TACR3-selective endogenous ligand) significantly enhanced calcium response in a broad range of CXCL12 concentrations compared with the sum values calculated by adding the responses obtained by Neurokinin B alone and CXCL12 alone at indicated doses. Maximal calcium response evoked by CXCL12 alone was taken as 100%. Sum represents the calculated additive value of the responses evoked by 0.4 nM of Neurokinin B alone and CXCL12 alone at indicated doses. Similarly, as shown inFIG.25B, addition of CXCL12 significantly enhanced downstream response in a broad range of Neurokinin B concentrations compared with the sum values calculated by adding the responses obtained by CXCL12 alone and Neurokinin B alone at indicated doses. Maximal response evoked by Neurokinin B alone was taken as 100%. Sum represents the calculated additive value of the responses evoked by 30 nM of CXCL12 alone and Neurokinin B alone at indicated doses. Statistically significant differences between the sum and co-treatment at each point were determined by Student's t test. *P<0.05; **P<0.01; Mean±SD (n=3). These results suggest an enhanced response in cells co-expressing CXCR4 and TACR3 in vivo in the presence of small amounts of CXCL12 and Neurokinin B together. Example 17. Confirmation of a Loss of Heteromer-Specific Property Upon Deletion of One of the Protomers InFIGS.8F &8G, enhanced calcium signaling was observed in MDA-MB-231 cells overexpressing CXCR4 alone upon co-treatment with CXCL12 and histamine. Using RT-qPCR, MDA-MB-231 cells were pervasively shown to express HRH1 as well as low level of CXCR4 mRNAs, with about twice more HRH1 mRNA being expressed compared with CXCR4 mRNA (Example 9). To confirm if the enhanced calcium signaling shown inFIGS.8F &8Gis due to the presence of endogenous HRH1 expression, and not to the presence of other histamine receptors, HRH1 gene in MDACXCR4+cells was deleted using CRISPR/Cas9 system. MDA-MB-231 cells stably expressing CXCR4 (MDACXCR4+) were transduced with lentiviruses encoding Cas9 and guide RNA targeting HRH1. The presence of functional HRH1 was detected by measuring calcium responses upon exposure to histamine. While MDACXCR4+cells showed dose-dependent increase in calcium signaling upon exposure to histamine, MDACXCR4+, HRH1−cells did not show any calcium response even at 1 μM histamine (FIG.26A). The result implies almost complete absence of functional HRH1 in MDACXCR4+, HRH1−cells. When MDA-MB-231 cells stably overexpressing CXCR4 (MDACXCR4+cells) were treated with histamine, dose-dependent increase in calcium signaling was observed (FIG.27A). When the cells were treated with histamine in the presence of CXCL12 (50 nM), significantly enhanced calcium response was obtained in a broad range of histamine concentrations, resulting in increased potency and efficacy as evidenced by the changes in EC50and Emaxvalues. However, in MDACXCR4+cells depleted with HRH1 using CRISPR/Cas9 system (MDACXCR4+, HRH1−cells), no histamine-mediated response was obtained in the absence or presence of CXCL12 (50 nM), reconfirming the absence of functional HRH1 in these cells. Maximal calcium response evoked by histamine alone in MDACXCR4+cells was taken as 100%. Sum represents the calculated additive value of the responses evoked by 50 nM of CXCL12 alone and histamine alone at indicated doses. In MDACXCR4+cells, stimulation with CXCL12 produced calcium response dose-dependently (FIG.27B). Addition of non-signaling concentration of histamine (15 nM) significantly enhanced calcium response in a broad range of CXCL12 concentrations, resulting in greatly increased potency and efficacy as evidenced by the changes in EC50and Emaxvalues. CXCL12-mediated calcium response in MDACXCR4+, HRH1−cells was similar to the response observed in MDACXCR4+cells. However, addition of histamine failed to increase CXCL12-mediated calcium response in MDACXCR4+, HRH1−cells, demonstrating the loss of heteromer-specific property upon deletion of HRH1. Maximal response evoked by CXCL12 alone in MDACXCR4+cells was taken as 100%. Sum represents the calculated additive value of the responses evoked by 15 nM of histamine alone and CXCL12 alone at indicated doses. Statistically significant differences between the sum and co-treatment at each point were determined by Student's t test. *P<0.05; **P<0.01; ***P<0.001; Mean±SD (n=3). EC50and Emaxvalues were calculated using GraphPad Prism software. InFIGS.18A-18B, significant enhancement in calcium signaling was also observed in wildtype MDA-MB-231 cells in the presence of CXCL12 and histamine together although individual ligand alone only produced faint signals. To confirm if the enhanced response is due to the endogenous expression of CXCR4, CXCR4 gene was targeted using CRISPR/Cas9 system, and expression of CXCR4 was detected using immunoblotting. Expression of CXCR4 was significantly decreased in MDA-MB-231 cells treated with guide RNA targeting CXCR4 compared to the expression in cells treated with control non-targeting guide RNA (FIG.26B). Enhanced calcium response in MDA-MB-231 cells upon co-treatment with CXCL12 and histamine is abrogated in the absence of CXCR4. As shown inFIG.28A, in MDA-MB-231 cells (MDA-MB-231 cells (MDAWT), but not in MDAWTcells depleted with CXCR4 using CRISPR/Cas9 system (MDACXCR4−)), enhanced signaling in the presence of small amount of histamine (15 nM) was evident in a broad range of CXCL12 concentrations although CXCL12-mediated dose-dependent increase in calcium response was not observed due to the low level expression of CXCR. Maximal calcium response evoked by CXCL12 alone in MDAWTcells was taken as 100%. Sum represents the calculated additive value of the responses evoked by 15 nM of histamine alone and CXCL12 alone at indicated doses. Deletion of CXCR4 completely abrogate the enhanced signaling in the presence of histamine, demonstrating loss of heteromer-specific property upon deletion of CXCR4. Similarly, as shown inFIG.28B, addition of CXCL12 enhanced histamine-mediated response in a broad range of histamine concentrations in MDA-MB-231 cells compared with the sum values calculated by adding the responses obtained by CXCL12 alone and histamine alone at indicated doses. The enhanced signaling shown in MDA-MB-231 cells was not observed in MDACXCR4−cells, although MDACXCR4−cells retained histamine responses. Maximal response evoked by histamine alone in MDAWTcells was taken as 100%. Sum represents the calculated additive value of the responses evoked by 100 nM of CXCL12 alone and histamine alone at indicated doses. Statistically significant differences between the sum and co-treatment at each point were determined by Student's t test. **P<0.01; ***P<0.001; Mean±SD (n=3). EC50 and Emaxvalues were calculated using GraphPad Prism software. Taken together, these results demonstrate that CXCR4-HRH1 heteromer is responsible for the enhanced signaling upon co-treatment with CXCL12 and histamine in wildtype MDA-MB-231 cells. Example 18. Ca2+ Mobilization Inhibition of the Enhanced CXCR4 Downstream Signaling Upon CXCR4-GPCRx Heteromer Formation—Comparison of Single Inhibitor Treatment to Combination Inhibitor Treatment Overview: As illustrated below (see Tables 7-14), co-treatment by various combinations of a CXCR4 inhibitor and a GPCRx inhibitor in a series of CXCR4-GPCRx heteromer-expressing cells results in a significantly reduced calcium response compared to single treatment by a CXCR4 inhibitor alone. The GPCRx protomers of the series of CXCR4-GPCRx heteromers evaluated in Tables 7-14 are: ADRB2, HRH1, ADCYAP1R1, C5AR1, CALCR, EDNRB, MLNR, and TACR3, respectively. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 only, or CXCR4 and the specified GPCRx. The cells were cultured for 2 days and were treated CXCR4 antagonist alone or co-treatment of CXCR4 antagonist and the specified GPCRx antagonist. And then, cells were stained with Cal-6 for 2 hours and stimulated with CXCL12 (20 nM) and the specified GPCRx agonist. Calcium mobilization was measured using FlexStation3. A value in the column labeled “None” is the IC50 of Ca2+ response in the specified CXCR4-GPCRx heteromer-expressing MDA-MB-231 treated with CXCR4 inhibitor only (in the absence of a specified GPCRx antagonist). A value in the remaining columns is the IC50 of Ca2+ response in the specified CXCR4-GPCRx heteromer-expressing MDA-MB-231 from the simultaneous treatment with CXCR4 antagonist and the specified GPCRx antagonist. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-ADRB2 heteromer was measured in the absence or presence of the representative ADRB2 inhibitors Carazolol or Propranolol (Table 7). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and ADRB2, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and Salmeterol (1 μM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 7ADRB2 inhibitorsCXCR4NoneCarazolol (10 μM)Propranolol (10 μM)inhibitorsIC50 [nM]IC50 [nM]IC50 [nM]AMD310022.10 ± 8.532.69 ± 3.416.23 ± 4.90Ulocuplumab0.226 ± 0.1440.009 ± 0.0040.041 ± 0.024BKT140123.86 ± 76.9126.66 ± 19.3266.14 ± 53.77 CXCR4 inhibitors AMD-3100, Ulocuplumab and BKT140 suppressed the CXCR4-ADRB2 signaling more efficiently (greater potency) in the presence of 10 μM of Carazolol or Propranolol as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of ADRB2 inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-ADRB2 heteromer context decreased by up to about 25 times or more when treated in combination with an ADRB2 inhibitor (Carazolol or Propranolol), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and ADRB2 inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-ADRB2 heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-HRH1 heteromer was measured in the absence or presence of the representative HRH1 inhibitors Hydroxyzine, Prometazine, or Cyproheptadine (Table 8). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and HRH1, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and Histamine (1 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 8HRH1 inhibitorsHydroxyzinePromethazineCyproheptadineCXCR4None(10 μM)(10 μM)(10 μM)inhibitorsIC50 [nM]IC50 [nM]IC50 [nM]IC50 [nM]AMD310039.07 ± 14.51.58 ± 1.142.59 ± 2.060.75 ± 0.49Ulocuplumab0.5 ± 0.440.003 ± 0.0030.000098 ± 0.00010.003 ± 0.001BKT140232.3 ± 91.9553.75 ± 21.241.66 ± 1.139.71 ± 7.17 CXCR4 inhibitors AMD-3100, Ulocuplumab and BKT140 suppressed the CXCR4-HRH1 signaling more efficiently (greater potency) in the presence of 10 μM of Hydroxyzine, Prometazine, or Cyproheptadine as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of HRH1 inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-HRH1 heteromer context decreased by up to about 5,100 times or more when treated in combination with an HRH1 inhibitor (Hydroxyzine, Promethazine, or Cyproheptadine), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and HRH1 inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-HRH1 heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-ADCYAP1R1 heteromer was measured in the absence or presence of the representative ADCYAP1R1 inhibitors M65 or PACAP-(6-38) (Table 9). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and ADCYAP1R1, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and PACAP-38 (1 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 9ADCYAP1R1 inhibitorsCXCR4NoneM65 (1 μM)PACAP-(6-38) (1 μM)inhibitorsIC50 [nM]IC50 [nM]IC50 [nM]AMD3100546.8 ± 337.820.21 ± 12.832.42 ± 0.09BKT140213.08 ± 142.727.46 ± 1.9319.56 ± 3.1 CXCR4 inhibitors AMD-3100 and BKT140 suppressed the CXCR4-ADCYAP1R1 signaling more efficiently (greater potency) in the presence of 1 μM of M65 or 1 μM of PACAP-(6-38) as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of ADCYAP1R1 inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-ADCYAP1R1 heteromer context decreased by up to about 225 times or more when treated in combination with an ADCYAP1R1 inhibitor (M65 or PACAP-(6-38)), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and ADCYAP1R1 inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-ADCYAP1R1 heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-C5AR1 heteromer was measured in the absence or presence of the representative C5AR1 inhibitor W54011 (Table 10). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and C5AR1, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and C5a (0.03 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 10C5AR1 inhibitorsCXCR4NoneW54011 (10 μM)inhibitorsIC50 [nM]IC50 [nM]AMD3100769.34 ± 240.77434.00 ± 82.94Ulocuplumab3.80 ± 0.221.06 ± 0.25BKT140118.01 ± 56.529.72 ± 6.87 CXCR4 inhibitors AMD-3100, BKT140, and Ulocuplumab suppressed the CXCR4-C5AR1 signaling more efficiently (greater potency) in the presence of 10 μM of W54011, as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of C5AR1 inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-C5AR1 heteromer context decreased by up to about 12 times or more when treated in combination with an C5AR1 inhibitor (W54011), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and C5AR1 inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-C5AR1 heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-CALCR heteromer was measured in the absence or presence of the representative CALCR inhibitor CT-(8-32) (Table 11). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and CALCR, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and calcitonin (100 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 11CALCR inhibitorsCXCR4NoneCT-(8-32) (salmon) (10 μM)inhibitorIC50 [nM]IC50 [nM]AMD31002677.67 ± 953.2312.54 ± 14.7BKT1401041.6 ± 756.915.62 ± 3.71 CXCR4 inhibitors AMD-3100 and BKT140 suppressed the CXCR4-CALCR signaling more efficiently (greater potency) in the presence of 10 μM of CT-(8-32) as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of CALCR inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-CALCR heteromer context decreased by up to about 210 times or more when treated in combination with an CALCR inhibitor (CT-(8-32)), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and CALCR inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-CALCR heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-EDNRB heteromer was measured in the absence or presence of the representative EDNRB inhibitors ambrisentan or bosentan (Table 12). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and EDNRB, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and BQ3020 (0.5 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 12EDNRB inhibitorsAmbrisentanBosentanCXCR4None(10 μM)(10 μM)inhibitorIC50 [nM]IC50 [nM]IC50 [nM]AMD3100926.10 ± 346.23287.13 ± 88.75169.12 ± 147.30Ulocuplumab14.11 ± 2.312.24 ± 0.184.68 ± 2.81BKT1402680.5 ± 3927.44529.37 ± 122.268.50 ± 7.37 CXCR4 inhibitors AMD-3100, BKT140, and Ulocuplumab suppressed the CXCR4-EDNRB signaling more efficiently (greater potency) in the presence of 10 μM of ambrisentan or 10 μM of bosentan, as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of EDNRB inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-EDNRB heteromer context decreased by up to about 315 times or more when treated in combination with an EDNRB inhibitor (ambrisentan or bosentan), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and EDNRB inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-EDNRB heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-MLNR heteromer was measured in the absence or presence of the representative MLNR inhibitor MA-2029 (Table 13). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and MLNR, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and motilin (0.2 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 13MLNR inhibitorsCXCR4NoneMA-2029 (10 uM)inhibitorIC50 [nM]IC50 [nM]AMD3100946.1 ± 171.4199.23 ± 64.34Ulocuplumab25.51 ± 16.482.25 ± 1.30BKT140643.6 ± 162.993.39 ± 155.20 CXCR4 inhibitors AMD-3100, BKT140, and Ulocuplumab suppressed the CXCR4-MLNR signaling more efficiently (greater potency) in the presence of 10 μM of MA-2029 as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of MLNR inhibitor. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-MLNR heteromer context decreased by up to about 11 times or more when treated in combination with an MLNR inhibitor (MA-2029), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and MLNR inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-MLNR heteromer expressing cells. The efficiency of CXCR4 inhibitors in suppressing the enhanced signaling of CXCR4-TACR3 heteromer was measured in the absence or presence of the representative TACR3 inhibitors SB 222200, osanetant, or talnetant (Table 14). MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and TACR3, and Ca2+ signaling was measured upon co-stimulating the cells with CXCL12 (20 nM) and neurokinin B (0.3 nM). Inhibitors were treated 30 min before agonist stimulation. Ca2+ mobilization was measured as described inFIG.5, and IC50values were calculated using GraphPad Prism software. TABLE 14TACR3 inhibitorsSB-222200OsanetantTalnetantCXCR4None(10 μM)(10 μM)(10 μM)inhibitorIC50 [nM]IC50 [nM]IC50 [nM]IC50 [nM]AMD31002680.33 ± 210.49343.13 ± 199.54567.53 ± 322.61179.63 ± 52.11Ulocuplumab50.65 ± 23.573.07 ± 1.184.61 ± 4.363.30 ± 0.89BKT1401497.67 ± 419.98217.90 ± 92.86194.33 ± 91.07230.30 ± 33.86 CXCR4 inhibitors AMD-3100, BKT140, and ulocuplumab suppressed the CXCR4-TACR3 signaling more efficiently (greater potency) in the presence of 10 μM of SB-222200, 10 μM of osanetant, or 10 μM of talnetant as shown with the reduced IC50values compared to the IC50values of CXCR4 inhibitors in the absence of TACR3 inhibitors. Depending on the identity of the CXCR4 inhibitor, the IC50 value of Ca response in the CXCR4-TACR3 heteromer context decreased by up to about 16 times or more when treated in combination with an TACR3 inhibitor (SB-222200, osanetant, or talnetant), relative to single treatment with the CXCR4 inhibitor alone (“None” column). These results suggest that co-treating with a CXCR4 inhibitor and TACR3 inhibitor more effectively inhibits increased Ca response than single treatment with a CXCR4 inhibitor alone in CXCR4-TACR3 heteromer expressing cells. Example 19. Anti-Tumor Effect of CXCR4-ADRB2 Heteromer Inhibitors on Tumor Growth InFIG.22, we observed that the CXCR4 and ADRB2 overexpressing cells bearing mice showed a dramatic increase in tumor proliferation compared to the A549 parent cell bearing mice. This suggests that CXCR4-ADRB2 heteromer promotes tumor proliferation. To investigate the anti-tumor effect of CXCR4-ADRB2 heteromer inhibitors on tumor growth, the A549-CXCR4-ADRB2, stably overexpressing CXCR4-ADRB2 heteromer (1×107cell/head) was injected subcutaneously in nude mice. When tumor sizes were reached an average of 50-100 mm3, CXCR4 inhibitor, ADRB2 inhibitor alone or combination of CXCR4 and ADRB2 inhibitors were treated. FIG.29Ais a graph comparing tumor growth rates for the in vivo antitumor effect of CXCR4 inhibitor LY2510924 (3 mg/kg), ADRB2 inhibitor Carvedilol (30 mg/kg), or combination of LY2510924 (3 mg/kg) and Carvedilol (30 mg/kg).FIG.29Bis a graph comparing tumor growth rates for the antitumor effect of CXCR4 inhibitor AMD070 (10 mg/kg), ADRB2 inhibitor Carvedilol (30 mg/kg), or combination of AMD070 (10 mg/kg) and Carvedilol (30 mg/kg). The tumor growth was monitored every third or fourth day by measuring the length (L) and width (W) of the tumor and calculating tumor volume based on the following formula: Volume=0.5 LW2. As shown inFIG.29A, CXCR4-ADRB2 overexpressing mice were more inhibited tumor growth by the CXCR4 inhibitor, LY2510924 or the ADRB2 inhibitor, Carvedilol than the control mice. In addition, the combination of LY2510924 and Carvedilol demonstrated that the tumor growth inhibitory effect was superior to the single administration group. More specifically CXCR4-ADRB2 overexpressing A549 bearing mice treated with vehicle as a control reached an average tumor volume of 554.2±152.7 mm3at day 21 post-treatment, as compared to LY2510924, Carvedilol or LY2510924 and Carvedilol, which reached an average tumor volume of 488.9±135.2 mm3, and 432.0±206.4 mm3, 356.3±125.4 mm3respectively, in the same time period. A similar pattern was observed in the group treated with AMD070, another inhibitor of CXCR4. FIG.29Bshows CXCR4-ADRB2 heteromer-overexpressing cell lines were transplanted at nude mouse and when tumor size reached 50-100 mm3CXCR4 inhibitor, AMD070 or ADRB2 inhibitor, Carvedilol was administered orally alone or in combination for 23 days. As shown inFIG.29B, the size of the tumor on the 23 days after administration of the drug was 618.5±190.9 mm3in the control group, 543.2±260.4 mm3or 510.4±139.9 mm3in the group treated with AMD070 or Carvedilol, respectively. The tumor size of drug treated group is shown to be smaller than that of control group. In addition, in the combination of AMD070 and Carvedilol, the size of the tumor was 418.0±238.4 m3, which indicates that the antitumor effect is superior to that of the single administration group. These results suggest that co-treatment of CXCR4 and ADRB2 inhibitors may provide a better therapeutic effect for CXCR4 and ADRB2 beteromer expressing patients. All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. Exemplary Embodiments In an embodiment, a method for treating cancer in a subject having a cell containing a CXCR4-GPCRx heteromer, the method comprising: administering to the subject a therapeutically effective amount of an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer. In an embodiment, a method for treating cancer in a subject having CXCR4-GPCRx heteromer, the method comprising: administering to the subject a therapeutically effective amount of an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein the cell containing the CXCR4-GPCRx heteromer has enhanced downstream signaling. In an embodiment, a method for treatment, amelioration, or prevention of a cancer in a subject having CXCR4-GPCRx heteromer, the method comprising: administering to the subject a therapeutically effective amount of an inhibitor of a CXCR4-GPCRx heteromer, wherein: GPCRx heteromerizes with CXCR4 in the subject, the heteromerization of GPCRx with CXCR4 is accompanied by enhancement of signaling downstream of CXCR4; and the enhancement of signaling downstream of CXCR4 is suppressed by the inhibitor of the CXCR4-GPCRx heteromer. In an embodiment, a method for assessing response, or potential response, of a subject having CXCR4-GPCRx heteromer to treatment, amelioration, or prevention of a cancer, the method comprising: obtaining a sample from the subject; detecting heteromerization of CXCR4 and GPCRx in the sample; and based at least in part on detection of the heteromerization of CXCR4 and GPCRx, assessing the subject's response, or potential response, to the treatment, amelioration, or prevention of a cancer. In an embodiment, a method for treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, the method comprising: administering to the patient an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein:i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; andii) the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In an embodiment, a method of suppressing enhanced downstream signaling from a CXCR4-GPCRx heteromer in a cell of a patient suffereing from cancer, the method comprising: administering to the patient an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein:i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; andii) the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In an embodiment, a pharmaceutical kit for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, the pharmaceutical kit comprising: an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. In an embodiment, a pharmaceutical composition for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, the pharmaceutical composition comprising:i) an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; andii) a pharmaceutically acceptable carrier; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling, the method comprising:1) determining whether the patient has the cancer cell containing the CXCR4-GPCRx heteromer having enhanced downstream signaling by: obtaining or having obtained a biological sample from the patient; and performing or having performed an assay on the biological sample to determine if:i) the patient's cancer cell contains said CXCR4-GPCRx heteromer; orii) a CXCR4-GPCRx heteromer-selective reagent: alters heteromer-specific properties or function of said CXCR4-GPCRx heteromer in a patient derived cell(s); alters heteromer-specific properties of a patient derived cell(s) containing the CXCR4-GPCRx heteromer; or decreases cell proliferation of a patient derived cell(s) containing the CXCR4-GPCRx heteromer; and2) if the patient has a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer to the cancer patient. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, the method comprising:1) determining whether the patient's cancer cell contains the CXCR4-GPCRx heteromer by: obtaining or having obtained a biological sample from the patient and performing or having performed an assay on the biological sample to determine if said CXCR4-GPCRx heteromer is present in the patient's cancer cell; wherein:a) the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; andb) the assay performed on the biological sample is or comprises one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay; and2) if the patient's cancer cell contains said CXCR4-GPCRx heteromer, then internally administering an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer to the patient. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer having enhanced downstream signaling, the method comprising:1) determining whether the patient has the cancer cell containing the CXCR4-GPCRx heteromer having enhanced downstream signaling by: obtaining or having obtained a biological sample from the patient; and performing or having performed an assay on the biological sample to determine if:i) the patient's cancer cell contains said CXCR4-GPCRx heteromer; orii) a CXCR4-GPCRx heteromer-selective reagent: alters heteromer-specific properties or function of said CXCR4-GPCRx heteromer in a patient derived cell(s); alters heteromer-specific properties of a patient derived cell(s) containing said CXCR4-GPCRx heteromer; or decreases cell proliferation of a patient derived cell(s) containing said CXCR4-GPCRx heteromer; and2) if the patient has a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering to the cancer patient a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; and3) if the patient does not have a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering to the cancer patient either the CXCR4 inhibitor or the GPCRx inhibitor as a single inhibitor. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer having enhanced downstream signaling, the method comprising:1) determining whether the patient has the cancer cell containing the CXCR4-GPCRx heteromer having enhanced downstream signaling by: obtaining or having obtained a biological sample from the patient; and performing or having performed an assay on the biological sample to determine if:i) the patient's cancer cell contains said CXCR4-GPCRx heteromer; orii) a CXCR4-GPCRx heteromer-selective reagent: alters heteromer-specific properties or function of said CXCR4-GPCRx heteromer in a patient derived cell(s); alters heteromer-specific properties of a patient derived cell(s) containing said CXCR4-GPCRx heteromer; or decreases cell proliferation of a patient derived cell(s) containing said CXCR4-GPCRx heteromer; and2) if the patient has a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering to the cancer patient a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; and3) if the patient does not have a cancer cell containing said CXCR4-GPCRx heteromer, then internally administering to the cancer patient either the CXCR4 inhibitor or the GPCRx inhibitor as a single inhibitor; wherein:a) progression of the cancer in the patient having said cancer cell containing the CXCR4-GPCRx heteromer is decreased in the range of 5-100% more upon administration of the combination of inhibitors, relative to administering the CXCR4 inhibitor or GPCRx inhibitor as the single inhibitor to said patient;b) efficacy of a CXCR4 inhibitor is increased in the range of 5-2000% when administered in combination with the GPCRx inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the CXCR4 inhibitor when administered as a single inhibitor; and/orc) efficacy of a GPCRx inhibitor is increased in the range of 5-2000% when administered in combination with the CXCR4 inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the GPCRx inhibitor when administered as a single inhibitor. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, the method comprising:1) determining whether the patient's cancer cell contains the CXCR4-GPCRx heteromer by: obtaining or having obtained a biological sample from the patient and performing or having performed an assay on the biological sample to determine if said CXCR4-GPCRx heteromer is present in the patient's cancer cell; wherein:a) the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; andb) the assay performed on the biological sample is or comprises one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNAseq, qRT-PCR, microarray, or a fluorescent animal assay; and2) if the patient's cancer cell contains said CXCR4-GPCRx heteromer, then internally administering to the cancer patient a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; and3) if the patient's cancer cell does not contain said CXCR4-GPCRx heteromer, then internally administering to the cancer patient either the CXCR4 inhibitor or the GPCRx inhibitor as a single inhibitor. In an embodiment, a method for treating cancer in a patient having a cancer cell containing a CXCR4-GPCRx heteromer, the method comprising:1) determining whether the patient's cancer cell contains the CXCR4-GPCRx heteromer by: obtaining or having obtained a biological sample from the patient and performing or having performed an assay on the biological sample to determine if said CXCR4-GPCRx heteromer is present in the patient's cancer cell; wherein:a) the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; andb) the assay performed on the biological sample is or comprises one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNAseq, qRT-PCR, microarray, or a fluorescent animal assay; and2) if the patient's cancer cell contains said CXCR4-GPCRx heteromer, then internally administering to the cancer patient a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; and3) if the patient's cancer cell does not contain said CXCR4-GPCRx heteromer, then internally administering to the cancer patient either the CXCR4 inhibitor or the GPCRx inhibitor as a single inhibitor; wherein:a) progression of the cancer in the patient having said cancer cell containing the CXCR4-GPCRx heteromer is decreased in the range of 5-100% more upon administration of the combination of inhibitors, relative to administering the CXCR4 inhibitor or GPCRx inhibitor as the single inhibitor to said patient;b) efficacy of a CXCR4 inhibitor is increased in the range of 5-2000% when administered in combination with the GPCRx inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the CXCR4 inhibitor when administered as a single inhibitor; and/orc) efficacy of a GPCRx inhibitor is increased in the range of 5-2000% when administered in combination with the CXCR4 inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the GPCRx inhibitor when administered as a single inhibitor. In certain embodiments, one or more than one (including for instance all) of the following further embodiments may comprise each of the other embodiments or parts thereof. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer has, causes, or produces, the enhanced downstream signaling. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling results from the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling results from agonism of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling results from CXCR4 agonism of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling results from GPCRx agonism of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling results from CXCR4 agonism and GPCRx agonism of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling is downstream of the CXCR4, the respective GPCRx, or the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling is downstream of the CXCR4. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling is downstream of the respective GPCRx. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling is downstream of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling from the CXCR4-GPCRx heteromer is relative to downstream signaling from a CXCR4 protomer or a respective GPCRx protomer in their respective individual protomer context. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling from the CXCR4-GPCRx heteromer is relative to downstream signaling from a CXCR4 protomer in an individual protomer context. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling from the CXCR4-GPCRx heteromer is relative to downstream signaling from a respective GPCRx protomer in an individual protomer context. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling from the CXCR4-GPCRx heteromer is relative to downstream signaling from a CXCR4 protomer and a respective GPCRx protomer in their respective individual protomer context. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the patient's cancer cells. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises determining or diagnosing the presence of a CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises determining or diagnosing the presence of a CXCR4-GPCRx heteromer in a cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises determining or diagnosing the presence of a CXCR4-GPCRx heteromer in a cancer cell or cancer tissue. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises determining or diagnosing the presence of a CXCR4-GPCRx heteromer in a cancer cell or cancer tissue obtained from a cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises determining or diagnosing the presence of a CXCR4-GPCRx heteromer; and wherein the determining or diagnosing comprises:1) obtaining or having obtained a biological sample (e.g., cells or tissue, such as cells or tissue from a cancer patient); and2) performing or having performed an assay on the biological sample, wherin the assay is or comprises one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling from the CXCR4-GPCRx heteromer is determined by an intracellular Ca2+ assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced downstream signaling is an enhanced amount of calcium mobilization. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization from the CXCR4-GPCRx heteromer is a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization from the CXCR4-GPCRx heteromer is a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is between 10-100% greater than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization from the CXCR4-GPCRx heteromer is a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is between 25-100% greater than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization is determined by an intracellular Ca2+ assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the intracellular Ca2+ assay is a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization is a synergistic amount of calcium mobilization. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the synergistic amount of calcium mobilization from the cells containing the CXCR4-GPCRx heteromer is a calcium mobilization amount that, upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist, is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater, than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer has two or more of the following characteristics:1) the CXCR4-GPCRx heteromer components in a cell colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism, as determined via one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay;2) an enhanced amount of calcium mobilization, such that:a) either CXCR4 or the respective GPCRx in an individual protomer context in a cell, upon co-stimulation with CXCL12 and a respective selective GPCRx agonist, results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; andb) the CXCR4-GPCRx heteromer exhibits an enhanced calcium mobilization upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; as determined via a calcium mobilization assay; or3) a CXCR4-GPCRx heteromer-selective reagent:i) alters heteromer-specific properties of the CXCR4-GPCRx heteromer in a patient derived cell;ii) alters heteromer-specific function of the CXCR4-GPCRx heteromer in a patient derived cell;iii) alters heteromer-specific properties of a patient derived cell containing the CXCR4-GPCRx heteromer; oriv) decreases cell proliferation of a patient derived cell(s) containing the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of the CXCR4-GPCRx heteromer in a patient derived cell, as determined by at least one of the following methods: PLA, radioligand binding assays, [35S]GTP-rS Binding assays, Calcuim assay, cAMP assay, GTPase assay, PKA activation, ERK1/2 and/or Akt/PKB Phosphorylation assays, Src and STAT3 phosphorylation assays, CRE-reporter assay, NFAT-RE-reporter assay, SRE-reporter assay, SRF-RE reporter assay, NF-kB-RE reporter assay, Secreted alkaline phosphatase Assay, Inositol 1-Phosphate Production assay, Adenylyl Cyclase Activity assay, analysis of target gene expression by RT-PCR, RT-qPCR, RNAseq, or microarray. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific function of the CXCR4-GPCRx heteromer in a patient derived cell, as determined by at least one of the following methods: PLA, radioligand binding assays, [35S]GTP-rS Binding assays, Calcuim assay, cAMP assay, GTPase assay, PKA activation, ERK1/2 and/or Akt/PKB Phosphorylation assays, Src and STAT3 phosphorylation assays, CRE-reporter assay, NFAT-RE-reporter assay, SRE-reporter assay, SRF-RE reporter assay, NF-kB-RE reporter assay, Secreted alkaline phosphatase Assay, Inositol 1-Phosphate Production assay, Adenylyl Cyclase Activity assay, analysis of target gene expression by RT-PCR, RT-qPCR, RNAseq, next generation sequencing (NGS), or microarray. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of a patient derived cell containing the CXCR4-GPCRx heteromer, as determined by at least one of the following methods: assays on proliferation, migration, invasion, and drug resistance (survival) of cancer cells, modulation of immune cell function, angiogenesis, vasculogenesis, metastasis, drug resistance, tissue microarray (TMA), and cancer cell-tumor microenvironment (TME) interaction. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer components comprise individual protomers CXCR4 and GPCRx. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cell containing either the CXCR4 or the respective GPCRx in an individual protomer context comprises, independently:i) the individual protomer CXCR4 in the absence of the respective individual protomer GPCRx; orii) the respective individual protomer GPCRx in the absence of the individual protomer CXCR4; respectively. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cell containing the CXCR4 in an individual protomer context comprises said individual protomer CXCR4 in the absence of the respective individual protomer GPCRx. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cell containing the respective GPCRx in an individual protomer context comprises said respective individual protomer GPCRx in the absence of the individual protomer CXCR4. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer components in the cell colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism, as determined via one or more of the following: a co-internalization assay, a colocalization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the proximity-based assay is, or comprises, resonance energy transfer (RET), bioluminescence RET (BRET), fluorescence RET (FRET), time-resolved fluorescence RET (TR-FRET), antibody-aided FRET, ligand-aided FRET, bimolecular fluorescence complementation (BiFC), or a proximity ligation assay (PLA). In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer components in the cell colocalize and physically interact, either directly or via intermediate proteins acting as conduits for allosterism, as determined via one or more of the following: a co-internalization assay, bimolecular fluorescence complementation (BiFC), or a proximity ligation assay (PLA). In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the patient's cancer cell contains the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer exhibits the enhanced amount of calcium mobilization, such that:a) either the CXCR4 or the respective GPCRx in an individual protomer context in the cell upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; andb) the CXCR4-GPCRx heteromer exhibits an enhanced calcium mobilization upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist relative to the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist; as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein:i) the calcium mobilization from the protomer CXCR4 or GPCRx, in the individual protomer context in the cell, is non-synergistic, as determined via calcium mobilization assay; andii) the calcium mobilization from the CXCR4-GPCRx heteromer in the cell is synergistic, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein in the individual protomer context:a) the individual protomer CXCR4 in the cell, in the absence of the respective individual protomer GPCRx; orb) the respective individual protomer GPCRx in the cell, in the absence of the individual protomer CXCR4; upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein in the individual protomer context, independently:a) the individual protomer CXCR4 in the cell, in the absence of the respective individual protomer GPCRx; andb) the respective individual protomer GPCRx in the cell, in the absence of the individual protomer CXCR4; upon co-stimulation with CXCL12 and a respective selective GPCRx agonist results in a calcium mobilization amount that is equal to or less than the sum of calcium mobilization amounts resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer upon co-stimulation with the CXCL12 and the respective selective GPCRx agonist results in a calcium mobilization amount that is greater than the sum of calcium mobilization amounts resulting from single agonist stimulation of said cells with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the calcium mobilization amount resulting from the co-stimulation of the CXCR4-GPCRx heteromer is an enhanced amount of calcium mobilization, relative to the sum of calcium mobilizations resulting from single agonist stimulation of said CXCR4-GPCRx heteromer, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater, than the sum of calcium mobilizations resulting from single agonist stimulation with either the CXCL12 or the respective selective GPCRx agonist, as determined via a calcium mobilization assay. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the enhanced amount of calcium mobilization is a synergistic amount of calcium mobilization. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers a CXCR4-GPCRx heteromer-selective reagent. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent:i) alters heteromer-specific properties of the CXCR4-GPCRx heteromer in the patient derived cell;ii) alters heteromer-specific function of the CXCR4-GPCRx heteromer in the patient derived cell;iii) alters heteromer-specific properties of the patient derived cell containing the CXCR4-GPCRx heteromer; oriv) decreases cell proliferation of the patient derived cell containing the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of the CXCR4-GPCRx heteromer in the patient derived cell. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific function of the CXCR4-GPCRx heteromer in the patient derived cell. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent alters heteromer-specific properties of the patient derived cell containing the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent decreases cell proliferation of the patient derived cell containing the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent is a CXCR4 inhibitor, a GPCRx inhibitor, or a CXCR4-GPCRx heteromer inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent is a CXCR4 antagonist, a GPCRx antagonist, or a CXCR4-GPCRx heteromer antagonist. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent is administered as a pharmaceutical composition. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers an inhibitor selected from the group consisting of: the CXCR4 inhibitor, the GPCRx inhibitor, or the CXCR4-GPCRx heteromer inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the inhibitor is administered as a pharmaceutical composition. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administered inhibitor is the CXCR4 inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administered inhibitor is the GPCRx inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administered inhibitor is the inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers the combination of inhibitors selected from the group consisting of: the CXCR4 inhibitor, the GPCRx inhibitor, and the inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors is administered as a pharmaceutical composition. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors comprises the CXCR4 inhibitor and the GPCRx inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors comprises the CXCR4 inhibitor and the inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors comprises the GPCRx inhibitor and the inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors are administered sequentially, concurrently, or simultaneously. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors are administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors are administered as separate pharmaceutical compositions, wherein the separate pharmaceutical compositions independently further comprise a pharmaceutically acceptable carrier. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4 inhibitor is an antagonist of CXCR4, an inverse agonist of CXCR4, a partial antagonist of CXCR4, an allosteric modulator of CXCR4, an antibody of CXCR4, an antibody fragment of CXCR4, or a ligand of CXCR4. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx inhibitor is an antagonist of GPCRx, an inverse agonist of GPCRx, a partial antagonist of GPCRx, an allosteric modulator of GPCRx, an antibody of GPCRx, an antibody fragment of GPCRx, or a ligand of GPCRx. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the inhibitor of the CXCR4-GPCRx heteromer is an antagonist of the CXCR4-GPCRx heteromer, an inverse agonist of the CXCR4-GPCRx heteromer, a partial antagonist of the CXCR4-GPCRx heteromer, an allosteric modulator of the CXCR4-GPCRx heteromer, an antibody of the CXCR4-GPCRx heteromer, an antibody fragment of the CXCR4-GPCRx heteromer, a ligand of the CXCR4-GPCRx heteromer, or a protein-protein interaction (PPI) inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4 inhibitor, the GPCRx inhibitor, or the inhibitor of the CXCR4-GPCRx heteromer, is an antibody-drug conjugate. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a therapeutically effective amount of the inhibitor of the CXCR4-GPCRx heteromer is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a sub-therapeutically effective amount of the inhibitor of the CXCR4-GPCRx heteromer is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a therapeutically effective amount of the CXCR4 inhibitor is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a sub-therapeutically effective amount of the CXCR4 inhibitor is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4 inhibitor is selected from the group consisting of: AD-114, AD-114-6H, AD-114-Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI 2341, BKT140 (BL-8040; TF14016; 4F-Benzoyl-TN14003), CTCE-9908, CX549, D-[Lys3] GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[1C]Methyl-AMD3465, PF-06747143, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (Burixafor), USL311, ulocuplumab (MDX1338/BMS-936564), viral macrophage inflammatory protein-II (vMIP-II), WZ811, 12G5, 238D2, 238D4, [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga]pentixafor, [90Y]pentixather, [99mTc]O2-AMD3100, [177Lu]pentixather, and 508MCl (Compound 26). In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a therapeutically effective amount of the GPCRx inhibitor is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a sub-therapeutically effective amount of the GPCRx inhibitor is administered to the patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is ADRB2 or HRH1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is ADCYAP1R1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the ADCYAP1R1 inhibitor is selected from the group consisting of: M65, Max.d.4, MK-0893, N-stearyl-[Ne17] neurotensin-(6-11)/VIP-(7-28), PACAP-(6-38), and PG 97-269. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is ADORA2B. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the ADORA2B inhibitor is selected from the group consisting of: 3-isobutyl-8-pyrrolidinoxanthine, alloxazine, AS16, AS70, AS74, AS94, AS95, AS96, AS99, AS100, AS101, ATL802, BW-A1433, caffeine, CGS 15943, CPX, CSC, CVT-6883, DAX, DEPX, derenofylline, DPCPX, FK-453, I-ABOPX, istradefylline, KF26777, LAS38096, LUF5981, MRE 2029F20, MRE 3008F20, MRS1191, MRS1220, MRS1523, MRS1706, MRS1754, MSX-2, OSIP339391, pentoxifylline, preladenant, PSB-10, PSB-11, PSB36, PSB603, PSB-0788, PSB1115, rolofylline, SCH 58261, SCH442416, ST-1535, theophylline, tonapofylline, vipadenant, xanthine amine congener, XCC, and ZM-241385. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is ADORA3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the ADORA3 inhibitor is selected from the group consisting of: ATL802, BW-A1433, caffeine, CGS 15943, CSC, CVT-6883, derenofylline, dexniguldipine, DPCPX, FK-453, flavanone, flavone, galangin, I-ABOPX, istradefylline, KF26777, LAS38096, LUF5981, MRE 2029F20, MRE 3008F20, MRE 3010F20, MRS1041, MRS1042, MRS1067, MRS1088, MRS1093, MRS1097, MRS1177, MRS1186, MRS1191, MRS1191, MRS1220, MRS1476, MRS1486, MRS1505, MRS1523, MRS1754, MRS928, MSX-2, nicardipine, preladenant, PSB-10, PSB-11, PSB36, PSB603, PSB1115, rolofylline, sakuranetin, SCH 58261, SCH442416, ST-1535, theophylline, tonapofylline, vipadenant, visnagin, VUF5574, VUF8504, VUF8507, xanthine amine congener, and ZM-241385. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is ADRB2. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the ADRB2 inhibitor is selected from the group consisting of: Alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cicloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is C5AR1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the C5AR1 inhibitor is selected from the group consisting of: A8Δ71-73, AcPhe-Orn-Pro-D-Cha-Trp-Arg, avacopan, C089, CHIPS, DF2593A, JPE1375, L-156,602, NDT9520492, N-methyl-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H, PMX205, PMX53, RPR121154, and W54011. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is CALCR. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CALCR inhibitor is selected from the group consisting of: α-CGRP-(8-37) (human), AC187, CT-(8-32) (salmon), and olcegepant. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is CHRM1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CHRM1 inhibitor is selected from the group consisting of: 3-Quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ-RA 741, atropine, benzatropine, biperiden, darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydrodifenidol, hexahydrosiladifenidol, hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine, ML381, muscarinic toxin 1, muscarinic toxin 2, muscarinic toxin 3, N-methyl scopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine, propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl, tripitramine, UH-AH 37, umeclidinium, and VU0255035. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is EDNRB. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the EDNRB inhibitor is selected from the group consisting of: A192621, ambrisentan, atrasentan, bosentan (RO 470203, Tracleer); BQ788, IRL 2500, K-8794, macitentan, RES7011, Ro 46-8443, SB209670, SB217242 (enrasentan), TAK 044, and tezosentan (R0610612). In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is HRH1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the HRH1 inhibitor is selected from the group consisting of: (−)-chlorpheniramine, (+)-chlorpheniramine, (−)-trans-H2-PAT, (+)-cis-H2-PAT, (+)-trans-H2-PAT, (±)-cis-H2-PAT, (±)-trans-H2-PAT, (R)-cetirizine, (S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239, alimemazine, amitriptyline, aripiprazole, arpromidine, asenapine, astemizole, AZD3778, azelastine, BU-E 47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine, fexofenadine, fluphenazine, fluspirilene, haloperidol, hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen, loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine, pimozide, pipamperone, pitolisant, promethazine, pyrilamine, quetiapine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, trifluoperazine, tripelennamine, triprolidine, ziprasidone, and zotepine. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is MLNR. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the MLNR inhibitor is selected from the group consisting of: GM-109, MA-2029, and OHM-11526. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is NTSR1. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the NTSR1 inhibitor is selected from the group consisting of: Meclinertant, SR48527, SR48692, and SR142948A. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx is TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the TACR3 inhibitor is selected from the group consisting of: [Trp7, β-Ala8] neurokinin A-(4-10), AZD2624, FK 224, GR138676, GSK 172981, GSK 256471, N′,2-diphenylquinoline-4-carbohydrazide 8m, N′,2-diphenylquinoline-4-carbohydrazide, osanetant, PD 154740, PD 161182, PD157672, saredutant, SB 218795, SB 222200, SB 235375, SCH 206272, SSR 146977, and talnetant. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the respective selective GPCRx agonist is natural ligand of the respective GPCRx, respectively. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises detecting the CXCR4-GPCRx heteromer in the cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises identifying the CXCR4-GPCRx heteromer in the cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method further comprises:i) obtaining or having obtained a biological sample from the cancer patient;ii) conducting or having conducted a diagnostic assay to determine presence, identity, or presence and identity, of a CXCR4-GPCRx heteromer in the obtained biological sample from the cancer patient; andiii) selecting the inhibitor or combination of inhibitors to suppress enhanced downstream signaling from the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cancer is selected from the group consisting of: breast cancer, lung cancer, brain cancer, kidney cancer, pancreatic cancer, ovarian cancer, prostate cancer, melanoma, multiple myeloma, gastrointestinal cancers, renal cell carcinoma, soft tissue sarcomas, hepatocellular carcinoma, stomach cancer, colorectal cancer, esophageal cancer, and leukemia. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the patient's biological sample is a biological fluid sample. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a liquid biopsy is performed on the biological fluid sample. In some embodiments, the biological fluid sample includes circulating tumor cells (CTCs), tumor-derived cell-free DNA (cfDNA), circulating small RNAs, and extracellular vesicles including exosomes, from bodily fluids as disclosed, for example, in Campos C D M et al., “Molecular Profiling of Liquid Biopsy Samples for Precision Medicine,” Cancer J. 2018 March/April; 24(2):93-103, which is incorporated hereby in its entirety. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the biological fluid sample is a blood sample, a plasma sample, a saliva sample, a cerebral fluid sample, an eye fluid sample, or a urine sample. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the patient's biological sample is a biological tissue sample. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a liquid biopsy is performed on the biological tissue sample. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the biological tissue sample is an organ tissue sample, a bone tissue sample, or a tumor tissue sample. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cancer cell contains the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein a normal, non-cancerous cell, does not contain the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the cancer cell of the patient contains the CXCR4-GPCRx heteromer in a greater concentration than a normal, non-cancerous cell from said patient, for example, 10% greater concentration than a normal, non-cancerous cell from said patient, such as 25% greater, 50% greater, 100% greater, 200% greater, or 300% greater, than a normal, non-cancerous cell from said patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer contained in the cancer cell of the patient is in a greater concentration than a normal, non-cancerous cell from said patient, for example, 10% greater concentration than a normal, non-cancerous cell from said patient, such as 25% greater, 50% greater, 100% greater, 200% greater, or 300% greater, than a normal, non-cancerous cell from said patient; and wherein the GPCRx of the CXCR4-GPCRx heteromer contained in the cancer cell of the patient is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the presence of the CXCR4-GPCRx heteromer in the cancer cell of the patient identifies a sub-population of CXCR4-mediated cancer patients. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the presence of the CXCR4-GPCRx heteromer in the cancer cell of the patient is a biomarker of a sub-population of CXCR4-mediated cancer patients. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the biomarker of the sub-population of CXCR4-mediated cancer patients allows for precision medicine, patient stratification, or patient classification. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the biomarker of the sub-population of CXCR4-mediated cancer patients allows for GPCR-based precision cancer therapeutics. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the presence of the CXCR4-GPCRx heteromer in the cancer cell of the patient identifies a sub-population of CXCR4-mediated cancer patients; and wherein the GPCRx of the CXCR4-GPCRx heteromer present in the cancer cell of the patient is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the presence of the CXCR4-GPCRx heteromer in the cancer cell of the patient is a biomarker of a sub-population of CXCR4-mediated cancer patients; and wherein the GPCRx of the CXCR4-GPCRx heteromer present in the cancer cell of the patient is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the inhibitor is an antibody. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the CXCR4-GPCRx heteromer-selective reagent is an antibody. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the antibody is a bi-specific antibody of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the antibody is a heteromer-specific antibody of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the inhibitor is a bi-specific ligand(s) of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the antibody is an antibody-drug conjugate (ADC), as disclosed in, for example, Beck a et al., “Strategies and challenges for the next generation of antibody-drug conjugates”, Nature Reviews Drug Discovery, 16:315-337, (2017) and Lambert, et al., “Antibody-Drug Conjugates for Cancer Treatment”, Annual Review of Medicine, 69:191-207 (2018), each of which are incorporated hereby in its entirety. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method comprises: administering to the patient an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein: i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; and ii) the administered inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the pharmaceutical kit or pharmaceutical composition, such as for use in treating cancer in a patient having a cell containing a CXCR4-GPCRx heteromer, comprises: an inhibitor or a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; wherein the CXCR4-GPCRx heteromer has enhanced downstream signaling. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein progression of the cancer in the patient having said cancer cell containing the CXCR4-GPCRx heteromer is decreased in the range of 5-100% more, 10-100% more, 20-100% more, 30-100% more, 40-100% more, 50-100% more, 60-100% more, 75-100% more, 5-75% more, 5-50% more, or 5-25% more, upon administration of the combination of inhibitors, relative to administering the CXCR4 inhibitor or GPCRx inhibitor as the single inhibitor to said patient. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the efficacy of a CXCR4 inhibitor is increased in the range of 5-2000%, 5-1750%, 5-1500%, 5-1250%, 5-1000%, 5-900%, 5-800%, 5-700%, 5-500%, 5-400%, 5-250%, 5-200%, 5-100%, 5-75%, 5-50%, 5-40%, 5-30%, 5-25%, 100-2000%, 200-2000%, 300-2000%, 500-2000%, 750-2000%, 1000-2000%, 1250-2000%, 1500-2000%, 5-1500%, 25-1500%, 50-1500%, 75-1500%, 100-1500%, 200-1500%, 300-1500%, 500-1500%, 750-1500%, 1000-1500%, or 1250-1500%, when administered in combination with the GPCRx inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the CXCR4 inhibitor when administered as a single inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the efficacy of a GPCRx inhibitor is increased in the range of 5-2000%, 5-1750%, 5-1500%, 5-1250%, 5-1000%, 5-900%, 5-800%, 5-700%, 5-500%, 5-400%, 5-250%, 5-200%, 5-100%, 5-75%, 5-50%, 5-40%, 5-30%, 5-25%, 100-2000%, 200-2000%, 300-2000%, 500-2000%, 750-2000%, 1000-2000%, 1250-2000%, 1500-2000%, 5-1500%, 25-1500%, 50-1500%, 75-1500%, 100-1500%, 200-1500%, 300-1500%, 500-1500%, 750-1500%, 1000-1500%, or 1250-1500%, when administered in combination with the CXCR4 inhibitor to the patient having said cancer cell containing the CXCR4-GPCRx heteromer, relative to efficacy of the GPCRx inhibitor when administered as a single inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer; or the pharmaceutical kit or pharmaceutical composition comprises a combination of inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the combination of inhibitors is a combination of two inhibitors selected from the group consisting of: an inhibitor of CXCR4, an inhibitor of GPCRx, and an inhibitor of the CXCR4-GPCRx heteromer. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers a combination of a CXCR4 inhibitor and a GPCRx inhibitor; or the pharmaceutical kit or pharmaceutical composition comprises a combination of a CXCR4 inhibitor and a GPCRx inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the method administers a CXCR4-GPCRx heteromer inhibitor; or the pharmaceutical kit or pharmaceutical composition comprises a CXCR4-GPCRx heteromer inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administering of the combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-2000 fold, 5-1750 fold, 5-1500 fold, 5-1250 fold, 5-1000 fold, 5-900 fold, 5-800 fold, 5-700 fold, 5-500 fold, 5-400 fold, 5-250 fold, 5-200 fold, 5-100 fold, 5-75 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 100-2000 fold, 200-2000 fold, 300-2000 fold, 500-2000 fold, 750-2000 fold, 1000-2000 fold, 1250-2000 fold, 1500-2000 fold, 5-1500 fold, 25-1500 fold, 50-1500 fold, 75-1500 fold, 100-1500 fold, 200-1500 fold, 300-1500 fold, 500-1500 fold, 750-1500 fold, 1000-1500 fold, or 1250-1500 fold, relative to single inhibitor administration. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the administering of the inhibitor or combination of inhibitors suppresses the enhanced downstream signaling from said CXCR4-GPCRx heteromer in the cancer patient in the range of between 5-2000 fold, 5-1750 fold, 5-1500 fold, 5-1250 fold, 5-1000 fold, 5-900 fold, 5-800 fold, 5-700 fold, 5-500 fold, 5-400 fold, 5-250 fold, 5-200 fold, 5-100 fold, 5-75 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 100-2000 fold, 200-2000 fold, 300-2000 fold, 500-2000 fold, 750-2000 fold, 1000-2000 fold, 1250-2000 fold, 1500-2000 fold, 5-1500 fold, 25-1500 fold, 50-1500 fold, 75-1500 fold, 100-1500 fold, 200-1500 fold, 300-1500 fold, 500-1500 fold, 750-1500 fold, 1000-1500 fold, or 1250-1500 fold, relative to suppression of downstream signaling from either a CXCR4 protomer or a GPCRx protomer in their respective individual protomer context. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; for example, selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, CHRM1, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of: ADRB2, EDNRB, and HRH1; selected from the group consisting of: ADRB2, CHRM1, and HRH1; or selected from the group consisting of: ADRB2, HRH1, and TACR3. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; for example, selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, HRH1 inhibitor, and TACR3 inhibitor; selected from the group consisting of: ADRB2 inhibitor, EDNRB inhibitor, and HRH1 inhibitor; selected from the group consisting of: ADRB2 inhibitor, CHRM1 inhibitor, and HRH1 inhibitor; or selected from the group consisting of: ADRB2 inhibitor, HRH1, inhibitor and TACR3 inhibitor. In a further embodiment, the method of treating, method of suppressing, pharmaceutical composition, or pharmaceutical kit, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the initial concentration of the inhibitor is in the range of between 1-10 uM (or each of the inhibitors of the combination at concentrations in the range of between 1-10 uM), such as at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uM, in testing and/or evaluating said inhibitor, or combination of inhibitors, regarding whether effective, or therapeutically effective, in suppressing an enhanced downstream signaling from a CXCR4-GPCRx heteromer. Materials and Methods Reagents BAY 60-6583, CGS 21680, C5a, acetylcholine, motilin, neurotensin, senktide, salmeterol, Cl-IB-MECA, salmon calcitonin, BQ-3020, octreotide, VU0255035, cetirizine, pyrilamine, MA-2029, meclinertant, and SSR 146977 were purchased from Tocris Bioscience (Ellisville, MO, USA). Galanin, endothelin-1, histamine, Prostaglandin E2 (PGE2), SRIF-14 (somatostatin), and bethanechol were purchased from Sigma-Aldrich (St Louis, MO, USA). CXCL12, vasoactive intestinal peptide (VIP), and CCL2 were purchased from R&D systems (Minneapolis, Minnesota, USA). Formoterol, carvedilol, oxybutynin, bosentan, hydroxyzine, and loratadine were from Prestwick Chemical (Illkirch, France). Apelin-13, roxithromycin, AMD3100, umeclidinium were obtained from Cayman Chemical Company (Ann Arbor, MI, USA), Selleckchem (Huston, TX, USA), Medchem express (Princeton, NJ, USA), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Construction of Adenoviral Vectors Containing GPCR cDNAs. Vectors containing BiFC fragments, pCS2+VNm10 and pBiFC-VC155, were obtained from Chang-Deng Hu (Hu et al., 2002) and James Smith (Saka et al., 2007), respectively. The VNm10 is a Venus VN154 variant containing L46F and L64F mutations. To construct pAdBiFC-VN and pAdBiFC-VC vectors, DNAs containing VNm10 and VC155 were amplified by PCR and cloned into pShuttle-CMV pAdBiFC-VN and pAdBiFC-VC vectors were obtained through homologous recombination between pAdEasy-1 and either pShuttle-CMV-VNm10 or pShuttle-CMV-VC155 using AdEasy vector system according to the manufacturer's instructions (Qbiogene, Carlsbad, CA). Human GPCR cDNA clones were obtained from the Missouri S&T cDNA Resource Center (Rolla, MO, USA). GPCR cDNAs were amplified by PCR and cloned into pDONR201 vector. Adenoviruses encoding GPCR, GPCR-VN, GPCR-VC, and GPCR-EGFP were obtained through in vitro LR recombination between entry clones containing GPCR and either pAdHTS, pAdBiFC-VN, pAdBiFC-VC, or pAdHTS-GFPC vectors, and subsequent transfection into 293A cells using AdHTS system as described previously (Choi et al., 2012; Song et al., 2014). Cell Culture U-2 OS cells and MDA-MB-231 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and 293A cells were from Invitrogen (Carlsbad, CA, USA). U-2 OS cells, MDA-MB-231 cells, and 293A cells were grown in McCoy's 5A medium, RPMI 1640, and Modified Eagle's medium, respectively, in the presence of 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and 100 μg/ml of streptomycin. Cells were cultured with 5% CO2 at 37° C. BiFC Assay U-2 OS cells were seeded at a density of 3000 cells per well in a black 96-well clear-bottom plate in 100 μl of McCoy's 5A medium supplemented with 10% FBS. On the following day, the cells were transduced with 30 MOI each of adenoviruses encoding GPCR-VN and GPCR-VC. Three days post-transduction, cells were fixed with 2% formaldehyde and nuclei were stained with Hoechst 33342 (Invitrogen, Carlsbad, CA). Images were acquired using IN cell Analyzer 1000 and analyzed with IN Cell Developer ToolBox (GE Healthcare, Waukesha, WI). BiFC and nuclear images were visualized using a ×20 objective and 360-nm (Hoechst) and 480-nm excitation filters, and monitored through 460- and 535-nm emission filters, respectively, with a 61002 trichroic mirror. Co-Internalization Assay U-2 OS cells were seeded at a density of 3000 cells per well in a black 96-well clear-bottom plate in 100 μl of McCoy's 5A medium supplemented with 10% FBS. On the following day, the cells were co-transduced with adenoviruses encoding CXCR4-EGFP (10 MOI) and GPCR-VC or GPCR-VN (30 MOI). Two days post-transduction, cells were stimulated with GPCRx agonists for 30 min and fixed with 2% formaldehyde. Images were obtained using IN cell analyzer 2000 using an excitation wavelength of 480 nm and an emission wavelength of 535 nm. Calcium Mobilization Assay MDA-MB-231 human breast cancer cells were seeded at 20,000 cells per well in a black clear bottom 96-well plate (Corning Costar, #3340) in 100 μl of RPMI 1640 supplemented with 10% FBS. The next day, cells were co-transduced with 10 MOI of CXCR4 and 30 MOI of HA-VC, 10 MOI of HA-VC and 30 MOI of GPCRx, or 10 MOI of CXCR4 and 30 MOI of GPCRx. Adenoviruses encoding HA-VC were used to adjust the total amount of adenoviruses transduced. After 2 days, cells were washed twice with assay buffer (Hank's balanced salt solution without phenol red, supplemented with 0.1% BSA and 20 mM HEPES, pH7.4), and stained with 5 μg/ml of Cal-520 AM (AAT Bioquest, Sunnyvale, CA, USA) diluted in assay buffer for 2 hr. Cells were washed with assay buffer three times, and incubated at 37° C. for another 30 min. Plate was loaded on a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and GPCRx agonists were added. Intracellular Ca2+ mobilization was measured at 37° C. using an excitation wavelength of 490 nm and an emission wavelength of 525 nm. Antagonists or vehicles were added 30 min before agonist treatment. Internalization Inhibition Assay CXCR4-GFP expressed U-2 OS cells were seeded at a density of 5000 cells per well in a black 96-well clear-bottom plate in 100 μl of McCoy's 5A medium supplemented with 10% FBS. On the following day, the cells were transduced with adenoviruses encoding GPCRx (30 MOI). Two days post-transduction, cells were stimulated with SDF-1 and/or GPCRx agonists for 20 min and fixed with 4% paraformaldehyde. Images were obtained using IN cell analyzer 2500 using an excitation wavelength of 480 nm and an emission wavelength of 535 nm. Proliferation Assay PDCs were seeded in 384-well plate at 500 ea/well in 40 ul culture media. After overnight growth, the cells were cultured for 7 days in the presence of several dose of GPCRx antagonist or DMSO alone. After the 7-day incubation, 15 ul ATPlite (PerkinElmer, Cat. No. 6016739) was added into the each well and the plates were shaken for 5 minutes in an orbital shaker at 700 rpm. The luminescent signal was detected within 30 minutes at PerkinElmer TopCount detection instrument. The cell viability was calculated using the equation: Cell viability (%)=(OD of antagonist treatment/OD of DMSO only treatment)×100%. Proximity Ligation Assay in Cell CXCR4 overexpression cell lines, the U2OS-CXCR4 was infected with ADRB2 expressing adenovirus, Ad-ADRB2 at the dose of 0, 2.5, 10, 40 MOIs for 2 days. Proximity ligation assays (PLA) were performed as described previously (Brueggemann et al., 2014; Tripathi et al., 2014). To perform PLA, infected cells were fixed with 4% paraformaldehyde (PFA) on sixteen-well tissue culture slides. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santacruz, Sc-53534), Rabbit anti-ADRB2 (1:200, Thermoscientific, PA5-33333) at 37° C. for 1 h in a humidifying chamber. Slides were then washed and incubated (1 h at 37° C.) with secondary anti-rabbit and anti-mouse antibodies conjugated with plus and minus Duolink II PLA probes. Slides were washed again and then incubated with ligation-ligase solution (30 min at 37° C.) followed by incubation with amplification-polymerase solution (2 h at 37° C.). Slides were then mounted with minimal volume of Duolink II mounting medium with 4′,6-diamidino-2phenylindole (DAPI) for 15-30 min, and PLA signals [Duolink In Situ Detection Reagents Green (λ excitation/emission 495/527 nm) or Red (λ excitation/emission 575/623 nm)] were identified as fluorescent spots under a IN Cell analyzer 2500. Proximity Ligation Assay in PDC PDCs were seeded in 96-chamber slide at 100000 ea/well in 100 ul culture media. To perform PLA, PDCs were fixed with 4% paraformaldehyde (PFA) and were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santacruz, Sc-53534), Rabbit anti-ADRB2 (1:200, Thermoscientific, PA5-33333), Rabbit anti-CHRM1 (1:200, Ls bio, Ls-C313301) at 37° C. for 1 h in a humidifying chamber. Slides were then washed and incubated (1 h at 37° C.) with secondary anti-rabbit and anti-mouse antibodies conjugated with plus and minus Duolink II PLA probes. Slides were washed again and then incubated with ligation-ligase solution (30 min at 37° C.) followed by incubation with amplification-polymerase solution (2 h at 37° C.). Slides were then mounted with minimal volume of Duolink II mounting medium with 4′,6-diamidino-2phenylindole (DAPI) for 15-30 min, and PLA signals [Duolink In Situ Detection Reagents Green (λ excitation/emission 495/527 nm) or Red (λ excitation/emission 575/623 nm)] were identified as fluorescent spots under a IN Cell analyzer 2500. Proximity Ligation Assay in PDX To perform PLA with PDX samples, the glioblastoma patient derived FFPE samples were used (provided by Samsung Seoul hospital in Seoul, Korea). After FFPE sample were de-paraffinized and performed heat induced antigen retrieval for 15 minutes at 100° C. Slides were blocked with blocking solution provided by Duolink and incubated with rabbit anti-CXCR4 (1:200, Thermoscientific, PA3305), mouse anti-ADRB2 (1:200, Santacruz, Sc-271322), at 37° C. for 1 h in a humidifying chamber. The other process was same as described above (PLA with PDC). Chemotactic Migration Assay Transwell plates (8 μm pore size polycarbonate membrane, 6.5-mm diameter) were coated with 50 μg/mL collagen for 2 h at 37° C. (Corning Inc.). MDA-MB-231 cells were serum-starved 24 h and then plated in serum-free medium containing 0.5% BSA in the top chamber (20,000 cells/100 μL). Antagonists or inverse agonists were added to the cells 30 min before plating the cells in the transwell plates. Attractants were added in the bottom chamber. Antagonists or inverse agonists were also included in the bottom chamber. After 3 h at 37° C., cells on the top transwell membrane were removed using a cotton swab, fixed, and stained with 0.1% Crystal violet. Chemotaxis was quantified by counting the migrated cells on the lower surface of the membrane of 10 fields per chamber at 10× objective. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNA was extracted using TRIzol (Invitrogen) and cDNA was synthesized from 1 μg of total RNA after treatment with DNase I (Sigma). RT-qPCR was conducted using SensiFAST SYBR kit (Bioline). Primer sequences are as follows: (SEQ ID NO: 1)ADRB2-F: 5′-CTCTTCCATCGTGTCCTTCTAC-3′,(SEQ ID NO: 2)ADRB2-R: 5′-AATCTTCTGGAGCTGCCTTT-3′;(SEQ ID NO: 3)HRH1-F: 5′-CCTCTGCTGGATCCCTTATTTC-3′,(SEQ ID NO: 4)HRH1-R: 5′-GGTTCAGTGTGGAGTTGATGTA-3′;(SEQ ID NO: 5)CXCR4-F: 5′-CCACCATCTACTCCATCATCTTC-3′,(SEQ ID NO: 6)CXCR4-R: 5′-ACTTGTCCGTCATGCTTCTC-3′;(SEQ ID NO: 7)β-actin-F: 5′-GGAAATCGTGCGTGACATTAAG-3′,(SEQ ID NO: 8)β-actin-R: 5′-AGCTCGTAGCTCTTCTCCA-3′;(SEQ ID NO: 9)GAPDH-F: 5′-ATGACATCAAGAAGGTGGTGAA-3′,(SEQ ID NO: 10)GAPDH-R: 5′-GCTGTTGAAGTCAGAGGAGAC-3′. Threshold cycles (Ct) were calculated by QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Lentivirus Production pLenti CMV Hygro DEST (w117-1) was a gift from Eric Campeau & Paul Kaufman (Addgene plasmid #17454) (Campeau et al., 2009). CXCR4 cDNA was inserted into the lentiviral vector using LR recombination. Lentiviruses encoding CXCR4 were produced using ViraPower Lentiviral Expression Systems (Invirtogen). Construction of Stable Cell Lines Expressing CXCR4 Alone or CXCR4 and ADRB2 Heteromer To establish stable CXCR4 expressing cell lines, lentiviral stock (containing the packaged pLenti6-CXCR4 expression construct which inserted with CXCR4 gene in pLenti-CMV Hygro DEST (Addgene, #17454)), were produced by co-transfecting the ViraPower Lentiviral Packaging Mix (Invitrogen, K497500) and pLenti6-CXCR4 expression construct into 293FT producer cell line. Transduction of this lentiviral stock into A549 cell line was performed and followed by selection with hygromycin (100 μg/mL) and blasticitin (5 μg/mL). To establish stable CXCR4-ADRB2 heteromer expressing cell lines, lentiviral stock (containing the packaged pLenti6/V5-ADRB2 expression construct which inserted with ADRB2 gene in pLenti6/V5-DEST Gateway™ Vector (Invitrogen, V49610)), were produced by co-transfecting the ViraPower Packaging Mix and pLenti6/V5-ADRB2 expression construct into 293FT producer cell line. Transduction of this lentiviral stock into A549-CXCR4 cell line was performed and followed by selection with hygromycin (100 μg/mL) and blasticitin (5 μg/mL). Then clones resistant to antibiotics were selected and performed RT-qPCR and immunofluorescence to confirm the expression of the inserted gene, CXCR4 and ADRB2. Mouse Xenograft Model Five week-old, female Balb/c-nu/nu mice were obtained from Envigo (France) and maintained in specific pathogen-free animal facility. All protocols for animal use and euthanasia were approved by the Qubest Bio (South Korea) Animal Experimental Ethics Committee based on the Animal Protection Act. The 1×107cells of A549, A549-CXCR4 or A549-CXCR4-ADRB2 were suspended in 100 μL of phosphate-buffered saline (PBS) and were implanted by subcutaneously in axillary region between the clavicular and chest wall on the right side of the mouse. The tumor growth was monitored every third or fourth day by measuring the length (L) and width (W) of the tumor with an electronic caliper and calculating tumor volume in the basis of the following formula: Volume=0.5 LW2. Generation of CXCR4 or HRH1 Knockout Cells Using CRISPR/Cas9 System CRISPR guide RNAs targeting CXCR4 and HRH1, and non-targeting guide RNA cloned in lentiCRISPR v2 vector were purchased from GenScript (Piscataway, NJ) (Sanjana et al., 2014). The guide RNA sequences are as follows: (CXCR4 gRNA #1; (SEQ ID NO: 11))TGTTGGCTGCCTTACTACAT,(HRH gRNA #3; (SEQ ID NO: 12))CGATCAAGTCCGCCACCGAG,and(non-targeting gRNA; (SEQ ID NO: 13))ACGGAGGCTAAGCGTCGCAA. Lentiviruses were produced using ViraPower Lentiviral Expression Systems (Invirtogen). MDA-MB-231 cells and MDA-MB-231 cells overexpressing CXCR4 (MDACXCR4+) were transduced with lentiviruses encoding non-targeting gRNA, CXCR4 gRNA, or HRH1 gRNAs. Cells were selected with puromycin (3 μM) for 2 weeks and loss of CXCR4 and HRH1 was estimated with immunoblotting and calcium responses, respectively. For detecting CXCR4, anti-CXCR4 rabbit monoclonal antibody (Abcam, #ab124824) was used. Source of GPCRx Inhibitors Ulocuplumab was purchased from Creative Biolabs (Shirley, NY, USA). BKT140 was purchased from Chem Scene (Monmouth Junction, NJ, USA). Carazolol was purchased from Santacruz Biotech (Dallas, TX, USA). Osanetant was purchased from Axon Medchem, (Groningen, Netherland). M65 and CT-(8-32) (salmon) were from Bachem (San Diego, CA, USA). PACAP-(6-38), W54011, PMX205, PMX53, AC187, SB 222200, and Talnetant were from Tocris Bioscience (Ellisville, MO, USA). Propranolol, Prometazine, Cyproheptadine, Hydroxyzine, Ambrisentan and macitentan were from Prestwick Chemical (Illkirch, France), and Selleckchem (Huston, TX, USA), respectively. In Vivo Studies For the antitumor efficacy test using CXCR4 and ADRB2 inhibitor, A549-CXCR4-ADRB2 cell line overexpressing CXCR4 and ADRB2 in A549, lung cancer cell line was subcutaneously administered (1×107cell in 100 μL PBS/head) to BALB/c-nu. 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11857601 | BEST MODE FOR CARRYING OUT THE INVENTION Combination Therapy of Fusion Protein In an aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as active ingredients, a fusion protein dimer comprising a CD80 protein or a fragment thereof, and an IL-2 protein or a variant thereof, and an anticancer agent. Since the fusion protein dimer comprising an IL-2 protein and a CD80 protein increases the immune activity in the body, it may be used in combination with various anticancer treatment methods that have been conventionally used. Specifically, the conventional treatment method that can be used in combination may be selected from the group consisting of an anticancer chemotherapeutic agent for chemotherapy, a target anticancer agent, an anticancer virus, an antibody therapeutic agent, a cell therapeutic agent, an immune checkpoint inhibitor, and a combination thereof. As used herein, the term “anticancer chemotherapeutic agent” is also referred to as an antineoplastic agent or a cytotoxic agent. It is a generic term for drugs that exhibit anticancer activity mainly by acting directly on DNA to block DNA replication, transcription and translation processes, or by interfering with the synthesis of nucleic acid precursors in the metabolic pathway, and by inhibiting cell division. The antineoplastic agent exhibits cytotoxicity by acting not only on tumor cells but also on normal cells. The anticancer chemotherapeutic agent may be used in maintenance therapy. In addition, as used herein, the term “maintenance therapy” refers to treatment of cancer with drugs after initial anticancer treatment, and refers to a treatment method performed to prevent or delay recurrence of cancer. Specifically, an anticancer chemotherapeutic agent may be any one selected from the group consisting of an alkylating agent, a microtubule inhibitor, an anti-metabolite, and a topoisomerase inhibitor. The alkylating agent may be any one selected from the group consisting of Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, Lomustine, Dacarbazine, Cisplatin, Carboplatin, and Oxaliplatin. The microtubule inhibitor may be any one selected from the group consisting of Docetaxel, Velban, Oncovin, and Navelbine. The anti-metabolite may be any one selected from the group consisting of Fluorouracil, Capecitabine, Cytarabine, Gemcitabine, Fludarabine, Methotrexate, Pemetrexed, and Mercaptopurine. The topoisomerase inhibitor may be any one selected from the group consisting of Hycamtin, Camptosar, Vepesid, Paclitaxel, Blenoxane, Adriamycin, and Cerubidine. As used herein, the term “target anticancer agent” is a therapeutic agent that specifically kills cancer cells by blocking signals involved in the growth and development of cancer by targeting specific proteins or specific genetic changes that are frequently present only in cancer cells. It is classified into monoclonal antibodies that react outside the cell, and small molecule substances that act inside the cell. Monoclonal antibodies are anticancer agents that block cancer cell induction signals transmitted to the outside of cells, and act on initiation signals related to proliferation, death and the like; and small molecule substances act on complex signal transduction occurring inside the cells. Specifically, proteins to be targeted may be EGFR, VEGFR, CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR/FGFR family, MEK/RAF, HER2/Neu, Ubiquitin, JAK, ALK, PARP, TGFβR1, Proteasome, Bcl-2, C-Met, VR1, VR2, VR3, c-kit, AXL, RET, Braf, DNMT, CDK4/6, STING, and the like. The target anticancer agent may be any one selected from the group consisting of Cetuximab, Trastuzumab, Pertuzumab, Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, Aflibercept, Rituximab, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Nilotinib, Imatinib, Bosutinib, Galunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Pazopanib, Trametinib, Dabrafenib, Trastuzumab, Afatinib, Lapatinib, Neratinib, Lenalidomide, Ixazomib, Ruxolitinib, Lestaurtinib, Pacritinib, Cobimethinib, Selumetinib, Trametinib, Binimetinib, Alectinib, Crizotinib, Venetoclax, Crizotinib, Cabozantinib, Bemcentinib, Gilteritinib, Selpercatinib, Pralsetinib, Vemurafenib, Olaparib, Talazoparib, Niraparib, Rucaparib, Azacitidine, Decitabine, Guadecitabine, Abemaciclib, Ribociclib, Palbociclib, CDNs, SB11285, and DMXAA. As used herein, the term “epidermal growth factor receptor (EGFR)” is a cell membrane receptor that regulates cell growth, division, survival, and death. In various cancers, the expression of EGFR is increased in tumor tissues. It is known that tumor tissues with the increased EGFR are invasive, metastatic, and highly resistant to anticancer agents. The EGFR inhibitor may be a substance that inhibits the EGFR. In an embodiment, it may be Cetuximab, Trastuzumab, Pertuzumab, Gefitinib, Elotinib, or Panitumumab. As used herein, the term “vascular endothelial growth factor receptor (VEGFR)” is a cell membrane receptor of a vascular endothelial growth factor that induces angiogenesis, and a VEGFR inhibitor inhibits the angiogenesis to suppress tumor growth and metastasis. In an embodiment, the VEGFR inhibitor may be Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, or Aflibercept. As used herein, the term “CD20 (B lymphocyte antigen CD20)” is a protein expressed on the surface of B cells and is used as a target protein for the treatment of B cell lymphoma. The CD20 target inhibitor may be Rituximab or Obinutuzumab. As used herein, the term “CD38 (cluster of differentiation 38)” is a protein that regulates cell proliferation and death while acting as a signal transduction receptor in immune cells, and an inhibitor targeting it may be Daratumumab. As used herein, the term “RNAK-L (Receptor activator of nuclear factor kappa-B ligand)” is a RANK receptor expressed on the surface of osteoclasts, and when it is activated by binding to its ligand, it acts to cause bone destruction. The RANK-L inhibitor is mainly used for cancer patients suffering from bone metastasis or osteoporosis, and it may be specifically Denosumab. As used herein, the term “BTK (Bruton's tyrosine kinase)” is an enzyme involved in the proliferation of B cells and may develop into hematologic malignancy when overexpressed. In an embodiment, the BTK target inhibitor may be Ibrutinib. As used herein, the term “Bcr-abl” is a fusion protein that is highly expressed in chronic myelogenous leukemia patients, and is known to induce abnormal proliferation of blood cells. Specifically, the inhibitor of the protein may be Dasatinib, Nilotinib, Imatinib, or Bosutinib. As used herein, the term “tumor growth factor β receptor (TGFβR)” is a cell membrane receptor of a tumor growth factor, and regulates the growth, migration, differentiation, death and the like of epithelial cells and hematopoietic cells. The TGFβR target inhibitor includes, but is not limited to, Galunisertib, Vactosertib or the like. As used herein, the term “PDGFR (platelet derived growth factor receptor)” is a cell membrane receptor of PDGF that is frequently expressed in cancer cells, and is known to regulate cancer growth, metastasis, and drug resistance by participating in angiogenesis. FGFR (Fibroblast growth factor receptor) is a receptor of fibroblast growth factor (FGF), and regulates various biological processes including cell growth, differentiation, migration, and the like. The FGFR gene is easily mutated, and these variants are commonly observed in breast cancer, uterine cancer, ovarian cancer, cervical cancer, and the like. The Inhibitor targeting PDGFR or FGFR may be Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Lenvatinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Axitinib, or Pazopanib. As used herein, the term “MEK/RAF” is an intracellular signaling mediator involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, cell migration, and the like, and is overactivated in cancer cells. The inhibitor targeting MEK/RAF may be Trametinib or Dabrafenib. As used herein, the term “HER-2/neu (human epidermal growth factor receptor 2) regulates cell proliferation through activation of PI3K/AkT. It is known that it is overexpressed in metastatic breast cancer, and ovarian cancer and the like, and induces resistance against anticancer agents. The Her2/neu target anticancer agent may be Trastuzumab, Afatinib, Lapatinib, or Neratinib. As used herein, the term “ubiquitin” maintains cell homeostasis by binding to other proteins and inducing proteolysis (ubiquitin-proteasome system, UPS) by proteasome, which is a proteolytic enzyme. Abnormal expression or activity of the UPS is observed in various tumors, and its inhibitor exhibits anticancer activity. Specifically, the inhibitor targeting ubiquitin or proteasome may be Lenalidomide or Ixazomib. As used herein, the term “JAK (Janus kinase)” is an upstream protein of STAT, which is a transcription factor that regulates cell proliferation, cell survival, cell migration, and immune response. A JAK inhibitor is known to decrease cell proliferation and induce cell death by inhibiting the activity of STAT. The JAK target inhibitor may be Ruxolitinib, Lestaurtinib, or Pacritinib. As used herein, the term “MAP2K (Mitogen-activated protein kinase kinase)” is an intracellular signaling mediator involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, cell migration and the like by phosphorylating MAPK, and it is overactivated in cancer cells. The MAP2K target inhibitor may be Cobimethinib, Selumetinib, Trametinib, or Binimetinib. As used herein, the term “ALK (Anaplastic lymphoma kinase)” is a signaling mediator that promotes cell proliferation, cell migration and angiogenesis and inhibits cell death; and it is overactivated in various cancer tissues. The ALK target inhibitor may be Alectinib or Crizotinib. As used herein, the term “Bcl-2” is a protein that inhibits cell death, and it is overexpressed or overactivated in various cancer tissues. The inhibitor targeting Bcl-2 may be Venetoclax. As used herein, the term “C-Met” is a receptor of hepatocyte growth factor (HGF), and activates signal transduction related to cell growth, formation, motility, survival, angiogenesis and the like. The C-Met target anticancer agent may be Crizotinib or Cabozantinib. As used herein, the term “VR (vanilloid receptor)” is also known as TRPV (Transient receptor potential vanilloid), and exists in the form of VR1, VR2, VR3, VR4, VR5, and VR6. VR is known to regulate proliferation, death, migration, infiltration and angiogenesis of cancer cells at each stage in the process of cancer progression. As used herein, the term “c-kit” is also known as CD117, and induces signal transduction that activates cell survival, proliferation and differentiation. c-kit is a proto-oncogene, and overexpression or mutation of its gene is related to the onset of cancer. As used herein, the term “AXL (tyrosine-protein kinase receptor UFO)” is a tyrosine kinase receptor present on the cell surface, and mediates signal transduction involved in cell proliferation and survival. It is known to be involved in anticancer agent resistance in anticancer treatment. In an embodiment, the AXL target anticancer agent may be Bemcentinib or Gilteritinib. As used herein, the term “RET (REarragned during transfection)” is a receptor that mediates signals involved in cell proliferation, cell death, and survival; and mutations in RET are known to be involved in cancer development. The RET target inhibitor may be Selpercatinib or Pralsetinib, but is not limited thereto. As used herein, the term “Braf” is a MAPK signaling mediator involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, cell migration, and the like, and genetic mutations are observed in cancer cells. The inhibitor targeting Braf may be Vemurafenib. As used herein, the term “PARP (Poly[ADP-ribose]polymerase)” is a protein that recognizes damaged DNA in the nucleus and is activated, and then activates a DNA repair-related protein. The PARP target inhibitor suppresses proliferation of cancer cells by inhibiting DNA repair of cancer cells. In an embodiment, the PARP target inhibitor may be Olaparib, Talazoparib, Niraparib, or Rucaparib. As used herein, the term “DNA methyltransferase (DNMT)” is an enzyme that transfers a methyl group to DNA, and expression of a gene is inhibited through the above process. The DMNT target inhibitor exhibits anticancer activity by inhibiting hypermethylation of the cancer suppressor gene and inducing normal expression of the cancer suppressor gene. In an embodiment, the DNMT target inhibitor may be Azacitidine, Decitabine, or Guadecitabine. As used herein, the term “CDK (cyclin dependent kinase) 4/6” is a protein that regulates the cell cycle and promotes cell growth, and is overactivated in the development and progression stages of various malignant tumors. The CDK4/6 target inhibitor exhibits anticancer activity by inhibiting cell cycle of cancer cells, inhibiting cell proliferation, and inducing cell death. The CDK4/6 target inhibitor may be Abemaciclib or Palbociclib. As used herein, the term “STING (Stimulator of Interferon Genes)” is an in vivo sensor that recognizes DNA fragments derived from cancer cells, and activates immune cells in the body such as dendritic cells by stimulating interferon genes. The STING agonist exhibits an immune enhancing effect and a cancer angiogenesis inhibitory effect. For example, the STING agonist may be CDNs, SB11285, DMXAA, or the like. As used herein, the term “anticancer virus therapeutic agent” is a therapeutic agent that kills cancer by inserting a specific gene targeting cancer cells into a virus that is capable of proliferation and has infectivity. The anticancer virus therapeutic agent may be Talimogenem or Laherparepvec. As used herein, the term “antibody therapeutic agent” is a therapeutic agent that exhibits anticancer effect by using an antibody that recognizes a specific protein of cancer cells as an antigen. The antibody therapeutic agent may be Trastuzumab, Emtansine, Emtansine, Rituximab, Ibritumomab, Tositumomab, Brentuximab, Ofatumumab, Obinutuzumab, Necitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Ipilimumab, or the like. As used herein, the term “immune cell therapeutic agent” is a therapeutic agent that exhibits anticancer effect by activating an immune response in the body using immune cells such as dendritic cells, natural killer cells, and T cells. The immune cell therapeutic agent is used after extracting and potentiating immune cells in the body or genetically engineering them to be reinjected into the body. The representative immune cell therapeutic agent includes T cell receptor-modified T cells (TCR-T), chimeric antigen receptor-modified T cells (CAR-T), and the like. Specifically, it may be Tisagenlecleucel or Axicabtagene Ciloleucel, but is not limited thereto. As used herein, the term “immune checkpoint inhibitor” is a substance that inhibits the activity of an immune checkpoint protein that inhibits differentiation, proliferation, and activity of immune cells, and it is known to eliminate cancer cells by preventing them from exerting the function of evading the immune system. The immune checkpoint inhibitor may be any one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-TIM3 antibody, an anti-GAL9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-BTLA antibody, and an anti-TIGIT antibody. In an embodiment, the immune checkpoint inhibitor may be Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, Duralumab and the like, but is not limited thereto. As used herein, the term “ADC (antibody drug conjugate)” is a therapeutic agent that chemically binds an antibody and a cytotoxic drug to exhibit high anticancer effect through target delivery. It may be Gemtuzumab-Ozogamicin, Brentuximab-Vedotin, Trastuzumab-Emtansine, Inotuzumab-Ozogamicin, Eribulin-Mesylate, and the like. The fusion protein dimer comprising an IL-2 protein and a CD80 protein may be used in combination with an anticancer vaccine or the like. In addition, an anticancer agent may be used not only in combination with the anticancer agent described above, but also in combination with an anticancer vaccine or the like. Preferably, the anticancer agent may be any one selected from the group consisting of Cisplatin, Oxaliplatin, ALTIMA, Axitinib (VR1,2,3, PDGFR, c-kit), Galunisertib (TGFβR1), Lenvatinib (VR1,2,3), Ramucirumab (VR2), Cabozatinib (c-Met, VR2, AXL, RET), Olaparib (PARP), Guadecitabine (DNMT), Docetaxel, Paclitaxel, Pemetrexed, Vemurafenib (Braf), Abemaciclib (CDK4/6), Cetuximab (EGFR), Durvalumab (PD-L1), Trastuzumab (Her2), DMXAA, NK cell, T cell, and Keytruda (PD-1). In addition, the anticancer agent may include one or more anticancer agents. Specifically, the fusion protein dimer may be used commonly together with two anticancer agents. As an example, it may be an anticancer chemotherapeutic agent and a target anticancer agent; an anticancer chemotherapeutic agent and an anticancer virus; a target anticancer agent and an antibody therapeutic agent; an anticancer chemotherapeutic agent and a cell therapeutic agent; and an anticancer chemotherapeutic agent and an immune checkpoint inhibitor. In addition, it may be a target anticancer agent and an anticancer virus; a target anticancer agent and an antibody therapeutic agent; a target anticancer agent and a cell therapeutic agent; a target anticancer agent and an immune checkpoint inhibitor. In addition, it may be an anticancer virus and an antibody therapeutic agent; an anticancer virus and a cell therapeutic agent; and an anticancer virus and an immune checkpoint inhibitor. In addition, it may be an antibody therapeutic agent and a cell therapeutic agent; and an antibody therapeutic agent and an immune checkpoint inhibitor. In addition, the fusion protein dimer may be used together with three anticancer agents. In addition to the two anticancer agents, a different anticancer agent may be further included and used. In an embodiment, the anticancer agent may be an anticancer chemotherapeutic agent and a target anticancer agent; an anticancer chemotherapeutic agent and an immune checkpoint inhibitor; or an anticancer chemotherapeutic agent, a target anticancer agent and an immune checkpoint inhibitor. Use of Fusion Protein Dimer in Anticancer Maintenance Therapy In another aspect of the present invention, there is provided a composition for anticancer maintenance therapy, comprising, as an active ingredient, a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof. As described above, “maintenance therapy” refers to treating cancer after initial anticancer treatment. In particular, it is a treatment method that increases the effect of cancer treatment by preventing or delaying the recurrence of cancer. Here, it may further include at least one anticancer agent for maintenance therapy. Here, the anticancer agent is as described above. Kit Comprising Fusion Protein Dimer In another aspect of the present invention, there is provided a kit for preventing or treating cancer, comprising, as active ingredients, a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof, and an anticancer agent. In another aspect of the present invention, there is provided a kit for anticancer maintenance therapy, comprising, as active ingredients, a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof, and an anticancer agent. Fusion Protein Comprising IL-2 Protein and CD80 Protein As used herein, the term “IL-2” or “interleukin-2”, unless otherwise stated, refers to any wild-type IL-2 obtained from any vertebrate source, including mammals, for example, primates (such as humans) and rodents (such as mice and rats). IL-2 may be obtained from animal cells, and also includes one obtained from recombinant cells capable of producing IL-2. In addition, IL-2 may be wild-type IL-2 or a variant thereof. In the present specification, IL-2 or a variant thereof may be collectively expressed by the term “IL-2 protein” or “IL-2 polypeptide.” IL-2, an IL-2 protein, an IL-2 polypeptide, and an IL-2 variant specifically bind to, for example, an IL-2 receptor. This specific binding may be identified by methods known to those skilled in the art. An embodiment of IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Here, IL-2 may also be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence, and may have the amino acid sequence of SEQ ID NO: 10. Here, IL-2 may be used under a concept encompassing a fragment of wild-type IL-2 in which a portion of N-terminus or C-terminus of the wild-type IL-2 is truncated. In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids are truncated from N-terminus of a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids are truncated from C-terminus of a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. As used herein, the term “IL-2 variant” refers to a form in which a portion of amino acids in the full-length IL-2 or the above-described fragment of IL-2 is substituted. That is, an IL-2 variant may have an amino acid sequence different from wild-type IL-2 or a fragment thereof. However, an IL-2 variant may have activity equivalent or similar to the wild-type IL-2. Here, “IL-2 activity” may, for example, refer to specific binding to an IL-2 receptor, which specific binding can be measured by methods known to those skilled in the art. Specifically, an IL-2 variant may be obtained by substitution of a portion of amino acids in the wild-type IL-2. An embodiment of the IL-2 variant obtained by amino acid substitution may be obtained by substitution of at least one of the 38th, 42nd, 45th, 61st, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. Specifically, the IL-2 variant may be obtained by substitution of at least one of the 38th, 42nd, 45th, 61st, or 72ndamino acid in the amino acid sequence of SEQ ID NO: 10 with another amino acid. In addition, when IL-2 is in a form in which a portion of N-terminus in the amino acid sequence of SEQ ID NO: 35 is truncated, the amino acid at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, its IL-2 variant may be obtained by substitution of at least one of 58th, 62nd, 65th, 81st, or 92ndamino acid in the amino acid sequence of SEQ ID NO: 35 with another amino acid. These amino acid residues correspond to the 38th, 42nd, 45th, 61st, and 72ndamino acid residues in the amino acid sequence of SEQ ID NO: 10, respectively. According to an embodiment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted as long as such IL-2 variant maintains IL-2 activity. According to another embodiment, one to five amino acids may be substituted. In an embodiment, an IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may be obtained by substitution of the 38thand 42ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38thand 45thamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38thand 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38thand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 42ndand 45thamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 42ndand 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 42ndand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 45thand 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 45thand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 61stand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, an IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be obtained by substitution of the 38th, 42nd, and 45thamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 42nd, and 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 42nd, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 45th, and 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 45th, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 61stand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 42nd, 45th, and 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 42nd, 45th, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 45th, 61stand 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, an IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may be obtained by substitution of the 38th, 42nd, 45th, and 61stamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 42nd, 45th, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 45th, 61st, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of the 38th, 42nd, 61st, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. In addition, in an embodiment, the IL-2 variant may be obtained by substitution of 42nd, 45th, 61st, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10. Furthermore, an IL-2 variant may be in a form in which five amino acids are substituted. Specifically, the IL-2 variant may be obtained by substitution of each of the 38th, 42nd, 45th, 61st, and 72ndamino acids in the amino acid sequence of SEQ ID NO: 10 with another amino acid. Here, the “another amino acid” introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, regarding amino acid substitution for the IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38thamino acid cannot be substituted with arginine, the 42ndamino acid cannot be substituted with phenylalanine, the 45thamino acid cannot be substituted with tyrosine, the 61stamino acid cannot be substituted with glutamic acid, and the 72ndamino acid cannot be substituted with leucine. Regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38thamino acid, arginine, may be substituted with an amino acid other than arginine. Preferably, regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 38thamino acid, arginine, may be substituted with alanine (R38A). Regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 42ndamino acid, phenylalanine, may be substituted with an amino acid other than phenylalanine. Preferably, regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 42ndamino acid, phenylalanine, may be substituted with alanine (F42A). Regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 45thamino acid, tyrosine, may be substituted with an amino acid other than tyrosine. Preferably, regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 45thamino acid, tyrosine, may be substituted with alanine (Y45A). Regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 61stamino acid, glutamic acid, may be substituted with an amino acid other than glutamic acid. Preferably, regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 61stamino acid, glutamic acid, may be substituted with arginine (E61R). Regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 72ndamino acid, leucine, may be substituted with an amino acid other than leucine. Preferably, regarding amino acid substitution for an IL-2 variant, in the amino acid sequence of SEQ ID NO: 10, the 72ndamino acid, leucine, may be substituted with glycine (L72G). Specifically, an IL-2 variant may be obtained by at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G, in the amino acid sequence of SEQ ID NO: 10. Specifically, an IL-2 variant may be obtained by amino acid substitutions at two, three, four, or five positions among the positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G. In addition, an IL-2 variant may be in a form in which two amino acids are substituted. Specifically, an IL-2 variant may be obtained by the substitutions, R38A and F42A. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A and Y45A. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A and Y45A. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, E61R and L72G. Furthermore, an IL-2 variant may be in a form in which three amino acids are substituted. Specifically, an IL-2 variant may be obtained by the substitutions, R38A, F42A, and Y45A. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, F42A, and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, F42A, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, Y45A, and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, Y45A, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A, Y45A, and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A, Y45A, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A, E61R, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, Y45A, E61R, and L72G. In addition, an IL-2 variant may be in a form in which four amino acids are substituted. Specifically, an IL-2 variant may be obtained by the substitutions, R38A, F42A, Y45A, and E61R. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, F42A, Y45A, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, F42A, E61R, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, R38A, Y45A, E61R, and L72G. In addition, in an embodiment, an IL-2 variant may be obtained by the substitutions, F42A, Y45A, E61R, and L72G. Furthermore, an IL-2 variant may be obtained by the substitutions, R38A, F42A, Y45A, E61R, and L72G. Preferably, an embodiment of the IL-2 variant may contain which are any one selected from the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A/F42A (b) R38A/F42A/Y45A (c) R38A/F42A/E61R (d) R38A/F42A/L72G Here, when IL-2 has the amino acid sequence of SEQ ID NO: 35, an amino acid substitution may be present at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: 10. In addition, even when IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, an amino acid substitution may be present at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: 10. Specifically, an IL-2 variant may have the amino acid sequence of SEQ ID NO: 6, 22, 23, or 24. In addition, an IL-2 variant may be characterized by having low in vivo toxicity. Here, the low in vivo toxicity may be a side effect caused by binding of IL-2 to the IL-2 receptor alpha chain (IL-2Rα). Various IL-2 variants have been developed to ameliorate the side effect caused by binding of IL-2 to IL-2Rα, and such IL-2 variants may be those disclosed in U.S. Pat. No. 5,229,109 and Korean Patent No. 1667096. In particular, IL-2 variants described in the present application have low binding ability for the IL-2 receptor alpha chain (IL-2Rα) and thus have lower in vivo toxicity than the wild-type IL-2. As used herein, the term “CD80”, also called “B7-1”, is a membrane protein present in dendritic cells, activated B cells, and monocytes. CD80 provides co-stimulatory signals essential for activation and survival of T cells. CD80 is known as a ligand for the two different proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 is composed of 288 amino acids, and may specifically have the amino acid sequence of SEQ ID NO: 11. In addition, as used herein, the term “CD80 protein” refers to the full-length CD80 or a CD80 fragment. As used herein, the term “CD80 fragment” refers to a cleaved form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. An embodiment of the CD80 fragment may be obtained by elimination of the 1stto 34thamino acids from N-terminus which are a signal sequence of CD80. Specifically, an embodiment of the CD80 fragment may be a protein composed of the 35thto 288thamino acids in SEQ ID NO: 11. In addition, an embodiment of the CD80 fragment may be a protein composed of the 35thto 242ndamino acids in SEQ ID NO: 11. In addition, an embodiment of the CD80 fragment may be a protein composed of the 35thto 232ndamino acids in SEQ ID NO: 11. In addition, an embodiment of the CD80 fragment may be a protein composed of the 35thto 139thamino acids in SEQ ID NO: 11. In addition, an embodiment of the CD80 fragment may be a protein composed of the 142ndto 242ndamino acids in SEQ ID NO: 11. In an embodiment, a CD80 fragment may have the amino acid sequence of SEQ ID NO: 2. In addition, the IL-2 protein and the CD80 protein may be attached to each other via a linker or a carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be attached to each other via a linker or a carrier. In the present description, the linker and the carrier may be used interchangeably. The linker links two proteins. An embodiment of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, an Fc domain of an immunoglobulin, or the like. Here, the Fc domain of immunoglobulin refers to a protein that contains heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, and does not contain heavy and light chain variable regions and light chain constant region 1 (CH1) of an immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and may preferably be IgG4. Here, Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4. In addition, the Fc domain of an immunoglobulin may be an Fc domain variant as well as wild-type Fc domain. In addition, as used herein, the term “Fc domain variant” may refer to a form which is different from the wild-type Fc domain in terms of glycosylation pattern, has a high glycosylation as compared with the wild-type Fc domain, or has a low glycosylation as compared with the wild-type Fc domain, or a deglycosylated form. In addition, an aglycosylated Fc domain is included therein. The Fc domain or a variant thereof may be adapted to have an adjusted number of sialic acids, fucosylations, or glycosylations, through culture conditions or genetic manipulation of a host. In addition, glycosylation of the Fc domain of an immunoglobulin may be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be in a mixed form of respective Fc regions of immunoglobulins, IgG, IgA, IgE, IgD, and IgM. In addition, the Fc domain variant may be in a form in which some amino acids of the Fc domain are substituted with other amino acids. An embodiment of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12. The fusion protein may have a structure in which, using an Fc domain as a linker (or carrier), a CD80 protein and an IL-2 protein, or an IL-2 protein and a CD80 protein are linked to N-terminus and C-terminus of the linker or carrier, respectively. Linkage between N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 may optionally be achieved by a linker peptide. Specifically, a fusion protein may consist of the following structural formula (I) or (II): N′-X-[linker(1)]n-Fc domain-[linker(2)]m-Y-C′ (I) N′-Y-[linker(1)]n-Fc domain-[linker(2)]m-X-C′ (II) Here, in the structural formulas (I) and (II), N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is a CD80 protein, Y is an IL-2 protein, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1. Preferably, the fusion protein may consist of the structural formula (I). The IL-2 protein is as described above. In addition, the CD80 protein is as described above. According to an embodiment, the IL-2 protein may be an IL-2 variant with one to five amino acid substitutions as compared with the wild-type IL-2. The CD80 protein may be a fragment obtained by truncation of up to about 34 contiguous amino acid residues from the N-terminus or C-terminus of the wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having the activity of binding to the T cell surface receptors CTLA-4 and CD28. Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. According to another embodiment, the fusion protein includes a polypeptide having a sequence identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. Here, the identity is, for example, percent homology, and may be determined through homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI). The peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5 to 80 contiguous amino acids, 20 to 60 contiguous amino acids, 25 to 50 contiguous amino acids, or 30 to 40 contiguous amino acids. In an embodiment, the peptide linker (1) may consist of 30 amino acids. In addition, the peptide linker (1) may contain at least one cysteine. Specifically, the peptide linker (1) may contain one, two, or three cysteines. In addition, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In an embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3. The peptide linker (2) may consist of 1 to 50 contiguous amino acids, 3 to 30 contiguous amino acids, or 5 to 15 contiguous amino acids. In an embodiment, the peptide linker (2) may be (G4S)n(where n is an integer of 1 to 10). Here, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, the peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5. In another aspect of the present invention, there is provided a dimer obtained by binding of two fusion proteins, each of which comprises an IL-2 protein and a CD80 protein. The fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof is as described above. Here, the binding between the fusion proteins constituting the dimer may be achieved by, but is not limited to, a disulfide bond formed by cysteines present in the linker. The fusion proteins constituting the dimer may be the same or different fusion proteins from each other. Preferably, the dimer may be a homodimer. An embodiment of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9. Pharmaceutical Use The pharmaceutical composition for treating or preventing cancer of the present invention, the composition comprising, as an active ingredient, a fusion protein comprising an IL-2 protein and a CD80 protein, and an anticancer agent may enhance efficacy for treating and/or preventing cancer. The fusion protein comprising an IL-2 protein and a CD80 protein, or the fusion protein dimer where the two fusion proteins are attached is as described above. The cancer may be selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. A preferred dose of the pharmaceutical composition varies depending on the patient's condition and body weight, severity of disease, form of drug, route and duration of administration and may be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing cancer of the present invention, the active ingredient may be contained in any amount (effective amount) depending on application, dosage form, blending purpose, and the like, as long as the active ingredient can exhibit anticancer activity. A conventional effective amount thereof will be determined within a range of 0.001 wt % to 20.0 wt % by weight, based on the total weight of the composition. Here, the term “effective amount” refers to an amount of an active ingredient capable of inducing an anticancer effect. Such an effective amount can be experimentally determined within the scope of common knowledge of those skilled in the art. As used herein, the term “treatment” may be used to mean both therapeutic and prophylactic treatment. Here, prophylaxis may be used to mean that a pathological condition or disease of an individual is alleviated or mitigated. In an embodiment, the term “treatment” includes both application or any form of administration for treating a disease in a mammal, including a human. In addition, the term includes inhibiting or slowing down a disease or disease progression; and includes meanings of restoring or repairing impaired or lost function so that a disease is partially or completely alleviated; stimulating inefficient processes; or alleviating a serious disease. As used herein, the term “efficacy” refers to capacity that can be determined by one or parameters, for example, survival or disease-free survival over a certain period of time such as one year, five years, or ten years. In addition, the parameter may include inhibition of size of at least one tumor in an individual. Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Thus, “enhanced efficacy” (for example, improvement in efficacy) may be due to enhanced pharmacokinetic parameters and improved efficacy, which may be measured by comparing clearance rate and tumor growth in test animals or human subjects, or by comparing parameters such as survival, recurrence, or disease-free survival. As used herein, the term “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount of a compound or composition effective to prevent or treat the disease in question, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause adverse effects. A level of the effective amount may be determined depending on factors including the patient's health condition, type and severity of disease, activity of drug, the patient's sensitivity to drug, mode of administration, time of administration, route of administration and excretion rate, duration of treatment, formulation or simultaneously used drugs, and other factors well known in the medical field. In an embodiment, the therapeutically effective amount means an amount of drug effective to treat cancer. Here, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as the carrier is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax, and inert solid may be contained as the carrier. A pharmaceutically acceptable adjuvant (buffer, dispersant) may also be contained in the pharmaceutical composition. Specifically, by including a pharmaceutically acceptable carrier in addition to the active ingredient, the pharmaceutical composition may be prepared into a parenteral formulation depending on its route of administration using conventional methods known in the art. Here, the term “pharmaceutically acceptable” means that the carrier does not have more toxicity than the subject to be applied (prescribed) can adapt while not inhibiting activity of the active ingredient. When the pharmaceutical composition is prepared into a parenteral formulation, it may be made into preparations in the form of injections, transdermal patches, nasal inhalants, or suppositories with suitable carriers according to methods known in the art. In a case of being made into injections, sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof may be used as a suitable carrier; and an isotonic solution, such as Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, and 5% dextrose, or the like may preferably be used. Formulation of pharmaceutical compositions is known in the art, and reference may specifically be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. This document is considered part of the present description. A preferred dose of the pharmaceutical composition may range from 0.01 μg/kg to 10 g/kg, or 0.01 mg/kg to 1 g/kg, per day, depending on the patient's condition, body weight, sex, age, severity of the patient, and route of administration. The dose may be administered once a day or may be divided into several times a day. Such a dose should not be construed as limiting the scope of the present invention in any aspect. Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of the present application may further contain any compound or natural extract, which has already been validated for safety and is known to have anticancer activity or a therapeutic effect on an infectious disease, so as to boost or reinforce anticancer activity. Use of Composition Comprising Fusion Protein Dimer and Anticancer Agent In another aspect of the present invention, there is provided a use of a composition for combination administration comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof, and an anticancer agent for the treatment of cancer disease. In another aspect of the present invention, there is provided a use of a composition for combination administration comprising a fusion protein dimer comprising an IL-2 protein and a CD80 protein, and an anticancer agent for enhancing the therapeutic effect of cancer disease. In another aspect of the present invention, there is provided a use of a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof for maintenance therapy. Here, an anticancer agent may be further included. In another aspect of the present invention, there is provided a method for treating cancer disease and/or a method for enhancing therapeutic effect, comprising a step of administering, to a subject, a fusion protein comprising an IL-2 protein and a CD80 protein or a fusion protein dimer in which the two fusion proteins are bound to each other, and an anticancer agent. The subject may be a subject suffering from cancer. In addition, the subject may be a mammal, preferably a human. The fusion protein comprising an IL-2 protein and a CD80 protein or the fusion protein dimer in which the two fusion proteins are bound to each other is as described above. Route of administration, dose, and frequency of administration of the fusion protein or the fusion protein dimer may vary depending on the patient's condition and the presence or absence of side effects, and thus the fusion protein or the fusion protein dimer may be administered to a subject in various ways and amounts. The optimal administration method, dose, and frequency of administration may be selected in an appropriate range by those skilled in the art. In addition, the fusion protein or the fusion protein dimer may be administered in combination with other drugs (for example, the above described anticancer agent) or physiologically active substances whose therapeutic effect is known with respect to a disease to be treated, or may be formulated in the form of combination preparations with other drugs. Due to IL-2 activity, the fusion protein in an embodiment of the present invention can activate immune cells such as natural killer cells. Thus, the fusion protein can be effectively used for cancer disease. In particular, it was identified that as compared with the wild type, an IL-2 variant with two to five amino acid substitutions, in particular, an IL-2 variant that contains amino acid substitutions at two, three, four, or five positions among the positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, has low binding ability for the IL-2 receptor alpha chain and thus exhibits improved characteristics with respect to pharmacological side effects of conventional IL-2. Thus, such an IL-2 variant, when used alone or in the form of a fusion protein, can decrease incidence of vascular (or capillary) leakage syndrome (VLS), which is a conventionally known problem of IL-2. Pharmaceutical Composition Comprising, as Active Ingredients, IL-2 Protein or Variant Thereof, CD80 Protein or Variant Thereof, and Anticancer Agent In another aspect of the present invention, there is provided a composition for treating cancer, comprising, as active ingredients, IL-2 or a variant thereof, a CD80 protein or a variant thereof, and an anticancer agent. Here, the IL-2 or variant thereof is as described above. In addition, the IL-2 or variant thereof may further include an immunoglobulin Fc region. Here, the IL-2 or variant thereof may bind to the N-terminus or C-terminus of the Fc region. In an embodiment, the IL-2 or variant thereof may bind to the C-terminus of the Fc region. In addition, as described above, the variant of IL-2 may be a form in which two amino acids are substituted or a form in which three amino acids are substituted. Here, the IL-2 or variant thereof may be directly bound to the Fc region, but may be bound through a peptide linker. Here, the peptide linker may be any one of the linkers described above. In addition, the CD80 protein or variant thereof is as described above. In addition, the CD80 or variant thereof may further include an immunoglobulin Fc region. Here, the CD80 or variant thereof may bind to the N-terminus or C-terminus of the Fc region. Here, the CD80 may be in the form of a fragment, and may be a fragment of CD80 including a V domain. In an embodiment, the CD80 or variant may bind to the N-terminus of the Fc region. In addition, the variant of CD80 may be a variant in various forms as long as its activity is maintained. In addition, the anticancer agent may be any one selected from various types of the anticancer agents described above. MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail by way of the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto. I. Preparation of Fusion Protein Preparation Example 1. Preparation of hCD80-Fc-IL-2 Variant (2M): GI101 In order to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 8) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) having two amino acid substitutions, in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 9. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI101”. Purification was carried out using chromatography containing Mab Select SuRe protein A resin. The fusion protein was bound thereto under a condition of 25 mM Tris, 25 mM NaCl, pH 7.4. Then, elution was performed with 100 mM NaCl and 100 mM acetic acid at pH 3. 20% 1 M Tris-HCl at pH 9 was placed in a collection tube, and then the fusion protein was collected. For the collected fusion protein, the buffer was exchanged through dialysis with PBS buffer for 16 hours. Thereafter, absorbance at 280 nm wavelength was measured, over time, with size exclusion chromatography using a TSKgel G3000SWXL column (TOSOH Bioscience), to obtain a highly concentrated fusion protein. Here, the isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition, and stained with Coomassie Blue to check its purity (FIG.6). It was identified that the fusion protein was contained at a concentration of 2.78 mg/ml when detected with NanoDrop (FIG.7). In addition, the results obtained by analysis using size exclusion chromatography are provided inFIG.8. Preparation Example 2. Preparation of mCD80-Fc-IL-2 Variant (2M): mGI101 In order to produce a fusion protein comprising a mouse CD80, an Fc domain, and an IL-2 variant, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 14) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a mCD80 (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acid substitutions, in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 15. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “mGI101”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.9). It was found that the fusion protein was contained at a concentration of 1.95 mg/ml when detected by absorbance at 280 nm using NanoDrop. Preparation Example 3. Preparation of hCD80-Fc: GI101C1 In order to produce a fusion protein comprising a human CD80 fragment and an Fc domain, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 16) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4). The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 17. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI101C1”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.10). It was observed that the fusion protein was contained at a concentration of 3.61 mg/ml when detected by absorbance at 280 nm using NanoDrop. Preparation Example 4. Preparation of Fc-IL-2 Variant (2M): GI101C2 In order to produce a fusion protein comprising an Fc domain and an IL-2 variant, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 18) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acid substitutions, in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 19. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI101C2”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.11). It was found that the fusion protein was contained at a concentration of 4.79 mg/ml when detected by absorbance at 280 nm using NanoDrop. Preparation Example 5. Preparation of mCD80-Fc: mGI101C1 In order to produce a fusion protein comprising a mouse CD80 and an Fc domain, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 20) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a mouse CD80 (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 21. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “mGI101C1”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.12). It was observed that the fusion protein was contained at a concentration of 2.49 mg/ml when detected by absorbance at 280 nm using NanoDrop. The fusion proteins prepared in Preparation Examples 1 to 5 are summarized in Table 1 below. TABLE 1ItemN-terminusLinkerC-terminusPreparation ExamplehCD80 fragmentFc domainhIL-2m1 (GI101)Preparation ExamplemCD80 fragmentFc domainhIL-2m2 (mGI101)Preparation ExampleCD80 fragmentFc domain—3 (GI101C1)Preparation Example—Fc domainIL-2m4 (GI101C2)Preparation ExamplemCD80 fragmentFc domain—5 (mGI101C1) Preparation Example 6. Preparation of CD80-Fc-IL-2: GI101w In order to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and a human IL-2, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contais a nucleotide sequence (SEQ ID NO: 31) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 32. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI101w”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. Preparation Example 7. Preparation of hCD80-Fc-IL-2 Variant (3M): GI102-M45 In order to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, and Y45A) (GI102-M45) with three amino acid substitutions, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 25) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 22), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 26. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI102-M45”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.13). Preparation Example 8. Preparation of hCD80-Fc-IL-2 Variant (3M): GI102-M61 In order to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, and E61R) (GI102-M61) with three amino acid substitutions, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 27) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 23), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 28. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI102-M61”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.14). Preparation Example 9. Preparation of hCD80-Fc-IL-3M: GI102-M72 In order to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, and L72G) (GI102-M72) with three amino acid substitutions, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 29) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 24), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 30. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “GI102-M72”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) condition and stained with Coomassie Blue to check its purity (FIG.15). Preparation Example 10. Preparation of mCD80-Fc-IL-3M: mGI102-M61 In order to produce a fusion protein comprising a mouse CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, and E61R) (GI102-M61) with three amino acid substitutions, a polynucleotide was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Specifically, the polynucleotide contains a nucleotide sequence (SEQ ID NO: 33) which encodes a fusion protein that contains a signal peptide (SEQ ID NO: 1), a mCD80 fragment (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 23), in this order, from the N-terminus. The polynucleotide was inserted into pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein of SEQ ID NO: 34. After the vector was introduced, culture was performed for 7 days in an environment of 37° C., 125 rpm, and 8% CO2concentration. Then, the culture was harvested and the fusion protein was purified therefrom. The purified fusion protein was designated “mGI102-M61”. The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. II. Identification of Binding Affinity Between Fusion Protein and its Ligand In order to identify the binding affinity between the fusion protein and its ligand, the binding affinity was measured using Octet RED 384. Experimental Example 1. Identification of Binding Affinity Between hCTLA-4 and GI101 AR2G biosensor (Amine Reactive 2ndgen, ForteBio, Cat: 18-5092) was previously hydrated with 200 μl of distilled water in a 96-well microplate (GreinerBio-one, Cat: 655209). A ligand (CTLA-4, Human CTLA-4/CD152, His tag, Sino Biological, Cat: 11159-H08H) to be attached to the AR2G biosensor was diluted with 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 5 μg/ml. In addition, GI101 to be attached to the ligand was diluted with 1×AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM. Activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in distilled water. 80 μl of each reagent was placed in a 384-well microplate (GreinerBio-one, Cat: 781209) and the program was set up. As a result, the binding affinity between hCTLA-4 and GI101 was measured as illustrated inFIG.16. Experimental Example 2. Identification of Binding Affinity Between hPD-L1/GI101 and hPD-L1/PD-1 Ni-NTA (Nickel charged Tris-NTA, Ni-NTA Biosensors, ForteBio, 18-5101) was previously hydrated with 200 μl of 1×Ni-NTA kinetic buffer (10× Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. A ligand (Human PD-L1/B7-H1 protein, His-tag, Sino biological, Cat: 10084-H08H) to be attached to the Ni-NTA Biosensors was diluted with 1×Ni-NTA kinetic buffer to a concentration of 5 μg/ml. GI101 to be attached to the ligand was diluted with 1×Ni-NTA kinetic buffer to 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM. In addition, human PD-1/PDCD1 (Human PD-1/PDCD1, Fc Tag, Sino Biological, Cat: 10377-H02H) to be attached to the ligand was diluted with 1×Ni-NTA kinetic buffer to a concentration of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM. Then, 80 μl of each reagent was placed in a 384-well microplate and the program was set up. As a result, the binding affinity between hPD-L1 and GI101 was measured as illustrated inFIG.17. In addition, the binding affinity between hPD-L1 and hPD-1 was measured as illustrated inFIG.18. Experimental Example 3. Identification of Binding Affinity Between mCTLA-4 and mGI101 The binding affinity between mCTLA-4 and mGI101 was identified in the same manner as in Experimental Example 1. Here, the equipment used is as follows: Biosensor: AR2G, Ligand: mCTLA-4 (Recombinant Mouse CTLA-4 Fc chimera, R&D Systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM). As a result, the binding affinity between mCTLA-4 and mGI101 was measured as illustrated inFIG.19. Experimental Example 4. Identification of Binding Affinity Between mPD-L1 and mGI101 The binding affinity between mPD-L1 and mGI101 was identified in the same manner as in Experimental Example 1. Here, the equipment used is as follows. Biosensor: AR2G, Ligand: mPD-L1 (Recombinant Mouse B7-H1/PD-L1 Fc chimera, R&D Systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM). As a result, the binding affinity between mPD-L1 and mGI101 was measured as illustrated inFIG.20. Experimental Example 5. Identification of Binding Ability of GI-101 (hCD80-Fc-hIL-2v) to CTLA-4 Binding kinetics measurements were performed using the Octet RED 384 instrument (ForteBio, Pall Life Science) with agitation at 30° C. and 1,000 rpm. The binding ability for CTLA-4 was measured using the Amine Reactive 2 generation (AR2G) biosensor chip, and the binding ability for PD-L1 was measured using the Nickel charged Tris-NTA (Ni-NTA) biosensor chip. The AR2G biosensor chip was activated with a combination of 400 mM EDC and 100 mM sulfo-NHS. Then, Human CTLA-4-His Tag (Sino Biological, Cat: 11159-H08H) was diluted with 10 mM acetate buffer (pH 5) to 5 μg/ml, and loaded on the AR2G biosensor chip for 300 seconds and fixed. Then, binding of CTLA-4 to GI-101 (hCD80-Fc-hIL-2v), GI-101C1 (hCD80-Fc), Ipilimumab (Bristol-Myers Squibb), and GI-101C2 (Fc-hIL-2v) at various concentrations was measured for 300 seconds and dissociation thereof was also measured for 300 seconds. Binding kinetics analysis was performed using Octet Data Analysis HT software ver. 10 provided by Pall Corporation. The results are illustrated inFIG.21. Experimental Example 6. Identification of Binding Affinity Between IL-2Rα or IL-2Rβ, and GI101 The binding ability for IL-2Rα was measured using the AR2G biosensor, and the binding ability for IL-2Rβ was measured using the Ni-NTA biosensors (Nickel charged Tris-NTA, Ni-NTA Biosensors, ForteBio, 18-5101). A ligand (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) to be attached to the AR2G biosensor was diluted with 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 5 μg/ml. The AR2G biosensor was activated with a buffer prepared by mixing 400 mM EDC and 100 mM sulfo-NHS, and then the diluted ligand was loaded on the AR2G biosensor for 300 seconds and fixed. Meanwhile, a ligand (IL-2Rβ-His Tag, Acro, Cat: CD2-H5221) to be attached to the Ni-NTA biosensor was diluted with 1×Ni-NTA kinetic buffer to a concentration of 5 μg/ml. The diluted ligand was loaded on the Ni-NTA biosensor for 600 seconds and fixed. Thereafter, GI101, GI101w, or Proleukin (Novartis, hIL-2), at various concentrations, to be attached to the ligand was loaded thereon for 300 seconds. Then, binding thereof was measured and dissociation thereof was also measured for 300 seconds. Binding kinetics analysis was performed using Octet Data Analysis HT software ver. 10 provided by Pall Corporation. The results are illustrated inFIGS.22to24. As a result, it was identified that GI101 has low binding ability for the IL-2 receptor IL-2Rα and high binding ability for IL-2Rβ, as compared with GI101w and Proleukin. Experimental Example 7. Measurement of Binding Affinity Between Fusion Protein and Ligand In order to identify the binding affinity between the fusion protein and its ligand, the binding affinity was measured using Octet RED 384. Experimental Example 7.1. Identification of Binding Affinity Between IL-2 Alpha Receptor, and GI101-M45, GI101-M61, or GI101-M72 AR2G biosensor (Amine Reactive 2ndgen, ForteBio, Cat: 18-5092) was previously hydrated with 200 μl of distilled water (DW) in a 96-well microplate (GreinerBio-one, Cat: 655209). A ligand (Human IL-2 R alpha protein, His Tag, Acro, ILA-H52H9) to be attached to the biosensor was diluted with 10 mM acetate buffer (pH 5) (AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 5 μg/ml. An analyte (GI101-M45, GI101-M61, GI101-M72) to be attached to the ligand was diluted with 1×AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095) to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively. Activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in DW. 80 μl of each reagent was placed in a 384-well microplate (GreinerBio-one, Cat: 781209) and the program was set up. As a result, the binding affinity between IL-2 alpha receptor and GI101-M45 is illustrated inFIG.25. In addition, the binding affinity between IL-2 alpha receptor and GI101-M61 is illustrated inFIG.26, and the binding affinity between IL-2 alpha receptor and GI101-M72 is illustrated inFIG.27. Experimental Example 7.2. Identification of Binding Affinity of GI102-M45, GI102-M61, and GI102-M72 to IL-2Rβ Ni-NTA Biosensors were previously hydrated with 200 μl of 1×Ni-NTA kinetic buffer (10× Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. A ligand (Human IL-2 R beta protein, His-Tag, Acro, CD2-H5221) to be attached to the biosensor was diluted with 1×Ni-NTA kinetic buffer to a concentration of 2 μg/ml. GI102-M45, GI102-M61, or GI102-M72 to be attached to the ligand was diluted with 1×Ni-NTA kinetic buffer to a concentration of 500 nM, 250 nM, 125 nM, or 62.5 nM. 80 μl of each reagent was placed in a 384-well microplate and the program was set up. As a result, the binding affinity between IL-2Rβ and GI102-M45 was measured as illustrated inFIG.28, and the binding affinity between IL-2Rβ and GI102-M61 was measured as illustrated inFIG.29. In addition, the binding affinity between IL-2Rβ and GI102-M72 was measured as illustrated inFIG.30. III. Identification of Immune Activity of Fusion Protein Experimental Example 8. Identification of IFN-γ Production Caused by Fusion Protein Experimental Example 8.1. Culture of CFSE-Labeled PBMCs Peripheral blood mononuclear cells (PBMCs) isolated from a human were labeled with carboxyfluorescein succinimidyl ester (CFSE) by being reacted with 1 μM CellTrace CFSE dye at 37° C. for 20 minutes. CFSE not bound to the cells was removed by being reacted for 5 minutes with a culture medium having a 5-fold volume of the staining reaction solution and then by being centrifuged at 1,300 rpm for 5 minutes. The CFSE-labeled PBMCs were resuspended in the culture medium (RPMI1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acid, and 2 mM L-glutamine), and then added to a 96-well microplate at 1×105cells per well. Treatment with 5 μg/ml of PHA (Lectin fromPhaseolus Vulgaris, red kidney bean, Sigma-Aldrich, St. Louis, MO, USA, cat No. L1668-5MG), and GI101, GI101C1, GI101C2, or IL-2 (Aldesleukin; human recombinant IL-2, Novartis) was performed and incubation was performed in a 5% CO2incubator at 37° C. for 6 days. Here, the treatment with GI101, GI101C1, GI101C2, and IL-2 was performed at a concentration of 1 nM, 10 nM, or 100 nM. The cells were analyzed by FACS, and human IFN-γ present in the culture medium was measured using an ELISA kit (Biolegend, San Diego, CA, USA, cat No. 430103). Experimental Example 8.2. FACS Analysis The cell pellets obtained by removing the supernatant were washed with FACS buffer (3% fetal bovine serum, 10 mM EDTA, 1 M HEPES, 100 unit/ml penicillin, streptomycin, 1 mM sodium pyruvate), and then reacted with Fc blocker (Biolegend, cat NO. 422302) at 4° C. for 5 minutes. Then, treatment with APC anti-CD3 Ab (Biolegend, cat NO. 300412) and PE anti-CD8a Ab (Biolegend, cat NO. 300908) was performed and reaction was allowed to proceed at 4° C. for 20 minutes. Then, the resultant was washed with FACS buffer. The cell pellets were resuspended in FACS buffer and then analyzed using BD LSR Fortessa (BD biosciences, San Diego, CA, USA) and FlowJo Software. Experimental Example 8.3. Human IFN-γ ELISA The amount of human IFN-γ secreted into the supernatant of each sample in which the cells had been cultured was measured using a human IFN-γ ELISA kit (Biolegend, cat No. 430103). Briefly, anti-human-IFN-γ antibodies were added to an ELISA plate, and reaction was allowed to proceed overnight at 4° C. so that these antibodies were coated thereon. Then, blocking was performed at room temperature for 1 hour with a PBS solution to which 1% BSA had been added. Washing with a washing buffer (0.05% Tween-20 in PBS) was performed, and then a standard solution and each sample were properly diluted and added thereto. Then, reaction was allowed to proceed at room temperature for 2 hours. After the reaction was completed, the plate was washed and secondary antibodies (detection antibodies) were added thereto. Reaction was allowed to proceed at room temperature for 1 hour. Washing with a washing buffer was performed, and then an Avidin-HRP solution was added thereto. Reaction was allowed to proceed at room temperature for 30 minutes. A substrate solution was added thereto and color development reaction was induced in the dark at room temperature for 20 minutes. Finally, H2504 was added thereto to stop the color development reaction, and the absorbance at 450 nm was measured with Epoch Microplate Spectrophotometer (BioTek instruments, Winooski, VT, USA), and the concentration was calculated. As a result, it was found that cells treated with GI101 exhibited a remarkable increase in IFN-γ secretion, as compared with cells treated with GI101C1, GI101C2, or IL-2 (FIGS.31and32). Experimental Example 9. Identification of Effect of GI101 on Proliferation of CD8+ T Cells Peripheral blood mononuclear cells (PBMCs) isolated from a human were labeled with CFSE by being reacted with 1 μM CellTrace CFSE dye at 37° C. for 20 minutes. CFSE not bound to the cells was removed by being reacted for 5 minutes with a culture medium having a 5-fold volume of the staining reaction solution and then by being centrifuged at 1,300 rpm for 5 minutes. The CFSE-labeled PBMCs were resuspended in the culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acid, and 2 mM L-glutamine), and then added to a 96-well microplate at 1×105cells per well. Thereafter, treatment with 1 μg/ml of anti-CD3ε antibody (Biolegend cat No. L1668-5MG), and GI101, GI101C1, GI101C2, or Proleukin (Novartis) was performed and incubation was performed in a 5% CO2incubator at 37° C. for 6 days. Here, the cells were treated with GI101, GI101C1, GI101C2, and IL-2 at a concentration of 100 nM. The incubated cells were examined for their degree of proliferation by measuring, with FACS analysis using APC-TCRαβ and PE-CD8α antibodies, a proportion of CD8+ T cells that had not been labeled with CFSE. As a result, it was found that GI101 activated proliferation of CD8+ T cells in vitro to a to similar extent to the wild-type IL-2 Proleukin (FIGS.33and34). Experimental Example 10. Identification of Effect of GI101 and GI102 on Proliferation of CD8+ T Cells Human PBMCs were purchased from Allcells (Lot #3014928, USA). 1M CellTrace CFSE dye was used, which was reacted with the human PBMCs under a light-blocking condition at room temperature for 20 minutes. The cells were labeled with CFSE by being reacted with 1 μM CellTrace CFSE dye at 37° C. for 20 minutes. CFSE not bound to the cells was removed by being reacted for 5 minutes with a culture medium having a 5-fold volume of the staining reaction solution and then by being centrifuged at 1,300 rpm for 5 minutes. The CFSE-labeled PBMCs were resuspended in the culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acid, and 2 mM L-glutamine), and then added to a 96-well microplate at 1×105cells per well. Thereafter, the CFSE-labeled PBMCs were subjected to treatment with 1 μg/ml of anti-CD3ε antibody (OKT3, eBioscience, USA), and GI101, GI101C1, GI101C2, or Proleukin (Novartis), and incubation was performed in a 5% CO2incubator at 37° C. for 7 days. Here, the cells were subjected to treatment with GI101, GI101C1, GI101C2, and IL-2 at a concentration of 10 μM. The incubated cells were examined for their degree of proliferation by measuring, with FACS analysis using anti-human CD4-PE antibody (BioLegend, USA), anti-human CD8-PE/Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, USA), a proportion of CD8+ T cells that had not been labeled with CFSE. As a result, the GI101, GI102 M61, GI101C2, and Proleukin treatment groups exhibited a significant increase in proportion of CD8+ T cells, as compared with the control (no stimulus), the anti-CD3 antibody alone treatment group, and the GI101C1 treatment group. In addition, as compared with the negative control (no stimulus) and the anti-CD3 antibody alone treatment group, the GI101, GI101C2, and Proleukin treatment groups exhibited a significant increase in proliferation of CD4+/FoxP3+ Treg cells, whereas the GI102 and GI101C1 treatment groups did not exhibit a significant increase in proliferation of CD4+/FoxP3+ Treg cells (FIG.35). Experimental Example 11. Identification of Effect of GI101 or GI101w on Proliferation of CD8+ T Cells and NK Cells 7-week-old C57BL/6 mice purchased from Orient Bio (Korea) were divided into 3 groups, each group including 3 mice, and PBS, GI101, or GI101w was injected intraperitoneally thereinto. Here, GI101 and GI101w were respectively prepared to be at 40.5 μg in 200 μl of PBS, and injected intraperitoneally thereinto. Five days after the injection, the spleens were removed from the mice of each group. The cells were isolated therefrom, and the total number of cells was measured using a hematocytometer. Splenocytes were examined for proportions of CD8+ T cells and NK cells therein, with FACS analysis using staining with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific blue-CD8α antibody (BD; 53-6.7). As such, the numbers of CD8+ T cells and NK cells present in the spleen were calculated. As a result, it was identified that GI101 activated proliferation of CD8+ T cells and NK cells in vivo as compared with GI101w (FIGS.36and37). Experimental Example 12. Identification of Effect of GI101 on Function of T Cells An experiment was performed using a CTLA-4 blockade bioassay kit (Promega cat No. JA4005). The experiment is briefly described as follows. CTLA-4 effector cells kept in liquid nitrogen were thawed in a 37° C. constant temperature water bath for 3 minutes, and 0.8 ml of CTLA-4 effector cells were mixed well with 3.2 ml of pre-warmed assay buffer (90% RPMI+10% fetal bovine serum). Then, the mixture was added to a 96-well white cell culture plate (SPL, cat No. 30196) at 25 μl per well. Then, 25 μl of GI101 at various concentrations was added thereto. For a negative control, 25 μl of assay buffer was added thereto. Then, the 96-well white cell culture plate was covered and placed at room temperature until aAPC/Raji cells were prepared. aAPC/Raji cells kept in liquid nitrogen were thawed in a 37° C. constant temperature water bath for 3 minutes, and 0.8 ml of aAPC/Raji cells were mixed well with 3.2 ml of pre-warmed assay buffer. Then, 25 μl of the mixture was added to the plate at per well, and reaction was allowed to proceed in a 5% CO2incubator at 37° C. for 16 hours. After the reaction was completed, the resultant was allowed to stand at room temperature for 15 minutes, and then the Bio-Glo reagent was added thereto while taking care to avoid bubbles. The Bio-Glo reagent was also added to three of the outermost wells and the wells were used as blanks to correct the background signal. Reaction was allowed to proceed at room temperature for 10 minutes, and then luminescence was measured with Cytation 3 (BioTek instruments, Winooski, VT, USA). Final data analysis was performed by calculating RLU (GI101-background)/RLU (no treatment-background). As a result, it was found that GI101 binds to CTLA-4 expressed on effector T cells, and activats the function of T cells rather than inhibiting the same (FIGS.38and39). Experimental Example 13. Identification of Effect of mGI101 and mGI102 on Immune Cells 7-week-old C57BL/6 mice purchased from Orient Bio (Korea) were divided into 3 groups, each group including 3 mice, and PBS, 3 mg/kg, 6 mg/kg, or 12 mg/kg of GI101, or 3 mg/kg, 6 mg/kg, or 12 mg/kg of mGI102 (mGI102-M61) was administered intravenously thereinto. On days 1, 3, 5, 7, and 14 after the injection, the spleen tissues were removed from the mice of each group. Thereafter, for the spleen tissue, the numbers of effector CD8+ T cells, NK cells, and Treg cells were calculated with FACS analysis using respective antibodies, and proportions of effector CD8+ T cells and NK cells with respect to Treg cells were respectively calculated. The information on the antibodies used in each cell analysis is as follows: Effector CD8+ T cell: PB anti-mouse CD3ε antibody (Biolegend, #155612; KT3.1.1), FITC anti-mouse CD8α antibody (BD, #553031, 53-6.7), PE/Cy7 anti-mouse CD44 antibody (Biolegend, #103030; IM7), APC anti-mouse CD122 antibody (Biolegend, #123214; TM-β1) NK cell: PB anti-mouse CD3ε antibody (Biolegend, #155612; KT3.1.1), PE anti-mouse NK-1.1 (Biolegend, #108708; PK136) Treg cell: FITC anti-mouse CD3 antibody (Biolegend, #100204; 17A2), PB anti-mouse CD4 antibody (Biolegend, #100531; RM4-5), PE anti-mouse CD25 antibody (Biolegend, #102008; PC61), APC anti-mouse Foxp3 antibody (Invitrogen, #FJK-16s, 17-5773-82). As a result, the group having received mGI101 or mGI102 (mGI102-M61) exhibited a significant increase in numbers of CD8+ T cells and NK cells at the time points from the 3rdto 14thdays after administration, as compared with the PBS administration group. In addition, it was found that the group having received mGI102 exhibited a significant increase in proportions of activated CD8+ T cells/Treg cells and NK cells/Treg cells at the time points from the 3rdto 7thdays after administration, as compared with the PBS administration group (FIG.40). IV. Identification of Anticancer Effect of Fusion Protein Experimental Example 14. Identification of Effect of GI101 on T Cell Activity Inhibition by Cancer Cells Expressing PD-L1 and CTLA-4 NCl-H292 cancer cell line expressing PD-L1 and CTLA-4 was cultured for 3 hours in a culture medium containing 10 μg/ml Mitomycin C (Sigma), and then Mitomycin C was removed by washing with the culture medium. Thereafter, 5×104cells of the Mitomycin C-treated NCl-H292 cancer cell line were incubated with 1×105cells of human PBMCs in a 96-well microplate. Here, treatment with 5 μg/ml of PHA (Sigma) was performed for T cell activity. In addition, GI101C1 and GI101 at a concentration of 50 nM were reacted with IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at a concentration of 50 nM for 30 minutes at 4° C., and then the resultant was used to treat the NCl-H292 cancer cells. After 3 days, the supernatant of the cell culture was collected and the amount of IFN-γ was quantified using an ELISA kit (Biolegend). As a positive control, human PBMCs stimulated with PHA in the absence of the Mitomycin C-treated NCl-H292 cancer cell line were used; and as a negative control, human PBMCs stimulated with PHA in the presence of the Mitomycin C-treated NCl-H292 cancer cell line were used. An experimental method using the IFN-7 ELISA kit was carried out in the same manner as in Experimental Example 9.3. As a result, GI101 effectively activated the immune response that had been inhibited by the cancer cell line overexpressing PD-L1. In addition, it was identified that GI101 inhibited signaling of CTLA-4 expressed on effector T cells (FIGS.41and42). Experimental Example 15. Identification of Anticancer Effect of mGI101 in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells BALB/c mice (female, 7-week-old) purchased from Orient Bio were subjected to an acclimation period of 7 days. Then, 5×106cells of CT-26 cancer cell line (ATCC, USA) were mixed with 0.05 ml of MATRIGEL™ matrix phenol red-free (BD), and allotransplantation of the mixture was performed by subcutaneous administration at 0.1 ml in the right dorsal region of the mice. A certain period of time after the cancer cell transplantation, the tumor volume was measured and subjects that reached about 28 mm3were selected, and then the selected mice were grouped evenly based on tumor size and body weight, each group including 10 mice. Thereafter, using a disposable syringe (31G, 1 ml), hIgG4 was administered at a dose of 6 mg/kg to a negative control. For experimental groups, mGI101 at a dose of 3 mg/kg, 6 mg/kg, or 12 mg/kg was administered intravenously thereto. A total of three administrations were given once every three days after the first administration. The tumor size was measured daily. As a result, it was found that the experimental group having received mGI101 at a dose of 6 mg/kg and 12 mg/kg exhibited significant tumor growth inhibition at some measurement time points and at the end of the test, as compared with the negative control (FIG.43). In addition, as a result of measuring a survival rate, it was found that the experimental group having received mGI101 at a dose of 6 mg/kg exhibited significant improvement at some measurement time points and at the end of the test, as compared with the negative control (FIG.44). Experimental Example 16. Identification of Anticancer Effect of GI101 in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells Experimental Example 16.1. Identification of Tumor Inhibitory Effect BALB/c mice (female, 7-week-old) purchased from Orient Bio were subjected to an acclimation period of 7 days. Then, 5×106cells of CT-26 cancer cell line (ATCC, USA) were suspended in 0.1 ml PBS, and allotransplantation of the suspension was performed by subcutaneous administration at 0.1 ml in the right dorsal region of the mice. A certain period of time after the cancer cell transplantation, the tumor volume was measured and subjects that reached about 50 mm3to 200 mm3were selected, and then the selected mice were grouped evenly based on tumor size and body weight, each group including 10 mice. Thereafter, using a disposable syringe (31G, 1 mL), no drug was administered to a negative control, and an anti-PD-1 antibody at a dose of 5 mg/kg, or an anti-PD-1 antibody at a dose of 5 mg/kg and an anti-CTLA-4 antibody at a dose of 5 mg/kg were administered intravenously to positive controls. For experimental groups, GI101 at a dose of 0.1 mg/kg or 1 mg/kg was administered intravenously thereto. A total of three administrations were given once every three days after the first administration. The tumor size was measured daily. As a result, in the mice transplanted with CT-26 cancer cell line, all groups having received anti-PD-1 antibody; anti-PD-1 antibody and anti-CTLA-4 antibody; or GI101 at a dose of 0.1 mg/kg or 1 mg/kg exhibited significant tumor growth inhibition, as compared with the negative control. In particular, the experimental group having received GI101 at a dose of 0.1 mg/kg exhibited a significant tumor inhibitory effect, as compared with the group having received an anti-PD-1 antibody (* p<0.05) (FIG.45). Experimental Example 16.2. Immune Cell Analysis in Cancer Tissue The mice of each group in Experimental Example 16.1 were sacrificed when the tumor volume reached an average of 200 mm3, and cancer tissues were collected. Thereafter, the cancer tissues were separated to a single-cell level to analyze immune cells therein, and then FACS analysis was performed on immune cells in the cancer tissues using the following antibodies: Anti-mouse-CD3 (Biolegend, Cat. No. 100320), Anti-mouse-CD4 (Biolegend, Cat. No. 100526), Anti-mouse-CD8 (Biolegend, Cat. No. 100750), Anti-mouse-FoxP3 (eBioscience, Cat. No. 12-5773-82), Anti-mouse-CD25 (Biolegend, Cat. No. 102049), Anti-mouse-CD44 (eBioscience, Cat. No. 61-0441-82), Anti-mouse-PD-1 (Biolegend, Cat. No. 135218), Anti-mouse-IFN-gamma (Biolegend, Cat. No. 505832), Anti-mouse-CD49b (Biolegend, Cat. No. 108906), Anti-mouse-H2 (Invitrogen, Cat. No. A15443), Anti-mouse-CD11c (Biolegend, Cat. No. 117343), Anti-mouse-CD80 (eBioscience, Cat. No. 47-4801-82), Anti-mouse-CD86 (Biolegend, Cat. No. 104729), Anti-mouse-F4/80 (eBioscience, Cat. No. 47-4801-82), and Anti-mouse-CD206 (eBioscience, Cat. No. 17-2061-80). As a result, the experimental group having received GI101 at a dose of 0.1 mg/kg exhibited a significant increase in CD8+ T cells, as compared with the positive control having received anti-PD-1 antibody alone at a dose of 5 mg/kg (* p<0.05,FIGS.46and47). Furthermore, all experimental groups having received GI101 exhibited a significantly increased level of expression of IFN-γ in T cells, as compared with the negative control (* p<0.05,FIGS.46and47). In addition, the experimental group having received GI101 at a dose of 0.1 mg/kg exhibited an increase in M1 macrophages as compared with the negative control and the positive control having received anti-PD-1 antibody alone (FIGS.48and49). In addition, all experimental groups having received GI101 exhibited an increased level of CD86 expression in macrophages and dendritic cells (* p<0.05,FIGS.48to51). Experimental Example 17. Identification of Anticancer Effect of GI101 in Mice Transplanted with Mouse-Derived Lung Cancer Cells Experimental Example 17.1. Identification of Tumor Inhibitory Effect C57BL/6 mice (female, 7-week-old) purchased from Orient Bio were subjected to an acclimation period of 7 days. Then, 5×106cells of LL/2 cancer cell line (ATCC, USA) were suspended in 0.1 ml PBS, and allotransplantation of the suspension was performed by subcutaneous administration at 0.1 ml in the right dorsal region of the mice. A certain period of time after the cancer cell transplantation, the tumor volume was measured and subjects that reached about 50 mm3to 200 mm3were selected, and then the selected mice were grouped evenly based on tumor size and body weight, each group including 10 mice. Thereafter, using a disposable syringe (31G, 1 mL), no drug was administered to a negative control, and an anti-PD-1 antibody at a dose of 5 mg/kg, or an anti-PD-1 antibody at a dose of 5 mg/kg and an anti-CTLA-4 antibody at a dose of 5 mg/kg were administered intravenously to positive controls. For experimental groups, GI101 at a dose of 0.1 mg/kg or 1 mg/kg was administered intravenously thereto. A total of three administrations were given once every three days after the first administration. The tumor size was measured daily. As a result, all experimental groups exhibited a significant tumor inhibitory effect, as compared with the negative control (* p<0.05) (FIG.52). Experimental Example 17.2. Immune Cell Analysis in Cancer Tissue The mice of each group in Experimental Example 17.1. were sacrificed when the tumor volume reached an average of 200 mm3, and cancer tissues were collected. Thereafter, FACS analysis was performed in the same manner as Experimental Example 16.2. to analyze immune cells in the cancer tissues. As a result, the experimental group having received GI101 at a dose of 0.1 mg/kg exhibited a significant increase in CD8+ T cells, as compared with the positive control having received anti-PD-1 antibody alone (* p<0.05,FIG.59). Furthermore, all experimental groups having received GI101 exhibited a significantly increased level of expression of IFN-γ, as compared with the negative control (* p<0.05,FIG.59). In addition, all experimental groups having received GI101 exhibited an increased level of CD86 expression in macrophages and dendritic cells (* p<0.05,FIGS.53to55). Experimental Example 18. Identification of Anticancer Effect of mGI102-M61 in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells BALB/c mice (female, 7-week-old) purchased from Orient Bio were subjected to an acclimation period of 7 days. Then, 5×106cells of CT26 cancer cell line (ATCC, USA) were mixed with 0.05 ml of MATRIGEL™ matrix phenol red-free (BD), and allotransplantation of the mixture was performed by subcutaneous administration at 0.1 ml in the right dorsal region of the mice. A certain period of time after the cancer cell transplantation, the tumor volume was measured and subjects that reached about 28 mm3were selected, and then the selected mice were grouped evenly based on tumor size and body weight, each group including 10 mice. Thereafter, using a disposable syringe (31G, 1 mL), hIgG4 was administered at a dose of 6 mg/kg to a negative control. For experimental groups, mGI102-M61 at a dose of 3 mg/kg, 6 mg/kg, or 12 mg/kg was administered intravenously thereto. A total of three administrations were given once every three days after the first administration. The tumor size was measured daily. As a result, it was identified that the experimental group having received mGI102-M61 at a dose of 12 mg/kg exhibited significant tumor growth inhibition at some measurement time points and at the end of the test, as compared with the negative control (FIG.56). In addition, as a result of measuring a survival rate, it was identified that the experimental group having received mGI102-M61 at a dose of 12 mg/kg exhibited significant improvement at some measurement time points and at the end of the test, as compared with the negative control (FIG.57). Experimental Example 19. Identification of Anticancer Effect of mGI101 in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells BALB/c mice (female, 7-week-old) purchased from Orient Bio (Korea) were subjected to an acclimation period of 7 days. Then, 5×106cells of CT-26 cancer cell line (ATCC, USA) were mixed with 0.05 ml of MATRIGEL™ matrix phenol red-free (BD), and allotransplantation of the mixture was performed by subcutaneous administration at 0.1 ml in the right dorsal region of the mice. A certain period of time after the cancer cell transplantation, the tumor volume was measured and subjects that reached about 200 mm3to 250 mm3were selected, and then the selected mice were grouped evenly based on tumor size and body weight, each group including 10 mice. Thereafter, using a disposable syringe (31G, 1 mL), hIgG4 was administered at a dose of 4 mg/kg to a negative control. For experimental groups, mGI101 at a dose of 1 mg/kg, 4 mg/kg, or 6 mg/kg was administered intravenously thereto. Additionally, groups having received mCD80 at 4.9 mg/kg or Fc-IL-2v (GI101C2) at 2.8 mg/kg were set as controls. In addition, a group having simultaneously received mCD80 at 4.9 mg/kg and Fc-IL-2v (GI101C2) at 2.8 mg/kg was set as a control. In tumor volume measurement, it was identified that the group having received mGI101 at a dose of 6 mg/kg exhibited significant inhibition at some measurement time points and at the end of the test, as compared with the negative control. An excellent tumor growth inhibition rate was observed as compared with the group having received a combination of mCD80 and Fc-IL-2v (GI101C2) (FIGS.58and59). In conclusion, in the tumor growth-inhibitory efficacy test on BALB/c mice allotransplanted with CT-26, a BALB/c mouse-derived colorectal cancer cell line, it was demonstrated that the test substance mGI101 had tumor inhibitory efficacy under this test condition as compared with respective mCD80 and IL-2v single formulations; and it was identified that mGI101 exhibited excellent anticancer efficacy as compared with the group having received a combination of mCD80 and IL-2v (FIGS.58and59). In particular, the group having received mGI101 at a dose of 6 mg/kg exhibited significant inhibition of tumor size, as compared with the negative control and the group having received a combination of mCD8 and Fc-IL2v (GI101C2). V. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and Immune Checkpoint Inhibitor Experimental Example 20. Identification of Anticancer Effect by Administration of Combination of GI101 and Anti-PD-1 Antibody in Mice Transplanted with Human-Derived Breast Cancer Cells This test was to evaluate the tumor growth inhibitory effect after intraperitoneal administration of GI101 as a test substance alone or in combination with Keytruda (Pembrolizumab, MSD), which is an anti-PD-1 antibody, as a positive control substance in a tumor model xenotransplanted with MDA-MB-231 cells, which are human-derived breast cancer cells, using a humanized mouse model prepared by xenotransplanting human PBMCs into NSGb2m mice. The stock solution of the test substance, negative control substance, and positive control substance described in Table 2 was diluted by adding excipients according to each dose. TABLE 2Positive controlNegative control—Test substancesubstancesubstanceExcipientSubstanceGI101KEYTRUDAhIgG4PBSnameAppearanceclear liquidclear liquidclear liquidclearliquidComponentFc fusion proteinanti-PD-1 antibody——pH7.5———Storagerefriger-refriger-refriger-refriger-conditionatedatedatedatedstoragestoragestoragestorage(4° C.)(4° C.)(4° C.)(4° C.)Precautionskeep refrigerated untilkeep refrigerated untilkeep refrigerated until—for handlingadministration, andadministration, andadministration, andprepare and use on theprepare and use on theprepare and use on theday of administrationday of administrationday of administration Human-derived breast cancer cells, MDA-MB-231 (Homo sapiens, human mammary gland/breast; derived from metastatic site: pleural effusion), were purchased from the Korea cell line bank (Korea) and used for the test. The cell culture medium has a composition as shown in the table below. Fetal bovine serum (FBS, 16000-044, Thermofisher scientific, USA), penicillin-streptomycin; 10,000 units/ml penicillin and 10,000 μg/ml streptomycin (15140122, Thermofisher scientific, USA); and RPMI1640 (A1049101, Thermofisher scientific, USA) per 100 ml were mixed and used. TABLE 3NameComposition (ml)FBS10Penicillin-Streptomycin1RPMI164089Total volume100 The cells to be used for the test were thawed, placed in a cell culture flask, and cultured in a 37° C., 5% CO2incubator (MCO-170M, Panasonic, Japan). The cells were suspended using Trypsin-EDTA (Cat. 25200-072, Thermofisher scientific, USA). The suspended cells were collected by centrifugation (125×g, 5 minutes) using a centrifuge, transferred to a new medium and a new flask, and passage cultured. The cultured cells were put to a centrifugation tube on the day of cell line transplantation and then collected. Thereafter, centrifugation (125×g, 5 minutes) was performed to discard the supernatant, and a cell suspension (5×106cells/0.05 ml) was prepare with PBS (Cat. LB 001-04, Welgene, KOREA) and stored on ice until inoculation. 8-week-old female NSGb2m (NOD.Cg-B2mtm1UncPrkdcscidIl2rgtm1Wjl/SzJ) mice were purchased from Joongang Bio (Korea) and used for the test. The body weight was measured the next day after the end of the quarantine and acclimation period, and then the human-derived PBMC cell suspension (5×106cells/0.2 ml) prepared for healthy animals was filled into a disposable syringe and administered to the caudal vein of the animals. General symptoms were observed once a day after cell transplantation. MATRIGEL™ matrix phenol red-free (0.05 ml, 356237, BD, USA) was added to the prepared MDA-MB-231 cell suspension (5×106cells/0.05 ml) to prepared the solution, and the solution was filled into a disposable syringe, and transplantation of the solution was performed by subcutaneous administration at 0.1 ml/head in the right dorsal region of the animals transplanted with human PBMCs. General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. A certain period of time after the cell transplantation, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and 32 subjects were selected so that the average of each group reached 40 to 80 mm3. The selected animals were grouped into a total of 4 groups as evenly as possible based on tumor volume and body weight, each group including 8 animals. As shown in Table 4, the test groups were configured. The test substance was administered to the animals using a disposable syringe (31G, 1 ml), and the administration frequency was 2 times/week, a total of 4 administrations were performed. TABLE 4Dosage amountDosage volumeNumber ofGroup(mg/kg)(ml/kg)animalsG1hIgG46108G2GI1016108G3Keytruda5108G7GI101 + Keytruda6 + 5108 General symptoms such as appearance, behavior, and excrement were observed once a day during the observation period, and deceased animals were identified. Body weight was measured on the day of cell line transplantation, twice a week, and on the day of animal sacrifice. The major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, mitutoyo, Japan) three times a week during the observation period, and the tumor volume (TV) was calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping. As a result of administering the drugs shown in Table 4 on days 21, 25, 28, and 31 after tumor transplantation, respectively, the group having received each of GI101 and Keytruda exhibited tumor growth inhibition as compared with the control (hIgG4). The group having received a combination of GI101 and Keytruda exhibited tumor growth inhibition as compared with the control. The group having received a combination of GI101 and Keytruda exhibited tumor growth inhibition as compared with the groups having received each of GI101 and Keytruda (FIG.60). As a result of calculating the tumor growth inhibition rate at the end of the experiment (after tumor transplantation, day 42) as compared with on day 1 of drug treatment (after tumor transplantation, day 21), the group having received hIgG4 exhibited the tumor growth inhibition rate of 30% or more in 2 mice, 50% or more in 1 mouse, and 80% or more in 1 mouse. In addition, the group having received GI101 exhibited the tumor growth inhibition rate of 30% or more in 5 mice, 50% or more in 5 mice, and 80% or more in 2 mice, and the group having received Keytruda exhibited the tumor growth inhibition rate of 30% or more in 7 mice, 50% or more in 5 mice, and 80% or more in 3 mice. In addition, the group having received a combination of GI101 and Keytruda exhibited the tumor growth inhibition rate of 30% or more in 8 mice, 50% or more in 8 mice, and 80% or more in 6 mice (FIG.61). In addition, the degree of tumor growth of individual experimental animals of each treatment group when a combination of GI101 and Keytruda is used in mice transplanted with human-derived breast cancer cells is shown inFIGS.62to66. Experimental Example 21. Identification of Anticancer Effect by Administration of Combination of mGI101 and Anti-PD-1 Antibody in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This test was to evaluate the tumor growth inhibitory effect after intraperitoneal administration of mGI101 as a test substance alone or in combination with an anti-PD-1 antibody as a positive control substance in a tumor model allotransplanted with MC38 into C57BL/6 mice. Rodent-derived colorectal cancer cells, MC38, were purchased from Kerafast (USA) and used for the test. MC38 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. In order to establish an allotransplanted tumor model, 1×106MC38 cells were subcutaneously injected into the right flank of C57BL/6 female mice (7-week-old). The mice were randomly assigned based on tumor volume (30 mm3), each group including 5 mice. The tumor grafts were identified about day 2 after cell inoculation. As shown in Table 5, the test groups were configured and the test substances were administered. TABLE 5Route of administration,DosageNumberExperimental groupdosing cycleamountof animalG1Vehicle control (hIgG4)i.p. BIW × 16 days10mg/kg5G2mGI101i.p. day 1, 5, 96mg/kg6G3Anti-PD-1 antibodyi.p. BIW × 16 days5mg/kg5(cloneRMP1-14, InVivoMab)G4mGI101 + anti-PD-1 antibodyi.p. day 1, 5, 9 (mGI101)0.6mg/kg5i.p. BIW × 16 days (anti-5mg/kgPD-1 antibody) Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified. At the end of the test period, the animals were sacrificed. The size of the MC38 solid cancer was measured using a tumor 3D scanner (TM900, Peria, Belgium). For each experimental group, the average loss and percentage change of body weight and the average tumor growth inhibition were calculated. The anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through one-way analysis of variance (at the end of this test) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant. All test animals maintained a healthy state without signs of pathological abnormalities after administration of mGI101 alone and with combination in an anti-PD-1 antibody. The results of combination therapy using mGI101 and/or an anti-PD-1 antibody against the MC38 tumor are shown inFIG.67. The anticancer effect was observed in the group having received the drug as compared with the control, and the difference in tumor size was noticeable during the test period of 16 days. The MC38 tumor is known as a model responsive to an anti-PD-1 antibody in previous literature, and the anticancer effect was also observed in the group having received the anti-PD-1 antibody of this test (p>0.01). The anticancer effect was also shown in the group having received mGI101 (6 mpk) alone as well as the group having received the anti-PD-1 antibody (p>0.01). The group having received a combination of mGI101 (0.6 mpk)+anti-PD-1 (5 mpk) exhibited a remarkably excellent anticancer effect (p>0.0001). Individual tumor sizes for each test group are shown inFIGS.69to73. According to the results of individual tumor sizes, slight tumor regression was observed in some animals of the group having received the anti-PD-1 antibody. The group having received mGI101 (6 mpk) alone exhibited a more excellent tumor growth inhibitory effect as compared with the group having received the anti-PD-1 antibody. The tumor size was maintained at the same size from day 5 to day 7, but was regrown after day 7. The group having received the combination (GI101 (0.6 mpk)+an anti-PD-1 antibody (5 mpk)) exhibited a remarkably excellent tumor growth inhibition. In particular, two mice of the group having received the combination showed a complete remission (no tumor). MC38 cells were reinjected into the left flank of two mice of the group having received the combination that showed a complete remission (the site opposite to the first injection site of cancer cells). These mice maintained an anti-PD-1 antibody administration (5 mpk, BIW) until day 32 (FIG.74). A tumor with a small size (>30 mm3) was observed in one of the two mice, but the tumor size did not grow any more until day 35 (FIG.69). In another mouse, no tumor was observed after the tumor was reinjected (FIGS.69and74). In conclusion, as a result of testing the anti-tumor efficacy of mGI101 alone and in combination with an anti-PD-1 antibody in the MC38 allotransplanted tumor model, the most excellent anti-tumor efficacy was shown in the group having received the combination (GI101 (0.6 mpk)+anti-PD-1 (5 mpk)). Two of the experimental animals in the group having received the combination showed a complete remission, and the complete remission mice reinjected with MC38 showed a cancer resistance effect (Table 6). TABLE 6DaysCR mouseAfterNo. 1No. 2treatmentTumor Vol. (mm3)Tumor Vol. (mm3)D178.223.6D284.513.5D336.30D400D500D600D700D800D900D1000D1100D1200D1300D1400D1500D1600D1700D1800D1900D2000D2100D2200D2300D2400*D2500D2600D2700D2800D2900D3000D3100D3212.80 Experimental Example 22. Identification of Anticancer Effect by Administration of Combination of mGI101 and Anti-PD-L1 Antibody in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This test was to evaluate the tumor growth inhibitory effect after administration of mGI101 as a test substance alone or in combination with an anti-PD-L1 antibody (BioXcell, Cat #BE0101) as a positive control substance in a tumor model allotransplanted with CT26 cells (murine colon carcinoma cells) into BALB/c mice. CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. In order to establish an allotransplanted tumor model, 5×105CT26 cells were subcutaneously injected into the right flank of BALB/c female mice (7-week-old). The mice were randomly assigned based on tumor volume (50 to 120 mm3), each group including 4 mice. The tumor grafts were identified about day 2 after cell inoculation. As shown in Table 7, the test groups were configured and the test substances were administered. TABLE 7Route ofNumberadministration,DosageofExperimental groupdosing cycleamountanimalsG1Vehicle control (PBS)i.p. BIW × 9 days—4G2mGI101i.v. QW × 9 days3 mg/kg4G3Anti-PD-L1 antibodyi.p. BIW × 9 days10 mg/kg4(BioXcell, Cat#BE0101)G4mGI101 +i.v. QW × 9 days3 mg/kg4anti-PD-L1 antibody(mGI101)i.p. BIW × 9 days10 mg/kg(anti-PD-L1antibody) Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified. At the end of the test period, the animals were sacrificed. The size of the CT26 solid cancer was measured using a tumor 3D scanner (TM900, Peria, Belgium). For each experimental group, the average loss and percentage change of body weight and the average tumor growth inhibition were calculated. The anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through one-way analysis of variance (at the end of this test) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant. As a result of testing the anti-tumor efficacy of mGI101 alone and in combination with an anti-PD-L1 antibody in the CT26 allotransplanted tumor model, the most excellent anti-tumor efficacy was shown in the group having received the combination (mGI101 (3 mpk)+anti-PD-L1 (10 mpk)) (FIG.75). Experimental Example 23. Identification of Anticancer Effect by Administration of Combination of mGI101 and Anti-TIGIT Antibody in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This test was to evaluate the tumor growth inhibitory effect after administration of mGI101 as a test substance alone or in combination with an anti-TIGIT antibody specifically binding to an extracellular domain (ECD) of TIGIT as a positive control substance in a tumor model allotransplanted with CT26 cells (murine colon carcinoma cells) into BALB/c mice. CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. In order to establish an allotransplanted tumor model, 5×105CT26 cells were subcutaneously injected into the right flank of BALB/c female mice (7-week-old). The mice were randomly assigned based on tumor volume (50 to 120 mm3), each group including 5 mice. The tumor grafts were identified about day 2 after cell inoculation. As shown in Table 8, the test groups were configured and the test substances were administered. TABLE 8Route ofNumberadministration,DsageofExperimental groupdosing cycleamountanimalsG1Vehicle control (PBS)i.p. BIW × 9 days—5G2mGI101i.v. QW × 9 days3 mg/kg5G3Anti-TIGIT antibodyi.p. BIW × 9 days20 mg/kg5G4mGI101 + anti-i.v. QW × 9 days3 mg/kg5TIGIT antibody(mGI101)i.p. BIW × 9 days20 mg/kg(anti-TIGITantibody) Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified. At the end of the test period, the animals were sacrificed. The size of the CT26 solid cancer was measured using a tumor 3D scanner (TM900, Peria, Belgium). For each experimental group, the average loss and percentage change of body weight and the average tumor growth inhibition were calculated. The anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through one-way analysis of variance (at the end of this test) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant. As a result of testing the anti-tumor efficacy of mGI101 alone and in combination with an anti-TIGIT antibody in the CT26 allotransplanted tumor model, the most excellent anti-tumor efficacy was shown in the group having received the combination (mGI101 (3 mpk)+anti-TIGIT (20 mpk)) (FIG.76). No anti-tumor effect was observed in the group having received the anti-TIGIT antibody alone as compared with the control, but the group having received a combination with mGI101 exhibited a remarkably excellent anti-tumor effect as compared with the group having received mGI101 alone. VI. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and TGF-βR Inhibitor Experimental Example 24. Identification of Anticancer Effect by Administration of Combination of mGI-101 and TGF-βR Inhibitor (Galunisertib) in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This test was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Galunisertib as a positive control substance in a tumor model allotransplanted with CT26 (mouse colon carcinoma) cells into mice. The stock solution of the test substance, negative control substance, and positive control substance described in Table 9 was diluted by adding excipients according to each dose. TABLE 9—Test substancePositive control substanceNegative control substanceSubstancemGI-101GalunisertibhIgG4nameAppearanceclear liquidclear liquidclear liquidComponentFc fusion proteinTGF-β inhibitor—Excipienthistidine buffer (20 mM),1% CMCPBSpH 7.0, poloxamer 188(carboxymethylcellulose)-0.07 w/w %, arginine-HClNa15 mg/mL, sucrose 150mg/mLpH7.5——Storagerefrigerated storage (4° C.)refrigerated storage (4° C.)refrigerated storage (4° C.)conditionPrecautionskeep refrigerated untilkeep refrigerated untilkeep refrigerated untilfor handlingadministration, and prepareadministration, and prepareadministration, and prepareand use on the day ofand use on the day ofand use on the day ofadministrationadministrationadministration Mouse-derived colorectal cancer cells, CT26 (Mus musculus, Colon adenocarcinoma), were purchased from ATCC (USA) and used for the test. The cells to be used for the test were thawed, mixed with RPMI1640 (A1049101, Thermofisher scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), and then placed in a cell culture flask, and cultured in a 37° C., 5% CO2incubator. The cells were washed with PBS, and then the cells were isolated using Trypsin-EDTA (15090, Gibco), and centrifugation (125×g, 5 minutes) was performed to discard the supernatant, and then the cells were suspended in a new medium to obtain the cell suspension. The viability of the cells was identified, and then a cell line was prepared by diluting in a medium to a concentration of 5.0×106cells/mL. 6-week-old male BALB/cAnHsd mice were purchased and used for the test. After the end of the acclimation period of 7 days, the cells were transplanted into healthy animals. The cell suspension (5×106cells/mL) was dispensed, and filled into a disposable syringe, and transplantation of the suspension was performed by subcutaneous administration at 0.1 mL/head in the right dorsal region of the animals. General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. After the inoculation of the cell line was performed, when the tumor size at the site to which the cell line was transplanted reached about 50 mm3, the tumor size of each group was distributed as uniformly as possible according to the tumor size. As shown in Table 10, the test groups were configured. The test substance was administered orally or intraperitoneally, and administered for 3 weeks depending on the composition of the test group. In the case of oral administration, the animals were fixed with the cervical spine skin fixation method, and administered directly into the stomach using a sonde for oral administration. In the case of intraperitoneal administration, the animals were fixed with the cervical spine skin fixation method, and administered intraperitoneally using a syringe equipped with a 26 gauge needle. The administration rate was not to exceed 200 μl/min. TABLE 10AdministeredFrequency ofRoute ofDosage amountDosage volumeGroupSexsubstanceadministrationadministration(mg/kg/day)(mL/kg/day)G1MVehicle2 times/weeki.p.—5G2MmGI-1012 times/weeki.p.35G3MGalunisertib3 times/weekp.o.7510G4MmGI-1012 times/weeki.p.35Galunisertib3 times/weekp.o.7510 The type of general symptoms including death, the date of onset, and the severity of symptoms were observed once a day during the administration and observation period, and recorded for each subject. Body weight was measured on the day of grouping or on the day of start of administration of the test substance, and thereafter once a week. The tumor size was measured three times a week for 3 weeks from the day of start of administration of the test substance. The major axis and minor axis of the tumor were measured using a caliper, and the tumor size (tumor volume, TV) was calculated using the following equations. TV (mm3)=(W2×L)/2 [Equation 1] The tumor growth inhibition rate was calculated using the tumor size measurement results. The tumor growth inhibition rate was calculated as follows. % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] As a result of measuring the tumor size, the tumor size levels of G2 and G4 on day 4 after the start of administration of the test substance were statistically significantly lower than that of G1 (p<0.01, p<0.001), and the tumor size levels of G2 and G4 on day 7 after the start of administration of the test substance were statistically significantly lower than that of G1 (p<0.05 or p<0.01). From day 2 to day 7 after the start of administration of the test substance, the tumor size levels of G2 and G4 tended to be lower than that of G1. In addition, until day 7 after the start of administration of the test substance, the tumor size levels of G4 was lower than that of G2 or G3 (FIGS.77and79). TABLE 11Tumor sizeUnit: mm3Experimental groupDayG1G2G3G4061.97 ± 12.4161.96 ± 11.8161.98 ± 12.2361.98 ± 12.162210.34 ± 32.30177.83 ± 78.22187.20 ± 55.14149.16 ± 43.024363.47 ± 36.18224.56 ± 105.31**260.69 ± 78.70187.35 ± 60.13***7488.92 ± 81.97291.81 ± 155.23 (9)*398.78 ± 146.30232.95 ± 99.73**N10101010The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Galunisertib 75 mg/kg PO,G4: mGI-101 3 mg/kg IP + Galunisertib 75 mg/kg PO***/**/*A significant difference at p < 0.001/p < 0.01/p < 0.05 level compared to the G1 As a result of calculating the tumor growth inhibition rate, from day 4 to day 7 after the start of administration of the test substance, the tumor growth inhibition level of G2 was statistically significantly higher than that of G1. From day 2 to day 7 after the start of administration of the test substance, the tumor growth inhibition level of G4 was statistically significantly higher than that of G1. In addition, on day 7, the tumor growth inhibition level of G4 was statistically significantly higher than that of G3. At the end of the experiment, mice with the tumor growth inhibition rate of 30%, 50%, or 80% or more are as shown inFIG.78. TABLE 12Tumor growth inhibitionUnit: %Experimental groupDayG1G2G3G420.00 ± 20.7421.91 ± 53.4115.60 ± 38.2641.24 ± 25.98*40.00 ± 10.5046.07 ± 35.1**34.09 ± 27.1458.42 ± 17.88***70.00 ± 19.2146.48 ± 36.39 (9)**21.12 ± 34.9859.96 ± 21.45***’$N10101010The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Galunisertib 75 mg/kg PO,G4: mGI-101 3 mg/kg IP + Galunisertib 75 mg/kg PO***/**/*A significant difference at p < 0.001/p < 0.01/p < 0.05 level compared to the G1$A significant difference at p < 0.05 level compared to the G3 Experimental Example 25. Identification of Anticancer Effect by Combination of mGI-101 and TGF-βR Inhibitor (Vactosertib) in Breast Cancer Cell Line This experiment was to evaluate the effect of killing cancer cells by treating MDA-MB-231 cells (human breast cancer cells) with the test substance GI-101 alone or in combination with the TGF-beta signal inhibitor Vactosertib substance in an in vitro environment. MDA-MB-231 cells were purchased from the Korea cell line bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation or inhibition of the proliferation of cancer cells. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μl (1×104cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour. Human peripheral blood mononuclear cells (PBMCs) were used in order to identify the effect of killing cancer cells by GI-101. The human PBMCs were purchased from Zen-Bio, and the PBMCs stored frozen were placed in a 37° C. water bath, and thawed as quickly as possible, and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×105cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions. In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs. Vactosertib power was dissolved in DMSO (Sigma) to a concentration of 48.4 mM, and diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 12.1 nM (50 μL) per well of a 96-well microplate. GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM by 50 μl per well of a 96-well microplate. The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent. As a result, it was identified that the group having received a combination of GI-101 and Vactosertib exhibited the excellent effect of killing cancer cells as compared with the group having received each drug alone. VII. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and VEGFR Inhibitor Experimental Example 26. Identification of Anticancer Effect by Administration of Combination of mGI-101 and VEGFR Inhibitor (Axitinib) This test was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Axitinib as a positive control substance in a tumor model allotransplanted with CT26 cells (mouse colon carcinoma) or LL2 cells (mouse lung carcinoma) into mice. The stock solution of the test substance, negative control substance, and positive control substance described in Table 13 was diluted by adding excipients according to each dose. TABLE 13—Test substancePositive control substanceNegative control substanceSubstancemGI-101AxitinibhIgG4nameAppearanceclear liquidclear liquidclear liquidComponentFc fusion proteinVEGFR inhibitor—Excipienthistidine buffer (20 mM),0.5% CMCPBSpH 7.0, poloxamer 188(carboxymethylcellulose)0.07 w/w %, arginine-HCl15 mg/mL, sucrose 150mg/mLpH7.5——Storagerefrigerated storage (4° C.)refrigerated storage (4° C.)refrigerated storage (4° C.)conditionPrecautionskeep refrigerated untilkeep refrigerated untilkeep refrigerated untilfor handlingadministration, and prepareadministration, and prepareadministration, and prepareand use on the day ofand use on the day ofand use on the day ofadministrationadministrationadministration Mouse-derived colorectal cancer cells, CT26 (Mus musculus, Colon adenocarcinoma), and mouse-derived lung cancer cells, LL/2 (Mus musculus, Lung adenocarcinoma), were purchased from ATCC (USA) and used for the test. The cells to be used for the test were thawed, mixed with RPMI1640 (A1049101, Thermofisher scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), and then placed in a cell culture flask, and cultured in a 37° C., 5% CO2incubator. The cells were washed with PBS, and then the cells were isolated using Trypsin-EDTA (15090, Gibco), and centrifugation (125×g, 5 minutes) was performed to discard the supernatant, and then the cells were suspended in a new medium to obtain the cell suspension. The viability of the cells was identified, and then a cell line was prepared by diluting in a medium to a concentration of 5.0×106cells/mL. 6-week-old male BALB/cAnHsd mice or C57BL/6NHsd mice were purchased and used for the test. After the end of the acclimation period of 7 days, the cells were transplanted into healthy animals. The cell suspension (5×106cells/mL) was dispensed, and filled into a disposable syringe, and transplantation of the suspension was performed by subcutaneous administration at 0.1 mL/head in the right dorsal region of the animals. General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. After the inoculation of the cell line was performed, when the tumor size at the site to which the cell line was transplanted reached about 50 mm3, the tumor size of each group was distributed as uniformly as possible according to the ranked tumor size. As shown in Table 14, the test groups were configured. The test substance was administered orally or intraperitoneally, and administered for 3 weeks depending on the composition of the test group. In the case of oral administration, the animals were fixed with the cervical spine skin fixation method, and administered directly into the stomach using a sonde for oral administration. In the case of intraperitoneal administration, the animals were fixed with the cervical spine skin fixation method, and administered intraperitoneally using a syringe equipped with a 26 gauge needle. The administration rate was not to exceed 200 μl/min. TABLE 14AdministeredFrequency ofRoute ofDosage amountDosage volumeGroupSexsubstanceadministrationadministration(mg/kg/day)(mL/kg/day)G1MVehicle2 times/weekIP35G2MmGI-1012 times/weekIP35G3MAxitinib3 times/weekPO3010G4MmGI-1012 times/weekIP35Axitinib3 times/weekPO3010 The tumor size was measured in the same manner as described in Experimental Example 24. Experimental Example 26.1. Mouse Tumor Model Allotrasplanted with Colorectal Cancer (CT26) Syngeneic model was prepared by subcutaneously transplanting the CT26 cell line into Balb/c mice, and then the test substance was administered to evaluate the anticancer effect. As a result of measuring the tumor size, the tumor size level of G4 on day 18 and day 21 after the start of administration of the test substance was statistically significantly lower than that of G1 and G2 (p<0.001, p<0.01, or p<0.05). The tumor size level of G4 on day 21 after the start of administration of the test substance was statistically significantly lower than that of G3 (p<0.01). In addition, the tumor size level of G3 on day 21 after the start of administration of the test substance was statistically significantly lower than that of G1 and G2 (p<0.001, p<0.01) (FIGS.80and82). TABLE 15Tumor sizeUnit: mm3Experimental groupDayG1G2G3G4059.15 ± 11.6563.53 ± 11.5661.67 ± 12.5859.58 ± 11.142205.25 ± 31.92148.98 ± 55.26169.28 ± 32.19127.90 ± 24.995354.24 ± 32.53186.53 ± 76.61261.76 ± 61.86178.53 ± 84.437468.75 ± 75.04249.93 ± 149.08354.05 ± 104.90224.92 ± 158.219573.13 ± 101.41290.71 ± 165.17442.98 ± 148.70235.75 ± 158.3512789.72 ± 88.58352.46 ± 195.64529.85 ± 206.76254.91 ± 176.31*141206.38 ± 229.96520.04 ± 307.47**671.72 ± 303.14*254.76 ± 159.31****161693.18 ± 210.83962.28 ± 634.90**970.72 ± 475.99**381.38 ± 235.37****,#,$192037.27 ± 341.201395.48 ± 982.61**1129.12 ± 639.16****465.02 ± 285.88****,####,$$213082.01 ± 462.371987.45 ± 1320.62****1710.57 ± 1133.79****880.10 ± 632 96****,####,$$N8888The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Axitinib 30 mg/kg PO,G4: mGI-101 3 mg/kg IP + Axitinib 30 mg/kg PO****/**/*A significant difference at p < 0.0001/p < 0.01/p < 0.05 level compared to the Vehicle####/#A significant difference at p < 0.0001/p < 0.05 level compared to the mGI-101$$$/$$/$A significant difference at p < 0.001/p < 0.01/p < 0.05 level compared to the Axitinib As a result of calculating the tumor growth inhibition rate, from day 2 to day 21 after the start of administration of the test substance, the tumor growth inhibition levels of G2 and G4 were statistically significantly higher than that of G1 (p<0.0001, p<0.001, p<0.01 or p<0.05), and from day 9 to day 14 after the start of administration of the test substance, the tumor growth inhibition level of G4 was statistically significantly higher than that of G3 (p<0.05). In addition, on day 19 after the start of administration of the test substance, the tumor growth inhibition level of G4 was statistically significantly higher than that of G2 (p<0.05). At the end of the experiment, mice with the tumor growth inhibition rate of 30%, 50%, or 80% or more are as shown inFIG.81. TABLE 16Tumor growth inhibitionUnit: %Experimental groupDayG1G2G3G420.00 ± 22.7641.51 ± 36.11*26.34 ± 20.4753.24 ± 19.71***50.00 ± 10.6958.32 ± 24.95***32.19 ± 18.6459.69 ± 29.99***70.00 ± 19.6062.12 ± 38.89****28.62 ± 24.3559.63 ± 39.68***90.00 ± 20.2962.87 ± 34.95****25.81 ± 27.7765.72 ± 31.54****,#,$120.00 ± 11.9866.48 ± 29.50****35.92 ± 27.4473.26 ± 24.71****’$140.00 ± 20.3365.87 ± 29.12****46.82 ± 25.79**82.99 ± 14.19****’$160.00 ± 13.2052.36 ± 41.33**44.37 ± 28.66**80.31 ± 14.77****190.00 ± 17.3141.48 ± 52.17*46.04 ± 31.96**79.50 ± 14.72****,#210.00 ± 15.4544.57 ± 46.55**45.45 ± 37.30**72.86 ± 21.07****N8888The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Axitinib 30 mg/kg PO,G4: mGI-101 3 mg/kg IP + Axitinib 30 mg/kg PO***/***/**/*A significant difference at p < 0.0001/p < 0.001/p < 0.01/p < 0.05 level compared to the Vehicle#A significant difference at p < 0.05 level compared to the mGI-101 3 mg/kg$A significant difference at p < 0.05 level compared to the mGI-101 3 mg/kg + Axitinib 30 mg/kg Experimental Example 26.2. Mouse Tumor Model Allotrasplanted Lung Cancer (LL/2) Syngeneic model was prepared by subcutaneously transplanting the LL/2 cell line into C57BL/6 mice, and then the test substance was administered to evaluate the anticancer effect. As a result of measuring the tumor size, G3 and G4 maintained a lower tumor size level than that of G1 until the end of the test. In addition, the tumor size level of G4 during the entire test period was the lowest level among all test groups, and the tumor size level of G4 on day 19 after the start of administration of the test substance was statistically significantly lower than that of G1 (p<0.05) and G2 (p<0.01). The tumor size level of G4 on day 21 after the start of administration of the test substance was statistically significantly lower than that of all groups of G1 (p<0.0001), G2 (p<0.0001), and G3 (p<0.01). In addition, the tumor size level of G3 on day 21 after the start of administration of the test substance was statistically significantly lower than that of G1 (p<0.01) and G2 (p<0.001) (FIGS.83and85). TABLE 17Tumor sizeUnit: mm3Experimental groupDayG1G2G3G4055.75 ± 12.2955.70 ± 11.4855.77 ± 11.2555.73 ± 9.99296.34 ± 25.8799.18 ± 19.9688.73 ± 21.2698.80 ± 24.935181.94 ± 40.25179.58 ± 39.71179.31 ± 52.62179.21 ± 58.267460.71 ± 126.65409.37 ± 72.98362.30 ± 97.92341.77 ± 91.559791.60 ± 247.83749.00 ± 174.47584.17 ± 172.94544.37 ± 155.89121141.58 ± 236.271066.72 ± 307.18892.90 ± 256.88841.86 ± 267.92141637.35 ± 402.071608.00 ± 572.501292.27 ± 385.431242.66 ± 424.70162219.74 ± 442.052200.03 ± 850.121775.26 ± 462.50 (9)1693.31 ± 579.80193044.87 ± 518.053223.87 ± 1191.112551.08 ± 695.31 (9)2215.02 ± 667.76*,##215285.56 ± 1120.415431.08 ± 1673.604236.33 ± 1060.18 (9)**,##3255.38 ± 819.52****,####,$$N10101010The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Axitinib 30 mg/kg PO,G4: mGI-101 3 mg/kg IP + Axitinib 30 mg/kg PO****/*A significant difference at p < 0.0001/p < 0.05 level compared to the Vehicle####/##A significant difference at p < 0.0001/p < 0.01 level compared to the mGI-101 3 mg/kg$$A significant difference at p < 0.01 level compared to the mGI-101 3 mg/kg + Axitinib 30 mg/kg As a result of calculating the tumor growth inhibition rate, the tumor growth inhibition level of G4 on day 19 and day 21 after the start of administration of the test substance was statistically significantly higher than that of G2 (p<0.01, p<0.05), and the tumor growth inhibition level of G4 on day 21 after the start of administration of the test substance was statistically significantly higher than that of G1 (p<0.05). The lowest tumor size level was observed in G4, and the tumor growth inhibition rate also tended to be highest. At the end of the experiment, mice with the tumor growth inhibition rate of 30%, 50%, or 80% or more are as shown inFIG.84. TABLE 18Tumor growth inhibitionUnit: %DayExperimental groupG1G2G3G420.00 ± 46.91−7.12 ± 34.3118.79 ± 28.38−6.12 ± 53.1950.00 ± 26.041.83 ± 27.192.10 ± 36.272.15 ± 43.7770.00 ± 29.8912.67 ± 19.1524.30 ± 23.6329.36 ± 21.69*90.00 ± 33.795.78 ± 24.0828.19 ± 23.4633.59 ± 20.83120.00 ± 21.786.89 ± 28.4722.90 ± 23.4227.60 ± 24.47140.00 ± 25.381.85 ± 36.3921.82 ± 24.1224.95 ± 26.67160.00 ± 20.300.91 ± 39.3720.58 ± 21.15 (9)24.33 ± 26.59190.00 ± 17.10−5.99 ± 39.9116.55 ± 23.06 (9)27.76 ± 22.20#210.00 ± 21.32−2.78 ± 32.0420.08 ± 20.18 (9)38.82 ± 15.63*,##N10101010The results were expressed as mean ± standard deviation.N: number of animals,G1: Vehicle control IP,G2: mGI-101 3 mg/kg IP,G3: Axitinib 30 mg/kg PO,G4: mGI-101 3 mg/kg IP + Axitinib 30 mg/kg PO*A significant difference at p < 0.05 level compared to the Vehicle##/#A significant difference at p < 0.01/p < 0.05 level compared to the mGI-101 3 mg/kg Experimental Example 27. Identification of Anticancer Effect by Administration of Combination of mGI-101 and VEGFR Inhibitor (Lenvatinib) Experimental Example 27.1. Identification of Anticancer Effect by Administration of Combination of mGI-101 and Lenvatinib in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Lenvatinib substance in a tumor model allotransplanted with CT26 cells (murine colon carcinoma cells) into BALB/c mice. CT26 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. In order to establish an allotransplanted tumor model, 1×106CT26 cells were subcutaneously injected into the right flank of BALB/c female mice (7-week-old). General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. A certain period of time after the cell transplantation, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the mice were assigned so that the average tumor volume of each group was less than 70-100 mm3, each group including 10 mice. As shown in Table 19, the test groups were configured and the test substances were administered. In the case of Lenvatinib powder, the dose was calculated and weighed, and it was prepared to the dose concentration using a 0.5% methyl cellulose excipient. In order to minimize the loss of the test substance, the doses for 3 or 4 days were weighed, and prepared using the excipient on the day of administration and injected. TABLE 19Route ofDosageNumber ofExperimental groupadministrationDosing cycleamountanimalsG1Vehiclei.v.QW—mg/kg10control (PBS)(once/week)G2mGI-101i.v.QW3mg/kg10(once/week)G3Lenvatinibp.o.daily3mg/kg10G4mGI-101 +i.v. + p.o.mGI-101: QW3 mg/kg + 310Lenvatinib(once/week),mg/kgLenvatinib:daily Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the CT26 solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of tumor size upon administration of mGI-101 alone or in combination with Lenvatinib substance against the CT26 tumor are shown inFIG.86. As a result of measuring the tumor size, as compared with the control, the statistically significant anticancer effect was observed in the group having received Lenvatinib alone and the group having received a combination of mGI-101+Lenvatinib. The tumor size level of the group having received Lenvatinib alone on day 18 and day 21 after the start of administration of the test substance was statistically significantly lower than that of the control (p<0.5, p<0.1). The tumor size level of the group having received a combination of mGI-101+Lenvatinib on day 16, day 18, and day 21 after the start of administration of the test substance was statistically significantly lower than that of the control (p<0.5, p<0.001, p<0.0001), and was statistically significantly lower than that of the group having received mGI-101 alone on day 18 and day 21 (p<0.01, p<0.0001). Individual tumor sizes for each test group are shown inFIG.88. According to the results of individual tumor sizes, the group having received a combination of mGI-101+Lenvatinib exhibited an excellent tumor growth inhibitory effect as compared with the group having received mGI-101 alone. At the end of the experiment, mice with the tumor growth inhibition rate of 30%, 50%, or 80% or more are as shown inFIG.87. The vehicle control exhibited a tumor growth inhibition rate of 30% or more in 4 mice, 50% or more in 3 mice, and 80% or more in 1 mouse. The group having received mGI-101 alone exhibited a tumor growth inhibition rate of 30% or more in 6 mice, 50% or more in 2 mice, and 80% or more in 1 mouse. The group having received Lenvatinib alone exhibited a tumor growth inhibition rate of 30% or more in 6 mice, 50% or more in 4 mice, and 80% or more in 1 mouse. The group having received a combination of mGI-101+Lenvatinib exhibited a tumor growth inhibition rate of 30% or more in 10 mice, 50% or more in 6 mice, and 80% or more in no mouse. Experimental Example 27.2. Identification of Anticancer Effect by Administration of Combination of mGI-101 and Lenvatinib in Mice Transplanted with Mouse-Derived Renal Cancer Cell Line This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Lenvatinib substance in a tumor model allotransplanted with Renca cells (mouse renal cancer cells) into BALB/c mice. Renca cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. In order to establish an allotransplanted tumor model, 5×106Renca cells were subcutaneously injected into the back of BALB/c female mice (8-week-old). General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the mice were randomly selected and assigned, each group including 10 mice. As shown in Table 20, the test groups were configured and the test substances were administered. TABLE 20ExperimentalRoute ofDosingDosageNumber ofgroupadministrationcycleamountanimalsG1Vehiclei.p.QW—mg/kg10control (PBS)(once/week)G2mGI-101i.p.BIW (23mg/kg10times/week)G3Lenvatinibp.o.daily3mg/kg10G4mGI-101 +i.p. + p.o.mGI-101: BIW3 mg/kg + 310Lenvatinib(2 times/week),mg/kgLenvatinib:daily The death of the mouse, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day during the test period, and recorded for each subject. The size of the Renca solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a vernier caliper, and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping, and the anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of tumor size upon administration of mGI-101 alone or in combination with Lenvatinib substance against the Renca tumor are shown inFIG.89. As a result of measuring the tumor size, the tumor size of the group having received a combination of mGI-101 (BIW)+Lenvatinib on day 15 after the start of administration of the test substance was statistically significantly lower than that of vehicle control and the group having received mGI-101 (BIW) alone (p<0.05). Individual tumor sizes for each test group are shown inFIG.91. According to the results of individual tumor sizes, the group having received a combination of mGI-101 (BIW)+Lenvatinib exhibited an excellent tumor growth inhibitory effect. FIG.90illustrates a tumor growth inhibition rate when mGI-101 and Lenvatinib are administered in combination in mice transplanted with Renca. The vehicle control exhibited a tumor growth inhibition rate of 30% or more in 4 mice, 50% or more in 3 mice, and 80% or more in 2 mice. The group having received mGI-101 alone once a week exhibited a tumor growth inhibition rate of 30% or more in 5 mice, 50% or more in 2 mice, and 80% or more in 1 mouse. The group having received mGI-101 alone twice a week exhibited a tumor growth inhibition rate of 30% or more in 3 mice, 50% or more in 1 mouse, and 80% or more in no mouse. The group having received Lenvatinib alone exhibited a tumor growth inhibition rate of 30% or more in 4 mice, 50% or more in 2 mice, and 80% or more in no mouse. The group having received a combination of mGI-101 (BIW)+Lenvatinib exhibited a tumor growth inhibition rate of 30% or more in 8 mice, 50% or more in 6 mice, and 80% or more in 1 mouse. VIII. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and EGFR Inhibitor Experimental Example 28. Identification of Anticancer Effect of Combination of mGI-101 and EGFR Inhibitor (Cetuximab) This experiment was to evaluate the effect of killing cancer cells by treating HCT116 cells (human colon cancer cells) with the test substance GI-101 alone or in combination with Cetuximab substance in an in vitro environment. HCT116 cells were purchased from the Korea cell line bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in McCoy's 5A medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in McCoy's 5A medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation of cancer cells or inhibition of the proliferation. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μL (1×104cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour. In order to identify the effect of killing cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by the test substance, natural killer cells (NK cells) were isolated from human peripheral blood mononuclear cells (PBMCs) using a CD56+CD16+NK cell isolation kit (Miltenyi Biotec) and used. In isolated NK cells, dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions. In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs. Cetuximab was diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 68.6 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration 100 nM by 50 μl per well of a 96-well microplate. The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent. FIG.92illustrates the degree of killing cancer cells measured when the cancer cells were treated with GI-101 at a concentration of 100 nM. All groups having received GI-101 alone, Cetuximab alone, and a combination of GI-101+Cetuximab exhibited a high level of killing cancer cells, and the group having received a combination of GI-101+Cetuximab exhibited the most excellent effect of killing cancer cells. IX. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and PARP Inhibitor Experimental Example 29. Identification of Anticancer Effect by Administration of Combination of mGI-101 and PARP Inhibitor (Olaparib) This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Olaparib, a PARP inhibitor, in a tumor model allotransplanted with 4T1 cells (mouse breast cancer cells) into BALB/c mice. 4T1 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. In order to establish an allotransplanted tumor model, 1×105of 4T1 cells were subcutaneously injected into the back of BALB/c female mice (8-week-old). General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the mice were randomly selected and assigned, each group including 11 mice. As shown in Table 21, the test groups were configured and the test substances were administered. TABLE 21ExperimentalRoute ofDosingDosageNumber ofgroupadministrationcycleamountanimalsG1Vehiclei.p.QW (once/week)—mg/kg11control (PBS)G2mGI-101i.p.BIW (2 times/week)3mg/kg11G3OlaparibP.O.5 times/week30mg/kg11G4mGI-101 +i.p. + P.O.mGI-101: BIW (23 mg/kg +11Olaparibtimes/week),30 mg/kgOlaparib: 5 times/week Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the 4T1 solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping, and the anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of tumor size upon administration of mGI-101 alone or in combination with Olaparib substance against the 4T1 tumor are shown inFIG.94. As a result of measuring the tumor size, the anticancer effect was observed in the group having received the drug as compared with the control, and according to the results of tumor size levels, the group having received mGI-101 alone and in combination with Olaparib substance exhibited an excellent tumor growth inhibitory effect as compared with the group having received mGI-101 alone. Experimental Example 30. Identification of Anticancer Effect by Administration of Combination of mGI-101 and PARP Inhibitor (Talazoparib) This experiment was to evaluate the effect of killing cancer cells by treating MDA-MB-231 cells (human breast cancer cells) with the test substance GI-101 alone or in combination with the PARP inhibitor Talazoparib substance in an in vitro environment. MDA-MB-231 cells were purchased from the Korea cell line bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation of cancer cells or inhibition of the proliferation. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μl (1×104cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour. Human peripheral blood mononuclear cells (PBMCs) were used in order to identify the effect of killing cancer cells by GI-101. The human PBMCs were purchased from Zen-Bio, and the PBMCs stored frozen were placed in a 37° C. water bath, and thawed as quickly as possible, and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×105cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions. In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs. Talazoparib test substance was diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 0.57 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM by 50 μl per well of a 96-well microplate. The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent. As a result, it was identified that the group having received a combination of GO-101 and Talazoparib exhibited the excellent effect of killing cancer cells as compared with the group having received each drug alone. X. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and DNA Methyltransferase Inhibitor Experimental Example 31. Identification of Anticancer Effect by Administration of Combination of mGI-101 and Guadecitabine in Mice Transplanted with Mouse-Derived Colorectal Cancer Cells This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Guadecitabine substance that inhibits DNA methylation in a tumor model allotransplanted with CT26 cells (murine colon carcinoma cells) into BALB/c mice. CT26 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. In order to establish an allotransplanted tumor model, 5×105of CT26 cells were subcutaneously injected into the right flank of BALB/c female mice (8-week-old) (FIG.97). The tumor grafts of the mice were identified about day 7 after cell inoculation, and the mice were randomly assigned based on tumor volume (50-120 mm3), each group including 13 mice. As shown in Table 22, the test groups were configured and the test substances were administered. TABLE 22Route ofDosingDosageNumber ofExperimental groupadministrationcycleamountanimalsG1Vehicle control (PBS)i.p.QW—mg/kg13(once/week)G2mGI-101i.v.QW0.6mg/kg13(once/week)G3mGI-101i.v.QW3mg/kg13(once/week)G4Guadecitabinei.p.total 4 times, 450μg13consecutivedaysG5mGI-101 +i.v. + i.p.mGI-101: QW0.6 mg/kg +13Guadecitabine(once/week),50 μgG6mGI-101 +i.v. + i.p.Guadecitabine:3 mg/kg +13Guadecitabinetotal 4 times, 450 ngconsecutivedays Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the CT26 solid cancer was measured using a tumor 3D scanner (TM900, Peria, Belgium). For each experimental group, the average loss and percentage change of body weight and the average tumor growth inhibition were calculated. The anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of tumor size upon administration of mGI-101 alone or in combination with Guadecitabine substance against the CT26 tumor are shown inFIGS.97and100. As a result of measuring the tumor size, the anticancer effect was observed in the group having received the drug as compared with the control, and the group having received a combination of mGI-101 and Guadecitabine exhibited a more excellent tumor growth inhibitory effect as compared with the group having received mGI-101 alone. The tumor size of the group having received a combination of mGI-101 (3 mg/kg)+Guadecitabine on day 7 after the start of administration of the test substance was statistically significantly lower than that of the control (p<0.05). On day 10 after the start of administration of the test substance, the tumor size level of the group having received mGI-101 (0.6 mg/kg) alone tended to be lower than that of the control, and the tumor size level of the group having received mGI-101 (3 mg/kg) alone was statistically significantly lower than that of the control (p<0.05). The tumor size levels of the group having received a combination of mGI-101 (0.6 mg/kg)+Guadecitabine and the group having received a combination of mGI-101 (3 mg/kg)+Guadecitabine were statistically significantly lower than that of the control (p<0.01, p<0.001). Individual tumor sizes for each test group are shown inFIGS.99and102. According to the results of individual tumor sizes, the group having received mGI-101 alone also exhibited the tumor growth inhibitory effect, and the group having received a combination mGI-101 and Guadecitabine substance exhibited the most excellent tumor growth inhibitory effect. At the end of the experiment, mice with the tumor growth inhibition rate of 30%, 50%, or 80% or more are as shown inFIGS.99and101. It was identified that subjects with the tumor growth inhibition rate of 30%, 50%, 80% or more were the most in the group having received a combination of mGI-101 (3 mg/kg)+Guadecitabine. XI. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and Anticancer Chemotherapeutic Agent (Antineoplastic or Cytotoxic Agent) Experimental Example 32. Identification of Anticancer Effect by Administration of Combination of mGI-101, Anti-PD-L1 Antibody, and Docetaxel in Mice Transplanted with Mouse-Derived Breast Cancer Cells This experiment was to evaluate the anticancer efficacy according to administration of the test substance mGI-101 alone or in combination with Docetaxel (Selleck Chemicals, cat. no. S1148) and the anti-PD-L1 antibody (BioXcell, cat. no. BE0101) in a tumor model allotransplanted with 4T1 cells (mouse breast cancer cells) into BALB/c mice. 4T1 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco), and then a cell suspension was prepare with PBS and stored on ice until injected into mice. In order to establish an allotransplanted tumor model, after identifying the location of the second mammary fat pad from the upper right of the ventral region of BALB/c female mice (8-week-old), 4T1 cell line prepared inside the mammary fat pad was injected at 4×104cells/40 μl/head. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the subjects were selected so that the average of each group reached less than 70-100 mm3, and the selected animals were assigned as evenly as possible based on tumor volume and body weight, each group including 10 animals. As shown in Table 23, the test groups were configured and the test substances were administered. Docetaxel was dissolved in 100% DMSO, and then the volume was adjusted with 5% of DMSO, 30% of PEG300, 5% of Tween 80, and 60% of distilled water for injection (DMSO PEG300: Tween 80: distilled water for injection=5%: 30%: 5%: 60% (v:v:v:v)) and prepared. The Anti-PD-L1 antibody was prepared and administered using 1×PBS in consideration of dosage amount and volume. TABLE 23Route ofDosingDosageNumber ofExperimental groupadministrationcycleamountanimalsG1Vehiclei.p.QW—mg/kg10control (PBS)(once/week)G2mGI-101 +i.p. + i.p.mGI-101: QW3 mg/kg + 1010aPD-L1(once/week),mg/kgG3Docetaxeli.p.aPD-Ll: QW15mg/kg10G4Docetaxel + mGI-i.p. +(once/week)15 mg/kg + 310101 + aPD-Lli.p. + i.p.Docetaxel:mg/kg + 10QWmg/kg(once/week), Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the 4T1 solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping, and the anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. After administration of mGI-101 alone or in combination with Docetaxel and anti-PD-L1 antibody to mice transplanted with mouse-derived breast cancer cells, the results of measuring the tumor size are shown inFIG.103. As a result of measuring the tumor size, on day 14 after the start of administration of the test substance, as compared with the control, the statistically significant anticancer effect was observed in the group having received Docetaxel and the group having received a combination of Docetaxel+mGI-101+aPD-L1 (p<0.05, p<0.01). Individual tumor sizes for each test group are shown inFIG.105. FIG.104illustrates a tumor growth inhibition rate when mGI-101 is administered alone or in combination with Docetaxel and an anti-PD-L1 antibody in mice transplanted with 4T1 at the end of the test. The vehicle control exhibited a tumor growth inhibition rate of 30% or more, 50% or more in no mouse. The group having received a combination of mGI-101+aPD-L1 exhibited a tumor growth inhibition rate of 30% or more in 1 mouse, and 50% or more in no mouse. The group having received Docetaxel exhibited a tumor growth inhibition rate of 30% or more in 5 mice, and 50% or more in 1 mouse. The group having received a combination of Docetaxel+mGI-101+aPD-L1 exhibited a tumor growth inhibition rate of 30% or more in 6 mice, and 50% or more in 2 mice. Experimental Example 33. Identification of Anticancer Effect by Administration of Combination of mGI-101 and Paclitaxel in Mice Transplanted with Mouse-Derived Breast Cancer Cells This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Paclitaxel substance in a tumor model allotransplanted with EMT6 cells (mouse breast cancer cells) into BALB/c mice. EMT6 cells were purchased from ATCC (USA) and cultured in Waymouth MB 751/1 medium (WELGENE) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco), and then a high-concentration cell suspension (2×106cells/0.4 mL) for 10 mice was prepare with PBS and stored on ice until injected into mice. In order to establish an allotransplanted tumor model, the skin was incised slightly away from the 4thnipple position from the upper right of the ventral region of BALB/c female mice (7-week-old), and the location of mammary fat pad at the incised site was identified, and then EMT6 cell line prepared inside the mammary fat pad was injected at 2×105cells/40 uL/head. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the subjects were selected so that the average of each group reached less than 70-100 mm3, and the selected animals were assigned as evenly as possible based on tumor volume and body weight, each group including 10 animals. As shown in Table 24 andFIG.106, the test groups were configured and the test substances were administered. TABLE 24Route ofDosingDosageNumber ofExperimental groupadministrationcycleamountanimalsG1Vehiclei.v.QW—mg/kg10control (PBS)(once/week)G2mGI-101i.v.QW3mg/kg10(once/week)G3Paclitaxel (PTX)i.p.total 7 times, 710mg/kg10consecutivedaysG4mGI-101 +i.v. + i.p.QW3 mg/kg + 1010Paclitaxel(once/week) +mg/kgtotal 7 times, 7consecutivedays Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the EMT6 solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping, and the anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of tumor size upon administration of mGI-101 alone or in combination with Paclitaxel substance against the EMT6 tumor are shown inFIG.107. As a result of measuring the tumor size, the anticancer effect was observed in the group having received the drug as compared with the control, and the tumor size level of the group having received mGI-101 alone and in combination with Paclitaxel substance on day 11 after the start of administration of the substance was statistically significantly lower than that of the control (p<0.01, p<0.001). The tumor size of all the group having received Paclitaxel, the group having received mGI-101 alone, and the group having received a combination of mGI-101 and Paclitaxel substance on day 13 after the start of administration of the substance was statistically significantly lower than that of the control (p<0.05, p<0.01, p<0.0001). Individual tumor sizes for each test group are shown inFIG.109. According to the results of individual tumor sizes, on day 13 after the start of administration of the test substance, the group having received mGI-101 alone exhibited complete remission in 2 mice, and the group having received a combination of mGI-101 and Paclitaxel exhibited complete remission in 1 mouse. FIG.108illustrates a tumor growth inhibition rate when mGI-101 and Paclitaxel are administered in combination in mice transplanted with EMT6. The vehicle control exhibited a tumor growth inhibition rate of 30% or more in 2 mice, 50% or more in 1 mouse, and 80% or more in no mouse. The group having received mGI-101 exhibited a tumor growth inhibition rate of 30% or more in 6 mice, 50% or more in 5 mice, and 80% or more in 5 mice. The group having received Paclitaxel exhibited a tumor growth inhibition rate of 30% or more in 7 mice, 50% or more in 4 mice, and 80% or more in 2 mice. The group having received a combination of mGI-101+Paclitaxel exhibited a tumor growth inhibition rate of 30% or more in 7 mice, 50% or more in 5 mice, and 80% or more in 4 mice. XII. Identification of Anticancer Effect According to Administration of Combination: mGI-101+Anti-PD-1+Pemetrexed+Cisplatin (Chemotherapy, Maintaining Therapy) Experimental Example 34. Identification of Anticancer Effect by Administration of Combination of mGI-101 and Anticancer Chemotherapeutic Agent and Anti-PD-1 Antibody in Mice Transplanted with Mouse-Derived Lung Cancer Cells This experiment was to evaluate the tumor growth inhibitory effect after intraperitoneal administration of mGI-101 as a test substance alone or in combination with an anticancer chemotherapeutic agent, Cisplastin (Selleck Chemicals, cat. no. S1166), Pemetrexed (Selleck Chemicals, cat. no. S1135), and an anti-PD-1 antibody (BioXcell, cat. no. BE0146) as a standard therapeutic agent in a tumor model allotransplanted with TC1 cells, lung cancer cell line, into C57BL/6 mice. Mouse-derived lung cancer cell line, TC1, was purchased from ATCC (USA) and used for the test. TC1 cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco), and then 1×106of TC1 cells were subcutaneously injected into the right flank of C57BL/6 female mice (7-week-old) in order to establish an allotransplanted tumor model. The mice were randomly assigned based on tumor volume (˜100 mm3), each group including 13 to 19 mice. The tumor grafts were identified about day 2 after cell inoculation. The test groups were configured as shown in Table 25, and the test substances were administered according to the schedule shown inFIG.110. The test was divided into the 1stline treatment and the anticancer maintenance therapy. For the 1stline treatment, cisplatin (CDDP) at 5 mg/kg, pemetrexed at 100 mg/kg, and an anti-PD-1 antibody at 10 mg/kg were intraperitoneally administered twice a week, respectively, and in the case of the group having received a combination with mGI-101, it was intraperitoneally administered once a week, i.e., a total of 3 times, at 3 mg/kg. In the anticancer maintenance therapy, mGI-101 was administered alone or mGI-101 was administered in combination with an anticancer chemotherapeutic agent and an anti-PD-1 antibody. TABLE 25Route ofNumberadministration,ofExperimental groupdosing cycleDosage amountanimalsG1mouse IgG4i.p. BIW3 mg/kg13G21) 1stline: Cisplatin +mGI-101: i.p. QWmGI-101: 3 mg/kg19Pemetrexed + anti-PD-1Cisplatin: i.p. BIWCisplatin: 5 mg/kgantibodyPemetrexed: i.p. BIWPemetrexed: 100 mg/kg2) Maintenance therapy:anti-PD-1 antibody:anti-PD-1 antibody: 10Pemetrexed + anti-PD-1i.p. BImg/kantibodyG31) 1stline: Cisplatin +13Pemetrexed + anti-PD-1antibody2) Maintenance therapy:Pemetrexed + anti-PD-1antibody + mGI-101G41) 1stline: Cisplatin +13Pemetrexed + anti-PD-1antibody2) Maintenancetherapy: mGI-101G51) 1stline: Cisplatin +15Pemetrexed + anti-PD-1antibody + mGI-1012)Maintenance therapy:Pemetrexed + anti-PD-1antibody + mGI-101 Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the TC1 solid cancer was measured using a tumor 3D scanner (TM900, Peria, Belgium). For each experimental group, the average loss and percentage change of body weight and the average tumor growth inhibition were calculated. The anti-tumor efficacy was evaluated as compared with the mouse IgG4 control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant. The results of administration of mGI-101 alone or in combination with an anticancer chemotherapeutic agent and an anti-PD-1 antibody against the TC1 tumor are shown inFIG.111. The anticancer effect was observed in the group having received the drug as compared with the control, and the difference in tumor size was noticeable during the test period of 24 days. In the case of the 1stline treatment, the group having received mGI-101 in combination with an anticancer chemotherapeutic agent and an anti-PD-1 antibody exhibited a more excellent tumor growth inhibitory effect than that of the group having received only an anticancer chemotherapeutic agent and an anti-PD-1 antibody. In the case of the anticancer maintenance therapy, the group having received mGI-101 alone exhibited as much anticancer effect as the group having received an anticancer chemotherapeutic agent and an anti-PD-1 antibody, and the group having received mGI-101 in combination with an anticancer chemotherapeutic agent and an anti-PD-1 antibody exhibited a more excellent tumor growth inhibitory effect. Individual tumor sizes for each test group are shown inFIGS.112to117. According to the results of individual tumor sizes, in the 1stline treatment and the anticancer maintenance therapy, the group having received a combination with mGI-101 exhibited the most excellent tumor growth inhibitory effect. FIG.118illustrates a result obtained by analyzing the survival rate of the mice according to the administration of a combination with mGI-101 during the 1stline treatment and the anticancer maintenance therapy in mice transplanted with TC1 cells. During the 1stline treatment and the anticancer maintenance therapy, a survival rate of 100% was identified in the group having received a combination with mGI-101. The average body weight of each test group is shown in Table 26. TABLE 26TreatmentBody weight (g) on daysTreatment−21357101213152023261mouse IgG4Mean17.5817.7418.2417.5817.8018.2318.0218.9419.6220.3021.2620.481mouse IgG4SEM1.001.070.951.141.081.091.011.081.321.351.611.7121stline: Cisplatin +Mean17.5817.8118.1317.2417.4118.1017.7718.5719.2920.4220.6920.61Pemetrexed + anti-PD-1Maintenance:Pemetrexed + anti-PD-121stline: Cisplatin +SEM0.820.770.980.941.151.561.471.561.711.912.011.61Pemetrexed + anti-PD-1Maintenance:Pemetrexed + anti-PD-131stline: Cisplatin +Mean17.4217.7318.0617.4217.7818.5418.1119.0119.2319.7220.2820.18Pemetrexed + anti-PD-1Maintenance:Pemetrexed + anti-PD-1 +mGI-10131stline: Cisplatin +SEM1.251.081.391.661.701.571.421.701.441.881.881.90Pemetrexed + anti-PD-1Maintenance:Pemetrexed + anti-PD-1 +mGI-10141stline: Cisplatin +Mean17.1017.7017.5816.5816.8317.4117.4918.0618.6719.2919.1819.15Pemetrexed + anti-PD-1Maintenance: mGI-10141stline: Cisplatin +SEM1.051.041.041.181.191.021.021.221.391.231.631.59Pemetrexed + anti-PD-1Maintenance: mGI-10151stline: Cisplatin +Mean17.5318.6418.9218.7218.7119.8219.6720.5920.7621.5621.8520.50Pemetrexed + anti-PD-1 +mGI-101Maintenance:Pemetrexed + anti-PD-1 +mGI-10151stline: Cisplatin +SEM0.990.851.010.960.891.181.150.941.341.671.690.71Pemetrexed + anti-PD-1Maintenance:Pemetrexed + anti-PD-1 +mGI-101 XIII. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and Anti-HER Antibody Experimental Example 35. Identification of Anticancer Effect by Administration of Combination of GI-101 and Trastuzumab This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Herceptin (Trastuzumab) substance in a tumor model xenotransplanted with BT-474 cells (human breast cancer cells) into BALB/c nu/nu mice. BT-474 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Welgene) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco), and then a cell line was prepared by diluting in a medium to a concentration of 5.0×106cells/0.05 mL. In order to establish a xenotransplanted tumor model for BT-474 cells, it was subcutaneously injected into the left flank of BALB/c nu/nu female mice (7-week-old) by pulling the leather skin of the mice to make a space using a pellet transplant trochar (MP-182, Innovative Research of America, USA) so that it was located under the skin of the left flank. 7 days after the injection of the estrogen pellet, the prepared BT-474 cell suspension (5×106cells/0.05 mL) was dispensed, and 0.05 mL MATRIGEL™ matrix phenol red-free (356237, BD) was added, and the prepared solution was filled into a disposable syringe, and transplantation of the solution was performed by subcutaneous administration at 0.1 mL/head in the right dorsal region of the animals. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the subjects were selected so that the average of each group reached less than 60-120 mm3, and the selected animals were assigned as evenly as possible based on tumor volume and body weight, each group including 10 animals. As shown in Table 27, the test groups were configured and the test substances were administered. TABLE 27Route ofDosingDosageNumber ofExperimental groupadministrationcycleamountanimalsG1Vehiclei.v.QW—mg/kg10control (PBS)(once/week)G2mGI-101i.v.QW3mg/kg(once/week)G3Herceptini.p.QW1mg/kg10(once/week)G4mGI-101 + Herceptini.v. + i.p.mGI-101: QW3 mg/kg + 110(once/week),mg/kgHerceptin: QW(once/week) Clinical symptoms such as a disease and a behavioral change were observed once a day during the test period, and deceased animals were identified, and the mice were sacrificed when the tumor size reached a size of 4,000 mm3. The size of the solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of measuring the tumor size after administration of mGI-101 alone or in combination with Herceptin to mice transplanted with human-derived breast cancer cells are shown inFIG.119. In the case of the group having received mGI-101 alone, a statistically significant reduction in tumor size was shown on day 28 after the start of administration of the test substance as compared with the vehicle control (p<0.05). In the case of the group having received Herceptin alone, a statistically significant reduction in tumor size was shown on day 14, day 18, and day 21 after the start of administration of the test substance as compared with the vehicle control (in the case of day 14 and day 18, p<0.05; in the case of day 21, p<0.01), but the tumor size tended to be increased rapidly on day 25 and day 28. In the case of the group having received a combination of mGI-101+Herceptin, a statistically significant reduction in tumor size was shown from day 14 to day 28 after the start of administration of the test substance as compared with the vehicle control, and a statistically significant reduction in tumor size was shown on day 28 after the start of administration of the test substance as compared with the group having received Herceptin alone (p<0.01). Individual tumor sizes for each test group are shown inFIG.121. According to the results of individual tumor sizes, the group having received the test substance exhibited a tumor growth inhibitory effect as compared with the vehicle control, and in particular, the group having received a combination of mGI-101+Herceptin exhibited an excellent tumor growth inhibitory effect as compared with the group having received mGI-101 alone. FIG.120illustrates a tumor growth inhibition rate when mGI-101 is administered alone or in combination with Herceptin in tumor mice xenotransplanted with BT-474 at the end of the test. The vehicle control exhibited a tumor growth inhibition rate of 30% or more in 3 mice, 50% or more in 3 mice, and 80% or more in no mouse. The group having received mGI-101 alone exhibited a tumor growth inhibition rate of 30% or more in 5 mice, 50% or more in 1 mouse, and 80% or more in no mouse. The group having received Herceptin alone exhibited a tumor growth inhibition rate of 30% or more in 3 mice, 50% or more in 2 mice, and 80% or more in 1 mouse. The group having received a combination of mGI-101+Herceptin exhibited a tumor growth inhibition rate of 30% or more in 5 mice, 50% or more in 4 mice, and 80% or more in no mouse. Experimental Example 36. Identification of Anticancer Effect by Administration of Combination of GI-101 and Pertuzumab This experiment was to evaluate the effect of killing cancer cells by treating HCT116 cells (human colon cancer cells) with the test substance GI-101 alone or in combination with Pertuzumab substance in an in vitro environment. HCT116 cells were purchased from the Korea cell line bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in McCoy's 5A medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in McCoy's 5A medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation of cancer cells or inhibition of the proliferation. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μL (1×104cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour. In order to identify the effect of killing cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by the test substance, natural killer cells (NK cells) were isolated from human peripheral blood mononuclear cells (PBMCs) using a CD56+CD16+NK cell isolation kit (Miltenyi Biotec) and used. In isolated NK cells, dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions. In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs. Pertuzumab was diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 16.9 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM by 50 μl per well of a 96-well microplate. The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent. FIG.122illustrates results obtained by measuring the degree of killing cancer cells after the HCT116 cells were treated with GI-101 at a concentration of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM and 100 nM, respectively. In the case of co-culturing only cancer cells and NK cells and in the case of treatment of Pertuzumab alone, the effect of killing cancer cells was not shown as in the case of culturing only cancer cells. In the case of treatment with GI-101 alone and with a combination of GI-101+Pertuzumab, an excellent effect of killing cancer cells was shown, and in the case of treatment with a combination of GI-101+Pertuzumab, the most excellent effect of killing cancer cells was shown. XIV. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and CDK4/6 Inhibitor Experimental Example 37. Identification of Anticancer Effect by Administration of Combination of GI-101 and Abemaciclib This experiment was to evaluate the tumor growth inhibitory effect after administration of mGI-101 as a test substance alone or in combination with Abemaciclib substance in a tumor model allotransplanted with 4T1 cells (mouse breast cancer cells) into BALB/c mice. 4T1 cells were purchased from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. In order to establish an allotransplanted tumor model, 1×105of 4T1 cells were subcutaneously injected into the back of BALB/c female mice (8-week-old). General symptoms were observed once a day during the engraftment and growth period after cell line transplantation. A certain period of time after cell inoculation of the tumor grafts of the mice, the tumor volume was measured for animals with no abnormalities in the health condition of the animals, and the mice were randomly selected and assigned, each group including 11 mice. As shown in Table 28, the test groups were configured and the test substances were administered. TABLE 28Route ofDosingDosageNumber ofExperimental groupadministrationcycleamountanimalsG1Vehiclei.p.QW—mg/kg11control (PBS)(once/week)G2mGI-101i.p.BIW (23mg/kg11times/week)G3Abemaciclibp.o.6 times/week50mg/kg11G4mGI-101 +i.p. + p.o.mGI-101:3 mg/kg + 5011AbemaciclibBIW (2mg/kgtimes/week),Abemaciclib: 6times/week The death of the mouse, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day during the test period, and recorded for each subject. The size of the Renca solid cancer was measured twice a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a vernier caliper, and the tumor volume (TV) and the tumor growth inhibition rate (TGI) were calculated by substituting them into the following equations. TV (mm3)=(W2×L)/2 [Equation 1] % TGI (Tumor Growth Inhibition)=(1−(Ti−T0)/(Vi−V0))×100 [Equation 2] The tumor volume before administration of each subject was set as the value measured at the time of grouping, and the anti-tumor efficacy was evaluated as compared with the vehicle control. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was made through two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant. The results of measuring the tumor size after administration of mGI-101 alone or in combination with Abemaciclib substance in mice transplanted with mouse-derived breast cancer cells are shown inFIG.123. In the case of the group having received a combination of mGI-101 (BIW)+Abemaciclib, the tumor growth inhibition tended to be highest. Individual tumor sizes for each test group are shown inFIG.125. According to the results of individual tumor sizes, the group having received a combination of mGI-101 (BIW)+Abemaciclib exhibited the greatest tumor growth inhibitory effect. FIG.124illustrates a tumor growth inhibition rate when mGI-101 and Abemaciclib are administered in combination in mice transplanted with 4T1. The vehicle control exhibited a tumor growth inhibition rate of 30% or more in 3 mice, and 50% or more and 80% or more in no mouse. The group having received mGI-101 (BIW) exhibited a tumor growth inhibition rate of 30% or more in 3 mice, 50% or more in 1 mouse, and 80% or more in no mouse. The group having received Abemaciclib exhibited a tumor growth inhibition rate of 30% or more in 4 mice, 50% or more in 1 mouse, and 80% or more in 1 mouse. The group having received a combination of mGI-101 (BIW)+Abemaciclib exhibited a tumor growth inhibition rate of 30% or more in 7 mice, 50% or more in 5 mice, and 80% or more in 1 mouse. Experimental Example 38. Identification of Anticancer Effect by Administration of Combination of GI-101 and Ribociclib This experiment was to evaluate the effect of killing cancer cells by treating MDA-MB-231 cells (human breast cancer cells) with the test substance GI-101 alone or in combination with Ribociclib substance in an in vitro environment. MDA-MB-231 cells were purchased from the Korea cell line bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation of cancer cells or inhibition of the proliferation. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μl (1×104cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour. Human peripheral blood mononuclear cells (PBMCs) were used in order to identify the effect of killing cancer cells by GI-101. The human PBMCs were purchased from Zen-Bio, and the PBMCs stored frozen were placed in a 37° C. water bath, and thawed as quickly as possible, and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×105cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions. In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs. Ribociclib test substance was diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 913 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration 100 nM by 50 μl per well of a 96-well microplate. The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent. FIG.126illustrates the effect of killing cancer cells in a condition of GI-101 at 100 nM. In the case of co-culturing only cancer cells and PBMCs, the effect of killing cancer cells was identified, and tended to be higher than that of treatment with GI-101 alone. In the case of treatment with Ribociclib alone and treatment with a combination of GI-101+Ribociclib, an excellent effect of killing cancer cells was shown as compared with the case of co-culturing only cancer cells and PBMCs, and in the case of treatment with a combination of GI-101+Ribociclib, the most excellent effect of killing cancer cells was shown. XV. Identification of Anticancer Effect According to Administration of Combination of Fusion Protein Dimer and STING Agonist Experimental Example 39. Identification of Anticancer Effect by Administration of Combination of mGI-101 and DMXAA This experiment was to evaluate the anticancer efficacy according to administration of mGI-101 as a test substance alone or in combination with DMXAA substance, a STING agonist, in a tumor model allotransplanted with MC38 cells (mouse colon cancer cells) into C57BL/6 mice. In order to establish an allotransplanted tumor model, 5×105of MC38 cells were subcutaneously injected into C57BL/6 mice. The tumor grafts of the mice were identified to be a size of 100-200 mm3about day 10 after cell inoculation, and the mice were assigned, each group including 5 mice. The test groups were configured as shown in Table 29, and the test substances were administered according to the schedule shown inFIG.127. TABLE 29ExperimentalRoute ofDosingDosageNumber ofgroupadministrationcycleamountanimalsG1VehiclemGI-101: BIW—mg/kg5control (PBS)G2mGI-101i.p.(2 times/week),6mg/kg5G3DMXAAI.T.DMXAA: Day450μg5G4mGI-101 +i.p. + I.T.10 and 136 mg/kg +5DMXAA450 μg The size of the MC38 solid cancer was measured three times a week during the observation period, and the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor were measured using a caliper, and the tumor volume (TV) was calculated by substituting them into the following equation, and once the tumor size was no less than 2 cm, the mice were sacrificed. TV (mm3)=(W2×L)/2 [Equation 1] The survival rate upon administration of mGI-101 alone or in combination with DMXAA substance against the MC38 tumor is shown inFIG.128. In the case of the vehicle control, all mice died before 40 days after tumor injection. In the case of the group having received mGI-101 alone, the survival rate on day 60 after tumor injection was 20%; and in the case of the group having received DMXAA, a STING agonist, alone, the survival rate on day 60 after tumor injection was 60%; and in the case of the group having received a combination of mGI-101+DMXAA, the survival rate on day 60 after tumor injection was 80%, indicating that the survival rate was higher than that of the other groups. Individual tumor sizes for each test group are shown inFIGS.129to133. The group having received a combination of mGI-101+DMXAA exhibited the greatest tumor growth inhibitory effect. | 214,415 |
11857602 | DETAILED DESCRIPTION Example implementations are now described with reference to the Figures. Reference numerals are used throughout the detailed description to refer to the various elements and structures. Although the following detailed description contains many specifics for the purposes of illustration, a person of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the disclosed inventive subject matter. Accordingly, the following implementations are set forth without any loss of generality to, and without imposing limitations upon, the claimed subject matter. The following abbreviations, which are used throughout this application, have the following specific meanings. DPEG®-A refers to MAL-DPEG®12-Tris(DPEG®24-acid)3(Quanta BioDesign #1145). Compound G or G refers to hGHR antagonist hGH-G120K having T142 and H151 changed to cysteine and having both added cysteines conjugated with DPEW®-A. Compound D or D refers to hGHR antagonist hGH-G120K having T142 changed to cysteine and having the added cysteine conjugated with a 40 kDa branched polyethylene glycol. Peg refers to SOMAVERT® (pegvisomant) and Dox refers to doxorubicin. The disclosed technology includes compositions and methods for treating cancer patients who are identified by expression of GHR, PRLR, selected ABC drug efflux pumps, selected EMT modulators, IGF-1, IGFBP3, SOCS-1, or CISH., wherein treatment comprises administering to the patient an effective dose of a chemotherapeutic drug combined with an effective dose of Compound G. GHR Expression in Cancer Cell Lines The effectiveness of an hGHR antagonist for cancer treatment, either by itself or in combination with a cancer drug, is related closely to the expression of the hGHR by a particular cancer. When the hGHR antagonist is also an antagonist of the PRLR, then the level of PRLR expression will also determine the susceptibility of a cancer to this treatment. It was previously observed that most of 60 cell lines from nine cancer types expressed high levels of either the hGHR, the PRLR, or both receptors [5]. Analysis of the levels of GHR mRNA expression from 37 cancer types (seeFIG.1), with between 4 and 131 cell lines included for each cancer, indicated that almost all of the cancer types had high hGHR expression for at least a subset of the individual cancers tested. The level of hGHR expression across multiple human patient datasets correlates with decreased patient survival for HER2 enriched breast cancer and triple-negative breast cancer (seeFIGS.2A-2C).FIGS.3A-3Dillustrate that breast cancer patients with increased levels of both GHR+PRLR expression or GH+GHR+PRLR expression have lower percent survivals than high levels of GH or GHR or PRLR expression alone. GHR Expression and the Expression of ABC Transporters and EMT Markers FIG.4Ashows a correlation across 40 different cancer cell types between GHR expression and the expression of ABC transporters across all patients in the TCGA database.FIG.4Bshows the same correlation between GHR expression and the expression of EMT mediators. Because upregulation of ABC transporters and EMT mediators lead to chemotherapy resistance, these Figures shows that chemotherapy resistance is associated with GHR expression. The effect of GHR antagonists on the expression of six ABC transporters in a melanoma cell line is shown inFIGS.5A(1)-5A(6). When used as a monotherapy, Compound G significantly reduces the expression of four of the six ABC transporters. When GH or doxorubicin is present, Compound G also reduces the expression of four ABC transporters. When both GH and doxorubicin are added, Compound G greatly reduces the expression of all six ABC transporters. This observation is due to the fact that the expression of ABC transporters is enhanced in presence of GH or doxorubicin or both GH and doxorubicin. The effect of hGHR antagonists on the expression of nine EMT markers in a melanoma cell line is shown inFIGS.5B(1)-5B(9). When used as a monotherapy, Compound G significantly reduces the expression of multiple EMT mediators. When GH or doxorubicin or both are present, Compound G also reduces the expression of multiple EMT mediators, presumably because the expression of EMT mediators is significantly enhanced in presence of GH or doxorubicin or both GH and doxorubicin. FIGS.6A-6Dshow the effect of the hGHR antagonists Compound G and SOMAVERT® (pegvisomant) on a basement membrane invasion assay using three pancreatic cancer cell lines. This assay quantifies the ability of cells to migrate through a membrane, a property of cells that have transitioned from epithelial cells to mesenchymal cells. For all three cell lines, the addition of GH increases cell migration. This effect of GUI is blocked by the addition of either SOMAVERT® (pegvisomant) or Compound G. The Effect of hGHR Antagonists on Drug Efflux The effect of hGHR antagonists on the drug efflux rate, using DiOC2 dye as a drug surrogate, from pancreatic cancer cells is shown inFIGS.7A-7C. With reference toFIG.7A, after 120 minutes, the efflux rate with GH addition is approximately four-fold greater than the efflux rate with no additives (PBS). In the presence of GH+Pegvisomant or GH+Compound G, the efflux rate is markedly suppressed compared with GH alone by a factor of approximately two.FIG.7B, illustrates the effect of hGHR antagonists on the percentage of drug retention from pancreatic cancer cells. In the PBS control, approximately 85% of the drug is retained after 120 minutes. The retention after 120 minutes decreases to ˜54% in the presence of GH but increases to ˜83% in the presence of GH plus Compound G or GH plus Pegvisomant. Finally,FIGS.7C(1)-7C(8) provides fluorescent images of a labeled drug after being loaded into pancreatic cancer cells and incubated for 0 minutes and 120 minutes in PBS (FIG.7C(1)-FIG.7C(2)), GH (FIG.7C(3)-FIG.7C(4)), GH+Pegvisomant (FIG.7C(5)-FIG.7C(6)), or GH+Compound G (FIG.7C(7)-FIG.7C(8)). InFIGS.7C(1)-7C(8), the fluorescence of the cells at 0 minutes is different for the four different conditions, so the drug remaining after 120 minutes (fluorescence) was compared with its own 0 min control. It is clear from the images that the cells treated with GH+Compound G retain the greatest amount of drug. FIG.8illustrates the viability of pancreatic cancer cells when incubated in the presence of anti-cancer drugs and either buffer (PBS), GH, GH+SOMAVERT® (pegvisomant), or GH+Compound G. The anti-cancer drugs are doxorubicin (doxo), erlotnib (erlo), or gemcitabine (gemc) and the control is DMSO, the vehicle for the anti-cancer drugs. In all cases, the addition of GH increases the cell viability. However, the addition of either SOMAVERT′ (pegvisomant) or Compound G to the GH reduces the viability, in all cases, to below that of PBS. The Effect of GHR Antagonists+Anti-Cancer Drugs on Pancreatic Tumor Xenografts Specific volumes of pancreatic tumor xenografts implanted in male nude mice after treatment with hGHR antagonists (10 mg/kg/day), gemcitabine (20 mg/kg/3-days) and combinations of the antagonists+gemcitabine are shown inFIG.9A. Treatments started at day-17. Both of the hGHR antagonists ((SOMAVERT® (pegvisomant) and Compound G)) significantly decrease the tumor volume relative to that of the PBS control by day-30 (13 days after start of treatment). However, for both of these antagonists, the tumor volume continues to increase. Gemcitabine alone shows a greater reduction of tumor volume compared to the hGHR antagonists, but the absolute tumor volume appears to resume trending upwards by day-43. The combinations of gemcitabine+SOMAVERT® (pegvisomant) and gemcitabine+Compound G show the greatest reduction of tumor volume. After day-43 (26 days of treatment) there is no indication that the tumor volume has begun to increase using the combination treatments. The combination of Compound G+gemcitabine gave the greatest tumor volume reduction. FIG.9Bshows the volume of pancreatic tumor xenografts implanted in male nude mice after treatment with 80 mg/kg/3-days gemcitabine and Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine. The plots for PBS, Compound G (10 mg/kg/day) alone, gemcitabine (20 mg/kg/3-days) alone, or Compound G (10 mg/kg/day)+gemcitabine (20 mg/kg/3-days), which are taken fromFIG.9A, are also included inFIG.9B. Gemcitabine by itself at 80 mg/kg/3-days does not decrease the tumor volume significantly beyond that obtained with 20 mg/kg/3-day dose of the same drug and, for the 80 mg/kg/3-day regimen, the tumor volume appears to have plateaued by day-43 (26 days of treatment). The 80 mg/kg/3-days Gemcitabine+mg/kg/day Compound G caused the maximum tumor volume reduction of all conditions, and the tumor volume appears to be still decreasing at the final day of the study (day-43). FIG.10shows the volume of pancreatic tumor xenografts implanted in female nude mice after treatment with either Compound-G (10 mg/kg/day) alone, or gemcitabine (20 or 80 mg/kg/3-days) alone, or Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine, or Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine. Treatment started at day-17. After days of treatment (day-42 of study), the tumor reduction due to 20 mg/kg/3-days gemcitabine alone or Compound G (10 mg/kg/day) alone are almost equivalent but only the gemcitabine treated mice show tumor volumes that are trending higher, indicating onset of chemoresistance. The combination of Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine reduces the tumor volume growth drastically and the tumor volume does not appear to trend upwards through the end of the study (day-44). Gemcitabine at 80 mg/kg/3-days alone shows the same tumor reduction at day-44 as Compound G (10 mg/kg/day)+20 mg/kg/3-days gemcitabine. Compound G (10 mg/kg/day)+80 mg/kg/3-days gemcitabine shows the greatest inhibition of tumor growth, which appears to be still decreasing after 44 days (end of study), at which point 3 of 8 animals in the group were tumor-free. Personalized Medicine/Precision Medicine Preliminary Diagnostic Test One implementation of the disclosed technology includes a preliminary molecular analysis of a tumor biopsy sample to determine if a patient is a suitable candidate for treatment with the disclosed combination therapy. This analysis involves analyzing expression levels of a predetermined set genes where specific changes in the expression levels of these genes correlates with the biological activities affected by the disclosed combination therapy. More specifically, identification of elevated levels of expression of selected genes is used to identify patients that are proper candidates for treatment with the GHR antagonist plus cancer therapeutic agent. Genes whose expression levels are key indicators of effective responsiveness to the disclosed GHR antagonist plus cancer therapy treatment include GHR, PRLR or both GHR and PRLR. Expression levels in a tumor biopsy are measured and quantified by performing a diagnostic test that measures levels of mRNA encoding these proteins that is expressed by the tumor cells. For example, the tumor biopsy sample could be processed to isolate mRNA which is then reverse transcribed into cDNA. The amount of cDNA derived from genes that encode these two receptors could then be measured using a variety of standard assays including qPCR analysis or gene chip analysis. Patients whose tumors express elevated levels of GHR, PRLR or both GHR and PRLR are potential candidates for receiving treatment with the disclosed combination therapy. Alternatively, the levels of these target proteins could be measured using techniques that directly measure the amount of these proteins present in the tumor. This approach includes the use of assays such as Western blots or ELISA assays. Additional genes whose expression levels are key indicators of effective response to the disclosed hGHR antagonist plus cancer therapeutic combination therapy include a key set of ATP-binding cassette (ABC) drug efflux pumps; ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1 and ABCG2. As with the target receptors described above, elevated levels of expression of at least some of these proteins identifies patients for which the disclosed combination hGHR antagonist plus cancer therapeutic agent would be effective. The levels of expression of these key genes are determined using the analytical techniques described above on samples derived from patient biopsies. In addition to the drug efflux pump proteins discussed herein, expression levels of a selected set of genes involved in promoting the adverse progression of cancer driven by the Epithelial to Mesenchymal Transition (EMT) can be measured. The set of key EMT modulators analyzed in a preliminary diagnostic analysis of a patient biopsy include CDH1, CDH2, SNAIL SNAI2, TGFB1, TGFB2, TGFB3, TGFBR2, TWIST1, TWIST2, VIM, ZEB1 and ZEB2. Elevated levels of expression of these genes further identify patients that are candidates for effective treatment with the disclosed combination GHR antagonist plus cancer therapeutic agent. The levels of expression of these genes would be determined by the analytical methods described above. In addition to the target receptors GHR and PRLR, Insulin Like Growth Factor 1 (IGF-1), Insulin Like Growth Factor Binding Protein 3 (IGFBP3), suppressor of cytokine signaling (SOCS) -1, -2, -3, and cytokine inducible SH2 containing protein (CISH) are important genes whose (RNA or protein) expression levels in the tumor biopsy (all the above) or serum (IGF1 and IGFBP3) can be used to identify patients who will respond effectively to treatment with the disclosed GHR antagonist. As with the target receptor proteins, the levels of these GH inducible downstream signaling factors are determined by gene expression analysis using mRNA gene expression techniques or, preferably, serum protein quantification techniques. IGF-1, IGFBP3, SOCS-1, -2, -3 and CISH are particularly useful for identifying patients that would be effectively treated by continuing administration of the GHR antagonist following completion of a combination therapy using GHR antagonist plus cancer chemotherapeutic agent. All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. Should one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. As previously stated and as used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. Unless context indicates otherwise, the recitations of numerical ranges by endpoints include all numbers subsumed within that range. Furthermore, references to “one implementation” are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, implementations “comprising” or “having” an element or a plurality of elements having a particular property may include additional elements whether or not they have that property. The terms “substantially” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, these terms can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%, and/or 0%. Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the disclosed subject matter, and are not referred to in connection with the interpretation of the description of the disclosed subject matter. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the disclosed subject matter. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description. There may be many alternate ways to implement the disclosed inventive subject matter. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the disclosed inventive subject matter. Generic principles defined herein may be applied to other implementations. Different numbers of a given module or unit may be employed, a different type or types of a given module or unit may be employed, a given module or unit may be added, or a given module or unit may be omitted. Regarding this disclosure, the term “a plurality of” refers to two or more than two. Unless otherwise clearly defined, orientation or positional relations indicated by terms such as “upper” and “lower” are based on the orientation or positional relations as shown in the figures, only for facilitating description of the present invention and simplifying the description, rather than indicating or implying that the referred devices or elements must be in a particular orientation or constructed or operated in the particular orientation, and therefore they should not be construed as limiting the present invention. The terms “connected”, “mounted”, “fixed”, etc. should be understood in a broad sense. For example, “connected” may be a fixed connection, a detachable connection, or an integral connection; a direct connection, or an indirect connection through an intermediate medium. For an ordinary skilled in the art, the specific meaning of the above terms in the present invention may be understood according to specific circumstances. It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the disclosed inventive subject matter. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. While the disclosed inventive subject matter has been illustrated by the description of example implementations, and while the example implementations have been described in certain detail, there is no intention to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the disclosed inventive subject matter in its broader aspects is not limited to any of the specific details, representative devices and methods, and/or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept. The following references form part of the specification of the present application and each reference is incorporated by reference herein, in its entirety, for all purposes.1. Pasut, G. and Veronese, M. (2012) State of the Art in Pegylation: The Great Versatility Achieved After Forty Years of Research.J. Controlled Release161, 461-472.2. Parveen, S. and Sahoo, S. K. Nanomedicine: Clinical Applications of Polyethylene Glycol Conjugated to Proteins and DrugsClin. Pharmacokinet.45, 965-988.3. Alconcel, S. N. S., Baas, A. S. and Maynard, H. D. (2011) FDA-Approved Poly(ethylene glycol)-Protein Conjugate Drugs.Polymer Chemistry2, 1442-1448.4. Kling, J. (2013) Pegylation of Biologics: A Multipurpose Solution.Bioprocess International11, 35-43.5. Perry, J. K., Wu, Z.-S., Mertani, H. C., Zhu, T., and Lobie, P. E. (2017) “Tumour-Derived Human Growth Hormone as a Therapeutic Target in Oncology”Trends in Endocrinology and Metabolism28: 587-596.6. Basu, R., Qian, Y., and Kopchick, J. J. (2018) “Lessons from growth hormone receptor gene-disrupted mice: are there benefits of endocrine defects?”European Journal of Endocrinology178: R155-R181.7. Coffin, V. (2017) “Prolactin Receptor Targeting in Breast and Prostate Cancers: New Insights into an Old Challenge”Pharmacology and Therapeutics179: 111-126.8. Sustarsic, E. G., Junnila, R. K., and Kopchick, J. J. (2013) “Human Metastatic Melanoma Cell Lines Express High Levels of Growth Hormone Receptor and Respond to GH Treatment”Biochem Biophys Res Commun.441: 144-150.9. Bukowski, K., Kciuk, M., and Kontek, R. (2020) “Mechanisms of Multidrug Resistance in Cancer Chemotherapy”Int. J. Mol. Sci.21, 323310. Basu, R., and Kopchick, J. J. (2019) “The Effects of Growth Hormone on Therapy Resistance in Cancer”Cancer Drug Resistance2: 827-846,11. Wu, A. M. L., Dalvi, P., Lu, X., Yang, M., Riddick, D. S., et al. (2013) “Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells”Molecular Pharmacology83:377-88.12. Neradugomma, N. K., Subramaniam, D., Tawfik, O. W., Coffin, V., Kumar, T. R., et al. (2014) “Prolactin signaling enhances colon cancer sternness by modulating Notch signaling in a Jak2-STAT3/ERK manner”Carcinogenesis35:795-80613. Zatelli, M. C., Minoia, M., Mole, D., Cason, V., Tagliati, F., Margutti, A., Bondanelli, M., Ambrosio, M. R., and Uberti, E.d (2009) “Growth Hormone Excess Promotes Breast Cancer Chemoresistance”Journal of Clinical Endocrinology and Metabolism94: 3931-3938.14. Minoia, M., Gentilin, E., Mole, D., Rossi, M., Filieri, C., Tagliati, F., Baroni, A., Ambrosio, M. R., and Uberti, E.d, Zatelli, M. C. “Growth Hormone Receptor Blockade Inhibits Growth Hormone-Induced Chemoresistance by Restoring Cytotoxic-Induced Apoptosis in Breast Cancer Cells Independently of Estrogen Receptor Expression”Journal of Clinical Endocrinology and Metabolism97: E907-E916. | 22,705 |
11857603 | EXAMPLES Materials and Methods Side chain protected PTH(1-34) (SEQ ID NO: 51) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers. Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers. PEG 2×20 kDa maleimide, Sunbright GL2-400MA was purchased from NOF Europe N. V., Grobbendonk, Belgium. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany. Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, KY, USA. Fmoc-Aib-OH was purchased from Iris Biotech GmbH, Marktredwitz, Germany. All other chemicals and reagents were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany, unless a different supplier is mentioned. Compound 11a (examples 11-15) was synthesized following the procedure described in patent WO29095479A2, example 1. Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of peptide resins. General procedure for the removal of ivDde protecting group from side chain protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The ivDde group was removed by incubating the resin with DMF/hydrazine hydrate 4/1 (v/v, 2.5 mL/g resin) for 8×15 min. For each step fresh DMF/hydrazine hydrate solution was used. Finally, the resin was washed with DMF (10×), DCM (10×) and dried in vacuo. General procedure for the removal of Fmoc protecting group from protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The Fmoc group was removed by incubating the resin with DMF/piperidine/DBU 96/2/2 (v/v/v, 2.5 mL/g resin) for 3×10 min. For each step fresh DMF/piperidine/DBU hsolution was used. Finally, the resin was washed with DMF (10×), DCM (10×) and dried in vacuo. RP-HPLC purification: For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used, equipped with the following columns: Waters XBridge™ BEH300 Prep C18 5 m, 150×10 mm, flow rate 6 mL/min, or Waters XBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 mL/min. Linear gradients of solvent system A (water containing 0.1% TFA v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v) were used. HPLC fractions containing product were pooled and lyophilized if not stated otherwise. Flash Chromatography: Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane and ethyl acetate as eluents. Products were detected at 254 nm. Ion Exchange Chromatography: Ion exchange chromatography (IEX) was performed using an Amersham Bioscience AEKTAbasic system equipped with a MacroCap SP cation exchanger column (Amersham Bioscience/GE Healthcare). 17 mM acetic acid pH 4.5 (solvent A) and 17 mM acetic acid, 1 M NaCl, pH 4.5 (solvent B) were used as mobile phases. Size Exclusion Chromatography: Size exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with HiPrep 26/10 desalting columns (Amersham Bioscience/GE Healthcare). 0.1% (v/v) acetic acid was used as mobile phase. Analytical Methods Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH300 C18column (2.1×50 mm, 1.7 m particle size, flow: 0.25 mL/min; solvent A: water containing 0.04% TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v)) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific or coupled to a Waters Micromass ZQ. Quantification of Plasma Total PTH(1-34) Concentrations: Plasma total PTH(1-34) concentrations were determined by quantification of a signature peptide close to the N-terminus (sequence: IQLMHNLGK) and a C-terminal signature peptide (sequence: LQDVHNF) after plasma protein precipitation, followed by sequential digestion with Endoproteinase Lys-C (origin: Lysobacter enzymogenes) and Endoproteinase Glu-C (origin:Staphylococcus aureusV8) of the supernatant. Subsequently, analysis by reversed phase liquid chromatography and detection by mass spectrometry (RP-HPLC-MS) was performed. Calibration standards of PTH(1-34) conjugate in blank heparin or EDTA plasma were prepared in concentration ranges from 1 to 1000 ng/mL PTH(1-34) eq (dilution with rat plasma) and 1 to 1000 ng/mL PTH(1-34) eq (dilution with monkey plasma). These solutions were used for the generation of a calibration curve. For quality control, three samples independent from the calibration standard solutions were prepared accordingly. Concentrations at the lower end (3-5 fold concentration of the respective LLOQ), the middle range (0.05-0.1 fold concentration of the respective ULOQ) and the upper end (0.5-0.8 fold concentration of the respective ULOQ). Sample preparation volumes can be altered depending on the targeted signal response after sample preparation. Processing procedure of the protein precipitation is described here for the analysis of plasma samples originated in rat species. Protein precipitation was carried out first by addition of 100 μL of internal standard solution (625 ng/mL of deuterated conjugate) and then by addition of 400 μL of acetonitrile to 50 μL of the plasma sample. 2 times 150 μL of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 50° C.). 50 μL of reconstitution solvent (50 mM Tris 0.5 mM CaCl2buffer, adjusted to pH 8.0) were used to dissolve the residue. Proteolytic digestion was performed as follows: 20 μg of Lys-C (order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 μL of 10 mM acetic acid. 3 μL of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37° C. Afterwards 10 μg of Glu-C (order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μL water, and 1.5 μL of the Glu-C solution added to each cavity and incubation continued for 1.5 hours at 37° C. After incubation samples were acidified with 2 μL water/formic acid 4:6 (v/v) and 10 μL were injected into the UPLC-MS system. Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (1.7 μm particle size; column dimensions 50×2.1 mm). Water (UPLC grade) containing 0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade) with 0.1% formic acid as mobile phase B. Mass analysis was performed on an AB Sciex 6500+QTrap in multiple reaction monitoring (MRM) mode, monitoring the transition m/z 352.0 to 462.1 (signature peptide IQLMHNLGK), 355.4 to 467.1 (signature peptide IQLMHNLGK, internal standard), 436.9 to 631.4 (signature peptide LQDVHNF), and 441.9 to 631.4 (signature peptide LQDVHNF, internal standard). Quantification of Plasma PEG Concentrations: Plasma total PEG concentrations were determined by quantification of the polymeric part of PTH(1-34) conjugates after plasma protein precipitation and enzymatic digestion of the supernatant. Analysis by size exclusion chromatography and detection by mass spectrometry (SEC-MS) followed. Calibration standards of PTH(1-34) conjugate in blank heparinized monkey plasma were prepared in concentration ranges from 50 to 4500 ng/mL PEG equivalents. These solutions were used for the generation of a quadratic calibration curve. Calibration curves were weighted 1/x. For quality control, three samples independent from the calibration standard solutions were prepared accordingly. Concentrations at the lower end (2-4 fold concentration of the LLOQ), the middle range (0.1-0.2 fold concentration of the ULOQ) and the upper end (0.8 fold concentration of the ULOQ). Protein precipitation was carried out by addition of 200 μL of precooled (5-10° C.) methanol to 100 μL of the plasma sample. 180 μL of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 45° C.). 50 μL of reconstitution solvent (50 mM Tris 0.5 mM CaCl2buffer, adjusted to pH 8.0) were used to dissolve the residue. Proteolytic digestion was performed as follows: 20 μg of Lys-C (order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 μL of 10 mM acetic acid. 3 μL of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37° C. Afterwards 10 μg of Glu-C (order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μL water, and 1.5 μL of the Glu-C solution added to each cavity and incubation continued for 1.5 hours at 37° C. After incubation samples were acidified with 2 μL water/formic acid 4:6 (v/v) and 5 μL were injected into the SEC-MS system. SEC-MS analysis was carried out by using an Agilent 1290 UPLC coupled to an Agilent 6460 TripleQuad mass spectrometer via an ESI probe. Acquisition of a distinct precursor ion of the polymer was achieved by applying high voltage in-source fragmentation (200-300V) at the MS interface. Chromatography was performed on a TOSOH TSK Gel SuperAW3000 analytical column (4.0 μm particle size; column dimensions 150×6.0 mm) at a flow rate of 0.50 mL/min (T=65° C.). Water (UPLC grade) containing 0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade) with 0.1% formic acid as mobile phase B. The chromatographic setup for sample analysis comprises an isocratic elution of 50% B over 8 minutes. Mass analysis was performed in single reaction monitoring (SRM) mode, monitoring the transition m/z 133.1 to 45.1. Quantification of Plasma Free PTH Concentrations: Free PTH concentrations in acidified plasma were determined as the sum of peptide PTH(1-34) and peptide PTH(1-33) after plasma protein precipitation, followed by solid phase extraction. Subsequently, analysis using liquid chromatography separation and detection by mass spectrometry (LC-MS) was performed. Calibration standards of PTH(1-34) and PTH(1-33) in blank acidified EDTA rat plasma were prepared in concentrations ranging from 5.00 to 500 pg/mL in acidified plasma for each analyte. The corresponding concentration range is 7.00 to 700 pg/mL for both analytes in neat plasma, as plasma is acidified as volume ratio plasma: 0.5 M citrate buffer pH 4=1: 0.4 v/v. Standard solutions were used for the generation of a calibration curve. For quality control, three samples were prepared independent of the calibration standard solutions at 15.0, 150 and 400 pg/mL in acidified plasma. Protein precipitation was carried out by addition of 150 μL of cold acetonitrile to 150 μL of the plasma sample after addition of 50.0 μL of cold internal standard solution, followed by centrifugation. The supernatant was decanted into a new ploypropylene tube and 900 μL of cold water was added. After another centrifugation step, the tubes were kept in ice-water until loading on the SPE column. Solid phase extraction: the HLB μelution columns were conditioned with 200 μL methanol followed by 200 μL water. The columns were loaded 3 times with 420 μL of the diluted samples by applying positive pressure. The SPE-columns were washed with 200 μL of methanol:water 5:95 v/v. The samples were eluted with 40.0 μL SPE elution solvent (Aceonitrile:water:Trifluoroactic acid 60:40:1 v/v/v), followed by 40.0 μL of water. The elution solvent was left standing on the columns for 2 minutes, after which very gentle pressure was applied for elution. Separation between metabolites and interfering endogenous compounds was achieved by LC-MS using an Xselect CSH C18 column (2.1×100 mm, 2.5 μm) at 50° C. and using 0.2% Formic acid and 0.5% dimethyl sulfoxide in water as mobile phase A, 0.5% dimethyl sulfoxide in acetonitrile:methanol (75:25, v/v) as mobile phase B, and operating at a gradient with a flow rate of 0.500 mL/min. A triple 6500 quadrupole mass spectrometer equipped with a turbo ion spray source was used for detection in positive ion mode. For PTH(1-34), PTH(1-33) and PTH(1-33) (Leu-d10)3, quantification was done by counting 5 times the same SRM transition. Quantification is based on multiple reaction monitoring (MRM) of the transitions of m/z:687.3-787.3 for PTH(1-34)662.8-757.9 for PTH(1-33)692.3-793.3 for PTH(1-34) (Leu-d10)3667.8-763.9 for PTH(1-33) (Leu-d10)3 A linear calibration curve with a 1/x2weighing factor was used for both analytes. Concentrations of free PTH were determined in acidified plasma, as a means to stabilize the analytes. Acidified plasma was prepared by diluting rat EDTA plasma 1.4 times (in case of blank, zero, calibration and QC samples) or rat whole blood (in case of study samples) 1.2 times. Assuming approximately 50% (v/v) of whole blood being plasma, the neat plasma concentrations are approximately 1.4 times higher than the reported concentrations in acidified plasma. Free PTH concentrations are calculated as sum of Free PTH (1-34) and Free PTH (1-33) in PTH(1-34) equivalents). Due to the reversible nature of the attachment of -L1- to -D, measurements for PTH receptor activity were made using stable analogs of the PTH prodrugs of the present invention, i.e. they were made using similar structures to those of the PTH prodrugs of the present invention which instead of a reversible attachment of —Z to -D have a stable attachment. This was necessary, because the PTH prodrugs of the present invention would release PTH in the course of the experiment and said released PTH would have influenced the result. Example 1 Synthesis of Linker Reagent 1f Linker reagent if was synthesized according to the following scheme: To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH3(819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. The mixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) and stirred further 15 min. The reaction mixture was added to saturated NaHCO3solution (200 mL) and extracted 5× with DCM. The combined organic phases were dried over Na2SO4and the solvents were evaporated in vacuo. The resulting N-methyl-N-Boc-N′-Tmob-ethylenediamine 1a was dried in high vacuum and used in the next reaction step without further purification. Yield:3.76g(11.48 mmol,89% purity,1a:double Tmob protected product=8:1) MS: m/z355.22=[M+H]+, (calculated monoisotopic mass=354.21). To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at rt, diluted with DCM (250 mL) and washed 3× with 0.1 M H2SO4(100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4, filtrated and the residue concentrated to a volume of 24 mL.1bwas purified using flash chromatography. Yield: 5.31g(148%, 6.66 mmol) MS: m/z796.38=[M+H]+, (calculated monoisotopic mass=795.37). To a solution of 1b (5.31 g, max. 4.52 mmol ref. to N-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3% v/v) was added. The solution was stirred for 12 min at rt, diluted with DCM (400 mL) and washed 3× with 0.1 M H2SO4(150 mL) and 3× with brine (150 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4and filtrated. 1c was isolated upon evaporation of the solvent and used in the next reaction without further purification. MS: m/z574.31=[M+H]+, (calculated monoisotopic mass=573.30). 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred for 4 h at rt, diluted with DCM (400 mL) and washed 3× with 0.1 M H2SO4(100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4, filtered and 1d was isolated upon evaporation of the solvent. Product 1d was purified using flash chromatography. Yield: 2.63g(62%, 94% purity) MS: m/z856.41=[M+H]+, (calculated monoisotopic mass=855.41). To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at rt. The mixture was diluted with DCM (200 mL) and washed 3× with 0.1 M H2SO4(50 mL) and 3× with brine (50 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4, filtered and 1e was isolated upon evaporation of the solvent. 1e was purified using flash chromatography. Yield: 2.1g(88%) MS: m/z878.4=[M+Na]+, (calculated monoisotopic mass=837.40). To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) were added DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5 min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reaction mixture was stirred at rt for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo and the residue was taken up in 90% acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture was purified by RP-HPLC. Product fractions were neutralized with 0.5 M pH 7.4 phosphate buffer and concentrated. The remaining aqueous phase was extracted with DCM and if was isolated upon evaporation of the solvent. Yield: 154mg(81%) MS: m/z953.4=[M+H]+, (calculated monoisotopic mass=952.43). Example 2 Synthesis of Linker Reagent 2g 4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise under stirring to a solution of ethylenediamine 2a (6.5 mL, 97.3 mmol) in DCM (20 mL). The reaction mixture was stirred for 2 h at rt after which it was diluted with diethyl ether (300 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na2SO4and 2b was isolated upon evaporation of the solvent. Yield: 3.18g(98%) Mmt protected intermediate 2b (3.18 g, 9.56 mmol) was dissolved in DCM (30 mL). 6-(Tritylthio)-hexanoic acid (4.48 g, 11.5 mmol), PyBOP (5.67 g, 10.9 mmol) and DIPEA (5.0 mL, 28.6 mmol) were added and the mixture was stirred for 30 min at rt. The solution was diluted with diethyl ether (250 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na2SO4and the solvent was removed in vacuo. 2c was purified using flash chromatography. Yield: 5.69g(85%) MS: m/z705.4=[M+H]+, (calculated monoisotopic mass=704.34). Compound 2c (3.19 g, 4.53 mmol) was dissolved in abhydrous THF (50 mL), 1 M BH3·THF solution in THF (8.5 mL, 8.5 mmol) was added and the mixture was stirred for 16 h at rt. More 1 M BH3·THF solution in THF (14 mL, 14.0 mmol) was added and the mixture was stirred for further 16 h at rt. Methanol (8.5 mL) and N,N′-dimethyl-ethylendiamine (3.00 mL, 27.9 mmol) were added and the mixture was heated under reflux for 3 h. The mixture was allowed to cool down and ethyl acetate (300 mL) was added. The solution was washed 2× with aqueous Na2CO3and 2× with aqueous NaHCO3. The organic phase was dried over Na2SO4and the solvent was removed in vacuo to obtain 2d. Yield: 3.22g(103%) MS: m/z691.4=[M+H]+, (calculated monoisotopic mass=690.36). Di-tert-butyl dicarbonate (2.32 g, 10.6 mmol) and DIPEA (3.09 mL, 17.7 mmol) were dissolved in DCM (5 mL) and added to a solution of 2d (2.45 g, 3.55 mmol) in DCM (5 mL). The mixture was stirred for 30 min at rt. The solution was concentrated in vacuo and purified by flash chromatography to obtain product 2e. Yield: 2.09g(74%) MS: m/z791.4=[M+H]+, (calculated monoisotopic mass=790.42). Compound 2e (5.01 g, 6.34 mmol) was dissolved in acetonitrile (80 mL). 0.4 M aqueous HCl (80 mL) followed by acetonitrile (20 mL) was added and the mixture was stirred for 1 h at rt. The pH was adjusted to pH 5.5 by addition of aqueous 5 M NaOH. The organic solvent was removed in vacuo and the remaining aqueous solution was extracted 4× with DCM. The combined organic phases were dried over Na2SO4and the solvent was removed in vacuo to obtain product 2f. Yield: 4.77g(95%) MS: m/z519.3=[M+H]+, (calculated monoisotopic mass=518.30). Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL) and added to a solution of p-nitrophenyl chloroformate (2.01 g, 9.98 mmol) in DCM (25 mL). 2,4,6-trimethylpyridine (4.38 mL, 33.3 mmol) was added and the solution was stirred for 45 min at rt. The solution was concentrated in vacu and purified by flash chromatography to obtain product 2g. Yield: 4.04g(89%) MS: m/z706.32=[M+Na]+, (calculated monoisotopic mass=683.30). Example 3 Synthesis of Permanent S1 PTH(1-34) Conjugate 3 Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (62.5 mg, 160 μmol), PyBOP (80.1 mg, 154 μmol) and DIPEA (53 μL, 306 μmol) in DMF (2 mL) was added to 0.21 g (51 μmol) of the resin. The suspension was agitated for 80 min at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 10 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 3 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 36mg(14%), 3*8TFA MS: m/z1062.31=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+=1062.30). Example 4 Synthesis of Permanent K26 PTH(1-34) Conjugate 4 Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (107 mg, 273 μmol), PyBOP (141 mg, 273 μmol) and DIPEA (95 μL, 545 μmol) in DMF (3 mL) was added to 0.80 g (90.9 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 4 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 40mg(8%), 4*8TFA MS: m/z1062.30=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1062.30). Example 5 Synthesis of Transient S1 PTH(1-34) Conjugate Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Aib-OH (79 mg, 244 μmol), PyBOP (127 mg, 244 μmol) and DIPEA (64 μL, 365 μmol) in DMF (1.5 mL) was added to 0.60 g (61 μmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10× with DMF and Fmoc-deprotected as described above. A solution of 2g (167 mg, 244 μmol) and DIPEA (64 μL, 365 μmol) in DMF (1.5 mL) was added to the resin. The suspension was agitated for 24 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 7 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 5 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 78mg(24%), 5*9TFA MS: m/z1101.59=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1101.57). Example 6 Synthesis of Transient S1 PTH(1-34) Conjugate 6 Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ala-OH (32 mg, 102 μmol), PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to 0.25 g (25 μmol) of the resin. The suspension was shaken for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 6 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 25mg(18%), 6*9TFA MS: m/z1098.75=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1098.07). Example 7 Synthesis of Transient S1 PTH(1-34) Conjugate 7 Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ser(Trt)-OH (117 mg, 205 μmol), PyBOP (108 mg, 207 μmol) and DIPEA (53 μL, 305 μmol) in DMF (2 mL) was added to 0.50 g (51 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (144 mg, 211 μmol) and DIPEA (53 μL, 305 μmol) in DMF (1.8 mL) was added to the resin. The suspension was shaken for 7 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 7 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 54mg(20%), 7*9TFA MS: m/z1102.08=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1102.07). Example 8 Synthesis of Transient S1 PTH(1-34) Conjugate 8 Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Leu-OH (36 mg, 102 μmol), PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to 0.25 g (25 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 8 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 31mg(22%), 8*9TFA MS: m/z1109.32=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1108.58). Example 9 Synthesis of Transient S1 PTH(1-34) Conjugate 9 Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 1e (182 mg, 213 μmol), PyBOP (111 mg, 213 μmol) and DIPEA (93 μL, 532 μmol) in DMF (5 mL) was added to 2.00 g (107 μmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 9 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 47mg(8%), 9*9TFA MS: m/z1108.58=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1108.57). Example 10 Synthesis of Transient K26 PTH(1-34) Conjugate 10 Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of if (867 mg, 910 μmol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and shaking the suspension for 1 h at rt. Crude 10 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried. Yield: 92mg(7%), 10*9TFA MS: m/z1108.58=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+=1108.57). Example 11 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 11b 0.15 mL of a 0.5 M NaH2PO4buffer (pH 7.4) was added to 0.5 mL of a 20 mg/mL solution of thiol 5 (10 mg, 1.84 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v). The solution was incubated at rt for 10 min after which 238 μL of a 10 mg/mL solution of maleimide 11a (2.4 mg, 2.21 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added. The solution was incubated for 20 min at rt. 10 μL TFA was added and the mixture was purified by RP-HPLC. The product fractions were freeze-dried to obtain 11b. Yield: 3.1mg(26%), 11b*9TFA MS: m/z1097.00=[M+4H]4+, (calculated monoisotopic mass for [M+5H]+1096.99). Example 12 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 12 Conjugate 12 was synthesized as described for 11b by using thiol 6 (10 mg, 1.85 μmol) and maleimide 11a (2.4 mg, 2.21 μmol). Yield: 10mg(83%), 12*9TFA MS: m/z1094.20=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+-1094.19). Example 13 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 13 Conjugate 13 was synthesized as described for 11 b by using thiol 7 (10 mg, 1.84 μmol) and maleimide 11a (2.4 mg, 2.21 μmol). Yield: 8mg(67%), 13*9TFA MS: m/z1097.40=[M+5H]5+, (calculated monoisotopic mass for [M+5H]5+=1097.39). Example 14 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 14 Conjugate 14 was synthesized as described for 11b by using thiol 8 (10 mg, 1.83 μmol) and maleimide 11a (2.4 mg, 2.21 μmol). Yield: 4mg(33%), 14*9TFA MS: m/z1378.01=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+=1378.00). Example 15 Synthesis of Low Molecular Weight Transient K26 PEG Conjugate 15 Conjugate 15 was synthesized as described for 11b by using thiol 10 (5.2 mg, 0.95 μmol) and maleimide 11a (1.23 mg, 1.14 μmol). Yield: 2.1mg(33%), 15*9TFA MS: m/z1102.60=[M+5H]5+, (calculated monoisotopic mass for [M+5H]5+=1102.59). Example 16 Synthesis of Permanent 2×20 kDa S1 PEG Conjugate 16 772 μL of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 μmol) and 2.5 mg/mL Boc-L1-Met in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added to 1.87 mL of a solution containing PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol) and 2.5 mg/mL Boc-L1-Met in water containing 0.1% TFA (v/v). 0.5 M NaH2PO4buffer (0.66 mL, pH 7.0) was added and the mixture was stirred for 30 min at rt. 10 μL of a 270 mg/mL solution of 2-mercaptoethanol in water was added. The mixture was stirred for 5 min at rt and 0.33 mL 1 M HCl were added. Conjugate 16 was purified by IEX followed by RP-HPLC using a linear gradient of solvent system A (water containing 0.1% AcOH v/v) and solvent system B (acetonitrile containing 0.1% AcOH v/v). The product containing fractions were freeze-dried. Yield: 97mg(2.01 μmol,57%) conjugate 16*8 AcOH Example 17 Synthesis of Permanent 2×20 kDa K26 PEG Conjugate 17 Conjugate 17 was prepared as described for 16 by reaction of thiol 4 (15 mg, 3.53 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol). Yield: 80mg(1.79 μmol,51%) conjugate 17*8 AcOH Example 18 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 18 Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37 mg, 8.40 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 445 mg, 9.24 μmol). The reaction was quenched by addition of 50 μL TFA without prior addition of 2-mercaptoethanol. Conjugate 18 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried. Yield: 161mg(3.33 μmol,40%) conjugate 18*9 AcOH Example 19 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 19 Conjugate 19 was prepared as described for 16 by reaction of thiol 7 (27 mg, 6.14 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 325 mg, 7.50 μmol). Yield: 249mg(5.16 μmol,84%) conjugate 19*9 AcOH Example 20 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 20 Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38 mg, 8.59 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 455 mg, 9.45 μmol). The reaction was quenched by addition of 50 μL TFA without prior addition of 2-mercaptoethanol. Conjugate 20 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried. Yield: 194mg(4.01 μmol,47%) conjugate 20*9 AcOH Example 21 Synthesis of Transient 2×20 kDa K26 PEG Conjugate 21 Conjugate 21 was prepared as described for 16 by reaction of thiol 10 (34 mg, 7.58 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 401 mg, 9.26 μmol). Yield: 256mg(5.30 μmol,70%) conjugate 21*9 AcOH Example 22 In vitro release kinetics of transient low molecular weight PEG conjugates Conjugates 11b, 12, 13, 14, and 15 were dissolved in pH 7.4 phosphate buffer (60 mM NaH2PO4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 by NaOH) containing 0.05 mg/mL pentafluorophenol as internal standard at a concentration of approximately 1 mg conjugate/mL. The solutions were filtered sterile and incubated at 37° C. At time points, aliquots were withdrawn and analysed by RP-HPLC and ESI-MS. The fraction of released PTH at a particular time point was calculated from the ratio of UV peak areas of liberated PTH and PEG conjugate. The % released PTH was plotted against incubation time. Curve-fitting software was applied to calculate the corresponding half times of release. Results:For conjugate 11b a release half life time of 3.2 d was obtained.For conjugate 12 a release half life time of 8.7 d was obtained.For conjugate 13 a release half life time of 10.8 d was obtained.For conjugate 14 a release half life time of 25.3 d was obtained.For conjugate 15 a release half life time of 6.9 d was obtained. Example 23 In Vitro Release Kinetics of Transient 2×20 kDa PEG Conjugates Conjugates 18, 19, 20, and 21 were dissolved in pH 7.4 phosphate buffer (60 mM NaH2PO4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 by NaOH) containing 0.08 mg/mL pentafluorophenol as internal standard at a concentration of approximately 5 mg conjugate/mL. The solutions were filtered sterile and incubated at 37° C. At time points, aliquots were withdrawn and analysed by RP-HPLC. The fraction of released PTH at a particular time point was calculated from the ratio of UV peak areas of liberated PTH and PEG conjugate. The % released PTH was plotted against incubation time. Curve-fitting software was applied to calculate the corresponding half times of release. Results:For conjugate 18 a release half life time of 2.8 d was obtained.For conjugate 19 a release half life time of 13.4 d was obtained.For conjugate 20 a release half life time of 1.3 d was obtainedFor conjugate 21 a release half life time of 7.1 d was obtained Example 24 PTH Receptor Activity of Permanent 2×20 kDa PEG Conjugates 16 and 17 in Cell Based Assay The residual PTH activity of permanently PEGylated conjugates 16 and 17 was quantified by measuring cAMP production from HEK293 cells over-expressing the PTH/PTHrP1 receptor (Hohenstein A, Hebell M, Zikry H, El Ghazaly M, Mueller F, Rohde, J. Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH),Journal of Pharmaceutical and Biomedical AnalysisSeptember 2014, 98: 345-350). PTH(1-34) from NIBSC (National Institute for Biological Standards and Control, UK) was used as reference standard. Results:For conjugate 16 a receptor activity of 0.12% was found relative to PTH(1-34) referenceFor conjugate 17 a receptor activity of 0.11% was found relative to PTH(1-34) reference The results indicate an effective lowering of receptor activity in the permanent 2×20 kDa PEG conjugates 16 and 17. It can be concluded that similar conjugates with transiently Ser1 or Lys26 linked PTH (like e.g. 18 and 21) are suitable PTH prodrugs providing low residual receptor activity. Direct analysis of transient conjugates in the cell assay is not possible due to linker cleavage under the assay conditions. The released PTH would influence the assay result. Example 25 Pharmacokinetic study of permanent 2×20 kDa PEG conjugates 16 and 17 in rats Male Wistar rats (6 weeks, 230-260 g) received either a single intravenous (2 groups, n=3 animals each) or a single subcutaneous (2 groups, n=3 animals each) administration of 16 or 17 at doses of 29 μg/rat PTHeqand 31 μg/rat PTHeqrespectively. Blood samples were collected up to 168 h post dose, and plasma was generated. Plasma PTH(I-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods. Results: Dose administrations were well tolerated with no visible signs of discomfort during administration and following administration. No dose site reactions were observed any time throughout the study. After intravenous injection of 16 and 17 the total PTH(1-34) tmaxwas observed at 15 min (earliest time point analyzed), followed by a slow decay in total PTH(1-34) content with a half life time of approx. 13 h and 11 h respectively. After subcutaneous injection the total PTH(1-34) concentration peaked at a tmaxof 24 h for both 16 and 17, followed by a slow decay in total PTH(1-34) content with half life times of approx. 1.5 days for both conjugates. The bioavailability was approx. 40% and 60% respectively. Similar PK curves were obtained for the N- and the C-terminal signature peptide up to 168 h post dose, indicating the presence of intact PTH(1-34) in the conjugate. The favourable long lasting PK and the stability of PTH in the conjugates indicate the suitability of the permanent 2×20 kDa PEG model compounds as slow releasing PTH prodrugs after subcutaneous injection. It can be concluded that similar conjugates with transiently Ser (like e.g. 18) or Lys26 linked PTH are suitable PTH prodrugs providing long lasting levels of released bioactive PTH. Example 26 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 19 in Cynomolgus Monkeys Male non naïve cynomolgus monkeys (2-4 years, 3.7-5.4 kg) received a single subcutaneous (n=3 animals) administration of 19 at a dose of 70 μg/kg PTHeq. Blood samples were collected up to 504 h post dose, and plasma was generated. Total plasma PTH(1-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods. The PEG concentrations were determined using the method described in Materials and Methods. Results: Dose administrations were well tolerated with no visible signs of discomfort during administration. One animal showed visible signs of discomfort 72 h post dose, but recovered the days after. No dose site reactions were observed any time throughout the study. The total PTH(1-34) concentration peaked at a tmaxof 24 h, followed by a slow decay in total PTH(1-34) content with a half life time of approx. 2.5 d for the N-terminal signature peptide and 0.9 d for the C-terminal signature peptide. The PEG concentration peaked at tmaxof 24 h, followed by a slow decay in PEG concentration with a half life time of 3.5 d. It can be concluded that conjugate 19 is a suitable prodrug for sustained delivery of PTH. Example 27 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys Non naïve cynomolgus monkeys (2-3 years, 2.5-4 kg) received daily subcutaneous (n=2 animals—1 male/1 female) administration of 18 at dose levels of 0.2, 0.5, and 1 μg/kg PTHeqfor 28 days. Blood samples were collected up to 28 days (at days 1, 13, and, 27 samples were collected at pre-dose, 2 h, 4 h, 8 h, 12 h, and 24 h post-dose) and plasma was generated. Plasma PTH(1-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods. Results: All dose administrations were performed without incident. No dose site reactions were observed any time throughout the study. Dose linearity was observed in the three groups. Dose stacking was observed from day 1 compared with day 13 and day 27. Total PTH(1-34) concentrations were quantified via the N-terminal signature peptide (sequence: IQLMHNLGK) at steady state (during day 27). A low peak-to-trough ratio of total PTH(1-34) for all dose groups of below 3 was observed after daily subcutaneous application at steady state in cynomolgus monkeys. As free peptide concentrations at steady state are correlated to total PTH(1-34) concentration, the peak-to-trough ratio for the free peptide is below 4 in cynomolgus monkeys. Example 28 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys Naïve cynomolgus monkeys (2-3.5 years, 2-5 kg) (3-5 males/3-5 females) received daily subcutaneous administrations of 18 at dose levels of 0.2, 0.5 and 1.5 μg PTH/kg. Blood samples were collected at; Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose, at Day 8: pre-dose, at Day 14: predose, 8 h, and 12 h and at Day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, 72 h, 168 h, and 336 h) and plasma was generated. Total PTH plasma concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC digestion as presented earlier in Materials and Methods. Results: Systemic exposure expressed as Cmaxand AUC increased in an approximately dose proportional manner. Systemic exposure of Total PTH expressed as AUC accumulated approximately 3-fold from Day 1 to Day 28. A low mean peak-to-trough ratio of Total PTH for all dose groups of 1.5 was observed after daily subcutaneous administration in cynomolgus monkeys at Day 28 (steady state observed from Day 8). This low mean peak-to-trough ratio of Total PTH can also be translated to Free PTH. Example 29 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Sprague-Dawley Rats Sprague-Dawley Crl:CD(SD) rats (initiation of dosing at 8 weeks of age) received daily subcutaneous administrations of 18 at dose levels of 10, 30 and 60 μg PTH/kg for 28 days. A TK group containing of 9 males and 9 females per dose group was divided into 3 subgroup with 3 rats per subgroup. Blood samples were collected up to 28 days with 3 rats per sex, per sampling time point. Samples were collected at Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose, and at Day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, and 336 h and plasma was generated. The total PTH plasma concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC digestion as presented earlier in Materials and Methods. The free PTH plasma concentrations were determined by quantification as the sum of PTH(1-34) and PTH(1-33) by LC-MS/MS as presented earlier in Materials and Methods. Results: Systemic exposure of total PTH and free PTH expressed as mean Cmaxand AUC increased in an approximately dose proportional manner. Systemic exposure of total PTH expressed as mean AUC accumulated 3-6 fold from day 1 to day 28 and free PTH expressed as mean AUC accumulated 2-3 fold from day 1 to day 28. Systemic exposure of total PTH in the female rat was approximately 2-fold higher than in males. Systemic exposure of free PTH was slightly higher in the female rat than in males. A low mean peak-to-trough ratio of total PTH for all dose groups of 1.2 was observed after daily subcutaneous administration in Sprague-Dawley rats at day 28 (steady state observed from Day 8). A low mean peak-to-trough ratio of free PTH in the range 1.5-2.4 was observed after daily subcutaneous administration in Sprague-Dawley rats at day 28. Abbreviations:ACN acetonitrileAcOH acetic acidAib 2-aminoisobutyric acidBMD bone mineral densityBn benzylBoc tert-butyloxycarbonylCOMU (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphatecAMP cyclic adenosine monophosphated dayDBU 1,3-diazabicyclo[5.4.0]undeceneDCC N,N′-dicyclohexylcarbodiimideDCM dichloromethaneDIPEA N,N-diisopropylethylamineDMAP dimethylamino-pyridineDMF N,N-dimethylformamideDMSO dimethylsulfoxideDTT dithiothreitolEDTA ethylenediaminetetraacetic acideq stoichiometric equivalentESI-MS electrospray ionization mass spectrometryEt ethylFmoc 9-fluorenylmethyloxycarbonylGlu-C endoproteinase Glu-Ch hourHATU O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphateHPLC high performance liquid chromatographyivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutylLC liquid chromatographyLTQ linear trap quadrupoleLys-C endoproteinase Lys-CLLOQ lower limit of quantificationMal 3-maleimido propylMe methylMeOH methanolmin minutesMmt monomethoxytritylMS mass spectrum/mass spectrometrym/z mass-to-charge ratioOtBu tert-butyloxyPEG poly(ethylene glycol)pHPotentia HydrogeniiPK pharmacokineticsPr propylPTH parathyroid hormonePyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphateQ-TOF quadrupole time-of-flightRP-HPLC reversed-phase high performance liquid chromatographyrt room temperatureSIM single ion monitoringSEC size exclusion chromatographysc subcutaneoust1/2half lifeTCP tritylchloride polystyrolTES triethylsilaneTFA trifluoroacetic acidTHF tetrahydrofuranTmob 2,4,6-trimethoxybenzylTrt triphenylmethyl, tritylULOQ upper limit of quantificationUPLC ultra performance liquid chromatographyUV ultravioletZQ single quadrupole | 47,471 |
11857604 | EXAMPLES The examples below make it possible to illustrate the invention but are in no way limiting in nature. The aim of the various examples presented below is to illustrate the possibility of the polysiloxane nanoparticles acting as transporter of molecules used in chemotherapy. The intended molecules adsorb at the surface of the nanoparticles according to the mechanism proposed inFIG.1. The examples below also make it possible to determine the drug concentration limit above which the drugs are no longer retained at the surface of the nanoparticle. During the various examples, the maximum drug load content on polysiloxane nanoparticles was determined, when said particles are present at a concentration corresponding to a concentration used during clinical trials for the nanoparticle in question. Increasing concentrations of active substances were thus brought into contact with the nanoparticles. Solutions are purified by tangential filtration. The molecules which have not be able to adsorb to the surface of the nanoparticles pass through the membrane and are found in the subnatant, where they can be detected by spectroscopic techniques such as UV/Visible absorption or else fluorescence spectroscopy (FIG.2). Preparation of a Solution of Polysiloxane-Based Ultrafine Nanoparticles The solution of polysiloxane-based ultrafine nanoparticles (AGuIX®) was synthesized according to the procedure described in the publication G. Le Duc et al.,Cancer Nanotechnology,2014. A solution of AGuIX® at a gadolinium concentration of 10 mM is analysed by DLS with a laser at 633 nm. A number-average hydrodynamic diameter of 3.2 nm is obtained. Nanovectors for Doxorubicin Delivery Example 1 50 μmol (Gd3+) of AGuIX® nanoparticles were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM ([Gd3+]). 2.85 mg of doxorubicin are placed in a 2.5 ml flask. 1.1 ml of ultrapure water are added to the flask, which is stirred until the doxorubicin has completely dissolved. A solution at 2.6 g/l of doxorubicin is then obtained, and is protected from the light with aluminium. 215 μl of this solution are then added to the solution of AGuIX®, as are 160 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 112 mg/l of doxorubicin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out in order to obtain a supernatant of 200 μl. The subnatant is analysed by UV-visible analysis. The supernatant is diluted 50-fold and is analysed by UV-visible analysis. Example 2 (Comparative) A solution of doxorubicin at 112 mg/l is prepared according to the procedure described in Example 1, the solution of AGuIX® being replaced with ultrapure water. Example 3 50 μmol (Gd3+) of AGuIX® nanoparticles were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM ([Gd3+]). 2.85 mg of doxorubicin are placed in a 2.5 ml flask. 1.1 ml of ultrapure water are added to the flask, which is stirred until the doxorubicin has completely dissolved. A solution at 2.6 g/l of doxorubicin is then obtained, and is protected from the light with aluminium. 327 μl of this solution are then added to the solution of AGuIX®, as are 48 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 170 mg/l of doxorubicin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out in order to obtain a supernatant of 200 μl. The subnatant is analysed by UV-visible analysis. The supernatant is diluted 50-fold and is analysed by UV-visible analysis. Example 4 (Comparative) A solution of doxorubicin at 170 mg/l is prepared according to the procedure described in Example 3, the solution of AGuIX® being replaced with ultrapure water. Comparative Results Examples 1/2 and 3/4 FIGS.3and4represent, respectively, the absorption spectra of the subnatants of the solutions of Examples 1 and 2, and of the solutions of Examples 3 and 4. These data show that the doxorubicin interacts with the AGuIX® nanoparticles. Indeed, for the solution containing 100 mM ([Gd3+]) of AGuIX® and 112 mg/l, the doxorubicin is adsorbed at the surface of the nanoparticle and is not detected in the subnatant after tangential filtration, contrary to a solution of doxorubicin alone. For the solution containing 100 mM ([Gd3+]) of AGuIX® and 170 mg/l, a very weak signal is detected by UV/VIS spectrophotometry, indicating that the majority of the doxorubicin is adsorbed at the surface of the nanoparticles and that a small amount passes through the membrane. A solution obtained by the procedure of Example 3 (doxorubicin at 170 mg/l and AGuIX® at 100 mM [Gd3+]) is diluted 50-fold and analysed by DLS with a laser at 633 nm. A number-average hydrodynamic diameter of 3.7 nm is obtained, which is greater than the diameter of 3.2 nm obtained for the AGuIX® nanoparticles, indicating a surface interaction of the nanoparticles with the doxorubicin. For a solution of AGuIX® nanoparticles at 100 mM ([Gd3+]) corresponding to 100 g/l of nanoparticles, a retention of doxorubicin is observed up to a minimum concentration of 112 mg/l, which corresponds to a load content by weight greater than 1.12 mg/g of nanoparticles (FIG.5). Nanovectors for Delivery of TATE Peptide Example 6 50 μmol (Gd3+) of AGuIX® were dispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM ([Gd3+]). 14.94 mg of tyr3-octreotate (TATE) peptide are placed in a 2.5 ml flask. 498 μl of ultrapure water are added to the flask, which is stirred until the peptide has completely dissolved. A solution containing 30 g/l of peptide is then obtained. 48 μl of this solution are then added to the solution of AGuIX®, as are 328 μl of ultrapure water. The flask is stirred for 30 minutes. A solution containing 100 mM of gadolinium and 2.90 g/l of peptide is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 200 μl. The subnatant is analysed by UV-visible analysis and fluorometry after 20-fold dilution. Example 7 (Comparative) A solution of TATE peptide at 2.90 g/l is prepared according to the procedure described in Example 6, the solution of AGuIX® being replaced with ultrapure water. Example 8 50 μmol (Gd3+) of the AGuIX® nanoparticles were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM ([Gd3+]). 6.1 mg of tyr3-octreotate (TATE) peptide are placed in a 2.5 ml flask. 203.3 μl of ultrapure water are added to the flask, which is stirred until the peptide has completely dissolved. A solution containing 30 g/l of peptide is then obtained. 97 μl of this solution are then added to the solution of AGuIX®, as are 279 μl of ultrapure water. The flask is stirred for 30 minutes. A solution containing 100 mM of gadolinium and 5.80 g/l of peptide is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 320 μl. The subnatant is analysed by UV-visible analysis (20-fold dilution) and fluorometry (40-fold dilution). Example 9 50 μmol (Gd3+) of the AGuIX® nanoparticles were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM ([Gd3+]). 14.94 mg of tyr3-octreotate (TATE) peptide are placed in a 2.5 ml flask. 498 μl of ultrapure water are added to the flask, which is stirred until the peptide has completely dissolved. A solution containing 30 g/l of peptide is then obtained. 193 μl of this solution are then added to the solution of AGuIX®, as are 182 μl of ultrapure water. The flask is stirred for 30 minutes. A solution containing 100 mM of gadolinium and 11.60 g/l of peptide is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 200 μl. The subnatant is analysed by UV-visible analysis and fluorometry after 20-fold dilution. Example 10 (Comparative) A solution of TATE peptide at 11.60 g/l is prepared according to the procedure described in Example 5, the solution of AGuIX® being replaced with ultrapure water. Example 11 50 μmol (Gd3+) of the AGuIX® nanoparticles were redispersed in 250 μl of ultrapure water in order to obtain a solution at 200 mM ([Gd3+]). 0.6 mg of tyr3-octreotate (TATE) peptide are placed in a 2.5 ml flask. 20 μl of ultrapure water are added to the flask, which is stirred until the peptide has completely dissolved. A solution containing 30 g/l of peptide is then obtained. 20 μl of this solution are then added to the solution of AGuIX®, as are 230 μl of ultrapure water. The flask is stirred for 30 minutes. A solution containing 100 mM of gadolinium and 1.20 g/l of peptide is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 320 μl. The subnatant is analysed by UV-visible analysis (20-fold dilution) or fluorometry (40-fold dilution). Results of Examples 6 to 10 FIGS.6,7and8represent, respectively, the absorption spectra of the 20-fold diluted subnatants of the solutions of Examples 6 and 7, of the solutions of Examples 9 and 10 and of the solution of Example 8. These data show that the TATE peptide adsorbs at the surface of the nanoparticles up to a concentration of approximately 2 g·L1(FIG.12). Indeed, before this limiting concentration, the TATE peptide is not detected by UV/VIS spectrophotometry in the subnatants of the solutions purified by tangential filtration. FIGS.9,10and11represent, respectively, the fluorescence spectra of the 20-fold diluted subnatants of Examples 6 and 7, of the Examples 9 and 10 and of Example 8. In the same way as for the detection by UV/VIS spectrophotometry, these data show that the TATE peptide interacts with the AGuIX® nanoparticles. A solution obtained by the procedure of Example 8 (TATE at 2.90 g/l) is diluted 10-fold and analysed by DLS with a laser at 633 nm. A number-average hydrodynamic diameter of 3.4 nm is obtained, which is greater than the diameter of 3.2 nm obtained for the AGuIX® nanoparticles, indicating a surface interaction of the nanoparticles with the TATE peptide. For a solution of AGuIX® nanoparticles at 100 mM ([Gd3+]) corresponding to 100 g/l of nanoparticles, a retention of the TATE peptide is observed up to a minimum concentration of 2 g/l, which corresponds to a load content by weight of greater than 20 mg/g of nanoparticles (FIG.12). Nanovectors for Delivery of Cisplatin Example 12 50 μmol (Gd3+) of AGuIX® were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM [Gd3+]. 3.1 mg of cisplatin are placed in a 2.5 ml flask. 1.2 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat to 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 24 μl of this solution are then added to the solution of AGuIX®, as are 351 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 120 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 160 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. The resulting solution is heated at 100° C. for 15 min. Once the reaction is finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. Example 13 50 μmol (Gd3+) of AGuIX® were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM [Gd3+]. 3.1 mg of cisplatin are placed in a 2.5 ml flask. 1.2 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat to 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 36 μl of this solution are then added to the solution of AGuIX®, as are 339 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 180 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 160 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. The resulting solution is heated at 100° C. for 15 min. Once the reaction is finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV/VIS spectrophotometry. Example 14 50 μmol (Gd3+) of AGuIX® were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM [Gd3+]. 3.1 mg of cisplatin are placed in a 2.5 ml flask. 1.2 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat to 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 72 μl of this solution are then added to the solution of AGuIX®, as are 303 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 360 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 160 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. The resulting solution is heated at 100° C. for 15 min. Once the reaction is finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. Example 15 50 μmol (Gd3+) of AGuIX® were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM [Gd3+]. 2.8 mg of cisplatin are placed in a 2.5 ml flask. 1.1 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat to 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 142 μl of this solution are then added to the solution of AGuIX®, as are 233 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 720 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 140 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. The resulting solution is heated at 100° C. for 15 min. Once the reaction is finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. Example 16 (Comparative) A solution of cisplatin at 720 mg/l is prepared according to the procedure described in Example 15, the solution of AGuIX® being replaced with ultrapure water. Example 17 50 μmol (Gd3+) of AGuIX® were redispersed in 125 μl of ultrapure water in order to obtain a solution at 400 mM [Gd3+]. 2.8 mg of cisplatin are placed in a 2.5 ml flask. 1.1 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat to 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 229 μl of this solution are then added to the solution of AGuIX®, as are 146 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of gadolinium and 1160 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 160 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. The resulting solution is heated at 100° C. for 15 min. Once the reaction is finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. Results (Examples 12, 13, 14, 15 and 17) Examples 12, 13, 14, 15 and 17 (cisplatin at 120-180-360-720-1160 mg/l) are analysed by DLS (samples diluted 10-fold) with a laser at 633 nm. The respective number-average hydrodynamic diameters are: 3.8, 3.7, 3.8, 3.4, 3.7 nm. They are greater than the diameter of 3.2 nm obtained for the AGuIX® nanoparticles, indicating a surface interaction of the nanoparticles with the cisplatin. Example 18 (Comparative) A solution of cisplatin at 1160 mg/l is prepared according to the procedure described in Example 15, the solution of AGuIX® being replaced with ultrapure water. Results of Examples 15/16 and 17/18 FIG.13represents the absorbance of the subnatants of Examples 12, 13 and 14 after treatments as described in the examples. FIGS.14and15represent, respectively, the absorption spectra of the subnatants of the solutions of Examples 15 and 16, and of the solutions of Examples 17 and 18. These data show that the cisplatin adsorbs at the surface of the nanoparticles up to a concentration of approximately 240 mg·L−1(FIG.16). Indeed, before this limiting concentration, the signal detected in the ODPA-treated subnatants, by UV/VIS spectrophotometry at 706 nm, is not modified. For a solution of AGuIX® nanoparticles at 100 mM ([Gd3+]) corresponding to 100 g/l of nanoparticles, a retention of the cisplatin is observed up to a minimum concentration of 240 mg/l, which corresponds to a load content by weight of greater than 2.4 mg/g of nanoparticles (FIG.16). Example 19 Nanoparticles based on polysiloxane and on free chelates for cisplatin delivery. For the synthesis of these nanoparticles, 6.187 ml (26.17 mmol) of APTES are added to 90 ml of diethylene glycol. The solution is stirred for 1 h at ambient temperature before 10 g (17.45 mmol) of DOTAGA anhydride are added. The solution is left to stir for 5 days. At the end of this, 7.952 ml of TEOS (34.90 mmol) are added to the solution, which is stirred for 1 hour. 900 ml of ultrapure water are then added, before heating at 50° C. with stirring for 18 h. The solution is then concentrated to 200 ml on a Vivaflow cassette with membranes having a cut-off threshold of 5 kDa. The pH is adjusted to 2 by adding hydrochloric acid. The solution is purified by a factor of 50 by Vivaflow, before being neutralized to pH 7.4 by controlled addition of 1 M sodium hydroxide. The solution is filtered and then lyophilised. After redispersion in water, the nanoparticles have a hydrodynamic diameter of 5.2 nm. 50 μmol (DOTAGA) of silica nanoparticles (62.5 mg) were redispersed in 141 μl of ultrapure water in order to obtain a solution at 354 mM of DOTAGA and 443 mg/l. 3 mg of cisplatin are placed in a 2.5 ml flask. 1.2 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat at 40° C. until it has completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 229 μl of this solution are then added to the solution of silica nanoparticles, as are 130 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of free chelate (125 g/l of nanoparticles) and 1160 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 200 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this solution are added to 200 μl of buffer and 100 μl of ODPA. This new solution is heated at 100° C. for 15 min. Once the reaction has finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. FIG.17represents the absorption spectra of the subnatants of the solutions Examples 18 and 19. A weaker signal can be observed in the subnatants for the cisplatin added to the solution of silica nanoparticles. The signal at 706 nm of the two UV spectra makes it possible to estimate a retention of a cisplatin concentration of 260 mg/l for a solution of nanoparticles at 125 g/l, corresponding to a load content by weight of 2.1 mg/g of nanoparticles. Example 20 Possibility of varying the load content by modifying the surface of the nanoparticles by chelation of bismuth ions. The nanoparticles described in Example 19 are dispersed in water (283 mg, 227 μmol of DOTAGA) in order to have a DOTAGA concentration of approximately 200 mM. The pH of the solution is adjusted to 5.5 by adding NaOH. 817 μl of a solution of BiCl3at 250 mM in 6 M HCl are slowly added in 3 additions with stirring at a temperature of 70° C. to accelerate the complexation. Between each addition, the pH is readjusted to 5.5 by slowly adding a 10 M sodium hydroxide solution. The solution is then heated to 80° C. for 1 hour after the final addition. At the end of this, ultrapure water is added to reach a chelate concentration of 100 mM at a pH of 5.5. The solution is then heated at 80° C. for 18 h. The excess Bi3+is removed by tangential filtration, then the solution is neutralized to reach a pH of 7 by adding sodium hydroxide, before filtration on a 0.2 μm membrane and lyophilisation. After redispersion in the water, the nanoparticles have a hydrodynamic diameter of 6.0 nm. 30 μmol of AGuIX@DOTA@Bi (Bi3+) (67.8 mg) were redispersed in 75 μl of ultrapure water in order to obtain a solution at 400 mM (904 g/L of nanoparticles). 3 mg of cisplatin are placed in a 2.5 ml flask. 1.2 ml of ultrapure water are added to the flask, which is stirred. Since cisplatin is not very soluble at ambient temperature, it is necessary to heat at 40° C. until it is completely dissolved. A solution containing 2.5 g/l of cisplatin is then obtained, and is protected from the light with aluminium. 118 μl of this solution are then added to the solution of AGuIX@DOTA@Bi, as are 107 μl of ultrapure water. The flask is stirred for 30 minutes in the dark. A solution containing 100 mM of bismuth (226 g/l of nanoparticles) and 1000 mg/l of cisplatin is thus obtained. This solution is placed in a 3 kDa Vivaspin®, and a tangential filtration cycle is carried out so as to obtain a supernatant of 80 μl. The subnatant is analysed by UV-visible analysis. The cisplatin is detectable by UV/VIS absorption at a wavelength of 706 nm after reaction with ODPA. For the reaction with cisplatin, a solution of ODPA at 1.4 mg/ml and a phosphate buffer (pH 6.8) are prepared. The subnatant is diluted 5-fold. 140 μl of this concentration are added to 200 μl of buffer and 100 μl of ODPA. This new solution is heated at 100° C. for 15 min. Once the reaction has finished and the temperature has returned to ambient temperature, 560 μl of DMF are added. The final solution is filtered and then analysed by UV-visible analysis. Results of Examples 18 and 20 FIG.18represents the absorption spectra of the subnatants of the solutions of Examples 18 and 20. As shown inFIG.18, the chelation of bismuth at the surface of the nanoparticles leads to a non-retention of the cisplatin at the surface of the nanoparticles, proving that changes regarding the number of metal elements chelated at the surface of the nanoparticles make it possible to vary the load content of the nanoparticles. | 25,852 |
11857605 | DETAILED DESCRIPTION Definitions The following terms shall be used to describe the present invention. In the absence of a specific definition set forth herein, the terms used to describe the present invention shall be given their common meaning as understood by those of ordinary skill in the art. Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps. In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein. As used herein, the terms “treat”, “treating”, “treatment”, and the like refer to reducing or ameliorating a disorder/disease and/or symptoms associated therewith. It will be appreciated, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. In certain embodiments, treatment includes prevention of a disorder or condition, and/or symptoms associated therewith. The term “prevention” or “prevent” as used herein refers to any action that inhibits or at least delays the development of a disorder, condition, or symptoms associated therewith. Prevention can include primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications. The term “subject” as used herein, refers to an animal, typically a mammal or a human, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of a compound described herein, then the subject has been the object of treatment, observation, and/or administration of the compound described herein. The term “therapeutically effective amount” as used herein, means that amount of the compound or pharmaceutical agent that elicits a biological and/or medicinal response in a cell culture, tissue system, subject, animal, or human that is being sought by a researcher, veterinarian, clinician, or physician, which includes alleviation of the symptoms of the disease, condition, or disorder being treated. The term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. The term “pharmaceutically acceptable carrier” refers to a medium that is used to prepare a desired dosage form of a compound. A pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe ed. (American Pharmaceutical Assoc. 2000), disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. As used herein, unless otherwise indicated, the term “halo” or “halide” includes fluoro, chloro, bromo or iodo. As used herein, “alkyl” refers to a straight-chain or branched saturated hydrocarbon group. Examples of alkyl groups include methyl-, ethyl-, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, iso-butyl, sec-butyl, tert-butyl), pentyl groups (e.g., 1-methylbutyl, 2-methylbutyl, iso-pentyl, tert-pentyl, 1,2-dimethylpropyl, neopentyl, and 1-ethylpropyl), hexyl groups, and the like. In various embodiments, an alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group), for example, 1-30 carbon atoms (i.e., C1-30 alkyl group). In certain embodiments, an alkyl group can have 1 to 6 carbon atoms, and can be referred to as a “lower alkyl group.” Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butyl groups (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl). In certain embodiments, alkyl groups can be optionally substituted as described herein. An alkyl group is generally not substituted with another alkyl group, an alkenyl group, or an alkynyl group. As used herein, “alkenyl” refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene). In various embodiments, an alkenyl group can have 2 to 40 carbon atoms (i.e., C2-40 alkenyl group), for example, 2 to 20 carbon atoms (i.e., C2-20 alkenyl group). In certain embodiments, alkenyl groups can be substituted as described herein. An alkenyl group is generally not substituted with another alkenyl group, an alkyl group, or an alkynyl group. As used herein, “cycloalkyl” by itself or as part of another substituent means, unless otherwise stated, a monocyclic hydrocarbon having between 3-12 carbon atoms in the ring system and includes hydrogen, straight chain, branched chain, and/or cyclic substituents. Exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. As used herein, a “fused ring” or a “fused ring moiety” refers to a polycyclic ring system having at least two rings where at least one of the rings is aromatic and such aromatic ring (carbocyclic or heterocyclic) has a bond in common with at least one other ring that can be aromatic or non-aromatic, and carbocyclic or heterocyclic. These polycyclic ring systems can be highly p-conjugated and optionally substituted as described herein. As used herein, “heteroatom” refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium. As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused (i.e., having a bond in common with) together or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl group can have 6 to 24 carbon atoms in its ring system (e.g., C6-24 aryl group), which can include multiple fused rings. In certain embodiments, a polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure. Examples of aryl groups having only aromatic carbocyclic ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic), pentacenyl (pentacyclic), and like groups. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5,6-bicyclic cycloalkyl/aromatic ring system), cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl/aromatic ring system), imidazoline (i.e., a benzimidazolinyl group, which is a 5,6-bicyclic cycloheteroalkyl/aromatic ring system), and pyran (i.e., a chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like. In certain embodiments, aryl groups can be optionally substituted. In certain embodiments, an aryl group can have one or more halogen substituents, and can be referred to as a “haloaryl” group. Perhaloaryl groups, i.e., aryl groups where all of the hydrogen atoms are replaced with halogen atoms (e.g., —C6F5), are included within the definition of “haloaryl.” In certain embodiments, an aryl group is substituted with another aryl group and can be referred to as a biaryl group. Each of the aryl groups in the biaryl group can be optionally substituted. The term “aralkyl” refers to an alkyl group substituted with an aryl group. As used herein, “heteroaryl” refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se) or a polycyclic ring system where at least one of the rings present in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryl groups include those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkyl rings. A heteroaryl group, as a whole, can have, for example, 5 to 24 ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered heteroaryl group). The heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O—O, S—S, or S—O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiophene S,S-dioxide). Examples of heteroaryl groups include, for example, the 5- or 6-membered monocyclic and 5-6 bicyclic ring systems shown below: where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl), SiH2, SiH(alkyl), Si(alkyl)2, SiH(arylalkyl), Si(arylalkyl)2, or Si(alkyl)(arylalkyl). Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, IH-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like. In certain embodiments, heteroaryl groups can be substituted as described herein. In certain embodiments, heteroaryl groups can be optionally substituted. The term “optionally substituted” refers to a chemical group, such as alkyl, cycloalkyl aryl, and the like, wherein one or more hydrogen may be replaced with a substituent as described herein, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term “carbocycle” is art-recognized and refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon. The term “nitro” is art-recognized and refers to —NO2; the term “halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term “sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl” means —OH; and the term “sulfonyl” and “sulfone” is art-recognized and refers to —SO2—. “Halide” designates the corresponding anion of the halogens. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In certain embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions, such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In certain embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts. As used herein, the term “isolated” in connection with a compound described herein means the compound is not in a cell or organism and the compound is separated from some or all of the components that typically accompany it in a cell or organism. As used herein, the term “substantially pure” in connection with a sample of a compound described herein means the sample contains at least 60% by weight of the compound. In certain embodiments, the sample contains at least 70% by weight of the compound; at least 75% by weight of the compound; at least 80% by weight of the compound; at least 85% by weight of the compound; at least 90% by weight of the compound; at least 95% by weight of the compound; or at least 98% by weight of the compound. As used herein, the term “substantially stroma-free” used in connection with a sample of a compound described herein means the sample contains less than 5% by weight stroma. In certain embodiments, the samples contains less than 4% by weight stroma; less than 3% by weight stroma; less than 2% by weight stroma; less than 1% by weight stroma; less than 0.5% by weight stroma; less than 0.1% by weight stroma; less than 0.05% by weight stroma; or less than 0.01% by weight stroma. As used herein, the term water-soluble polymer includes those water-soluble polymers that are substantially biocompatible and substantially nonimmunogenic and specifically excludes any water-soluble polymers that are not biocompatible and substantially nonimmunogenic. With respect to biocompatibility, a substance is considered substantially biocompatible if the beneficial effects associated with use of the substance alone or with another substance in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician. With respect to substantially nonimmunogenic, a substance is considered substantially nonimmunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. In certain embodiments, that the water-soluble polymer described herein as well as thiosuccinyl-crosslinked hemoglobin conjugates comprising the same are substantially biocompatible and substantially nonimmunogenic. The present disclosure provides a thiosuccinyl-crosslinked hemoglobin conjugate comprising a tetrameric hemoglobin; at least one water-soluble polymer covalently attached to the tetrameric hemoglobin via an optional linker; and at least one thiosuccinyl crosslinking moiety of Formula 1: or a pharmaceutically acceptable salt or zwitterion thereof, wherein each N* independently represents a nitrogen selected from the group consisting of a nitrogen in a lysine residue side chain in the tetrameric hemoglobin and a nitrogen at a N-terminus in the tetrameric hemoglobin; and R1is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or —(CR2)nY, wherein n is an integer number selected from 0-10; R for each instance is independently hydrogen, alkyl, aralkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; or two instances of R taken together form a 3-6 membered cycloalkyl or heterocycloalkyl containing 1, 2, or 3 heteroatoms selected from N, O, and S; Y is selected from the group consisting of OR4, SR4, N(R4)2, —(C═O)R4, —(C═O)OR4, —O(C═O)R4, —O(C═O)OR4, —(C═O)N(R4)2, —(NR4)(C═O)R4, —(NR4)(C═O)OR4, —O(C═O)N(R4)2, —O(C═NR4)N(R4)2, —(NR4)(C═O)N(R4)2, —(C═NR4)N(R4)2, —(NR4)(C═NR4)N(R4)2, —(S═O)R4, —S(O)2R4, —S(O)2OR4, —S(O)2N(R4)2, —OS(O)2R4, —(NR4)S(O)2R4, —OS(O)2OR4, —OS(O)2N(R4)2, —(NR4)S(O)2N(R4)2, —(NR4)S(O)2OR4, and —(CRR2R3), wherein R2is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR4, SR4, N(R4)2, —(C═O)R4, —(C═O)OR4, —O(C═O)R4, —O(C═O)OR4, —(C═O)N(R4)2, —(NR4)(C═O)R4, —(NR4)(C═O)OR4, —O(C═O)N(R4)2, —O(C═NR4)N(R4)2, —(NR4)(C═O)N(R4)2, —(C═NR4)N(R4)2, —(NR4)(C═NR4)N(R4)2, —(S═O)R4, —S(O)2R4, —S(O)2OR4, —S(O)2N(R4)2, —OS(O)2R4, —(NR4)S(O)2R4, —OS(O)2OR4, —OS(O)2N(R4)2, —(NR4)S(O)2N(R4)2, or —(NR4)S(O)2OR4; R3is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR4, SR4, N(R4)2, —(C═O)R4, —(C═O)OR4, —O(C═O)R4, —O(C═O)OR4, —(C═O)N(R4)2, —(NR4)(C═O)R4, —(NR4)(C═O)OR4, —O(C═O)N(R4)2, —O(C═NR4)N(R4)2, —(NR4)(C═O)N(R4)2, —(C═NR4)N(R4)2, —(NR4)(C═NR4)N(R4)2, —(S═O)R4, —S(O)2R4, —S(O)2OR4, —S(O)2N(R4)2, —OS(O)2R4, —(NR4)S(O)2R4, —OS(O)2OR4, —OS(O)2N(R4)2, —(NR4)S(O)2N(R4)2, or —(NR4)S(O)2OR4; and R4for each instance is independently selected from the group consisting of hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; or R1is a moiety selected from the group consisting of: and N5-(1-((carboxymethyl)amino)-1-oxo-3λ3-propan-2-yl)glutamine or a pharmaceutically acceptable salt thereof, wherein m is a whole number selected from 1-1,000. The water-soluble polymer may be a saccharide (e.g., a dextran, an amylose, a hyalouronic acid, a poly(sialic acid), a heparan, a heparin, etc.); a poly(amino acid), e.g., a polyaspartic acid and a polyglutamic acid; a synthetic polymer (e.g., a polyacrylic acid, a polyether, e.g., polyethylene glycol); and copolymers and combinations thereof. The water-soluble polymer can be a linear polymer or a branched polymer. Branched polymer backbones are generally known in the art. The branched polymer can have a central branch core moiety and a group of linear polymer chains linked to the central branch core. In certain embodiments, the central branch core is a polyol, such as glycerol, pentaerythritol, or sorbitol and one or more poly(alkylene glycol) moieties are covalently bonded to the central branch core. In certain embodiments, the water-soluble polymer is a poly(alkylene glycol), such as a PEG, a polypropylene glycol (PPG), a copolymer of ethylene glycol and propylene glycol, and the like. The PEG can be a multi-armed PEG, a forked PEG, or a branched PEG, The PEG can be an alkoxy terminated PEG (such as methoxy-PEG) or a hydroxyl terminated PEG (hydroxy-PEG). The water-soluble polymer can have an average molecular weight between 1,000-50,000 Daltons, 1,000-40,000 Daltons, 1,000-30,000 Daltons, 1,000-20,000 Daltons, 2,000-20,000 Daltons, 1,000-10,000 Daltons, 2,000-10,000 Daltons, 3,000-10,000 Daltons, 3,000-8,000 Daltons, 4,000-8,000 Daltons, or 4,000-6,000 Daltons. In exemplary embodiments, the water-soluble polymer has an average molecular weight of 1,000, 2,000, 5,000, 10,000, 20,000, or 40,000 Daltons. The thiosuccinyl-crosslinked hemoglobin conjugate may comprise between 1-50, 10-50, 1-40, 10-40, 1-30, 10-30, 1-25, 10-25, 1-20, 2-20, 3-20, 4-20, 5-20 6-20, 7-20, 8-20, 9-20, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 11-13, 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 12-14, or 12-13 water-soluble polymers. The water-soluble polymer can be the same or a mixture of different water-soluble polymers. The water-soluble polymer can be directly attached to the tetrameric hemoglobin or attached via an optional linker. Any linker known in the art can be used to attach the water-soluble polymer to the tetrameric hemoglobin. In certain embodiments, the linker is represented by the formula: A(CH2)p(C═O)*, A(CH2)pN-succinimide*, A(CH2)pNH*, A(CH2)pS*, A(CH2)p(SO2)*, A(CH2)pNH(C═O)*, A(CH2)pNH(C═S)*, A(CH2)pPh(C═O)*, A(CH2)pPh(*C═C(H)(CN)), or A(CH2)pNH(C═O)CH2* as shown below: wherein A represents the water-soluble polymer, p is a whole number between 1-20, and * represents the tetrameric hemoglobin. In certain embodiments, p is a whole number between 1-18, 1-16, 1-14, 1-12, 1-10, 1-9, 2-10, 3-10, 2-9, 4-10, 5-9, 2-8, 2-6, 4-8, or 4-6. In exemplary embodiments, the linker is represented by the formula: A(CH2)p(C═O)*, wherein p is 1-10, 1-9, 2-10, 3-10, 2-9, 4-10, 5-9, 2-8, 2-6, 4-8, or 4-6. The water-soluble polymer can be covalently attached to the tetrameric hemoglobin via an optional linker to a nitrogen selected from the group consisting of a nitrogen in a lysine residue side chain in the tetrameric hemoglobin and a nitrogen at a N-terminus in the tetrameric hemoglobin; or the water-soluble polymer can be covalently attached to the tetrameric hemoglobin via an optional linker to a sulfur in a cysteine reside side chain in the tetrameric hemoglobin. While the examples below are generally directed to thiosuccinyl-crosslinked hemoglobin conjugate comprising a α2β2tetrameric hemoglobin, other forms of hemoglobin are also contemplated by the present disclosure, such as other tetrameric hemoglobin, e.g., α2γ2; trimeric hemoglobin, e.g., αβ2, αβ3, αγ2, and α2γ; dimeric hemoglobin, e.g., αβ and αγ; and the like; as well as polymeric forms of hemoglobin comprising one or more monomeric forms of hemoglobin; and hemoglobin derivatives that have been subjected to other methods of chemical modification including, but not limited to, methods for conjugation to polyalkylene oxide, reaction with pyridoxal phosphate, reaction with a dialdehyde, reaction with bis-diaspirin ester, reaction with iodoacetamide or other thiol-blocking reagents, or reaction in the presence of reagents such as 2,3-diphosphoglycerate (2,3-DPG) or chemically similar compounds, or genetically crosslinked hemoglobin derivatives, such as 2αβ2(dialpha beta hemoglobin), wherein the dialpha moiety comprises two alpha chains that are genetically crosslinked with, e.g., a glycine linker covalently linking the N-terminus and the C-terminus of each alpha chain. The tetrameric hemoglobin can comprise naturally occurring and/or non-naturally occurring α, β, and γ globin chain polypeptide sequences. The tetrameric hemoglobin can be human hemoglobin, bovine hemoglobin, porcine hemoglobin, ovine hemoglobin, equine hemoglobin, or blood from other invertebrates and recombinant and/or transgenically produced hemoglobin. In instances in which the tetrameric hemoglobin is human hemoglobin [e.g., comprising two α globin chain (UniProt Accession Number: P69905); and two β globin chains (UniProt Accession Number: P68871)], each N* may independently represent a nitrogen present in any one or more of amino acid residues at position 1, 8, 12, 17, 41, 57, 61, 62, 91, 100, 128, and 140 of the α globin chains; or at position 1, 9, 18, 60, 62, 66, 67, 83, 96, 121, 133, and 145 of the β globin chains. In certain embodiments, each N* independently represents a nitrogen present in the amino acid residues at position 100 of the α globin chains. In instances in which the tetrameric hemoglobin is bovine hemoglobin [e.g., comprising two α globin chain (UniProt Accession Number: P01966); and two β globin chains (UniProt Accession Number: P02070)], each N* may independently represent a nitrogen present in any one or more of amino acid residues at position 1, 8, 12, 17, 41, 57, 62, 69, 91, 100, 128, and 140 of the α globin chains; or at position 1, 7, 16, 18, 58, 60, 64, 65, 75, 81, 94, 103, 119, and 131 of the β globin chains. In certain embodiments, each N* independently represents a nitrogen present in any one or more of amino acid residues and 1 and 81 of the β globin chains. In instances in which the tetrameric hemoglobin is porcine hemoglobin [e.g., comprising two α globin chain (UniProt Accession Number: P01965); and two β globin chains (UniProt Accession Number: P02067)], each N* may independently represent a nitrogen present in any one or more of amino acid residues at position 1, 7, 11, 16, 40, 56, 61, 68, 90, 99, 127, and 139 of the α globin chains; or at position 1, 9, 18, 60, 62, 66, 67, 77, 83, 88, 133 and 145 of the β globin chains. In certain embodiments, R1is alkyl or —(CR2)nY; or R1is a moiety selected from the group consisting of: and N5-(1-((carboxymethyl)amino)-1-oxo-3λ3-propan-2-yl)glutamine or a pharmaceutically acceptable salt thereof, wherein m is a whole number selected from 1-1,000. In instances in which R1is: m can be 1-1,000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-25, 1-20, 1-15, 1-10, or 1-5. In instances in which R1is —(CR2)nY, n can be 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In certain embodiments, each R is independently hydrogen or alkyl. In certain embodiments, R1is —(CH2)nY. In certain embodiments, Y is —(CRR2R3), wherein R for each instance is independently selected from the group consisting of hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; R2is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —N(R4)2, or —NH(C═O)R4; R3is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —CO2R4, —(C═O)NHR4, —OR4, or —N(R4)2; and R4for each instance is independently selected from the group consisting of hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In certain embodiments, R is hydrogen. In certain embodiments, R2is —N(R4)2or —NH(C═O)R4; and R3is —CO2R4, —(C═O)NHR4, —OR4, or —N(R4)2. In certain embodiments, Y is —(CRR2R3), wherein, R is hydrogen; R2is hydrogen, —N(R4)2, —NH(C═O)R4, or —NH(C═O)N(R4)2; and R3is —CO2R4, —(C═O)NHR4, —OR4, or —N(R4)2. In certain embodiments, R1is —(CH2)n(CHR2R3), wherein n is 1, 2, 3, or 4; R2is —N(R4)2or —NH(C═O)R4; R3is —CO2R4or —(C═O)NHR4; and each R4is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. In certain embodiments, R1is —(CH2)n(CHR2R3), wherein n is 1, 2, 3, or 4; R2is —N(R4)2or —NH(C═O)R4; R3is —CO2H; and each R4is independently selected from the group consisting of hydrogen or alkyl. In certain embodiments, R1is selected from the group consisting of: or a pharmaceutically acceptable salt of zwitterion thereof, wherein m is a whole number selected from 1-1,000. In certain embodiments, the thiosuccinyl-crosslinked hemoglobin conjugate comprises a α2β2tetrameric bovine hemoglobin comprising two α globin chains (UniProt Accession Number: P01966) and two β globin chains (UniProt Accession Number: P02070), wherein the β globin chains are crosslinked with at least one thiosuccinyl crosslinking moiety of Formula 1, wherein R1is selected from the group consisting of: or a pharmaceutically acceptable salt or zwitterion thereof, wherein at least one N* represents the nitrogen at the N-terminus of a β globin chain and at least one N* represents the nitrogen in the lysine side chain at position 81 of a β globin chain. In certain embodiments, the thiosuccinyl-crosslinked hemoglobin conjugate is isolated and/or substantially pure. In certain embodiments, the thiosuccinyl-crosslinked hemoglobin conjugate is substantially stroma-free. In alternative embodiments, the present disclosure also provides analogs in which the sulfur depicted in Formula 1 is replaced with a moiety selected from the group consisting of selenium, disulfide, and diselenide, wherein R1and each N* are as defined in any one or more embodiments described herein. The thiosuccinyl-crosslinked hemoglobin conjugate described herein and their pharmaceutically acceptable salts can be administered to a subject either alone or in combination with pharmaceutically acceptable carriers or diluents in a pharmaceutical composition according to standard pharmaceutical practice. The thiosuccinyl-crosslinked hemoglobin conjugate can be administered parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical, the preferred method being intravenous administration. Accordingly, the present disclosure provides pharmaceutically acceptable compositions, which comprise a therapeutically-effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions of the present disclosure may be specially formulated for administration in liquid form, including those adapted for the following: (1) parenteral administration, for example, by intravenous as, for example, a sterile solution or suspension. As set out herein, certain embodiments of the thiosuccinyl-crosslinked hemoglobin conjugate described herein may contain a basic functional group, such as amino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term “pharmaceutically-acceptable salts” in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of thiosuccinyl-crosslinked hemoglobin conjugate of the present disclosure. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified thiosuccinyl-crosslinked hemoglobin conjugate of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the bromide, chloride, sulfate, bisulfate, carbonate, bicarbonate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. The pharmaceutically acceptable salts of the compounds of the present disclosure include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from nontoxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, the thiosuccinyl-crosslinked hemoglobin conjugate described herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the thiosuccinyl-crosslinked hemoglobin conjugate of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, solubilizing agents, buffers and antioxidants can also be present in the compositions. Methods of preparing the pharmaceutical comprising the thiosuccinyl-crosslinked hemoglobin conjugate include the step of bringing into association a thiosuccinyl-crosslinked hemoglobin conjugate described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound described herein with liquid carriers (liquid formulation), liquid carriers followed by lyophilization (powder formulation for reconstitution with sterile water or the like), or finely divided solid carriers, or both, and then, if necessary, shaping or packaging the product. Pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise one or more thiosuccinyl-crosslinked hemoglobin conjugate described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars (such as sucrose), alcohols, non-ionic surfactants (such as Tween 20), antioxidants, buffers, bacteriostats, chelating agents, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the compounds of the present disclosure may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. The pharmaceutical composition may comprise between 1-10 g/dL of the thiosuccinyl-crosslinked hemoglobin conjugates. In certain embodiments, the pharmaceutical composition comprises between 1-10 g/dL; 1-9 g/dL; or 1-8 g/dL; 1-7 g/dL; 1-6 g/dL; 2-6 g/dL; 3-6 g/dL; 4-6 g/dL or 4.5-5.5 g/dL of the thiosuccinyl-crosslinked hemoglobin conjugates. In certain embodiments, the pharmaceutical composition comprises an isolated and/or substantially pure thiosuccinyl-crosslinked hemoglobin conjugate. The concentration of the thiosuccinyl-crosslinked hemoglobin conjugates in samples described herein (in g/dL) is based solely on the mass of the thiosuccinyl-crosslinked hemoglobin content in the pharmaceutical composition and does not account for the mass of the water soluble polymers conjugated to the thiosuccinyl-crosslinked hemoglobin conjugates. The molecular weight difference arising from the weight of various numbers and types of water-soluble polymer conjugated to the thiosuccinyl-crosslinked hemoglobin conjugates does not substantially contribute to the overall mass of the thiosuccinyl-crosslinked hemoglobin. Consequently, there is a negligible difference in the thus approximated hemoglobin concentration. In certain embodiments, the pharmaceutical composition comprises one or more thiosuccinyl-crosslinked hemoglobin conjugates selected from the group consisting of thiosuccinyl-crosslinked hemoglobin conjugates comprising one, two, and three thiosuccinyl crosslinking moieties of Formula 1. The number of different thiosuccinyl-crosslinking moieties present in the hemoglobin conjugate and their relative amounts can be readily controlled by modifying the reaction conditions of the crosslinking reaction and/or by separating undesired fumaryl-crosslinked hemoglobin crosslinking and/or thiosuccinyl-crosslinked hemoglobin thiol addition products by purification. In certain embodiments, the pharmaceutical composition comprises a thiosuccinyl-crosslinked hemoglobin conjugate having one thiosuccinyl crosslinking moiety of Formula 1; a thiosuccinyl-crosslinked hemoglobin conjugate having two thiosuccinyl crosslinking moieties of Formula 1; and a thiosuccinyl-crosslinked hemoglobin conjugate having three thiosuccinyl crosslinking moieties of Formula 1. In certain embodiments, the pharmaceutical composition comprises a thiosuccinyl-crosslinked hemoglobin conjugate having one thiosuccinyl crosslinking moiety of Formula 1; a thiosuccinyl-crosslinked hemoglobin conjugate having two thiosuccinyl crosslinking moieties of Formula 1; and a thiosuccinyl-crosslinked hemoglobin conjugate having three thiosuccinyl crosslinking moieties of Formula 1 in a mass ratio of 2.5-3.5:5.5-6.5:0.5-1.5, respectively. In certain embodiments, the pharmaceutical composition comprises a thiosuccinyl-crosslinked hemoglobin conjugate having one thiosuccinyl crosslinking moiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 0.1-80%; 0.1-70%; 0.1-60%; 0.1-50%; 10-50%; 20-50%; 20-40%; 25-45%; or 25-35% w/w with respect to the total weight of all of the thiosuccinyl-crosslinked hemoglobin conjugate present in the pharmaceutical composition (e.g., relative to the total weight of the thiosuccinyl-crosslinked hemoglobin conjugate having one thiosuccinyl crosslinking moiety of Formula 1; the thiosuccinyl-crosslinked hemoglobin conjugate having two thiosuccinyl crosslinking moieties of Formula 1; and the thiosuccinyl-crosslinked hemoglobin conjugate having three thiosuccinyl crosslinking moieties of Formula 1 present in the pharmaceutical composition). In certain embodiments, the pharmaceutical composition comprises a thiosuccinyl-crosslinked hemoglobin conjugate having two thiosuccinyl crosslinking moiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 10-90%; 20-90%; 20-80%; 20-70%; 30-70%; 40-70%; 50-70%; 50-60%; or 55-65% w/w with respect to the total weight of all of the thiosuccinyl-crosslinked hemoglobin conjugates present in the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a thiosuccinyl-crosslinked hemoglobin conjugate having three thiosuccinyl crosslinking moiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 0.1-80%; 0.1-70%; 0.1-60%; 0.1-50%; 0.1-40%; 0.1-30%; 0.1-20%; 5-20%; or 5-15% w/w with respect to the total weight of all of the thiosuccinyl-crosslinked hemoglobin conjugates present in the pharmaceutical composition. The pharmaceutical composition can comprise the fumaryl crosslinked hemoglobin conjugate in less than 10%, less than 9%, less 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% by weight, less than 0.5%, or less than 0.1% by weight; or substantially no fumaryl crosslinked hemoglobin conjugate. The thiosuccinyl-crosslinked hemoglobin conjugate and the fumaryl crosslinked hemoglobin conjugate may be present in the pharmaceutical composition in a mass ratio of 90:10 to 99.99:0.01. In certain embodiments, the thiosuccinyl-crosslinked hemoglobin conjugate and the fumaryl crosslinked hemoglobin conjugate may be present in the pharmaceutical composition in a mass ratio of 90:10 to 99:1; 90:10 to 98:2; 90:10 to 97:3; 90:10 to 96:4; 90:10 to 95:5; 91:9 to 95:5; 92:8 to 95:5; 93:7 to 95:5; 94:6 to 95:5; 93:7 to 97:3; 94:6 to 96:4; 91:9 to 99.99:0.01; 92:8 to 99.99:0.01; 93:7 to 99.99:0.01; 94:6 to 99.99:0.01; 95:5 to 99.99:0.01; 96:4 to 99.99:0.01; 97:3 to 99.99:0.01; 98:2 to 99.99:0.01; 99:1 to 99.99:0.01; 99.5:0.5 to 99.99:0.01; or 99.9:0.1 to 99.99:0.01, respectively. In certain embodiments, the pharmaceutical composition comprises substantially no fumaryl crosslinked hemoglobin conjugate. In certain embodiments, the pharmaceutical composition further comprises an antioxidant. Exemplary antioxidants include, but are not limited to, cysteine, N-acetyl cysteine, γ-glutamyl-cysteine, glutathione, 2,3-dimercapto-I-propanol, 1,4-butanedithiol, sodium dithionite, other biologically compatible thiols and ascorbate. The antioxidant can inhibit or reverse the formation of methemoglobin. In certain embodiments, the pharmaceutical composition comprises 5% (w/w) or less of the antioxidant. In certain embodiments, the pharmaceutical composition comprises 4.5% (w/w) or less; 4.0% (w/w) or less; 3.5% (w/w) or less; 3.0% (w/w) or less; 2.5% (w/w) or less; 2.0% (w/w) or less; 1.5% (w/w) or less; 1.0% (w/w) or less; 0.9% (w/w) or less; 0.8% (w/w) or less; 0.7% (w/w) or less; 0.6% (w/w) or less; 0.5% (w/w) or less; 0.4% (w/w) or less; 0.3% (w/w) or less; 0.2% (w/w) or less; or 0.1% (w/w) or less of the antioxidant. In certain embodiments, the pharmaceutical composition comprises between 0.001 to 1% (w/w); 0.01 to 1% (w/w); 0.01 to 1% (w/w); 0.01 to 0.9% (w/w); 0.01 to 0.8% (w/w); 0.01 to 0.7% (w/w); 0.01 to 0.6% (w/w); 0.01 to 0.5% (w/w); 0.01 to 0.4% (w/w); 0.01 to 0.3% (w/w); 0.05 to 0.3% (w/w); 0.1 to 0.3% (w/w); or 0.15 to 0.25% (w/w) antioxidant. In certain embodiments, the pharmaceutical composition includes less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% by weight methemoglobin. In certain embodiments, provided herein is a solid pharmaceutical composition comprising a thiosuccinyl-crosslinked hemoglobin conjugate as described herein, NAC, sucrose, and Tween 20. In certain embodiments, provided herein is a pharmaceutical composition comprising a thiosuccinyl-crosslinked hemoglobin conjugate as described herein, NAC, NaCl, and NaCH3COO. In certain embodiments, the pharmaceutical composition comprising a thiosuccinyl-crosslinked hemoglobin conjugate as described herein, NAC, NaCl, NaCH3COO, sucrose, and Tween 20. The present disclosure also provides methods of preparing the thiosuccinyl-crosslinked hemoglobin conjugate described herein. The thiosuccinyl-crosslinked hemoglobin can readily be prepared by any number of well-known methods known to those of ordinary skill in the art. In certain embodiments, the method for preparing the thiosuccinyl-crosslinked hemoglobin conjugate comprises: contacting a tetrameric hemoglobin with a fumaryl crosslinking agent thereby forming a fumaryl-crosslinked hemoglobin; contacting the fumaryl-crosslinked hemoglobin with a thiol or a pharmaceutically acceptable salt or zwitterion thereof thereby forming a thiosuccinyl-crosslinked hemoglobin; and contacting the thiosuccinyl-crosslinked hemoglobin with a reactive water-soluble polymer reagent comprising a water-soluble polymer, a reactive functional group and optionally a linker, wherein the linker is covalently attached to the water-soluble polymer and the reactive functional group, thereby forming the thiosuccinyl-crosslinked hemoglobin conjugate. Any fumaryl crosslinking agent that is capable of intramolecularly crosslinking hemoglobin known in the art can be used in the methods described herein. In certain embodiments, the fumaryl crosslinking agent can be represented by a compound of Formula 4: wherein each LG1can independently be any leaving group in the art. Exemplary leaving groups include, but are not limited to, Cl, Br, I, 3,5-dibromosalicylate, salicylate, or the like. In certain embodiments, LG1is selected from the group consisting of: The compound of Formula 4 can be performed or formed in situ, e.g., by reaction of fumaric acid with a carbonyl activating agent and optionally a coupling additive. Exemplary carbonyl activating agents include, but are not limited to, carbodiimide, such as DCC, DIC, EDC, CIC, BMC, CPC, BDDC, PIC, PEC, and BEM, a uronium/aminium salt, such as HATU, HBTU, TATU, TBTU, HAPyU, TAPipU, HAPipU, HBPipU, HAMBU, HBMDU, HAMTU, 5,6-B(HATU), 4,5-B(HATU), HCTU, TCTU, and ACTU, phosphonium salts, such as AOP, BOP, PyAOP, PyBOP, PyOxm, PyNOP, PyFOP, NOP, and PyClock, immonium salts, such as BOMI, BDMP, BMMP, BPMP, and AOMP. Exemplary coupling additives include, but are not limited to, HOBt. 6-NO2—HOBt, 6-Cl—HOBt, 6-CF3—HOBt, HOAt, HODhbt, HODhat, HOSu, and Oxyma. In certain embodiments, the crosslinking agent is a salicyl fumarate analog, wherein the aryl ring of each of the salicyl groups is independently optionally substituted. In certain embodiments, the crosslinking agent is selected from the group consisting of bis-3,5-dibromosalicyl fumarate (DBSF), fumaryl chloride and bis(salicyl) fumarate. In the step of contacting the crosslinking agent and the tetrameric hemoglobin, the molar ratio of the crosslinking agent and the tetrameric hemoglobin can be between 0.8:1 to 20:1, respectively. In certain embodiments, the crosslinking agent and the tetrameric hemoglobin are present in a molar ratio between 0.8:1 to 19:1; 0.8:1 to 18:1; 0.8:1 to 17:1; 0.8:1 to 16:1; 0.8:1 to 15:1; 0.8:1 to 14:1; 0.8:1 to 13:1; 0.8:1 to 12:1; 0.8:1 to 11:1; 0.8:1 to 10:1; 0.8:1 to 9:1; 0.8:1 to 8:1; 0.8:1 to 7:1; 0.8:1 to 6:1; 0.8:1 to 5:1; 0.8:1 to 4:1; 0.8:1 to 3.5:1; 0.8:1 to 3:1; 0.8:1 to 2.5:1; 0.8:1 to 2:1; 0.8:1 to 1.5:1; 1:1 to 3:1; 1.1:1 to 3:1; 1.5:1 to 3:1; 2:1 to 3:1; or 2.25:1 to 2.75:1, respectively. In the step of contacting the crosslinking agent and the tetrameric hemoglobin, the concentration of the tetrameric hemoglobin can be between 5-25 g/dL. In certain embodiments, the concentration of the tetrameric hemoglobin in the step of contacting the crosslinking agent and the tetrameric hemoglobin can be between 5-20 g/dL; 10-20 g/dL; 10-18 g/dL; 10-16 g/dL; 10-15 g/dL; 11-15 g/dL; 12-15 g/dL; or 13-15 g/dL. The tetrameric hemoglobin can be reacted with the crosslinking agent in a polar protic solvent, such as in an aqueous solution. In certain embodiments, the crosslinking reaction takes place in water. In order to facilitate the crosslinking reaction, the pH of the reaction solvent can be maintained at a pH greater than 7. In certain embodiments, the pH of the crosslinking reaction solvent has a pH between 7-10; 8-10; 8.5 to 9.5; 8.7 to 9.3; or 8.9 to 9.1. The thus formed fumaryl-crosslinked hemoglobin can optionally purified using any method known to those skilled in the art, such as by filtration, heat-induced precipitation, centrifugation, chromatography, and the like. The presence of oxygen in the crosslinking reaction is also known to affect the p50 value of the resulting crosslinked hemoglobin. Depending on the oxygen content in the fumaryl crosslinking reaction, the p50 value of the resulting fumaryl-crosslinked hemoglobin can have a value ranging from 5-70 mmHg. In certain embodiments, the hemoglobin is crosslinked under oxygenated conditions, to give a fumaryl-crosslinked hemoglobin with a p50 value of 5-20 mmHg or 10-20 mmHg. In certain embodiments, the hemoglobin is crosslinked under deoxygenated conditions to give a fumaryl-crosslinked hemoglobin with a p50 value of 20-70 mmHg; 30-70 mmHg; 40-70 mmHg; 40-60 mmHg; 38-50 mmHg; 45-65 mmHg; or 55-65 mmHg. In instances in which the hemoglobin is first thio-blocked by reaction of the hemoglobin with iodoacetamide thereby forming a thio-blocked hemoglobin; crosslinking the thus formed thio-blocked hemoglobin with a fumaryl crosslinking agent thereby forming a fumaryl-crosslinked thio-blocked hemoglobin; and contacting the fumaryl-crosslinked thio-blocked hemoglobin with a thiol or a pharmaceutically acceptable salt or zwitterion thereof thereby forming a thiosuccinyl-crosslinked thio-blocked hemoglobin, the p50 value of the resulting thiosuccinyl-crosslinked thio-blocked hemoglobin crosslinked under deoxygenated conditions can range from 15-70 mmHg; 25-50 mmHg; or 35-50 mmHg, while the p50 value of the resulting thiosuccinyl-crosslinked thio-blocked hemoglobin and thiosuccinyl-crosslinked thio-blocked hemoglobin conjugate crosslinked under oxygenated conditions can range from 5-25 mmHg; 5-15 mmHg, 5-10 mmHg or 10-15 mmHg. The fumaryl-crosslinked hemoglobin can then reacted with the thiol thereby forming the thiosuccinyl-crosslinked hemoglobin. The thiol can be represented by the formula R1SH as defined in any embodiment described herein. The fumaryl-crosslinked hemoglobin can be present in the reaction with the thiol at a concentration between 5-20 g/dL. In certain embodiments, the fumaryl-crosslinked hemoglobin is present in the reaction with the thiol at a concentration between 5-18 g/dL; 5-16 g/dL; 5-14 g/dL; 5-12 g/dL; 7-12 g/dL; 8-12 g/dL; or 9-11 g/dL. The thiol can be present in the reaction with the fumaryl-crosslinked hemoglobin at a concentration between 1-500 mM. In certain embodiments, the thiol can be present in the reaction with the fumaryl-crosslinked hemoglobin at a concentration between 1-450 mM; 1-400 mM; 1-350 mM; 1-300 mM; 1-250 mM; 1-200 mM; 1-180 mM; 1-160 mM; 1-140 mM; 1-120 mM; 1-100 mM; 10-100 mM; 20-100 mM; 30-100 mM; 30-90 mM; 40-80 mM; 77.5-310 mM, 174-310 mM, 9.7-77.5 mM; 19.4-77.5 mM; or 38.8-77.5 mM. The reaction of the thiol and the fumaryl-crosslinked hemoglobin can be conducted at a pH between 7-11. In certain embodiments, the reaction of the thiol and the fumaryl-crosslinked hemoglobin is conducted at a pH between 7-11; 7-10; 7.4 to 10; 7.4 to 9, 7.4 to 8.2, or 8.2 to 9. The pH of the thiol addition reaction solvent can be maintained at the desired pH by use of pH buffer within the desired range or the addition of a Brønsted base to the reaction mixture, as needed. The selection of the appropriate Brønsted base or pH buffer is well within the skill of a person of ordinary skill in the art. Useful Brønsted bases include, but are not limited to Group I and Group II hydroxides, carbonates, and bicarbonates; organic amines, and the like. The fumaryl-crosslinked hemoglobin can be reacted with the thiol in a polar protic solvent, such as in an aqueous solution. In certain embodiments, the thiol addition reaction takes place in water. The reaction of the thiol with the fumaryl-crosslinked hemoglobin can generally conducted until all of the fumaryl-crosslinked hemoglobin starting material is converted to the desired thiosuccinyl-crosslinked hemoglobin, the fumaryl-crosslinked hemoglobin no longer is being converted to the desired thiosuccinyl-crosslinked hemoglobin, and/or the concentration of impurities and/or side products increases beyond a desired amount. Depending on the reaction conditions, the reaction of the thiol with the fumaryl-crosslinked hemoglobin can take between 1-72 hr; 6-72 hr, 12-72 hr, 24-72 hr, 36-72 hr, 48-72 hr, 60-72 hr, 12-48 hr, or 24-48 hr. In cases in which the rate of reaction of the thiol with the fumaryl-crosslinked hemoglobin is very slow (e.g., such as in the case of certain high molecular weight PEGylated thiols), the reaction of the thiol with the fumaryl-crosslinked hemoglobin can take up to one month. The thus formed thiosuccinyl-crosslinked hemoglobin can optionally purified using any method known to those skilled in the art, such as by filtration, heat-induced precipitation, centrifugation, chromatography, and the like. The reactive water-soluble polymer reagent can comprise any reactive functional group that is capable of covalently conjugating the water-soluble polymer and optionally the linker to hemoglobin. The reactive functional group can be any reactive functional group used for bioconjugation, such as succinimidyl ester, maleimide, 2-thiopyrridine, iodoacetamide, an arylpropionlonitrile, isocyanate, blocked isocyanate isothiocyanate, benzoyl fluoride, and the like. In certain embodiments, the reactive water-soluble polymer reagent is selected from the group consisting of: wherein A represents the water-soluble polymer; LG2is a leaving group; and p is a whole number between 1-20. In certain embodiments, p is a whole number between 1-18, 1-16, 1-14, 1-12, 1-10, 1-9, 2-10, 3-10, 2-9, 4-10, 5-9, 2-8, 2-6, 4-8, or 4-6. In exemplary embodiments, the linker is represented by the formula: A(CH2)p(C═O)LG2, wherein p is 1-10, 1-9, 2-10, 3-10, 2-9, 4-10, 5-9, 2-8, 2-6, 4-8, or 4-6. In certain embodiments, LG2is a leaving group selected from the group consisting of F, Cl, Br, I, In certain embodiments, the reactive water-soluble polymer reagent is wherein A is PEG; LG2is and p is 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-9, 2-10, 3-10, 2-9, 4-10, 5-9, 2-8, 2-6, 4-8, or 4-6. In the step of contacting the thiosuccinyl-crosslinked hemoglobin and the reactive water-soluble polymer reagent, the molar ratio of the reactive water-soluble polymer reagent and the thiosuccinyl-crosslinked hemoglobin are contacted in a molar ratio between 1:1-150:1, 1:1-100:1, 1:1-50:1, 1:1-40:1, 1:1-30:1, 5:1-30:1, 8:1-30:1, 5:1-25:1, 5:1-20:1, 10:1-20:1, 15:1-20:1, 16:1-20:1, 16:1-19:1, 16:1-18:1, respectively. The step of contacting the reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin can comprise combining a solution comprising the reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin or combining the neat reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin. The step of contacting the reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin can comprise combining one, two, three, four, or more portions of the reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin. The step of contacting the reactive water-soluble polymer reagent with the thiosuccinyl-crosslinked hemoglobin can comprise adding the reactive water-soluble polymer reagent to a solution comprising the thiosuccinyl-crosslinked hemoglobin or adding the thiosuccinyl-crosslinked hemoglobin to a solution comprising the reactive water-soluble polymer reagent. In instances in which a solution comprising the reactive water-soluble polymer reagent is combined with the thiosuccinyl-crosslinked hemoglobin, any solvent in which the reactive water-soluble polymer reagent is at least partially soluble can be used. The selection of the appropriate solution is well within the skill of a person of ordinary skill in the art. In certain embodiments, the solution is a phosphate buffered saline aqueous solution or a saline solution. The thiosuccinyl-crosslinked hemoglobin can be reacted with the reactive water-soluble polymer reagent in a polar protic solvent, such as in an aqueous solution. In certain embodiments, the reaction between the reactive water-soluble polymer reagent and the thiosuccinyl-crosslinked hemoglobin takes place in phosphate buffer saline aqueous solution. The thus formed thiosuccinyl-crosslinked hemoglobin conjugate can optionally purified using any method known to those skilled in the art, such as by filtration, heat-induced precipitation, centrifugation, chromatography, and the like. Advantageously, the p50 value of the thiosuccinyl-crosslinked hemoglobin conjugates described herein can be controlled by the reaction conditions under which the reactive water-soluble polymer reagent conjugation takes place. Depending on the oxygen content in the reactive water-soluble polymer reagent conjugation, the p50 value of the resulting thiosuccinyl-crosslinked hemoglobin conjugate can have a value ranging from 5-70 mmHg. In certain embodiments, the p50 value of the thiosuccinyl-crosslinked hemoglobin conjugate is 5-20 mmHg; 10-20 mmHg; 15-25 mmHg; 10-30 mmHg; 10-40 mmHg; 20-40 mmHg; 20-50 mmHg; 10-70 mmHg; 20-70 mmHg; 30-70 mmHg; 40-70 mmHg; 40-60 mmHg; 50-60 mmHg; 35-55 mmHg; 38-50 mmHg; 45-65 mmHg; 45-60 mmHg; or 55-65 mmHg. Surprisingly, if the reactive water-soluble polymer reagent conjugation of the thiosuccinyl-crosslinked hemoglobin occurs under deoxygenated conditions, the p50 value of the resulting thiosuccinyl-crosslinked hemoglobin conjugate can be substantially unchanged relative to the p50 value of the unconjugated thiosuccinyl-crosslinked hemoglobin starting material. In certain embodiments, when the reactive water-soluble polymer reagent conjugation of the thiosuccinyl-crosslinked hemoglobin occurs under deoxygenated conditions, the p50 value of the resulting thiosuccinyl-crosslinked hemoglobin conjugate can be within about ±10% or less, about ±9% or less, about ±8% or less, about ±7% or less, about ±6% or less, about ±5% or less, about ±4% or less, about ±3% or less, about ±2% or less, or about ±1% or less of the p50 value of the unconjugated thiosuccinyl-crosslinked hemoglobin starting material. In certain embodiments, the p50 value of the resulting thiosuccinyl-crosslinked hemoglobin conjugate can be substantially the same as the p50 value of the thiosuccinyl-crosslinked hemoglobin. In certain embodiments, the p50 value of the thiosuccinyl-crosslinked hemoglobin conjugate can be 5-70 mmHg; 10-70 mmHg; 20-70 mmHg; 30-70 mmHg; 40-70 mmHg; 40-60 mmHg; 35-55 mmHg; 38-50 mmHg; 45-65 mmHg; 45-60 mmHg; or 55-65 mmHg. If the reactive water-soluble polymer reagent conjugation of the thiosuccinyl-crosslinked hemoglobin occurs under oxygenated conditions, the resulting thiosuccinyl-crosslinked hemoglobin conjugate can have a p50 value about 10-20% less, about 15-20% less, about 12-18% less, or about 15% less than the p50 value of the thiosuccinyl-crosslinked hemoglobin. In certain embodiments, the p50 value of the thiosuccinyl-crosslinked hemoglobin conjugate can is 5-70 mmHg, 5-60 mmHg, 5-50 mmHg, 5-40 mmHg, 5-35 mmHg, be 5-30 mmHg, 10-70 mmHg, 10-60 mmHg, 10-50 mmHg, 10-40 mmHg, 10-35 mmHg, 5-30 mmHg, 5-20 mmHg, 15-25 mmHg, or 10-20 mmHg. The present disclosure also provides therapeutic methods of using the thiosuccinyl-crosslinked hemoglobin conjugate described herein. The thiosuccinyl-crosslinked hemoglobin conjugate can be used in any therapeutic methods that hemoglobin based oxygen carriers can be used. The present disclosure provides a method for increasing the volume of the blood circulatory system in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. In certain embodiments, the subject suffers from hemorrhagic shock. The present disclosure provides a method of supplying oxygen to the tissues and organs in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. In certain embodiments, the subject suffers from ischemia, including for example myocardial ischemia-reperfusion injury. The ischemia can be global or regional. The present disclosure provides a method of treating cancer in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. The thiosuccinyl-crosslinked hemoglobin conjugate can be administered alone or in combination with one or more cancer therapeutics and/or radiotherapy to treat cancer. In certain embodiments, the cancer is selected from the group consisting of leukemia, head and neck cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, nasopharyngeal cancer, esophageal cancer and brain cancer. In certain embodiments, the cancer is triple-negative breast cancer or colorectal cancer. The cancer therapeutic can be bortezomib, 5-fluorouracil, doxorubicin, or cisplatin. The present disclosure also provides a method of treating systemic lupus erythematosus in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. The present disclosure also provides a method of treating peripheral artery disease in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. The present disclosure also provides a method of treating traumatic brain injury in a subject in need thereof, wherein the method comprises transfusing into the system of the subject a therapeutically effective amount of the thiosuccinyl-crosslinked hemoglobin conjugate according to any embodiment or combination of embodiments described herein. EXAMPLES Example 1: Preparation of Pegylated Cysteinyl-succinyl Crosslinked Hemoglobin Conjugate An exemplary schematic flow diagram of the process of making pegylated cysteinyl-crosslinked hemoglobin is illustrated inFIG.1. The preparation steps included (1) generation of highly purified hemoglobin solution from bovine whole blood, (2) hemoglobin stabilization by crosslinking solution with bis(3,5-dibromosalicyl) fumarate (DBSF), (3) modification of fumaryl moieties in stabilized fumaryl-crosslinked hemoglobin by thiols, (4) pegylation of thiosuccinyl crosslinked hemoglobin with 5000 Molecular Weight (MW) PEG and (5) formulation of pegylated thiosuccinyl crosslinked hemoglobin with 0.05%-0.2% (w/v) NAC. In an exemplary embodiment, cysteine was used to modify the fumaryl moieties in stabilized fumaryl-crosslinked hemoglobin and pegylated cysteinyl-succinyl crosslinked hemoglobin was obtained after PEG conjugation. In brief, bovine whole blood collected from a slaughter house was processed, lysed and purified by ultrafiltration and column chromatography steps to produce highly purified hemoglobin solution. To prevent the dissociation of the hemoglobin into heterodimers, the tetrameric hemoglobin was stabilized by crosslinking reaction with DBSF. The residual DBSF and hydrolyzed derivatives, such as 3,5-dibromosalicylic acid (DBSA) were then removed by ultrafiltration. The stabilized hemoglobin crosslinked by fumaryl bridges (fumaryl-crosslinked hemoglobin) was then modified by cysteine through 1,4-addition reaction of thiol to the fumaryl moieties present in the fumaryl-crosslinked hemoglobin to give cysteinyl-succinyl crosslinked hemoglobin. Ultrafiltration purification step was then carried out to bring the cysteine and cystine levels to below 0.03% (w/w). In the pegylation step, surface-exposed lysine residues of the cysteinyl-succinyl crosslinked hemoglobin were conjugated with PEG chains through its reaction with PEG-NHS ester (MW 5000, named as PEG-5K-HS) in PBS for 2 hours. Subsequent quenching reaction and MetHb reduction step with cysteine for 16 hours provided pegylated cysteinyl-succinyl crosslinked hemoglobin with <5% MetHb. The solution containing the above-mentioned pegylated cysteinyl-succinyl crosslinked hemoglobin was further purified by ultrafiltration to achieve PEG and cysteine levels in the purified product below 0.2 mg/mL and 0.03% (w/w), respectively. The solution containing the purified pegylated cysteinyl-succinyl crosslinked hemoglobin was formulated with NAC at a concentration of 0.05% to 0.2% (w/v), to maintain low MetHb levels (<5%) throughout long-term storage. Example 2: Preparation of Highly Purified Bovine Hemoglobin Solution Blood cells were separated from whole bovine blood through centrifugation and the collected blood cells were subjected to a cell washing step (Lima, M. C., 2007, Artif Cells Blood Substit Immobil Biotechnol, 35(4):431-47). Methods for the isolation and purification of hemoglobin from blood cells described in the literature can be used to prepare the hemoglobin used in the current method (Houtchens, R. A. & Rausch, C. W., 2000, U.S. Pat. No. 6,150,507; Wong, B. L. & Kwok, S. Y, 2011, U.S. Pat. No. 7,989,593 B1). The residual amount of plasma was further removed from the collected blood cells by hollow fiber filtration step. A hypotonic solution was mixed with the washed blood cells to release the intracellular hemoglobin through a tightly controlled process. The cell debris were removed from cell lysate via a 0.2 μm filtration step and followed by additional ultrafiltration steps to partially remove the impurities to form a partially purified hemoglobin solution (PHS). To further purify the PHS, the PHS was buffer exchanged to contain minimal salt concentration prior to the negative mode anion column chromatography step. The flow through fraction containing highly purified PHS was collected for which the pH, tHb and salt concentration were adjusted, sterile filtered and stored at 2-8° C. prior to the downstream process. The highly purified hemoglobin solution ismycoplasmafree and contains very low levels of contaminants such as bovine plasma proteins (≤1 ppm), phospholipids (≤9.2 nM), residual bovine DNA (≤0.025 pg/μL) and endotoxin (≤0.1 EU/mL). Example 3: Preparation of Cysteinyl-Succinyl Crosslinked Hemoglobin Example 3A: Preparation of Fumaryl-Crosslinked Tetrameric Hemoglobin The highly purified hemoglobin solution was deoxygenated to less than 0.1 mg/L dissolved oxygen level in 0.9% (w/v) aqueous NaCl solution prior to the crosslink reaction. The crosslinking reaction was carried out by incubating the deoxygenated highly purified PHS (tHb=13-15 g/dL) with 2.5 molar equivalents of DBSF at pH 9.0 for a period of 4 hours at 10-30° C. under an inert atmosphere of nitrogen (dissolved oxygen level maintained at less than 0.1 mg/L). The deoxygenated environment maintains the hemoglobin molecules in tensed state for reaction and prevents oxidation of the hemoglobin, which results in the formation of MetHb. MetHb is physiologically inactive and doesn't carry oxygen. During the crosslinking reaction, the reaction pH was maintained by the addition of deoxygenated 0.1-0.5 M NaOH aqueous solution. The reaction mixture was then purified using tangential flow filtration (TFF) system with 30 kDa NMWCO membrane. The purification was completed after undergoing 10-16 diafiltration volume (DV). The concentration of the hemoglobin solution was maintained at 9.5-10.5 g/dL through a continuous feeding of acetate buffer (99 mM NaCl, 46 mM NaCH3COO, pH 8.2-8.4) into the reaction tank throughout the purification process. Example 3B: Preparation of Cysteinyl-Succinyl Crosslinked Hemoglobin The fumaryl moieties of the crosslinker bridges of the stabilized hemoglobin were modified by cysteine through 1,4-thiol-ene addition reaction. The reaction was carried out by the addition of 40-80 mM cysteine at pH 8.0-8.3 to fumaryl-crosslinked hemoglobin (tHb=7-10 g/dL) in acetate buffer (99 mM NaCl, 46 mM NaCH3COO, pH 8.2-8.4) for a period of 15-30 hours at 10-30° C. under deoxygenated conditions for which the dissolved oxygen levels maintained below 0.1 mg/L. After the reaction, the residual cysteine/cystine in the reaction mixture was removed by a filtration step using a 30 kDa NMWCO membrane. The concentration of the hemoglobin solution was maintained at 9.5-10.5 g/dL through a continuous feeding of acetate buffer (99 mM NaCl, 46 mM NaCH3COO, pH 8.2-8.4) into the reaction tank. After going through 10-16 diafiltration volume (DV), the cysteine/cystine levels in the cysteinyl-succinyl crosslinked hemoglobin solution were found below 0.03% (w/w). After cysteine modification, up to 95% of the fumaryl-crosslinked hemoglobin was modified to give cysteinyl-succinyl crosslinked hemoglobin with relatively low oxygen-carrying properties. Example 4: Preparation of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Example 4A: Optimization of Pegylation Conditions A 5 kDa PEG chain equipped with NHS ester group (including hexanoate NHS ester (PEG-5K-HS; CH3O(CH2CH2O)n(CH2)5COONHS) or acetate NHS ester (PEG-5K-AS; CH3O(CH2CH2O)n(CH2)COONHS) were used as model pegylation reagents to study the effect of various reaction parameters, including reactant equivalents, reaction time, spacer length, reaction medium and reaction atmosphere, on the pegylation efficiency of cysteinyl-succinyl crosslinked hemoglobin. i) Effects of PEG Equivalent and Reaction Time The effects of reactant equivalents and reaction time were investigated using PEG-5K-HS. After the conjugation reaction of PEG-5K-HS (9 and 17 equivalents) with cysteinyl-succinyl crosslinked hemoglobin in phosphate buffer saline (PBS; 0.9 w/v % NaCl, 0.1 M sodium phosphate, pH=7.7) under deoxygenated condition for 2 hours, additional protein bands with increased molecular weight were visualized on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel by both iodine and Coomassie stain, as shown inFIG.2. This suggests the successful conjugation of PEG side chains onto the cysteinyl-succinyl crosslinked hemoglobin. Upon increasing the reaction time from 2 to 4 hours, there was no substantial change of the protein band intensity. A noticeable amount of unpegylated hemoglobin was found in the reaction mixture with the 9 equivalents reaction, as shown from the protein band at ca. 15 kDa inFIG.2a. In contrast, the increase of PEG equivalent from 9 to 17 prominently enhanced the conjugation efficiency as indicated by the absence of unpegylated hemoglobin band (ca. 15 kDa) and concomitant increase in band intensity at higher molecular weight, as shown inFIG.2b. These results were in agreement with the dynamic light scattering results showing that the increase of PEG equivalent from 9 to 17 increased the average hydrodynamic diameter of the pegylated hemoglobin from 12.36 to 14.27 nm. Under both conditions, the average hydrodynamic diameter of the resulting pegylated hemoglobin was almost doubled when compared to unpegylated cysteinyl-succinyl crosslinked hemoglobin (6.35 nm). ii) Effects of PEG Spacer Length and Reaction Medium The effect of PEG spacer length on the pegylation efficiency was investigated using PEG-5K-HS and PEG-5K-AS. The SDS-PAGE analysis of the pegylation of cysteinyl-succinyl crosslinked hemoglobin with PEG-5K-HS and PEG-5K-AS is shown inFIG.3, respectively. Similar to the study with PEG equivalents and reaction time, PEG-5K-HS effectively attached to the cysteinyl-succinyl crosslinked hemoglobin with full consumption of monomeric hemoglobin chains, as shown inFIG.3a. Under similar reaction conditions, limited pegylation was observed for the reaction with PEG-5K-AS and most of the crosslinked hemoglobin remained in the unpegylated form, as shown inFIG.3b. Without wishing to be bound by thereof, it is believed that the difference can be ascribed to the increased hydrolysis rate of the NHS ester with a shorter spacer unit, leading to the deactivation of NHS under aqueous conditions, hence the loss of its conjugation ability toward the primary amine of lysine residue for conjugation. In addition, the effect of reaction medium on the conjugation efficiency was also investigated by changing the buffer component from phosphate to borate. The pegylation efficiency of cysteinyl-succinyl crosslinked hemoglobin by PEG-5K-HS in borate buffer was found to be reduced, as indicated by the increased band intensity of unpegylated hemoglobin in SDS-PAGE, as shown inFIG.3a. Nevertheless, compared to spacer length and reactant equivalents, the effect of reaction medium towards the pegylation efficiency was relatively minimal. iii) Effect of Reaction Atmosphere Hemoglobin has strong binding affinity toward oxygen and the binding of oxygen results in the conformation of relaxed state (R-state, oxygenated conditions), which may expose different subset of surface amino acids when compared to the tense state (T-state, deoxygenated conditions) in the absence of oxygen. As NHS ester mainly reacts with primary amine on the protein surface, different degree and sites of pegylation may be expected from the reactions carrying out under R- and T-state of hemoglobin, respectively. In order to examine the effect of hemoglobin state on the pegylation efficiency, the reaction shown in Example 4A (i) was repeated under oxygenated conditions. As shown inFIG.4, similar SDS-PAGE patterns were obtained in the pegylation reaction of cysteinyl-succinyl crosslinked hemoglobin under oxygenated conditions, compared with those carried out under deoxygenated conditions, as shown inFIG.2. In general, the degree of pegylation followed a PEG-concentration dependent manner that conjugates with higher molecular weight were formed in the reaction with higher PEG equivalents, either from the reaction carried out under R- or T-state of hemoglobin. Although the efficiency of pegylation reaction under oxygenated conditions was comparable to that under deoxygenated conditions, the pegylation reaction under deoxygenated environment yielded better results. This can be attributed to the increase in MetHb levels in the reaction product, which increased from 9.1% to 22.5% after conjugation reaction with 17 equivalents of PEG-5K-HS under oxygenated conditions for 2 hours while that under deoxygenated conditions was only 15.9%, as shown in Table 1. TABLE 1The Change of MetHb levels in Different Reaction Steps (HS9 andHS17 = Reactions with 9 and 17 Equivalent of PEG-5K-HS,respectively).PEGEquiv-MetHbO2HbConditionsalentReaction Step[%][%]Cysteinyl-succinyl9.10.7Crosslinked HemoglobinDe-HS9Pegylation, 2 hours12.30.4oxygenatedCysteine Reduction,7.01.116 hoursHS17Pegylation, 2 hours15.90.0Cysteine Reduction,5.80.916 hoursOxygenatedHS9Pegylation, 2 hours17.874.2Cysteine Reduction,12.01.516 hoursHS17Pegylation, 2 hours22.569.5Cysteine Reduction,12.82.016 hours Nevertheless, subsequent introduction of cysteine for reaction quenching reduced the MetHb in the pegylated crosslinked hemoglobin solution to a lower level (oxygenated conditions: 12.8% and deoxygenated conditions: 5.8%, Table 1). Consequently, deoxygenated conditions were maintained throughout the production process to eliminate the repetitive oxygenation and deoxygenation steps in the production process for cost reduction and minimize MetHb impurities for quality enhancement. Example 4B: Pegylation of Cysteinyl-Succinyl Crosslinked Hemoglobin The conditions used in the pegylation of cysteinyl-succinyl crosslinked hemoglobin prepared in Example 3 are shown inFIG.5. Once the purified pegylated cysteinyl-succinyl crosslinked hemoglobin was prepared, the composition containing 4.5-5.5 g/dL pegylated cysteinyl-succinyl crosslinked hemoglobin was formulated with NAC with a final concentration of 0.05% to 0.2% (w/v) NAC. In pegylation reaction, PEG-5K-HS (17 equivalents with respect to molar amount of hemoglobin) was dissolved in deoxygenated 0.1 M PBS at pH 7.7 and immediately added into an equal volume of a solution containing cysteinyl-succinyl crosslinked hemoglobin (hemoglobin content=9.0 g/dL, RA-buffer at pH 7.7) for conjugation. After reaction for 2 hours, a reducing reagent (77.5 mM cysteine) was immediately added to the hemoglobin mixture and incubated for 16-18 hours. Apart from its reducing properties, cysteine also acts as a reaction quencher to stop the pegylation reaction by reacting with residual PEG-5K-HS. Therefore, cysteine functions not only as a reducing agent to convert the non-functional MetHb to functional hemoglobin, but also as a reaction quencher to tightly control the pegylation process for enhancing the product and process consistency. Example 4C: Purification of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin After reaction with cysteine, the pegylated cysteinyl-succinyl crosslinked hemoglobin was purified through TFF using 30 kDa NMWCO membrane. Residual PEG was found to flow through the membrane and removed from the pegylated cysteinyl-succinyl crosslinked hemoglobin reaction mixture as undetectable level of residual PEG was revealed by SDS-PAGE, as shown inFIG.6. After 12 DV, purified pegylated cysteinyl-succinyl crosslinked hemoglobin with free PEG and cysteine level of the mixture below 0.2 mg/mL and 0.03% (w/w), respectively, was obtained. To maintain the low MetHb level (<5%) throughout storage, NAC at a concentration of 0.05% to 0.2% (w/v) was added to the solution containing the above-mentioned purified pegylated cysteinyl-succinyl crosslinked hemoglobin. Example 5: Purity of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Composition Example 5A: Quantification of Free Hemoglobin in Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The amount of free hemoglobin in the pegylated cysteinyl-succinyl crosslinked hemoglobin was quantified using size-exclusion chromatography (Yarra™ 3 μm SEC-2000, LC Column 300×7.8 mm) with phosphate buffer (20 mM sodium phosphate, 0.9% NaCl, pH 6.8) as mobile phase. The proteins eluting from the column were monitored by the UV-absorption signal at 220 nm. As shown inFIG.7, cysteinyl-succinyl crosslinked hemoglobin displayed two elution peaks corresponding to a majority of stabilized tetrameric hemoglobin (retention time: 7.20 min) with small amount of stabilized octameric hemoglobin (retention time: 6.35 min). After pegylation, the peak retention time shifted to 5.17 min with concomitant disappearance of the peaks eluted at 6.35 and 7.20 min for the cysteinyl-succinyl crosslinked hemoglobin. This suggests that the size of the crosslinked hemoglobin molecules is increased by the conjugation of PEG on the hemoglobin and at least 95% of hemoglobin was found to be covalently attached with PEG chains after conjugation process. Example 5B: Determination of Residual PEG in the Composition of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin After pegylation, the pegylated cysteinyl-succinyl crosslinked hemoglobin solution containing excess free PEG was further purified by TFF equipped with 30 kDa NMWCO membrane. To evaluate the residual amount of PEG, which is considered a process-related impurity, the same methodology for the estimation of the average PEG side chain per cysteinyl-succinyl crosslinked hemoglobin molecule was deployed. It was found that the pegylated cysteinyl-succinyl crosslinked hemoglobin solution contained 0.088±0.051 mg/mL residual PEG. Example 6: Characterization of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The properties of pegylated cysteinyl-succinyl crosslinked hemoglobin were analyzed by different biochemical methods, as shown below. Example 6A: Size-Exclusion Chromatography of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Versus Cysteinyl-Succinyl Crosslinked Hemoglobin The average PEG side chain per cysteinyl-succinyl crosslinked hemoglobin molecule and the molecular weight of pegylated cysteinyl-succinyl crosslinked hemoglobin were measured by running a reverse phase column on an Ultra Performance Liquid Chromatography (UPLC) coupled with Evaporative Light Scattering Detector (ELSD). Briefly, an Acquity™ UPLC Peptide BEH C18 column (2.1 mm×150 mm) was used to quantify the amount of free PEG in the sample. The average PEG side chain per cysteinyl-succinyl crosslinked hemoglobin molecule was calculated by subtracting the remaining amount of free PEG after 2 hours reaction from the initial PEG addition. Subsequently, the estimated molecular weight of pegylated cysteinyl-succinyl crosslinked hemoglobin was calculated by multiplying the average number of PEG side chains to the molecular weight of PEG-5K-HS (5000 Da). After the pegylation process, the average PEG side chain per cysteinyl-succinyl crosslinked hemoglobin molecule was found to be 13.22±0.72, giving an estimated molecular weight for the pegylated cysteinyl-succinyl crosslinked hemoglobin as 131±3 kDa (compared to 65 kDa for cysteinyl-succinyl crosslinked hemoglobin). The result clearly indicates that the pegylation process increased the molecular weight of the cysteinyl-succinyl crosslinked hemoglobin. Example 6B: Light Scattering and SDS-PAGE Analysis of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Versus Cysteinyl-Succinyl Crosslinked Hemoglobin The size of pegylated cysteinyl-succinyl crosslinked hemoglobin was also studied by measuring the hydrodynamic diameter using light scattering. Briefly, the non-invasive back scattering at 1730 was measured for the sample at 25° C. The hydrodynamic diameter increased from 6.52±0.18 nm for cysteinyl-succinyl crosslinked hemoglobin to 13.98±0.21 nm for the pegylated cysteinyl-succinyl crosslinked hemoglobin. In addition, SDS-PAGE analysis was also performed under reducing condition, as shown inFIG.8. It is seen that the band intensity at higher molecular weight was increased when comparing pegylated cysteinyl-succinyl crosslinked hemoglobin to the unpegylated cysteinyl-succinyl crosslinked hemoglobin, which is in agreement and further supports the above findings. Example 6C: In-Vitro Stability of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The stability, in terms of auto-oxidation rate (MetHb formation), for the pegylated cysteinyl-succinyl crosslinked hemoglobin, was evaluated. The initial linear formation rate of MetHb was calculated for both pegylated cysteinyl-succinyl crosslinked hemoglobin and cysteinyl-succinyl crosslinked hemoglobin, respectively. Briefly, the absorbance at 560, 576 and 630 nm were recorded every 15 minutes for 3 hours at 30° C. using a spectrometer. The following equation was used for calculating the amount of MetHb: [MetHb]=(2.6828A630−0.174A576−0.3414A560)*10−4Mol The amount of MetHb was plotted against time, and the slope on a linear curve fit for the first 3 hours of MetHb change was calculated. The results show that the auto-oxidation rate of pegylated cysteinyl-succinyl crosslinked hemoglobin (4.69±0.43 Met %/hr) is slightly higher than that of unpegylated cysteinyl-succinyl crosslinked hemoglobin (3.44±0.56 Met %/hr). Example 6D: Oxygen Affinity Properties of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The oxygen affinity properties of hemoglobin can be described by its p50 value, where the fraction of hemoglobin saturated with oxygen (O2) is plotted against a range of partial pressure O2(pO2). p50 is defined as the O2partial pressure where 50% of the hemoglobin is saturated with O2, and is often used as a descriptor of oxygen affinity. The oxygen dissociation curve for the cysteinyl-succinyl crosslinked hemoglobin and pegylated cysteinyl-succinyl crosslinked hemoglobin solution were obtained using a Hemox analyzer (TCS Scientific, New Hope, Pa.), as shown inFIG.9. Oxygen tension was measured with a Clark oxygen electrode, and the hemoglobin saturation was measured using a built-in dual wavelength spectrophotometer. The measurement was carried out in Hemox solution (135 mM NaCl, 5 mM KCl and 30 mM TES, pH 7.4) with a final hemoglobin concentration of 0.05 g/dL and the temperature maintained at 37° C. throughout the measurement. A computer-based analysis of oxygen dissociation curve was performed yielding p50 for oxygen binding. Oxygen dissociation parameters were further derived by fitting the Adair equations to each oxygen dissociation curve by nonlinear least-squares procedure included in the Hemox analyzer software (TCS Hemox DAQ System, Version 2.0). The Adair p50 for cysteinyl-succinyl crosslinked hemoglobin and pegylated cysteinyl-succinyl-crosslinked hemoglobin is 56.40±8.12 mmHg and 53.50±8.91 mmHg, respectively. This indicates that the conditions used in the pegylation process surprisingly do not alter the oxygen affinity of the cysteinyl-succinyl crosslinked hemoglobin molecule. It is worth mentioning that the pegylation of hemoglobin normally results in a change of oxygen binding affinity compared with its unmodified counterpart. For example, the p50 value of a PEGylated hemoglobin product Sanguinate™ is reported to be 9-14 mmHg (Abuchowski, A. et. al., 2017, US Patent 20170072023 A1), which is significantly lower than that of its parent bovine hemoglobin (24-26 mmHg). Since the use of pegylated hemoglobin as an oxygen-carrying therapeutic is undoubtedly related to its oxygen-offloading ability, the pegylation strategy described herein on the one hand can provide improved physiochemical and pharmacokinetics profiles properties to pegylated cysteinyl-succinyl crosslinked hemoglobin. On the other hand, the oxygen-binding affinity of the cysteinyl-succinyl crosslinked hemoglobin was found comparable even after pegylation and thus its therapeutic efficacy for different indications can be retained after pegylation. In this way, the p50 value of the pegylated cysteinyl-succinyl crosslinked hemoglobin can be controlled by the p50 value of the cysteinyl-succinyl crosslinked hemoglobin. To date, there are limited/no examples of pegylated hemoglobin molecules with high p50 values, particularly in the range of 30-65 mmHg. The high oxygen-offloading ability of the pegylated cysteinyl-succinyl crosslinked hemoglobin renders it as an efficient oxygen delivery agent in vivo and critical to certain clinical applications which require rapid and efficient tissue oxygen supply. Example 6E: Aggregation Properties of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin To understand the role of pegylation on aggregation properties of cysteinyl-succinyl crosslinked hemoglobin, the levels of aggregates ranging from 1-10 μm in size were measured in pegylated and unpegylated crosslinked hemoglobin by Multisizer 4e, respectively. In brief, the hemoglobin samples were taken out from the container and diluted with Isoton II Diluent at 1:1 ratio (10 mL: 10 mL) with gentle mixing. Two separate samples were prepared form each hemoglobin sample for triplicate measurement using 50 μm aperture tube, according to the instruction manual. The levels of aggregates, in term of particulate counts of 1-1.66 μm, 1.66-5 μm and 5-10 μm were added up, for comparison. The level of aggregates in the hemoglobin samples from 2 separate batches were measured, as shown inFIG.10. The results revealed that there was a 6 to 15-fold decrease in aggregation level in cysteinyl-succinyl crosslinked hemoglobin after pegylation, implying that the aggregation quality is significantly enhanced in the pegylated cysteinyl-succinyl crosslinked hemoglobin, compared to the unpegylated ones. Example 7: Impact of Different Polyethylene Glycol (PEG) Chain Lengths, Spacer-Arm Lengths and Pegylation Reaction Conditions on the Physiological Properties of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin As shown from Example 6A and 6B, the pegylation of cysteinyl-succinyl crosslinked hemoglobin using PEG-5K-HS under the presented reaction conditions resulted in a significant increase of molecular weight and hydrodynamic diameter, while the oxygen affinity of the hemoglobin remained unchanged. The following study was conducted to examine the impact on pegylation efficiency, molecular weight, hydrodynamic diameter, colloid osmotic pressure (COP) and oxygen-binding affinity (p50 values), imposed by using various chain lengths, spacer-arm lengths and molar equivalents of the PEG-NHS esters in the production of pegylated cysteinyl-succinyl crosslinked hemoglobin. Compared to the Example 4A, the focus of this study is to investigate the structural effects of PEG reagents on the properties of the resulting pegylated cysteinyl-succinyl crosslinked hemoglobin. With increased understanding about the effects of various reagents on the physiochemical properties of the pegylated cysteinyl-succinyl crosslinked hemoglobin, the developed pegylation process can potentially function as a mean to customize the properties of a pegylated hemoglobin product to fit specific pharmaceutical needs. An illustration of the PEG-NHS esters examined in this study was shown inFIG.11. Example 7A: Varying PEG-Chain Length to Control the Hydrodynamic Diameter The workflow of this study is similar to that shown inFIG.5. In general, PEG chains equipped with a hexanoate NHS ester (PEG-HS) with chain lengths of 1, 2, 5 and 10 kDa were dissolved in PBS (0.9 w/v % NaCl, 0.1 M sodium phosphate, pH=7.7, [PEG]=23.7 mM) and added to a freshly prepared cysteinyl-succinyl crosslinked hemoglobin (tHb=9.0 g/dL; 1.4 mM) under nitrogen atmosphere. Upon mixing of the two, the reaction mixture consisted of 4.5 g/dL of the hemoglobin with 17 molar equivalents of PEG-NHS ester and the reaction was allowed to proceed for 2 hours before quenching with 77.5 mM cysteine. Pegylation efficiency of the reaction was examined using reverse-phase UPLC-ELSD method, after which the reaction mixture was purified by ultrafiltration (MWCO=30 kDa) and subsequently formulated with 0.2% (w/v) NAC. The pegylated crosslinked hemoglobin was stored at 4° C. prior to characterization. As reflected from UPLC-ELSD analysis, comparable pegylation efficiency was found across all the PEG chain lengths examined (1-10 kDa) as reflected from the similar PEG conjugation numbers (PEG:Hb=13.0-13.6) and conjugation yield (76-80%) in their reaction with cysteinyl-succinyl crosslinked hemoglobin, as shown in Table 2. In contrast, the molecular weight and hydrodynamic diameter of the pegylated crosslinked hemoglobin increased by approximately one-fold as chain length increased from 1 to 10 kDa (77 and 197 kDa; and 9.56 and 17.30 nm, respectively) and the data recorded from samples with 2 and 5 kDa chains aligned with the trends. Notably, the variation of PEG chain length imposed negligible effect toward the oxygen binding affinity of the resulting pegylated crosslinked hemoglobin, in term of Adair's p50. All these results fell within expectations as the pegylation process modified the surface exposed lysine residues or specifically primary amine from the hemoglobin. Increasing the polymer chain length would concomitantly increase the molecular weight and hydrodynamic diameter of the crosslinked hemoglobin, but the oxygen binding sites, which are present at the core of protein structures, remained unaltered. TABLE 2A Summary of Physical Properties and p50 Values of Cysteinyl-succinyl Crosslinked Hemoglobin Pegylated with PEG-NHS withDifferent Chain Lengths (1K, 2K, 5K and 10K).AdairPegylationMWHydrodynamicp50PEG/HbYield [%][kDa]Diameter [nm][mmHg]XLHbaNANA646.52 ± 0.1855.9PEG-1K-HS13.076779.56 ± 0.0755.1PEG-2K-HS13.680929.95 ± 0.0655.6PEG-5K-HS13.47913113.57 ± 0.0852.6PEG-10K-HS13.37819717.30 ± 0.0852.1acysteinyl-succinyl crosslinked hemoglobin; NA—Not Applicable A similar study was carried out upon further expanding the PEG chain length from 10 to 40 kDa. The reactions were carried out by mixing equal volume of PEG-NHS ester ([PEG-HS]=118.6 mg/mL) and cysteinyl-succinyl crosslinked hemoglobin ([Hb]=9.0 g/dL). After reaction for 2 hours, the mixture was purified with Q-column using 50 mM Tris buffer (pH 9) with increasing NaCl content (0-0.3 M) as the elution buffer. The fractions contained pegylated cysteinyl-succinyl crosslinked hemoglobin were eluted and characterized. As shown in Table 3, although PEG-40K-HS can effectively attach to the hemoglobin with slightly lower yield, no significant difference was found for the hydrodynamic diameter of the resulting pegylated crosslinked hemoglobin. Nevertheless, these results clearly demonstrated that PEG-NHS ester with different chain lengths (from 1 kDa-40 kDa) can be effectively attached to the hemoglobin under the conditions examined. By the use of PEG-NHS ester with different PEG chain lengths, the hydrodynamic diameter of the pegylated hemoglobin can be selectively controlled and customized. TABLE 3A Summary of Physical Properties and p50 Values of Cysteinyl-succinyl Crosslinked Hemoglobin Pegylated with PEG-NHSwith Different Chain Lengths (10K, 20K and 40K).PegylationHydrodynamicAdairPEGYieldMWDiameterp50EquivalentPEG/Hb[%][kDa][nm][mmHg]XLHbaNANANA646.52 ± 0.1855.9PEG-10K-HS8.56.47612914.74 ± 0.0153.5PEG-20K-HS4.32.86612114.83 ± 0.0353.2PEG-40K-HS2.11.36311813.0 ± 0.0256.0acysteinyl-succinyl crosslinked hemoglobin;NA—Not Applicable Example 7B: Varying Reaction Conditions to Control the COP Values This study aims to establish a possible correlation between reaction equivalents of PEG and the COP values of the resulting pegylated crosslinked hemoglobin. In this study, pegylation reaction was conducted in a similar fashion to that shown inFIG.5and the cysteinyl-succinyl crosslinked hemoglobin samples were allowed to react with PEG-5K-HS under different reaction equivalents (3, 9, 13, 17 and 25). The pegylation efficiency was examined using reverse-phase UPLC-ELSD method and the COP value was measured via a colloid osmometer (OSMOMAT 050). As shown in Table 4, an increase in reaction equivalents from 3 to 25 was directly reflected by a 9-fold increase of their corresponding product PEG:Hb ratios from 2.2 to 18.5. Notably, the pegylation efficiency across all conditions examined was found to be around 75%, suggesting that the reaction equivalent is not a determining factor toward the conjugation yield in the concentration range of PEG examined. With increasing the molar equivalents of PEG, it is expected that the conjugation number can be further increased. The COP of the resulting pegylated crosslinked hemoglobin are shown in Table 4. Generally, all samples with concentration of 4.5 g/dL displayed high COP values which exceeded the measurement limit of OSMOMAT 050 (i.e. 73.5 mmHg). Therefore, the samples were one-fold diluted with 0.9% NaCl and the measurement was repeated. The result revealed that the COP values of the diluted hemoglobin samples at 2.25 g/dL increased in a linear fashion when the PEG:Hb ratio is increased, as shown in Table 4 andFIG.12. This result suggests that the COP values of pegylated crosslinked hemoglobin are mainly dependent on the number of PEG chains attached. TABLE 4A Summary of COP Values of Pegylated Cysteinyl-succinyl CrosslinkedHemoglobin with Different Reaction Molar Equivalent of PEG-5K-HS.COPCOPPEGPegylation[mmHg] @[mmHg] @EquivalentPEG:HbYield [%]4.5 g/dL2.25 g/dLXLHbaNANA17.77.932.27325.39.296.77473.5b17.61310.27873.5b20.71713.47873.5b26.32518.57473.5b33.8acysteinyl-succinyl crosslinked hemoglobin;bexceed the detection limit of the osmometer. Apart from controlling the COP value by adjusting the number of PEG chains, the COP value of the pegylated crosslinked hemoglobin was found to be regulated by varying the PEG chain length. As shown in Table 5, the attachment of short PEG chains (1 and 2 kDa) did not give rise to a significant increase to the COP values of the resulting pegylated hemoglobin. A substantial increase of the COP value of the pegylated hemoglobin has only been found upon attachment of longer PEG chains (5 and 10 kDa). In general, the COP value of pegylated hemoglobin is positively correlated to the chain length of PEG chain attached. For example, increasing the PEG chain length from 2K to 10K brought a 8-fold enhancement of the COP value from 5.5 to 43.8 mmHg (tHb=1.5 g/dL). All these results suggest that with a tight control of reaction equivalent and PEG chain length TABLE 5A Summary of COP Values of Cysteinyl-succinyl CrosslinkedHemoglobin Pegylated with PEG-NHS with Different ChainLengths (1K, 2K, 5K and 10K, 17 molar equivalents) withDifferent Chain Lengths.COPCOPCOP[mmHg][mmHg][mmHg]Pegylation@ 4.5@ 2.25@ 1.5PEG:HbYield [%]g/dLg/dLg/dLXLHbaNANA17.77.95.2PEG-1K-12.77521.88.45.1HSPEG-2K-13.68026.59.55.5HSPEG-5K-13.47973.5b29.48.3HSPEG-10K-13.37873.5b73.5b43.8HSacysteinyl-succinyl crosslinked hemoglobin;bexceeded the detection limit of the osmometer. Example 7C: Varying Reaction Atmosphere to Control the p50 Value Hemoglobin generally occurs in two different states, the relaxed state (R-state, oxygenated conditions), when bound to oxygen, and the tense state (T-state, deoxygenated conditions), in the absence of oxygen. As NHS ester mainly reacts with primary amines on the protein surface, different degree and sites of pegylation may be expected from the reactions carrying out under the R- and T-states of hemoglobin. In order to examine the effect of hemoglobin state on the pegylation efficiency and the properties of the resultant pegylated crosslinked hemoglobin, reactions using 17 equivalents of PEG-5K-HS were performed under oxygenated conditions using cysteinyl-succinyl crosslinked hemoglobin with two different starting p50 levels (high and low p50 values; 56 and 20 mmHg, respectively), and the results were compared with those carried out under deoxygenated conditions. In general, the pegylation efficiency and the resultant hydrodynamic diameters for the reactions performed under oxygenated conditions were comparable to those performed under deoxygenated conditions (approximately 11-12 PEG/Hb and a hydrodynamic diameter of approximately 13-14 nm, Table 6). In contrast, the reaction atmosphere imposed a notable difference toward the oxygen binding affinity of the pegylated crosslinked hemoglobin. The results revealed that the p50 value of the pegylated cysteinyl-succinyl crosslinked hemoglobin when pegylation performed under oxygenated conditions was found to be approximately 15% lower than the corresponding unpegylated crosslinked hemoglobin (decreased from 55.9 mmHg to 42.2 mmHg for the hemoglobin with high p50 value; and from 19.5 mmHg to 15.2 mmHg for the hemoglobin with low p50 value), whereas the p50 value of the pegylated crosslinked hemoglobin when pegylation performed under deoxygenated conditions remained unchanged, as shown in Table 6. Given that the crosslink reaction is carried out in deoxygenated conditions, pegylation under deoxygenated conditions is a critical factor to maintain an unchanged oxygen affinity, regardless of the starting oxygen affinity of the unpegylated hemoglobin. In contrast, pegylation under oxygenated condition provides a mean to alter the oxygen affinity of the hemoglobin. TABLE 6Properties of Cysteinyl-succinyl Crosslinked Hemoglobinunder Pegylation at Different Reaction Atmosphere.StartingAdairO2Hb atHydrodynamicAdairPegylationp50PegylationMWDiameterp50Condition[mmHg][%]PEG/Hb[kDa][nm][mmHg]Deoxygenated55.9 ± 0.2−0.411.612314.4 ± 0.154.4 ± 0.1Oxygenated87.512.311613.7 ± 0.142.2 ± 1.2Deoxygenated19.5 ± 0.36.111.712314.1 ± 0.219.7 ± 1.1Oxygenated82.412.012412.8 ± 0.115.2 ± 0.2 Example 7D: The Reaction Studies of PEG Reagents with Variation in their Spacer-Arm Length The pegylation efficiency on using PEG with different spacer-arm lengths, including PEG-5K-AS (acetate NHS ester), PEG-5K-PS (propionate NHS ester) PEG-5K-HS (hexanoate NHS ester) and PEG-5K-DCS (decanoate NHS ester), in the reaction with 17 equivalents, were examined. As shown in Table 7, using reaction conditions as shown for PEG-5K-HS, similar pegylation efficiency and resultant hydrodynamic diameter were observed when cysteinyl-succinyl crosslinked hemoglobin was conjugated by PEG-5K-PS and PEG-5K-DCS (12.6 and 13.5 PEG/Hb with the yield >70%; hydrodynamic diameter=13.2 and 13.1 nm, respectively), whereas limited pegylation was observed for the reaction using PEG-5K-AS (2.5 PEG/Hb with 15% yield; hydrodynamic diameter=7.8 nm). Without wishing to be bound by theory, it is believed that this difference can be ascribed to the increased hydrolysis rate of the NHS ester with a shorter spacer-arm unit, leading to the deactivation of NHS under aqueous conditions and hence the loss of its conjugation ability toward the primary amine of lysine residues. Regardless of the PEG reagents with different spacer-arm length used, there was no obvious difference for the p50 values of all pegylated hemoglobin, suggesting that the conjugation of PEG chains with different spacer-arm length would also not affect the oxygen binding of the heme group and thus not result in a change of p50 value. In contrast, the spacer-arm length had prominent effects on the stability and reactivity of the reagents and affected the conjugation efficiency of the pegylation reaction. Studies concerning the hydrolysis rate of different PEG reagents and the reaction optimization for PEG-5K-AS have been conducted and the results were shown in Example 8A and 8C, respectively. TABLE 7Properties of Cysteinyl-succinyl Crosslinked Hemoglobin Pegylated withPEG with Different Spacer-arm Lengths.PegylationMWHydrodynamicAdair p50PEG/HbYield [%][kDa]Diameter [nm][mmHg]XLHbaNANA646.52 ± 0.1855.9PEG-5K-AS2.515777.77 ± 0.0750.4PEG-5K-PS12.67412813.16 ± 0.0255.5PEG-5K-HS13.57913212.64 ± 0.0852.6PEG-5K-DCS12.97612913.10 ± 0.0250.5acysteinyl-succinyl crosslinked hemoglobin; NA—Not Applicable Example 8: Further Optimization of Pegylation Reaction with Different PEG-NHS Esters In the above examples, the pegylation reactions with PEG-5K-HS were performed in a relatively small reaction scale (<4 L). However, depending on the chemical properties of PEG-NHS ester, the reaction conditions could be optimized to achieve maximum pegylation yield for cost reduction. In the following examples, the reactivity and stability of various PEG reagents in pegylation reaction were examined. With an understanding of the chemical properties of the PEG reagents, different optimization processes were developed as shown below. Example 8A: Studying the Hydrolysis Rate of PEG-NHS Ester with Different Spacer-Arm Lengths This study aims to study the hydrolysis rate of PEG-NHS ester with varying spacer-arm lengths via UPLC-ELSD analysis. PEG-NHS ester losses its conjugation ability upon hydrolysis in aqueous solution to give non-reactive carboxyl-PEG (Lim, C. Y, 2014, Langmuir, 30:12868-78). Given that the retention time of active PEGs is different compared with their hydrolyzed counterparts in UPLC-ELSD analysis, monitoring the relative proportion of the active PEG-NHS ester over time gives a depiction of PEG degradation progression, thus allowing the measurement of the hydrolysis half-life of PEG-NHS ester under various reaction conditions. In this study, four species with fixed PEG chain length (5 kDa), but with acetyl-, pentyl-, hexyl- and decanoyl-carbon chains as their respective spacer-arms, were investigated. Each type of PEG-NHS ester was dissolved in 0.1 M PBS and in 0.9% NaCl in separate trials. Samples were collected every 30 minutes for a total of 5 hours, and the amount of active and inactive form of PEG was determined by the peak area of each corresponding species in the chromatograms.FIG.13shows a representative elution profile of PEG-5K-HS as an example. Peak area of active PEG (retention time=6.7 min) shrunk over the course of 5 hours, as peaks of hydrolyzed PEGs (retention time=5.7-6.5 min) gradually increased at the same rate. The half-life of active PEG-5K-HS was estimated from the plot of time versus the quantified amount of inactive PEG in the reaction mixture, the half-lives regarding the hydrolysis rate of the 4 types of PEG-NHS ester in PBS and in 0.9% NaCl, respectively, are summarized in Table 8. The results showed that the half-life of PEG-5K-PS (spacer-arm 3 carbons) in PBS was determined to be 1 hour while lengthening the spacer-arm to 10 carbons (PEG-5K-DCS) significantly increased the half-life to 3.7 hours. This suggests that the spacer-arm length is negatively correlated with the rate of PEG hydrolysis, where the half-life of active PEG increases with the length of the spacer-arm. Notably, the results also revealed that PEG displayed enhanced stability from the dissolution in 0.9% NaCl compared with that in PBS. For instance, the stability of PEG-5K-PS was increased by 7-fold from the dissolution in 0.9% NaCl (t1/2in PBS=1 hr vs. t1/2in 0.9% NaCl=7.2 hr). Similar finding was also observed for PEG-5K-HS (t1/2in PBS=2 hr vs. t1/2in 0.9% NaCl>8.0 hr). TABLE 8Hydrolysis Rate and Half-life for PEG-5K-AS, PEG-5K-PS, PEG-5K-HSand PEG-5K-DCS in 0.9% NaCl and 0.1M PBS, respectively, at roomtemperature.PEG-NHS EsterDissolutionActive PEG afterSpeciesConditions5 hr [%]Half-life (t1/2)PEG-5K-AS0.9% NaCl0≤5 minPEG-5K-PS64.87.2 hrPEG-5K-HS84.8>8 hrPEG-5K-DCS94.6>8 hrPEG-5K-ASPBS0SpontaneousPEG-5K-PS01 hrPEG-5K-HS11.02 hrPEG-5K-DCS33.13.7 hr Example 8B: Reaction Optimization Upon Changing the PEG Stock Solution Medium In light of the increased stability of PEG-NHS ester in 0.9% NaCl solution compared with that in 0.1 M PBS, a reaction trial was carried out by preparing the PEG solution in 0.9% NaCl and the stock solution was kept at room temperature for 0, 1 and 2 hours before the conjugation reaction so as to study the effect of extended processing time on the pegylation yield, which may also provide insight on the flexibility of the pegylation process. The pegylation reaction was conducted in a similar manner as shown in the previous examples with 4.5 g/dL Hb containing 59.3 mg/mL PEG-5K-HS for the conjugation, and a control study was also carried out by the dissolution of PEG-5K-HS in PBS. As shown in Table 9, no significant difference was found in the PEG conjugation number when the reactions were carried out immediately after dissolution in either 0.9% NaCl or in PBS (t=0 h; 14.0 and 13.9 PEG/Hb, respectively; pegylation yield=82%). In contrast, when the reaction was delayed by 2 hours, there was a slight decrease in conjugation number from 14.0 to 12.4 PEG/Hb in 0.9% NaCl (pegylation yield=82% and 73%, respectively). However, a massive reduction of conjugation efficiency was found for that in PBS (decreased from 13.9 to 5.1 PEG/Hb; pegylation yield=82% and 30%, respectively) when the reaction was delayed for 2 hours. The reduction of conjugation number and pegylation efficiency under PBS solution suggests that a higher reaction equivalent, and thus higher production cost, will be required in order to compensate for the loss of PEG reagent through hydrolysis. Therefore, the preparation of PEG stock solution in 0.9% NaCl would minimize the hydrolysis of the PEG reagents and hence improve the pegylation yield of the reaction, thus reducing the cost of the production process. TABLE 9Effects of Dissolution Medium and Dissolution Time on the PegylationEfficiency.ReactionDelay afterDeterminedDissolutionDissolutionPEG/HbPegylationHydrodynamicBufferof PEG [hr]RatioYield [%]Diameter [nm]PEG in 0.9%014.08214.17 ± 0.07NaCl112.97614.09 ± 0.07212.47313.69 ± 0.08PEG in 0.1M013.98213.92 ± 0.02PBS17.54412.34 ± 0.1225.13011.38 ± 0.04 Example 8C: Pegylation Reaction Through Addition of PEG in Powder Form As shown in the above examples, the conjugation yield of PEG-5K-AS was found to be inefficient (PEG/Hb=2.5, pegylation yield=14%, Table 7) owing to its high hydrolysis rate. The pegylation efficiency of PEG-5K-AS is enhanced by direct addition of PEG reagent in its powder form so as to minimize the loss of active PEG in the solution preparation step. Notably, upon changing the reagent addition method, there is significant improvement in the pegylation efficiency for the reaction using PEG in powdered form compared to that in aqueous form (Powdered: ˜7.7 PEG/Hb; PBS: ˜2.5 PEG/Hb; Table 10), although the conjugation number is still lower than those observed in other PEG-NHS esters examined. In order to further increase the conjugation number, a two-step pegylation reaction, each step with 17 equivalents of PEG, was performed using PEG-5K-AS. As shown in Table 10, the pegylation efficiency was found to be similar among the first and second pegylation reactions (7.7 and 7.4 PEG/Hb, respectively), indicating that the amount of active PEG is the limiting factor governing the pegylation efficiency of the reaction. Additionally, this result suggests that the efficiency can be increased by removing the initial PEG dissolution step; thus, the impact of PEG hydrolysis toward the conjugation yield can be minimized, leading to an effective conjugation of hemoglobin with the use of PEG reagent having high hydrolysis rate. TABLE 10Properties of Pegylated Cysteinyl-succinyl crosslinked Hemoglobinwith PEG Reagents with Different Spacer-arm Chain Lengths underDifferent Conditions. PEG equipped with different spacer-arm lengths(17 molar equivalents, dissolved in 0.1M of PBS or remained in solidform) were reacted with hemoglobin (p50 = 55.9 ± 0.2 mmHg) underdeoxygenated conditions for 2 hours.PEG-NHSPegylationMWHydrodynamicEster SpeciesConditionsPEG/Hb[kDa]Diameter [nm]PEG-5K-AS17 Equivalent2.5777.8 ± 0.1PEG-5K-PSPEG in PBS12.612813.2 ± 0.1PEG-5K-HS13.513212.6 ± 0.1PEG-5K-DCS12.912913.1 ± 0.1PEG-5K-AS17 Equivalent7.710311.4 ± 0.1PEG-5K-PSPEG in14.213515.1 ± 0.6PEG-5K-HSPowder14.913914.3 ± 0.2PEG-5K-DCS14.213514.4 ± 0.1PEG-5K-ASPowder PEG15.114013.9 ± 0.1(×2)a(7.7 + 7.4)atwo successive cycles of pegylation reactions with 17 molar equivalents of PEG reagent used in each reaction. Similar pegylation reaction was conducted with other PEG-NHS esters with longer spacer-arm chain length. However, the improvement of conjugation efficiency was less prominent for the PEG examined, as shown in Table 10. Additionally, regardless of the different spacer-arm lengths used, given that a similar number of PEG was attached, the hydrodynamic diameters of the resulting pegylated hemoglobin were found to be similar (13.9-15.1 nm). Nonetheless, all the results as shown in EXAMPLE 8B and 8C indicated that the efficiency of pegylation reaction can be enhanced by (1) preparation of PEG stock solution in 0.9% NaCl solution and (2) addition of PEG reagent in powdered form. With the selection of suitable method for the pegylation process, PEG chains with various properties can be effectively attached to cysteinyl-succinyl crosslinked hemoglobin to obtain pegylated crosslinked hemoglobin with desired properties. Example 9: Determination of Maximum Pegylation Number on Cysteinyl-Succinyl Crosslinked Hemoglobin In the pegylation process of cysteinyl-succinyl crosslinked hemoglobin, PEG-NHS ester reacts with the surface-exposed amines to give a stable amide bond between PEG and hemoglobin. Since the lysine side chain contributes the majority of the primary amines in a protein, the maximum conjugation number of PEG chain on the hemoglobin roughly equals the number of lysine residues in the protein's amino acid sequence (i.e., 48 lysine residues for bovine hemoglobin and 4 amino groups from the N-terminus of the hemoglobin subunits), although the actual empirical numbers would be lower due to the steric hindrance originated from PEG chains and amino acid side-chains. With an aim to determine the maximum conjugation number between the reactions of PEG-NHS ester and cysteinyl-succinyl crosslinked hemoglobin, a pegylation reaction of cysteinyl-succinyl crosslinked hemoglobin was carried out using PEG-1K-HS dissolved in 0.1 M PHS, under deoxygenated conditions for 2 hours. As shown in Table 11, upon varying the reaction equivalents of the PEG-1K-HS from 17 to 85, the number of PEG conjugated significantly increased from 12.7 to 48.2. Notably, this value showed a negligible increase upon further increasing the reaction equivalent of PEG to 143, probably suggesting 48 PEG chains are the maximum number of conjugation to cysteinyl-succinyl crosslinked hemoglobin under the reaction conditions examined. In the reaction with PEG-5K-HS, a total of 43 PEG/Hb was achieved with increased molecular weight (280 kDa), but only with a slightly increased hydrodynamic diameter (from 14.1 to 15.9 nm). TABLE 11Examination of Maximum Conjugation Number of PEG Chainstowards Cysteinyl-succinyl Crosslinked Hemoglobin usingPEG-1K-HS.Reaction AmountDeterminedof PEG-1K-HSPEGPEG/Hb[mg/mL of reaction]EquivalentRatio11.91712.759.38548.210014348.3 Example 10: Specifications of Composition of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The specifications of the pegylated cysteinyl-succinyl crosslinked hemoglobin used for the below safety, pharmacokinetics and tissue oxygenation studies, are shown in Table 12. TABLE 12Physical Properties of Cysteinyl-succinyl CrosslinkedHemoglobin Conjugate.Pegylated Cysteinyl-succinyl CrosslinkedHbtHb [g/dL]4.5-5.5pH7.4-8.4MetHb [%]≤8%Endotoxin [EU/mL]≤0.25Colloid Osmotic Pressure [mmHg]>73Estimated PEG no./Hb12-14Estimated MW [kDa]125-135Average Hydrodynamic Size [nm]13.5-14.5Free Dimer [%]0Unpegylated Hemoglobin≤5%Residual PEG [mg/mL]≤0.2 Example 11: Safety of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Example 11A: Reduced Immunogenic Responses in Rat with Infusion of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Immunogenic safety in patients is crucial for successful protein therapeutics development, especially for those used for repeated dosing and prolonged exposure. Therefore, the immunogenicity of the pegylated cysteinyl-succinyl crosslinked hemoglobin was evaluated using a rat immunization model established by Chang TMS and Varma R (Chang, T. M. S & Varma, R., 1998, Artif Cells Blood Substitut Biotechnol, 16(1-3): 205-215). This screening platform can differentiate the host immune response and possible adverse effects of hemoglobin products used for repeated dosing. Male Sprague-Dawley (SD) rats aged 6-8 weeks (250 g±25 g) were used for this study. The immunization schedule followed Chang TMS and Varma R (Chang, T. M. S & Varma, R., 1998, Artif Cells Blood Substitut Biotechnol, 16(1-3): 205-215) with minimum modification, as shown inFIG.14. Each rat received three immunization doses of 1 mg/mL pegylated cysteinyl-succinyl crosslinked hemoglobin at Day 0, 14 and 28. The subcutaneous (s.c.) injection of 1 mg/mL/rat inoculum of equal volume of pegylated cysteinyl-succinyl crosslinked hemoglobin and Freund's complete (for first immunization) or incomplete (for second and third immunization) adjuvant to induce immunogenic response. Subsequently, rats were challenged by intravenous (i.v.) injection of 2 mL of 100 mg pegylated cysteinyl-succinyl crosslinked hemoglobin per rat biweekly for a total of 4 rounds of challenge. Body weights were recorded prior to each immunization and after each challenge dose. Survival and clinical signs were recorded after each challenge dose. Blood was collected via retro-orbital route before each immunization and post-2 hour and at different time points after the challenge under anesthesia for blood analysis. Organs were harvested for histopathological analyses at 24 hour post-challenge. i) Immunoglobulin and Immune Complex Profile Blood samples collected in K2-EDTA tubes underwent centrifugation to separate plasma for immunoglobulin (IgG) detection. The levels of total IgG in plasma were quantified by rat IgG ELISA kits following manufacture's recommendation. Immunization of rats with pegylated cysteinyl-succinyl crosslinked hemoglobin induced an increasing level of IgG from Day 0, Day 14 to Day 28, as shown inFIG.15a. Blood was also collected every 3 to 5 days after the challenge dose for the total level of IgG detection in plasma.FIG.15bshowed an increased total IgG level after third immunization doses and peaked at Day 10 after each challenge dose. The increased IgG level after each high dose (100 mg/2 mL) challenge was lower than that after immunization. This suggested a higher dose did not trigger greater IgG levels. In addition, the level of IgG after each challenge did not increase significantly when compared to prior challenge and the increased IgG level after the 4thchallenge was also reduced by 14 days, as shown inFIG.15b. This implies that repeated doses of pegylated cysteinyl-succinyl crosslinked hemoglobin did not trigger a hyper immune response and that the increased level of IgG can only sustain for a period of 14-day. To study the profile of anti-drug antibody (ADA) response against pegylated cysteinyl-succinyl crosslinked hemoglobin, sample containing 5 μg pegylated cysteinyl-succinyl crosslinked hemoglobin (antigen) was mixed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer and boiled at 95-100° C. for 10 minutes. Samples and pre-stained marker were loaded to 7.5% SDS-PAGE gel. The gel was run at 80V for 15 minutes followed by 120V for approximately 60 minutes in 1× Running Buffer (BioRad). Proteins were then transferred to polyvinylidene fluoride (PVDF) membrane in 1× Transfer Buffer (BioRad) with 20% ethanol using the Trans-Blot Turbo Transfer System (BioRad) for 10 minutes at room temperature. After protein transfer, the membrane was then blocked by 5% non-fat milk for 1 hr at room temperature with agitation, followed by 3 washes with 1×TBST (Tris-buffered saline (TBS) with 0.05% Tween-20). The membrane was incubated in rat plasma collected (primary antibody; diluted with 5% non-fat milk to 0.01 g/dL total protein concentration) with agitation overnight at 4° C. The membrane was then washed with TBST for 3 times with agitation for 10 minutes, followed by incubating it in anti-rat IgG HP (secondary antibody, diluted in 5% non-fat milk to 1:10000 dilution) for 1 hour with agitation at room temperature. After the 2ndantibodies incubation, the membrane was washed with TBST for 3 times with agitation for 10 minutes and incubated with peroxide and luminol/enhance solution mixture and imaged the blot using ChemiDoc imaging system. The western blot results showed that the intensity corresponding to ADA increased gradually after the 1stchallenge dose and reached peak at post 10 day after 1stchallenge, and dropped before the 2rdchallenge dose, as shown inFIG.16. A similar trend was observed in each challenge dose, the signal was maintained for nearly 14 days and decreased over the time. Although the level of specific ADA was increased after each challenge dose, its level was relatively low, compared to that observed for unpegylated hemoglobin. Apart from the detection of ADA in circulation, the level of specific immune complex (IC) between the ADA and pegylated hemoglobin was evaluated after each challenge dose. It is reported that most adverse effects related to immunogenicity for therapeutic proteins is a consequence of circulating and cell surface bound drug bearing IC (Krishna, M. & Nadler, S. G, 2016, Front Immunol, 7:21). Therefore, the levels of specific IC triggered by the challenge dose of pegylated cysteinyl-succinyl crosslinked hemoglobin in immunized rats were measured using an in-house developed ELISA. The Pierce™ Protein A/G coated microtiter plate (ThermoScientific) was coated with 1 μg/mL rabbit anti-cysteinyl-succinyl crosslinked hemoglobin antibody in 100 mM bicarbonate buffer (pH 9.6) overnight at 4° C. Wells were washed with 10 mM phosphate-buffered saline pH 7.4 in 0.05% (v/v) Tween® 20 (PBST) 3 times and blocked by Starting Block™ buffer (ThermoFisher) for 1 hr at 37° C. After the wells were washed with PBST for 4 times, 100 μL of 4-fold serially-diluted rat plasma in Starting Block™ solution starting at 1:25 was added to the wells and incubated for 1 hr at 37° C. Plates were washed 4 times with PBST and 100 μL of anti-rat IgG conjugated with horseradish peroxidase (HRP) diluted in 1:12000 with Starting Block™ solution were added to the wells and incubated for 1 hr at 37° C. followed by 4 PBST washes. 100 μL of 3,3′,5,5′ tetramethylbenzidine (TBM) was added to the wells. 100 μL of 0.1 M hydrochloric acid was added after the color was developed. The absorbance at 450 nm was measured using FLUOstar Omega microplate reader (BMG LABTECH). The results showed that the repeated challenge of high dose (100 mg/rat) of pegylated cysteinyl-succinyl crosslinked hemoglobin in the immunized rats, the IC titer at 2 hr after each challenge was slightly higher than previous challenge except for the 3rdchallenge, as shown inFIG.17. However, this increase was still low relative to the one time challenge of unpegylated hemoglobin-immunized rats. ii) Body Weight, Survival Rate, Adverse Clinical Symptoms and Histopathological Changes The body weight of rats was recorded before each immunization dose and 2 hour after the challenge dose. Clinical signs and survival rate of rats were observed after each challenge.FIG.18ashowed that the body weight of rats immunized with pegylated cysteinyl-succinyl crosslinked hemoglobin increased from Day 0 to Day 42. Multiple challenges of rats with high dose also do not affect the body weight of the rats, as shownFIG.18b. Importantly, all rats survived with challenge of 100 mg/rat of pegylated cysteinyl-succinyl crosslinked hemoglobin for 4 bi-weekly challenge doses and only a few rats had cyanosis at ears and feet (25%, 3 out of 12 rats) and displayed hypo-activity (58%, 7 out of 12 rats) immediately after challenge, as summarized in Table 3. Moreover, the rats recovered from such mild adverse clinical symptoms within 20 minutes after the challenge and there was a progressive decrease in the percentage of hypo-activity in rats after each challenge dose, as shown in Table 13. TABLE 13Survival Rate and Adverse Clinical Symptoms Observed in Rat after Challenge.Challenge DosePercentage1st2nd3rd4thDeath0%(0/12)AdvEar & Feet Cyanosis25%(3/12)25% (3/12)20% (1/5)Diarrhea0%(0/12)Hypo-activity58%(7/12)58% (7/12)50% (4/8)20% (1/5)50% (1/2)Rapid Breathing0%(0/12)Limbs Swelling0%(0/12)Gait Instability0%(0/12) The challenged rats were further analyzed for histopathological changes. The rat organs including kidney, liver, spleen and heart were harvested at 24 hours post-challenge with pegylated cysteinyl-succinyl crosslinked hemoglobin. Only mild alveolar infiltrates was found in the lung and no significant histopathological changes was observed in other organs, as shown inFIG.19. In summary, the results revealed that four times repeated high dose (100 mg/rat) challenge of pegylated cysteinyl-succinyl crosslinked hemoglobin in immunized rats would not trigger an immediate increase of specific ADA, though an increase of total IgG level was observed. Importantly, a relatively low IC titer was detected post 2 hours in every challenge dose and even after 4 high dose challenges. Multiple challenge of rats with pegylated cysteinyl-succinyl crosslinked hemoglobin do not reduce the survival rate of rats, and histopathological assessment suggested no clinically significant finding in heart, liver, kidney and spleen, only a mild alveolar infiltration was found in the lung. Thus, it is believed that the pegylated cysteinyl-succinyl crosslinked hemoglobin only elicits mild host humoral response and would not trigger high immunogenic response for repeated dosing, or cause any significant adverse effects. As such, it is more antigenically safe to administer, especially for repeated dosing and prolonged exposure. Example 11B: Elimination of Renal Toxicity by Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The hematoxylin and eosin (H&E)-stained kidney tissues showed intact tubules and no loss of tubule cells after pegylated cysteinyl-succinyl crosslinked hemoglobin challenge (FIG.19d). No hemorrhage and blood clot were observed in the kidney tissues. This suggested that the IV-infusion of pegylated cysteinyl-succinyl crosslinked hemoglobin would not cause any kidney structural damage or kidney injury at 24 hr-post challenge. Example 11C: Reduced Cardiac Toxicity of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Cardiotoxicity, namely myocardial infarction, has been reported from a number of HBOC products (Estep, T. P., 2019, Artif Cells Nanomed Biotechnol, 47: 593-601). To assess the cardiac toxicity of pegylated cysteinyl-succinyl crosslinked hemoglobin, Sprague Dawley (SD) rats were used. Following brief anesthesia with isoflurane, antibiotics and analgesics were administrated before surgery. After the surgical site was cleaned and slaved, an incision was made to the right of the midline of the ventral surface of the neck, along the jugular groove, and the right jugular vein was isolated. Following the cannulation of the vessel, the sterile catheter primed with normal saline, was tunneled using a sterile I.V. catheter to a position on the dorsal surface of the rat, whereupon it was connected to a harness. The neck wound was closed using Michel clips. The harness was connected to a swivel tether, and the rat, removed from anesthesia, was then placed in a single housed cage. The tether was then connected to a syringe pump and saline pumped at a slow rate to ensure the potency. The rat was allowed to recover for at least 4 days before dosing. On the dosing day, the weight of the rat was measured and the pegylated cysteinyl-succinyl crosslinked hemoglobin was given via a syringe pump at 1250 mg/kg with infusion rate at 6 mL/kg/hr, respectively. Same volume of buffer was infused in the control group in parallel. Hearts were harvested at 72 hours post administration and examined macroscopically, and then they were immersed in neutral buffered 10% formalin solution for tissue fixation, following by histopathological analysis. The histopathological results showed that rats infused with pegylated cysteinyl-succinyl crosslinked hemoglobin (n=6, per group) at 1250 mg/kg appeared to be well tolerated and no significant lesion of heart was observed, while infusion of unpegylated hemoglobin to the rat resulted in cardiomyopathy in 4 out of 6 rats, as shown in Table 14. Severity of heart lesions raging from very mild to moderate were defined in the rat infused with unpegylated crosslinked hemoglobin. This suggests that the pegylated cysteinyl-succinyl crosslinked hemoglobin is less cardiotoxic than the unpegylated ones. TABLE 14Incidence of Cardiomyopathy in Rats with Single Infusion ofPegylated Cysteinyl-succinyl Crosslinked Hemoglobin.Dose LevelIncidence ofGroup(mg/kg)CardiomyopathyControl Group00/12Pegylated Hemoglobin12500/6Unpegylated Hemoglobin12504/6 Example 12: Pharmacokinetics of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin Example 12A: Enhanced In Vivo Circulation Stability of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The half-life of pegylated cysteinyl-succinyl crosslinked hemoglobin in SD rat was investigated. Male SD rats (250-280 g) were anesthetized by 1.5% isoflurane and were undergone cardiac and femoral catheterization 3 days before infusion. Rats were infused with 620 mg/kg pegylated cysteinyl-succinyl crosslinked hemoglobin with an infusion rate of 3 mL/kg/hr. Rat plasma samples were collected at pre-dose, 1, 2, 4, 15, 24, 28 and 44 hours post-infusion. Total hemoglobin of samples and standards were measured using HemoCue® Plasma/Low Hb System and the half-lives were calculated using PKSolver 2.0 (Linear Trapezoidal). The results showed that the half-life of pegylated cysteinyl-succinyl crosslinked hemoglobin was almost triple (t½=19.9 hr), compared to that of unpegylated cysteinyl-succinyl crosslinked (t½=7.1 hr), as shown in Table 15. This implies that the pegylation of cysteinyl-succinyl crosslinked hemoglobin increases in-vivo blood circulation times by changing the pharmacokinetics properties of the crosslinked hemoglobin itself. HBOC product with enhanced in vivo circulation stability can increase its bioavailability to achieve higher therapeutic effect. TABLE 15In vivo Circulation Stability of Pegylated Cysteinyl-succinyl CrosslinkedHemoglobin vs. Cysteinyl-succinyl Crosslinked Hemoglobin.Half-life (t½)Pegylated Cysteinyl-succinyl Crosslinked Hemoglobin19.9 hrCysteinyl-succinyl Crosslinked Hemoglobin7.1 hr Example 12B: In Vivo Distribution of Pegylated Cysteinyl-Succinyl Crosslinked Hemoglobin The in vivo distribution of pegylated cysteinyl-succinyl crosslinked hemoglobin was studied in male Balb/c mice. All mice were supplied with low-fluorescence diet throughout the study. Male Balb/c mice (20-25 g) were individually administered with 10 mg/0.25 mL/mouse of pegylated cysteinyl-succinyl crosslinked hemoglobin or cysteinyl-succinyl crosslinked hemoglobin, respectively. For the treatment group, mice were administered with a mixture of hemoglobin conjugated to Alexa Fluor 647 fluorescent dye and unconjugated hemoglobin in a ratio of 1:80 (c-hemoglobin), while for the control group, mice were administered with unconjugated hemoglobin only. All mice were injected intravenous through tail vein with an interval of 60 minutes according to Table 16. TABLE 16Illustration of Injection Sequence of Pegylated Cysteinyl-succinylCrosslinked Hemoglobin.Time (hour)Treatmentn0c-hemoglobin (Treatment Group)11121313Unconjugated Hemoglobin (Control Group)1c-hemoglobin: mixture of hemoglobin conjugated to Alexa Fluor 647 fluorescent dye and unconjugated hemoglobin in a ratio of 1:80. After the injections, mice were anesthetized by 3% isoflurane inhalant within an induction chamber and subsequently transferred to the IVIS® Spectrum in vivo imaging system (PerkinElmer) with continuous supply of isoflurane/oxygen. Fluorescence signals were measured with filters at 620 nm (excitation) and 670 nm (emission) and serial images were taken at every 10 minutes for 1 hour. The imaging procedure was repeated at 4, 24 and 72 hours after the first recording. A separate experiment was also performed to measure the fluorescence signals in vital organs (liver, kidney, spleen, lungs and heart) of the mice as well as the biochemical changes in urine. Intravenous injections were made at time 0, according to Table 16 and the mice were sacrificed at 6 hours after injection. Sampled organs from the groups were subsequently measured for their fluorescence levels using IVIS® Spectrum in vivo imaging system, while the fluorescence level from urine was measured using FLUOstar Omega microplate reader (BMG LABTECH). An illustration of the study setup was shown in Table 17. TABLE 17Urine Collection and Organ Imaging Study Setup.GroupInjectionn1No injection (Control group)22c-Hemoglobin (cysteinyl-succinyl crosslinked hemoglobin)23c-Hemoglobin (pegylated cysteinyl-succinyl crosslinked2hemoglobin)c-hemoglobin: mixture of hemoglobin conjugated to Alexa Fluor 647 fluorescent dye and unconjugated hemoglobin in a ratio of 1:80. The IVIS spectra of pegylated cysteinyl-succinyl crosslinked hemoglobin and unpegylated ones are shown inFIG.20. Both pegylated and unpegylated cysteinyl-succinyl crosslinked hemoglobin was gradually distributed to the whole body within 2 hours, but IVIS signal of unpegylated hemoglobin was undetectable after 24 hours post-injection, as shown inFIG.20A, while IVIS signal of pegylated cysteinyl-succinyl crosslinked hemoglobin was still detectable after 72 hours post-injection, as shown inFIG.20B. This indicates that the pegylated hemoglobin can impart longer circulating time by modification of the physicochemical properties of crosslinked hemoglobin. This can enhance localization to sites of interest for potential treatment compared with unpegylated ones. Moreover,FIG.21showed that the IVIS signals at bladder increased with time, indicating there is an accumulation of the succinyl crosslinked hemoglobin in the bladder. The renal clearance of drug was highly possibly through urination. The collected urine shows strong fluorescent intensity (>80 fold stronger than the control group). This result confirms the drug was cleared and broken down through kidney. Importantly, the results also revealed that pegylated cysteinyl-succinyl crosslinked hemoglobin was well distributed in all vital organs at 6 hours post-injection, while the unpegylated cysteinyl-succinyl crosslinked hemoglobin was mainly accumulated in the liver at 3 hours post-injection, as shown inFIG.22. This reflects that the pegylation changes the pharmacokinetics of cysteinyl-succinyl crosslinked hemoglobin, which may widen the application of pegylated cysteinyl-succinyl crosslinked hemoglobin. In sum, the results suggest that the pegylated cysteinyl-succinyl crosslinked hemoglobin has outstanding pharmacokinetic and pharmacodynamics properties, resulting in enhanced in vivo circulation stability and specific organ/tissue bioavailability for different therapeutic indications. Example 13: Restoration of Liver Tissue Oxygenation in Hemorrhagic Shock A fixed-pressure of hemorrhagic shock model was used for evaluating the efficacy of in vivo liver tissue oxygenation (TO2) of pegylated cysteinyl-succinyl crosslinked hemoglobin and the unpegylated ones. Sprague-Dawley male rats ranging from 300-320 g were put under anesthesia. Sterile catheters primed with saline were tunneled into left femoral vein, left femoral artery and right femoral vein, and a sensor with pressure measurement function was tunneled into the right femoral artery. A fiber optic probe was placed between right and triangular lobes and the real-time tissue oxygen (TO2) was measured. In this model, the drop of mean arterial pressure (MAP) was induced by removing 0.1 mL blood for every 10 seconds from the femoral artery catheter and maintained at the margin of 60 mmHg or below during hemorrhagic shock. Subsequently, the resuscitation process was preceded only when the arterial lactate concentration reached 8.00-11.00 mmol/L. Blood was collected at baseline, during hemorrhagic shock, 60 and 120 min after resuscitation for measuring the lactate concentration. The results showed an increased tissue oxygen (TO2) was observed after resuscitation of either pegylated or unpegylated crosslinked hemoglobin in this rat hemorrhagic shock model, as shown inFIG.23. While the increased TO2level for pegylated cysteinyl-succinyl crosslinked hemoglobin was higher than dextran (negative control) but below that of the whole blood (positive control), the TO2level for the unpegylated ones was below both controls. The TO2level remained stable for at least 60 minutes after completing the resuscitation for the pegylated cysteinyl-succinyl crosslinked hemoglobin, but gradually dropped for the unpegylated ones. This suggested that pegylated hemoglobin might have better tissue oxygenation in rat liver than unpegylated hemoglobin. Importantly, the results also revealed that the lactate concentration was back to almost baseline level at 120 min after resuscitation for pegylated crosslinked hemoglobin compared to unpegylated ones, as shown inFIG.24. These findings suggested that the resuscitation with the pegylated cysteinyl-succinyl crosslinked hemoglobin would improve the TO2restoring ability and also the metabolic function in rats with hemorrhagic shock condition. | 138,369 |
11857606 | DETAILED DESCRIPTION OF THE INVENTION I. Introduction The present invention links a receptor-mediated translocation function (e.g., derived from a bacteriocin) to another functional cargo domain, e.g., a killing domain, as in a bacteriocin to achieve an entity which can attack the Gram-negative bacteria targets. The chimeric (and related) “bacteriocin” constructs described herein combine an bacteriocin-derived receptor-mediated translocation function linked to a labeling or killing function, which may be a peptidoglycan degrading enzyme activity. The bacteriocin-derived receptor-mediated translocation function is achieved with a protein segment which recognizes an outer membrane receptor on the bacteria, typically a protein, which assists in mediating the translocation. Generally, the receptor recognition function provides selectivity and specificity in target cell into which the translocation is effected. Thus, the translocation may be characterized as a “receptor-mediated” process. In many embodiments, the translocation involves two “functional” steps and domains within the bacteriocin, a binding step (involving receptor binding segment or RBS) and a translocation step (involving a translocation segment or TS), though the two steps may not necessarily be separable physically or temporally. The binding step often involves some specificity of binding of the bacteriocin to its cognate receptor, which then may take some conformational shape which allows the bacteriocin and the cargo domain to be transported or flipped across the lipid bilayer membrane. In certain embodiments, the receptor-mediated translocation is only across the outer membrane, and the cargo domain is accessible to the periplasmic space of the bacterial host. The peptidoglycan (murein) sacculus is an essential structural component of the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the bacteria cytoplasmic membrane. The sacculus must withstand up to 25 atmospheres of osmotic pressure. The sacculus is flexible, allowing reversible expansion under pressure, which allows diffusion of even large protein molecules. See, e.g., Silhavy et al. (2010)CSH Persp. Biol.,2:a000414; Vollmer et al. (2008)FEMS Microbio. Revs32:149-167; Bos et al. (2007)Ann. Rev. Microbiol.61:191-214; and Costerton et al. (1974) Bact. Revs. 38:87-110. Many antibiotics act on the peptidoglycan layer of a target bacteria species. This structure is thus a critical component in the survival of a bacterial target. Attack of the peptidoglycan is a rational strategy for killing target bacterial hosts. Although the peptidoglycan layer is typically about 1-3 layers thick, the outer membrane of a Gram-negative bacterium serves as a permeability barrier that prevents externally applied enzymes from reaching their substrate. The receptor-mediated translocation domain or segment allows the protein, e.g., with muralytic activity, to be transferred across the bacterial outer membrane. For example, the receptor-mediated translocation segment itself may mediate a membrane transfer event, thereby moving the muralytic activity from outside of the bacterial outer membrane to the inside, and allowing contact between the enzyme and its peptidoglycan substrate. The receptor-mediated translocation segment may take advantage of an endogenous translocation system in the outer membrane by presenting earmark motifs which signal the system to import the molecule into the periplasmic space. In some embodiments, the receptor-mediated translocation segment directs the construct polypeptide to the receptor expressing outer leaflet of the outer membrane, and the muralytic polypeptide flips from the outer leaflet of the outer membrane to the inner leaflet, thereby delivering the muralytic segment to the peptidoglycan substrate. II. Gram-Negative Bacteria A. Outer Membrane The cell envelope of gram-negative bacteria consists of two membranes, the inner membrane (IM) and the outer membrane (OM), which are separated by the periplasm containing the peptidoglycan layer. The two membranes have an entirely different structure and composition. Whereas the IM is a phospholipid bilayer, the OM is an asymmetrical bilayer, consisting of phospholipids and lipopolysaccharides (LPS) in the inner and outer leaflet, respectively. Additionally, these membranes differ with respect to the structure of the integral membrane proteins. Whereas integral IM proteins typically span the membrane in the form of hydrophobic α-helices, integral OM proteins (OMPs) generally consist of antiparallel amphipathic β-strands that fold into cylindrical β-barrels with a hydrophilic interior and hydrophobic residues pointing outward to face the membrane lipids (Koebnik et al. (2000) “Structure and function of bacterial outer membrane proteins: barrels in a nutshell” Mol. Microbiol. 37:239-53). Both membranes also contain lipoproteins, which are anchored to the membranes via an N-terminal N-acyl-diacylglycerylcysteine, with the protein moiety usually facing the periplasm in the case ofEscherichia coli(Pettersson et al. (1997) “Response ofNeisseria meningitidisto iron limitation” Antonie van Leeuwenhoek 71:129-36). The LPS molecule can be divided into three parts: lipid A, core polysaccharides, and O-antigen repeats. Lipid A represents the hydrophobic component of LPS which locates in the outer leaflet of the outer membrane, while core polysaccharides and O-antigen repeats are displayed on the surface of the bacterial cells (Raetz et al. (2007) “Lipid A modification systems in Gram-negative bacteria” Annu Rev Biochem 76:295-329). The detailed structure of LPS varies from one bacterium to another, and this variation could affect the virulence of the bacterium. See, e.g., Galanos et al. (1985) “Synthetic and naturalEscherichia colifree lipid A express identical endotoxic activities” Eur J Biochem 148:1-5; and Wilkinson (1996) “Bacterial lipopolysaccharides-themes and variations” Prog Lipid Res 35:283-343. B. Peptidoglycan Layer Peptidoglycan (murein) is an essential and specific component of the bacterial cell wall found on the outside of the cytoplasmic membrane of almost all bacteria (Rogers et al., (1980); Park, (1996); Nanninga, (1998); Mengin-Lecreulx & Lemaitre, (2005)). Its main function is to preserve cell integrity by withstanding the internal osmotic pressure. Any inhibition of its biosynthesis or its specific degradation during cell growth will result in cell lysis. Peptidoglycan also contributes to the maintenance of a defined cell shape and serves as a scaffold for anchoring other cell envelope components such as proteins (Dramsi et al., 2008) and teichoic acids (Neuhaus & Baddiley, (2003)). The peptidoglycan structure of both Gram-positive and Gram-negative bacteria comprises repeating disaccharide backbones of N-acetylglucosamine (NAG) and β-(1-4)-Nacetylmuramic acid (NAM) that are cross-linked by peptide stem chains attached to the NAM residues. In gram-negative bacteria, the stem peptide attached to the carboxyl group of each muramic acid usually consists of L-Ala-_-D-Glu-(L)-meso-diaminopimelic acid (Dap)-D-Ala, although the stem peptide often lacks D-Ala or, more rarely, terminates in D-Ala-D-Ala. About one-half of the stem peptides are involved in cross-links between neighboring glycan strands (Rogers et al., (1980)). Muralytic domains are known in the art. Among these are the class of lysozyme proteins. See, e.g., Salazar and Asenjo (2007)Biotechnol. Lett.29:985-94. Breakdown of the peptidoglycan structure occurs naturally in at least four contexts. One is biosynthesis of the structure itself; as the bacterial cell grows and divides, it must necessarily must break down the structure. See, e.g., Vollmer (2008)FEMS Microbiol Rev.32:287-306; Scheurwater et al. (2008)Int. J. Biochem. Cell Biol.40:586-91; Keep et al. (2006)Trends Microbiol.14:271-276; and Baba and Schneewind (1998)EMBO J.17:4639-4646. There are additional situations when the cell itself must rearrange or modify structure which was synthesized earlier. Second, eukaryotic hosts degrade the structure upon clearing of an infection, e.g., using mutanolysin or lysozymes. See, e.g., Callewaert and Michiels (2010)J. Biosci.35:127-60; Harder et al. (2007)Endocr. Metab. Immune Disord Drug Targets7:75-82; and Lichtman et al. (1992)J. Clin. Invest.90:1313-1322. A third area is in phage replication, where the phage typically employs an endolysin to release the replicated phages and lyse the bacterial host cell. See, e.g., Srividhya and Krishnaswamy (2007)J. Biosci.32:979-90; and Loessner (2005)Curr. Opin. Microbiol.8:480-487. This is a lysis of the peptidoglycan layer of cells from within. The fourth context is where phage infection requires that the peptidoglycan barrier be traversed, as described in Padmanabhan et al. WO2007/130655. This is degradation of the peptidoglycan layer from the exterior of the cell. Each of these mechanisms involves some means to disassemble the peptidoglycan structure. Thus, muralytic activities are found in genomes of eukaryotic hosts for bacteria, in bacteria genomes themselves, and in phage (and related prophages) which target bacteria as hosts. Muralytic domains can be found by homology to any of these sources, and informatics can be used to identify candidate genes with their respective canonical motifs. While the muralytic activity is one class of killing domains encompassed by the invention, many of the examples are described using this example and the invention is not to be limited to these embodiments, but many other killing or toxic segments may be substituted. Peptidoglycan “degrading activities” can be converted into highly effective bactericidal activities for use against Gram-negative bacterial pathogens under therapeutic conditions, and can include muraminidase, glucosaminidase, amidase, or endopeptidase activities. Exemplary muralytic domains can be identified, incorporated into chimeric constructs to be delivered to the peptidoglycan substrate, produced, purified, and confirmed to have bactericidal activity against bacterial hosts with an outer membrane. Recombinant constructs comprising such activities have significant advantageous properties as antimicrobial compositions and formulations. An example of the linked polypeptides of the invention uses a muralytic fragment, e.g., comprising a lysozyme domain fromPseudomonasphage P134, which is closely related to phage phiKMV. The ORF36 in phage P134 that corresponds to that in phiKMV lyses Gram-negative bacterial cells whose outer membrane has been removed. Contacting the construct to a variety of different Gram-negative bacteria after the outer membrane was removed resulted in the cells being broken down. These results demonstrate that the peptidoglycans from different Gram-negative bacteria species are susceptible to the muralytic activity. Sequence homology searches identify various other similar domains which can be used as alternative sources for peptidoglycan degrading activities. The small size of the polypeptides exhibiting these activities affords efficient large scale production. Accessibility to relevant cell wall target components, e.g., peptidoglycans, at the bacterial target is provided, as are pharmacological distribution upon in vivo administration. Relevant muralytic activities can be found within the lysozyme-like superfamily, lytic transglycosylase (LT), goose egg white lysozyme (GEWL); the Superfamily C100442 containing Lysozyme like domain, which contains several members including the Soluble Lytic Transglycosylases (SLT), Goose Egg-White Lysozymes (GEWL), Hen Egg-White Lysozymes (HEWL), Chitinases, Bacteriophage lambda lysozymes, Endolysins, Autolysins, Chitosanases. All these members are involved in the hydrolysis of beta-1,4-linked polysaccharides. The Cysteine Histidine dependent Amidohydrolase/Peptidase (CHAP) domain is found in phage endolysins and bacterial autolysins. Most proteins containing a CHAP domain function as peptidoglycan hydrolases and are commonly associated with amidases. See Bateman and Rawlings (2003)Trends Biochem. Sci.5:234-237; and Pritchard et al. (2004)Microbiology150:2079-2087. See also the Carbohydrate-Active enZYmes Database found at cazy.org. The CAZY database describes the families of structurally related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds. Another source for endopeptidases is the database from the website found at merops.sanger.ac.uk/cgi-bin/clan_index?type=P. Analogous strategies can be used to identify and use other killing domains from muralytic domains, based, e.g., on the killing functions described below. Certain functional killing domains may be identified, and analogous or homologous alternative substitutions may be constructed. C. Cell Membrane Lipases and other functional activities which degrade the lipid bilayer of the prokaryote host can kill the cell. Additional toxic or toxin segments which will kill the target Gram-negative cells might be substituted, as could smaller molecule toxins conjugated to a cargo peptide for translocation into the cell. Preferably activities which act only on prokaryotes and would have no effect on a eukaryote will be highly selective in effect, only acting on the target but having little or no effect on a host organism being infected by a Gram-negative bacteria. I. Bacteriocin Polypeptides A. Bacteriocins Bacteriocins are a diverse family of protein antibiotics produced by bacteria, which are naturally used to kill members of the same or closely related species. Bacteriocins produced byE. coli, called the colicins, were the first ones to be identified and are well studied and many of them are characterized. Almost all of the colicins characterized so far exhibit a three domain architecture with an N-terminal translocation domain, a receptor binding domain and a C-terminal killing domain. The killing domains are usually either nucleases or membrane damaging pore formers. The bacteriocin producing bacteria is protected from its own action by immunity protein that is produced by the bacteriocin expressing strain and functions by stochiometrically binding to the killing domain and inhibiting its activity. Examples of bacteriocins polypeptides useful in the invention, along with their domain boundaries, are presented in Table 1. TABLE 1Domain boundaries of Bacteriocinsand Chimeric Bacteriocin ConstructsBacteriocinSEQ ID NO:PolypeptideDomain2Klebicin CCLTranslocation domain: 1-320Receptor binding domain: 322-457Killing domain: 475-5594Klebicin BTranslocation domain: 1-490Receptor binding domain: 492-631Killing domain: 632-7656Klebicin CTranslocation domain: 1-239Receptor binding domain: 376-517Killing domain: 533-6168Klebicin DTranslocation domain: 1-315Receptor binding domain: 467-609Killing domain: 626-71012Klebicn CCLKlebicin CCL:TD RD-Translocation domain: 1-320Klebicin B KDReceptor binding domain: 321-473Klebicin B killing domain: 474-61514P623 S5 TD-S5 translocation domain: 1-150RD-Linker-S5 receptor binding domain: 151-300GP36 CD-hisLinker: 301-306GP36 CD: 307-521XhoI site: 522-5236X his: 524-52916P624 S5 TD-S5 translocation domain: 1-150RD-Linker-S5 receptor binding domain: 151-300GP36 CDLinker: 301-306GP36 CD: 307-52118P625 S5 TD-S5 translocation domain: 1-150RD-Linker-S5 receptor binding domain: 151-300Phi29CDLinker: 301-306Phi29 CD: 307-45420P626 S5 TD-S5 translocation domain: 1-150RD-Linker-S5 receptor binding domain: 151-300BP7eLinker: 301-306BP7e: 307-46722P638 S5 PyocinS5 translocation domain: 1-150with 6X-His tagS5 receptor binding domain: 151-300S5 killing domain: 301-4986X his: 499-50424P652 S5 PyocinS5 translocation domain: 1-150without His tagS5 receptor binding domain: 151-300S5 killing domain: 301-49826Fyu A BD- T4Translocation domain- 1-25lysozyme fusionReceptor binding domain- 1-67T4 lysozyme domain: 168-32928Fyu A BD -Translocation domain- 1-25GP36 fusionReceptor binding domain- 1-167T4 lysozyme domain: 168-38330PelB-FyuAPel B: 1 to 22receptorFyuA receptor: 23 to 675 Klebicins: Bacteriocins produced byKlebsiellaare called klebicins. Klebicins have similar domain architecture as that of the colicins isolated fromE. coli. Four different types of klebicins were reported and whose DNA sequence was described—Klebicin B, Klebicin C, Klebicin CCL and Klebicin D (Riley et al. (2001) and Chavan et al. (2005)) S-Type Pyocins: Soluble or S-type pyocins are protease- and heat-sensitive, chromosome-encoded bacteriocins fromP. aeruginosathat are able to kill cells from the same species. These antibacterials are secreted a binary protein complexes consisting of large protein that harbors the killing function and a smaller immunity protein that remains tightly bound to the cytotoxic domain of the former. Several types of S-type pyocins have been described and characterized: pyocins 51, S2, AP41, S3, S4 and S5. Pyocin Sa turned out to be identical to pyocin S2. To kill a target cell, a S-type pyocin would first bind to a specific receptor located on the outer membrane of the bacterial cells and it would then be further translocated to exert its inhibitory function. Pesticin: Pesticin fromYersinia pestisis a toxin that killsY. pestis, Yersinia enterocolitica, and certainEscherichia colistrains (Hu and Brubaker (1974)), which is encoded by a 9.5 kb plasmid, pYP (Kol'tsova et al. (1973); Ferber and Brubaker, (1981)). Pesticin exhibits N-acetylglucosaminidase activity (Ferber and Brubaker (1979)). Pesticin can utilize the FyuA receptor that is responsible for the transport of the yersiniae iron chelator, yersiniabactin (Heesemann et al. (1993); Rakin et al. (1994); Fetherston et al. (1995)). The expression of pesticin is thought to be controlled by the SOS system (Hu et al. (1972)), and its transport through the outer membrane and interaction with the cognate FyuA receptor is TonB-dependent (Ferber et al. (1981)). B. Cargo Domain To prepare chimeric constructs of the invention, a bacteriocin-derived receptor-mediated translocation domain is linked to a heterologous cargo domain that provides a desired function (e.g., labelling or killing). For example, a killing segment will comprise a segment, which may be less than the complete “domain” and include variations which retain function but differ from a classically defined “domain”, which will kill the target cell. The domain may be a component of a protein, e.g., of a bacteriocin, which naturally operates to kill the target cell. That domain may be substituted or replaced with another domain which can kill the target cell, which may be a catalytic activity which can kill the cell, or may be some structural feature which functions to block or interfere with normal cell activity to effect killing. Yet another option is for actual toxic chemicals or structures to be conjugated or attached to carrier peptide or other chemical linkages which are operably linked to the receptor-mediated translocation domain. Examples may be toxic conjugates analogous to those used as targeted toxins in chemotherapies, which might be taken up into the target cells and released from the carrier inside the cell, with a stoichiometry which may interfere in many different copies of target enzyme or substrate. Examples of killing segments are provided in Table 2. TABLE 2Bacteriocin-derived Killing Segments1DNaseCytoplasmPyocin S1, S2, S3,Klebicin B2rRNaseCytoplasmPyocin S6, Colicin E3,E4, E6, Klebicin C,CCL, Cloacin DF133tRNaseCytoplasmPyocin S4, Colicin E5,Colicin D, Klebicin D4Pore formation (CellPeriplasmPyocin S5, Colicin 1amembrane damage)5Peptidoglycan degradationPeriplasmColicin M, Pesticin(muraminidase)6Inhibitors of periplasmicPeriplasmPyocin PaeMenzymes Large bacteriocins (>60 kDa) are protein toxins that kill bacteria closely related to the producing organism by targeting either nucleic acids (e.g., DNA, and RNA, tRNA or rRNA) in the cytoplasm or cell membrane components of susceptible bacteria. Genes coding for bacteriocins are located either on plasmids or genomes of the producing organism and could be identified for the whole genome sequence using various bioinformatic tools. Whole genome information available from a database, e.g., the NCBI Genome database, can be mined to identify putative bacteriocins and multiple sequence alignment and sequence identity searches will help in narrowing down on the possible bacteriocins. For example, more than 3000 nuclease bacteriocins were identified using a Hidden Markov Model (HMM) from 53 different bacterial species distributed across diverse ecological niches, including human, animals, plants, and the environment (Sharp et al. (2017) Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models. PLoS Comput Biol 13(7): e1005652). In addition to nucleases and pore forming activity, bacteriocins can also be lipases; decouplers of oxidation; activatable mutagens; blockers of transcription/translation; inducers of apoptosis; interference with critical cell functions such as cdc, energy metabolism, cell wall and membrane biogenesis and maintenance, etc. In addition to killing domains derived from bacteriocins, antimicrobial peptides derived from a number of sources can be used. Examples are provided in Table 3. TABLE 3Antimicrobial peptides (AMPs) for fusion to bacteriocinsAntimicrobialpeptideAmino acid SequenceSalient featuresReferenceWLBU2RRWVRRVRRWVRRVde novo design of modularDeslouches et al. (2005)VRVVRRWVRRcationic amphipathic peptidesActivity of the De Novo(CAPs) reported to be activeEngineered Antimicrobialin human serumPeptide WLBU2 againstPseudomonas aeruginosainHuman Serum and WholeBlood: Implications forSystemic ApplicationsAntimicrobial Agents andChemother.49:3208-3216CathelicidinGLLRKGGEKIGEKLKKDerived from mouseMishra et al. (2015) EvaluationrelatedIGQKIKNFFQKLVPQPEanalogue of cathlelicidinof the antibacterial andantimicrobialQantimicrobial peptide (CAP)antibiofilm activities of novelpeptideCRAMP-vancomycin(CRAMP)conjugates with diverse linkersOrg. Biomol. Chem.13(27):7477-86SushiHAEHKVKIGVEQKYGCorresponds to residues 268Li et al. (2004) Perturbation ofQFPQGTEVTYTCSGNYto 301 of the factor C Sushi 3Lipopolysaccharide (LPS)FLMdomain designated S3Micelles By Sushi 3 (S3)Antimicrobial PeptideJ. Biol.Chem.279:50150-50156.RI18RKKTRKRLKKIGKVLKDerived from PorcineLyu et al. (2016) AntimicrobialWImyeloid antimicrobialactivity, improved cellpeptide-36 (PMAP-36)selectivity and mode of actionof short PMAP-36-derivedpeptides against bacteria andCandidaScientific Reports,article number: 27258Cecropin-beeKWKLFKKIGIGAVLKVResistant to salt up to 300Friedrich et al. (1999) Salt-melittin hybridLTTGLPALISmMResistant Alpha-HelicalpeptideCationic Antimicrobial(CEME)PeptidesAntimicrobial Agentsand Chemotherapy43:1542-1548SyntheticGRRRRSVQWCACorresponds to the N-Brouwer et al. (2011)peptide hLF1-terminal eleven residues ofDiscovery and development of11human lactoferrina synthetic peptide derivedfrom lactoferrin for clinical usePeptides32:1953-1963.MagaininGIGKFLHSAKKFGKAFIsolated fromXenopusskin,Matsuzaki et al. (1997)VGEIMNShave broad spectra ofInteractions of an Antimicrobialantimicrobial activity andPeptide, Magainin 2, Withlow toxicities to normalOuter and Inner Membranes ofeukaryotic cellsGram-Negative BacteriaBiochim. Biophys. Acta1327:119-130OmigananILRWPWWPWRRKIsolated from the cytoplasmicSader et al. (2004) Omiganangranules of bovinePentahydrochloride (Mbi 226),neutrophilsA Topical 12-Amino-AcidCationic Peptide: Spectrum ofAntimicrobial Activity andMeasurements of BactericidalActivityAntimicrob AgentsChemother.48(8):3112Arenicin-3GFCWYVCYRNGVRVCIsolated from the lugwormAndra et al. (2008) StructureYRRCNArenicola marina.Exhibitand Mode of Action of thepotent, rapid antimicrobialAntimicrobial Peptide Arenicinactivity in vitro against aBiochem J.410(1):113-22broad range of multi-resistantpathogenic Gram-negativebacteriaLBP peptideSDSSIRVQGRWKVRASCorresponds to the NTaylor et al. (1995)FFKLQGSFDVSVKGterminal region ofLipopolysaccharideslipopolysaccharide bindingNeutralizing Peptides Reveal aprotein (LBP) that has highLipid A Binding Site of LPSaffinity toBinding ProteinJ. Biol. Chem.Lipopolysaccharide (LPS)270:17934-17938ProtaminePRRRRSSSRPVRRRRRPA polycationic peptide foundAspedon et al. (1996) TheRVSRRRRRRGGRRRRin the nuclei of sperm ofAntibacterial Action ofdifferent animal speciesProtamine: Evidence forDisruption of CytoplasmicMembrane Energization inSalmonella TyphimuriumMicrobiology142:3389-3397ApidaecinsGNNRPVYIPQPRPPHPRProline-rich AMPsCzihal et al. (2009) Mapping ofLexpressed in insects as part ofApidaecin Regions Relevant forthe innate immune system.Antimicrobial Activity andThey are very active againstBacterial InternalizationGram-negative bacteria,Internatl J. Peptide Res. Andespecially EnterobactericeaeTherapeutics15(2):57-164membersSheep myeloidRGLRRLGRKIAHGVKKα-helical cathelicidin derivedSkerlavaj et al. (1999) Smap-antimicrobialYGPTVLRIIRIAGpeptide deduced from sheep29: A Potent Antibacterial andpeptidemyeloid mRNAAntifungal Peptide from Sheep(SMAP29)LeukocytesFEBS Letters463:58-62Sheep myeloidRGLRRLGRKIAHGVKKSynthetic α-helicalJacob B et.al. (2016) TheantimicrobialYGcathelicidin derived peptidestereochemical effect ofpeptide -18deduced from sheep myeloidSMAP-29 and SMAP-18 on(SMAP18)mRNAbacterial selectivity, membraneinteraction andanti-inflammatory activity.Amino acidsDOI10.1007/s00726-016-2170-y C. Linkers, Other Components; Immunity Proteins Many of the chimeric constructs of the invention will have linkers which attach the different components as a single polypeptide. Alternatively, the construct may comprise multiple polypeptides, often synthesized as a single polypeptide but may be cleaved and maintain structural integrity by secondary or tertiary structure. Rates of transfer across the outer membrane can be measured by a number of methods. One method is to indirectly evaluate the results of transfer, e.g., the effects of a killing segment reaching its periplasmic substrate. The criteria of measurement can be release of measureable cell contents, substrate release, or cell lysis. Cell killing can also be a measure of peptidoglycan digestion. A more direct method is to track the number of molecules transferred into the periplasmic space, e.g., using a detectable label. The efficiency of transfer of a particular transfer segment will often be evaluated by measuring an amount of passenger segment transferred. A detectable label can be used to differentiate between the periplasmic space conditions (more oxidizing than outside the OM) and the extracellular environment. See Rajarao et al. (2002) FEMS Microbiology Letters 215:267-272. An efficient receptor-mediated translocation segment will effect at least a 3 fold increase in the level of killing of target host by the killing segment, or at least a 3-fold increase in the level of transfer, as compared to absence of the membrane transfer segment. In some embodiments, the receptor-mediated translocation segment will increase the level of killing or transfer by at least about 5, 7, 10, 15, 20, 30, 50, 80, 100, 150, 250 or more fold compared to the absence of the membrane transfer segment. The assay is typically carried out under conditions which approximate the concentrations which might be used according to the application. The assay will typically measure transfer over a time period ranging from minutes, e.g., about 1, 2, 5, 10, 15, or 30 minutes, to an hour or two. II. Definitions “Receptor Mediated Translocation Domain” (RMTD) is the domain, typically derived from a bacteriocin or related protein, which functions to provide receptor specific translocation of the bacteriocins and chimeric constructs of the invention across the Gram-negative Outer Membrane. Generally domain structure considers secondary or tertiary protein structure in setting boundaries. The identified segments have been described above. Various forms of mutagenesis or means to test variability in the necessary matching of sequence can be empirically tested. Generally, the RMTD will exhibit at least about 60% matching when optimally aligned to a natural sequence, but will preferably have greater matching, e.g., about 65%, 70%, 75%, 80%, preferably 85%, 90%, 95%, or more over the region of alignment. Segments will typically be regions exhibiting particularly higher matching rates than over the entire domain, over regions which may be generally at least about 65%, 70%, 75%, preferably 80%, 85%, 90% or more of the length. The segment matching will be a selected higher matching number over a shorter segment of alignment. In some embodiments, the receptor-mediated translocation domain (RMTD) can comprise two distinct segments. The first is a “Receptor Binding Segment” (RBS), typically derived from a bacteriocin or related protein, which confers selectivity or specificity of interaction of the chimeric construct with a cognate receptor. This interaction is important in the initial interaction between the construct and the target, and generally provides selectivity, which then allows the temporal steps of the translocation process to take place. The RBS will likely be testable for maintaining function as the sequence of the domain is modified, e.g., with substitutions or modification, to evade claim scope. The matching to natural sequence will typically be at least about, e.g., 65% of the natural, about 70%, 75%, 80%, preferably about 85%, 90%, 95%, or more over the region of alignment. Receptor Binding Segments will be regions of particularly high matching over shorter segments. The length of alignment may be generally at least about 65%, 70%, 75%, preferably 80%, 85%, 90% or more of the length of the domain, with any combination of the matching measures. The second segment is a “translocation segment,” (TS) also referred to as a TMD (transmembrane domain), translocating domain, transfer segment, and like terms, which can affect transfer of an operably linked cargo domain across the outer membrane of Gram negative bacteria. Such a domain may itself have the ability to translocate the associated segment across the membrane, or be recognized by an endogenous translocation system which will effect transport of the linked catalytic segment. The chimeric polypeptide can be transferred intact across the membrane, or be modified during translocation. The membrane transfer domain can itself further have the ability to compromise the inner membrane, thereby killing by this additional mechanism. “Cargo Domain” will typically be a functional protein domain which will be translocated when operably linked to the RMTD. The “cargo” descriptor emphasizes that the domain, or segment, may be passive or active. In certain embodiments, the segment may have function, e.g., a killing domain or segment, which effects killing of the target cell upon translocation. The killing may be catalytic, e.g., enzymatic, as a nuclease, protease, muralytic enzyme, metabolic disruptor, structural disassembler, or any of many active functions which can effect toxicity or killing, whether directly or indirectly. The segment or domain may be passive, e.g., as a labelling segment, like GFP or carrier of various chemically attached entities. Thus, the cargo domain may be a polypeptide used as a carrier for toxic conjugates which are chemically transported to the cell compartment, and there released, which may act in a stoichiometric manner. Chemical attachment of antibiotics, antimicrobials, or the like may be delivered into the appropriate cell compartment by the translocation process and released at the appropriate site within the target cell. “Operably linked” refers to functional linkage of elements. Thus two elements are operably linked if the function of the first segment (e.g., translocation domain) operates to translocate a cargo domain, e.g., a muralytic or other functional (killing) segment or domain. A “killing activity” may include an enzymatic activity that kills or decreases the viability or growth rate of the target bacteria. An “environment” of a bacterium can include an in vitro or an in vivo environment. In vitro environments can include a reaction vessel, e.g., holding isolated or purified bacteria, a surface to be sterilized (e.g., in a public health facility), equipment, surfaces in animal quarters, or public health facilities such as water, septic, or sewer facilities. Other in vitro conditions can provide mixed species populations, e.g., including a number of symbiotically or interacting species in close proximity. An in vivo environment can be a host organism infected by a target bacterium. In vivo environments include organs, such as bladder, kidney, lung, skin, heart and blood vessels, stomach, fur, intestine, liver, brain or spinal cord, sensory organs, such as eyes, ears, nose, tongue, pancreas, spleen, thyroid, etc. In vivo environments include tissues, such as gums, nervous tissue, lymph tissue, glandular tissue, and biological fluids, e.g., blood, sputum, etc. Catheter, tubing, implant, and monitoring or treatment devices which are introduced into or attached to the body may be sources of infection under normal usage. Environments also include the surface of food, e.g., fish, meat, or plant materials. Meats include, e.g., beef, pork, chicken, turkey or other poultry. Plant materials include vegetable, fruits, or juices made from fruits and/or vegetables, or may include clothing or shelter. In some embodiments, surfaces that have come in contact with a bacterially-infected food product are treated with a protein of the invention, including a VAME construct or chimera. “Introducing” a composition to an environment includes applying or administering a compound or composition, and such that a targeted bacteria is exposed to the compound or composition. Introducing said compound or composition can be effected by live or dead bacteria which may produce or release such. A “cell wall degrading protein” is a protein that has detectable, e.g., substantial, degrading activity on an accessible cell wall or components thereof “Muralytic” activity can be a result of the degrading activity. Cell wall degrading domains can be derived, e.g., from the tail plates of myoviridae phage or ends of tails from siphoviridae phage, and other phage virion muralytic polypeptides. “GMP conditions” refers to good manufacturing practices, e.g., as defined by the Food and Drug Administration of the United States Government. Analogous practices and regulations exist in Europe, Japan, and most developed countries. The term “substantially” in the above definitions of “substantially pure” generally means at least about 60%, at least about 70%, at least about 80%, or more preferably at least about 90%, and still more preferably at least about 92%, 95%, 97%, or 99% pure, whether protein, nucleic acid, or other structural or other class of molecules. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain a basic chemical structure as a naturally occurring amino acid. Amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. “Protein”, “polypeptide”, or “peptide” refers to a polymer in which most or all of the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used. Additionally, unnatural amino acids, e.g., β-alanine, phenylglycine, and homoarginine, are also included. Amino acids that are not gene-encoded may also be used in the present invention. Furthermore, amino acids that have been modified to include appropriate structure or reactive groups may also be used in the invention. The amino acids used in the present invention may be theD- orL-isomer, or mixtures thereof. TheL-isomers are generally preferred. In addition, other peptidomimetics are also useful in the present invention. For a general review, see, Spatola, A. F., in Weinstein et al. (eds. 1983) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Marcel Dekker, New York, p. 267. The term “recombinant” when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques. In particular, fusions of sequence may be generated, e.g., incorporating an upstream secretion cassette upstream of desired sequence to generate secreted protein product. A “fusion protein,” “chimeric protein,” “protein conjugate,” and like terms refer to a protein comprising amino acid sequences that are in addition to, in place of, less than, and/or different from the amino acid sequences encoding the original or native full-length protein or subsequences thereof. More than one additional domain can be added to a cell wall muralytic protein as described herein, e.g., an epitope tag or purification tag, or multiple epitope tags or purification tags. Additional domains may be attached, e.g., which may add additional killing activities (on the target or associated organisms of a mixed colony or biofilm), targeting functions, or which affect physiological processes, e.g., vascular permeability or integrity of biofilm. Alternatively, domains may be associated to result in physical affinity between different polypeptides to generate multichain polymer complexes. The term “nucleic acid” refers to a deoxyribonucleotide, ribonucleotide, or mixed polymer in single- or double-stranded form, and, unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated or by context, a particular nucleic acid sequence includes the complementary sequence thereof. A “recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of affecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes typically include at least promoters and/or transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors for effecting expression can be included. In certain embodiments, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette. In certain embodiments, a recombinant expression cassette encoding an amino acid sequence comprising a muralytic activity on a cell wall is expressed in a bacterial host cell. A “heterologous sequence” or a “heterologous nucleic acid”, as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. Modification of the heterologous sequence may occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence. The term “isolated” refers to material that is substantially or essentially free from components which interfere with the activity of an enzyme. For a saccharide, protein, or nucleic acid of the invention, the term “isolated” refers to material that is substantially or essentially free from components which normally accompany the material as found in its native state. Typically, an isolated saccharide, protein, or nucleic acid of the invention is at least about 80% pure, usually at least about 90%, or at least about 95% pure as measured by band intensity on a silver stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art. For example, a protein or nucleic acid in a sample can be resolved by polyacrylamide gel electrophoresis, and then the protein or nucleic acid can be visualized by staining. For certain purposes high resolution of the protein or nucleic acid may be desirable and, e.g., HPLC or mass spectroscopy or a similar means for purification may be utilized. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms or by visual inspection. In certain alignments of identity, no gaps are permitted, while in other algorithms, gaps are allowed with appropriate penalty measures. The phrase “substantially identical,” in the context of two nucleic acids or proteins, refers to two or more sequences or subsequences that have, over the appropriate segment, at least greater than about 60% nucleic acid or amino acid sequence identity, about 65%, 70%, 75%, 80%, 85%, 90%, preferably about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over one or more region of the sequences that corresponds to at least about 13, 15, 17, 23, 27, 31, 35, 40, 50, or more amino acid residues in length, more preferably over a region of at least about 60, 70, 80, or 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the entire length of the reference sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these and related algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 and Supplements) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov) or similar sources. A further indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with the protein encoded by the second nucleic acid, as described below. Thus, a protein is typically substantially identical to a second protein, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below. The phrases “specifically binds to a protein” or “specifically immunoreactive with”, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. “Conservatively modified variations” of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at each position where an arginine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Each polynucleotide sequence described herein which encodes a protein also describes possible silent variations, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and UGG which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid which encodes a protein is typically implicit in each described sequence. Those of skill recognize that many amino acids can be substituted for one another in a protein without affecting the function of the protein, e.g., a conservative substitution can be the basis of a conservatively modified variant of a protein such as the disclosed cell wall muralytic proteins. An incomplete list of conservative amino acid substitutions follows. The following eight groups each contain amino acids that are normally conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Alanine (A); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T), Cysteine (C); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton (1984) Proteins). Furthermore, one of skill will recognize that individual substitutions, deletions, or additions which alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are effectively “conservatively modified variations” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. One of skill will appreciate that many conservative variations of proteins, e.g., killing segments or cell wall muralytic proteins, and nucleic acids which encode proteins yield essentially identical products. For example, due to the degeneracy of the genetic code, “silent substitutions” (e.g., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded protein) are an implied feature of each nucleic acid sequence which encodes an amino acid. As described herein, sequences are preferably optimized for expression in a particular host cell used to produce the killing segment, e.g., cell wall muralytic proteins (e.g., yeast, human, and the like). Similarly, “conservative amino acid substitutions,” in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention. See also, Creighton (1984) Proteins, W. H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence generally are also “conservatively modified variations”. The practice of this invention can involve the construction of recombinant nucleic acids and the expression of genes in host cells, preferably bacterial host cells. Optimized codon usage for a specific host will often be applicable. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1999 Supplement) (Ausubel). Suitable host cells for expression of the recombinant polypeptides are known to those of skill in the art, and include, for example, prokaryotic cells, such asE. coli, and eukaryotic cells including insect (baculovirus), mammalian (CHO cells), fungal cells (e.g., yeast,Pichia, Aspergillus niger), and bacteriophage expression systems. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem. 35:1826; Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. III. Commercial Applications Various applications of the bacteriocin polypeptides described herein can be immediately recognized. The proteins can be used for antibacterial treatment of articles which may be contaminated in normal use. Locations, surfaces, equipment, or environments where target bacteria are public health hazards can be treated using the bacteriocin polypeptides described herein. Locations of interest include public health facilities where target bacteria containing materials exist. These materials may include waste products, e.g., liquid, solid, or air. Aqueous waste treatment plants may incorporate the described chimeric bacteriocin constructs to eliminate target bacteria from effluent, whether by treatment with the chimeric bacteriocin constructs or cells that express and release these polypeptides. Solid waste sites can introduce these polypeptides to minimize possibility of target host outbreaks. Food preparation areas and equipment can be regularly treated using the described bacteriocin compositions, thereby providing means to effectively eliminate target bacteria. Medical and other public environments subject to contamination can use similar means to minimize growth and spread of target microorganisms. The present methods can be used in contexts where elimination of target bacteria is desired, including air filtration systems, e.g., for an intensive care unit. The described bacteriocin polypeptides can be used as a protein stabilizer or a preservative, i.e., where the target bacteria are destabilizing agents. Such compositions can be used as part of the formulation for drugs, or preservative for meat or other food products. In some embodiments, these chimeric bacteriocin constructs can be used in aquatic food products, e.g., as a stabilizer or as a component of preservative formulations. Such applications are particularly useful for materials that must be kept antiseptic but cannot contain classical antibiotics. Alternative applications include use in a veterinary or medical context. Means to determine the presence of particular bacteria, or to identify specific targets may utilize the effect of selective agents on the population or culture. Inclusion of bacteriostatic activities to cleaning agents, including washing of animals and pets, may be desired. The bacteriocin polypeptides described herein can be used to treat bacterial infections of, e.g., humans, mammals, animals, and plants. These polypeptides can be administered to a subject prophylacticly or where the subject has a bacterial infection. In addition, the present methods can be applied to display (e.g., zoo or performing), companion (e.g., dogs, cats, other pets), racing (e.g., horses), or farm (e.g., dairy and beef cattle, sheep, goats, pigs, chicken, fish, shrimp, lobster, and the like) animals where the composition is applied to reduce the presence of bacteria. These chimeric bacteriocin constructs can be used to treat infections caused by bacteria that replicate slowly, as the killing mechanism does not depend upon host cell replication. Many current antibacterial agents, e.g., antibiotics, are most useful against replicating bacteria. For example, these bacteriocin polypeptides can be used to target bacteria that replicate with doubling times of about, e.g., 1-72 hours, 1-48 hours, 1-24 hours, 1-12 hours, 1-6 hours, 1-3 hours, or 1-2 hours. Medically relevant Gram-negative cocci species includeNeisseria gonorrhoeaeand spirochaetes (causing a sexually transmitted disease);Neisseria meningitides(causing meningitis); andMoraxella catarrhalis(causing respiratory symptoms). Relevant Gram-negative bacilli species includeHemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Burkholderia, andPseudomonas aeruginosa(respiratory problems);Escherichia coli, Proteus mirabilis, Enterobacter cloacae, andSerratia marcescens(urinary problems), andHelicobacter pylori, Salmonella enteritidis, Salmonella typhi(gastrointestinal problems), and spirochaetes (sexually transmitted disease). Gram-negative bacteria associated with nosocomial infections includeAcinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia, e.g., in intensive-care units of hospital establishments. Other relevant that can be targeted using the presently described bacteriocin polypeptides include Gram-negative species includeStenotrophomonas, Bdellovibrio, acetic acid bacteria, and alpha-proteobacteria such asWolbachia, the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria. Gram-variable organisms, which may have an outer membrane under certain conditions (display a Gram-variable pattern with Gram staining), can also be targeted using the present bacteriocin polypeptides. Gram-variable bacteria include e.g., the generaActinomyces, Arthobacter, Corynebacterium, Mycobacterium, andPropionibacterium, which have cell walls particularly sensitive to breakage during cell division, and display Gram-negative staining. In cultures ofBacillus, Butyrivibrio, andClostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative. In addition, the age of the bacterial culture can influence the results of the Gram stain. IV. Administration The route of administration and dosage of these bacteriocin polypeptides chimeric bacteriocin constructs described herein vary with the infecting bacteria strain(s), the site and extent of infection (e.g., local or systemic), and the subject being treated. The routes of administration include but are not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous (IV), intramuscular, intraperitoneal, intrathecal, intraocular, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and/or meninges. Excipients which can be used as a vehicle for the delivery of the therapeutic will be apparent to those skilled in the art. For example, the muralytic polypeptide can be in lyophilized form and dissolved (resuspended) prior to administration (e.g., by IV injection). The dosage is contemplated to be in the range of about 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 or more chimeric bacteriocin construct molecules per bacterium in the host infection. Depending upon the size of the protein, which may itself be tandemly associated, or in multiple subunit form (dimer, trimer, tetramer, pentamer, etc.) or in combination with one or more other entities, e.g., enzymes or fragments of different specificity, the dose may be about 1 million to about 10 trillion/per kg/per day, and preferably about 1 trillion/per kg/per day, and may be from about 106killing units/kg/day to about 1013killing units/kg/day. Methods to evaluate killing capacity may be similar to methods used by those of skill to evaluate intact replicating phage, e.g., plaque forming units or pfu, though killing units may be better evaluated by determining the number of surviving bacteria after titration of the killing units. Quantification of killing is distinct, since non-replicating phage will not form plaques on bacterial host lawns. Thus, serial dilution methods can be used to evaluate the quantity of “killing” units in place of standard pfu. Serial dilutions of bacterial cultures exposed to the killing compositions can be used to quantify killing units. Total bacterial counts can be compared with viable colony units can establish the viable fraction of bacteria and what fraction was susceptible to the killing constructs. Other means for evaluating stasis activity may include release of intracellular contents, whether natural or loaded, or enzymatic activity on defined or prepared substrates which correspond to natural cell wall structures. The therapeutic(s) are typically administered until successful elimination of the pathogenic bacteria is achieved. The invention contemplates single dosage forms, as well as multiple dosage forms of the compositions of the invention, as well as methods for accomplishing sustained release means for delivery of such single and multi-dosages forms. Broad spectrum formulations can be used while specific diagnosis of the infecting strain is determined. With respect to the aerosol administration to the lungs or other mucosal surfaces, the therapeutic composition is incorporated into an aerosol formulation specifically designed for administration. Many such aerosols are known in the art, and the present invention is not limited to any particular formulation. An example of such an aerosol is the Proventil™ inhaler manufactured by Schering-Plough, the propellant of which contains trichloromonofluoromethane, dichlorodifluoromethane, and oleic acid. Other embodiments include inhalers that are designed for administration to nasal and sinus passages of a subject or patient. The concentrations of the propellant ingredients and emulsifiers are adjusted if necessary based on the specific composition being used in the treatment. The number of enzyme killing units to be administered per aerosol treatment will typically be in the range of about 106to 1013killing units, e.g., about 1012killing units. Typically, the killing will decrease the host replication capacity by at least about 3 fold, e.g., 10, 30, 100, 300, etc., to many orders of magnitude. Slowing the rate of host replication without killing can also have significant therapeutic or commercial value. Genetic inactivation efficiencies may be about 4, 5, 6, 7, 8, or more log units. V. Formulations The invention further contemplates pharmaceutical compositions comprising at least one bacteriocin polypeptide of the invention provided in a pharmaceutically acceptable excipient. The formulations and pharmaceutical compositions of the invention thus contemplate formulations comprising an isolated bacteriocin polypeptide specific for a bacterial host; a mixture of two, three, five, ten, or twenty or more enzymes that affect the same or typical bacterial host; and a mixture of two, three, five, ten, or twenty or more enzymes that affect different bacterial hosts or different strains of the same bacterial host, e.g., a cocktail mixture of bacteriocin polypeptides that collectively inhibit the growth of multiple Gram-negative bacterial species. In this manner, the compositions of the invention can be tailored to the needs of the patient. The compounds or compositions can be sterile or near sterile. A “therapeutically effective dose” is a dose that produces the effects, bacteriostatic (reducing bacterial growth) or bactericidal (killing bacteria), for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. See, e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery;Lieberman(1992)Pharmaceutical Dosage Forms(vols.1-3), Dekker; Lloyd (1999)The Art, Science and Technology of Pharmaceutical Compounding; and Pickar(1999)Dosage Calculations. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the condition may be necessary, and will be ascertainable by those skilled in the art. Various pharmaceutically acceptable excipients are well known in the art. As used herein, “pharmaceutically acceptable excipient” includes a material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive reactions with the subject's immune system. Such excipients include stabilizers, preservatives, salt or sugar complexes or crystals, and the like. Exemplary pharmaceutically carriers include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples include, but are not limited to, standard pharmaceutical excipients such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. In other embodiments, the compositions will be incorporated into solid matrix, including slow release particles, glass beads, bandages, inserts on the eye, and topical forms. Further included are formulations for liposomal delivery, and formulations comprising microencapsulated enzymes, including sugar crystals. Compositions comprising such excipients are formulated by well known conventional methods (see, e.g.,Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Col). The proteins may be subjected to PEGylation to achieve advantages often deriving therefrom. See, e.g., Jevsevar et al. (2010)Biotechnol. J.5:113-128; Brocchini et al. (2008)Adv. Drug Delivery Revs.60:3-12; Jain and Jain (2008)Crit. Rev. Ther. Drug Carrier Syst.25:403-47, PMID: 190626331; and Shaunak et al. (2006)Nature Chemical Biology2:312-313. Alternatives exist for achieving similar stabilizing results. See, e.g., Schellenberger et al. (2009)Nature Biotechnology27:1186-1192. In general, pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, capsules (e.g., adapted for oral delivery), suppositories, microbeads, microspheres, liposomes, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions comprising the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Formulations may incorporate stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value. The pharmaceutical composition can comprise other components in addition to the bacteriocin polypeptide, e.g., more than one active ingredient, e.g., two or more, three or more, five or more, or ten or more different enzymes, where the different enzymes may be specific for the same, different, or accompanying bacteria. For example, the pharmaceutical composition can contain multiple (e.g., at least two or more) defined killing activities, wherein at least two of them in the composition have different bacterial host specificity or different specificity. In this manner, the therapeutic composition can be adapted for treating a mixed infection of different bacteria, or may be a composition selected to be effective against various types of infections found commonly in a particular institutional environment. A select combination may result, e.g., by selecting different groups of killing entities derived from various sources of differing specificity so as to target multiple strains present, or potentially present in the infection. As noted above, the killing activity can be administered in conjunction with other agents, such as a conventional antimicrobial agent or a reagent which provides for efficacy against biofilm or capsule forming cultures. Various materials are described, e.g., in Davies and Marques (2009) J. Bacteriology 191:393-403; Kimura and Itoh (2002) Appl. and Env. Microbiology 69:2491-2497; Kim and Geider (2000) Phytopathology 90:1263-1268; Hughes et al. (1998) J. Appl. Microbiology 85:583-590; and Bartell and Orr (1969) J. Virology 4:580-584. In some embodiments, an additive (e.g., fatty acid) or biofilm depolymerase may be added as an additional domain to the chimeric construct, as an additional component in a formulation, or administered in combination, simultaneously or sequentially, with the described bacteriocin killing activity. Combinations may improve or complement the killing activity selected. VI. Methodology Some aspects of practicing the present invention involve well-known methods general clinical microbiology, general methods for handling bacteriophage, and general fundamentals of biotechnology, principles and methods. References for such methods are listed below. A. General Clinical Microbiology General microbiology is the study of the microorganisms. See, e.g., Sonenshein et al. (ed. 2002)Bacillus Subtilis and Its Closest Relatives: From Genes to CellsAmer. Soc.Microbiol.; Alexander and Strete (2001)Microbiology: A Photographic Atlas for the LaboratoryBenjamin/Cummings; Cann (2001)Principles of Molecular Virology(3d ed.); Garrity (ed. 2005)Bergey's Manual of Systematic Bacteriology(2 vol. 2d ed.) Plenum; Salyers and Whitt (2001)Bacterial Pathogenesis: A Molecular Approach(2d ed.) Amer. Soc. Microbiol.; Tierno (2001)The Secret Life of Germs: Observations and Lessons from a Microbe HunterPocket Star; Block (ed. 2000)Disinfection, Sterilization, and Preservation(5th ed.) Lippincott Williams & Wilkins Publ.; Cullimore (2000)Practical Atlas for Bacterial IdentificationLewis Pub.; Madigan et al. (2000)Brock Biology of Microorganisms(9th ed.) Prentice Hall; Maier et al. (eds. 2000)Environmental MicrobiologyAcademic Pr.; Tortora et al. (2000)Microbiology: An Introductionincluding Microbiology Place (TM) Website, Student Tutorial CD-ROM, and Bacteria ID CD-ROM (7th ed.), Benjamin/Cummings; Demain et al. (eds. 1999)Manual of Industrial Microbiology and Biotechnology(2d ed.) Amer. Soc. Microbiol.; Flint et al. (eds. 1999)Principles of Virology: Molecular Biology, Pathogenesis, and ControlAmer. Soc. Microbiol.; Murray et al. (ed. 1999)Manual of Clinical Microbiology(7th ed.) Amer. Soc. Microbiol.; Burlage et al. (eds. 1998)Techniques in Microbial EcologyOxford Univ. Press; Forbes et al. (1998)Bailey&Scott's Diagnostic Microbiology(10th ed.) Mosby; Schaechter et al. (ed. 1998)Mechanisms of Microbial Disease(3d ed.) Lippincott, Williams & Wilkins; Tomes (1998)The Gospel of Germs: Men, Women, and the Microbe in American LifeHarvard Univ. Pr.; Snyder and Champness (1997)Molecular Genetics of BacteriaAmer. Soc.Microbiol., ISBN: 1555811027; Karlen (1996)MAN AND MICROBES: Disease and Plagues in History and Modern TimesTouchstone Books; and Bergey (ed. 1994)Bergey's Manual of Determinative Bacteriology(9th ed.) Lippincott, Williams & Wilkins. More recent editions may be available. B. General Methods for Handling Bacteriophage General methods for handling bacteriophage are well known, see, e.g., Snustad and Dean (2002)Genetics Experiments with Bacterial VirusesFreeman; O'Brien and Aitken (eds. 2002)Antibody Phage Display: Methods and ProtocolsHumana; Ring and Blair (eds. 2000)Genetically Engineered VirusesBIOS Sci. Pub.; Adolf (ed. 1995)Methods in Molecular Genetics: Viral Gene Techniquesvol. 6, Elsevier; Adolf (ed. 1995)Methods in Molecular Genetics: Viral Gene Techniquesvol. 7, Elsevier; and Hoban and Rott (eds. 1988)Molec. Biol. of Bacterial Virus Systems(Current Topics in Microbiology and Immunology No. 136) Springer-Verlag. C. General Fundamentals of Biotechnology, Principles and Methods General fundamentals of biotechnology, principles and methods are described, e.g., in Alberts et al. (2002)Molecular Biology of the Cell(4th ed.) Garland; Lodish et al. (1999)Molecular Cell Biology(4th ed.) Freeman; Janeway et al. (eds. 2001)Immunobiology(5th ed.) Garland; Flint et al. (eds. 1999)Principles of Virology: Molecular Biology, Pathogenesis, and Control, Am. Soc. Microbiol.; Nelson et al. (2000)Lehninger Principles of Biochemistry(3d ed.) Worth; Freshney (2000)Culture of Animal Cells: A Manual of Basic Technique(4th ed.) Wiley-Liss;Arias and Stewart(2002)Molecular Principles of Animal Development, Oxford University Press; Griffiths et al. (2000)An Introduction to Genetic Analysis(7th ed.) Freeman;Kierszenbaum(2001)Histology and Cell Biology, Mosby; Weaver (2001)Molecular Biology(2d ed.) McGraw-Hill; Barker (1998)At the Bench: A Laboratory NavigatorCSH Laboratory; Branden and Tooze (1999)Introduction to Protein Structure(2d ed.), Garland Publishing; Sambrook and Russell (2001)Molecular Cloning: A Laboratory Manual(3 vol., 3d ed.), CSH Lab. Press; and Scopes (1994)Protein Purification: Principles and Practice(3d ed.) Springer Verlag. More recent editions may be available. D. Mutagenesis; Site Specific, Random, Shuffling Based upon the structural and functional descriptions provide herein, homologs and functional variants can be generated. Segments with penetration functions can be found by structural homology. These may also serve as the starting points to screen for variants of the structures, e.g., mutagenizing such structures and screening for those which have desired characteristics, e.g., broader substrate specificity. Standard methods of mutagenesis may be used, see, e.g., Johnson-Boaz et al. (1994)Mol. Microbiol.13:495-504; U.S. Pat. Nos. 6,506,602, 6,518,065, 6,521,453, 6,579,678. E. Screening Screening methods can be devised for evaluating mutants or new candidate killing segments. Killing activity screens can use crude bacteria cultures, isolated substrate components, reactant preparations, synthetic substrates, or purified reagents to determine the affinity and number of substrate sites on target cells. Penetration assays can be incorporated to evaluate integrity of the outer membranes of target strains, lawn inhibition assays, viability tests of cultures, activity on target substrate preparations or other substrates, or release of components may be evaluated. For example, in a cell wall muralytic function assay, amidase activity may be measured by release of soluble N-acetyl hexose amines (e.g., modified Morgan-Elson reaction) or endopeptidase activity by assay for free amino groups (L-alanine for ala-gly endopeptidases, L-glycine for gly-gly endopeptidases) using a DNFB assay), all three of these assays based on Petit et al. (1966) Biochemistry 5:2764-76. Gly-gly endopeptidase activity can also be measured as the release of free amino groups from N-acetylated hexaglycine (acetyl-Gly6), see Kline et al. (1994) Anal. Biochem. 217:329-331. Linkers can be tested to compare the effects on membrane transfer or degradation, or to compare the activities of various orientations of the active fragments. Panels of targets (e.g., Gram-negative, Gram-positive, mycobacteria and spores) can be screened using killing segments to determine which fragments are critical or efficient on a broader or narrower spectrum of targets. One method to test for, e.g., a cell wall degrading activity is to treat phage with mild detergents or denaturants to release proteins associated with the virion. These proteins are further tested for wall degrading or muralytic activity on bacterial cells. Another method is to determine cell wall degradation activity or lysis from without (LO) on a phage resistant host. A third method to assess wall degrading or muralytic activity associated with phage structural component is to perform Zymogram assays, e.g., where a pure phage preparation is electrophoresed on SDS-polyacrylamide gel incorporating autoclaved host cells. Proteins on the gels are allowed to renature in situ and then act upon the cell wall components giving rise to clear “muralytic” zones when the rest of the gel stains blue with methylene blue dye. See, e.g., Lepeuple et al, (1998)Appl. Environ. Microbiol.64:4142-428. The clear zones are visualized and the protein band from each zone is eluted. The protein can be identified, e.g., by N-terminal sequencing or by Mass spectrometry. The coding sequence for the degrading protein can then be isolated. VII. Isolation of Nucleic Acids Encoding Bacteriocins; Component Domains The invention further provides nucleic acids that encode the killing segment or membrane transfer proteins. Such polynucleotides may encode, e.g., bacteriocins described herein, and other killing domains as described above. Nucleic acids that encode killing segment polypeptides are relevant to the nucleic acid embodiments of the invention. These nucleic acids (e.g., cDNA, genomic, or subsequences (probes)) can be cloned, or amplified by in vitro methods such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), or the self-sustained sequence replication system (SSR). Besides synthetic methodologies, a wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc.; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., 1994 Supplement); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0246864. A DNA that encodes a cargo domain can be prepared by a suitable method described above, including, e.g., cloning and restriction of appropriate sequences with restriction enzymes. Nucleic acids encoding a desired killing segment can be isolated by routine cloning methods. An exemplary nucleotide sequence of, e.g., a cell wall degrading polypeptide, e.g., in Accession Number YP_024486, can be used to design probes that specifically hybridize to a gene; or to an mRNA, encoding a killing protein or segment, in a total nucleic acid sample (e.g., in a Southern or Northern blot). Once the target nucleic acid encoding the killing protein or segment is identified, it can be isolated according to standard methods known to those of skill in the art. Further, the isolated nucleic acids can be cleaved with restriction enzymes to create nucleic acids encoding the full-length killing polypeptide, or subsequences thereof, e.g., containing subsequences encoding at least a subsequence of a catalytic domain of a killing polypeptide. These restriction enzyme fragments, encoding a killing polypeptide or subsequences thereof, can then be ligated, for example, to produce a nucleic acid encoding a killing polypeptide. Similar methods can be used to generate appropriate linkers between fragments. A nucleic acid encoding an appropriate polypeptide, or a subsequence thereof, can be characterized by assaying for the expressed product. Assays based on the detection of the physical, chemical, or immunological properties of the expressed polypeptide can be used. For example, one can identify a killing segment polypeptide by the ability of a polypeptide encoded by the nucleic acid to kill target bacterial cells, e.g., as described herein Also, a nucleic acid encoding a desired polypeptide, or a subsequence thereof, can be chemically synthesized. Suitable methods include the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill recognizes that while chemical synthesis of DNA is often limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences. Nucleic acids encoding a desired polypeptide, or subsequences thereof, can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid sequence or subsequence is PCR amplified, using a sense primer containing one restriction enzyme site (e.g., NdeI) and an antisense primer containing another restriction enzyme site (e.g., HindIII). This will produce a nucleic acid encoding the desired polypeptide or subsequence and having terminal restriction enzyme sites. This nucleic acid can then be easily ligated into a vector containing a nucleic acid encoding the second molecule and having the appropriate corresponding restriction enzyme sites. Suitable PCR primers can be determined by one of skill in the art using the sequence information provided in GenBank or other sources. Appropriate restriction enzyme sites can also be added to the nucleic acid encoding the cargo polypeptide or a polypeptide subsequence thereof by site-directed mutagenesis. The plasmid containing a cargo polypeptide-encoding nucleotide sequence or subsequence is cleaved with the appropriate restriction endonuclease and then ligated into an appropriate vector for amplification and/or expression according to standard methods. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds) Academic Press Inc. (1990); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomeli et al. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. Some nucleic acids encoding cargo polypeptides can be amplified using PCR primers based on the sequence of the identified polypeptides. Other physical properties, e.g., of a recombinant cargo polypeptide expressed from a particular nucleic acid, can be compared to properties of known desired polypeptides to provide another method of identifying suitable sequences or domains, e.g., of the cargo proteins that are determinants of bacterial specificity, binding specificity, and/or catalytic activity. Alternatively, a cargo polypeptide encoding nucleic acid or recombinant cargo polypeptide gene can be mutated, and its role as a cargo polypeptide, or the role of particular sequences or domains established by detecting a variation in bacterial “function” normally enhanced by the unmutated, naturally-occurring, or control cargo polypeptide. Those of skill will recognize that mutation or modification of killing polypeptides of the invention can be facilitated by molecular biology techniques to manipulate the nucleic acids encoding the polypeptides, e.g., PCR. Other mutagenesis or gene shuffling techniques may be applied to the functional fragments described herein, including linker features compatible with chimeric constructs. Functional domains of newly identified killing polypeptides can be identified by using standard methods for mutating or modifying the polypeptides and testing them for activities such as acceptor substrate activity and/or catalytic activity, as described herein. The sequences of functional domains of the various killing proteins can be used to construct nucleic acids encoding or combining functional domains of one or more killing polypeptides. These multiple activity polypeptide fusions can then be tested for a desired bacteriostatic or bacteriolytic activity. Particular examples of sources for killing polypeptides include prophage sequences, including incomplete remnants of functional phage genomes, or pyocin-like structures, including particles derived from phage-like genetic segments, e.g., deletion or mutated genetic remnants of phage remaining in the DNA of a bacterium. Nucleic acids encoding killing polypeptides can be identified by alignment and comparison with known nucleic acid or amino acid sequences of killing polypeptides, e.g., to determine the amount of sequence identity between them. This information can be used to identify and select polypeptide domains that confer or modulate killing polypeptide activities, e.g., target bacterial or binding specificity and/or degrading activity based on the amount of sequence identity between the polypeptides of interest. For example, domains having sequence identity between the killing polypeptides of interest, and that are associated with a known activity, can be used to construct polypeptides containing that domain and other domains, and having the activity associated with that domain (e.g., bacterial or binding specificity and/or killing activity). Similar strategies may be applied to isolate appropriate domains or motifs, or to linkers for spacing between domains. VIII. Expression of Desired Polypeptides in Host Cells The proteins described herein can be expressed in a variety of host cells, includingE. coli, other bacterial hosts, and yeast. The host cells can be microorganisms, such as, for example, yeast cells, bacterial cells, or filamentous fungal cells. Examples of suitable host cells include, for example,Azotobactersp. (e.g.,A. vinelandii),Pseudomonassp.,Rhizobiumsp.,Erwiniasp.,Escherichiasp. (e.g.,E. coli),Bacillus, Pseudomonas, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus, Staphylococcus, andKlebsiellasp., among many others. The cells can be of any of several genera, includingSaccharomyces(e.g.,S. cerevisiae),Candida(e.g.,C. utilis, C. parapsilosis, C. krusei, C. versatilis, C. lipolytica, C. zeylanoides, C. guilliermondii, C. albicans, andC. humicola),Pichia(e.g.,P. farinosaandP. ohmeri),Torulopsis(e.g., Tcandida, T. sphaerica, T. xylinus, T. famata, andT. versatilis), Debaryomyces (e.g.,D. subglobosus, D. cantarellii, D. globosus, D. hansenii, andD. japonicus),Zygosaccharomyces(e.g.,Z. rouxiiandZ. bailii),Kluyveromyces(e.g.,K. marxianus),Hansenula(e.g.,H. anomalaandH. jadinii), and Brettanomyces (e.g.,B. lambicusandB. anomalus). Examples of useful bacteria include, but are not limited to,Escherichia, Enterobacter, Azotobacter, Erwinia, Klebsielia, Bacillus, Pseudomonas, Proteus, andSalmonella. Eukaryotic cells, e.g., CHO or yeast cells, can also be used for production. Once expressed in a host cell, the chimeric bacteriocin constructs can be used to prevent growth or kill target bacteria. In some embodiments, the described bacteriocin construct is used to decrease growth of a Gram-negative bacterium. In some embodiments, the protein is used to decrease growth of aKlebsiella, Pseudomonas, e.g.,Pseudomonas aeruginosa, orEscherichiabacterium. Fusion constructs combining such fragments can be generated, including fusion proteins comprising a plurality of killing activities. Typically, a polynucleotide that encodes the bacteriocin or chimeric bacteriocin construct is placed under the control of a promoter that is functional in the desired host cell. An extremely wide variety of promoters is well known, and can be used in expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites, etc., can be included. Constructs that include one or more of these control sequences are termed “expression cassettes.” Accordingly, the invention provides expression cassettes into which the nucleic acids that encode fusion proteins, e.g., combining a killing fragment with an outer membrane translocating fragment, are incorporated for expression in a desired host cell. Expression control sequences that are suitable for use in a particular host cell can be obtained by cloning a gene that is expressed in that cell. Commonly used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PLpromoter and N-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128. For expression of bacteriocins or chimeric bacteriocin constructs in prokaryotic cells other thanE. coli, a promoter that functions in the particular prokaryotic production species is used. Such promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used. For example, the hybrid trp-lac promoter functions inBacillusin addition toE. coli. A ribosome binding site (RBS) is conveniently included in the expression cassettes of the invention. An exemplary RBS inE. coliconsists of a nucleotide sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine and Dalgarno (1975)Nature254:34; Steitz, In Biological regulation and development: Gene expression (ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY). For expression of proteins in yeast, convenient promoters include GAL1-10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), and MFα (Herskowitz and Oshima (1982) in The Molecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209). Another suitable promoter for use in yeast is the ADH2/GAPDH hybrid promoter as described in Cousens et al., Gene 61:265-275 (1987). For filamentous fungi such as, for example, strains of the fungiAspergillus(McKnight et al., U.S. Pat. No. 4,935,349), examples of useful promoters include those derived fromAspergillus nidulansglycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093 2099 (1985)) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al.). Either constitutive or regulated promoters can be used in the present invention. Regulated promoters can be advantageous because the host cells can be grown to high densities before expression of the fusion proteins is induced. High level expression of heterologous polypeptides slows cell growth in some situations. An inducible promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals. Such promoters are referred to herein as “inducible” promoters, which allow one to control the timing of expression of the desired polypeptide. ForE. coliand other bacterial host cells, inducible promoters are known to those of skill in the art. These include, for example, the lac promoter, the bacteriophage lambda PLpromoter, the hybrid trp-lac promoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc. Nat'l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat'l. Acad. Sci. USA 82: 1074-8). These promoters and their use are discussed in Sambrook et al., supra. The construction of polynucleotide constructs generally requires the use of vectors able to replicate in bacteria. A plethora of kits are commercially available for the purification of plasmids from bacteria (see, e.g., EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech; StrataCleanJ, from Stratagene; and, QIAexpress Expression System, Qiagen). The isolated and purified plasmids can then be further manipulated to produce other plasmids, and used to transfect cells. Cloning inStreptomycesorBacillusis also possible. Selectable markers are often incorporated into the expression vectors used to express the polynucleotides of the invention. These genes can encode a gene product, such as a polypeptide, necessary for the survival or growth of transformed host cells grown in a selective culture medium. A number of selectable markers are known to those of skill in the art and are described for instance in Sambrook et al., supra. Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques as described in the references cited above. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required. To confirm correct sequences in plasmids constructed, the plasmids can be analyzed by standard techniques such as by restriction endonuclease digestion, and/or sequencing according to known methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are well-known to persons of skill. A variety of common vectors suitable for use as starting materials for constructing the expression vectors of the invention are well known in the art. For cloning in bacteria, common vectors include pBR322 derived vectors such as pBLUESCRIPT™, and λ-phage derived vectors. In yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses). Expression vectors can be introduced into a chosen host cell using standard methods known to those of skilled in the art. For example, the expression vectors can be introduced into prokaryotic cells, includingE. coli, by calcium chloride transformation, and into eukaryotic cells by calcium phosphate treatment or electroporation. Translational coupling can be used to enhance expression. The strategy uses a short upstream open reading frame derived from a highly expressed gene native to the translational system, which is placed downstream of the promoter, and a ribosome binding site followed after a few amino acid codons by a termination codon. Just prior to the termination codon is a second ribosome binding site, and following the termination codon is a start codon for the initiation of translation. The system dissolves secondary structure in the RNA, allowing for the efficient initiation of translation. See Squires, et al. (1988), J. Biol. Chem. 263: 16297-16302. The various polypeptides of the invention can be expressed intracellularly, or can be secreted from the cell. Intracellular expression often results in high yields. If necessary, the amount of soluble, active fusion polypeptide may be increased by performing refolding procedures (see, e.g., Sambrook et al., supra.; Marston et al. (1984) Bio/Technology 2:800; Schoner et al. (1985) Bio/Technology 3:151). In embodiments in which the polypeptide is secreted, either into the periplasm or into the extracellular medium, the DNA sequence is often linked to a cleavable signal peptide sequence. The signal sequence directs translocation of the fusion polypeptide through the cell membrane. An example of a suitable vector for use inE. colithat contains a promoter-signal sequence unit is pTA1529, which has theE. coliphoA promoter and signal sequence (see, e.g., Sambrook et al., supra.; Oka et al. (1985) Proc. Natl. Acad. Sci. USA 82:7212; Talmadge et al. (1980) Proc. Natl. Acad. Sci. USA 77:3988; Takahara et al. (1985) J. Biol. Chem. 260:2670). In another embodiment, the fusion polypeptides are fused to a subsequence of protein A or bovine serum albumin (BSA), for example, to facilitate purification, secretion, or stability. Affinity methods, e.g., using substrate for the catalytic fragment may be appropriate. The bacteriocin polypeptides of the invention can also be further linked to other polypeptide segments, e.g., biofilm depolymerase segments. This approach often results in high yields, because normal prokaryotic control sequences direct transcription and translation. InE. coli, lacZ fusions are often used to express heterologous proteins. Suitable vectors are readily available, such as the pUR, pEX, and pMR100 series. For certain applications, it may be desirable to cleave extraneous sequence from the fusion polypeptide after purification. This can be accomplished by any of several methods known in the art, including cleavage by cyanogen bromide, a protease, or by Factor Xa(see, e.g., Sambrook et al., supra.; Itakura et al. (1977) Science 198:1056; Goeddel et al. (1979) Proc. Natl. Acad. Sci. USA 76:106; Nagai et al. (1984) Nature 309:810; Sung et al. (1986) Proc. Natl. Acad. Sci. USA 83:561). Cleavage sites can be engineered into the gene for the fusion polypeptide at the desired point of cleavage. More than one recombinant polypeptide may be expressed in a single host cell by placing multiple transcriptional cassettes in a single expression vector, or by utilizing different selectable markers for each of the expression vectors which are employed in the cloning strategy. A suitable system for obtaining recombinant proteins fromE. coliwhich maintains the integrity of their N-termini has been described by Miller et al (1989)Biotechnology7:698-704. In this system, the gene of interest is produced as a C-terminal fusion to the first 76 residues of the yeast ubiquitin gene containing a peptidase cleavage site. Cleavage at the junction of the two moieties results in production of a protein having an intact authentic N-terminal reside. IX. Purification of Desired Polypeptides A crude cellular extract containing the expressed intracellular or secreted polypeptides described herein can be used in the methods of the present invention. The bacteriocin polypeptides can also be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Because the degrading segments, at least, derive from phage proteins selected for stability, purification can involve denaturation of contaminating materials. Substantially pure compositions are typically about 70, 75, 80, 85, 90, 92, 95, 98 to 99% or higher homogeneous. The purified polypeptides can also be used, e.g., as immunogens for antibody production, which antibodies may be used in immunoselection purification methods. To facilitate purification of the polypeptides of the invention, the nucleic acids that encode them can also include a coding sequence for an epitope or “tag” for which an affinity binding reagent is available, e.g., a purification tag. Examples of suitable epitopes include the myc and V-5 reporter genes; expression vectors useful for recombinant production of fusion polypeptides having these epitopes are commercially available (e.g., Invitrogen (Carlsbad CA) vectors pcDNA3.1/Myc-His and pcDNA3.1/V5-His are suitable for expression in mammalian cells). Additional expression vectors suitable for attaching a tag to the polypeptides of the invention, and corresponding detection systems are known to those of skill in the art, and several are commercially available (e.g., FLAG, Kodak, Rochester NY). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity ligands. Typically, six adjacent histidines are used, although one can use more or fewer than six. Suitable metal chelate affinity ligands that can serve as the binding moiety for a polyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli (1990) Genetic Engineering: Principles and Methods, J. K. Setlow, Ed., Plenum Press, NY; commercially available from Qiagen (Santa Clarita, CA)). Purification tags also include maltose binding domains and starch binding domains. Purification of maltose binding domain proteins is known to those of skill in the art. Other haptens that are suitable for use as tags are known to those of skill in the art and are described, for example, in the Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene OR). For example, dinitrophenol (DNP), digoxigenin, barbiturates (see, e.g., U.S. Pat. No. 5,414,085), and several types of fluorophores are useful as haptens, as are derivatives of these compounds. Kits are commercially available for linking haptens and other moieties to proteins and other molecules. For example, where the hapten includes a thiol, a heterobifunctional linker such as SMCC can be used to attach the tag to lysine residues present on the capture reagent. One of skill would recognize that certain modifications can be made to the catalytic or functional domains of the bacteriocin polypeptides without diminishing their biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the catalytic domain into a fusion polypeptide. Such modifications are well known to those of skill in the art and include, for example, the addition of codons at either terminus of the polynucleotide that encodes the catalytic domain, e.g., a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction enzyme sites or termination codons or purification sequences. The following discussion of the invention is for the purposes of illustration and description, and is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. All publications, patents, patent applications, Genbank numbers, and websites cited herein are hereby incorporated by reference in their entireties for all purposes. Later versions of textbooks may include more recent methodologies. EXAMPLES Example I:KlebsiellaType Bacteriocins; Klebicins Klebicins are high molecular weight (>30 kDa) bacteriocins produced byKlebsiellaspp. Like other bacteriocins, klebicins are also modular proteins having three domains. Although Klebicins such as Klebicin B, C, CCL, and D were sequenced and some of them were proposed to be used for epidemiological typing ofKlebsiellastrains, very little is known about their antibacterial properties. P628 (Wild-type Klebicin CCL): Klebicin CCL is identical to bacteriocin Cloacin DF13, which is produced byEnterobacter cloacae. Cloacin DF13 utilizes the Tol-ABQR pathway for translocation and employs LutA as a cell surface receptor. The receptor for the Klebicin is expected to be modulated by the presence of iron. The bacteria uses siderophores to scavenge iron from the environment and these siderophores enter the cell using the receptor expressed on the cell surface. The near-identity of DF13 and klebicin CCL suggest that the Tol pathway and the LutA receptor are shared between these species. The klebicin CCL is expected to be a nuclease with specific degradation of rRNA. Since LutA is distributed as a cell surface receptor in many Enterobacteriacea, the klebicin CCL might have broad killing range. The receptor for the Klebicin is expected to be modulated by the presence of iron. The bacteria uses these receptors to scavenge iron from the environment by releasing siderophores and these siderophores enter the cell using the receptor expressed on the cell surface. Based on the published DNA sequence (AF190857.1), we isolated Klebicin CCL fromKlebsiellaspp. in GangaGen bacterial collection and cloned into anE. coliexpression vector along with its immunity gene for heterologous expression. Screening of Klebcin CCL Immunity Gene inKlebsiellaStrains: Primers were designed to screen for the presence of klebicin CCL, using the sequence available form the database. Since the immunity gene is a small product and always associated with the klebicin, immunity gene PCR was done. Several clinicalKlebsiellaspp. isolates were screened by colony PCR. Out of the 19 isolates tested, 4 were positive for immunity gene and these four strains are expected to harbour the klebicin CCL gene. The results are shown in Table 4. Strain B2092 was used for isolating the CCL gene for cloning. TABLE 4Klebicin CCLStrainsImmunity PCRB2092+B2093−NDM KL1−NDM KL2+B2095−MTCC 109−B2091−B2107−NDM KL3+NDM KL5−B2063+B2062−B2023−B2058−B236−B2007−B2108−B2094−NDM KL7− Cloning and Expression of Klebicin CCL: The gene encoding the klebicin CCL along with its immunity gene was PCR amplified fromKlebsiellastrain B2092 and cloned intoE. coliexpression vector pET26b at NdeI-XhoI site, for expression in native form without any affinity tags.E. colitransformants were screened by PCR, plasmid DNA isolated from the positive clones and presence of the insert confirmed by restriction digestion analysis. 5 out of the 6 clones tested released the cloned insert of ˜1.9 kb. The clones were sequence confirmed and test protein expression was done. Protein Expression: Test protein expression was performed inE. coliER2566 by inducing with 1 mM IPTG at 37° C. for 4 hours. The expected size of fusion protein is ˜60 kDa. After 4 hrs of IPTG induction, the cells were pelleted, resuspended in 20 mM sodium phosphate buffer pH 7 and sonicated to lyse the cells. The soluble and insoluble fraction of the cells was separated by centrifugation at 10000 rpm for 15 minutes. The supernatant and pellets were analyzed on a 12% acrylamide gel. Clones 1, 3, and 4 expressed the protein of interest and is exclusively present in the soluble form. Clone #1 was designated as pGDC 628. Purification of P628: Since the P628 was expressed without any affinity tag, it was purified by conventional ion-exchange chromatography. Briefly, the sonicated supernatant fration was passed through anion exchange chromatography matrix, UnoQ and the flowthrough was collected. The collected flowthough was then loaded onto a cation exchange chromatography matrix, UnoS. The protein bound matrix was washed and the protein was eluted with increasing concentration of NaCl containing buffer. A step gradient elution with 100 mM, 300 mM, 500 mM and 1M NaCl was done and the samples were analyzed on a 12% acrylamide gel. P628 bound to the cation exchange matrix and the bound protein was eluted in 300 and 500 mM NaCl. These fractions were dialyzed against 20 mM SPB pH 7.0 separately overnight to remove NaCl. Protein concentration was estimated by Bradford assay, 1 mg/ml and 1.3 mg/ml. Activity of Purified P628: The antibacterial activity of the purified P628 was determined by three assays—a) lawn inhibition assay, b) CFU drop assay and c) MIC assay. a) Lawn Inhibition Assay Lawn inhibition assay is a simple qualitative assay to determine the antibacterial activity of a test protein. In this assay, a bacterial lawn using a test isolate is made on LB agar plate and a defined concentration of the test protein is placed on the lawn, air dried, and incubated at 37° C. for 16-18 hrs. A positive result would indicate a clear inhibition zone on the lawn. Since the cell surface receptor LutA is present in Enterobacteriacea family, the P628 was tested on lawns ofKlebsiellaspp. isolates andE. coliisolates. P628 was tested on 69Klebsiellaspp. clinical isolates and 41E. coliclinical isolates. 20 μL of 1 mg/mL (20 μg) P628 was placed on the lawns of the clinical isolates made on LB agar. The plates were incubated at 37° C. for 16-18 hrs. The P628 showed inhibition zone on 85Klebsiellaisolates corresponding to 70% of the total tested isolates and 6E. coliisolates corresponding to 15% of the tested isolates. The lysis zone on lawns were variable with very clear lysis zones (rated 3+), moderate lysis zones (rated 2+) and turbid lysis zones (1+). The percentage is represented in table 5 below. TABLE 5Total Klebsiella isolates tested - 102Sensitive isolates - 78/102 (76%)E. coliisolates tested - 71Sensitive isolates - 20/71 (28%) P628 shows lysis on 76% of the testedKlebsiellaspp., suggesting that this could be a potent protein. Although the LutA receptor is distributed inE. colias well, only 28% of the testedE. colistrains are sensitive to P628. b) CFU Drop Assay: The antibacterial activity of P628 was tested againstKlebsiella pneumoniaeclinical isolate B2094 in both LB media and Fetal Bovine Serum (FBS). Briefly, ˜106cells/mL of B2094 were resuspended in LB or FBS and treated with 100 and 200 μg/mL of P628 in 20 mM SPB pH 7.0 in a volume of 200 μL. The reaction mixture was incubated at 37° C. for 2 hours and the remaining number of viable cells were enumerated by dilution plating on LB plates and incubated at 37° C. for 18 hrs. P628 killedK. pneumoniaein both LB and FBS. However, the activity was much better in FBS with ˜4 logs cell killing obtained in FBS and 1 log cell killing in LB media. The results are shown inFIG.1. P628 has potent antibacterial activity against clinicalK. pneumoniaestrain B2094 and it is active in serum. c) CFU Drop Assay with Additional Strains: CFU drop assay with additional strains were done in growth media and FBS. In this assay, antibacterial activity of P628 was tested on two additional clinicals isolates ofK. pneumoniae, B2064 and B2065. These strains were treated with 200 μg/mL of P628 in Cation adjusted Muller Hinton Broth (CA-MHB medium), 50% FBS and 75% FBS. The reaction mixture was incubated at 37° C. for 2 hours and the remaining number of viable cells were enumerated by dilution plating on LB plates and incubated at 37° C. for 18 hrs. P628 is active in both CA-MHB and FBS on tested isolates with at least 2 logs cell killing obtained in both media (FIGS.2A and2B) P628 demonstrates potent antibacterial activity against testedK. pneumoniaeclinical isolates. d) MIC: Minimum inhibitory concentration (MIC) was determined using a modified Clinical and Laboratory Standards Institute (CLSI) broth microdilution procedure onK pneumoniaestrain 2094 in CA-MHB, Casamino acids media (CAA) and FBS. A 10-point MIC was set up in microtitre plates in duplicates with two-fold dilutions starting at 875 μg/mL. Each well was inoculated with 5×105cells of the test isolate. Microtiter plates were incubated at 35° C. for 18-20 hrs. The endpoint for this assay was complete inhibition of growth at the end of incubation as determined by colorless wells after addition of Iodonitro tetrazolium (INT) dye. MIC was obtained at 100 μg/mL in CA-MHB, 14 μg/mL in CAA and 219 μg/mL in FBS on strain B2094. Better MIC was obtained with CAA and FBS, indicating that P628 works better in iron replete conditions. e) MIC on Additional Clinical Isolates: 16 additional clinical strains that are resistant to several antibiotics were tested for sensitivity to P628 by MIC in both CAMHB and FBS. The results are shown in Table 6 TABLE 6MIC at 6 h (μg/mL)IsolatesAntibiogramCAMHB50% FBSB2135Amp2.71.4B2437Amp, Amox,1.4<0.3Cefuroxime, Ceftriaozone,CefepimeB2138Ampicillin, cefuroxime,5.41.4CeftriaozoneB2139Ampicillin, Amoxicillin,1.4<0.4Cefuroxime, Ceftriaozone,Gentamicin, Ciprofloxacin,TrimithoprimB2143Ampicillin1.4<0.4B2152Ampicillin, Trimithoprim5.51.4B2153Ampicillin442.7/1.4B2154Ampicillin220.3B2157Ampicillin0.680.08ATCCQC strain0.3<0.0213883B2107Ampicillin, Amoxicillin,87.544Cefozitin, Cefilotine, Gentamicin,cefixime, Trimithoprim,Ticaricillin, Pipericillin,ceftazidime, Ceftriaxome,Ertapenem, Amikacin,Ciprofloxacin, NorfloxaccinB2128Ampicillin3505.4B2129Ampicillin, Ticaricillin, cefalotin,110.7Cefixime, Ceftrioxone,Gentamicin, Nalidixic acid,Ciprofloxacin, Norfloxacin,TrimethoprimB2162Not available0.7<0.3B2105Ampicillin, Amoxicillin,17511Cefozitin, Cefilotine, Gentamicin,Cefixime, Trimithoprim,Ticaricillin, Pipericillin,Ceftazidime, Ceftriaxome,Ertapenem, Amikacin,Ciprofloxacin, NorfloxaccinB2163Not available1144 Drug-resistant clinicalK. pneumoniaeclinical isolates are sensitive to P628. f) Dose Response of P628 onK. pneumoniae: The dose response of P628 in fetal calf serum (FCS) was evaluated with twoK. pneumoniaestrains using the CFU drop assay. Briefly, ˜106cells in 50% FCS at varying concentrations of protein was incubated at 37° C. for 2 hours and remaining number of viable cells were enumerated by plating on LB plates. The experiment was setup in duplicates and the results plotted as average of duplicates. A dose response was performed on a clinical isolate ofK. pneumoniae, B2094, isolated from a patient. P628 in the concentration range of 100 μg/ml to 1 μg/ml was used. The results are shown inFIG.3. While 1 μg/mL demonstrated a static effect, 3 log cell killing was obtained with 10 μg/mL of P628. With this strain, the killing seemed to be saturated at 10 μg/mL with similar killing obtained with 25, 50 and 100 μg/mL. K. pneumoniaeATCC 13883 is a quality control strain for testing antibiotics and is highly sensitive to P628. P628 concentration of 100 μg/ml to 0.25 μg/ml was used. The results are shown inFIG.4. A dose-depended killing was obtained on ATCC 13883 with ˜1 log cell killing obtained with 0.25 μg/ml. More than 5 log cell killing was obtained with 10 μg/ml of P628. Example II: Evaluation of In Vivo Efficacy of P628 in Neutropenic Mouse Model of K.PneumoniaeLung Infection A standard neutropenic mouse model ofKlebsiella pneumoniaelung infection model was used for this study (W. A. Craig and D. R. Andes. 2008. In Vivo Pharmacodynamics of Ceftobiprole against Multiple Bacterial Pathogens in Murine Thigh and Lung Infection Models. Antimicrob. Agents And Chemother. 52, [10] 3492-3496) Six to eight weeks old female BALB/c mice were rendered neutropenic by administration of cyclophosphamide. These immunocompromised mice were challenged intranasally with 106CFU ofKlebsiella pneumoniaestrain ATCC13883. At 2 hours post-infection, a group of animals were treated with P628 at 27 mg/kg via intravenous (IV) route, another group treated with 50 microliters of P628 at 0.27 mg via intranasal route and another group treated with ciprofloxacin at 10 mg/kg by oral route. In groups treated with IV P628 and ciprofloxacin, the treatment regimen was once in 12 hours for three days and the treatment regimen for group treated with intranasal P628 was once a day up to three days. All the animals in the infection control succumb to lung infection by 72 hours. While treatment of animals with intranasal administration of P628 completely protected the animals from lethal lung infection giving 100% protection, only one animal died in the group treated with IV P628 giving 83% protection. Treatment with oral ciprofloxacin also completely protected the mice from lethal infection. The results are presented in Table 7. TABLE 7Survival (%)at 72 hoursGroupDosage and routepost-infectionInfection Control [~106Vehicle: IV0CFU/animal, intranasal]Infection + ReferenceCiprofloxacin (10 mg/kg,100standardp.o.)P628 Only, IV5 ml/kg [~27 mg/kg], IV100Infection plus P628 [IV]5 ml/kg [~27 mg/kg], IV83Infection plus P62850 μl per dose [~270 μg],100[Intranasal]intranasal P628 administered via both intranasal and intravenous routes protected the mice fromK. pneumoniaeinduced lethal lung infection. P628 is efficacious in this animal model. Example III: P636: Klebicn CCL TD Rd—Klebicin B KD Introduction Bacteriocins are a diverse family of protein antibiotics produced by bacteria, which kill members of the same or closely related species. There are few reports of bacteriocins (klebicins) fromKlebsiellaspp., none of them have been characterized and nothing is known about their antibacterial properties. Klebicins have been used for the purpose of typingKlebsiellaspp for many decades, but have not been characterized in terms of their antibacterial properties in vitro or in vivo. These proteins exert their antibacterial activity in a very specific manner by binding to a receptor and translocating into periplasm or cytoplasm where the killing domain of the klebicin exerts bactericidal effect by virtue of its DNAse/RNase activity. The domain organization in klebicins comprises of translocation domain, receptor binding domain and killing domain. The reason behind lack of killing in certain strains is due to either absence of a receptor or presence of an immunity protein. Hence, it should be possible to extend the host range by replacing the killing domain of the klebicin by a similar domain which cannot be neutralized by the immunity protein. Klebicin CCL has RNase activity and is produced byKlebsiellaspp. It has greater than 99% sequence homology with a bacteriocin, cloacin DF13 fromEnterobacter cloacae. Klebicin B has DNase activity and is produced byKlebsiellaspp. The strategy was to replace the killing domain of Klebicin CCL with a killing domain of Klebicin B to overcome the immunity problem thus increasing the antibacterial host range with this chimeric molecule. Generating Klebicin CCL (Translocating Domain-Receptor Binding Domain)—Klebicin B (Killing Domain): Cloning Strategy: The klebicin CCL translocating domain (TD)-receptor binding domain (RBD) was PCR amplified and fused to the PCR amplified product of Klebicin B killing domain (KD) along with the klebicin B immunity protein (The immunity protein is only transcriptionally fused and is essential for the expression of the fusion protein) by overlap extension PCR. The resulting PCR product was cloned into pET26b as NdeI-XhoI. The clones were sequence confirmed and labelled as pGDC 636, Klebicin CCL (translocating domain-receptor binding domain)—Klebicin B (Killing domain) Protein Expression Studies: Protein expression was done inE. coliER2566 by inducing with 1 mM IPTG at 37° C. at 0.8° Da) for 4 hours and checked on SDS-PAGE. To determine if the protein was soluble, the cell pellet was sonicated, the supernatant and pellet separated by centrifugation and loaded on SDS-PAGE. The protein was observed in the supernatant fraction Induced protein cell pellet was resuspended in buffer, sonicated to lyse the cells, separated supernatant and pellet by centrifugation at 10,000 rpm. Protein purification was done from soluble fraction by anion exchange chromatography (unoQ) with Sodium phosphate buffer (pH 7) to retain the contaminating proteins on the matrix and allowing the protein of interest to flow through followed by cation Exchangechromatography (unoS) Sodium phosphate buffer (pH 7) with elution with sodium chloride. The protein purified to ˜90% homogeneity Bactericidal Activity of P636 onK. pneumoniae2094: The antibacterial activity of P636 was tested using the CFU drop assay. ˜106cells in Cas amino acid (CAA) broth and 50% FCS at 200 μg/ml, was incubated at 37° C. for 2 hours and remaining number of viable cells were enumerated. The experiment was setup in duplicates and the results tabulated as average of duplicates. The results are shown inFIG.5. P636 was active in CAA and showed 4 logs drop, however in 50% it did not show any significant drop in cfu. Cell Binding Activity of P636: Cell binding assays were carried out to determine the binding potential of P636 toK. pneumoniaecells. Cells ofKlebsiella pneumoniaeB2094 (108cells) in 10 mM SPB containing 150 mM saline were incubated with protein P636 at 10 μg and incubated at 37° C. for 30 minutes, vials were centrifuged at 10,000 rpm to pellet cells and the cell pellet was washed with buffer. The supernatant and pellet were loaded on SDS-PAGE. Protein alone without cells were maintained as controls. P636 was observed in the supernatant indicating that the protein was soluble in the assay buffer. In addition, P636 observed in the supernatant indicating that the protein did not bind to cells under the conditions tested. Example IV S5 Pyocin—Lysozyme Chimeric Fusions Introduction Bacteriocins are proteinaceous molecules produced by bacteria to kill closely related bacteria. Several bacteriocins are known, e.g.: Colicins, pyocins, pesticins, etc. Pyocins are bacteriocins produced by more than 70% ofPseudomonasspp. The high molecular weight pyocins are the R-type and F-type pyocins and the small molecular weight pyocins are the S-type pyocins. The specificity for the entry of S-type pyocins is determined by a receptor present on the cell surface. These receptors are utilized by the cell for the uptake of iron and referred to as iron-siderophore receptors. The domain organization of S-type pyocins are receptor binding domain (RD), translocation domain (TD), and killing domain (KD). Cloning of S-Type Pyocins and S-Type Pyocin—Lysozyme Chimeric Fusions: S-type pyocins and fusions of S-type pyocin translocation domain and binding domain with lysozyme domains (peptidoglycan degrading domains) were achieved by cloning into pET26b plasmid and sequence confirmed. The source of lysozyme domains were from:a. GP36 CD fromP. aeruginosaphage P134b. Phi29 lysozyme fromB. subtilisphage Phi29c. BP7e lysozyme fromE. coliphage BP7 Physical map of constructs is presented inFIG.6 Protein Purification: Protein expression was done inE. coliER2566 by inducing at 37° C. with 1 mM IPTG at OD600of 0.8 for 4 hours. Induced cell pellet was resuspended in 20 mM sodium phosphate buffer, sonicated to lyse the cells, separated supernatant and pellet by centrifugation at 10,000 rpm. Proteins P624, P625, P626, and P652 were purified from the soluble fraction using two-step ion exchange chromatography. Briefly, the clarified cell lysate was passed through an anion exchange chromatography using unosphere Q matrix (Biorad) and the flow through that contained the protein of interest was collected. The flow through was then passed through a cation exchange chromatography using unosphere S matrix (Biorad) and the bound protein was eluted with a step gradient of NaCl. The protein of interest was eluted in 300 mM NaCl for P624, P625, P626, and P652. The proteins were dialysed against 20 mM SPB, pH 7.0+150 mM NaCl for P624, P626, and P652, and with 20 mM SPB, pH 7.0 for P625. His tagged proteins P623 and P638 were purified by Ni-NTA chromatography, eluted in 300 mM Imidazole and dialysed against 20 mM SPB, pH 7.0+150 mM NaCl for P638 and 20 mM SPB, pH 7.0 for P623. All proteins were purified to −80% homogeneity. OD fall assay: The catalytic activity of all lysozyme domains—GP36 CD, Phi29 lysozyme, and BP7e lysozyme were determined by a turbidity reduction OD fall assay using chloroform treatedP. aeruginosaPA01 cells as a substrate. 50 μg/ml of purified proteins were used in this assay. An active protein by OD fall assay will also suggest the correct folding of the lysozyme domain in the fusion proteins. All the three lysozyme domains were catalytically active. The results are shown inFIG.7. Lawn Inhibition Assay: P. aeruginosaKGN 1665 lawn was prepared by growing colonies in LB broth to an OD600of 0.8 and a lawn was prepared on an LB agar plate. The fusion proteins were spotted at the below mentioned concentrations. P626 was spotted on CAA agar onP. aeruginosaPAO1, and P652 on LB agar onP. aeruginosaDSMZ 50071. P623: 20 μg; P624: 38 μg; P625: 32 μg; P626: 60 μg; P638: 12 μg; P652: 30 μg. Inhibition zone was observed with all the tested proteins except P625 Bactericidal Activity: The antibacterial activity of S5 pyocin and chimeric fusions P623, P624, P625, P626, P638, and P652 were tested againstP. aeruginosaPAO1, using the CFU drop assay. Briefly, ˜106cells in CAA broth and 50% fetal calf serum (FCS) at 200 μg/ml were incubated at 37° C. for 2 hours and enumerated remaining number of viable cells by plating appropriate dilutions on LB agar plates. The experiment was set up in duplicates and the results tabulated as average of duplicates. The respective lysozymes (P200, P198, and P501) were used as negative controls. The results are shown inFIG.8A-8C. P623 and P624 (S5 pyocin-GP36 fusion) were showing bactericidal activity on PA01 in CAA None of the proteins were bactericidal on PA01 in 50% FCS. Example V Using Klebicin and Pyocin to Target Mixed Infections (K.PneumoniaeandP. Aeruginosa) Introduction Klebsiella pneumoniaeandPseudomonas aeruginosaare two biofilm-forming organisms that can coexist during infections of the urinary tract, respiratory tract, and burn wounds and associated with foreign bodies (Childers et al. (2013)). Bacteriocins are proteinaceous molecules naturally produced by bacteria to kill closely related bacteria. Several bacteriocins are known, e.g., Klebicins, pyocins, colicins, pesticins, etc. Klebicins have been used for the purpose of typingKlebsiellaspp for many decades, but have not been characterized in terms of their antibacterial properties in vitro or in vivo. Pyocins are bacteriocins produced by more than 70% ofPseudomonasspp. The high molecular weight pyocins are the R-type and F-type pyocins and the small molecular weight pyocins are the S-type pyocins. The specificity for the entry of S-type pyocins is determined by a receptor present on the cell surface. Cloning of Klebicin CCL and S5 Pyocin Klebicn CCL gene was PCR amplified from the genome ofK. pneumoniae, with its immunity gene, and cloned into pET26b plasmid, expressed inE. coliER2566, and purified by conventional chromatography (anion and cation exchange chromatography). The construct was sequence confirmed and labeled (designated) pGDC 628. S5 type pyocin was PCR amplified from the genome ofP. aeruginosaand cloned into pET26b plasmid, expressed inE. coliER2566, and purified by conventional chromatography (anion and cation exchange chromatography). The construct was sequence confirmed and designated pGDC 652. Lawn Inhibition Assay: A lawn ofK. pneumoniaeB2094 andP. aeruginosaKGN 1665 was prepared on an LB agar plate. Both proteins at 25 μg concentration were spotted on a CAA agar plate. The combination of P628 and P652 showed lawn inhibition in mixed cultures. Bactericidal Activity of P628 and P652 onP. aeruginosaKGN 1665 andK. pneumoniaeB2094 The antibacterial activity of P628 and P652 were tested using the CFU drop assay. ˜106cells ofP. aeruginosaKGN 1665(˜1×106) andK. pneumoniaeB2094 (˜1×106) were mixed in CAA broth at 200 μg/mL and 400 μg/mL, was incubated at 37° C. for 2 hours and remaining number of viable cells were enumerated. The experiment was set up in duplicates and the results tabulated as average of duplicates. The results are shown inFIGS.9A and9B. The combination of P628 and P652 exhibit bactericidal activity in mixed cultures at 400 μg/ml and 200 μg/ml A dose-dependent study with mixed cultures was done to determine the minimum amount of P628 and P652 required to kill the cells by at least 3 orders of magnitude. The results are shown inFIG.10A. Combination of P628 and P652 exhibit bactericidal activity in mixed cultures even at 10 μg/ml in both CAA and FCS. Bactericidal Activity of P628 and P652 onP. aeruginosaKGN 1665 andE. coliB563 The antibacterial activity of P628 and P652 was tested using the CFU drop assay. ˜106cells ofP. aeruginosaKGN 1665 (˜1×106) andE. coliB563 (˜1×106) were mixed in CAA broth and proteins added individually and in combination at 10 μg/ml, was incubated at 37° C. for 2 hours and remaining number of viable cells were enumerated. The experiment was setup in duplicates and the results tabulated as average of duplicates. The results are shown inFIG.10B. The Combination of P628 and P652 exhibit bactericidal activity in mixed cultures at 10 μg/ml. Example VI FYU a Binding Domain—Lysozyme Domain Fusions Introduction Bacteria utilize Iron through receptors on the cell surface for the uptake of iron. The uptake is mediated by molecules called siderophores wherein the siderophore binds to free iron and enters through the receptors following which the iron is released from the siderophore and utilized. Pesticins are bacteriocins produced byYersinia pestisand the receptor for pesticin uptake is the iron uptake receptor FyuA present inYersinia pseudotuberculosisand certain pathogenic strains ofE. coli. Pesticin contains a Fyu A binding domain (FyuA BD) and a peptidoglycan degrading domain (PGD). Lukacik et al. (2012) “Structural engineering of a phage lysin that targets Gram-negative pathogens” Proc Natl Acad Sci USA, 109:9857-62. The authors demonstrated that replacing the PGD domain with a heterologous lysozyme domain from the T4 lysozyme that is structurally similar to its native lysozyme domain was able to enter and kill bacterial cells. Generating Fyu a Binding Domain—T4 Lysozyme and Fyu a Binding Domain—P. aeruginosaPhage P134 Virion Associated Lysozyme GP36 (Cloning Strategy) Fyu A binding domain was fused with T4 lysozyme as NdeI-XhoI site in pET26b as synthetic construct. Fyu A binding domain was fused to theP. aeruginosaphage P134 virion associated lysozyme GP36 in theE. coliexpression vector pET26b into the cloning sites NdeI-XhoI. The clones were sequence confirmed and designated as pGDC 558 (Fyu A BD—T4 lysozyme fusion) and pGDC 567 (Fyu A BD—GP36 fusion) Protein Expression Studies: Test protein expression was performed inE. coliER2566 by inducing with 1 mM IPTG at 37° C. for 4 hours induced at OD600of 0.8. Induced cells were pelleted, resuspended in 20 mM Sodium phosphate buffer and sonicated to lyse the cells. The lysate was then pelleted by centrifugation at 10,000 rpm for 15 minutes and the supernatants and pellets were collected separately and analyzed on an SDS-PAGE gel. Protein expression was observed at −37 kDa for P558 and 42 kDa for P567 on acrylamide gel in soluble fraction of the cells. Purification of Proteins: Protein expression was done inE. coliER2566 by inducing with 1 mM IPTG at 37° C. at 0.8 OD600for 4 hours. Induced cell pellet was resuspended in 20 mM sodium phosphate buffer, sonicated to lyse the cells, separated supernatant and pellet by centrifugation at 10,000 rpm. Protein was purified from the soluble fraction using two-step ion exchange chromatography. Briefly, the clarified cell lysate was passed through an anion exchange chromatography using unosphere Q matrix (Biorad) and the flow through that contained the protein of interest was collected. The flow through was then passed through a cation exchange chromatography using unosphere S matrix (Biorad) and the bound protein was eluted with a step gradient of NaCl. The protein of interest was eluted in 500 mM NaCl. The proteins were dialysed against 20 mM SPB, pH 7.0+300 mM NaCl. OD Fall Assay The catalytic activity of T4 lysozyme and GP36 lysozyme in the fusion proteins were determined by a turbidity reduction OD fall assay using chloroform treatedP. aeruginosaPA01 cells as substrate. 50 μg/ml of purified proteins were used in this assay. An active protein by OD fall assay will also suggest the correct refolding of the lysozyme domain in the fusion proteins. The results are shown inFIG.11. The purified proteins P558 and P567 were catalytically active. Cloning and Expression of FyuA Receptor inE. coliER2566 The FyuA BD fusions utilize FyuA receptor for entry into bacteria. Lab strains ofE. colido not harbor this receptor and hence are not sensitive to these proteins. However, if the receptor could be expressed heterologously from a plasmid in labE. coli, the strain may become sensitive to the fusion proteins. To this end, the FyuA receptor was isolated from anE. coliclinical isolate and cloned into pET26b as NcoI-XhoI for expression as a PelB signal sequence fusion tag for periplasmic localization of the receptor. Protein Expression Studies Test protein expression was performed inE. coliER2566 by inducing with 1 mM IPTG at 37° C. for 4 hours induced at OD600of 0.8. Protein of expected size was observed in the induced cells. The clones were sequence confirmed and designated as pGDC 571. Testing of P558 and P567 on FyuA Expressing ER2566/pGDC571 pGDC571 and pET26b were transformed intoE. coliER2566 and the resulting colonies were grown to an OD600of 0.8 and a lawn prepared on an LB plate. 50 μg of P558 and P567 were spotted on ER2566/pGDC 571+ and ER2566 pET26b (control). Lawn inhibition observed with P558 and P567 indicating that these proteins were active on a FyuA expressingE. colistrain. Effect of P558 and P567 on FyuA ExpressingE. coli The antibacterial activities of P558 and P567 were tested against FyuA expressingE. coliusing the CFU drop assay. ˜107cells of ER2566/pGDC 571 in LB broth were treated with 30 μg/ml and 300 μg/ml of P558 and with 300 μg/ml of P567, incubated at 37° C. for 2 and for 4 hours and enumerated remaining number of viable cells. The experiment was set up in duplicates and the results tabulated as average of duplicates. The results are shown inFIG.12. A static effect observed with P558 at 300 μg/ml until 4 hours. Viability of the cells at respective time points were determined by plating appropriate dilutions on LB plates and incubated these plates at 37° C., for 16-18 hrs. The results are shown inFIG.13. A bacteriostatic effect was observed with P558 (300 μg/ml, with the cell numbers remaining constant even after 4 hours. The effect of P558 and P567 on FyuA expressingE. colias described above was carried out at protein concentrations of 300 μg/ml and 1350 μg/ml for P558 and 1250 μg/ml for P567. As a control, the ER2566 with the vector control (ER2566/pET26b) also was treated with P558 and P567 at the same concentrations. The results are shown inFIG.14. P558 inhibited growth of ER2566 cells expressing FyuA receptor and no growth inhibition observed with control (ER2566/pET26b). Activity of P558 onE. coliER2566/FyuA+ The antibacterial activity of P558 was tested against FyuA expressingE. coliusing the CFU drop assay. Briefly, ˜107cells of ER2566/FyuA in 50% LB broth and 50% fetal calf serum (FCS) were treated with P558 at 300 μg/ml, incubated at 37° C. for 2 and 4 hours and the cell killing was determined by enumerating the remaining number of viable cells. The experiment was set up in duplicates and the results tabulated as average of duplicates. The results are shown inFIGS.15A and15B. Activity of P558 onYersinia pseudotuberculosis: The antibacterial activity of P558 was tested againstYersinia pseudotuberculosisusing the CFU drop assay. Briefly, ˜107cells ofY. pseudotuberculosisin 50% LB broth and 50% fetal calf serum (FCS) were treated with P558 at 300 μg/ml, incubated at 37° C. for 2 and 4 hours and the cell killing was determined by enumerating the remaining number of viable cells. The experiment was set up in duplicates and the results tabulated as average of duplicates. The results are shown inFIG.16. P558 showed static effect onY. pseudotuberculosisin both 50% LB medium and 50 FCS. Activity of P558 onE. coliSLC-6 The antibacterial activity of P558 was tested againstE. coliSLC-6, a urinary tract infection isolate using the CFU drop assay. UTI isolates are known to harbor FyuA gene and express the receptor in the urinary tract that would aid the bacteria to colonize and survive. Briefly, ˜107cells in 50% LB broth and 50% fetal calf serum (FCS) at 300 μg/ml, incubated at 37° C. for 2 and 4 hours and enumerated remaining number of viable cells. The experiment was setup in duplicates and the results tabulated as average of duplicates. The results are shown inFIG.17. P558 showed static effect onE. coliSLC-6 in both 50% LB medium and 50% FCS. Activity of P558 onE. coliUTI Isolates Positive for fyuA Gene PCR ClinicalE. colistrains isolated from urine was screened for the presence of fyuA gene by PCR. Few of the positive ones were taken as test strains for determining the activity of P558. Assay Conditions: 50% LB broth and 50% Fetal calf serum (FCS), Reaction volume: 2 ml. Duration: 2 and 4 hours at 37° C., 200 rpm. Strains tested:E. coliER2566/FyuA, B5031, B5113 (E. coliUTI isolate). The results are shown inFIGS.18A and18B. P558 showed static effect onE. coliB5031 in 50% LB and 50% FCS Activity of P558 onKlebsiellaClinical Isolates Positive for fyuA Gene PCR: (FyuA+) ClinicalKlebsiellastrains isolated from urine were screened for the presence of fyuA gene by PCR. Few of the positive ones were taken as test strains for determining the activity of P558. Assay Conditions: 50% LB broth and 50% Fetal calf serum (FCS), Reaction volume: 2 ml. Duration: 2 and 4 hours at 37° C., 200 rpm. Strains tested:E. coliER2566/FyuA,Klebsiellaspp B2103, Klebsiellaspp B2096 (KlebsiellaPCR positive for FyuA+). The results are shown inFIG.19. P558 showed static effect onE. coliB2103 in 50% LB. MIC of P558 in LB (50%) and FCS (50%) MIC assay was done with P558 in 50% LB and 50% FCS by the CLSI method onE. coliER2566/FyuA,E. coliER2566/pET26b,Y. pseudotuberculosisandE. coliSLC-6. MIC was observed at both 6 hours and 18 hours. The results are shown in Tables 8 and 9. P558 showed very low MIC onE. coliER2566 (FyuA) only at 6 h, however no MIC observed on other strains tested. TABLE 8P558 MIC in μg/mL at 6 hSl. NoIsolates50% MHB50% FCS1E. coliER2566/FyuA0.090.092E. coliER2566/pET26b>925>9253Y. pseudotuberculosis>925>9254E. coliSLC-6>925>925 TABLE 9P558 MIC in μg/mL at 18 hSl. NoIsolates50% MHB50% FCS1E. coliER2566/FyuA>950>9502E. coliER2566/pET26b>950>9503Y. pseudotuberculosis>950>9504E. coliSLC-6>950>950 Other FyuABD Fusions: Fusions of FyuA binding domain and peptidoglycan degrading domains were generated by cloning into pET26b plasmid and sequence confirmed.a. FyuA BD—Phi29 lysozyme fromB. subtilisphage Phi29b. FyuA BD—BP7e lysozyme fromE. coliphage BP7c. FyuA BD—Phi6 P5 lytic enzyme fromP. syringiaephage Phi6d. FyuA BD—GS linker—GP36 CD The proteins were purified by ion exchange chromatography to 90% homogeneity. OD Fall Assay: The catalytic activity of the FyuA fusions were determined by OD fall assay using chloroform treatedP. aeruginosacells as substrate. 50 μg/ml of purified proteins were used in this assay. An active protein by OD fall assay will also suggest the correct refolding of the lysozymes. The results are shown inFIG.20. The purified proteins P581, P583, and P580 were catalytically active as observed by the OD fall obtained. P578 was not active indicating that the catalytic domain was non functional. Effect of FyuA BD Fusions on FyuA ExpressingE. coli: The antibacterial activity of the fusion proteins were tested against FyuA expressingE. coliusing the CFU drop assay. Briefly, ˜107cells of ER2566/FyuA in 50% LB broth were treated with P558 at 300 μg/ml, incubated at 37° C. for 2 and 4 hours and the cell killing was determined by enumerating the remaining number of viable cells. The experiment was set up in duplicates and the results tabulated as average of duplicates. The results are shown inFIGS.21. P558 and P581 inhibited growth of ER2566 cells expressing FyuA receptor. No inhibition was observed with other proteins. Viability of the cells at respective time points were determined by plating appropriate dilutions on LB plates and incubated these plates at 37° C., for 16-18 hrs. The results are shown inFIG.22. P558 showed ˜1 log drop and P581 ˜2 logs drop in 50% LB medium Lawn Inhibition Assay: The fyuA construct pGDC571 was transformed intoE. coliER2566 and the resulting colonies were grown in LB broth to an OD600of 0.8 and a lawn was prepared on an LB agar plate. The fusion proteins were spotted on ER2566/pGDC 571 andY. pseudotuberculosis. Clear inhibition zone observed with P581 on FyuA expressing ER2566 andY. pseudotuberculosis. Activity of P581 on Clinical UTI Strains (FyuA+) in LB and FCS Yersinia pseudotuberculosis, E. coliB5501, B5503, and B5504. Assay Conditions: 50% LB broth and 50% Fetal calf serum (FCS). Reaction volume: 2 ml. Duration: 2 and 4 hours at 37° C., 200 rpm. Cells: 105CFU/mL. Protein: 300 μg/mL. Incubation: 37° C., 200 rpm, 2 h, 4 h. The results are shown inFIG.23. P581 was active onY. pseudotuberculosisin both LB and FCS. Exampe VII Transfer of a Selected Bacteriocin Receptor to a TargeTEscherichiaBacteria The gene encoding the FyuA receptor is PCR amplified fromYersinia pseudotuberculosisgenome (Accession: Z35107.1) using primers containingE. colisignal sequence (e.g., pelB). A broad host range conjugative plasmid (e.g., pLM2) is isolated fromSalmonella typhimuriumLT2 and the above PCR product is cloned at a suitable restriction site, transformed by electroporation intoE. colilab strain and screened by PCR for recombinant clones. The colony containing the gene of interest is the “donor” bacteria. 5 ml of donor and recipient cells (E. coliin which the FyuA receptor has to be expressed) are grown to OD600of 0.5-0.7. 100 microliters of donor and recipient cultures are mixed (Controls: 100 microliters of donor and recipient cells alone), centrifuged to wash cells with 0.85% saline twice. The pellet is resuspended in 20 microliters of saline, and spotted on a well-dried LB agar petri plate. The plate is allowed to dry and incubated overnight at degrees centigrade following which the culture is scraped into 500 microliters saline and vortexed to disrupt mating pairs. The suspension is plated at various appropriate dilutions on respective selection plates, e.g., dual antibiotic plates. Appropriate colonies are typically confirmed for conjugation by PCR for the presence of conjugative plasmid. The transconjugant colony is grown in LB broth to an OD600of 0.8. The culture is diluted to OD600of 0.2 and spread plated on LB agar plate and allowed to dry. Protein P558 (FyuA binding domain— T4 lysozyme) fusion is spotted (10 μg) on the lawn and plate incubated at 37° C. for 17 hours. A zone of inhibition seen as clearance indicates the susceptibility of the bacteria due to the expression of the FyuA receptor. A control culture of the recipient bacteria is also spotted with P558. Example VIII Construction of Bacteriocins Fused with Amps The genes encoding bacteriocins are cloned intoE. coliexpression vectors such as pET plasmids and the expression of the recombinant bacteriocins are confirmed. DNA sequences encoding the AMPS are cloned either at the 5′ or 3′ end of the bacteriocins by PCR based methods to obtain a fusion gene. Different AMP sequences as listed in the table above are fused to various bacteriocins. These fusion genes are cloned into bacterial expression vectors and DNA sequence are confirmed. Alternatively, the DNA sequence encoding the AMPS is synthesized as oligos with appropriate restriction enzyme recognition sites to clone into plasmids that already harbor bacteriocin genes. Protein Expression, Purification and Refolding: All DNA sequence confirmed chimeric bacteriocins are expressed in appropriate laboratoryE. coli. For example,E. coliER2566 carrying the plasmids are grown, e.g., at 37° C. till OD600reached ˜0.8 to 1.0 and the protein expression is induced by addition of IPTG to a final concentration of 1 mM and the induction is done, e.g., at 37° C. for 4 hours. After 4 hours of IPTG induction, the cells are harvested and protein expression checked on an acrylamide gel. Once the expression of the test recombinant chimeric bacteriocin is confirmed, it is purified, e.g., by affinity chromatography. Proteins that are expressed in the soluble fraction of the cells are purified, e.g., using native purification conditions and proteins expressed as inclusion bodies (IBs) are purified under denaturing conditions, using either urea or guanidine hydrochloride to denature the IBs. Refolding of the denatured proteins is done, e.g., by removal of the denaturant, e.g., by dialyzing against appropriate buffer at 4° C. for 16-18 hrs. After refolding the homogeneity of the purified, refolded proteins is analyzed on an acrylamide gel and the protein concentration determined by Bradford's assay. Bactericidal Assays: a) CFU drop assay in buffer and buffered saline: Gram-negative cells grown, e.g., in LB medium, until mid-log phase (OD600of 0.6) are diluted 100-fold in appropriate buffer such as 20 mM HEPES pH 7.0 or 20 mM SPB pH 7.0 with and without 150 mM NaCl to a final density of ˜106CFU/ml. 100 μL of cells are treated with different concentrations (e.g., 50-200 μg/mL) of purified test proteins. The final volume of the reaction mixture is adjusted, e.g., to 200 μL with appropriate buffers. The reaction mixture is incubated, e.g., at 37° C. for 2 hours and enumerated remaining number of viable cells by plating of appropriate dilutions on LB plate followed by overnight incubation at 37° C. The antibacterial activity is calculated by dividing initial number of untreated cells with number of residual cells in log units and plotting the data as bar graph.b) CFU drop assay in growth media: Gram-negative cells grown, e.g., in LB medium, until mid-log phase (OD600of 0.6) are diluted 100-fold in either LB or CA-MHB media to a final density of ˜106CFU/ml. 100 μL of cells are treated with different concentrations (e.g., 50-200 μg/mL) of purified test proteins. The final volume of the reaction mixture is adjusted, e.g., to 200 with appropriate buffers. The reaction mixture is incubated, e.g., at 37° C. for 2 hours, and enumerated remaining number of viable cells by plating of appropriate dilutions on LB plate followed by overnight incubation at 37° C. The antibacterial activity is calculated by dividing initial number of untreated cells with number of residual cells in log units and plotting the data as bar graph.c) CFU drop assay in Fetal Bovine Serum (FBS): Gram-negative cells grown, e.g., in LB medium until mid-log phase (OD600of ˜0.6) are diluted 100-fold in FBS to a final density of ˜106CFU/ml. 100 μL of cells are treated with different concentrations (e.g., 100-400 μg/mL) of purified test proteins. The final volume of the reaction mixture is adjusted, e.g., to 200 μL with CA-MHB media. The reaction mixture is incubated, e.g., at 37° C. for 2 hours and enumerated remaining number of viable cells by plating of appropriate dilutions on LB plate followed by overnight incubation at 37° C. The antibacterial activity is calculated by dividing initial number of untreated cells with number of residual cells in log units and plotting the data as bar graph. Minimum Inhibitory Concentration (MIC) Determination: MIC is determined, e.g., using a modified Clinical and Laboratory Standards Institute (CLSI) broth microdilution procedure on Gram-negative cells in Cation-adjusted Mueller Hinton Broth (CA-MHB media) or in 50% FBS. A 10-point MIC is set up in microtitre plates in duplicates with two fold dilutions. Wells of 96-well polystyrene plated are coated, e.g., with 0.5% BSA for 1 hour at 37° C. and each well is inoculated, e.g., with 5×105cells/mL Gram-negative bacteria. A positive control for growth which is devoid of test proteins is included in the assay. The microtiter plates are incubated, e.g., at 35° C. for 18-20 hrs. The MIC is defined as the minimum concentration that completely inhibits bacterial growth at the end of incubation, e.g., as determined by colorless wells after addition of Iodonitro tetrazolium (INT) dye. INFORMAL SEQUENCE LISTINGNCBI accession AF190857DNA sequence:SEQ ID NO: 1ATGAGTGGTG GAGACGGTCG AGGTCCGGGT AATTCAGGTC TGGGACATAA TGGTGGTCAGGCCAGTGGGA ATGTGAACGG TACGTCTGGT AAAGGCGGCC CTTCATCAGG TGGGGGTACGGATCCAAACA GCGGGCCGGG CTGGGGTACG ACGCATACGC CTAACGGAGA TATTCATAACTACAATCCGG GGGAGTTTGG TCACGGAGGG AATAAACCCG GTGGCAATGG CGGTAACAGCGGCAATCATC CCGGTAGTTC TGGTGGCAGA CAGTCTTCGG CCACAGCGAT GGCCTTCGGTCTGCCTGCTC TGGCTACTCC GGGCTCCGGG GGGCTGGCTT TAGCCGTTTC CGGCGATGCGTTGTCGGCAG CCGTTGCTAG TGTGCTGGCT GCCCTGAAAG GGCCGTTTAA GTTTGGTCTGTGGGGGATTG CGATCTACGG TGTGCTGCCT TCTGAGATTG CAAAAGATGA TCCGAAAATGATGTCAAAAA TTATGACGTC ATTACCGGCC GATGCGGTGA CGGAGACTCC GGCAAGTACTTTACCACTGG ACCAGGCGAC GGTTCGTGTC AGACAACGGG TTGTGGATGT GGTGAAGGATGAGCGGCAGC ATATTGCGGT TGTCGCAGGT CGGCCAATGA GTGTCCCTGT GGTGGATGCGAAACCGACAA AACGTCCGGG GGTATTCAGT GTGTCGATTC CGGGTCTCCC GTCTCTGCAGGTGAGCGTAC CGAAAGGTGT TCCGACAGCG AAAGCCCCGC CAAAAGGCAT TGTTGCTGAAAAAGGTGATT CACGTCCGGC TGGTTTTACA GCCGGTGGTA ACTCCCGTGA GGCCGTTATTCGTTTCCCGA AAGAGACCGG ACAGAAGCCG GTTTATGTGT CGGTGACAGA TGTTCTTACCCCGGCACAGG TAAAACAGCG TCAGGAGGAA GAAAAGCGTC GCCAGCAGGC ATGGGACGCCGCTCATCCGG AAGAGGGGCT GAAAAGAGAC TATGATAAAG CGAAAGCCGA GCTGGATGCCGAAGATAAAA ATATTGCGAC CTTAAACAGC CGCATTGCAT CGACAGAGAA GGCGCTCCCCGGTGCAAGGG CTGCTGTTCA GGAAGCCGAT AAAAAGGTGA AAGAGGCAGA GGCGAATAAGGATGATTTTG TGACTTATAA CCCTCCTCAT GAATATGGCT CCGGGTGGCA GGATCAGGTTCGCTATCTTG ATAAGGATAT TCAGAATCAG AATGCGAAAT TAAAAGCGGC TCAGGCATCTTTAAACGCAA TGAATGAATC CTTATCCAGA GATAAGGCTT GCACTTCCCG GGCGATGGAGAGCCGGAAAC AAAAGGAGAA AAAAGCGAAG GATGCAGAAA ATAAGTTAAA TGAGGAAAAGAAAAAACCTC GCAAGGGAGC TAAAGACTAC GGCCATGATT ATCATCCAGC CCCGAAAACTGAAGACATAA AGGGACTGGG TGACCTCAAA AAAGGTACAC CTAAAACACC AATGCAGGGATATCTTTAA GGCGTAAACG CTGGATTGGT GATAAAGGCC GTAAGATTTA TGAATGGGACGGTGGAGGTA GTGAGCTTGA AGGGTATCGT GCCAGTGATG GCGAACACCT CGGGGCATTTTCCCAGCACG CGGGTAAGCA AATTAAAGGT CCGGATCCGA AAGGGCGAAA CATTAAAAAAGATCCTAAAAAmino acid sequence:SEQ ID NO: 2MSGGDGRGPG NSGLGHNGGQ ASGNVNGTSG KGGPSSGGGT DPNSGPGWGT THTPNGDIHNYNPGEFGHGG NKPGGNGGNS GNHPGSSGGR QSSATAMAFG LPALATPGSG GLALAVSGDALSAAVASVLA ALKGPFKFGL WGIAIYGVLP SEIAKDDPKM MSKIMTSLPA DAVTETPASTLPLDQATVRV RQRVVDVVKD ERQHIAVVAG RPMSVPVVDA KPTKRPGVFS VSIPGLPSLQVSVPKGVPTA KAPPKGIVAE KGDSRPAGFT AGGNSREAVI RFPKETGQKP VYVSVTDVLTPAQVKQRQEE EKRRQQAWDA AHPEEGLKRD YDKAKAELDA EDKNIATLNS RIASTEKALPGARAAVQEAD KKVKEAEANK DDFVTYNPPH EYGSGWQDQV RYLDKDIQNQ NAKLKAAQASLNAMNESLSR DKACTSRAME SRKQKEKKAK DAENKLNEEK KKPRKGAKDY GHDYHPAPKTEDIKGLGDLK KGTPKTPMQG GGGRRKRWIG DKGRKIYEWD SQHGELEGYR ASDGEHLGAFDPKTGKQIKG PDPKGRNIKK YLNCBI accession: NC_002610.1DNA sequence:SEQ ID NO: 3ATGGGTGGTG GATTTAACTA TAACGGGGAA GGTGCTACTG GCACCGGATT GGATCGTGATCCATATGTTC GCGACAGCAA TGGTAATGCT ATTGGTGTTA AATCACGCTA TCACGCGGAGTCCTATGGGA CGTCAAGTCC AGCCTTAGGG CCTAATGGCG CTATTCAGAT TACTGCTGGGGTTATTGCTG TGCCTGGAGA TAAGCCCCGA CCTGACGGTG GTAGTGGTGG TGGGAATACTGTTAACACCG GACCTGCAGG ACAGCTCCTG GTGATGAATA AAGGTCAGCT TGGATACTGGGAAACTCGTT CTACGGGCGC GGGTAACAAT GAGCATAATA CGAGTGTATT TGTTGCTGTAGGTCCTTCTG AGGCAGAAAA AACTGCTTCT GCAGAGAAGG CGTTAAAGGA AAAACAGCAGGCAGAAGCAG CAGCAAAAGA CTTTGCGGCT AAAACTGCTG CCGCATCAGC CACGGCGGAAAAAGAACGCC AGCAGGCAAT TGCTGCTGCA ACTGCAGCGG GTCAGCATCA GTCAGTTTCTGATGCCCGTA ATAGCCTGAA TAACGCGACG TCAGATGTGT CTCGTCTGAA ATCAGCTGCAGACAGTGCAC TGCAGGAGGC AAAGGCAAAA CGGAAGGCTG CTATTGATGC TGTACCTGTTGCAACCCAGG CGGAAAATAA ATATCAGGAG CTGCAGCAGA AGATTAAAGG CCTGAAGCTGAAAAATGGTG AGTATGGTAC GGATAAATGG GAAATAATTG GCTCTAATAA GGAGCATGATCACTGGGGAT ACAGGTTTTA TCCATCCGGA ATTACCAAAG CTCAGGTTGA TGCGGCGCAGATCGATGCTG TGAATAAGCG AAATCAGGCT ACCAGTCTTG CCAGTCAGGC AACAGCAGCAGAACAGGACA GCCTGAAAGC TACAGCTGCC TATAATGCGG CAGAAACGCG CCGTCAGGCTGCTCAGGCGG CGTTAAATTC TGCTGAACAG GCTGCTGCTG CTGAACGTAA GCGGCAGGAAGCTGAGGCGG CTGCAGCAGC TGCTGCTGAG AAAAAACGTC AGGCAGACGC AGCAGCAAAAGCTGCAGAGG AAGCGCGTGC GGCAGCGGAA AAAGCCAGGC TGATGCAGGA GCGTCAGGCAGCAGCAGATA AGCTGAAATC CACAGATATT CAGTCTGTTC GCGGGATCCC GTCTACGGCTGCGCCTGCAG CGTCACCCAT TTCCTGGGCC GTTGCATCAC TTGGTGGTAT ATCGCTGGATAGTGTTACTG CAGGGAAAGC ATGGACGCAG ATTGCTGAGG TGATGGCTAA ACTACGAGGTATTGCCGGTG CGAGTCTTGT TGGTCCCGTG GTGGCAACTG CTGTAGGGCT GTTTTGGTCACGTGATGTTG GTATTGGCAG TGATGTGGTG CCCGGACGTG ACATCAGCGG GCTGATGCCGGGTGATGCAC TGTCATTACC TGATCTGGCC ACTTTGATTA AAGCTGCTGA CAGTAAAACGGGTGTCAGTA TGCCGGTTCG AGGCCGGATT ATCGTGCGTG AAGGCGATTA TCTGGAGTCTCAGTTCGTTC GAACACCTGT TGCCGGTAGT GTTCCGGTTG TTCGGGCTGC TCTGGATAAAGCTACTGGTT ACTGGGGATA TACGTTGCCG GCGATACAGG GTGTGCCCGG ACAGACAATACTGGTGAGTC CGTCAGATGC GCCGGGCGTT AATGGTCCTC TGGGACTTGC TGGGCCGGTTCCTTTGCCTG AAACTATTAT ACATACCGGT GGGCAAACTA CGGTTCCTCA GGGGGGGACTGTGACAGTTT CECCGGCAGA AGACGATATT GATTTCAATG ATTTGATTCT GGTATTTCCGCCGGAGTCCG GTCTTAAACC GTTGTATGTG ATGTACCGTA GCCCTCGTAA CATGCCGGGGACAGCCAGTG GTAAAGGTCA GAACGTTGGA AATAACTGGA TGGGGGGGAC CAGTACCGGGGATGGTGCTC CTGTTCCTTC CCAGATTGCA GATAAATTAC GTGGGAAGGC TTTCGGTAGTTTTGATTCTT TCTGTCGGGC TTTCTGGAAA GCGGTTGCTG CTGATCCGGA CCTCAGTAAGCAGTTTTATC CTGATGATAT AGAGCGAATG AAATTAGGGC GAGCTCCAAC AGTTCGATTCCGAGATTCTG TAGGTAAAAG GGTTAAGGTT GAACTACACC ATAAAGTTGA AATTTCTAAAGGTGGTGATG TCTATAACGT AGATAACCTG AATGCATTAA CACCTAAACG TCATATTGAAATTCATAAGG GGAACTGAAmino acid sequence:SEQ ID NO: 4MGGGFNYNGE GATGTGLDRD PYVRDSNGNA IGVKSRYHAE SYGTSSPALG PNGAIQITAGVIAVPGDKPR PDGGSGGGNT VNTGPAGQLL VMNKGQLGYW ETRSTGAGNN EHNTSVFVAVGPSEAEKTAS AEKALKEKQQ AEAAAKDFAA KTAAASATAE KERQQAIAAA TAAGQHQSVSDARNSLNNAT SDVSRLKSAA DSALQEAKAK RKAAIDAVPV ATQAENKYQE LQQKIKGLKLKNGEYGTDKW EIIGSNKEHD HWGYRFYPSG ITKAQVDAAQ IDAVNKRNQA TSLASQATAAEQDSLKATAA YNAAETRRQA AQAALNSAEQ AAAAERKRQE AEAAAAAAAE KKRQADAAAKAAEEARAAAE KARLMQERQA AADKLKSTDI QSVRGIPSTA APAASPISWA VASLGGISLDSVTAGKAWTQ IAEVMAKLRG IAGASLVGPV VATAVGLEWS RDVGIGSDVV PGRDISGLMPGDALSLPDLA TLIKAADSKT GVSMPVRGRI IVREGDYLES QFVRTPVAGS VPVVRAALDKATGYWGYTLP AIQGVPGQTI LVSPSDAPGV NGPLGLAGPV PLPETIIHTG GQTTVPQGGTVTVSPAEDDI DFNDLILVFP PESGLKPLYV MYRSPRNMPG TASGKGQNVG NNWMGGTSTGDGAPVPSQIA DKLRGKAFGS FDSFCRAFWK AVAADPDLSK QFYPDDIERM KLGRAPTVRFRDSVGKRVKV ELHHKVEISK GGDVYNVDNL NALTPKRHIE IHKGNNCBI accession: AY578793.1DNA sequence:SEQ ID NO: 5ATGGCAGATA ATCAACCGGT TCCTCTTACC CCCGCACCAC CTGGAATGGT ATCACTTGGCGTCAATGAAA ACGGCGAAGA GGAGATGACT GTCATTGGTG GAGATGGCAG CGGCACAGGGTTTTCTGGGA ATGAAGCACC TATTATTCCT GGAAGTGGTA GCCTCCAGGC CGACTTAGGTAAAAAGTCTC TAACCCGACT ACAGGCTGAA AGTTCAGCAG CAATTCATGC GACTGCAAAATGGACTACAG AGAATCTTGC AAAAACGCAG GCTGCGCAGG CTGAAAGGGC CAAGGCTGCCATGCTTTCTC AGCAGGCAGC AAAAGCAAAA CAGGCCAAAC TCACGCTACA TCTGAAAGATGTAGTGGATC GCGCGCTTCA GAACAACAAA ACGCGGCCTA CTGTTATTGA TCTTGCTCATCAGAATAACC AACAAATGGC CGCAATGGCC GAGTTTATAG GCCGTCAAAA GGCAATTGAAGAAGCTCGTA AAAAGGCTGA AAGGGAAGCC AAAAGGGCTG AAGAAGCTTA TCAGGCTGCTTTGAGAGCGC AGGAAGAAGA ACAGCGCAAG CAGGCAGAAA TTGAACGGAA ATTGCAGGAGGCAAGGAAGC AAGAGGCAGC GGCAAAAGCA AAAGCTGAAG CTGACAGAAT TGCGGCTGAGAAAGCTGAAG CAGAGGCAAG AGCTAAAGCG GAAGCTGAGC GACGGAAAGC AGAGGAGGCTCGAAAGGCGC TTTTTGCAAA GGCTGGGATT AAGGACACTC CTGTTTATAC ACTGGAGAAGACAAAAGCAG CCACTACGTT GTTTTTAACA CCGGGTGTTA GGTTACTGAA TCGTGCTCCAGCGATGATAC AGTTATCCGC TTTGGCTGCA GAAATTAATG GCGTCTTAAC TACTGCTGCTAGTGCAGTAA TGACGGCTAC TGCTGAATTC TCAGGTTGGA TTGCCTCAGC GTTATGGCGAGGTGTAGCTG GTGTTGCAAC AGCTAGTACT GTTGGTCCCA TGGTTGCCGC AGCATCGACGCTATTCTTTT CACCTCGCGC AGGTGGCGGA AGCGACAGTA AGGTTCCTGG TAGGGATATCGAGATGTTGG CTGCGCAAGC CAGGTTGTTC ACGGCGGGTA AGCTGAGTAT CGAACATGAAGAGCGTCAAC CTCCCGGTAC GTGGCTTCAT CTCTTCGGAA ACTGATGGGC GCCAGTCTCTGATGCTTGTA AAAACCGGTT CTGATGGAGT ACCTTCCACT GTTCCTGTAT TGGATGCTGTACGTGACAGT ACTACTGGCC TTGATAAAAT AACGGTACCG GCGATGTCTG GTGCGCCGTCGCGGACCATC CTCGTGAATC CGGTTCCAAT TGGACCTGCT GCTCCGTGGC ATACCGGCAATAGCGGGCCA GTGCCAGTAA CACCTGTTCA CACCGGTACA GAGGTGAAGC AGGCTGACAGTATCGTCACG ACAACTTTGC CGATTGCAGA CATTCCGCCA CTACAGGACT TCATCTACTGGCAGCCGGAT GCTTCTGGGA CAGGTGTTGA ACCTATTTAT GTAATGACTA GTCAACCCAGGAAAGGAGTA AAAGACTACG GACATGATTA TCATCCGGCT CCAAAAACTG AAGAAATTAAGGGGTTGGGG GAGTTGATTG AGTCTCGGAA AAAAACTCCA AAACAAGGGG GAGGTGGACGACGAGATCGA TGGGTGGGAG ATAAAGGACG AAAAATCTAT GAGTGGGATT CGCAGCATGGAGAACTTGAA GGTTACAGAG CTAGCGACGG CTCTCATCTT GGAGCATTTG ATCCAAACACCGGCAAGCAA CTTAAAGGTC CGGACCCTAA ACGTAACATC AAAAAATATC TTTGAAmino acid sequence:SEQ ID NO: 6MADNQPVPLT PAPPGMVSLG VNENGEEEMT VIGGDGSGTG FSGNEAPIIP GSGSLQADLGKKSLTRLQAE SSAAIHATAK WTTENLAKTQ AAQAERAKAA MLSQQAAKAK QAKLTLHLKDVVDRALQNNK TRPTVIDLAH QNNQQMAAMA EFIGRQKAIE EARKKAEREA KRAEEAYQAALRAQEEEQRK QAEIERKLQE ARKQEAAAKA KAEADRIAAE KAEAEARAKA EAERRKAEEARKALFAKAGI KDTPVYTLEK TKAATTLFLT PGVRLLNRAP AMIQLSALAA EINGVLTTAASAVMTATAEF SGWIASALWR GVAGVATAST VGPMVAAAST LFFSPRAGGG SDSKVPGRDIEMLAAQARLF TAGKLSIEPG MKSVNLPVRG FISSETDGRQ SLMLVKTGSD GVPSTVPVLDAVRDSTTGLD KITVPAMSGA PSRTILVNPV PIGPAAPWHT GNSGPVPVTP VHTGTEVKQADSIVTTTLPI ADIPPLQDFI YWQPDASGTG VEPIYVMTSQ PRKGVKDYGH DYHPAPKTEENTGKQLKGPD PKRNIKKYL GRRDRWVGDK GRKIYEWDSQ HGELEGYRAS DGSHLGAFDPIKGLGELIES RKKTPKQGGGNCBI accession: AY578792DNA sequence:SEQ ID NO: 7ATGAGTGATA TCACATATAA TCCTGAGGAC TATAACAATG GTATACCACC TGAGCCGGGTCTGGTGTGGA AGCCGGGAGG CTCATTCCCC AATGGCAGTT ACGTTCCCGG CTCATGGGGCTGGCCAACGC GCGGATACGA TGTTCCTCCG CTGCCGGGTG ATACCGAAAT GCTGACGGTCACCCCAAAAG GAACGCCGGC TGATACCTGG CCTAAAAGAC CTGATATCAA AGAGTGGTATGTTCCGGGTG AAAAACCCTT CGACCCGTCA ACAGGTAATG GATGGGTGCC TGATGTGGACGGCTACGCCG AATCGCTGCC TGCCGGTATA CCGGCTGTGG TACAGGCTGC AATCAGTAAGGTGAAAGGTG CGCCACTGAA AGGCGGCATG TCCGCCGTGG ATATCTGGAA ACTGAAACCCGCAACGGAGT ACCCGGGGAG ATTTAACAGC ACCGACCCGG CATTCAGCTG GTTTCCGGTTCGGGCGCTGA CTGACACTGA TATATCTGCG ATGCCCGTTG CCCCTGAGAC TGTTCCGGTGCATACCCGTA TCCTTGATAA TGTTCATGAT GGTGTACAGT TTGTTTCTGC GGTGTTTGCGGGTAGCATGC AGTACAATCT GCCGGTGGTG AAAGCGCAGG CCACTGCCGG CAGTGATTATTACACTATCG GACGTCTGCC GGGCATCATG AGTGCTTTCA CATTCTCTTT CTACACAAAAGGAACACCAC AGGACTCCCG TTTCTTCCGG GATACAGTGA AAGCCGGGGG AGATTTACGCGAAGCAGGCT TCACTGTGGG GGCCAATACC AGCGATTTTA TCATCTGGTT TCCGCAGGGGAGCGGACTGG AGCCGCTGTA TTTTTCCATG ACCATGAATA TGCCGGCTGG GCCGCTGCAGCGTCGCCAGG AAGCCGAAAA CAAGGCCAGA GCAGAAGCTG ACAGGCTCCG GGCAGAGGCGGAGGCAAAAA TTCGCGCTGA AGCAGAGGCC CGGGCGAAAG CAGAAGCAGA ACGCAAAGCCCTGTTCGCTA AGGCCGGTAT TCAGGATACA CCGGTTTACA CGCCGGAGAT GGTGAAGGCGGCAAATGCGG CGCTGTCTGC CGGAGGCTCA ATGGCGCTCA GCCGGGCCCC GGGGATGATACAGCACTCTG CTGCAGGCGT GGGGACGCTA CCCTTCAACA GTAGTCTGGC GGGATGGGAAGCCGGCGCGC TCTGGCGCGG TGTCGACGTG CTTGCCAGGA TCGCGCCGGT CGCGTCCGCCGTGGCCACGG TTGCCACAGT GCTCACCCTT GTCAGGGCTG CACTGGATAT CCCTGCAGCCGGCGAGGGCA GTGACAGGGT TCCCGGACGC AACATTGACA TGCTTGCCGC CCAGGCCAGCCTGTACACGG CCATGAAGAC GAACATTCAG CCGGGGATGA AGACCGTTGA CCTGCCAGTCAGGGGATATA TCTCGTATGA CGGCAACGGC CGGCAGTCGG TCAACTTGGT CAGGACGGGGACGGGCGGGG TTTCGGCCAC GGTGCCGGTG CTGAGTGCCG TGCGTGACAA AACCACCGGCCTGGATAAAA TCACGGTACC GGCCGTGGCG GGCGCCCCGT CGCGGACCAT CCTGATTAACCCTGTACCGG TCGGTCCTGC GACACCATCG CATACCGGCA GCAGTACGCC GGTTCCGGTGACGCCGGTGC ACACTGGTAC CGATGTTAAG CAGGCGGACA GCATCGTCAC CACAACGTTGCCGGCGGCAG ATATTCCTGC GCTGCAGGAC TTCATCTACT GGCAGCCGGA TGCAACCGGGACGGGCGTGG AACCCATCTA TGTCATGCTG AGTGATCCGT TGGATTCGGG GAAATATACCCGCAGGCAGC TCCAGAAGAA GTACAAGCAT GCTATCGATT TTGGTATCAC AGATACGAAGATAAATGGTG AAACACTTAC TAAGTTCCGG GATGCAATTG AAGCACATCT TTCAGATAAGGATACCTTTG AAAAAGGAAC ATATCGGCGT GATAAGGGAT CGAAGGTTTA TTTCAATCCTAAAACAATGA ATGCTGTTAT TATTCAGGCT AATGGTGACT TTCTGTCTGG ATGGAAAATTAATCCTGCGG CAGATAATGG TAGAATTTAT TTAGAAACGG GTGATTTATG AAmino acid sequence:SEQ ID NO: 8MSDITYNPED YNNGIPPEPG LVWKPGGSFP NGSYVPGSWG WPTRGYDVPP LPGDTEMLTVTPKGTPADTW PKRPDIKEWY VPGEKPFDPS TGNGWVPDVD GYAESLPAGI PAVVQAAISKVKGAPLKGGM SAVDIWKLKP ATEYPGRENS TDPAFSWFPV RALTDTDISA MPVAPETVPVHTRILDNVHD GVQFVSAVFA GSMQYNLPVV KAQATAGSDY YTIGRLPGIM SAFTFSFYTKGTPQDSRFFR DTVKAGGDLR EAGFTVGANT SDFIIWFPQG SGLEPLYFSM TMNMPAGPLQRRQEAENKAR AEADRLRAEA EAKIRAEAEA RAKAEAERKA LFAKAGIQDT PVYTPEMVKAANAALSAGGS MALSRAPGMI QHSAAGVGTL PFNSSLAGWE AGALWRGVDV LARIAPVASAVATVATVLTL VRAALDIPAA GEGSDRVPGR NIDMLAAQAS LYTAMKTNIQ PGMKTVDLPVRGYISYDGNG RQSVNLVRTG TGGVSATVPV LSAVRDKTTG LDKITVPAVA GAPSRTILINPVPVGPATPS HTGSSTPVPV TPVHTGTDVK QADSIVTTTL PAADIPALQD FIYWQPDATGTGVEPIYVML SDPLDSGKYT RRQLQKKYKH AIDFGITDTK INGETLTKFR DAIEAHLSDKDTFEKGTYRR DKGSKVYFNP KTMNAVIIQA NGDFLSGWKI NPAADNGRIY LETGDLDNA sequence of Klebicin CCL along with immunity gene:>AF190857.1:166-1854Klebsiellapneumoniaecloacin operon, completesequence, 1956 basesSEQ ID NO: 9ATGAGTGGTG GAGACGGTCG AGGTCCGGGT AATTCAGGTC TGGGACATAA TGGTGGTCAGGCCAGTGGGA ATGTGAACGG TACGTCTGGT AAAGGCGGCC CTTCATCAGG TGGGGGTACGGATCCAAACA GCGGGCCGGG CTGGGGTACG ACGCATACGC CTAACGGAGA TATTCATAACTACAATCCGG GGGAGTTTGG TCACGGAGGG AATAAACCCG GTGGCAATGG CGGTAACAGCGGCAATCATC CCGGTAGTTC TGGTGGCAGA CAGTCTTCGG CCACAGCGAT GGCCTTCGGTCTGCCTGCTC TGGCTACTCC GGGCTCCGGG GGGCTGGCTT TAGCCGTTTC CGGCGATGCGTTGTCGGCAG CCGTTGCTAG TGTGCTGGCT GCCCTGAAAG GGCCGTTTAA GAAAAGAGACTATGATAAAG CGAAAGCCGA GCTGGATGCC GAAGATAAAA ATATTGCGAC CTTAAACAGCCGCATTGCAT CGACAGAGAA GGCGCTCCCC GGTGCAAGGG CTGCTGTTCA GGAAGCCGATAAAAAGGTGA AAGAGGCAGA GGCGAATAAG GATGATTTTG TGACTTATAA CCCTCCTCATGAATATGGCT CCGGGTGGCA GGATCAGGTT CGCTATCTTG ATAAGGATAT TCAGAATCAGAATGCGAAAT TAAAAGCGGC TCAGGCATCT TTAAACGCAA TGAATGAATC CTTATCCAGAGATAAGGCTT GCACTTCCCG GGCGATGGAG AGCCGGAAAC AAAAGGAGAA AAAAGCGAAGGATGCAGAAA ATAAGTTAAA TGAGGAAAAG AAAAAACCTC GCAAGGGAGC TAAAGACTACGGCCATGATT ATCATCCAGC CCCGAAAACT GAAGACATAA AGGGACTGGG TGACCTCAAAAAAGGTACAC CTAAAACACC AATGCAGGGA GGTGGAGGTA GGCGTAAACG CTGGATTGGTGATAAAGGCC GTAAGATTTA TGAATGGGAC TCCCAGCACG GTGAGCTTGA AGGGTATCGTGCCAGTGATG GCGAACACCT CGGGGCATTT GATCCTAAAA CGGGTAAGCA AATTAAAGGTCCGGATCCGA AAGGGCGAAA CATTAAAAAA TATCTTTAAg aggtaagtat gggacttaaattaaatttaa cctggtttga taagaaaact gaagagttta aaggggaaga gtattctaaagactttggtg atgatggttc tgtcattgaa agtcttggga tgcctttaaa ggataatattaacaatggtt gttttgatgt gaaaaatgag tgggtttcat tattgcaacc ctactttaaacataaaatca atctttctga tagttcatat tttgtttcat ttgattatcg ggatggtaactggtaaAmino acid sequence of klebicin CCL:SEQ ID NO: 10MSGGDGRGPG NSGLGHNGGQ ASGNVNGTSG KGGPSSGGGT DPNSGPGWGT THTPNGDIHNYNPGEFGHGG NKPGGNGGNS GNHPGSSGGR QSSATAMAFG LPALATPGSG GLALAVSGDALSAAVASVLA ALKGPFKFGL WGIAIYGVLP SEIAKDDPKM MSKIMTSLPA DAVTETPASTLPLDQATVRV RQRVVDVVKD ERQHIAVVAG RPMSVPVVDA KPTKRPGVFS VSIPGLPSLQVSVPKGVPTA KAPPKGIVAE KGDSRPAGFT AGGNSREAVI RFPKETGQKP VYVSVTDVLTPAQVKQRQEE EKRRQQAWDA AHPEEGLKRD YDKAKAELDA EDKNIATLNS RIASTEKALPGARAAVQEAD KKVKEAEANK DDFVTYNPPH EYGSGWQDQV RYLDKDIQNQ NAKLKAAQASLNAMNESLSR DKACTSRAME SRKQKEKKAK DAENKLNEEK KKPRKGAKDY GHDYHPAPKTEDIKGLGDLK KGTPKTPMQG GGGRRKRWIG DKGRKIYEWD SQHGELEGYR ASDGEHLGAFDPKTGKQIKG PDPKGRNIKK YL6. Klebicin CCL TDRD + Kleb B KD + Imm: 2107 basesDNA Sequence:SEQ ID NO: 11ATGAGTGGTG GAGACGGTCG AGGTCCGGGT AATTCAGGTC TGGGACATAA TGGTGGTCAGGCCAGTGGGA ATGTGAACGG TACGTCTGGT AAAGGCGGCC CTTCATCAGG TGGGGGTACGGATCCAAACA GCGGGCCGGG CTGGGGTACG ACGCATACGC CTAACGGAGA TATTCATAACTACAATCCGG GGGAGTTTGG TCACGGAGGG AATAAACCCG GTGGCAATGG CGGTAACAGCGGCAATCATC CCGGTAGTTC TGGTGGCAGA CAGTCTTCGG CCACAGCGAT GGCCTTCGGTCTGCCTGCTC TGGCTACTCC GGGCTCCGGG GGGCTGGCTT TAGCCGTTTC CGGCGATGCGTTGTCGGCAG CCGTTGCTAG TGTGCTGGCT GCCCTGAAAG GGCCGTTTAA GTTTGGTCTGTGGGGGATTG CGATCTACGG TGTGCTGCCT TCTGAGATTG CAAAAGATGA TCCGAAAATGATGTCAAAAA TTATGACGTC ATTACCGGCC GATGCGGTGA CGGAGACTCC GGCAAGTACTTTACCACTGG ACCAGGCGAC GGTTCGTGTC AGACAACGGG TTGTGGATGT GGTGAAGGATGAGCGGCAGC ATATTGCGGT TGTCGCAGGT CGGCCAATGA GTGTCCCTGT GGTGGATGCGAAACCGACAA AACGTCCGGG GGTATTCAGT GTGTCGATTC CGGGTCTCCC GTCTCTGCAGGTGAGCGTAC CGAAAGGTGT TCCGACAGCG AAAGCCCCGC CAAAAGGCAT TGTTGCTGAAAAAGGTGATT CACGTCCGGC TGGTTTTACA GCCGGTGGTA ACTCCCGTGA GGCCGTTATTCGTTTCCCGA AAGAGACCGG ACAGAAGCCG GTTTATGTGT CGGTGACAGA TGTTCTTACCCCGGCACAGG TAAAACAGCG TCAGGAGGAA GAAAAGCGTC GCCAGCAGGC ATGGGACGCCGCTCATCCGG AAGAGGGGCT GAAAAGAGAC TATGATAAAG CGAAAGCCGA GCTGGATGCCGAAGATAAAA ATATTGCGAC CTTAAACAGC CGCATTGCAT CGACAGAGAA GGCGCTCCCCGGTGCAAGGG CTGCTGTTCA GGAAGCCGAT AAAAAGGTGA AAGAGGCAGA GGCGAATAAGGATGATTTTG TGACTTATAA CCCTCCTCAT GAATATGGCT CCGGGTGGCA GGATCAGGTTCGCTATCTTG ATAAGGATAT TCAGAATCAG AATGCGAAAT TAAAAGCGGC TCAGGCATCTTTAAACGCAA TGAATGAATC CTTATCCAGA GATAAGGCTT GCACTTCCCG GGCGATGGAGAGCCGGAAAC AAAAGGAGAA AAAAGCGAAG GATGCAGAAA ATAAGTTAAA TGAGGAAAAGAAAAAACCTC GCAAGGGAGC TAAAGACTAC GGCCATGATG GTCTTAAACC GTTGTATGTGATGTACCGTA GCCCTCGTAA CATGCCGGGG ACAGCCAGTG GTAAAGGTCA GAACGTTGGAAATAACTGGA TGGGGGGGAC CAGTACCGGG GATGGTGCTC CTGTTCCTTC CCAGATTGCAGATAAATTAC GTGGGAAGGC TTTCGGTAGT TTTGATTCTT TCTGTCGGGC TTTCTGGAAAGCGGTTGCTG CTGATCCGGA CCTCAGTAAG CAGTTTTATC CTGATGATAT AGAGCGAATGAAATTAGGGC GAGCTCCAAC AGTTCGATTC CGAGATTCTG TAGGTAAAAG GGTTAAGGTTGAACTACACC ATAAAGTTGA AATTTCTAAA GGTGGTGATG TCTATAACGT AGATAACCTGAATGCATTAA CACCTAAACG TCATATTGAA ATTCATAAGG GGAACTGAaa tcgctaataaaactttggct gactatacag agcaggaatt tattgagttt atcgaaaaaa ttaaaaaggcagactttgct actgagtctg agcatgatga ggctatttat gagttcagcc agttgactgagcatccagat gcttgggatc ttatttatca tectcaagca ggagccgata actctcctgctggtgttgta aaaacagtaa aagagtggcg agcagctaac ggtaagccag gttttaaaaaatcgtgaKleb CCL TD RBD (1-1419); Klebicib B KD (1420-1849);Klebicibn B Immunity protein (1850-end)SEQ ID NO: 12Amino Acid sequence; Theoretical pI/Mw: 9.54/65021.95MSGGDGRGPG NSGLGHNGGQ ASGNVNGTSG KGGPSSGGGT DPNSGPGWGT THTPNGDIHNYNPGEFGHGG NKPGGNGGNS GNHPGSSGGR QSSATAMAFG LPALATPGSG GLALAVSGDALSAAVASVLA ALKGPFKFGL WGIAIYGVLP SEIAKDDPKM MSKIMTSLPA DAVTETPASTLPLDQATVRV RQRVVDVVKD ERQHIAVVAG RPMSVPVVDA KPTKRPGVFS VSIPGLPSLQVSVPKGVPTA KAPPKGIVAE KGDSRPAGFT AGGNSREAVI RFPKETGQKP VYVSVTDVLTPAQVKQRQEE EKRRQQAWDA AHPEEGLKRD YDKAKAELDA EDKNIATLNS RIASTEKALPGARAAVQEAD KKVKEAEANK DDFVTYNPPH EYGSGWQDQV RYLDKDIQNQ NAKLKAAQASLNAMNESLSR DKACTSRAME SRKQKEKKAK DAENKLNEEK KKPRKGAKDY GHDGLKPLYVMYRSPRNMPG TASGKGQNVG NNWMGGTSTG DGAPVPSQIA DKLRGKAFGS FDSFCRAFWKAVAADPDLSK QFYPDDIERM KLGRAPTVRF RDSVGKRVKV ELHHKVEISK GGDVYNVDNLNALTPKRHIE IHKGNP623: S5 TD-RD-Linker-GP36 CD-his: 1590 basesSEQ ID NO: 13ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACggaggaggag gatcaGGTGT GGCCCTGGAC CGCACGCGGG TTGATCCCCA GGCAGTCGGCAACGAGGTGC TCAAGCGCAA CGCGGATAAG CTGAATGCGA TGCGGGGCGC CGAGTACGGTGCCAACGTCA AGGTCAGCGG CACGGACATT CGCATGAACG GGGGTAACAG TGCCGGCATGCTGAAGCAGG ACGTGTTCAA CTGGCGGAAG GAACTGGCTC AGTTCGAGGC TTACCGAGGGGAGGCGTATA AGGATGCCGA TGGTTATAGT GTGGGCCTGG GGCATTACCT GGGCAGTGGCAATGCTGGGG CAGGTACTAC AGTCACGCCT GAGCAAGCCG CGCAGTGGTT CGCCGAGGACACCGACCGCG CACTCGACCA GGGTGTGAGG TTGGCCGACG AGCTGGGCGT TACGAACAATGCCTCTATCC TGGGATTGGC CGGTATGGCC TTCCAGATGG GCGAAGGACG TGCCCGGCAGTTCCGTAACA CCTTCCAGGC GATCAAGGAT CGCAACAAGG AAGCCTTCGA GGCTGGTGTGCGAAACAGCA AGTGGTACAC GCAGACGCCC AACCGGGCCG AGGCATTCAT CAAGCGCATGGCGCCCCACT TCGATACACC GAGTCAAATC GGTGTCGATT GGTACAGCGC CGCAACAGCGGAGCTCGAGC ACCACCACCA CCACCACTAAProtein; Theoretical pl/Mw: 6.56/59443.77SEQ ID NO: 14MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKATY NGELLVDEIA SLQARLVKLNGGGGSGVALD RTRVDPQAVG NEVLKRNADK LNAMRGAEYG ANVKVSGTDI RMNGGNSAGMLKQDVFNWRK ELAQFEAYRG EAYKDADGYS VGLGHYLGSG NAGAGTTVTP EQAAQWFAEDTDRALDQGVR LADELGVTNN ASILGLAGMA FQMGEGRARQ FRNTFQAIKD RNKEAFEAGVRNSKWYTQTP NRAEAFIKRM APHFDTPSQI GVDWYSAATA ELEHHHHHHP624: S5 TD-RD-Link-GP36 CD without his tag: 1572 basesSEQ ID NO: 15ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACggaggaggag gatcaGGTGT GGCCCTGGAC CGCACGCGGG TTGATCCCCA GGCAGTCGGCAACGAGGTGC TCAAGCGCAA CGCGGATAAG CTGAATGCGA TGCGGGGCGC CGAGTACGGTGCCAACGTCA AGGTCAGCGG CACGGACATT CGCATGAACG GGGGTAACAG TGCCGGCATGCTGAAGCAGG ACGTGTTCAA CTGGCGGAAG GAACTGGCTC AGTTCGAGGC TTACCGAGGGGAGGCGTATA AGGATGCCGA TGGTTATAGT GTGGGCCTGG GGCATTACCT GGGCAGTGGCAATGCTGGGG CAGGTACTAC AGTCACGCCT GAGCAAGCCG CGCAGTGGTT CGCCGAGGACACCGACCGCG CACTCGACCA GGGTGTGAGG TTGGCCGACG AGCTGGGCGT TACGAACAATGCCTCTATCC TGGGATTGGC CGGTATGGCC TTCCAGATGG GCGAAGGACG TGCCCGGCAGTTCCGTAACA CCTTCCAGGC GATCAAGGAT CGCAACAAGG AAGCCTTCGA GGCTGGTGTGCGAAACAGCA AGTGGTACAC GCAGACGCCC AACCGGGCCG AGGCATTCAT CAAGCGCATGGCGCCCCACT TCGATACACC GAGTCAAATC GGTGTCGATT GGTACAGCGCC GCAACAGCGGAGTAAProtein; Theoretical pl/Mw: 6.19/58620.92SEQ ID NO: 16MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKAIY NGELLVDEIA SLQARLVKLNGGGGSGVALD RTRVDPQAVG NEVLKRNADK LNAMRGAEYG ANVKVSGTDI RMNGGNSAGMLKQDVFNWRK ELAQFEAYRG EAYKDADGYS VGLGHYLGSG NAGAGTTVTP EQAAQWFAEDTDRALDQGVR LADELGVTNN ASILGLAGMA FQMGEGRARQ FRNTFQAIKD RNKEAFEAGVRNSKWYTQTP NRAEAFIKRM APHFDTPSQI GVDWYSAATA EP625: S5 TD-RD-Link-Phi29CD: 1365 basesSEQ ID NO: 17ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACggaggaggag gatcaCAAAT TTCACAAGCG GGTATCAACT TAATTAAGAG CTTTGAGGGTTTACAACTGA AAGCATATAA AGCTGTTCCG ACTGAGAAGC ATTACACCAT TGGTTACGGTCATTACGGTT CCGATGTTTC ACCTAGGCAG GTTATCACTG CTAAACAGGC TGAAGACATGTTGCGTGATG ATGTGCAGGC TTTTGTGGAT GGTGTAAATA AAGCATTAAA AGTATCTGTCACCCAAAATC AATTTGATGC ACTTGTCTCA TTCGCTTACA ACGTTGGGTT AGGGGCTTTCAGGTCTTCTT CTCTACTGGA ATACTTGAAT GAAGGAAGAA CAGCTCTAGC GGCGGCTGAATTCCCTAAAT GGAATAAGTC AGGCGGTAAA GTTTATCAAG GGTTGATTAA CCGTAGAGCACAGGAGCAAG CCTTGTTTAA TAGTGGAACA CCTAAAAATG TTTAAProtein; Theoretical pl/Mw: 8.71 QYAYEVPPID GLLKQITQGK/51160.99SEQ ID NO: 18MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKATY NGELLVDEIA SLQARLVKLNGGGGSQISQA GINLIKSFEG LQLKAYKAVP TEKHYTIGYG HYGSDVSPRQ VITAKQAEDMLRDDVQAFVD GVNKALKVSV TQNQFDALVS FAYNVGLGAF RSSSLLEYLN EGRTALAAAEFPKWNKSGGK VYQGLINRRA QEQALFNSGT PKNVP626: S5 TD-RD-Link-BP7e: 1422 basesSEQ ID NO: 19ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACggaggaggag gatcaGGTGA CATTTTTGAT ATGCTGCGCC AAGACGAAGG CCTGGACCTGAACCTGTATA AAGACACGGA AGGCTACTGG ACGATTGGTA TTGGTCAGCT GGTCACCAAAAACCCGAGTA AAGATGTGGC ACGTGCTGAA CTGGACAAAC TGATGGGTCG TGTGTGCAATGGCCGCATTA CGATGGCGGA AGCCGAACAA CTGTTTAACC GTAGCGTTGA AAATGCACGTCGCGCTATCC TGCGCAACCC GAAACTGAAA CCGGTGTATG ATGTTCTGGA CGAAGTGCGTCGCTGTGCGC TGATCAACAT GGTTTTTCAG ATGGGCGAAG CGGGTGTCGC CGGCTTCACCAATAGCCTGC GTATGCTGCA GCAAAAACGC TGGAACGATG CGGCCGTCAA TCTGGCACAGTCTCGCTGGT ACAAACAAAC GCCGAATCGT GCGAAACGCG TTATTGCTAC CTTCAAAACGGGCACCTGGG CGGCGTATCG TTGAProtein; Theoretical pI/Mw: 9.05/53373.88SEQ ID NO: 20MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKATY NGELLVDEIA SLQARLVKLNGGGGSGDIFD MLRQDEGLDL NLYKDTEGYW TIGIGQLVTK NPSKDVARAE LDKLMGRVCNGRITMAEAEQ LFNRSVENAR RAILRNPKLK PVYDVLDEVR RCALINMVFQ MGEAGVAGFTNSLRMLQQKR WNDAAVNLAQ SRWYKQTPNR AKRVIATFKT GTWAAYRP638: S5 Pyocin with 6X-His tag: 1497 basesSEQ ID NO: 21ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACGCCGAAACGA CACGACGCAG GACAGAAGCA GAACGCAAGG CGGCCGAGGA ACAAGCGTTGCAAGATGCTA TTAAATTTAC TGCCGACTTT TATAAGGAAG TAACTGAGAA ATTTGGCGCACGAACATCGG AGATGGCGCG CCAACTGGCC GAAGGCGCCA GGGGGAAAAA TATCAGGAGTTCGGCGGAAG CAATCAAGTC GTTTGAAAAG CACAAGGATG CGTTAAATAA AAAACTTAGCCTTAAAGATA GGCAAGCCAT TGCCAAAGCC TTTGATTCTC TAGACAAGCA GATGATGGCGAAGAGCCTTG AGAAATTTAG CAAAGGCTTT GGAGTTGTAG GCAAAGCTAT TGACGCCGCCAGCCTGTACC AAGAGTTCAA GATATCTACG GAAACCGGGG ACTGGAAACC ATTCTTTGTAAAAATTGAAA CACTAGCTGC TGGTGCGGCC GCCAGTTGGC TTGTGGGTAT TGCATTTGCCACGGCAACAG CCACTCCTAT AGGCATTCTG GGGTTCGCAC TGGTAATGGC AGTTACCGGGGCGATGATTG ACGAAGACCT TCTAGAAAAA GCAAACAATC TTGTAATATC CATTCACCACCACCACCACC ACTAATheoretical pI/Mw: 8.50/56075.04SEQ ID NO: 22MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKATY NGELLVDEIA SLQARLVKLNAETTRRRTEA ERKAAEEQAL QDAIKFTADF YKEVTEKFGA RTSEMARQLA EGARGKNIRSSAEAIKSFEK HKDALNKKLS LKDRQAIAKA FDSLDKQMMA KSLEKFSKGF GVVGKAIDAASLYQEFKIST ETGDWKPFFV KIETLAAGAA ASWLVGIAFA TATATPIGIL GFALVMAVTGAMIDEDLLEK ANNLVISIHH HHHHP652: S5 Pyocin without His tag: 1497 basesSEQ ID NO: 23ATGTCCAATG ACAACGAAGT ACCTGGTTCC ATGGTTATTG TCGCACAAGG TCCAGACGATCAATACGCAT ACGAGGTTCC CCCTATCGAT AGCGCGGCCG TTGCCGGGAA TATGTTTGGCGACTTAATTC AAAGAGAAAT ATATCTACAG AAAAACATTT ATTATCCAGT CCGATCTATTTTTGAACAAG GAACAAAAGA AAAGAAGGAG ATCAACAAGA AAGTATCTGA TCAAGTCGATGGCTTGCTAA AGCAGATCAC TCAAGGAAAA AGGGAGGCCA CAAGGCAAGA GCGAGTCGATGTCATGTCGG CAGTCCTGCA CAAGATGGAA TCTGATCTTG AAGGATACAA AAAGACCTTTACCAAAGGCC CATTCATTGA CTACGAAAAG CAGTCAAGCC TCTCCATCTA TGAGGCCTGGGTCAAGATCT GGGAGAAGAA CTCTTGGGAA GAAAGAAAGA AGTACCCTTT TCAGCAGCTTGTTAGAGATG AACTGGAGCG GGCGGTTGCC TACTACAAAC AAGATTCACT CTCTGAAGCGGTAAAAGTGC TAAGACAGGA GCTCAACAAG CAAAAAGCGC TAAAGGAAAA AGAGGACCTCTCTCAACTGG AGCGGGACTA CAGAACCCGA AAGGCGAATC TCGAGATGAA AGTACAATCCGAGCTTGATC AAGCGGGAAG TGCTTTGCCT CCATTGGTCA GTCCAACGCC AGAGCAATGGCTTGAACGTG CCACAAGACT GGTTACGCAA GCAATTGCTG ATAAAAAGCA GCTGCAGACCACAAACAATA CTCTTATCAA GAATTCCCCA ACCCCTCTAG AAAAGCAGAA AGCCATCTACAATGGTGAGC TACTTGTGGA TGAGATAGCC AGTCTACAGG CCCGCTTAGT TAAGCTGAACGCCGAAACGA CACGACGCAG GACAGAAGCA GAACGCAAGG CGGCCGAGGA ACAAGCGTTGCAAGATGCTA TTAAATTTAC TGCCGACTTT TATAAGGAAG TAACTGAGAA ATTTGGCGCACGAACATCGG AGATGGCGCG CCAACTGGCC GAAGGCGCCA GGGGGAAAAA TATCAGGAGTTCGGCGGAAG CAATCAAGTC GTTTGAAAAG CACAAGGATG CGTTAAATAA AAAACTTAGCCTTAAAGATA GGCAAGCCAT TGCCAAAGCC TTTGATTCTC TAGACAAGCA GATGATGGCGAAGAGCCTTG AGAAATTTAG CAAAGGCTTT GGAGTTGTAG GCAAAGCTAT TGACGCCGCCAGCCTGTACC AAGAGTTCAA GATATCTACG GAAACCGGGG ACTGGAAACC ATTCTTTGTAAAAATTGAAA CACTAGCTGC TGGTGCGGCC GCCAGTTGGC TTGTGGGTAT TGCATTTGCCACGGCAACAG CCACTCCTAT AGGCATTCTG GGGTTCGCAC TGGTAATGGC AGTTACCGGGGCGATGATTG ACGAAGACCT TCTAGAAAAA GCAAACAATC TTGTAATATC CATTTAATheoretical pl/Mw: 8.50/56075.04SEQ ID NO: 24MSNDNEVPGS MVIVAQGPDD QYAYEVPPID SAAVAGNMFG DLIQREIYLQ KNIYYPVRSIFEQGTKEKKE INKKVSDQVD GLLKQITQGK REATRQERVD VMSAVLHKME SDLEGYKKTFTKGPFIDYEK QSSLSIYEAW VKIWEKNSWE ERKKYPFQQL VRDELERAVA YYKQDSLSEAVKVLRQELNK QKALKEKEDL SQLERDYRTR KANLEMKVQS ELDQAGSALP PLVSPTPEQWLERATRLVTQ AIADKKQLQT TNNTLIKNSP TPLEKQKAIY NGELLVDEIA SLQARLVKLNAETTRRRTEA ERKAAEEQAL QDAIKFTADF YKEVTEKFGA RTSEMARQLA EGARGKNIRSSAEAIKSFEK HKDALNKKLS LKDRQAIAKA FDSLDKQMMA KSLEKFSKGF GVVGKAIDAASLYQEFKIST ETGDWKPFFV KIETLAAGAA ASWLVGIAFA TATATPIGIL GFALVMAVTGAMIDEDLLEK ANNLVISIFyu A BD- T4 lysozyme fusion:DNA sequence:SEQ ID NO: 25ATGAGCGACA CGATGGTTGT GAATGGCAGC GGCGGCGTTC CGGCGTTCCT GTTTAGCGGCAGCACCCTGA GCAGCTATCG TCCGAATTTC GAGGCGAACA GCATCACCAT TGCGCTGCCGCACTATGTGG ACCTGCCGGG CCGTAGCAAC TTCAAGCTGA TGTATATCAT GGGTTTTCCGATTGACACCG AGATGGAAAA GGATAGCGAG TACAGCAACA AAATCCGTCA AGAAAGCAAGATTAGCAAAA CCGAGGGCAC CGTGAGCTAC GAACAGAAAA TCACCGTTGA GACCGGCCAAGAAAAGGATG GTGTGAAAGT TTATCGTGTG ATGGTTCTGG AGGGCACCAT CGCGGAGAGCATTGAACACC TGGACAAGAA AGAGAACGAA GACATCCTGA ACAACAACCG TAACCGTATTGTGCTGGCGG ACAACACCGT TATCAACTTC GATAACATTA GCCAGCTGAA GGAATTTCTGCGTCGTAGCG TGAACATCGT TATTTTCGAG ATGCTGCGTA TCGACGAACG TCTGCGTCTGAAGATTTATA AAGATACCGA GGGCTACTAT ACCATCGGTA TTGGCCACCT GCTGACCAAAAGCCCGAGCC TGAACGCGGC GAAGAGCGAA CTGGACAAAG CGATCGGCCG TAACTGCAACGGTGTGATTA CCAAGGATGA GGCGGAAAAA CTGTTTAACC AGGACGTGGA TGCGGCGGTTCGTGGTATCC TGCGTAACGC GAAGCTGAAA CCGGTGTACG ACAGCCTGGA TGCGGTTCGTCGTTGCGCGC TGATTAACAT GGTGTTCCAA ATGGGCGAGA CCGGCGTTGC GGGTTTTACCAACAGCCTGC GTATGCTGCA GCAAAAGCGT TGGGACGAAG CGGCGGTTAA CCTGGCGAAAAGCATCTGGT ATAACCAGAC CCCGAACCGT GCGAAACGTG TGATTACCAC CTTCCGTACCGGCACCTGGG ATGCGTATAA AAACCTGTAAAmino acid sequence:SEQ ID NO: 26MSDTMVVNGS GGVPAFLFSG STLSSYRPNF EANSITIALP HYVDLPGRSN FKLMYIMGFPIDTEMEKDSE YSNKIRQESK ISKTEGTVSY EQKITVETGQ EKDGVKVYRV MVLEGTIAESIEHLDKKENE DILNNNRNRI VLADNTVINF DNISQLKEFL RRSVNIVIFE MLRIDERLRLKIYKDTEGYY TIGIGHLLTK SPSLNAAKSE LDKAIGRNCN GVITKDEAEK LFNQDVDAAVRGILRNAKLK PVYDSLDAVR RCALINMVFQ MGETGVAGFT NSLRMLQQKR WDEAAVNLAKSIWYNQTPNR AKRVITTFRT GTWDAYKNLTheoretical pI/Mw: 8.62/37291.55 Fyu A BD-GP36 fusion:DNA sequence:SEQ ID NO: 27ATGAGCGACA CGATGGTTGT GAATGGCAGC GGCGGCGTTC CGGCGTTCCT GTTTAGCGGCAGCACCCTGA GCAGCTATCG TCCGAATTTC GAGGCGAACA GCATCACCAT TGCGCTGCCGCACTATGTGG ACCTGCCGGG CCGTAGCAAC TTCAAGCTGA TGTATATCAT GGGTTTTCCGATTGACACCG AGATGGAAAA GGATAGCGAG TACAGCAACA AAATCCGTCA AGAAAGCAAGATTAGCAAAA CCGAGGGCAC CGTGAGCTAC GAACAGAAAA TCACCGTTGA GACCGGCCAAGAAAAGGATG GTGTGAAAGT TTATCGTGTG ATGGTTCTGG AGGGCACCAT CGCGGAGAGCATTGAACACC TGGACAAGAA AGAGAACGAA GACATCCTGA ACAACAACCG TAACCGTATTGTGCTGGCGG ACAACACCGT TATCAACTTC GATAACATTA GCCAGCTGAA GGAATTTCTGCGTCGTAGCG TGAACATCGT TGGTGTGGCC CTGGACCGCA CGCGGGTTGA TCCCCAGGCAGTCGGCAACG AGGTGCTCAA GCGCAACGCG GATAAGCTGA ATGCGATGCG GGGCGCCGAGTACGGTGCCA ACGTCAAGGT CAGCGGCACG GACATTCGCA TGAACGGGGG TAACAGTGCCGGCATGCTGA AGCAGGACGT GTTCAACTGG CGGAAGGAAC TGGCTCAGTT CGAGGCTTACCGAGGGGAGG CGTATAAGGA TGCCGATGGT TATAGTGTGG GCCTGGGGCA TTACCTGGGCAGTGGCAATG CTGGGGCAGG TACTACAGTC ACGCCTGAGC AAGCCGCGCA GTGGTTCGCCGAGGACACCG ACCGCGCACT CGACCAGGGT GTGAGGTTGG CCGACGAGCT GGGCGTTACGAACAATGCCT CTATCCTGGG ATTGGCCGGT ATGGCCTTCC AGATGGGCGA AGGACGTGCCCGGCAGTTCC GTAACACCTT CCAGGCGATC AAGGATCGCA ACAAGGAAGC CTTCGAGGCTGGTGTGCGAA ACAGCAAGTG GTACACGCAG ACGCCCAACC GGGCCGAGGC ATTCATCAAGCGCATGGCGC CCCACTTCGA TACACCGAGT CAAATCGGTG TCGATTGGTA CAGCGCCGCAACAGCGGAGT GAAmino acid sequence:SEQ ID NO: 28MSDTMVVNGS GGVPAFLFSG STLSSYRPNF EANSITIALP HYVDLPGRSN FKLMYIMGFPIDTEMEKDSE YSNKIRQESK ISKTEGTVSY EQKITVETGQ EKDGVKVYRV MVLEGTIAESIEHLDKKENE DILNNNRNRI VLADNTVINF DNISQLKEFL RRSVNIVGVA LDRTRVDPQAVGNEVLKRNA DKLNAMRGAE YGANVKVSGT DIRMNGGNSA GMLKQDVFNW RKELAQFEAYRGEAYKDADG YSVGLGHYLG SGNAGAGTTV TPEQAAQWFA EDTDRALDQG VRLADELGVTNNASILGLAG MAFQMGEGRA RQFRNTFQAI KDRNKEAFEA GVRNSKWYTQ TPNRAEAFIKRMAPHFDTPS QIGVDWYSAA TAETheoretical pI/Mw: 5.51/42394.38pelB-FyuA receptor: 2028 basesSEQ ID NO: 29ATGAAATACC TGCTGCCGAC CGCTGCTGCT GGTCTGCTGC TCCTCGCTGC CCAGCCGGCGATGGCCATGG GCCAGACTTC ACAGCAAGAC GAAAGCACGC TGGTGGTTAC CGCCAGTAAACAATCTTCCC GCTCGGCATC AGCCAACAAC GTCTCGTCTA CTGTTGTCAG CGCGCCGGAATTAAGCGACG CCGGCGTCAC CGCCAGCGAC AAACTCCCCA GAGTCTTGCC CGGGCTCAATATTGAAAATA GCGGCAACAT GCTTTTTTCG ACGATCTCGC TACGCGGCGT CTCTTCAGCGCAGGACTTCT ATAACCCCGC CGTCACCCTG TATGTCGATG GCGTCCCTCA GCTTTCCACCAACACCATCC AGGCGCTTAC CGATGTGCAA AGCGTGGAGT TGCTGCGAGG CCCACAGGGAACGTTATATG GCAAAAGCGC TCAGGGCGGG ATCATCAACA TCGTCACCCA GCAGCCGGACAGCACGCCGC GCGGCTATAT TGAAGGCGGC GTCAGTAGCC GCGACAGTTA TCGAAGTAAGTTCAACCTGA GCGGCCCCAT TCAGGATGGC CTGCTGTACG GCAGCGTCAC CCTGTTACGCCAGGTTGATG ACGGCGACAT GATTAACCCC GCGACGGGAA GCGATGACTT AGGCGGCACCCGCGCCAGCA TAGGGAATGT GAAACTGCGT CTGGCGCCGG ACGATCAGCC CTGGGAAATGGGCTTTGCCG CCTCACGCGA ATGTACCCGC GCCACCCAGG ACGCCTATGT GGGATGGAATGATATTAAGG GCCGTAAGCT GTCGATCAGC GATGGTTCAC CAGACCCGTA CATGCGGCGCTGCACTGACA GCCAGACCCT GAGTGGGAAA TACACCACCG ATGACTGGGT TTTCAACCTGATCAGCGCCT GGCAGCAGCA GCATTATTCG CGCACCTTCC CTTCCGGTTC GTTAATCGTCAATATGCCTC AGCGCTGGAA TCAGGATGTG CAGGAGCTGC GCGCCGCAAC CCTGGGCGATGCGCGTACCG TTGATATGGT GTTTGGGCTG TACCGGCAGA ACACCCGCGA GAAGTTAAATTCAGCCTACG ACATGCCGAC AATGCCTTAT TTAAGCAGTA CCGGCTATAC CACCGCTGAAACGCTGGCCG CATACAGTGA CCTGACCTGG CATTTAACCG ATCGTTTTGA TATCGGCGGCGGCGTGCGCT TCTCGCATGA TAAATCCAGT ACACAATATC ACGGCAGCAT GCTCGGCAACCCGTTTGGCG ACCAGGGTAA GAGCAATGAC GATCAGGTGC TCGGGCAGCT ATCCGCAGGCTATATGCTGA CCGATGACTG GAGAGTGTAT ACCCGTGTAG CCCAGGGATA TAAACCTTCCGGGTACAACA TCGTGCCTAC TGCGGGTCTT GATGCCAAAC CGTTCGTCGC CGAGAAATCCATCAACTATG AACTTGGCAC CCGCTACGAA ACCGCTGACG TCACGCTGCA AGCCGCGACGTTTTATACCC ACACCAAAGA CATGCAGCTT TACTCTGGCC CGGTCGGGAT GCAGACATTAAGCAATGCGG GTAAAGCCGA CGCCACCGGC GTTGAGCTTG AAGCGAAGTG GCGGTTTGCGCCAGGCTGGT CATGGGATAT CAATGGCAAC GTGATCCGTT CCGAATTCAC CAATGACAGTGAGTTGTATC ACGGTAACCG GGTGCCGTTC GTACCACGTT ATGGCGCGGG AAGCAGCGTGAACGGTGTGA TTGATACGCG CTATGGCGCA CTGATGCCCC GACTGGCGGT TAATCTGGTCGGGCCGCATT ATTTCGATGG CGACAACCAG TTGCGGCAAG GCACCTATGC CACCCTGGACAGCAGCCTGG GCTGGCAGGC AACGGCAGCA GCGCCGTCGC GCAGGTCAAT ATGGGTCGCACCGTCGGTAT CAATACGCGA ATTGATTTCT TCTGATheoretical pl/Mw: 5.35/73772.10SEQ ID NO: 30MKYLLPTAAA GLLLLAAQPA MAMGQTSQQD ESTLVVTASK QSSRSASANN VSSTVVSAPELSDAGVTASD KLPRVLPGLN IENSGNMLFS TISLRGVSSA QDFYNPAVTL YVDGVPQLSTNTIQALTDVQ SVELLRGPQG TLYGKSAQGG IINIVTQQPD STPRGYIEGG VSSRDSYRSKFNLSGPIQDG LLYGSVTLLR QVDDGDMINP ATGSDDLGGT RASIGNVKLR LAPDDQPWEMGFAASRECTR ATQDAYVGWN DIKGRKLSIS DGSPDPYMRR CTDSQTLSGK YTTDDWVFNLISAWQQQHYS RTFPSGSLIV NMPQRWNQDV QELRAATLGD ARTVDMVFGL YRQNTREKLNSAYDMPTMPY LSSTGYTTAE TLAAYSDLTW HLTDRFDIGG GVRFSHDKSS TQYHGSMLGNPFGDQGKSND DQVLGQLSAG YMLTDDWRVY TRVAQGYKPS GYNIVPTAGL DAKPFVAEKSINYELGTRYE TADVTLQAAT FYTHTKDMQL YSGPVGMQTL SNAGKADATG VELEAKWRFAPGWSWDINGN VIRSEFTNDS ELYHGNRVPF VPRYGAGSSV NGVIDTRYGA LMPRLAVNLVGPHYFDGDNQ LRQGTYATLD SSLGWQATER MNISVYVDNL FDRRYRTYGY MNGSSAVAQVNMGRTVGINT RIDFF | 186,751 |
11857607 | DETAILED DESCRIPTION OF THE INVENTION Since the present invention can have adaptability for diverse transformation and examples of practical application, below is a more detailed description of the present invention. Nevertheless, this is no means to limit the form of practical application; it should be understood that the intention is to include the concept and the extent of technology in all of the transformation, equivalents to alternatives. In describing the present invention, if any detailed description about the prior art is considered to deteriorate the fundamental principles of the present invention, the description will be omitted. A telomere is known as a repetitive sequence of genetic material at the ends of chromosomes that prevent chromosomes from damage or merging of other chromosomes. The length of a telomere is shortened at each cell division, and after a certain number of cell division, the telomere length is extremely shortened to the extent in which the cell stops dividing and dies. On the other hand, the elongation of telomeres is known to extend the life span of a cell. For an example, cancer cells excrete an enzyme called telomerase, which prevents shortening of telomeres, thus resulting in proliferation of cancer cells. The present invention was accomplished upon the discovery of telomerase-derived peptides with anti-inflammatory effects. In one embodiment of the present invention, a peptide with anti-inflammatory activities is provided. The peptide comprises at least one amino acid sequence of SEQ ID NO: 1, the peptide has above 80% homology with above-mentioned sequence, or the peptide is a fragment of the above-mentioned peptides. Peptide with anti-inflammatory activity in the present invention is a peptide having an amino acid sequence to SEQ ID NO: 1. Peptide of SEQ ID NO: 1 is a peptide consisting of 16 amino acids in the location of telomerase-[611-626]. SEQ ID NO: 1EARPALLTSRLRFIPK In one embodiment of the present invention, a polynucleotide that codes a peptide with anti-inflammatory activities is provided. The polynucleotide codes a peptide comprising at least one amino acid sequence of SEQ ID NO: 1, a peptide having above 80% homology with above-mentioned sequence, or a peptide being a fragment of the above-mentioned peptides. The polynucleotide mentioned above enables production of the peptides in large quantities. For example, cultivation of vectors that include polynucleotides encoding peptides allows production of peptides in large quantities. The peptides disclosed herein can include a peptide comprising amino acid sequence above 80%, above 85%, above 90%, above 95%, above 96%, above 97%, above 98%, or above 99% homology. Moreover, the peptides disclosed in the present invention can include a peptide comprising SEQ ID NO: 1 or its fragments, and a peptide with more than 1 transformed amino acid, more than 2 transformed amino acid, more than 3 transformed amino acid, more than 4 transformed amino acid, more than 5 transformed amino acid, more than 6 transformed amino acid, or more than 7 transformed amino acid. In the present specification and claims, the terms “homology” and “sequence identity” are used interchangeably to indicate the degree of sequence overlap between two amino acid (or if relevant: nucleic acid) sequences. Unless otherwise stated the term “Sequence identity” for peptides as used herein refers to the sequence identity calculated as (nref−ndif)·100/nref, wherein ndifis the total number of non-identical residues in the two sequences when aligned so that a maximum number of amino acids are identical and wherein nrefis the number of residues in the shortest of the sequences. Hence, the DNA sequence agtcagtc will have a sequence identity of 75% with the sequence aatcaatc (ndif=2 and nref=8). In some embodiments the sequence identity is determined by conventional methods, e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, using the CLUSTAL W algorithm of Thompson et al., 1994, Nucleic Acids Res 22:467380, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). The BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) for which software may be obtained through the National Center for Biotechnology Information www.ncbi.nlm.nih.gov/) may also be used. When using any of the aforementioned algorithms, the default parameters for “Window” length, gap penalty, etc., are used. In one embodiment of the present invention, changes in amino acid sequence belong to the modification of peptide's physical and chemical characteristics. For example, amino acid transformation can be performed by improving thermal stability of the peptide, altering substrate specificity, and changing the optimal pH. In one embodiment of the present invention, a peptide comprising amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence or a peptide fragment of above-mentioned peptides is preferably made of 30 or less amino acids. In one embodiment of the present invention, a peptide comprising amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence or a peptide fragment of above-mentioned peptides comprises a peptide originates from telomerase, more specifically, telomerase ofHomo sapiens. The term “amino acid” herein includes not only the 22 standard amino acids that are naturally integrated into peptide but also the D-isomers and transformed amino acids. Therefore, in a specific embodiment of the present invention, a peptide herein includes a peptide having D-amino acids. On the other hand, a peptide may include non-standard amino acids such as those that have been post-translationally modified. Examples of post-translational modification include phosphorylation, glycosylation, acylation (including acetylation, myristorylation, plamitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, transformation in chemical properties (e.g. β-removing deimidation, deamidation) and structural transformation (e.g. formation of disulfide bridge). Also, changes of amino acids are included and the changes of amino acids occur due to chemical reaction during the combination process with crosslinkers for formation of a peptide conjugate. A peptide disclosed herein may be a wild-type peptide that has been identified and isolated from natural sources. On the other hand, when compared to peptide fragments of SEQ ID NO: 1, the peptides disclosed herein may be artificial mutants that comprise one or more substituted, deleted and/or inserted amino acids. Amino acid alteration in wild-type polypeptide—not only in artificial mutants—comprises conservative substitution of amino acids that do not influence protein folding and or activation. Examples of conservative substitution belong to the group consisting of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagines), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). The amino acid substitutions that do not generally alter the specific activity are known in the art of the present invention. Most common occurred alteration are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and the opposite alterations. Another example of conservative substitutions are shown in the following table 1. TABLE 1OriginalExamples of residuePreferable residueamino acidsubstitutionsubstitutionAla (A)val; leu; ileValArg (R)lys; gln; asnLysAsn (N)gln; his; asp, lys; argGlnAsp (D)glu; asnGluCys (C)ser; alaSerGln (Q)asn; gluAsnGlu (E)asp; glnAspGly (G)alaAlaHis (H)asn; gln; lys; argArgIle (I)leu; val; met; ala; phe; norleucineLeuLeu (L)norleucine; ile; val; met; ala; pheIleLys (K)arg; gln; asnArgMet (M)leu; phe; ileLeuPhe (F)leu; val; ile; ala; tyrTyrPro (P)alaAlaSer (S)thrThrThr (T)serSerTrp (W)tyr; pheTyrTyr (Y)trp; phe; thr; serPheVal (V)ile; leu; met; phe; ala; norleucineLeu The substantial transformation of the biological properties of peptides are performed by selecting significantly different substitution in the following efficacies: (a) the efficacy in maintaining the structure of the polypeptide backbone in the area of substitution, such as sheet or helical three-dimensional structures, (b) the efficacy in maintaining electrical charge or hydrophobicity of the molecule in the target area, or (c) the efficacy of maintaining the bulk of the side chain. Natural residues are divided into groups by general side chain properties as the following:(1) hydrophobicity: Norleucine, met, ala, val, leu, ile;(2) neutral hydrophilicity: cys, ser, thr;(3) acidity: asp, glu;(4) basicity: asn, gln, his, lys arg;(5) residue that affects chain orientation: gly, pro; and(6) aromaticity: trp, tyr, phe. Non-conservative substitutions may be performed by exchanging a member of the above classes to a different class's. Any cysteine residues that are not related in maintaining the proper three-dimensional structure of the peptide can typically be substituted into serine, thus increasing the oxidative stability of the molecule and preventing improper crosslinkage. Conversely, improvement of stability can be achieved by adding cysteine bond(s) to the peptide. Altered types of amino acids variants of peptides are those that antibody glycosylation pattern changed. The term “change” herein relates to deletion of carbohydrate residues and/or addition of at least one glycosylated residues that do not exist within a peptide. Glycosylation in peptides are typically N-connected or O-connected. The term “N-connected” herein relates to that carbohydrate residues are attached to the side chain of asparagine residues. As tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (where the X is any amino acid except proline) are the recognition sequence for attaching carbohydrate residue enzymatically to the side chain of asparagine. Therefore, with the presence of one of these tripeptide sequences in a polypeptide, the potential glycosylation sites are created. “O-connected glycosylation” means attaching one of sugar N-acetylgalactosamine, galactose, or xylose to hydroxyl amino acids. The hydroxyl amino acids are most typically serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can be used. Addition of glycosylation site to a peptide is conveniently performed by changing amino acid sequence to contain tripeptide sequence mentioned above (for N-linked glycosylation sites). These changes may be made by addition of at least one serine or threonine residues to the first antibody sequence, or by substitution with those residues (for O-linked glycosylation sites). In one embodiment of the present invention, a polynucleotide is a nucleic acid molecule that can be spontaneous or artificial DNA or RNA molecules, either single-stranded or double-stranded. The nucleic acid molecule can be one or more nucleic acids of same type (for example, having a same nucleotide sequence) or nucleic acids of different types. The nucleic acid molecules comprise one or more DNA, cDNA, decoy DNA, RNA, siRNA, miRNA shRNA, stRNA, snoRNA, snRNA PNA, antisense oligomer, plasmid and other modified nucleic acids, but not limited to those. A HMGB1 protein is known as a cytokine. It first undergoes acetylation and translocation to cytoplasm by external stimulation. Then it is secreted out of the cell, therefore serving the role of inflammation-causing cytokine. Because when one has an inflammation due to such activity, HMGB1 protein is secreted out of the cell, and patients with inflammatory diseases such as Churg strauss syndrome, rheumatoid arthritis and Sjogren's syndrome will present with elevated serum levels of HMGB1. Hence, if nucleus contains large amount of HMGB1 even when there is a stimulus that causes inflammation, it is suggestive of the fact that HMGB1 is not being secreted out of the cell, which means inflammation is being suppressed. In one embodiment of the present invention, when treated a cell with a peptide comprising amino acid sequence of SEQ ID NO: 1, a peptide having above 80% homology of amino acid sequence with above-mentioned sequence, or a fragment of the above-mentioned peptides, amount of HMGB1 within the nucleus increases. This represents that the peptides mentioned above have excellent inflammation preventive or suppressive effects. Also, in specific embodiments of the present invention, a peptide comprising amino acid sequence of SEQ ID NO: 1, a peptide having above 80% homology of amino acid sequence with above-mentioned sequence, or a fragment of the above-mentioned peptides, has an advantage in that it has high feasibility due to its low toxicity within a cell. In the present invention, an “inflammatory disease” is a broad indication that refers to any disease that designates inflammation as a main cause or inflammation caused by disease. Specifically, an inflammatory disease includes (1) general or localized inflammatory disease (for example, allergies; immune-complex disease; hayfever; hypersensitive shock; endotoxin shock; cachexia, hyperthermia; granulomatosis; or sarcoidosis); (2) gastro-intestinal related diseases (for example, appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatitis; ulcerative, acute, or ischemic colitis; cholangitis; cholecystitis, steatorrhea, hepatitis, Crone's disease; or Whipple's Disease); (3) dermal related diseases (for example, psoriasis; burns; sunburns; dermatitis; Urticarial warts or wheal); (4) vascular related diseases (for example, angiitis; vasculitis; endocarditis; arteritis; atherosclerosis; thrombophlebitis; pericarditis; congestive heart failure; myocarditis; myocardial ischemia; periarteritis nodosa; recurrent stenosis; Buerger's disease; or rheumatic fever); (5) respiratory diseases (for example, asthma; epiglottitis; bronchitis; emphysema; rhinitis; cystic fibrosis; interstitial pneumonitis; COPD (chronic obstructive pulmonary disease); adult respiratory distress syndrome; coniosis; alveolitis; bronchiolitis; pharyngitis; pleurisy; or sinusitis); (6) bone, joint, muscle and connective tissue related diseases (for example, eosinophilic granuloma; arthritis; arthralgia; osteomyelitis; dermatomyositis; fasciitis; Paget's disease; gout; periodontal disease; rheumatoid arthritis; myasthenia gravis; ankylosing spondylitis; or synovitis); (7) urogenital disorders (for example, epididymitis; vaginitis; prostatitis; or urethritis); (8) central or peripheral nervous system related diseases (for example, Alzheimer's disease; meningitis; encephalitis; multiple sclerosis; cerebral infarction; cerebral embolism; Guillain-Barre syndrome; neuritis; neuralgia; spinal cord injury; paralysis; or uveitis); (9) virus (for example, influenza; respiratory syncytial virus; HIV; hepatitis B; hepatitis C; or herpes virus), infectious disease (for example, Dengue fever; or septicemia), fungal infection (for example, candidiasis); or bacterial, parasitic, and similar microbial infection (for example, disseminated bacteremia; malaria; onchocerciasis; or amebiasis); (10) autoimmune disease (for example, thyroiditis; lupus; Goodpasture's syndrome; allograft rejection; graft versus host disease; or diabetes); and (11) cancer or tumor disease (for example, Hodgkin's disease), but not limited to those. Treating the inflammatory component of such diseases has been a major goal of the global pharmaceutical industry for a number of decades, and a wide variety of useful treatments have been developed. Examples include the corticosteroids (a range of natural, semisynthetic and synthetic agents designed to mimic the effect of cortisol, including prednisolone, methylprednisolone, dexamethasone, betamethasone, fluticasone and so forth), cyclooxygenase inhibitors (both non-selective or cox-1 selective, such as indomethacin, sulfasalzine and aspirin, and more recently cox-2 selective, such as celecoxib), leukotriene blockers (such as monteleukast) and anti-TNFs (such as modified monoclonal neutralising antibodies, including infliximab (Remicade™) and adalimumab (Humira™), TNF receptor fusion proteins, such as etanercept (Enbrel™), as well as small molecule TNF-α synthesis inhibitors like thalidomide). In one embodiment of the present invention, an anti-inflammatory composition comprising a peptide as an active ingredient is provided. The peptide comprises amino acid sequence of SEQ ID NO: 1, the peptide has above 80% homology with above-mentioned sequence, or the peptide is a fragment of the above-mentioned peptides. In one embodiment of the present invention, the anti-inflammatory composition may contain 0.1 μg/mg to 1 mg/mg, specifically 1 μg/mg to 0.5 mg/mg, more specifically 10 μg/mg to 0.1 mg/mg of a peptide comprising of amino acid sequence SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or peptide fragment of above-mentioned peptides. When the peptide is contained in the above mentioned range, all the safety and stability of the composition may be satisfied and appropriate in terms of cost-effectiveness. In one embodiment of the present invention, the composition may have application with all animals including human, dog, chicken, pig, cow, sheep, guinea pig, and monkey. In one embodiment of the present invention, the pharmaceutical composition for the use of treatment or prophylaxis of inflammatory disease with an active ingredient that is comprised of a peptide consisting of an amino acid of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or peptide fragment of SEQ ID NO:1, is provided. In one embodiment of the present invention, the pharmaceutical composition may be administered through oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal, in the bone marrow, epidural or subcutaneous means. Forms of oral administration may be, but not limited to, tablets, pills, soft or hard capsules, granules, powders, solution, or emulsion. Forms of non-oral administration may be, but not limited to, injections, drips, lotions, ointments, gels, creams, suspensions, emulsions, suppository, patch, or spray. In one embodiment of the present invention, the pharmaceutical composition, if necessary, may contain additives, such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, coloring agents, aromatics or sweeteners. In one embodiment of the present invention, the pharmaceutical composition can be manufactured by conventional methods of the industry in the art. In one embodiment of the present invention, the active ingredient of the pharmaceutical composition may vary according to the patient's age, sex, weight, pathology and state, administration route, or prescriber's judgment. Dosage based on these factors is determined within levels of those skilled in the art, and the daily dose for example may be, but not limited to, 0.1 μg/kg/day to 1 g/kg/day, specifically 1 μg/kg/day to 10 mg/kg/day, more specifically the 10 μg/kg/day to 1 mg/kg/day, more specifically the 50 μg/kg/day to 100 μg/kg/day. In one embodiment of the present invention, the pharmaceutical composition may be administered, but not limited to, 1 to 3 times a day. In one embodiment of the present invention, a skin external composition for improvement or prevention of skin inflammation is provided. The skin external composition may contain an active ingredient that is a peptide comprising of an amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or a peptide fragment of above-mentioned peptides. In another embodiment of the present invention, a cosmetic composition for improvement or prevention of skin inflammation is provided. The cosmetic composition may contain an active ingredient that is a peptide comprising of an amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or peptide fragment of above-mentioned peptides. In one embodiment of the present invention, external application composition or cosmetic composition may be provided in all forms appropriate for topical applications. For example, forms can be provided as solutions, emulsions obtained by dispersion of oil phase in water, emulsion obtained by dispersion of water in oil phase, suspension, solid, gel, powder, paste, foam or aerosol. These forms can be manufactured by conventional methods of the industry in the art. In one embodiment of the present invention, the cosmetic composition may include, within levels that will not harm the main effect, other ingredients that can desirably increase the main effect. In one embodiment of the present invention, the cosmetic composition may additionally include moisturizer, emollient agents, surfactants, UV absorbers, preservatives, fungicides, antioxidants, pH adjusting agent, organic or inorganic pigments, aromatics, cooling agent or antiperspirant. The formulation ratio of the above-mentioned ingredients can be decided by those skilled in the art within levels that will not harm the purpose and the effects of the present invention, and the formulation ratio based on total weight of the cosmetic composition can be 0.01 to 5% by weight, specifically 0.01 to 3% by weight. In one embodiment of the present invention, a food composition for inflammation prevention or suppression is provided. The food composition may contain with an active ingredient that is a peptide comprising of an amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or peptide fragment of above-mentioned peptides. In one embodiment of the present invention, food composition is not limited to forms, but for example may be granules, powder, liquid, and solid forms. Each form can be formed with ingredients commonly used in the industry appropriately chosen by those skilled in the art, in addition to the active ingredient, and can increase the effect with other ingredients. Decision for dosage on the above-mentioned active ingredient is within the level of those skilled in the art, and daily dosage for example may be 1 μg/kg/day to 10 mg/kg/day, more specifically the 10 μg/kg/day to 1 mg/kg/day, more specifically the 50 μg/kg/day to 100 μg/kg/day, but not limited to these numbers and can vary according to age, health status, complications and other various factors. In one embodiment of the present invention, a use of prevention or treatment of inflammatory disease with a peptide comprising of an amino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acid sequence above 80% homology with above-mentioned sequence, or peptide fragment of above-mentioned peptides, is provided. In one embodiment of the present invention, the method of prevention or treatment of inflammatory disease with applying peptides mentioned above in patients is provided. In one embodiment of the present invention, a kit for prophylaxis or treatment of inflammatory diseases is provided. The kit may contain: a peptide with anti-inflammatory activity or a composition comprising of the peptide, wherein the peptide comprises amino acid sequence of SEQ ID NO: 1, the peptide has above 80% homology with above-mentioned sequence, or the peptide is a fragment of the above-mentioned peptides; and instructions including at least one of administration dose, administration route, administration frequency, and indication of the peptide or composition. The terms used herein is intended to be used to describe the embodiments, not to limit the present invention. Terms without numbers in front are not to limit the quantity but to show that there may be more than one thing of the term used. The term “including”, “having”, “consisting”, and “comprising” shall be interpreted openly (i.e. “including but not limited to”). Mention of range of numbers is used instead of stating separate numbers within the range, so unless it is explicitly stated, each number can be read as separate numbers integrated herein. The end values of all ranges are included in the range and can be combined independently. Unless otherwise noted or clearly contradicting in context, all methods mentioned herein can be performed in the proper order. The use of any one embodiment and all embodiment, or exemplary language (e.g., that use “like ˜”), unless included in the claims, is used to more clearly describe the present invention, not to limit the scope of the present invention. Any language herein outside of the claims should not be interpreted as a necessity of the present invention. Unless defined otherwise, technical and scientific terms used herein have meaning normally understood by a person skilled in the art that the present invention belongs to. The preferred embodiments of the present invention are the best mode known to the inventors to perform the present invention. It may become clear to those skilled in the art after reading the statements ahead of the variations in the preferred embodiments. The present inventors hope that those skilled in the art can use the variations adequately and present invention be conducted in other ways than listed herein. Thus, the present invention, as allowed by the patent law, includes equivalents, and variations thereof, of the key points of the invention stated in the appended claims. In addition, all possible variations within any combination of the above-mentioned components are included in the present invention, unless explicitly stated otherwise or contradicting in context. Although the present invention is described and shown by exemplary embodiments, those skilled in the art will understand well that there can be various changes in the form and details without departing from the spirit of the invention and range, defined by the claims below. Tumor necrosis factor (TNF), particularly TNF-α, is known to be released from inflammatory cells and cause various cytotoxic reactions, immunological reactions and inflammatory reactions. TNF-α is known to be involved in the occurrence and prolongation of many inflammatory and autoimmune diseases and further cause serious septicemia and septic shock when it is released into the blood and acts systemically. Because TNF-α is a factor associated widely with the immune system of a living body, the development of agents inhibiting TNF-α is actively carried out. TNF-α is biosynthesized in an inactive form and becomes an active form by being cleaved by protease; the enzyme responsible for the activation is called a tumor necrosis factor-converting enzyme (TACE). Thus, a substance inhibiting this TACE can treat, improve, or prevent diseases, pathologic conditions, abnormal conditions, troubles, adverse symptoms and the like ascribed to TNF-α (KR2011-0060940A). High-mobility group box 1 (HMGB1) protein exists in high concentrations in thymus, lymph nodes, testes, and in fetal liver, and with exception to liver and brain cells, usually exists inside of the nucleus. The said HMGB1 protein has 3 domains consisting of A-box, B-box, and C-terminal. It was reported by Tracey et al., 1999 that HMGB1 protein has a role as a cytokine which induces inflammation, and the mechanism of said HMGB1's inflammation induction is by an external stimulus causing acetylation of HMGB1 which then moves from the nucleus into the cytoplasm. Afterward, it is known to be secreted out of the cell, or secreted out from the cell in necrosis. (Bonaldi T et al., EMBO 3, (22)5551-60, 2003). The invention is further described by the figures, the following examples and experiments, which are solely for the purpose of illustrating specific embodiments of this invention, and are not to be construed as limiting the scope of the invention in any way. Example 1 Synthesis of PEP-1 and Measurement of Anti-Inflammatory Activities of PEP-1 (SEQ ID NO: 1) Experiment 1-1: Synthesis of PEP-1 (SEQ ID NO: 1) A peptide comprised of 16 amino acids with the chemical structure 1 as below having the sequence SEQ ID: 1 (PEP-1) derived from human telomerase was synthesized: SEQ ID NO: 1 (PEP-1) was synthesized according to the existing method of solid phase peptide synthesis. In detail, the peptides were synthesized by coupling each amino acid from C-terminus through Fmoc solid phase peptide synthesis, SPPS, using ASP48S (Peptron, Inc., Daejeon ROK). Those peptides with their first amino acid at the C-terminus being attached to resin were used as follows:NH2-Lys(Boc)-2-chloro-Trityl ResinNH2-Ala-2-chloro-Trityl ResinNH2-Arg(Pbf)-2-chloro-Trityl Resin All the amino acid materials to synthesize the peptide were protected by Fmoc at the N-terminus, and the amino acid residues were protected by Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl) that can be dissolved in acid. Such as:Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid. HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate]/HOBt [N-Hydroxybenzotriazole]/NMM [4-Methylmorpholine] were used as the coupling reagents. In 20% of DMF, piperidine was used to remove Fmoc. In order to remove the protection from residue or to separate the synthesized peptide from Resin, cleavage cocktail [TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/EDT (ethanedithiol)/H2O=92.5/2.5/2.5/2.5] was used. Synthesized the peptide by using the solid phase scaffold combined to starting amino acid with the amino acid protection, reacting the corresponding amino acids separately, washing with solvent and deprotected, and repeating the process. After cutting off the synthesized peptide from the resin, it was purified by HPLC and verify for synthesis by MS, and then freeze-dried. Specific synthesis process of PEP 1 is described by the following. 1) Coupling Melted the amino acid (8 equivalent) protected with NH2-Lys(Boc)-2-chloro-Trityl Resin, and coupling agent HBTU (8 equiv.)/HOBt (8 equiv.)/NMM (16 equiv.) and added to DMF, then let react in room temperature for 2 hours, then washed with DMF, MeOH, and DMF in that order. 2) Fmoc deprotection Added 20% piperidine in DMF and reacted in room temperature for 5 minutes 2 times, then washed with DMF, MeOH, and DMF in that order. 3) Make the basic framework of peptide by repeating reactions 1 and 2 repeatedly. 4) Cleavage: Add Cleavage Cocktail to the completely synthesized peptide and separated the peptide from the resin. 5) Add cooling diethyl ether into obtained mixture, and then centrifugation is used to precipitate gathered peptide. 6) After purification by Prep-HPLC, check the molecular weight by LC/MS and lyophilize to produce in powder form. Example 2 Anti-Inflammatory Activity Measurement of PEP-1 Cell Lines Culture Raw 264.7 macrophage cell (KCBL, 40071) from Korea Cell Bank was maintained in Dulbecco's modified Eagle's medium (DMEM; PAA, Austria) containing 10% fetal bovine serum (FBS; Gibco Laboratories), 100 unit/mL of streptomycin, and penicillin (Gibco Laboratories) at 37° C. with 5% CO. Raw264.7 cells were seeded into a 96-well plate at a density of 1×106cells/mL and incubated overnight. On the following day, the medium was replaced with fresh medium and 5 μg/mL of peptide (obtained as described in Experiment example 1) was added to the cells. After 30 min incubation of cells with the peptide 50 μL of LPS (to a final concentration of 1 μg/mL) was added, and cells were incubated for additional 24 hr. The experimental sample with the induction of inflammatory response was treated with 1 μg/mL mL lipopolysaccharide (LPS; Sigma, USA) and control sample was treated with phosphate buffered saline (PBS; pH 7.2). The supernatant samples from each condition was collected in eppendorf tubes and subjected to further analysis. Experiment 2-1. NO Level Analysis The level of nitric oxide (NO) was measured in Raw 264.7 cell (1×106cell/ml) using Griess reagent system (Promega, USA). Culture medium of 50 μl was added to a 96-well plate and Griess reagent I (NED) solution and Griess reagent II (Sulfanilamide solution) are added in the same amount. After 10 min incubation of cells with the reagents, the optical density at 540 nm was measured within 30 min using a microplate reader (Molecular Devices, USA). The concentration of NO was calculated by using a standard curve (0˜100 μM) of sodium nitrite. As shown in Table 2 below, stimulation of cells with LPS increased the expression of NO, but in co-treatment with LPS and PEP-1, the expression level of NO mentioned above decreased. NO is produced during inflammation, and the result showing PEP-1 reduced NO level to 65% of the control strongly support the anti-inflammatory effect of PEP-1. TABLE 2The measurement of anti-inflammatory effectof human telomerase derived PEP 1NO ExpressionDecreasedLevel ofNO ExpressioncontrolLevelTest sample(%)(%)PBS0—LPS 1 μg/mLPBS1000PEP 1 (0.5 μg/mL)3565 Experiment 2-2. Analysis of Cytokine Inhibitory Effect To investigate the effect of PEP-1 on inhibiting proinflammatory cytokine production RAW 264.7 cell were pre-treated with PEP 1 at a concentration of 5 μg/mL challenged with LPS at a concentration of 1 μg/mL, and cells were further incubated for 24 hr. The supernatant samples containing cell culture medium was collected and analyzed for the cytokine levels using ELISA kits (eBioscience, San Diego). 96 wells plates were coated with 100 μL of capture antibodies (diluted in coating buffer to the concentration recommended by manufacturer's protocol) overnight at 4° C. Then, after washing the plates 5 times, 200 μL of assay diluents was added to each well and incubated for 1 hr at room temperature for blocking. After washing each well with wash buffer five times, cell culture sample or each cytokine standard protein sample was diluted and 100 μL of each added into each well. The plate containing samples were incubated overnight at 4° C. Then, after washing the plate five times with the wash buffer, 100 μL of secondary antibody conjugated to avidin was added and incubated for 1 hr at room temperature. Following incubation with the secondary antibody, the plate was washed five times and incubated with 100 μL of avidin-HRP (BD Bioscience) for 30 min at room temperature. After washing the plate seven times, 100 μL of TMB solution (Pierce) was added and incubated for 15 min at room temperature. The reaction was stopped by adding 50 μl of 2N H2SO4in each well The optical density at 450 nm was measured using a microplate reader. Statistical analysis was performed by variance analysis using ANOVA procedure of SPSS program, and verified the significance between analyses using Duncan's multiple range test. Experiment 2-3. IL-6 Secretion Measurement As shown in Table 3 below, treatment with LPS alone increased the cytokine IL-6 (interleukin-6) secretion. However, co-treatment with LPS and PEP-1 showed a decrease in the level of the proinflammatory cytokine IL-6 secretion. More importantly, after the treatment with PEP-1, the level of proinflammatory cytokine secretion decreased by more than 70%, which indicates a robust anti-inflammatory effect of PEP-1. TABLE 3Cytokine IL-6 production inhibition by PEP-1cytokine IL-6 productionTest sample% of controlinhibition %PBS0—LPS 1 μg/mlPBS1000PEP 1 (5 μg/ml)2872 Experiment 2-4. HMGB1, TNF-α, COX-2 Expression Inhibition Protein expression level was determined by Western blot analysis. Cells grown in PEP-1 containing medium were washed with PBS, treated with 0.05% trypsin-EDTA, and collected by centrifugation. The collected cells were dissolved in an appropriate volume of lysis buffer. Intracellular debris was pelleted by centrifugation, and equal amount of protein from each sample was separated by SDS-polyacrylamide gel electrophoresis. The separated protein was transferred to nitrocellulose membrane (Schleicherand Schuell, Keene, NH, USA), then was tested for the antibody specific for each protein. The membrane was incubated with ECL (enhanced chemiluminoesence) solution (Amersham Life Science Corp., Arlington Heights, IL, USA), exposed to X-ray, and the level of protein expression was analyzed according to the exposure level shown on the X-ray film. Western blot analysis was performed to determine the inhibitory effect of PEP-1 on the cytokine protein expression. As shown in Table 4 below, stimulation of cells with LPS increased the expression of cytokines; HMGB1, TNF-α and COX. However, if cells were treated with both LPS and PEP-1, the expression level of pro-inflammatory cytokines mentioned above decreased. The result showing the treatment with PEP-1 decreased pro-inflammatory cytokine levels by more than 70% provide strong evidence supporting the anti-inflammatory effect of PEP-1. TABLE 4The measurement of inhibitory effect of PEP-1on pro-inflammatory cytokine expression level.Cytokine Expression Level(band intensity) % of controlTest sampleHMGB1TNF-αCOX-2PBS———LPS 1 μg/mlPBS100100100PEP 1 (5 μg/ml)302522 Example 3 Investigation of the Inhibitory Effect of PEP-1 on TNF-α Level in HepG2 Cells Experiment 3-1: Cell Culture PBMC (peripheral blood mononuclear cell) was separated from the blood samples (50 ml) collected from healthy subjects using Ficoll-Paque™ PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). PBMCs were then enriched in complete RPMI 1640 medium containing 20% of human serum, followed by transferring to 100 mm polystyrene cell culture plate coated with human serum for 30 mins. After 2 hr incubation at 37° C. and 5% CO2, the monocytes were detached from the bottom of cell culture plate using cold PBS (Phosphate Buffered Saline) (Gibco/Life Technologies, Carlsbad, CA, USA), and 1×105cells were cultured in each well of 96-well plate in RPMI 1640 medium (supplemented with penicillin-streptomycin; 100 mg/ml, human serum; 20%) over night. For Luciferase Analysis, HEK293/null (human embryonic kidney) cells and HEK293/TRL stably expressing TLR2 (toll-like receptor2) obtained from Seoul National University School of Dental Medicine were used. One day before the luciferase experiment, 2.5×105cells were seeded into each well of 12-well plate and cultured overnight in DMEM (Dulbecco's modified Eagle's medium) medium (supplemented with blasticidin; 10 μg/ml, fetal bovine serum; 10%)(Invitrogen/Life Technologies, Carlsbad, CA, USA) Experiment 3-2: Cytokine Assay To see the effect of PEP-1 on TNF-α level in terms of protein expression level, ELISA (enzyme linked immunosorbent assay) was performed. 1×105PBMC-derived monocytes were cultured in 96-well plate over night. After then, LPS (lipopolysaccharide; 10 ng/ml, Sigma) was treated for 2 hours, followed by 3 times washes with PBS. OPTI-MEM medium (Invitrogen/Life Technologies, Carlsbad, CA, USA) was then treated for an hour to induce the starvation, and 4 μM of FITC (Fluorescein Isothiocyanate), FITC-TAT, PEP-1-FITC, and FITC-PEP-1 were treated for 2 hours before measuring the TNF-α level. After culturing, cell soup was collected, and the amount of TNF-α was measured using ELISA kit (R&D, Minneapolis, MN, USA) as follows: TNF measurement uses sandwich ELISA method. 100 ul of TNF-α primary antibody was added into each well of pre-coated 96-well plate, and the plate was incubated at 4° C. overnight. On next day, the plate was washed 3 times with 0.5% Tween20 wash solution for 5 min each, and then 100 μl of each sample and standard solution was added and left at room temperature for 2 hrs. After washing the plate like above, 100 μl of HRP-conjugated secondary antibody was added into each well and left at room temperature for 2 hrs. Again, plate was washed, and avidin/biotin was added for measuring the absorbance. TNF-α level of each sample was quantified using the standard graph calculated from the absorbance of standard solution. PBMC-derived monocytes were stimulated with endotoxin LPS (10 ng/ml) for 2 hrs, starved for 1 hr using OPTI-MEM, and then 4 uM of FITC, FITC-TAT, PEP 1-FITC and FITC-PEP 1 were treated for 2 hrs. After incubation, TNF-α level was measured with cell culture medium using ELISA. As a result, in case of FITC and FITC-TAT, TNF-α level increased due to LPS (6.2 and 6.7 ng/ml, respectively), but TNF-α level significantly decreased in case of PEP-1-FITC and FITC-PEP-1 (0.17 and 0.25 ng/ml, respectively) and the difference was statistically significant (P<0.01) (FIG.1). Experiment 3-3: Luciferase Assay To investigate the role of PEP 1 in inflammatory response, we evaluated NF-κB expression patterns through luciferase analysis. First, we incubated HEK293/null and HEK293/TLR2 (Graduate School of Dentistry, Seoul National University) in a 12-well plate for 24 hours, so that we would get 2.5×105cells/well. After washing three times with PBS, medium was replaced with OPTI-MEM (Invitrogen/Life Technologies, Carlsbad, CA, USA) and incubated for 4 hours, and then a mixture of 3 μl lipofectamine (Invitrogen/Life Technologies), 1 μg NF-κB luciferase and long renilla luciferase (Promega, Madison, WI, USA) was added into each well and again incubated for 4 hours. Lipoprotein pam3cys (10 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) was put into all of the wells except for those of negative control, and FITC (4 μM) and FITC-PEP 1 (4 μM) were treated for 18 hours before it was washed with PBS for three times. We confirmed the activation of NF-kB through TD-20/20 luminometer (Turner designs, Sunnyvale, CA, USA) after dissolving (lysis) of cells by putting 50 μl of passive lysis buffer—provided by dual-luciferase reporter assay system (Promega)—into each well. Transfection efficacy was confirmed by cotransfection of pCMV-renilla luciferase (Promega), and we analyzed results by calibrating the luciferase values. After transfecting NF-κB luciferase to HEK293/null and HEK293/TLR2 cell lines, pam3cys, a synthetic lipoprotein, and FITC (4 μM), a negative control were treated together, and pam3cys with FITC-PEP 1 (4 μM) were again treated together to be cultured for 18 hours. The measurement of NF-κB expression patterns by luciferase strength through lysis of cells with passive lysis buffer—provided by dual-luciferase reporter assay system (Promega)—showed that there was no difference in lipoprotein or FITC-PEP-1 treated or non-treated HEK293/null. However, when lipoprotein, an agonist of TLR2, was treated to HEK293/TLR2 cell line, NF-κB expression increased (P<0.01) compared to that in untreated, confirming occurrence of inflammatory responses. Also, NF-κB expression increased when FITC-PEP 1 was treated together compared to that in untreated; and expression decreased compared to the negative control in which lipoprotein and FITC were treated together (P<0.01) (FIG.2). Ultimately, we were able to confirm that inflammatory response that can be caused by TLR 2 is reduced when PEP 1 is treated together. Experiment 3-4: Reanalysis of Peptides that Affect Levels of Cytokines in THP1 Cell Line As a Human acute monocytic leukemia cell line, THP-1 monocyte cell line (American Type Culture Collection (Manassas, VA, USA) was used to reconfirm the effects of PEP 1. Cells were grown at a density of 0.5-7×105cells/mL in RPMI 1640 containing 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/mL streptomycin, and maintained at 37° C. under 5% CO2. THP-1 cells were differentiated into macrophages by treating cells with phorbol myristate acetate (PMA) at 100 ng/mL for 24 hr at 37° C. for 24 hr. All reagents and medium were purchased from Gibco BRL. PMA, LPS and 2-mercaptoethanol were purchased from Sigma (St. Louis, MO, USA). Peptide RIA was synthesized from Peptron (Daejeon, Republic of Korea). Reverse transcription PCR kit was purchased from Promega (Madison, WI, USA). RT2SYBR® Green qPCR Mastermix_reagents and QIAzol were purchased from QIAGEN (Valencia, CA, USA). Following differentiation into macrophages, THP-1 cells were washed two times using complete RPMI 1640 (5 min/wash). Then, cells were treated for 4 hr. with 10 ng/ml LPS and/or 4 μM peptide RIA in FBS free RPMI 1640. Total RNA samples were isolated from peptide-treated THP-1 cells by using Trizol (QIAzol) reagent and, and cDNA was synthesized by reverse transcriptase PCR using reverse transcription PCR kit from Promega following manufacturer's protocol. Then, real-Time qPCR was performed using CFX96 (Bio-Rad) instrument with SYBR Green system. Primers used in the experiments are found in Table 5. The PCR cycling conditions were 95° C. for 10 min for activation of HotStart DNA Taq Polymerase, followed by 45 cycles of 95° C. for 10 sec, 55° C. for 30 sec, and 72° C. for 30 sec. All samples were measured in triplicate and differences in gene expression were calculated using the 2-cycle threshold method. All the data were normalized against β actin (housekeeping gene) and presented as means of +/−S.E. from at least three independent experiments. TABLE 5Primers used for qRT-PCR analysis.Gene NameDNA sequencetnf-alpha(forward) 5′-CTATCTGGGAGGGGTCTTCC-3′(reverse) 5′-ATGTTCGTCCTGCTCACAGG-3′il-1 beta(forward) 5′-GGACAAGCTGAGGAAGATGC-3′(reverse) 5′-TCGTTATCCCATGAGTCGAA-3′il-6(forward) 5′-AAAAGTCCTGATCCAGTTCCTG-3′(reverse) 5′-TGAGTTGTCATGTCCTGCAG-3′il-8(forward) 5′-GTGCAGTTTTGCCAAGGAGT-3′(reverse) 5′-AATTTCTGTGTTGGCGCAGT-3′inos(forward) 5′-CACCATCCTGGTGGAACTCT-3′(reverse) 5′-TCCAGGATACCTTGGACCAG-3′beta actin(forward) 5′-AGAAAATCTGGCACCACACC-3′(reverse) 5′-GGGGTGTTGAAGGTCTCAAA-3′ As shown inFIG.3, the cytokines involved in the inflammatory responses decreased noticeably by treating with PEP 1. Example 4 Analysis of Inflammatory Response Induced by Amyloid-β Protein HMGB1 first undergoes acetylation and translocation to cytoplasm by external stimulation. Then it is secreted out of the cell, therefore serving the role of inflammation-causing cytokine. Because when one has an inflammation due to such activity, HMGB1 protein is secreted from the cell, and patients with inflammatory diseases such as Churg strauss syndrome, rheumatoid arthritis and Sjogren's syndrome will present with elevated serum levels of HMGB1. Hence, if nucleus contains large amount of HMGB1 even when there is a stimulus that causes inflammation, it is suggestive of the fact that HMGB1 is not being secreted out of the cell, which means inflammation is being suppressed. Experiment 4-1: Analysis of Survival and Proliferation of Neural Stem Cells by Anti-Inflammatory Effects of PEP-1 First of all, PEP-1 was prepared according to the manufacturing methods described in Example 1. Experiment 4-2: Neural Stem Cell Culture and Amyloid-β Toxicity Assessment After removing the cortex from the head of an embryonic rat that had been pregnant for 13 days, it was cultured for a week with Basic Fibroblast Growth Factor (bFGF) to obtain the neural stem cells. To analyze the effects of the amyloid-β protein on the neural stem cells, the pre-oligomerized amyloid-β protein of concentrations 0 to 40 μM was treated on neural stem cells for 48 hours, then CCK-8 assay, BrdU, and TUNEL assay were used for cytotoxicity assessment (refer from BA Yankner et al, 1990 and KN Dahlgren et al, 2002). We used the same concentration of amyloid-β protein in subsequent experiments after we confirmed that cell survival was reduced to 60% when processed with 20 μM of amyloid-β protein (Refer toFIGS.4and5). Experiment 4-3: Cell Toxicity Assessment by Treatment with PEP-1 To evaluate the impact of PEP-1 on the cultured neural stem cells, the neural stem cells were firstly cultured by a well-known method (BA Yankner et, al, 1990 and KN Dahlgren et al, 2002). Then, different concentrations (0, 1, 10, 50, 100, 200 μM) of PEP-1 were treated for 48 hours, followed by cell viability and proliferation assessments using MTT assay, BrdU and TUNEL assay. PEP-1's concentrations from 0 to 200 μM appeared stable in the neuronal system since they did not inhibit both survival and proliferation of neural stem cells (Refer toFIGS.6and7). Experiment 4-4: Cell Toxicity Assessment by Co-Treatment of Amyloid-β Protein and Telomerase Peptide To determine whether PEP 1 has the effect of suppressing the neurotoxicity caused by amyloid-β protein, 20 μM amyloid-β protein and various concentrations of PEP-1 were co-treated for 48 hours. The cell viability and apoptosis were measured using MMT assay, CCK-8 assay, LDH assay and TUNEL assay, and neural stem cell proliferation by BrdU assay. The results of MMT assay and CCK-8 assay confirmed that 10 μM of PEP-1 began to protect neural stem cells from neurotoxicity by amyloid-β, and the most effective protection was provided in 100 μM. (Refer toFIG.8). LDH assay was carried out for assessment of the degree of cell death as another method, and we confirmed that the increase in cell death by amyloid-β decreased by PEP-1, and efficacy was seen starting at 1 μM concentration (Refer toFIG.9). We also confirmed with BrdU assay that the decreased cell proliferation due to amyloid-β protein was restored when processed with PEP-1 (Refer toFIG.10). Cell mobility is a vital matter due to the nature of neural stem cell. According to the experimental results of cell mobility, we confirmed that the decreased cell proliferation due to amyloid-β protein was restored when processed with PEP-1, and that it increased even more when in 10 μM concentration, compared to control. This suggests that in the future clinical trials, processing prior to stem cell transplantation may draw more effective results. (Refer toFIG.11). To confirm the degree of neuronal stem cells damage, TUNEL assay was performed. Neuronal stem cell death was observed to be significantly increased in 20 μM amyloid-β protein treatment group, and neuronal stem cell death decreased when treated with 1 to 100 μM of PEP 1. (Refer toFIG.12) The mechanism of action of PEP-1's protective effect on apoptosis by amyloid-β protein was investigated. First, it was investigated whether PEP-1 is capable of minimizing the oxidative damage caused by amyloid-β protein. Change in generation of reactive oxygen species after treatment with amyloid-β protein and PEP-1 was observed by using DCF-DA staining (Molecular Probes, Eugene, OR). In the group in which reactive oxygen species increased due to 20 μM of amyloid-β protein, the increased reactive oxygen species decreased by PEP-1 treatment (1 μM, 10 μM, 50 μM) (Refer toFIG.13). Experiment 4-5: Comparative Analysis of Protein Expression Levels Between the Groups Treated with and without PEP-1 Protein expression level of PEP-1 treated group and untreated group was analyzed by 2D-electrophoresis technique and antibody microarray technique. Prepared 200 μg by extracting proteome from the neural stem cells cultured in Experiment 3-1 of Example 3. In addition, the group in which PEP-1 was not treated was used as the comparison group in the same condition. 2D-electrophoresis was performed using 12% acrylamide gels. First gel electrophoresis was performed at PI 4˜10N, using a gel size of 8.5×7 cm. After electrophoresis, it was dyed with Colloidal Coomassie Blue, and then compared expression by using PDQuest software to analyze each spot. Difference in the expression levels of more than 1.5 times was identified using MALDI-TOF MS (Matrix Desoprtion/lionization Time of Flight Mass Spectromestry). Among these, proteins correlated with inflammation-related signaling, such as i-NOS and HMGB-1 were identified (Refer to Table 6). The changes in protein expression levels either increased or decreased by 1.5 times by amyloid-β protein, but it was confirmed that expression level was regulated close to that of negative control when PEP-1 was added (Refer toFIG.14). Antibody microarray was carried out by using cell signaling kit (CSAA1, Panorama™ Ab Microarray Cell Signaling kit), array slides were scanned by GenePix Personal 4100A scanner (Molecular Devices) and the data were analyzed by GenePix Pro 5.0 (Molecular Devices). The Table 6 below is an analysis of expression levels of proteins associated with inflammation by 2D electrophoresis technique. The control group represents protein expression level of cells that were not treated with neither amyloid-β protein nor PEP-1. It shows increased or decreased multiple of protein expression based on the control group's expression level. We confirmed with the results of analysis that like the suggested in Table 6 below, inflammation related protein over-expression or under-expression was controlled by PEP-1; the protein expression level was close to that of negative control group. TABLE 620 ug20 ugβ - amyloidβ - amyloid + PEP 1Negativetreated grouptreated groupProteinControl(fold)(fold)HSP 701.0−2.31.2HSP 901.0−1.81.0HMGB11.0−1.52.8GADD 1531.01.61.2i-NOS1.01.9−1.1e-NOS1.01.9−1.1Pyk21.02.01.2MAP Kinase1.02.21.0 Phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway serves a crucial role in the growth and survival of neuronal stem cells. PI3K pathway is activated by growth factors and regulatory factors, and is involved in the normal regulation of neuronal stem cell growth and survival. AKT signaling pathways disable several pro-apoptotic factors, including a well-known apoptotic signaling molecule, GSK3β. To further investigate the anti-inflammatory effects of PEP-1, we performed Western blot on HMGB1, since it showed a major change in protein analysis. As a result, the processing of the PEP-1 increased the protein expression levels in anti-apoptotic proteins such as Ki67, pAKT, PI3K, HSTF-1 and Bcl-2, and decreased the protein expression levels of apoptotic signals such as Bax, GSK3β, Cytochrom-c, caspase-3 (Refer toFIG.15). HMGB1, a non-histone structure protein that binds to DNA, serves diverse roles within a cell; such as stabilizing nucleosome structure and regulating gene expression. As one of the inflammation-causing substance that is excreted in the late phase of inflammatory response, it is excreted by macrophages and monocytes when inflammation is stimulated, but when neuron is significantly damaged and leads to cell necrosis, it will be excreted out of the cell, causing an intense inflammatory response. The increase of HMGB1 by PEP-1 treatment after the decrease by amyloid-β treatment in the cytoplasm of the nerve cells reflects the fact that PEP-1 inhibits secretion of HMGB1 out of the cell caused by neuron cell death; therefore suggesting that PEP-1 has powerful anti-inflammatory effects (Refer toFIG.15). In addition, we investigated the response of PEP-1 to the amyloid-β aggregation. Aggregation of protein was inhibited when treated with PEP-1 (Refer toFIG.16(A)) in induction of aggregation of amyloid-β, and protein underwent degradation when PEP-1 was treated on the amyloid-β protein that was already induced for aggregation (Refer toFIG.16(B)). In the mechanism of action of PEP-1, we have previously confirmed the increase in cell survival signaling and decrease in apoptosis signaling of PI3K. To investigate whether these effects are direct or indirect, we treated PI3K-inhibitor, LY294002 (Promega). As a result, the increased cell viability after treating with PEP 1 decreased when treated with LY294002. Thus, we can conclude that PI3K is directly associated with PEP 1's neuroprotective effect (Refer toFIG.17). PEP-1 inhibits apoptosis of neural stem cells by amyloid-β protein. Also, the improvement of cell mobility of neural stem cells was confirmed, therefore suggesting a variety of possibilities in clinical application. The inhibition effects from neurotoxicity caused by beta-amyloid protein was verified by the anti-inflammatory effect of the mechanism of action of PEP 1, increased survival factors of neuro stem cells and decreased apoptotic factors, especially activation of PI3K signaling pathway and antioxidant effects. Example 5 qPCR Array MATERIALS AND METHODS THP-1 Cell Culture THP-1 cells (human monocytic leukemia derived cell line) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in RPMI-1640 (Life technologies, Carlsbad, CA, USA) medium supplemented with 10% FBS (Life technologies), 1% penicillin/streptomycin (Life technologies), and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) at 37° C. in 5% CO2. THP-1 cells that normally grow in suspension were differentiated into an adherent macrophage-like phenotype in differentiation medium (complete growth medium containing 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich)) for 24 hr. For differentiation, THP-1 cells (3×106cells/plate, ˜95% confluency) were seeded into 10-cm tissue culture plates and incubated in differentiation medium. Treatment of THP-1 Cells with Anti-Inflammatory Peptide, PEP-1 Following differentiation, the macrophage-like THP-1 cells were washed twice using complete growth medium. Then, cells were treated with 10 ng/mL lipopolysaccharide (Sigma-Aldrich) and/or 4 μM PEP-1 for 4 h at 37° C. RNA Isolation and cDNA Synthesis from THP-1 Cells Total RNA was extracted and purified using RNeasy mini kit from Qiagen (Valencia, CA, USA) following manufacturer's protocol. cDNAs were synthesized by reverse transcription using Reverse Transcription System from Promega (Madison, WI, USA) according to the manufacturer's protocol. PCR Arrays Then, cDNA samples from THP-1 cells were used as template for real-time quantitative PCR (qPCR) analysis. For qPCR analysis, RT2Profiler PCR Array kits were purchased from SABiosciences/Qiagen (Valencia, CA, USA). Four different PCR array kits analyzing separate signaling pathways used in the experiment are as follows: human signal transduction pathway finder, human inflammatory cytokines & receptors, human transcription factors, human NF-κB signaling pathway. PCR was performed with SYBR Green detection system (Qiagen) using a Bio-Rad (Mercules, CA, USA) CFX 96 real-time PCR instrument. Thermocycling conditions were: 95° C. for 10 sec; 55° C. for 30 sec; 95° C. for 10 min; 95° C. for 10 sec, 55° C. for 30 sec, and 72° C. for 30 sec for 50 amplification cycles. Data represents the average value from three independent experiments, and % decrease was determined by the target gene expression in LPS treated samples vs. LPS+PEP-1 treated samples. Among 336 genes analyzed, only those showing statistically significant (p<0.05, student's t-test) % decrease were shown in table 7. RESULTS TABLE 7% Decrease by PEP 1Signalling(LPS + PEP-1/LPS),pathwayGenep < 0.05TNFα *↓↓ 34%IL10 *↓↓ 32%ILlRa *↓ 20%IL17C↓↓ 35%G-CSF *↓↓↓ 48%GM-CSF *↓↓ 29%CCL4/MIP1β *↓ 22%CCL26/MIP4α↓↓↓ 42%TNF ReceptorTNFR11B↓↓↓ 38%FamilyCD40Ligand↓ 18%Lipid biosynthesisACSL5 (acyl-coA synthase)↓ 17%ApoptosisBCL10↓↓ 32%NFkBIkBα↓↓ 27%PEP-1 inhibits the genes shown in the table above (*, NF-κB target genes containing consensus NF-κB binding sites in the promoter region). PEP-1 inhibited transcription of genes shown in Table 7 with THE % inhibition calculated as the ratio in the level of transcription between LPS treated vs. LPS+PEP-1-treated samples (THP-1 cells). Among the 336 genes analyzed, only the 13 genes in Table 7 showed statistically significant decrease following PEP-1 treatment. Those genes can be grouped into different functional categories, which include chemokines & cytokines, TNFα receptor signaling, lipid metabolism, apoptosis, and NF-κB signaling. More importantly, genes in the chemokines & cytokines category have been known as NF-κB target genes, having NF-κB consensus DNA-binding sites in their promoter regions. Taken together, data from PCR arrays support that PEP-1 may exert anti-inflammatory effects by modulating the master regulator of inflammation NF-κB, and by doing so PEP-1 can be used as anti-inflammatory therapeutic agents in a wide range of inflammatory diseases. | 60,699 |
11857608 | DETAILED DESCRIPTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. As used herein, the term “amino acid” is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally-occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics. The terms “endotoxin free” or “substantially endotoxin free” relate generally to dosage forms, compositions, solvents, devices, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such asListeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects. Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and/or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300° C. are typically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250° C. and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art. Also included are methods of producing KLK1 polypeptides in and isolating them from eukaryotic cells such as mammalian cells to reduce, if not eliminate, the risk of endotoxins being present in a composition of the invention. Preferred are methods of producing KLK1 polypeptides in and isolating them from recombinant cells grown in chemically defined, serum free media. Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Ameobocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/ml, or EU/mg protein. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU. The “half-life” of an agent such as KLK1 polypeptide of dosage form can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. “Half-life” can also refer to the time it takes for the levels of agent to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and/or any one or more selected tissues. The terms “modulating” and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount or level produced by a control composition, sample or test subject. A “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount or level produced a control composition, sample or test subject. As one non-limiting example, the comparison can be between the amount or level of a pharmacokinetic parameter/profile or biological/therapeutic response produced by administration of a lower-dosage form (e.g., 1-10 μg/kg) of KLK1 relative to administration of a higher dosage form of KLK1. Other examples of comparisons and “statistically significant” amounts are described herein. The terms “polypeptide,” “protein” and “peptide” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. In certain embodiments, the polypeptide is a “recombinant” polypeptide, produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell. The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing. A result is typically referred to as “statistically significant” if it is unlikely to have occurred by chance. The significance level of a test or result relates traditionally to the amount of evidence required to accept that an event is unlikely to have arisen by chance. In certain cases, statistical significance may be defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true (a decision known as a Type I error, or “false positive determination”). This decision is often made using the p-value: if the p-value is less than the significance level, then the null hypothesis is rejected. The smaller the p-value, the more significant the result. Bayes factors may also be utilized to determine statistical significance (see Goodman,Ann Intern Med.130:1005-13, 1999). The term “solubility” refers to the property of a KLK1 polypeptide provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 6.0, pH 7.0, pH 7.4, pH 8.0 or pH 9.0. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (for example, pH 6.0) and relatively higher salt (for example, 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (for example, about 20, about 21, about 22, about 23, about 24, or about 25° C.) or about body temperature (37° C.). In certain embodiments, a KLK1 polypeptide has a solubility of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, or at least about 60 mg/ml at room temperature or at 37° C. “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity. “Treatment” or “treating,” as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. “Treatment” or “treating” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art. As used herein, the terms “therapeutically effective amount”, “therapeutic dose,” “prophylactically effective amount,” or “diagnostically effective amount” is the amount of an agent (e.g., KLK1 polypeptide or dosage form thereof) needed to elicit the desired biological response following administration. A “subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with a KLK1 polypeptide or a dosage form thereof. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances such as host cell proteins or nucleic acids. A “wild type” or “reference” sequence or the sequence of a “wild type” or “reference” protein/polypeptide may be the reference sequence from which variant polypeptides are derived through the introduction of changes. In general, the “wild type” amino acid sequence for a given protein is the sequence that is most common in nature. Similarly, a “wild type” gene sequence is the polynucleotide sequence for that gene which is most commonly found in nature. Mutations can be introduced into a “wild type” gene (and thus the protein it encodes) either through natural processes or through human induced means. Each embodiment in this specification is to be applied to every other embodiment unless expressly stated otherwise. Dosage Forms Embodiments of the present disclosure relate to dosage forms of one or more tissue kallikrein (KLK1) polypeptides, which are formulated at a total KLK1 polypeptide dosage of about 0.1 μg/kg to about 5 μg/kg or to about 10.0 μg/kg. In some instances, the dosage forms are suitable for (or adapted for) subcutaneous or intravenous administration to a subject, for example, a human subject. Certain dosage forms comprise, consist, consist essentially of, or are composed of a total KLK1 polypeptide dosage of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg/kg, including all ranges in between. For instance, certain dosage forms comprise, consist, consist essentially of, or are composed of a total KLK1 polypeptide dosage of about 0.1 μg/kg to about 10.0 μg/kg, about 0.1 μg/kg to about 9.0 μg/kg, about 0.1 μg/kg to about 8.0 μg/kg, about 0.1 μg/kg to about 7.0 μg/kg, about 0.1 μg/kg to about 6.0 μg/kg, about 0.1 μg/kg to about 5.0 μg/kg, or about 0.1 μg/kg to about 4.0 μg/kg, or about 0.1 μg/kg to about 3.0 μg/kg, or about 0.1 μg/kg to about 2.0 μg/kg, or about 0.01 μg/kg to about 1.0 μg/kg, or about 0.5 μg/kg to about 10.0 μg/kg, about 0.5 μg/kg to about 9.0 μg/kg, about 0.5 μg/kg to about 8.0 μg/kg, about 0.5 μg/kg to about 7.0 μg/kg, about 0.5 μg/kg to about 6.0 μg/kg, about 0.5 μg/kg to about 5.0 μg/kg, or about 0.5 μg/kg to about 4.0 μg/kg, or about 0.5 μg/kg to about 3.0 μg/kg, or about 0.5 μg/kg to about 2.0 μg/kg, or about 0.5 μg/kg to about 1.0 μg/kg, or about 1.0 μg/kg to about 10.0 μg/kg, about 1.0 μg/kg to about 9.0 μg/kg, about 1.0 μg/kg to about 8.0 μg/kg, about 1.0 μg/kg to about 7.0 μg/kg, about 1.0 μg/kg to about 6.0 μg/kg, about 1.0 μg/kg to about 5.0 μg/kg, or about 1.0 μg/kg to about 4.0 μg/kg, or about 1.0 μg/kg to about 3.0 μg/kg, or about 1.0 μg/kg to about 2.0 μg/kg, or about 2.0 μg/kg to about 10.0 μg/kg, or about 2.0 μg/kg to about 9.0 μg/kg, or about 2.0 μg/kg to about 8.0 μg/kg, or about 2.0 μg/kg to about 7.0 μg/kg, or about 2.0 μg/kg to about 6.0 μg/kg, or about 2.0 μg/kg to about 5.0 μg/kg, or about 2.0 μg/kg to about 4.0 μg/kg, or about 2.0 μg/kg to about 3.0 μg/kg, or about 3.0 μg/kg to about 10.0 μg/kg, or about 3.0 μg/kg to about 9.0 μg/kg, or about 3.0 μg/kg to about 8.0 μg/kg, or about 3.0 μg/kg to about 7.0 μg/kg, or about 3.0 μg/kg to about 6.0 μg/kg, or about 3.0 μg/kg to about 5.0 μg/kg, or about 3.0 μg/kg to about 4.0 μg/kg, or about 4.0 μg/kg to about 10.0 μg/kg, or about 4.0 μg/kg to about 9.0 μg/kg, or about 4.0 μg/kg to about 8.0 μg/kg, or about 4.0 μg/kg to about 7.0 μg/kg, or about 4.0 μg/kg to about 6.0 μg/kg, or about 4.0 μg/kg to about 5.0 μg/kg, or about 5.0 μg/kg to about 10.0 μg/kg, or about 5.0 μg/kg to about 9.0 μg/kg, or about 5.0 μg/kg to about 8.0 μg/kg, or about 5.0 μg/kg to about 7.0 μg/kg, or about 5.0 μg/kg to about 6.0 μg/kg, or about 6.0 μg/kg to about 10.0 μg/kg, or about 6.0 μg/kg to about 9.0 μg/kg, or about 6.0 μg/kg to about 8.0 μg/kg, or about 6.0 μg/kg to about 7.0 μg/kg, or about 7.0 μg/kg to about 10.0 μg/kg, or about 7.0 μg/kg to about 9.0 μg/kg, or about 7.0 μg/kg to about 8.0 μg/kg, or about 8.0 μg/kg to about 10.0 μg/kg, or about 8.0 μg/kg to about 9.0 μg/kg, or about 9.0 μg/kg to about 10.0 μg/kg. Specific dosage forms have a total KLK polypeptide dosage of about 2.5 μg/kg to about 3.5 μg/kg, or about 3 μg/kg, including dosage forms suitable for subcutaneous administration. Particular dosage forms have a total KLK polypeptide dosage of about 0.5 μg/kg to about 1.0 μg/kg, or about 0.75 μg/kg, including dosage forms suitable for intravenous administration. Tissue Kallikrein-1 (KLK1) Polypeptides. As noted above, certain dosage forms comprise one or more tissue kallikrein-1 or KLK1 polypeptides. Tissue kallikreins are members of a gene super family of serine proteases comprising at least 15 separate and distinct proteins (named tissue kallikrein 1 through 15) (Yousef et al., 2001, Endocrine Rev;22:184-204). Tissue kallikrein-1 is a trypsin-like serine protease. In humans and animal tissues, tissue kallikrein-1 cleaves kininogen into lysyl-bradykinin (also known as kallidin), a decapeptide kinin having physiologic effects similar to those of bradykinin. Bradykinin is a peptide that causes blood vessels to dilate and therefore causes blood pressure to lower. Kallidin is identical to bradykinin with an additional lysine residue added at the N-terminal end and signals through the bradykinin receptor. The KLK1 gene encodes a single pre-pro-enzyme that is 262 amino acid residues in length and that includes the “pre-” sequence (residues 1-18) and the “pro-” sequence (residues 19-24), which is activated by trypsin-like enzymes. The “mature” and “active” form human KLK1 is a glycoprotein of about 238 amino acid residues (residues 25-262) with a molecular weight of 26 kDa and a theoretical pI of 4.6. KLK1 has five disulfide bonds in its tertiary structure that are believed to be responsible for the protein's high stability, both against trypsin digestion and heat inactivation. The amino acid sequence of tissue kallikrein-1 is available for a wide variety of species, including, but not limited to, human (SEQ ID NO:1 and SWQ ID NO:2), mouse (see, for example, GenBank: AAA39349.1, Feb. 1, 1994); domestic cat (see, for example, NCBI Reference Sequence: XP_003997527.1, Nov. 6, 2012); gorilla (see, for example, NCBI Reference Sequence: XP_004061305.1, Dec. 3, 2012); cattle (see, for example, GenBank: AA151559.1, Aug. 2, 2007); dog (see, for example, CBI Reference Sequence: NP_001003262.1, Feb. 22, 2013); rat (see, for example, GenBank: CAE51906.1, Apr. 25, 2006); and olive baboon (see, for example, NCBI Reference Sequence: XP_003916022.1, Sep. 4, 2012). KLK1 is functionally conserved across species in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. A tissue kallikrein-1 polypeptide of the present invention may have any of the known amino acid sequences for KLK1, or a fragment or variant thereof. In certain embodiments, the KLK1 polypeptide is a “mature” KLK1 polypeptide. In certain embodiments, the KLK1 polypeptide is a human KLK1 polypeptide, optionally a mature human KLK1 polypeptide. In particular embodiments, the KLK1 polypeptide is a recombinant human polypeptide, for example, a recombinant human KLK1 polypeptide, optionally in the mature form. Recombinant human KLK1 (rhKLK1) can provide certain advantages over other sources of KLK1, such as urinary KLK1 (e.g., human KLK1 isolated from human urine), including a homogenous preparation of rhKLK1, simpler regulatory path to licensure, and options to alter the amino acid sequence or glycosylation pattern based on cell culture conditions. Exemplary amino acid sequences of human tissue kallikrein-1 (hKLK1) polypeptides are provided in Table K1 below. TABLE K1Exemplary KLK1 SequencesSEQ IDSourceSequenceNO:HumanMWFLVLCLALSLGGTGAAPPIQSRIVGGWECEQHSQPWQAALYHFSTFQCGGILVH1KLK1RQWVLTAAHCISDNYQLWLGRHNLFDDENTAQFVHVSESEPHPGFNMSLLENHTRQADEDYSHDLMLLRLTEPADTITDAVKVVELPTEEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECKKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENSHumanMWFLVLCLALSLGGTGAAPPIQSRIVGGWECEQHSQPWQAALYHFSTFQCGGILVH2KLK1RQWVLTAAHCISDNYQLWLGRHNLFDDENTAQFVHVSESEPHPGFNMSLLENHTRQvariantADEDYSHDLMLLRLTEPADTITDAVKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECKKVHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENSHumanIVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTAAHCISDNYQLWLGRHNL3matureFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDLMLLRLTEPADTITDAKLK1VKVVELPTEEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECKKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENSHumanIVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTAAHCISDNYQLWLGRHNL4matureFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDLMLLRLTEPADTITDAKLK1VKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECKKVHVQKvariantVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS In certain embodiments, a KLK1 polypeptide comprises, consists, or consists essentially of SEQ ID NO:1-3 or 4, or residues 1-262, residues 19-262, or residues 25-262 of SEQ ID NO:1 or SEQ ID NO:2, including fragments and variants thereof. Amino acids 1 to 18 of SEQ ID NO:1 and 2 represent the signal peptide, amino acids 19 to 24 represent propeptide sequences, and amino acids 25 to 262 represent the mature peptide. Thus, the preproprotein includes a presumptive 17-amino acid signal peptide, a 7-amino acid proenzyme fragment and a 238-amino acid mature KLK1 protein. A comparison between SEQ ID NO:1 and SEQ ID NO:2 (or SEQ ID NO:3 and SEQ ID NO:4) shows two amino acid differences between the two hKLK1 amino acid sequences. Single-nucleotide polymorphism (SNPs) between the two individuals within a species account for an E to Q substitution at amino acid residue 145 of 262 and an A to V substitution at position 188 of 262. SEQ ID NO:1 has an E (glutamic acid) at position 145 and an A (alanine) at position 188, while SEQ ID NO:2 has a Q (glutamine) at position 145 and a V (valine) at position 188. In some embodiments, KLK1 polypeptide has an Eat position 145; a Q at position 145; an A at position 188; an A at position 188; an Eat position 145 and an A at position 188; a Q at position 145 and a V at position 188; a Q at position 145 and an A at position 188; or an E at position 145 and a V at position 188. As noted above, certain embodiments include active variants and fragments of reference KLK1 polypeptide. A “variant” of a starting or reference polypeptide is a polypeptide that has an amino acid sequence different from that of the starting or reference polypeptide. Such variants include, for example, deletions from, insertions into, and/or substitutions of residues within the amino acid sequence of the polypeptide of interest. A variant amino acid, in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence. Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. The amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites. In some embodiments, a KLK polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to a reference sequence, such as, for example, an amino acid sequence described herein (for example, SEQ ID NOs: 1-4). In some aspects, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:1 or 3, or to a fragment of SEQ ID NO:1 or 3, such as for example, residues 25-262 or residues 78-141 of SEQ ID NO:1. Such a KLK1 polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a Vat position 188. In some aspects, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:2 or 4, or to a fragment of SEQ ID NO:2 or 4, such as for example, residues 25-262 or residues 78-141 of SEQ ID NO:2. Such a KLK1 polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a Vat position 188. “Percent (%) amino acid sequence identity” with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Variants may also include heterologous sequences or chemical modifications which are added to the reference KLK1 polypeptide, for example, to facilitate purification, improve metabolic half-life, or make the polypeptide easier to identify. Examples include affinity tags such as a His-tag, Fc regions, and/or a PEGylation sequence and PEG. The term “fragment” includes smaller portions of a KLK1 polypeptide (or variants thereof) that retain the activity of a KLK1 polypeptide. Fragments includes, for example, a KLK1 polypeptide fragment that ranges in size from about 20 to about 50, about 20 to about 100, about 20 to about 150, about 20 to about 200, or about 20 to about 250 amino acids in length. In other embodiments, a KLK1 polypeptide fragment ranges in size from about 50 to about 100, about 50 to about 150, about 50 to about 200, or about 50 to about 250 amino acids in length. In other embodiments, a KLK1 polypeptide fragment ranges in size from about 100 to about 150, about 100 to about 200, about 100 to about 250, about 150 to about 175, about 150 to about 200, or about 150 to about 250 amino acids in length. In other illustrative embodiments, a KLK1 polypeptide fragment ranges in size from about 200 to about 250 amino acids in length. Certain embodiments comprise a polypeptide fragment of a full-length KLK1 of about, up to about, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more (e.g., contiguous) amino acid residues. In some embodiments, a fragment may have residues 25-262 or residues 78-141 of a preproprotein sequence. In some embodiments, a fragment may be any such fragment size, as described above, of SEQ D NO:1 or SEQ ID NO:2. In some instances, fragments and variants of a KLK1 polypeptide retain the enzymatic capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. In some embodiments, an active variant or fragment retains serine protease activity of a KLK1 polypeptide that releases kallidin from a higher molecular weight precursor such as kininogen, or that cleaves a substrate similar to kininogen such as D-val-leu-arg-7 amido-4-trifluoromethylcoumarin to release a colorimetric or fluorometric fragment. The protease activity of KLK1 polypeptides can be measured in an enzyme activity assay by measuring either the cleavage of low-molecular-weight kininogen, or the generation of lys-bradykinin. In one assay format, a labeled substrate is reacted with the KLK1 glycoform, and the release of a labeled fragment is detected. One example of such a fluorogenic substrate suitable for KLK1 measurement of activity is D-val-leu-arg-7 amido-4-trifluoromethylcoumarin (D-VLR-AFC, FW 597.6) (Sigma, Cat #V2888 or Ana Spec Inc Cat #24137). When D-VLR-AFC is hydrolyzed, the free AFC produced in the reaction can be quantified by fluorometric detection (excitation 400 nm, emission 505 nm) or by spectrophotometric detection at 380 nm (extinction coefficient=12,600 at pH 7.2). Other methods and substrates may also be used to measure KLK1 proteolytic activity. Glycoforms and Mixtures Thereof. In certain embodiments, the dosage form comprises a mixture of one or more KLK1 polypeptide glycoforms, including dosage forms that comprise defined ratios of double and triple glycosylated KLK1 polypeptides (see U.S. application Ser. No. 14/677,122, incorporated by reference in its entirety). Human kallikrein has three potential Asn-linked (N-linked) glycosylation sites at residues 78, 84, and 141, relative to the mature amino acid sequence shown, for example, in SEQ ID NO: 3 or 4, as well as putative O-linked glycosylation sites. However, O-linked glycosylation is not detected in naturally-occurring KLK1. By SDS-PAGE analysis, KLK1 polypeptides glycosylated at all three positions (positions 78, 84, and 141) are detected as the high molecular weight band and are referred to herein as the high-molecular weight, triple glycosylated glycoform of KLK1 (or “high glycoform” or “triple glycoform” KLK1). By SDS-PAGE analysis, KLK1 polypeptides glycosylated at only two of three available positions (positions 78 and 84) are detected as a low molecular weight band and are referred to herein as the low-molecular weight, double glycosylated glycoform of KLK1 (or as “low glycoform” or “double glycoform” KLK1). Certain dosage forms therefore comprise a mixture of KLK1 glycoforms at a defined ratio, for example, comprising a first KLK1 polypeptide and a second KLK1 polypeptide, wherein the first KLK1 polypeptide has three glycans attached at the three different positions available for glycosylation in the polypeptide, and wherein the second KLK1 polypeptide has two glycans attached at only two of the three different positions available for glycosylation in the polypeptide. In certain embodiments, the first and second KLK1 polypeptides are present in the dosage form at a ratio of about 45:55 to about 55:45, including, for example, about 46:54, about 47:53, about 48:52, about 49:51, about 51:49, about 52:48, about 53:47, and about 54:46, including all integers and decimal points in between. In specific embodiments, the first and second KLK1 polypeptides are present in the dosage form at a ratio of about 50:50. In some embodiments, the ratio of the first and second KLK1 polypeptides is not about 60:40. In some embodiments, the ratio of the first and second KLK1 polypeptides is not about 40:60. In certain embodiments, the dosage form is free or substantially free of other glycosylated isoforms (glycoforms) of KLK1. Some dosage forms comprise a triple glycoform of a KLK1 polypeptide and a double glycoform of a KLK1 polypeptide, wherein the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 45:55 to about 55:45 including, for example, about 46:54, about 47:53, about 48:52, about 49:51, about 51:49, about 52:48, about 53:47, and about 54:46. In some embodiments, the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 50:50. In some embodiments, the ratio of the triple glycoform and double glycoform is not about 60:40. In some embodiments, the ratio of the triple glycoform and double glycoform is not about 40:60. In certain embodiments, the dosage form is free or substantially free of other glycosylated isoforms (glycoforms) of KLK1. The ratios of the double and triple glycosylated isoforms of KLK1 can be detected and quantitated by a variety of methods, including high performance liquid chromatography (HPLC), which may include reversed phase (RP-HPLC), lectin affinity chromatography and lectin affinity electrophoresis. The preparation and characterization of KLK1 glycoform mixtures is described in U.S. application Ser. No. 14/677,122, incorporated by reference in its entirety. Additional Agents. In certain embodiments, the dosage form comprises one more additional therapeutic agents, for example, a second therapeutic agent. In some embodiments, the additional agent is selected from one or more of an angiotensin receptor blocker, edavarone, finerenone, and bardoxalone, including combinations thereof. Examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof. Purity. In some embodiments, the “purity” of a dosage form is characterized, for example, by the amount (e.g., total amount, relative amount, percentage) of host cell protein(s), host cell DNA, endotoxin, and/or percentage single peak purity by SEC HPLC. In some instances, the purity of a dosage form is characterized by the amount (e.g., percentage) of KLK1 polypeptide relative to other components, for example, any one or more of the foregoing. In some embodiments, purity of a dosage form is characterized relative to or by the levels or amount of host cell proteins. The host cells used for recombinant expression may range from bacteria and yeast to cell lines derived from mammalian or insect species. The cells contain hundreds to thousands of host cell proteins (HCPs) and other biomolecules that could contaminate the final product. The HCP may be secreted along with the protein of interest, or released by accidental lysing of the cells, and may contaminate the protein of interest. Two types of immunological methods may be applied to HCP analysis: Western blotting (WB) and immunoassay (IA), which includes techniques such as ELISA and sandwich immunoassay or similar methods using radioactive, luminescent, or fluorescent reporting labels. Compositions of the present invention may include host cell protein of less than about 500, less than about 400, less than about 300, less than about 200, less than about 100 or less than about 50 ng/mg total protein. In some instances, purity is characterized relative to or by the levels or amount of host cell DNA. Detection of residual host cell DNA may be performed by Polymerase Chain Reaction (PCR) with a variety of primers for sequences in the host cell genome. Residual host cell DNA is generally reported as being below a certain threshold level, but may also be quantitated with a rPCR method. Compositions of the present invention may include host cell deoxyribonucleic acid (DNA) of less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 10 pg/mg total protein. In certain embodiments, purity is characterized relative to or by the amount or levels of endotoxin. As noted herein, endotoxin is extremely potent, heat stable, passes sterilizing membrane filters, and is present everywhere bacteria are or have been present. An Endotoxin Unit (EU) is a unit of biological activity of the USP Reference Endotoxin Standard. The bacterial endotoxins test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate (white blood cells) from the horseshoe crab (Limulus polyphemusorTachypleus tridentatus). Limulus amoebocyte lysate (LAL) reagent, FDA approved, is used for all USP endotoxin tests. There are at least three methods for this test: Method A, the gel-clot technique, which is based on gel formation; Method B, the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and Method C, the chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex. At least two types of endotoxin tests are described in the USP <85> BET. Photometric tests require a spectrophotometer, endotoxin-specific software and printout capability. The simplest photometric system is a handheld unit employing a single-use LAL cartridge that contains dried, pre-calibrated reagents; there is no need for liquid reagents or standards. The FDA-approved unit is marketed under the name of Endosafe®-PTS™. The device requires about 15 minutes to analyze small amounts of sample, a 25 μL aliquot from CSP diluted in a sterile tube, and to print out results. In contrast, gel-clot methods require a dry-heat block, calibrated pipettes and thermometer, vortex mixer, freeze-dried LAL reagents, LAL Reagent Water (LRW) for hydrating reagents and depyrogenated glassware. In this clot test, diluted sample and liquid reagents require about an hour for sample and positive-control preparation and an hour's incubation in a heat block; results are recorded manually. Thus, the simplicity and speed of the automated system make it ideally suited to the pharmacy setting. In some instances, the purity of a dosage form is characterized by the degree of aggregation. For instance, the degree of aggregation of KLK1 can be determined by Size-exclusion chromatography (SEC), which separates particles on the basis of size. It is a generally accepted method for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. Certain compositions are also substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC). Certain embodiments are free of aggregates with greater than about 96%, about 97%, about 98%, or about 99%, appearing as a single peak by SEC HPLC. In certain embodiments, the “purity” of the KLK1 polypeptide(s) in a dosage form is specifically defined. For instance, certain dosage forms comprise one or more hKLK1 polypeptides that are at least about 80, at least about 85, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, or 100% pure, including all decimals in between, relative to other components in the dosage form. Purity can be measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. In certain embodiments, the dosage form has one or more of the following determinations of purity: less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC HPLC. In some instances, the dosage forms are formulated with pharmaceutically acceptable excipients, diluents, adjuvants, or carriers, for instance, to optimize stability and achieve isotonicity. In certain aspects, the pH of the dosage form is near physiological pH or about pH 7.4, including about pH 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5, or any range thereof. In some embodiments, a dosage form comprises a KLK1 polypeptide in combination with a physiologically acceptable carrier. Such carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., Edition 21 (2005). The phrase “physiologically-acceptable” or “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce a significant allergic or similar untoward reaction when administered to a human. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparations can also be emulsified. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. The dosage forms described herein may be formulated for administered by a variety of techniques, including, for example, subcutaneous and intravenous administration. Particular embodiments include administration by subcutaneous injection. In some instances, a subcutaneous injection (abbreviated as SC, SQ, sub-cu, sub-Q or subcut with SQ) is administered as a bolus into the subcutis, the layer of skin directly below the dermis and epidermis, collectively referred to as the cutis. Exemplary places on the body where people can inject SC most easily include, without limitation, the outer area of the upper arm, just above and below the waist, excepting in certain aspects the area right around the navel (a˜2-inch circle), the upper area of the buttock, just behind the hip bone, and the front of the thigh, midway to the outer side, about 4 inches below the top of the thigh to about 4 inches above the knee. These areas can vary with the size of the person. Also, changing the injection site can prevent lumps or small dents called lipodystrophies from forming in the skin. Subcutaneous injections usually go into the fatty tissue below the skin and in certain instances can utilize a smaller, shorter needle. In specific instances, a needle that is about ½ inch to about ⅝ of an inch in length with a gauge of about 25 to about 31 is sufficient to subcutaneously administer the medication. As will be appreciated by someone skilled in the art, these are general recommendations and SC injections may be administered with needles of other sizes. In some embodiments SC administration is performed by pinching-up on the tissue to prevent injection into the muscle, and/or insertion of the needle at a˜45° angle to the skin. Also included are methods of treating a subject in need thereof, comprising administering to the subject an effective amount of a dosage form as described herein. For instance, certain embodiments include methods of treating an ischemic condition, vascular dementia, a hemorrhagic condition, traumatic brain injury (TBI), diabetes, or kidney disease, among others. Thus, in some embodiments, the subject has an ischemic condition. Non-limiting examples include brain ischemia (ischemic stroke), transient ischemic attack (TIA), cardiac ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and cutaneous ischemia. In some embodiments, the subject has vascular dementia. In some embodiments, the subject has a hemorrhagic condition, for example, a hemorrhagic stroke, including intracerebral (within the brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. In some embodiments, the subject has diabetes, for example, type 2 diabetes (T2D). In particular embodiments, the subject has a traumatic brain injury (TBI). In some embodiments, the subject has a kidney disease, for example, chronic kidney disease, diabetic kidney disease, or polycystic kidney disease. In some embodiments, the subject has systemic lupus erythematosus (SLE) or a related condition or complication such as lupus nephritis. In particular embodiments, the subject has pulmonary arterial hypertension (PAH), focal segmental glomerulosclerosis, or essential hypertension. These and related medical conditions can be diagnosed according to routine techniques in the art. In certain instances, administration of the dosage form achieves in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides. In some instances, administration of the dosage form achieves a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours following administration. In some instances, the dosage form is administered intravenously or subcutaneously. In some instances, the therapeutically-effective serum level is about or at least about 1.0 to about or at least about 5.0 ng/ml, or about or at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/ml, including all ranges in between. In some instances, administration of the dosage form achieves and maintains in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides. For instance, in some embodiments, administration of the dosage forms achieves a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, and maintains in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides for about or at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the administration (e.g., a single subcutaneous or intravenous administration). In some instances, the therapeutically-effective serum level is about or at least about 1.0 to about or at least about 5.0 ng/ml, or about or at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/ml, including all ranges in between. In some instances, administration of the dosage form achieves or results in an improved pharmacokinetic profile or biological (e.g., therapeutic) effect relative to a higher KLK1 polypeptide dosage form. For example, in some instances, subcutaneous administration of the dosage form achieves an improved pharmacokinetic profile or biological (e.g., therapeutic) effect relative to a dosage form having a total KLK1 polypeptide dosage of at least about 15 μg/kg, at least about 20 μg/kg, or at least about 50 μg/kg, or at least about 100 μg/kg, or at least about 400 μg/kg, or more. In some instances, the improved pharmacokinetic profile includes increased serum half-life following a single subcutaneous or intravenous administration, which is measured, for example, at about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration (e.g., a single subcutaneous administration). Certain embodiments include a dosage regimen of administering one or more KLK1 dosage forms at defined intervals over a period of time. For example, certain dosage regimens include administering a KLK1 dosage form once or twice a day, once or twice every two days (e.g., once a day every other day), once or twice every three days (e.g., once a day every third day following an initial or earlier administration), once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week, once or twice every other week. Specific dosage regimens include administering a KLK1 dosage form once a day every three days (e.g., once a day every third day following an initial or earlier administration), including wherein the dosage form is administered subcutaneously. Specific embodiments include intravenously administering at least one intravenous dosage form to the subject, followed by subcutaneously administering one or more subcutaneous dosages form to the subject, for example, as a dosing regimen of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. In particular embodiments, the intravenous administration or dosage form achieves in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, or 4 hours following the intravenous administration, and the subcutaneous administration or dosage form maintains the therapeutically-effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. In some instances, preparation are substantially endotoxin-free or pyrogen-free, as described herein. According to the FDA Guidance for Industry; Estimating the Maximum Safe Starting Dose in Initial Clinical Trial for Therapeutics in Adult Healthy Volunteers (July 2005), Appendix D: Converting animal doses to human equivalent doses. A human equivalent dose is 1/7 the rat dose and a human equivalent dose is 1/12 a mouse dose. In some embodiments, a dosage form describe herein is administered with one or more additional therapeutic agents or modalities. In some aspects, administration of the dosage form allows for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic modalities alone, providing relief from the toxicity observed with the administration of higher doses of the other modalities. One or more additional therapeutic agents may be administered before, after, and/or coincident (e.g., together with) to the administration of a dosage form described herein. A dosage form and any additional therapeutic agents can be administered separately or as part of the same mixture or cocktail. As used herein, an additional therapeutic agent includes, for example, an agent whose use for the treatment of a condition (e.g., an ischemic or hemorrhagic condition) is known to persons skilled in the art. Examples of additional agents include angiotensin receptor blockers, edavarone, finerenone, and bardoxalone, including combinations thereof. Particular examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof. Devices. Also included are devices that comprise a dosage form described herein, including devices suitable for subcutaneous or intravenous delivery. In some embodiments, the device is a syringe. In some embodiments, the syringe is attached to a hypodermic needle assembly, optionally comprising a protective cover around the needle assembly. In some embodiments, the needle may be about ½ inch to about ⅝ of an inch in length and has a gauge of about 25 to about 31. Certain embodiments thus include devices that attached or attachable to a needle assembly that is suitable for subcutaneous administration, comprising a dosage form described herein. For example, certain devices include a vial or syringe, optionally where the vial or syringe is attachable to or is attached to a hypodermic needle assembly. Also included are vials having a rubber cap, where a needle/syringe can be inserted into the vial via the rubber cap to withdraw the dosage form for subcutaneous administration. In particular aspects, the device is a syringe that is attachable or attached to a hypodermic needle, and is packaged with one or more removable and/or permanent protective covers around the needle or needle assembly. For instance, a first removable protective cover (which is removed during administration) can protect a user or other person from the needle prior to administration, and a second protective cover can be put (i.e., snapped) into place for safe disposal of the device after administration. The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. EXAMPLES Example 1 Pharmacokinetics of Low-Dosage KLK1 Formulations in Humans The pharmacokinetics of low-dosage KLK1 formulations were evaluate in humans. The formulations are composed of mixture of double and triple glycosylation isoforms of mature human KLK1, which were prepared as described in U.S. application Ser. No. 14/677,122 (incorporated by reference). A single dose comparison study was designed to establish safety, tolerability and pharmacokinetics of the KLK1 formulation after 30 minute IV infusion and a single subcutaneous injection. The IV dose was 0.75 μg/kg and the subcutaneous dose was 3 μg/kg. Study groups consisted of 12 volunteers with 6 women and 6 men in each. The intravenous dose was supplied to the clinical site as a 104.4 mg/ml drug product dissolved in phosphate-buffered saline (pH=7.2). The volume of KLK1 polypeptide required for dosing was calculated based on the body weight of each study volunteer and the dose level. The subcutaneous dose was provided as a 26.1 mg/ml solution in phosphate-buffered saline. Prior to administration this dosage was diluted to 2.61 mg/ml in normal saline and the volume of injection adjusted according to the body weight of the study participant. The concentration of KLK1 in plasma was measured by ELISA using a KLK1-specific antibody. This method has been developed and validated for use in human clinical trials. As shown inFIGS.2A-2B, subcutaneous administration of low-dosage (3 μg/kg; n=12) formulations of mature human KLK1 glycoforms not only achieved effective plasma levels of KLK1, but also resulted in significantly prolonged serum half-file, even relative to intravenous administration of low-dosage formulations (0.75 μg/kg; n=12), in healthy human subjects. Intravenous administration of low-dosage formulations of mature human KLK1 glycoforms rapidly achieved effective plasma levels of KLK1.FIG.2Ashows the serum levels at 80 hours andFIG.2Bshows serum levels at 4 hours post-administration. The pharmacokinetic (PK) profile of two KLK1 subcutaneous dosing strategies (3 μg/kg; and 15/25 μg/kg) in healthy volunteers was also evaluated in an phase I clinical study. The results are shown inFIG.3. The points represent the mean values derived from six participants per group. The arrows represent times of dosing. The 3 μg/kg dose, which is considered the target dose from the most recent patent, maintained fairly steady drug levels at the desired or therapeutically-effective serum/plasma concentration of about 3-5 ng/ml. In contrast, the higher dosage caused levels to be proportionally higher with a greater fluctuation between doses. A dosage test of KLK1 was performed in patients with Type 2 diabetes. The results are shown inFIGS.4A-4D. The points are results of a meal tolerance test conducted˜2 hours after dosing, using the HOMA2-IR measure of insulin resistance three hours after dosing. The numbers are a derived measure of insulin resistance where higher numbers represent greater insulin resistance and denote greater illness.FIG.4Dshows the results from the placebo group and theFIG.4Cshows the results of the drug groups (DM199). The Day 1 test was run as a baseline followed by single doses of the indicated dose level on subsequent days. The insulin resistance improved on Day 5 when a low dose was tested compared to the results of Day 8 when a higher dose was tested (FIG.4C), suggesting that the low dosage was more effective. A 28-day multiple dose study in Type 2 Diabetic patients (n=12-13 per group) was also performed. The results are shown inFIGS.5A-5B. The bars represent the mean±SEM change from baseline serum creatinine and urea concentration. These are typical measures of kidney function where above normal values indicate kidney impairment. This result indicates that the low dose (3 μg/kg) had a significantly greater effect than the higher dose on improving measurements of kidney function. The P values are based on comparisons between the 3 μg/kg group and placebo. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. | 62,972 |
11857609 | DETAILED DESCRIPTION The present disclosure relates to compositions and method for treating DME and/or RVO in a patient. For example, but not by way of limitation, the present disclosure relates to methods and compositions for inhibiting caspase-9 signaling activity associated with the induction and/or exacerbation of DME and/or RVO in a patient. As used herein, the term “DME” refers to clinically detectable diabetic macular edema. DME occurs in patients having clinically detectable diabetes mellitus (also referred to herein simply as diabetes), frequently in type 2 diabetes mellitus but also in type 1 diabetes mellitus. Clinical symptoms of DME include retinal edema and diabetic retinopathy with macular edema. DME may be detected using optical coherence tomography (OCT). DME is the major cause of blindness in working age adults (20-70 years old). As used herein, the term “RVO” refers to clinically detectable retinal vein occlusion. RVO can occur in any patients, but is more common in those also having clinically detectable atherosclerosis, diabetes, hypertension, glaucoma, macular edema, or vitreous hemorrhage. RVO is more common in elderly patients. RVO can cause glaucoma and macular edema, including DME. RVO may be detected using angiography and/or OCT. RVO is the second leading cause of blindness in working age adults. As used herein, the term “patient” refers to any animal, including any mammal, including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like). In particular, the patient is a human. As used herein, an “effective amount” is an amount sufficient to cause a beneficial or desired clinical result in a patient. An effective amount can be administered to a patient in one or more doses. It is typically administered to the retina of the patient. In terms of treatment, an effective amount is an amount that is sufficient to ameliorate the impact of and/or inhibit the induction and/or exacerbation of DME and/or RVO in a patient, or otherwise reduce the pathological consequences of the disease(s). The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors may be taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, the condition being treated, the severity of the condition, prior responses, type of inhibitor used, the caspase-9 signaling pathway member to be inhibited, the cell type expressing the target, and the form and effective concentration of the composition (also referred to herein as a “treatment,” “inhibitor,” or “conjugate”) being administered. As used herein, “treat,” “treating” and similar verbs refer to of ameliorating the impact of and/or inhibiting the induction and/or exacerbation of DME and/or RVO in a patient. Without being limited to a particular mode of action, the caspase-9 signaling pathway as discussed herein may include the pathway shown inFIG.1. In this pathway, RVO induces activation of caspase-9 in blood vessels which leads to activation of caspase-7. Caspase-7 cleaves the co-chaperone protein p23. p23 is a negative regulator of HIF-1 a, thus cleavage of p23 leads to an increase in HIF-la, the rate-limiting step in the formation of the HIF-1 transcription factor, which increases VEGF levels leading to edema. The caspase-9 signaling pathway inhibitor, shown as Penl-XBIR3 inFIG.1as an illustrative example, may act by blocking the action of caspase-9 on caspase-7. Methods of Inhibiting DME and/or RVO In certain embodiments, the instant disclosure is directed to methods of or uses of treatments disclosed herein in ameliorating the impact of and/or inhibiting the induction and/or exacerbation of DME and/or RVO in a patient by administering an effective amount of a caspase-9 signaling pathway inhibitor, or conjugate thereof. In certain embodiments, the methods of the present disclosure are directed to the administration of a caspase-9 signaling pathway inhibitor, or conjugate thereof, via eye drops in order to inhibit DME and/or RVO. The treatment, when used to treat the effects of DME and/or RVO, may be administered as a single dose or multiple doses. For example, but not by way of limitation, where multiple doses are administered, they may be administered at intervals of 6 times per 24 hours or 4 times per 24 hours or 3 times per 24 hours or 2 times per 24 hours or 1 time per 24 hours or 1 time every other day or 1 time every 3 days or 1 time every 4 days or 1 time per week, or 2 times per week, or 3 times per week. In certain embodiments, the initial dose may be greater than subsequent doses or all doses may be the same. In certain embodiments, the inhibitor used in connection with the methods and uses of the instant disclosure is a Pen l-XBIR3 conjugate as disclosed herein. In certain embodiments, the Penl-XBIR3 conjugate is administered to a patient suffering from DME and/or RVO either as a single dose or in multiple doses. The concentration of the Penl-XBIR3 composition administered is, in certain embodiments: 0.1 μM to 1,000 μM; 1 μM to 500 μM; 10 μM to 100 μM; or 20 μM to 60 μM. In certain embodiments, a specific human equivalent dosage can be calculated from animal studies via body surface area comparisons, as outlined in Reagan-Shaw et al., FASEB J., 22; 659-661 (2007). In certain embodiments, eye size comparisons can be employed to calculate a specific human equivalent dosage. In certain embodiments, the caspase-9 signaling pathway inhibitor, either alone or in the context of a membrane-permeable conjugate, is administered in conjunction with one or more additional therapeutics. In certain of such embodiments the additional therapeutics include, but are not limited to an anti-VEGF therapeutic and/or a steroidal therapeutic. In certain embodiments the method involves the administration of one or more additional caspase-9 signaling pathway inhibitors either alone or in the context of a membrane-permeable conjugate. Compositions Caspase-9 Signaling Pathway Inhibitors In certain embodiments, the caspase-9 signaling pathway inhibitors of the present disclosure are peptide inhibitors of caspase-9. In certain embodiments, the caspase-9 signaling pathway inhibitors include, but are not limited to the class of protein inhibitors identified as Inhibitors of (“IAPs”). IAPs generally contain one to three BIR (baculovirus IAP repeats) domains, each consisting of approximately 70 amino acid residues. In addition, certain IAPs also have a RING finger domain, defined by three cysteines, followed by one histidine, followed by four additional cysteines that can coordinate two zinc atoms. Exemplary mammalian IAPs suitable for use herein, include, but are not limited to c-IAP1 (Accession No. Q13490.2), cIAP2 (Accession No. Q13489.2), and XIAP (Accession No. P98170.2), each of which have three BIRs in the N-terminal portion of the molecule and a RING finger at the C-terminus. NAIP (Accession No. Q13075.3), another suitable mammalian IAP, contains three BIRs without RING, and survivin (Accession No. 015392.2) and BRUCE (Accession No. Q9H8B7), which are two additional suitable IAPs, both of which contain just one BIR. In certain embodiments the peptide inhibitor of caspase-9 is XBIR3 having the sequence (SEQ ID NO. 11)MGSSHHHHHHSSGLVPRGSHMSTNTCLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGFYTDW ALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLLEQRGQEYINNIHL THS. In certain embodiments the peptide inhibitor of caspase-9 is XBIR3 having the sequence (SEQ ID NO. 12)MGSSHHHHHHSSGLVPRGSHMSTNTLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGFYTDW ALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLLEQRGQEYINNIHLTHS. In certain embodiments the peptide inhibitors of caspase-9 include, but are not limited to EG Z-VEID-FMK (“VEID” disclosed as SEQ ID NO: 1) (WO 2006056487); Z-VAD-FMK, CrmA, and Z-VAD-(2, 6-dichlorobenzoyloxopentanoic acid) (Garcia-Calvo, et al., J. Biol. Chem, 273, 32608-32613 (1998)). Peptide inhibitors of caspase-9 include those amino acid sequences that retain certain structural and functional features of the identified caspase-9 inhibitor peptides, yet differ from the identified inhibitors' amino acid sequences at one or more positions. Such variants can be prepared by substituting, deleting, or adding amino acid residues from the original sequences via methods known in the art. In certain embodiments, such substantially similar sequences include sequences that incorporate conservative amino acid substitutions. As used herein, a “conservative amino acid substitution” is intended to include a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including: basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); P-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Other generally preferred substitutions involve replacement of an amino acid residue with another residue having a small side chain, such as alanine or glycine. Amino acid substituted peptides can be prepared by standard techniques, such as automated chemical synthesis. In certain embodiments, a peptide inhibitor of caspase-9 of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the original peptide inhibitor of caspase-9, such as an IAP, and is capable of caspase-9 inhibition. As used herein, the percent homology between two amino acid sequences may be determined using standard software such as BLAST or FASTA The effect of the amino acid substitutions on the ability of the synthesized peptide inhibitor of caspase-9 to inhibit caspase-9 can be tested using the methods disclosed in Examples section, below. Inhibitor-Cell Penetrating Peptide Conjugates In certain embodiments of the present disclosure, the caspase-9 signaling pathway inhibitor is conjugated to a cell penetrating peptide to form an inhibitor-cell penetrating peptide conjugate. As used herein, a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. In certain embodiments, the cell-penetrating peptide used in the membrane-permeable complex of the present disclosure preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with the caspase-9 signaling pathway inhibitor, which has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of the present disclosure may include, but are not limited to, Penetratinl, transportan, pisl, TAT(48-60), pVEC, MTS, and MAP. The cell-penetrating peptides of the present disclosure include those sequences that retain certain structural and functional features of the identified cell-penetrating peptides, yet differ from the identified peptides' amino acid sequences at one or more positions. Such polypeptide variants can be prepared by substituting, deleting, or adding amino acid residues from the original sequences via methods known in the art. In certain embodiments, such substantially similar sequences include sequences that incorporate conservative amino acid substitutions, as described above in connection with peptide caspase-9 inhibitors. In certain embodiments, a cell-penetrating peptide of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the identified peptide and is capable of mediating cell penetration. The effect of the amino acid substitutions on the ability of the synthesized peptide to mediate cell penetration can be tested using the methods in Examples section, below. In certain embodiments of the present disclosure, the cell-penetrating peptide of the membrane-permeable complex is Penetratinl, comprising the peptide sequence C(NPys)-RQIKIWFQNRRMKWKK (SEQ ID NO: 2), or a conservative variant thereof. As used herein, a “conservative variant” is a peptide having one or more amino acid substitutions, wherein the substitutions do not adversely affect the shape—or, therefore, the biological activity (i.e., transport activity) or membrane toxicity—of the cell-penetrating peptide. Penetratinl is a 16-amino-acid polypeptide derived from the third alpha-helix of the homeodomain of Drosophila antennapedia. Its structure and function have been well studied and characterized: Derossi et al., Trends Cell Biol., 8(2):84-87, 1998; Dunican et al., Biopolymers, 60(1):45-60, 2001; Hallbrink et al., Biochim. Biophys. Acta, 1515(2):101-09, 2001; Bolton et al., Eur. J. Neurosci., 12(8):2847-55, 2000; Kilk et al., Bioconjug. Chem, 12(6):911-16, 2001; Bellet-Amalric et al., Biochim Biophys. Acta, 1467(1):131-43, 2000; Fischer et al., J. Pept. Res., 55(2):163-72, 2000; Thoren et al., FEBS Lett., 482(3):265-68, 2000. It has been shown that Penetratinl efficiently carries avidin, a 63-kDa protein, into human Bowes melanoma cells (Kilk et al., Bioconjug. Chem, 12(6):911-16, 2001). Additionally, it has been shown that the transportation of Penetratinl and its cargo is non-endocytotic and energy-independent, and does not depend upon receptor molecules or transporter molecules. Furthermore, it is known that Penetratinl is able to cross a pure lipid bilayer (Thoren et al., FEBS Lett., 482(3):265-68, 2000). This feature enables Penetratinl to transport its cargo, free from the limitation of cell-surface-receptor/-transporter availability. The delivery vector previously has been shown to enter all cell types (Derossi et al., Trends Cell Biol., 8(2):84-87, 1998), and effectively to deliver peptides (Troy et al., Proc. Natl. Acad. Sci. USA, 93:5635-40, 1996) or antisense oligonucleotides (Troy et al., J. Neurosci., 16:253-61, 1996; Troy et al., J. Neurosci., 17:1911-18, 1997). Other non-limiting embodiments of the present disclosure involve the use of the following exemplary cell permeant molecules: RL16 (H-RRLRRLLRRLLRRLRR-OH) (SEQ ID NO: 3), a sequence derived from Penetratinl with slightly different physical properties (Biochim Biophys Acta. 2008 July-;1 780(7-8):948-59); and RVG-RRRRRRRRR (SEQ ID NO: 4), a rabies virus sequence which targets neurons see P. Kumar, H. Wu, J. L. McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P. Shankar and N. Manjunath, Transvascular delivery of small interfering RNA to the central nervous system, Nature 448 (2007), pp. 39-43. In certain alternative non-limiting embodiments of the present disclosure, the cell-penetrating peptide of the membrane-permeable complex is a cell-penetrating peptide selected from the group consisting of: transportan, pIS1, Tat(48-60), pVEC, MAP, and MTS. Transportan is a 27-amino-acid long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin, and the 14-residue sequence of mastoparan in the carboxyl terminus, connected by a lysine (Pooga et al., FASEB J., 12(1):67-77, 1998). It includes the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 5), or a conservative variant thereof. pIs1 is derived from the third helix of the homeodomain of the rat insulin 1 gene enhancer protein (Magzoub et al., Biochim Biophys. Acta, 1512(1):77-89, 2001; Kilk et al., Bioconjug. Chem, 12(6):911-16, 2001). pisl includes the amino acid sequence PVIRVW FQNKRCKDKK (SEQ ID NO: 6), or a conservative variant thereof. Tat is a transcription activating factor, of 86-102 amino acids, that allows translocation across the plasma membrane of an HIV-infected cell, to transactivate the viral genome (Hallbrink et al., Biochem Biophys. Acta., 1515(2):101-09, 2001; Suzuki et al., J. Biol. Chem, 277(4):2437-43, 2002; Futaki et al., J. Biol. Chem, 276(8):5836-40, 2001). A small Tat fragment, extending from residues 48-60, has been determined to be responsible for nuclear import (Vives et al., J. Biol. Chem, 272(25):16010-017, 1997); it includes the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 7), or a conservative variant thereof. pVEC is an 18-amino-acid-long peptide derived from the murine sequence of the cell-adhesion molecule, vascular endothelial cadherin, extending from amino acid 615-632 (Elmquist et al., Exp. Cell Res., 269(2):237-44, 2001). pVEC includes the amino acid sequence LLIILRRRIRKQAHAH (SEQ ID NO: 8), or a conservative variant thereof. MTSs, or membrane translocating sequences, are those portions of certain peptides which are recognized by the acceptor proteins that are responsible for directing nascent translation products into the appropriate cellular organelles for further processing (Lindgren et al., Trends in Pharmacological Sciences, 21(3):99-103, 2000; Brodsky, J. L., Int. Rev. Cyt., 178:277-328, 1998; Zhao et al., J. Immunol. Methods, 254(1-2):137-45, 2001). An MTS of particular relevance is MPS peptide, a chimera of the hydrophobic terminal domain of the viral gp41 protein and the nuclear localization signal from simian virus 40 large antigen; it represents one combination of a nuclear localization signal and a membrane translocation sequence that is internalized independent of temperature, and functions as a carrier for oligonucleotides (Lindgren et al., Trends in Pharmacological Sciences, 21(3): 99-103, 2000; Morris et al., Nucleic Acids Res., 25:2730-36, 1997). MPS includes the amino acid sequence GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 9), or a conservative variant thereof. Model amphipathic peptides, or MAPs, form a group of peptides that have, as their essential features, helical amphipathicity and a length of at least four complete helical turns (Scheller et al., J. Peptide Science, 5(4):185-94, 1999; Hallbrink et al., Biochim. Biophys. Acta., 1515(2):101-09, 2001). An exemplary MAP comprises the amino acid sequence KLALKLALKALKAALKLA (SEQ ID NO: 10)-amide, or a conservative variant thereof. In certain embodiments, the cell-penetrating peptides and the caspase-9 signaling pathway inhibitors described above are covalently bound to form conjugates. In certain embodiments the cell-penetrating peptide is operably linked to a peptide caspase-9 inhibitor via recombinant DNA technology. For example, in embodiments where the caspase-9 signaling pathway inhibitor is a peptide caspase-9 inhibitor, a nucleic acid sequence encoding that peptide caspase-9 inhibitor can be introduced either upstream (for linkage to the amino terminus of the cell-penetrating peptide) or downstream (for linkage to the carboxy terminus of the cell-penetrating peptide), or both, of a nucleic acid sequence encoding the peptide caspase-9 inhibitor of interest. Such fusion sequences including both the peptide caspase-9 inhibitor encoding nucleic acid sequence and the cell-penetrating peptide encoding nucleic acid sequence can be expressed using techniques well known in the art. In certain embodiments the caspase-9 signaling pathway inhibitor can be operably linked to the cell-penetrating peptide via a non-covalent linkage. In certain embodiments such non-covalent linkage is mediated by ionic interactions, hydrophobic interactions, hydrogen bonds, or van der Waals forces. In certain embodiments the caspase-9 signaling pathway inhibitor is operably linked to the cell penetrating peptide via a chemical linker. Examples of such linkages typically incorporate 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P. Exemplary linkers include, but are not limited to, a substituted alkyl or a substituted cycloalkyl. Alternately, the heterologous moiety may be directly attached (where the linker is a single bond) to the amino or carboxy terminus of the cell-penetrating peptide. When the linker is not a single covalent bond, the linker may be any combination of stable chemical bonds, optionally including, single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds. In certain embodiments, the linker incorporates less than 20 nonhydrogen atoms and are composed of any combination of ether, thioether, urea, thiourea, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. In certain embodiments, the linker is a combination of single carbon-carbon bonds and carboxamide, sulfonamide or thioether bonds. A general strategy for conjugation involves preparing the cell-penetrating peptide and the caspase-9 signaling pathway inhibitor components separately, wherein each is modified or derivatized with appropriate reactive groups to allow for linkage between the two. The modified caspase-9 signaling pathway inhibitor is then incubated together with a cell-penetrating peptide that is prepared for linkage, for a sufficient time (and under such appropriate conditions of temperature, pH, molar ratio, etc.) as to generate a covalent bond between the cell-penetrating peptide and the caspase-9 signaling pathway inhibitor molecule. Numerous methods and strategies of conjugation will be readily apparent to one of ordinary skill in the art, as will the conditions required for efficient conjugation. By way of example only, one such strategy for conjugation is described below, although other techniques, such as the production of fusion proteins or the use of chemical linkers is within the scope of the present disclosure. In certain embodiments, when generating a disulfide bond between the caspase-9 signaling pathway inhibitor molecule and the cell-penetrating peptide of the present disclosure, the caspase-9 signaling pathway inhibitor molecule can be modified to contain a thiol group, and a nitropyridyl leaving group can be manufactured on a cysteine residue of the cell-penetrating peptide. Any suitable bond (e.g., thioester bonds, thioether bonds, carbamate bonds, etc.) can be created according to methods generally and well known in the art. Both the derivatized or modified cell-penetrating peptide, and the modified caspase-9 signaling pathway inhibitor are reconstituted in RNase/DNase sterile water, and then added to each other amounts appropriate for conjugation (e.g., equimolar amounts). The conjugation mixture is then incubated for 60 min at 37° C., and then stored at 4° C. Linkage can be checked by running the vector-linked caspase-9 signaling pathway inhibitor molecule, and an aliquot that has been reduced with DTT, on a 15% non-denaturing PAGE. Caspase-9 signaling pathway inhibitor molecules can then be visualized with the appropriate stain. In certain embodiments, the present disclosure is directed to a Penetratinl-XBIR3 (Penl-XBIR3) conjugate. In certain of such embodiments, the sequence of the Pen-l-XBIR3 is: C(NPys)-RQIKIWFQNRRMKWKK-s-s-MGSSHHHHHHSSGLVPRGSHMSTNTCLPRNPSMADYEARIFTFGTWIYSVNK EQLARAGFYTDW ALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLL EQRGQEYINNIHL THS (SEQ ID NO 2 and 11, respectively, linked by a disulfide bond). In other of such embodiments, the sequence of the Penl-XBIR3 is: C(NPys)-RQIKIWFQNRRMKWKK-s-s-MGSSHHHHHSSGLVPRGSHMSTNTLPRNPSMADYEARIFTFGTWIYSVNKE QLARAGFYTDW ALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLLE QRGQEYINNIHLTHS (SEQ ID NO 2 and 12, respectively, linked by a disulfide bond). Pharmaceutical Compositions In certain embodiments, the caspase-9 signaling pathway inhibitors or conjugates of the present disclosure are formulated for retinal administration. For administration via eye drops, a solution or suspension containing the caspase-9 signaling pathway inhibitor or conjugate can be formulated for direct application to the retina by conventional means, for example with a dropper, pipette or spray. In certain embodiments, the caspase-9 signaling pathway inhibitor or conjugate of the present disclosure is formulated in isotonic saline. In certain embodiments, the caspase-9 signaling pathway inhibitor or conjugate of the present disclosure is formulated in isotonic saline at or about pH 7.4. To facilitate delivery to a cell, tissue, or subject, the caspase-9 signaling pathway inhibitor, or conjugate thereof, of the present disclosure may, in various compositions, be formulated with a pharmaceutically-acceptable carrier, excipient, or diluent. The term “pharmaceutically-acceptable”, as used herein, means that the carrier, excipient, or diluent of choice does not adversely affect either the biological activity of the caspase-9 signaling pathway inhibitor or conjugate or the biological activity of the recipient of the composition. Suitable pharmaceutical carriers, excipients, and/or diluents for use in the present disclosure include, but are not limited to, lactose, sucrose, starch powder, talc powder, cellulose esters of alkonoic acids, magnesium stearate, magnesium oxide, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, gum arabic, acacia gum, sodium and calcium salts of phosphoric and sulfuric acids, polyvinylpyrrolidone and/or polyvinyl alcohol, saline, and water. Specific formulations of compounds for therapeutic treatment are discussed in Hoover, J. E., Remington's Pharmaceutical Sciences (Easton, Pa.: Mack Publishing Co., 1975) and Liberman and Lachman, eds. Pharmaceutical Dosage Forms (New York, N.Y.: Marcel Decker Publishers, 1980). In accordance with the methods of the present disclosure, the quantity of the caspase-9 signaling pathway inhibitor or conjugate thereof that is administered to a cell, tissue, or subject should be an effective amount. EXAMPLES Example 1: Eyedrops Deliver Penl-XBIR3 to Mouse and Rabbit Retinas The ability of eyedrops to deliver Penl-XBIR3 in mice and rats was tested. Results are presented inFIG.2andFIG.3. In mice, Penl-XBIR3 (10 μg) eyedrops were applied, then the animals were sacrificed at the indicated times. In rabbits, 200 μg Penl-XBIR3 eyedrops or a saline vehicle were administered BID for 4.5 days. The final dose given 5 h prior to harvest of retinas. Plasma from rabbits obtained at baseline and harvest. Retinal lysates were immunoprecipitated with XIAP, followed by western blotting for anti-His. XBIR3 contains a His tag, so uptake of XBIR3 is detectable using anti-His. Blots for the mouse and rabbit samples, along with graphs quantifying the results, are presented inFIG.2. XBIT3 uptake was observed in both mouse and rabbit samples. Uptake in the mouse samples was detected by 1 h and maintained through 24 h. In rabbit there was significant XBIR3 in retina at 5d. Baseline and post-treatment plasma from rabbits was analyzed by immunoprecipitation with XIAP followed by western blot with anti-His. A Ponceau protein stain was used to show input protein amounts. XBIR3 was not detected in rabbit plasma (FIG.3), indicating that it remains localized in the eye. Example 2: Mouse Model of RVO In the following experiments, a mouse model of RVO, which induces reproducible retinal edema was used. RVO is the model that was used for testing anti-VEGF therapies for DME. Brown et al., Ophthalmology 117, 1124-1133 el 121 (2010); and Campochiaro et al., Ophthalmology 117, 1102-1112 e1101 (2010). I n this model, Rose Bengal, a photoactivatable dye, is injected into the tail veins of adult C57B16 mice and photoactivated by laser of retinal veins around the optic nerve head. A clot is formed and edema or increased retinal thickness develops rapidly. Inflammation, also seen in diabetes, also develops. Fluorescein leakage and maximal retinal edema, measured by fluorescein angiography and optical coherence tomography (OCT), respectively, using the Phoenix Micron IV, is observed 24 h after RVO. Retinal edema is maintained over the first 3 days RVO. By day 4 the edema decreases and the retina subsequently thins out. In addition to edema formation there is evidence of cell death in the photoreceptor cell layer by day 2 after RVO. In this example, mice were anesthetized with intra-peritoneal (IP) injection of ketamine and xylazine. One drop of 0.5% alcaine was added to the eye as topical anesthetic. The retina was imaged with the Phoenix Micron IV to choose veins for laser ablation using the Phoenix Micron IV image guided laser. One to four veins around the optic nerve head were ablated by delivering a laser pulse (power 50 mW, spot size 50 μm, duration 3 seconds) to each vein. Example 3: Target Activation and Engagement Penl-XBIR3 eyedrops were delivered immediately after RVO and at 24 h. At 48 h, the eyes were imaged via OCT. FIG.4presents images from individual animals (2 control, 2 RVO, 4 RVO+Penl-XBIR3). For each animal there are three sets of OCT and brightfield images. The brightfield image has a horizontal line showing the level of the OCT. Four hours after RVO, mouse retinas were harvested for western blot to detect activated caspase-9 (c1Casp9) (FIG.5, left panel). The blot showed a 10-fold induction of c1Casp9. To show target engagement, after RVO, mice were given Penl-XBIR3 and retinas were harvested and immunoprecipitated with anti-His followed by western blot for clCasp9 (FIG.5, right panel). There was a 21-fold increase in binding of XBIR3 and clCasp9 by 4 h and a 45-fold increase by 24 h. Example 4: Penl-XBIR3 Provided Significant Protection in RVO The efficacy of Penl-XBIR3 eyedrops in RVO was evaluated. Penl-XBIR3 eyedrops were given immediately after RVO and at 24 h. At 48 h OCT images showed significant protection against RVO (FIG.6) with less increase in retinal thickness and abrogation of retinal detachment (**P<0.01). Individual retinal layers were also examined, as they are not affected equally by RVO. Retinal layers include the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL), the inner segments (IS), the outer segments (OS), and the retinal pigment epithelium (RPE), which is located next to the choroid. Penl-XBIR3 decreased retinal detachment (FIG.7, **P<0.01), protected the inner retinal layers (FIG.8, **P<0.01), decreased swelling of the outer retina layers, such as the outer plexiform layer (FIG.9, **P<0.01) and protected the photoreceptors (FIG.10, *P<0.05, **P<0.01). Example 5: Penl-XBIR3 Blocked Cell Death after RVO TUNEL staining is a marker of cell death. RVO induces TUNEL staining by 24 h in the INL. Retinas were harvested at 48 h from mice treated with Penl-XBIR3 or untreated mice, then processed for immunohistochemistry. Analysis of samples showed that TUNEL positive cells were decreased by Penl-XBIR3 eyedrops and that the eyedrops maintained INL thickness (FIG.11). Example 6: Penl-XBIR3 Provided Functional Protection in RVO RVO induces a decrease in A and B waves on an electroretinogram (ERG). Treatment with Penl-XBIR3 immediately after RVO and at 24 h provided ERG improvement (scoptic focal ERG, spot size 1500 μm, flash intensity −2.3 log cd/m2) up to 7d post-RVO (FIG.12). Example 7: Penl-XBIR3 Prevented an Increase in Cleaved Caspase-7 after RVO RVO induces activation of caspase-7, a target of active caspase-9, in blood vessels. Penl-XBIR3 prevents this increase at 24 h as shown by a comparison of retinal section obtained 24 h post-RVO and stained as indicated (FIG.13.) Example 8: Penl-XBIR3 Prevented Induction of VEGF and HIF-1a by RVO RVO leads to induction of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor I-alpha (HIF-1a) within 4 h of induction of RVO. Treatment with Penl-XBIR3 after RVO abrogated the increase in VEGF and HIF-1a. Retinas were harvested at 4 h post-RVO and analyzed by western blot for VEGF expression (FIG.14, left) and by immunohistochemistry for HIF-1a expression (FIG.14, right). The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are to fall within the scope of the appended claims. Patents, patent applications, publications, procedures, and the like are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties. | 33,377 |
11857610 | DETAILED DESCRIPTION The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entireties for any purpose. Definitions Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Measured values are understood to be approximate, taking into account significant digits and the error associated with the measurement. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: “Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. General routes of administration for protein therapeutics include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, orally, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. “Alpha-1 antitrysin” abbreviated (A1AT), (A1-PI) or (AAT) herein includes a polypeptide comprising the full length human A1AT, which may be obtained from pooled human plasma, or which may be recombinant. It also includes a shorter human A1AT that retains a biological function such as protease inhibition (WO2010/088415) of the full length protein as well as fusion molecules comprising a human A1AT polypeptide. In some embodiments it includes AATs having no significant serine protease inhibitor activity (WO2010/088415). In some embodiments, the A1AT is a fusion molecule comprising an A1AT polypeptide and a fusion partner, optionally an Fc molecule, (e.g., an Fc fragment, an Fc analog, etc.), PEG, or albumin, such as an A1AT-Fc fusion molecule described in WO2013/106589 and WO2014/160768. In cases where the subject is non-human, the appropriate non-human A1AT may be used in lieu of human A1AT. In some embodiments, the A1AT comprises a signal polypeptide whereas in other embodiments it does not. In some embodiments, the A1AT comprises Zemaira® (CSL Behring), Prolastin® (Grifols), Prolastin® C (Grifols), Aralast® (Shire), Aralast NP® (Shire), Glassia® (Kamada), Trypsone® (Grifols), Alfalastin® (LFB Biomedicaments), or other commercial formulation or any combination thereof. The terms “subject” and “patient” are used interchangeably herein to refer to a human unless the context makes it clear that a non-human subject or patient is intended (e.g. a “canine subject” or the like). In some embodiments, methods of treating other mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided. “Treatment,” as used herein, refers to therapeutic treatment, for example, wherein the object is to slow down (lessen) an existing disease or disorder, as well as, for example, wherein the object is to prevent or delay the onset of symptoms of the disease or disorder in a patient who is at risk for developing the disease or disorder or to reduce the severity of the disease or disorder once it has begun. “Reducing the risk of onset of” a disease or disorder is a type of treatment for the disease or disorder intended to reduce the risk that a subject who does not presently have the disease or disorder will develop the disease or disorder in the future, such as after an event like HCT or another non-organ or cellular transplantation. The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a subject or to reduce the risk of onset of the disease or disorder. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of A1AT for example may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibodies to elicit a desired response in the individual. “Hematopoietic cell transplantation” abbreviated HCT or HSCT is a procedure that comprises transplantation of hematopoietic stem cells or progenitor cells from a donor to a host or recipient, for example, with the goal of reestablishing immune cell function or bone marrow function. “Graft versus host disease” abbreviated GVHD, occurs when immune cells such as immunocompetent T cells and natural killer (NK) cells in the donor transplant recognize host antigens as foreign and target them. Symptoms may range from rashes of the skin to systemic complications involving organs such as the gastrointestinal tract and liver. GVHD may be either acute or chronic. “Acute GVHD” abbreviated aGVHD, may occur within the first 100 days after a transplantation procedure while chronic GHVD (cGVHD) may occur at later time points such as up to 12 months following a transplantation procedure. Acute GVHD may be graded depending upon its severity and the extent of systemic involvement, such as Stage I, II, III, or IV (also called Grade I, II, III, or IV), with Stage IV being the most severe and having the highest risk of mortality. An “immunosuppressive agent” herein broadly refers to any therapeutic agent intended to reduce an immune reaction in a patient. GVHD may also be steroid-refractory. “Steroid-refractory acute GVHD” as used herein, for example, refers to aGVHD that does not improve despite treatment with steroids such as methylprednisone and methylprednisolone. Hematopoietic Cell Transplantation (HCT) HCT involves transplantation of hematopoietic cells such as stem cells and progenitor cells from a donor to a patient, for example, to reestablish immune cell function or bone marrow function in the patient. For example, hematopoietic stem and/or progenitor cells may be collected from bone marrow, peripheral blood or umbilical cord blood and infused into the patient. The source of the transplanted cells may be from the patient to be treated (“autologous HCT”), for example, collected prior to a therapy that may act to destroy these cells such as myeloablative therapy or chemotherapy, and used where the patient's hematopoietic cells are otherwise healthy. In such cases, immunoreactions following treatment are relatively rare since the transplanted cells were originally taken from the patient. Alternatively, where the patient's own hematopoietic cells are diseased, for example, the donor cells are taken from a different human donor (“allogeneic HCT”), who may or may not be genetically related to the patient. Appropriate donors for allogeneic HCT may be identified, for example, through comparison of human leukocyte antigens (HLAs) of the donor versus recipient cells. A sibling or relative with an identical set of HLAs to the recipient is ideal, but, of course, not always available. A “syngeneic HCT” is a procedure in which the donor is an identical twin of the recipient. An unrelated donor, for example, identified through a database ideally will have no or only one HLA mismatch compared to the recipient. A greater degree of mismatch may be tolerated if circumstances otherwise warrant. In some cases, umbilical cord blood may be used as donor as it may be considered immunologically naïve compared to cells from an adult donor. HCT procedures may be used as treatments for a variety of medical conditions such as cancers of immune origin, e.g. leukemia, lymphoma, and myeloma, and other diseases that result in abnormal hematopoiesis such as thalassemia, sickle cell anemia, severe combined immunodeficiency, aplastic anemia, myelodysplastic syndrome, and HIV-associated lymphomas, as well as for patients who are undergoing treatment (e.g. chemotherapy) for disorders where hematopoietic cells may be damaged by the disease treatment, such as neuroblastoma and germ cell tumors. Accordingly, in embodiments herein, the patient may suffer from a leukemia, lymphoma, or myeloma. In other embodiments, the patient may suffer from a genetic hematopoietic disorder, such as thalassemia, sickle cell anemia, severe combined immunodeficiency, aplastic anemia, myelodysplastic syndrome. Furthermore, in embodiments herein, the patient may suffer from one or more of the following diseases or disorders, which may be treated with allogeneic HCT: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoblastic leukemia (CLL), a myeloproliferative disorder, a myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism such as mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophy, adrenoleukodystrophy, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, or leukocyte adhesion deficiency. In some embodiments, patients having one of the above-listed diseases or disorders may receive an allogeneic HCT. To attempt to avoid complications following an HCT procedure, the HCT patient may undergo various post-HCT drug regimens as well as particular pre-conditioning procedures. In some cases, the cellular transplant may be prepared in such a way as to reduce risks of transplanting immunocompetent T cells or cells that may trigger adverse reactions such as GVHD. Such procedures are described further below. GVHD Following Transplantation Both acute (a) and chronic (c) GVHD are potential complications of an HCT procedure, particularly an allogeneic HCT procedure. In spite of immunosuppressive treatments following HCT procedures, about 20-80% of allogeneic HCT recipients develop aGVHD following the procedure. (See, e.g., P. J. Martin et al.Biol. Blood Marrow Transplant.18: 1150-63 (2012).) In general, GVHD is often considered acute if it occurs within 100 days of the HCT procedure. The risk of GVHD is related to the source of the transplanted cells. In particular, the risk of GVHD may increase if there is an HLA mismatch between the donor and recipient and the risk is greater in the case of an unrelated donor compared to a matched, sibling donor. (See Id.) The risk of GVHD is also lower in transplants from umbilical cord blood compared to transplants taken from adult bone marrow or adult peripheral blood, perhaps due to the immaturity of the immune cells in umbilical cord blood. However, the use of umbilical cord blood may be limited due to its relatively low volume. Acute GVHD following HCT may occur in various degrees of severity and may involve different bodily organs to different degrees. aGVHD may frequently affect the skin, liver, and gastrointestinal (GI) tract. aGVHD may be graded according to criteria set by the IMBTR, for example, depending on the severity of symptoms and whether the liver and/or GI tract are involved. (P. A. Rowlings et al.,Br. J. Haematol.97: 855-64 (1997).) For example, the grading system involves first grading the involvement of different organs. The impact on the skin may be graded on a 4-level scale by the extent of a skin rash (% of the body surface impacted) and whether a generalized erythroderma is present with or without bullae formation. The concentration of bilirubin may indicate extent of liver involvement. The extent to which diarrhea and abdominal pain occur may be used to indicate GI tract involvement. In general, Stage I aGVHD does not involve the liver or GI tract and comprises a skin rash but not generalized erythroderma or more serious symptoms. Stage II or higher aGVHD either involves generalized erythroderma (exfoliative dermatitis involving 90% or more of the skin) and/or liver and/or GI tract involvement. For example, a Stage II patient may have generalized erythroderma but no involvement of liver or the GI tract, or may have a systemic reaction including a less serious skin reaction along with liver and/or GI tract symptoms such as a high bilirubin concentration or significant diarrhea symptoms. A Stage III aGVHD patient may have both a generalized erythroderma and more significant liver and GI tract symptoms, while a Stage IV aGVHD patient may have severe skin rash involving bullae along with abdominal pain, severe diarrhea, and high bilirubin concentration, for instance. The following table summarizes how aGVHD may be staged in some embodiments, herein: TABLE 1Exemplary determination of aGVHD stage (IMBTR criteria)OrganLiverLower GIStageSkin(Bilirubin)Upper GI(stool/day)0No active<2 mg/dLNone or<500 mL/dayerythematousintermittentGVHD rashnausea oranorexia1Maculopapular2-3 mg/dLPersistent500-999 mL/dayrash <25% ofnausea,body surfacevomiting orarea (BSA)anorexia(not relatedto drugtoxicity)2Maculopapular3.1-6 mg/dL1000-1500 mL/dayrash 25-50% BSA3Maculopapular6.1-15 mg/dL>1500 mL/dayrash >50% BSA4Generalized>15 mg/dLSevererash (>50% BSA)abdominalplus bullouspain with orformation andwithout ileusdesquamationor grossly>5% BSAbloody stool,regardless ofstool volumeOverall clinical stage0No stage 1-4 in any organ1Stage 1-2 skin and stage 0 liver, upper GI, and lower GI2Stage 3 skin; and/or stage 1 liver; and/or stage 1 upperGI; and/or stage 1 lower GI3Stage 2-3 liver and/or stage 2-3 lower GI, with stage 0-3skin and/or stage 0-1 upper GI4Stage 4 skin, liver, or upper GI, with stage 0-1 upper GI GVHD in the upper GI may be confirmed in some cases by upper GI biopsy or colonoscopy. Patient prognosis also significantly worsens with grade of aGVHD, with patients having Stage IV aGVHD having a less than 10% survival rate. (MC Pasquini, 2008.) In some embodiments, aGVHD may be scored by criteria developed at the University of Minnesota, and published in M. L. MacMillan et al.,Biol. Blood Marrow Transplant.,21(4): 761-767 (2015). According to this scoring system, initial high-risk (HR) aGVHD is defined as either skin stage 4 (see table above), lower GI stage 3-4 (see table above) or liver stage 3-4 (see table above), or skin stage 3+ and either lower GI stage 2-4 or liver stage 2-4. Symptoms that do not meet these criteria may be classified as standard risk (SR) aGVHD. Accordingly, this system uses the IMBTR stages above to place aGVHD subjects into “high risk (HR)” or non-high risk, i.e., standard risk (SR) categories. In spite of immunosuppressive treatments following HCT procedures, about 20-80% of allogeneic HCT recipients develop aGVHD following the procedure. (See, e.g., P. J. Martin et al.Biol. Blood Marrow Transplant.18: 1150-63 (2012).) In any event, several immunosuppressive treatments are commonly used to treat GVHD in post-HCT patients. For example, current first-line therapy for aGVHD includes steroid treatment with methylpredisone or methylprednisolone, which may be administered for Grade II and higher aGVHD. There are various second-line therapies for patients who do not respond sufficiently to steroid treatment and are, thus, steroid-refractory. These include mycophenolate mofetil (MMF), anti-TNF antibodies or other antibody drugs such as antibodies binding to CD3, CD147 and IL-2, antilymphocyte globulin (ATG), mesenchymal stem cells, and methotrexate (MTX). Given the severity of higher grade aGVHD, however, further treatment options as well as procedures to reduce the risk of onset of aGVHD in HCT patients are needed. Exemplary Methods of Reducing the Risk of Onset of aGVHD Encompassed herein are methods of reducing the risk of onset of aGVHD in a subject by administering A1AT both before and after an HCT procedure. For example, in some embodiments, administration of A1AT begins one, two, or three days prior to the HCT procedure and continues for at least 4 weeks after the procedure, such as at least 8 weeks, at least 12 weeks, or at least 100 days, or at least 120 days following the HCT procedure. In some embodiments, administration of A1AT begins prior to the HCT procedure such as one, two, three, seven, ten, or fourteen days prior or 1 month, 2 months, or 3 months prior to the procedure, and then continues for at least 4 weeks after the procedure. As administration of A1AT begins prior to the HCT procedure, in some embodiments the subject does not have GVHD symptoms at the start of the administration period. Similarly, in some embodiments, the subject's individual risk of developing aGVHD after the HCT procedure cannot be determined prior to the procedure. In some embodiments, the subject is administered A1AT according to the following schedule: (a) administering a dose of at least 120 mg/kg A1AT, such as 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, or 300 mg/kg, to the subject at least one day prior to an HCT procedure; and (b) administering a dose of at least 90 mg/kg A1AT, such as 90, 100, 110, 120, 130, 140, 150, 160, 180, or 200 mg/kg A1AT, to the subject twice weekly following HCT for at least 4 weeks. In some embodiments, this regime is then followed by a dose of at least 90 mg/kg A1AT, such as 90, 100, 110, 120, 130, 140, 150, 160, 180, or 200 mg/kg A1AT, once weekly for at least an additional 4 weeks. This procedure may optionally also involve administration of at least one immunosuppressive agent. Specific administration schedules include (a) administering a dose of 120 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 90, 100, 110, or 120 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks, optionally followed by a dose of 90, 100, 110, or 120 mg/kg A1AT once weekly for at least an additional 4 weeks; as well as (a) administering a dose of 180 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 100, 110, 120, 130, or 140 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks, optionally followed by a dose of 100, 110, 120, 130, or 140 mg/kg A1AT once weekly for at least an additional 4 weeks; as well as (a) administering a dose of 150 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 90, 100, 110, or 120 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks, optionally followed by a dose of 90, 100, 110, or 120 mg/kg A1AT once weekly for at least an additional 4 weeks; as well as (a) administering a dose of 120 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 90 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks; as well as (a) administering a dose of 120 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 90 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks followed by a dose of 90 mg/kg A1AT once weekly for at least an additional 4 weeks; as well as (a) administering a dose of 150 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 100 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks; as well as (a) administering a dose of 150 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 100 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks followed by a dose of 100 mg/kg A1AT once weekly for at least an additional 4 weeks; as well as (a) administering a dose of 180 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 120 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks; as well as (a) administering a dose of 180 mg/kg A1AT to the subject one day prior to an HCT procedure; and (b) administering a dose of 120 mg/kg A1AT to the subject twice weekly following HCT for at least 4 weeks followed by a dose of 120 mg/kg A1AT once weekly for at least an additional 4 weeks. Any of these above schedules may be performed in combination with administration of at least one immunosuppressive agent. Immunosuppressive agents administered following HCT may include steroids such as prednisone, methylprednisone or methylprednisolone, which are currently first-line therapies for treating aGVHD following HCT, additional steroids such as budesonide and beclomethasone, which may be administered when there is GI tract involvement, as well as other agents such as calcineurin inhibitors like tacrolimus, sirolimus, and cyclosporine or others, which may be administered with methotrexate in some cases. Other therapies such as pentostatin, ruxolitinib, brenbuximab vedotin (anti-CD30 antibody), tocilizumab (anti-IL6R antibody), an IL6 signaling inhibitor, mycophenolate mofetil (MMF), an anti-TNF antibody, basiliximab, daclizumab, inolimomab, alemtuzumab, etanercept, infliximab, a leukotriene antagonist, antilymphocyte globulin (ATG) such as horse ATG, and/or mesenchymal stem cells may also be administered in combination with A1AT in some embodiments. In some embodiments, subjects may be administered a combination of A1AT with steroid and an IL6 signaling inhibitor such as tocilizumab or another IL6 signaling inhibitor. In some embodiments, where methylprednisone or methylprednisolone are administered, they are administered at 1-3 mg/kg/day, or 1-2 mg/kg/day. In some embodiments, the subject may have also undergone a conditioning regimen such as a myeloablative conditioning regimen or a reduced intensity conditioning regimen. In some embodiments, the HCT procedure is an allogeneic HCT procedure comprising cells from (a) a related donor with at least one HLA mismatch or (b) an unrelated donor with or without at least one HLA mismatch. In some embodiments, the donor is a related donor with one HLA mismatch. In some embodiments, the donor is an unrelated donor without an HLA mismatch. In some embodiments, the donor is an unrelated donor with one HLA mismatch. In some embodiments, the donor cells are not from umbilical cord blood. In some embodiments, the donor cells are derived from bone marrow. In some embodiments, the cells are derived from peripheral blood. In some embodiments, the subject suffers from a disease or disorder such as leukemia, lymphoma, or myeloma. In other embodiments, the patient may suffer from a genetic hematopoietic disorder, such as thalassemia, sickle cell anemia, severe combined immunodeficiency, aplastic anemia, myelodysplastic syndrome. Furthermore, in embodiments herein, the patient may suffer from one or more of the following diseases or disorders, which may be treated with allogeneic HCT: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoblastic leukemia (CLL), a myeloproliferative disorder, a myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism such as mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophy, adrenoleukodystrophy, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, or leukocyte adhesion deficiency. In some embodiments, the subject is at risk of developing Stage III or IV aGVHD following HCT. In some embodiments, the median serum A1AT levels in the subject are above the normal human physiological levels on the day of the HCT procedure and remain above those levels for at least 28 days after the HCT procedure. In some embodiments, the peak serum A1AT levels in the subject are above normal human physiological levels on the day of the HCT procedure and remain above those levels for at least 28 days after the HCT procedure. AAT levels in subjects without genetic deficiency are generally in the range of 1.5 to 3.5 mg/mL, but, as AAT is an acute-phase reactant, these levels can be increased transiently by at least 2-fold or even more, for example, in a physiological response to inflammation. (Silverman and Sandhaus,N Engl J Med.2009 Jun. 25; 360(26):2749-57.) In some embodiments, the median serum A1AT levels in the subject remain above 2.5 mg/mL on the day of the HCT procedure and for at least 28 days after the HCT procedure. In some embodiments, the peak serum A1AT levels in the subject remain above 2.5 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the median serum A1AT levels in the subject remain above 2.0 mg/mL on the day of the HCT procedure and for at least 28 days after the HCT procedure. In some embodiments, the peak serum A1AT levels in the subject remain above 2.0 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the administration of A1AT is performed such that the median serum A1AT levels in the subject are above 3.0 mg/mL on the day of the HCT procedure and remain above 3.0 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the administration is performed such that the peak serum A1AT levels in the subject are above 3.0 mg/mL on the day of the HCT procedure and remain above 3.0 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the administration of A1AT is performed such that the median serum A1AT levels in the subject are above 3.5 mg/mL on the day of the HCT procedure and remain above 3.5 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the administration is performed such that the peak serum A1AT levels in the subject are above 3.5 mg/mL on the day of the HCT procedure and remain above 3.5 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the median serum A1AT levels in the subject remain above 4.0 mg/mL on the day of the HCT procedure and for at least 28 days after the HCT procedure. In some embodiments, the peak serum A1AT levels in the subject remain above 4.0 mg/mL for at least 28 days after the HCT procedure. In some embodiments, the median serum A1AT levels in the subject remain above 5.0 mg/mL on the day of the HCT procedure and for at least 28 days after the HCT procedure. In some embodiments, the peak serum A1AT levels in the subject remain above 5.0 mg/mL for at least 28 days after the HCT procedure. In some embodiments, dosage levels of A1AT given to a patient are chosen so as to be at or above a dosage level that has been shown to provide an average or median peak serum A1AT level in a group of previously tested clinical subjects of greater than or equal to a particular threshold, such as 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, or 5 mg/mL. In some embodiments, pre- and post-HCT A1AT dosages are designed so as to be at or above a dosage level that has been shown to provide an overall average or median serum A1AT level in a group of previously tested clinical subjects of greater than 1.5 mg/mL, greater than 2.0 mg/mL, or between 1.5 and 3.5 mg/mL, or between 2 and 4 mg/mL. In other methods herein, pre- and post-HCT A1AT dosages are designed to ensure that median serum A1AT levels remain above 2.0 mg/mL both on the day of the HCT procedure and for at least 28 days after the HCT procedure. For example, in some embodiments, A1AT dosing following the HCT procedure may be daily, every 2 days, every 3 days, every 7 days, every 10 days or every 14 days so long as the median serum levels of A1AT remain above 2.0 mg/mL for at least 28 days after the procedure. For instance, in some embodiments patients may experience a depletion of serum proteins such as A1AT, for example, as a result of developing aGVHD symptoms. In such a case, the portion of the dosage regime following the HCT procedure may need to be adjusted so as to maintain serum A1AT levels above the normal human serum level following the HCT procedure. In other cases A1AT levels may be relatively stable such that a less frequent dosing following the procedure can be used. For example, in some embodiments, A1AT dosing following the HCT procedure may be daily, every 2 days, every 3 days, every 7 days, every 10 days or every 14 days so long as the median serum levels of A1AT remain above 3.0 mg/mL for at least 28 days after the procedure. In other embodiments, A1AT dosing following the HCT procedure may be daily, every 2 days, every 3 days, every 7 days, every 10 days or every 14 days so long as the median serum levels of A1AT remain above 3.5 mg/mL for at least 28 days after the procedure. In other embodiments, A1AT dosing following the HCT procedure may be daily, every 2 days, every 3 days, every 7 days, every 10 days or every 14 days so long as the median serum levels of A1AT remain above 4.0 mg/mL for at least 28 days after the procedure. In other embodiments, A1AT dosing following the HCT procedure may be daily, every 2 days, every 3 days, every 7 days, every 10 days or every 14 days so long as the median serum levels of A1AT remain above 5.0 mg/mL for at least 28 days after the procedure. In methods herein, the A1AT may be derived from pooled human plasma or may be recombinant. The A1AT may also be a fusion protein, for example, comprising a fusion partner of albumin, Fc, or polyethylene glycol. Certain A1AT products, including A1AT-Fc fusion proteins, are described in patent publications WO 2013/106589, WO 2006/133403, U.S. Pat. Nos. 9,457,070, 9,884,096, and WO 2013/003641. Several commercial therapeutic A1AT products are available, including Prolastin®, Prolastin® C, Glassia®, Aralast®, Aralast® NP, Zemaira®/Respreeza®, and Alfalastin® (LFB). Exemplary Methods of Treating aGVHD Following HCT with A1AT and Steroids The disclosure herein also includes methods of treating aGVHD following an HCT procedure, for example, upon initial diagnosis of aGVHD, with a combination of A1AT and at least one steroid. Acute GVHD may be diagnosed clinically at any time from completion of the HCT procedure to 100 days from the procedure. Late-onset aGVHD can also occur more than 100 days after HCT. The at least one steroid may comprise, for example, prednisone, methylprednisone or methylprednisolone, and/or a non-absorbable oral steroid such as budesonide or beclomethasone. The A1AT may be administered twice weekly at a dose of, for example, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 220, or 240 mg/kg A1AT for at least 4 weeks following the initial aGVHD diagnosis, along with the steroid administration. In some embodiments, this twice-weekly regime is followed by administration of, for example, 90, 100, 110, 120, 130, 140, 150, 160, 180, or 200 mg/kg A1AT, once weekly for at least an additional 4 weeks, along with continuation of steroid administration. In some embodiments, the A1AT is administered twice weekly at a dose of at least 90 mg/kg for at least 4 weeks following the initial aGVHD diagnosis, along with the steroid administration. In some embodiments, the A1AT is administered twice weekly at a dose of at least 100 mg/kg for at least 4 weeks following the initial aGVHD diagnosis, along with the steroid administration. In some embodiments, the A1AT is administered twice weekly at a dose of at least 110 mg/kg for at least 4 weeks following the initial aGVHD diagnosis, along with the steroid administration. In some embodiments, the A1AT is administered twice weekly at a dose of at least 120 mg/kg for at least 4 weeks following the initial aGVHD diagnosis, along with the steroid administration. In some embodiments, upon at least a partial response (PR) after the first 4 weeks of treatment, the dosage frequency is reduced to once weekly at either the same dosage level or a reduced dosage level. In some embodiments, the patient's blood or plasma is monitored to ensure that peak serum levels of A1AT remain at least at 3.5 mg/mL over the first 4 weeks (or 28 days) of A1AT administration, and a higher or more frequent dose of A1AT may be administered if peak A1AT levels fall below that threshold. In some embodiments, the first dose of A1AT or the first two doses may be a loading dose, i.e., a higher dose level than the subsequent doses. In some embodiments, a loading dose may be, for example, 120, 130, 140, 150, 160, 180, 200, or 220 mg/kg A1AT. In some embodiments, the combination of twice-weekly A1AT and steroid is continued for longer than 4 weeks, such as for 8, 10, 12, 14, 16, or 18 weeks, or for 60, 80, 100, 120, 140, 160, or 180 days. In some such embodiments, upon at least a partial response (PR) after the first 4 weeks of treatment, the dosage frequency is reduced to once weekly at either the same dosage level or a reduced dosage level. And in some such embodiments, upon at least a PR, the frequency of steroid dosage is also made longer and/or the steroid dosage reduced so as to taper the subject off from the A1AT and steroid. In some embodiments, the overall treatment regime lasts for 100, 120, 140, 160, or 180 days. In some embodiments, the patient's peak serum levels of A1AT remain at least 3.0 mg/mL over the first 4 weeks (or 28 days) of treatment. In some embodiments, if the patient's peak serum levels of A1AT fall below 3.0 mg/mL during the first 4 weeks (or 28 days) of treatment, the dose of A1AT is increased until peak serum levels remain above 3.0 mg/mL. In some embodiments, peak serum levels of A1AT remain at least 3.5 mg/mL over the first 4 weeks (or 28 days) of treatment. In some embodiments, if peak serum levels of A1AT fall below 3.5 mg/mL during the first 4 weeks (or 28 days) of treatment, the dose of A1AT is increased until peak serum levels remain above 3.5 mg/mL. In some embodiments, peak serum levels of A1AT remain at least 4.0 mg/mL over the first 4 weeks (or 28 days) of treatment. In some embodiments, if peak serum levels of A1AT fall below 4.0 mg/mL during the first 4 weeks (or 28 days) of treatment, the dose of A1AT is increased until peak serum levels remain above 4.0 mg/mL. In some embodiments, peak serum levels of A1AT remain at least 4.5 mg/mL over the first 4 weeks (or 28 days) of treatment. In some embodiments, if peak serum levels of A1AT fall below 4.5 mg/mL during the first 4 weeks (or 28 days) of treatment, the dose of A1AT is increased until peak serum levels remain above 4.5 mg/mL. In some embodiments, peak serum levels of A1AT remain at least 5.0 mg/mL over the first 4 weeks (or 28 days) of treatment. In some embodiments, if peak serum levels of A1AT fall below 5.0 mg/mL during the first 4 weeks (or 28 days) of treatment, the dose of A1AT is increased until peak serum levels remain above 5.0 mg/mL. In some embodiments, dosage levels of A1AT given to a patient are chosen so as to be at or above a dosage level that has been shown to provide an average or median peak serum A1AT level in a group of previously tested clinical subjects of greater than or equal to a particular threshold, such as 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, or 5 mg/mL. In some embodiments, A1AT dosages are designed so as to be at or above a dosage level that has been shown to provide an overall average or median serum A1AT level in a group of previously tested clinical subjects of greater than 1.5 mg/mL, greater than 2.0 mg/mL, or between 1.5 and 3.5 mg/mL, or between 2 and 4 mg/mL. Any of the above schedules may also be performed in combination with administration of at least one immunosuppressive agent. Immunosuppressive agents administered following HCT may include a second steroid such as prednisone, methylprednisone, methylprednisolone, budesonide or beclomethasone, as well as other agents such as calcineurin inhibitors like tacrolimus, sirolimus, and cyclosporine or others, which may be administered with methotrexate in some cases. For example, the above A1AT dosing schedules may be added to a steroid treatment plan that is based on the patient's grade or stage of aGVHD and the location and type of symptoms (e.g. whether there is GI tract involvement or whether symptoms are primarily localized to the skin). For example, patients presenting Stage II aGVHD are often treated with systemic steroids such as methylprednisolone. Non-absorbable oral steroids may be added as local therapy or substituted for systemic steroids in cases where there is GI tract involvement or suspected GI tract infection. In contrast, patients presenting Stage I aGVHD, which, for example, may mostly present as a maculopapular rash without liver or GI tract involvement, may be treated with topical steroids or a combination of topical and systemic steroids. Steroid regimens for Stage II to Stage IV aGVHD generally involve treatment with glucocorticoids such as prednisone, methylprednisone, methylprednisolone, and/or the non-absorbable glucocorticoids such as budesonide or beclomethasone, which may be added to the regimen or substituted for other steroids for patients with GI tract involvement. Oral beclomethasone, however, is generally not given if patients have a GI infection such as cytomegalovirus (CMV) colitis, or are suspected of having such an infection. Accordingly, in methods herein, the at least one steroid may comprise, for example, prednisone, methylprednisone, or methylprednisolone, budesonide or beclomethasone, administered alone or in combination. Prednisone, methylprednisone, or methylprednisolone may be administered at a dose of, for example, between 0.5 and 3.0 mg/kg per day, such as 1-2.5 mg/kg/day. For example, prednisone may be dosed for systemic administration at 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, or 3 mg/kg per day, or for example at 2-2.5 mg/kg per day. Methylprednisolone may be dosed systemically at a range of moderate to high dosages such as 1-20 mg/kg or 1-10 mg/kg per day, or at more moderate dosages or dose ranges such as 0.5-3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, or 3 mg/kg per day, or 1.5-2.5 mg/kg per day. In some embodiments, methylprednisolone may be administered at 2 mg/kg/day. In some embodiments, methylprednisolone may be administered at 1.5 mg/kg/day. In some embodiments, methylprednisolone may be administered at 1 mg/kg/day. Beclomethasone may be administered in dosages such as 5-10 mg/day, or 5, mg/day, 6 mg/day, 7 mg/day, 8 mg/day, 9 mg/day, or 10 mg/day. As noted above, once at least a partial response is achieved after 4 weeks, the dosage and frequency of the steroid may be slowly tapered down. Other therapies may also be added to a regimen of steroid plus A1AT in some embodiments, such as pentostatin, ruxolitinib, brenbuximab vedotin (anti-CD30 antibody), tocilizumab (anti-IL6R antibody), an IL6 signaling inhibitor, mycophenolate mofetil (MMF), an anti-TNF antibody, basiliximab, daclizumab, inolimomab, alemtuzumab, etanercept, infliximab, a leukotriene antagonist, antilymphocyte globulin (ATG) such as horse ATG, and/or mesenchymal stem cells. In some embodiments, patients may be administered a combination of A1AT with steroid and an IL6 signaling inhibitor such as tocilizumab or another IL6 signaling inhibitor. In some embodiments, the subject may have also undergone a conditioning regimen such as a myeloablative conditioning regimen or a reduced intensity conditioning regimen. In some embodiments, the HCT procedure is an allogeneic HCT procedure comprising cells from (a) a related donor with at least one HLA mismatch or (b) an unrelated donor with or without at least one HLA mismatch. In some embodiments, the donor is a related donor with one HLA mismatch. In some embodiments, the donor is an unrelated donor without an HLA mismatch. In some embodiments, the donor is an unrelated donor with one HLA mismatch. In some embodiments, the donor cells are not from umbilical cord blood. In some embodiments, the donor cells are derived from bone marrow. In some embodiments, the cells are derived from peripheral blood. In some embodiments, the subject suffers from a disease or disorder such as leukemia, lymphoma, or myeloma. In other embodiments, the patient may suffer from a genetic hematopoietic disorder, such as thalassemia, sickle cell anemia, severe combined immunodeficiency, aplastic anemia, myelodysplastic syndrome. Furthermore, in embodiments herein, the patient may suffer from one or more of the following diseases or disorders, which may be treated with allogeneic HCT: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoblastic leukemia (CLL), a myeloproliferative disorder, a myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism such as mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophy, adrenoleukodystrophy, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, or leukocyte adhesion deficiency. In some embodiments, the subject is at risk of developing Stage III or IV aGVHD following HCT. In methods herein, the A1AT may be derived from pooled human plasma or may be recombinant. The A1AT may also be a fusion protein, for example, comprising a fusion partner of albumin, Fc, or polyethylene glycol. Several commercial therapeutic A1AT products are available, including Prolastin®, Glassia®, Aralast®, and Zemaira®/Respreeza®. EXAMPLES The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1: A Phase 2/3 Open-Label and Double-Blind Clinical Studies of A1AT for Reducing Risk of Onset of Acute GVHD in HCT Recipients A human clinical trial of A1AT in HCT recipients is planned in two parts, an open-label portion with two cohorts of 20 patients, cohorts 1 and 2, given two particular dosage regimes, followed by a double-blind and placebo-controlled trial of A1AT vs. placebo in 160 patients using the dosage regime chosen in the open-label portion of the trial. In the open-label portion, cohort 1 will be given 120 mg/kg A1AT (Zemaira®/Respreeza®) one day prior to an HCT procedure (day −1) and then will be given 90 mg/kg A1AT twice weekly until day 28, then 90 mg/kg A1AT once weekly until day 56. Cohort 2 will be given 180 mg/kg A1AT (Zemaira®/Respreeza®) one day prior to an HCT procedure (day −1) and then will be given 120 mg/kg A1AT twice weekly until day 28, then 120 mg/kg A1AT once weekly until day 56. Data from the two cohorts will be reviewed at least 100 days after the HCT procedures for all patients and a dosage regime for the double-blind portion of the trial will be selected based on the previously tested regimes. In these dosage regimes, sufficient A1AT may be given both prior to and following HCT such that either median or peak A1AT levels in patients exceed levels of A1AT in normal human peripheral blood. Patients selected for the trial must have an age of at least 18 years for the open-label portion and at least 12 years for the double-blind portion. The trial will be restricted to patients who have received a myeloablative conditioning regimen. Since the conditioning regimen can impact risk of developing aGVHD, limiting variation in conditioning regimen may help to evaluate results. Patients must also receive HCT from either a) a related donor where there is one or two HLA mismatches (e.g., 6/8 or 7/8) or b) an unrelated donor with either an HLA match or a mismatch (e.g. 7/8). Patients receiving an umbilical cord blood transplant are excluded, as are patients given anti-T cell antibody therapy, and patients who have previously had an HCT. Patients will also be treated with a standard immunosuppression regimen of tacrolimus and methotrexate in addition to the A1AT or placebo. The observed rate of aGVHD in patients receiving tacrolimus and methotrexate is about 40-60%. (M. Jagasia et al.Blood119(1): 296-307 (2012); Pavletic & Fowler, 2012.) Patients who develop aGVHD in spite of the treatment regimen may further be treated with steroids. In the second phase of the trial, patients on placebo who develop aGVHD and who are treated with steroids but are refractory to the steroid treatment may be unblended so as to receive further treatment with A1AT. The primary endpoint for both parts of the trial will be the frequency of Grade II or higher aGVHD within 100 days following HCT. aGVHD in patients is graded according to Table 1 provided earlier in this disclosure. Secondary endpoints will be the frequency of each of Grades II, III, and IV aGVHD within 100 days following HCT, incidence of chronic GVHD at days 180 and 365 after HCT, incidence of systemic infections at days 28, 60, 180, and 365, days to non-relapse mortality, and overall mortality at days 180 and 366. Other secondary endpoints include frequency of recurrence of primary malignancies at days 180 and 365, incidence of discontinuation of immune suppression at days 180 and 365, time to neutrophil engraftment, frequency of steroid-refractory aGVHD (Grade II-IV) that respond to treatment with A1AT at day 28, overall response rate (ORR) for subjects with steroid-refractory aGVHD at day 56, incidence of related adverse events, and pharmacokinetic parameters such as AUC, Cmax and trough. Example 2: A Phase III Clinical Trial of A1AT for Treatment of High Risk aGVHD after HCT in Combination with Methylprednisolone A Phase III, multi-center, randomized, placebo-controlled clinical trial of A1AT plus methylprednisolone or placebo plus methylprednisolone is planned for patients in need of initial treatment following an HCT procedure for high risk aGVHD (see the clinical high risk aGVHD features under the Minnesota standards at the following URL: http://www (followed by) z (dot) umn (dot) edu (dot) MNAcuteGVHDRiskScore). Newly diagnosed adult (>12 years) male and non-pregnant female patients with high-risk aGVHD following allogeneic HCT will be included in the trial. A1AT is contraindicated in IgA deficient patients, however. Patients with newly diagnosed aGVHD after HCT will be randomized to receive 120 mg/kg A1AT (Zemaira®) or placebo twice weekly in addition to methylprednisolone (MP) at 2 mg/kg/day. If the subject has a response (either complete response (CR) or partial response (PR)) at Day 28 after the start of treatment, the subject may receive an additional 4 weeks of treatment with AAT once weekly. If a subject has a response (either CR or PR) at Day 28 after the start of treatment, the subject may also receive less frequent doses of methylprednisolone. Patients will remain in follow up through a primary end-point of 6 months (180 days). Patients will be assessed for GVHD through 8 weeks (on treatment), then a minimum of every two weeks through 12 weeks (120 days), then monthly through to the 6 month end-point. The trial will include 110 patients, with 55 per treatment arm. For this trial, A1AT (Zemaira®) is supplied as a sterile, white lyophilized powder in 1 g vials and is reconstituted in sterile water for injection at 50 mg/mL. The placebo product (AlbuRX®5), a commercial albumin product, is similarly diluted to a 1.2% albumin solution in 5% dextrose, and has been demonstrated to be a visual match for the A1AT solution at 50 mg/mL. An unblinded pharmacist will prepare the two solutions. The A1AT will be dosed at 120 mg/kg twice weekly, followed optionally by once weekly dosing after the first 4 weeks (28 days). Dose modeling estimates that a dose of 120 mg/mL twice weekly should achieve plasma A1AT levels of at least 3.5 mg/mL, which may be sufficient to attenuate the inflammatory process of GVHD. Dosing is intended to achieve a targeted steady-state AAT level of greater than or equal to 3.5 mg/mL. Study assessments will include assessments for safety, clinical activity, pharmacokinetics, and pharmacodynamics. Clinical assessments will include scoring of GVHD symptoms, for example, including assessment of skin rash, gastrointestinal symptoms such as diarrhea, vomiting, and nausea, as well as liver function. The primary objectives of the trial include assessing the efficacy of A1AT in combination with MP in patients with newly diagnosed GVHD. Secondary objectives include assessing the safety of A1AT in the treatment of patients with newly diagnosed GVHD and assessing the pharmacokinetic profile. Primary end-points include overall response rate (ORR), complete response (CR), and partial response (PR) at day 28 following start of treatment, as well as GVHD-free, relapse-free survival (GRFS) at six months in patients with newly diagnosed GVHD receiving A1AT compared to placebo in combination with the standard of care MP treatment, such as a 25% increase in GRFS at 6 months compared to MP standard of care. Additional endpoints include non-relapse mortality at day 180, incidence of recurrence of primary malignancies through day 180, pharmacokinetic parameters, incidence of chronic GVHD at day 100 and at day 180, incidence of discontinuation of immune suppression at day 28, day 60, and day 180, incidence of systemic infections at day 28, day 60, and day 180, and incidence of related adverse events. | 53,434 |
11857611 | DETAILED DESCRIPTION OF THE INVENTION Compositions and methods are provided to produce an immune response to a Plasmodium antigen, in a subject in need thereof. The compositions and methods of the present invention can be used to prevent or delay symptoms of malaria infection or to treat malaria in a subject in need thereof. Ideal immunogenic compositions or vaccines have the characteristics of safety, efficacy, scope of protection and longevity, however, compositions having fewer than all of these characteristics may still be useful in preventing malaria or limiting symptoms or disease progression in an exposed subject treated prior to the development of symptoms. In one embodiment the present invention provides a vaccine that permits at least partial, if not complete, protection after a single immunization. In one embodiment, the composition is a recombinant vaccine or immunogenic vector that comprises one or more nucleic acid sequences Plasmodium antigens or immunogenic fragments thereof. In one embodiment, the vector expresses proteins that form VLPs and generate an immune response to a Plasmodium antigen or immunogenic fragment thereof. In exemplary embodiments, the immune responses are long-lasting and durable so that repeated boosters are not required, but in one embodiment, one or more administrations of the compositions provided herein are provided to boost the initial primed immune response. I. Definitions Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise, e.g., “a peptide” includes a plurality of peptides. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure. The term “antigen” refers to a substance or molecule, such as a protein, or fragment thereof, that is capable of inducing an immune response. The term “binding antibody” or “bAb” refers to an antibody which either is purified from, or is present in, a body fluid (e.g., serum or a mucosal secretion) and which recognizes a specific antigen. As used herein, the antibody can be a single antibody or a plurality of antibodies. Binding antibodies comprise neutralizing and non-neutralizing antibodies. The term “cell-mediated immune response” refers to the immunological defense provided by lymphocytes, such as the defense provided by sensitized T cell lymphocytes when they directly lyse cells expressing foreign antigens and secrete cytokines (e.g., IFN-gamma), which can modulate macrophage and natural killer (NK) cell effector functions and augment T cell expansion and differentiation. The cellular immune response is the 2ndbranch of the adaptive immune response. The term “conservative amino acid substitution” refers to substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in substantially altered immunogenicity. For example, these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide. The term “deletion” in the context of a polypeptide or protein refers to removal of codons for one or more amino acid residues from the polypeptide or protein sequence, wherein the regions on either side are joined together. The term deletion in the context of a nucleic acid refers to removal of one or more bases from a nucleic acid sequence, wherein the regions on either side are joined together. The term “Ebola virus” refers to a virus of species Zaire ebolavirus and has the meaning given to it by the International Committee on Taxonomy of Viruses as documented in (Kuhn, J. H. et al. 2010 Arch Virol 155:2083-2103). The term “fragment” in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment the fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference polypeptide. In one embodiment, a fragment of a full-length protein retains activity of the full-length protein. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein. The term “fragment” in the context of a nucleic acid refers to a nucleic acid comprising an nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least contiguous 80 nucleotides, at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In one embodiment the fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid sequence. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains activity of the full-length protein. In another embodiment, the fragment encodes a peptide or polypeptide that of the full-length protein does not retain the activity of the full-length protein. As used herein, the phrase “heterologous sequence” refers to any nucleic acid, protein, polypeptide, or peptide sequence which is not normally associated in nature with another nucleic acid or protein, polypeptide, or peptide sequence of interest. As used herein, the phrase “heterologous gene insert” refers to any nucleic acid sequence that has been or is to be inserted into the recombinant vectors described herein. The heterologous gene insert may refer to only the gene product encoding sequence or may refer to a sequence comprising a promoter, a gene product encoding sequence (such as GP, VP, or Z), and any regulatory sequences associated or operably linked therewith. The term “homopolymer stretch” refers to a sequence comprising at least four of the same nucleotides uninterrupted by any other nucleotide, e.g., GGGG or TTTTTTT. The term “humoral immune response” refers to the stimulation of Ab production. Humoral immune response also refers to the accessory proteins and events that accompany antibody production, including T helper cell activation and cytokine production, affinity maturation, and memory cell generation. The humoral immune response is one of two branches of the adaptive immune response. The term “humoral immunity” refers to the immunological defense provided by antibody, such as neutralizing Ab that can directly bind a Plasmodium antigen; or, binding Ab that identifies a neoplastic cell for killing by such innate immune responses as complement (C′)-mediated lysis, phagocytosis, and natural killer cells. The term “immunogenic composition” is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest. The term “immune response” refers to any response to an antigen or antigenic determinant by the immune system of a subject (e.g., a human). Exemplary immune responses include humoral immune responses (e.g., production of antigen-specific antibodies) and cell-mediated immune responses (e.g., production of antigen-specific T cells). Assays for assessing an immune response are known in the art and may comprise in vivo assays, such as assays to measure antibody responses and delayed type hypersensitivity responses. In an embodiment, the assay to measure antibody responses primarily may measure B-cell function as well as B-cell/T-cell interactions. For the antibody response assay, antibody titers in the blood may be compared following an antigenic challenge. As used herein, “antibody titers” can be defined as the highest dilution in post-immune sera that resulted in a value greater than that of pre-immune samples for each subject. The in vitro assays may comprise determining the ability of cells to divide, or to provide help for other cells to divide, or to release lymphokines and other factors, express markers of activation, and lyse target cells. Lymphocytes in mice and man can be compared in in vitro assays. In an embodiment, the lymphocytes from similar sources such as peripheral blood cells, splenocytes, or lymph node cells, are compared. It is possible, however, to compare lymphocytes from different sources as in the non-limiting example of peripheral blood cells in humans and splenocytes in mice. For the in vitro assay, cells may be purified (e.g., B-cells, T-cells, and macrophages) or left in their natural state (e.g., splenocytes or lymph node cells). Purification may be by any method that gives the desired results. The cells can be tested in vitro for their ability to proliferate using mitogens or specific antigens. The ability of cells to divide in the presence of specific antigens can be determined using a mixed lymphocyte reaction (MLR) assay. Supernatant from the cultured cells can be tested to quantitate the ability of the cells to secrete specific lymphokines. The cells can be removed from culture and tested for their ability to express activation antigens. This can be done by any method that is suitable as in the non-limiting example of using antibodies or ligands which bind to the activation antigen as well as probes that bind the RNA coding for the activation antigen. The term “improved therapeutic outcome” relative to a subject diagnosed as having malaria refers to a slowing or diminution in the symptoms, or detectable symptoms associated with malaria. The term “inducing an immune response” means eliciting a humoral response (e.g., the production of antibodies) or a cellular response (e.g., the activation of T cells) directed against a Plasmodium antigen in a subject to which the composition (e.g., a vaccine) has been administered. The term “insertion” in the context of a polypeptide or protein refers to the addition of one or more non-native amino acid residues in the polypeptide or protein sequence. Typically, no more than about from 1 to 6 residues (e.g., 1 to 4 residues) are inserted at any one site within the polypeptide or protein molecule. The term “Marburg virus” refers to a virus of species Marburg marburgvirus and has the meaning given to it by the International Committee on Taxonomy of Viruses as documented in (Kuhn, J. H. et al. 2010 Arch Virol 155:2083-2103). The term “marker” refers to is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. The term “modified vaccinia Ankara,” “modified vaccinia ankara,” “Modified Vaccinia Ankara,” or “MVA” refers to a highly attenuated strain of vaccinia virus developed by Dr. Anton Mayr by serial passage on chick embryo fibroblast cells; or variants or derivatives thereof. MVA is reviewed in (Mayr, A et al. 1975 Infection 3:6-14; Swiss Patent No. 568,392). The term “Plasmodium antigen immunogenic fragment” as used herein means an immunogenic poly amino acid containing at least 10 consecutive amino acids of a Plasmodium antigen sequence. The term “neutralizing antibody” or “NAb” refers to an antibody which either is purified from, or is present in, a body fluid (e.g., serum or a mucosal secretion) and which recognizes a specific antigen and inhibits the effect(s) of the antigen in the subject (e.g., a human). As used herein, the antibody can be a single antibody or a plurality of antibodies. The term “non-neutralizing antibody” or “nnAb” refers to a binding antibody that is not a neutralizing antibody. “Operably linked.” A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. The term “prevent”, “preventing” and “prevention” refers to the inhibition of the development of malaria or symptoms thereof. The term “promoter” refers to a polynucleotide sufficient to direct transcription. The term “prophylactically effective amount” refers to the amount of a composition (e.g., the recombinant MVA vector or pharmaceutical composition) which is sufficient to result in the prevention of the development, recurrence, or onset of malaria or a symptom thereof. The term “recombinant” means a polynucleotide of semisynthetic, or synthetic origin that either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature. The term “recombinant,” with respect to a viral vector, means a vector (e.g., a viral genome that has been manipulated in vitro, e.g., using recombinant nucleic acid techniques to express heterologous viral nucleic acid sequences. The term “regulatory sequence” “regulatory sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence. Not all of these control sequences need always be present so long as the selected gene is capable of being transcribed and translated. The term “shuttle vector” refers to a genetic vector (e.g., a DNA plasmid) that is useful for transferring genetic material from one host system into another. A shuttle vector can replicate alone (without the presence of any other vector) in at least one host (e.g.,E. coli). In the context of MVA vector construction, shuttle vectors are usually DNA plasmids that can be manipulated inE. coliand then introduced into cultured cells infected with MVA vectors, resulting in the generation of new recombinant MVA vectors. The term “silent mutation” means a change in a nucleotide sequence that does not cause a change in the primary structure of the protein encoded by the nucleotide sequence, e.g., a change from AAA (encoding lysine) to AAG (also encoding lysine). The term “subject” means any mammal, including but not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, rats, mice, guinea pigs and the like. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, marker history, and the like). The term “Sudan virus” refers to a virus of species Sudan ebolavirus and has the meaning given to it by the International Committee on Taxonomy of Viruses as documented in (Kuhn, J. H. et al. 2010 Arch Virol 155:2083-2103). The term “surrogate endpoint” means a clinical measurement other than a measurement of clinical benefit that is used as a substitute for a measurement of clinical benefit. The term “surrogate marker” means a laboratory measurement or physical sign that is used in a clinical or animal trial as a substitute for a clinically meaningful endpoint that is a direct measure of how a subject feels, functions, or survives and is expected to predict the effect of the therapy (Katz, R., NeuroRx 1:189-195 (2004); New drug, antibiotic, and biological drug product regulations; accelerated approval-FDA. Final rule. Fed Regist 57: 58942-58960, 1992.). The term “synonymous codon” refers to the use of a codon with a different nucleic acid sequence to encode the same amino acid, e.g., AAA and AAG (both of which encode lysine). Codon optimization changes the codons for a protein to the synonymous codons that are most frequently used by a vector or a host cell. The term “therapeutically effective amount” means the amount of the composition (e.g., the recombinant MVA vector or pharmaceutical composition) that, when administered to a mammal for treating malaria, is sufficient to effect such treatment for malaria. The term “treating” or “treat” refer to the eradication or control of malaria, the reduction or amelioration of the progression, severity, and/or duration of a condition or one or more symptoms caused by malaria resulting from the administration of one or more therapies. The term “vaccine” means material used to provoke an immune response and confer immunity after administration of the material to a subject. Such immunity may include a cellular or humoral immune response that occurs when the subject is exposed to the immunogen after vaccine administration. The term “vaccine insert” refers to a nucleic acid sequence encoding a heterologous sequence that is operably linked to a promoter for expression when inserted into a recombinant vector. The heterologous sequence may encode a glycoprotein or matrix protein described here. The term “virus-like particles” or “VLP” refers to a structure which resembles the native virus antigenically and morphologically. II. Plasmodium Antigens The compositions of the present invention are useful for inducing an immune response to a Plasmodium antigen. In one embodiment, the plasmodium antigen sequence is selected from Plasmodium blood or liver stage antigen or a combination thereof. In one embodiment, the plasmodium antigen is a sporozite stage antigen selected from CSP, TRAP, or STARP, or a combination thereof. In one embodiment, the plasmodium antigen is a merozoite stage antigen selected from MSP-1, MSP-2, MSP-3, GLURP, EBA-140, EBA-175, RAP1, RAP2, or AMA-1, or a combination thereof. In one embodiment, the plasmodium antigen is a gametocyte stage antigen selected from Pfs25, Pfs230, PfsSEA-1, Pfs45/48, Pfs SEA-1, or a combination thereof. In one embodiment, the plasmodium antigen is selected from CPBAgl, AgAPN1, SGS, or a combination thereof. In one embodiment, the plasmodium antigen is a liver stage antigen selected from LSA1, LSA3, SALSA, or a combination thereof. The nucleic acid sequences of many Plasmodium antigens are published and are available from a variety of sources, including, e.g., GenBank and PubMed. In certain embodiments, the one or more genes encodes a polypeptide, or fragment thereof, that is substantially identical (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% identical) to the selected Plasmodium antigen over at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 contiguous residues of the selected Plasmodium antigen or immunogenic fragment thereof that retain immunogenic activity. In certain embodiments, the polypeptide, or the nucleic acid sequence encoding the polypeptide, may have a mutation or deletion (e.g., an internal deletion, truncation of the amino- or carboxy-terminus, or a point mutation). III. Recombinant Viral Vectors In one aspect, the present invention is a recombinant viral vector comprising one or more nucleic acid sequences encoding Plasmodium antigens or immunogenic fragments thereof. In certain embodiments, the recombinant viral vector is a vaccinia viral vector, and more particularly, an MVA vector, comprising one or more nucleic acid sequences encoding Plasmodium antigens or immunogenic fragments thereof. Vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al 1982 PNAS USA 79:7415-7419; Smith, G. L. et al. 1984 Biotech Genet Engin Rev 2:383-407). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 110,385). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells. Several such strains of vaccinia virus have been developed to avoid undesired side effects of smallpox vaccination. Thus, a modified vaccinia Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A et al. 1975 Infection 3:6-14; Swiss Patent No. 568,392). The MVA virus is publicly available from American Type Culture Collection as ATCC No.: VR-1508. MVA is distinguished by its great attenuation, as demonstrated by diminished virulence and reduced ability to replicate in primate cells, while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the parental CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The resulting MVA virus became severely host cell restricted to avian cells. Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr A. et al. 1978 Zentralbl Bakteriol [B] 167:375-390; Stickl et al. 1974 Dtsch Med Wschr 99:2386-2392). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851). Additionally, novel vaccinia vector vaccines were established on the basis of MVA having foreign DNA sequences inserted at the site of deletion III within the MVA genome (Sutter, G. et al. 1994 Vaccine 12:1032-1040). Recombinant MVA vaccinia viruses can be prepared as set out in PCT publication WO2017/120577 incorporated by reference herein. A DNA-construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a predetermined insertion site (e.g., between two conserved essential MVA genes such as I8R/G1L; in restructured and modified deletion III; or at other non-essential sites within the MVA genome) is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker. The DNA-construct to be inserted can be linear or circular. A plasmid or polymerase chain reaction product is preferred. Such methods of making recombinant MVA vectors are described in PCT publication WO/2006/026667 incorporated by reference herein. The DNA-construct contains sequences flanking the left and the right side of a naturally occurring deletion. The foreign DNA sequence is inserted between the sequences flanking the naturally occurring deletion. For the expression of a DNA sequence or gene, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385). The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J. 1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by means of spheroplasts (Schaffher 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art. The MVA vectors described in WO2017/120577 are immunogenic after a single prime or a homologous prime/boost regimen. Other MVA vector designs require a heterologous prime/boost regimen while still other published studies have been unable to induce effective immune responses with MVA vectors. Conversely, these MVA vector are useful in eliciting effective T-cell and antibody immune responses. Furthermore, the utility of an MVA vaccine vector capable of eliciting effective immune responses and antibody production after a single homologous prime boost is significant for considerations such as use, commercialization and transport of materials especially to affected third world locations. In one embodiment, the present invention is a recombinant viral vector (e.g., an MVA vector) comprising one or more nucleic acid sequences encoding Plasmodium antigens or immunogenic fragments thereof. The viral vector (e.g., an MVA vector) may be constructed using conventional techniques known to one of skill in the art. The one or more heterologous gene inserts encode a polypeptide having desired immunogenicity, i.e., a polypeptide that can induce an immune reaction, cellular immunity and/or humoral immunity, in vivo by administration thereof. The gene region of the viral vector (e.g., an MVA vector) where the gene encoding a polypeptide having immunogenicity is introduced is flanked by regions that are indispensable. In the introduction of a gene encoding a polypeptide having immunogenicity, an appropriate promoter may be operatively linked upstream of the gene encoding a polypeptide having desired immunogenicity. In one aspect, the present invention is a composition comprising a) a recombinant modified vaccinia Ankara (MVA) vector comprising a Plasmodium antigen-encoding sequence under the control of a promoter compatible with poxvirus expression systems. In one embodiment, the Plasmodium antigen assembles into virus-like-particles (VLPs) when expressed. In one embodiment, the present invention is a composition comprising a) a recombinant modified vaccinia Ankara (MVA) vector comprising a Plasmodium antigen-encoding sequence and a matrix protein-encoding sequence (matrix protein sequence), wherein both the plasmodium antigen sequence and matrix protein sequence are under the control of promoters compatible with poxvirus expression systems. In one embodiment, the plasmodium antigen is a sporozite stage antigen selected from CSP, TRAP or STARP, or a combination thereof. In one embodiment, the plasmodium antigen is a merozoite stage antigen selected from MSP-1, MSP-2, MSP-3, GLURP, EBA-140, EBA-175, RAP1, RAP2, or AMA-1, or a combination thereof. In one embodiment, the plasmodium antigen is a gametocyte stage antigen selected from Pfs25, Pfs230, PfsSEA-1, Pfs45/48, Pfs SEA-1, or a combination thereof. In one embodiment, the plasmodium antigen is selected from CPBAgl, AgAPN1, SGS, or a combination thereof. In one embodiment, the plasmodium antigen is a liver stage antigen selected from LSA1, LSA3, SALSA, or a combination thereof. In one embodiment, the matrix protein is selected from Marburg virus VP40 matrix protein, Ebola virus VP40 matrix protein, human immunodeficiency virus type 1 (HIV-1) matrix protein, or Lassa virus matrix Z protein. In one embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into the MVA vector in a natural deletion site, a modified natural deletion site, or between essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into the same natural deletion site, a modified natural deletion site, or between the same essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen sequence is inserted into a deletion site selected from I, II, III, IV, V, or VI and the matrix protein sequence is inserted into a deletion site selected from I, II, III, IV, V, or VI. In another embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into different natural deletion sites, different modified deletion sites, or between different essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen is inserted in a first deletion site and matrix protein sequence is inserted into a second deletion site. In one embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into one or more deletion sites of the MVA vector. In one embodiment, the matrix protein is selected from Marburg virus VP40 matrix protein, Ebola virus VP40 matrix protein, human immunodeficiency virus type 1 (HIV-1) matrix protein (Clade A, B or C), or Lassa virus matrix Z protein. In one embodiment, the Ebola virus VP40 matrix protein is selected from Zaire Ebola virus VP40 or Sudan Ebola virus VP40. In one embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into the MVA vector in a natural deletion site, a modified natural deletion site, or between essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into the same natural deletion site, a modified natural deletion site, or between the same essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen sequence is inserted into a deletion site selected from I, II, III, IV, V, or VI and the matrix protein sequence is inserted into a deletion site selected from I, II, III, IV, V, or VI. In another embodiment, the Plasmodium antigen sequence and the matrix protein sequence are inserted into different natural deletion sites, different modified deletion sites, or between different essential or non-essential MVA genes. In another embodiment, the Plasmodium antigen is inserted in a first deletion site and matrix protein sequence is inserted into a second deletion site. In a particular embodiment, the Plasmodium antigen is inserted between two essential and highly conserved MVA genes; and the matrix protein sequence is inserted into a restructured and modified deletion III. In a particular embodiment, the Plasmodium antigen sequence is inserted between two essential and highly conserved MVA genes to limit the formation of viable deletion mutants. In a particular embodiment, the Plasmodium antigen protein sequence is inserted between MVA genes, I8R and G1L. In a particular embodiment, the Plasmodium antigen protein sequence is inserted between MVA genes, A50R and B1R. In a particular embodiment, the matrix protein sequence is inserted between MVA genes, I8R and G1L. In a particular embodiment, the matrix protein sequence is inserted between MVA genes, A50R and B1R. In one embodiment, the Plasmodium antigen protein sequence is expressed with a matrix protein sequence as a fusion protein. In a particular embodiment, the Plasmodium antigen/matrix protein fusion protein sequence is inserted between MVA genes, I8R and G1L. In a particular embodiment, the Plasmodium antigen/matrix protein fusion protein sequence is inserted between MVA genes, A50R and B1R. In one embodiment, the promoter is selected from the group consisting of Pm2H5, Psyn II, and mH5 promoters, or combinations thereof. In one embodiment, the recombinant MVA viral vector expresses a Plasmodium antigen and matrix proteins that assemble into VLPs. In one embodiment, the deletion III site is restructured and modified to remove non-essential flanking sequences. In one embodiment the Plasmodium antigen is CSP and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is MSP-1 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is MSP-2 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is MSP-3 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is GLURP and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is EBA-175 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is Pfs25 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is Pfs230 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is Pfs SEA-1 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is Pfs45/48 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is Pfs SEA-1 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is CPBAgl and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is AgAPN1 and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. In one embodiment the Plasmodium antigen is SGS and the matrix protein is Zaire Ebola VP40, Sudan Ebola VP40, Marburg virus VP40, Lassa virus Z protein, or HIV Gag protein. The one or more genes introduced into the recombinant viral vector are under the control of regulatory sequences that direct its expression in a cell. The nucleic acid material of the viral vector may be encapsulated, e.g., in a lipid membrane or by structural proteins (e.g., capsid proteins), that may include one or more viral polypeptides. In exemplary embodiments, the present invention is a recombinant viral vector (e.g., a recombinant MVA vector) comprising one or more genes, or one or more polypeptides encoded by the gene or genes, from a Plasmodium spp. In one embodiment, the sequence encoding a Plasmodium antigen or immunogenic fragment thereof is inserted into deletion site I, II, III, IV, V, or VI of the MVA vector. In one embodiment, the sequence encoding a Plasmodium antigen or immunogenic fragment thereof is inserted between I8R and G1L of the MVA vector, or into restructured and modified deletion III of the MVA vector; and a second sequence encoding a Plasmodium antigen or immunogenic fragment thereof is inserted between 18R and G1L of the MVA vector, or into restructured and modified deletion site III of the MVA vector. In one embodiment, the recombinant vector comprises in a first deletion site, a nucleic acid sequence encoding a Plasmodium antigen or immunogenic fragment thereof operably linked to a promoter compatible with poxvirus expression systems, and in a second deletion site, a nucleic acid sequence encoding a VLP-forming protein operably linked to a promoter compatible with poxvirus expression systems. In exemplary embodiments, the present invention is a recombinant MVA vector comprising at least one heterologous nucleic acid sequence (e.g., one or more sequences) encoding a Plasmodium antigen or immunogenic fragment thereof which is under the control of regulatory sequences that direct its expression in a cell. The sequence may be, for example, under the control of a promoter selected from the group consisting of Pm2H5, Psyn II, or mH5 promoters. The recombinant viral vector of the present invention can be used to infect cells of a subject, which, in turn, promotes the translation into a protein product of the one or more heterologous sequence of the viral vector (e.g., a Plasmodium antigen or immunogenic fragment thereof). As discussed further herein, the recombinant viral vector can be administered to a subject so that it infects one or more cells of the subject, which then promotes expression of the one or more viral genes of the viral vector and stimulates an immune response that is therapeutic or protective against malaria. In one embodiment, the recombinant MVA vaccine expresses proteins that assemble into virus-like particles (VLPs) comprising the Plasmodium antigen or immunogenic fragment thereof. While not wanting to be bound by any particular theory, it is believed that the Plasmodium antigen is provided to elicit a protective immune response and the matrix protein is provided to enable assembly of VLPs and as a target for T cell immune responses, thereby enhancing the protective immune response and providing cross-protection. In one embodiment, the matrix protein is a Marburg virus matrix protein. In one embodiment, the matrix protein is an Ebola virus matrix protein. In one embodiment, the matrix protein is a Sudan virus matrix protein. In one embodiment, the matrix protein is a human immunodeficiency virus type 1 (HIV-1) matrix protein. In one embodiment, the matrix protein is a human immunodeficiency virus type 1 (HIV-1) matrix protein encoded by the gag gene. In one embodiment, the matrix protein is a Lassa virus matrix protein. In one embodiment, the matrix protein is a Lassa virus Z protein. In one embodiment, the matrix protein is a fragment of a Lassa virus Z protein. In one embodiment, the matrix protein is a matrix protein of a virus in the Filoviridae virus family. In one embodiment, the matrix protein is a matrix protein of a virus in the Retroviridae virus family. In one embodiment, the matrix protein is a matrix protein of a virus in the Arenaviridae virus family. In one embodiment, the matrix protein is a matrix protein of a virus in the Flaviviridae virus family. One or more nucleic acid sequences may be optimized for use in an MVA vector. Optimization includes codon optimization, which employs silent mutations to change selected codons from the native sequences into synonymous codons that are optimally expressed by the host-vector system. Other types of optimization include the use of silent mutations to interrupt homopolymer stretches or transcription terminator motifs. Each of these optimization strategies can improve the stability of the gene, improve the stability of the transcript, or improve the level of protein expression from the sequence. In exemplary embodiments, the number of homopolymer stretches in the Plasmodium antigen sequence will be reduced to stabilize the construct. A silent mutation may be provided for anything similar to a vaccinia termination signal. An extra nucleotide may be added in order to express the transmembrane, rather than the secreted, form of any Plasmodium antigen. In exemplary embodiments, the sequences are codon optimized for expression in MVA; sequences with runs of ≥5 deoxyguanosines, ≥5 deoxycytidines, ≥5 deoxyadenosines, and ≥5 deoxythymidines are interrupted by silent mutation to minimize loss of expression due to frame shift mutations; and the GP sequence is modified through addition of an extra nucleotide to express the transmembrane, rather than the secreted, form of the protein. In one embodiment, the present invention provides a vaccine vector composition that is monovalent. As used herein the term monovalent refers to a vaccine vector composition that contains sequences from one Plasmodium antigen. In another embodiment, the present invention provides a vaccine that is bivalent. As used herein the term bivalent refers to a vaccine vector composition that contains two vectors having sequences from different Plasmodium antigens. In another embodiment, the present invention provides a vaccine that is trivalent. As used herein the term trivalent refers to a vaccine vector composition that contains three vectors having sequences from different Plasmodium antigens. In another embodiment, the present invention provides a vaccine that is quadrivalent. As used herein the term quadrivalent refers to a vaccine vector composition that contains four vectors having sequences from different Plasmodium antigens. As used herein, the terms tetravalent and quadrivalent are synonymous. The present invention also extends to host cells comprising the recombinant viral vector described above, as well as isolated virions prepared from host cells infected with the recombinant viral vector. IV. Pharmaceutical Composition The recombinant viral vectors or immunogenic peptides described herein are readily formulated as pharmaceutical compositions for veterinary or human use, either alone or in combination. The pharmaceutical composition may comprise a pharmaceutically acceptable diluent, excipient, carrier, or adjuvant. In one embodiment, the present invention is a vaccine effective to protect and/or treat malaria comprising a recombinant MVA vector that expresses at least one Plasmodium antigen (e.g., a Plasmodium antigen) or an immunogenic fragment thereof. The vaccine composition may comprise one or more additional therapeutic agents. The pharmaceutical composition may comprise 1, 2, 3, 4 or more than 4 different recombinant MVA vectors described herein. In one embodiment, the present invention provides a vaccine vector composition that is monovalent. As used herein the term monovalent refers to a vaccine vector composition that contains one Plasmodium antigen. In another embodiment, the present invention provides a vaccine that is bivalent. As used herein the term bivalent refers to a vaccine vector composition that contains two vectors having sequences from different Plasmodium antigens. In another embodiment, the present invention provides a vaccine that is trivalent. As used herein the term trivalent refers to a vaccine vector composition that contains three vectors having sequences from different Plasmodium antigens. In another embodiment, the present invention provides a vaccine that is quadrivalent. As used herein the term quadrivalent refers to a vaccine vector composition that contains four vectors having sequences from different Plasmodium antigens. As used herein, the terms tetravalent and quadrivalent are synonymous. As used herein, the phrase “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as those suitable for parenteral administration, such as, for example, by intramuscular, intraarticular (in the joints), intravenous, intradermal, intraperitoneal, and subcutaneous routes. Examples of such formulations include aqueous and non-aqueous, isotonic sterile injection solutions, which contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable diluents, excipients, carriers, or adjuvants and their formulations are known to those skilled in the art. In one embodiments, adjuvants are used as immune response enhancers. In various embodiments, the immune response enhancer is selected from the group consisting of alum-based adjuvants, oil based adjuvants, Specol, RIBI, TiterMax, Montanide 1SA50 or Montanide ISA 720, GM-CSF, nonionic block copolymer-based adjuvants, dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants AS-1, AS-2, Ribi Adjuvant system based adjuvants, QS21, Quil A, SAF (Syntex adjuvant in its microfluidized form (SAF-m), dimethyl-dioctadecyl ammonium bromide (DDA), human complement based adjuvants m. vaccae, ISCOMS, MF-59, SBAS-2, SBAS-4, Enhanzyn®, RC-529, AGPs, MPL-SE, QS7, Escin; Digitonin; and Gypsophila, Chenopodium quinoa saponins. The compositions utilized in the methods described herein can be administered by a route selected from, e.g., parenteral, intramuscular, intraarterial, intravascular, intravenous, intraperitoneal, subcutaneous, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, topical administration, and oral administration. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated). Formulations suitable for oral administration may consist of liquid solutions, such as an effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets or tablets, each containing a predetermined amount of the vaccine. The pharmaceutical composition may also be an aerosol formulation for inhalation, e.g., to the bronchial passageways. Aerosol formulations may be mixed with pressurized, pharmaceutically acceptable propellants (e.g., dichlorodifluoromethane, propane, or nitrogen). For the purposes of this invention, pharmaceutical compositions suitable for delivering a therapeutic or biologically active agent can include, e.g., tablets, gelcaps, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Any of these formulations can be prepared by well-known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21sted.), ed. A R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, lnforma Healthcare, 2006, each of which is hereby incorporated by reference. The immunogenicity of the composition (e.g., vaccine) may be significantly improved if the composition of the present invention is co-administered with an immunostimulatory agent or adjuvant. Suitable adjuvants well-known to those skilled in the art include, e.g., aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM-Matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof. Pharmaceutical compositions according to the invention described herein may be formulated to release the composition immediately upon administration (e.g., targeted delivery) or at any predetermined time period after administration using controlled or extended release formulations. Administration of the pharmaceutical composition in controlled or extended release formulations is useful where the composition, either alone or in combination, has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, Tl, is defined as the ratio of median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain a therapeutic level. Many strategies can be pursued to obtain controlled or extended release in which the rate of release outweighs the rate of metabolism of the pharmaceutical composition. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Suitable formulations are known to those of skill in the art. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the vaccine dissolved in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the vaccine, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; (d) suitable emulsions; and (e) polysaccharide polymers such as chitins. The vaccine, alone or in combination with other suitable components, may also be made into aerosol formulations to be administered via inhalation, e.g., to the bronchial passageways. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Suitable formulations for rectal administration include, for example, suppositories, which consist of the vaccine with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the vaccine with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons. The vaccines of the present invention may also be co-administered with cytokines to further enhance immunogenicity. The cytokines may be administered by methods known to those skilled in the art, e.g., as a nucleic acid molecule in plasmid form or as a protein or fusion protein. This invention also provides kits comprising the vaccines of the present invention. For example, kits comprising a vaccine and instructions for use are within the scope of this invention. V. Method of Use The compositions of the invention can be used as vaccines for inducing an immune response to a Plasmodium antigen. In exemplary embodiments, the present invention provides a method of inducing an immune response to a Plasmodium antigen in a subject in need thereof, said method comprising administering a recombinant viral vector that encodes at least one Plasmodium antigen or immunogenic fragment thereof to the subject in an effective amount to generate an immune response to a Plasmodium antigen. The result of the method is that the subject is partially or completely immunized against the Plasmodium antigen. In one embodiment, invention provides methods for activating an immune response in a subject using the compositions described herein. In some embodiments, the invention provides methods for promoting an immune response in a subject using a composition described herein. In some embodiments, the invention provides methods for increasing an immune response in a subject using a composition described herein. In some embodiments, the invention provides methods for enhancing an immune response in a subject using a composition described herein. In exemplary embodiments, the present invention provides a method of treating, reducing, preventing or delaying malaria in a subject in need thereof, said method comprising administering the composition of the present invention to the subject in a therapeutically effective amount a recombinant MVA described herein. The result of treatment is a subject that has an improved therapeutic profile for malaria. In exemplary embodiments, the present invention provides a method of treating malaria in a subject in need thereof, said method comprising administering the composition of the present invention to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profile for malaria. In one embodiment, the immune response is a humoral immune response, a cellular immune response or a combination thereof. In a particular embodiment, the immune response comprises production of binding antibodies against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of neutralizing antibodies against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of non-neutralizing antibodies against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of a cell-mediated immune response against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of neutralizing and non-neutralizing antibodies against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of neutralizing antibodies and cell-mediated immunity against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of non-neutralizing antibodies and cell-mediated immunity against the Plasmodium antigen. In a particular embodiment, the immune response comprises production of neutralizing antibodies, non-neutralizing antibodies, and cell-mediated immunity against the Plasmodium antigen. In certain embodiments, the compositions of the invention can be used as vaccines for treating a subject at risk of developing malaria, or a subject already having malaria. The recombinant viral vector comprises genes or sequences encoding Plasmodium antigens, viral proteins to promote assembly of virus-like particles (VLPs) or additional enzymes to facilitate expression and glycosylation of the Plasmodium antigen. Typically, the vaccines will be in an admixture and administered simultaneously, but may also be administered separately. A subject to be treated according to the methods described herein may be one who has been diagnosed by a medical practitioner as having such a condition. (e.g., a subject having malaria). Diagnosis may be performed by any suitable means. One skilled in the art will understand that a subject to be treated according to the present invention may have been identified using standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors. Prophylactic treatment may be administered, for example, to a subject not yet having malaria but who is susceptible to, or otherwise at risk of developing malaria. Therapeutic treatment may be administered, for example, to a subject already having malaria in order to improve or stabilize the subject's condition. The result is an improved therapeutic profile. In some instances, as compared with an equivalent untreated control, treatment may ameliorate a disorder or a symptom thereof by, e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as measured by any standard technique. In other embodiments, treatment may result in amelioration of one or more symptoms of malaria. According to this embodiment, confirmation of treatment can be assessed by detecting an improvement in or the absence of symptoms. In one embodiment, the present invention is a method of inducing an immune response in a subject (e.g., a human) by administering to the subject a recombinant viral vector that encodes at least one Plasmodium antigen or immunogenic fragment thereof. The immune response may be a cellular immune response or a humeral immune response, or a combination thereof. The composition may be administered, e.g., by injection (e.g., intramuscular, intraarterial, intravascular, intravenous, intraperitoneal, or subcutaneous). It will be appreciated that more than one route of administering the vaccines of the present invention may be employed either simultaneously or sequentially (e.g., boosting). In addition, the vaccines of the present invention may be employed in combination with traditional immunization approaches such as employing protein antigens, vaccinia virus and inactivated virus, as vaccines. Thus, in one embodiment, the vaccines of the present invention are administered to a subject (the subject is “primed” with a vaccine of the present invention) and then a traditional vaccine is administered (the subject is “boosted” with a traditional vaccine). In another embodiment, a traditional vaccine is first administered to the subject followed by administration of a vaccine of the present invention. In yet another embodiment, a traditional vaccine and a vaccine of the present invention are co-administered. While not to be bound by any specific mechanism, it is believed that upon inoculation with a pharmaceutical composition as described herein, the immune system of the host responds to the vaccine by producing antibodies, both secretory and serum, specific for one or more Plasmodium antigen or immunogenic fragments thereof; and by producing a cell-mediated immune response specific for one or more Plasmodium antigen or immunogenic fragments thereof. As a result of the vaccination, the host becomes at least partially or completely immune to one or more Plasmodium antigen or immunogenic fragments thereof, or resistant to developing moderate or severe diseases caused by malaria. In one aspect, methods are provided to alleviate, reduce the severity of, or reduce the occurrence of, one or more of the symptoms associated with malaria comprising administering an effective amount of a pharmaceutical composition comprising a recombinant MVA viral vector that comprises Plasmodium antigen and matrix protein sequences optionally co-expressing sequences that facilitate expression of and desired glycosylation the Plasmodium antigen. In another aspect, the invention provides methods of providing anti-Plasmodium antigen immunity comprising administering an effective amount of a pharmaceutical composition comprising a recombinant MVA vaccine expressing Plasmodium antigen and a viral matrix protein to permit the formation of VLPs. It will also be appreciated that single or multiple administrations of the vaccine compositions of the present invention may be carried out. For example, subjects who are at particularly high risk of malaria may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored by measuring amounts of binding and neutralizing secretory and serum antibodies as well as levels of T cells, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. In one embodiment, administration is repeated at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or more than 8 times. In one embodiment, administration is repeated twice. In one embodiment, about 2-8, about 4-8, or about 6-8 administrations are provided. In one embodiment, about 1-4-week, 2-4 week, 3-4 week, 1 week, 2 week, 3 week, 4 week, or more than 4 week intervals are provided between administrations. In one specific embodiment, a 4-week interval is used between 2 administrations. In one embodiment, the invention provides a method of monitoring treatment progress. In exemplary embodiments, the monitoring is focused on biological activity, immune response, and/or clinical response. In one embodiment, the biological activity is a T-cell immune response, regulatory T-cell activity, or molecule response (MRD). In one embodiment, immune response is monitored for example, by an immune assay such as a cytotoxicity assay, an intracellular cytokine assay, a tetramer assay, or an ELISPOT assay. In one embodiment, upon improvement of a subject's condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. A. Dosage The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the immune system of the individual to synthesize antibodies, and, if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be monitored on a patient-by-patient basis. However, suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 5.0×106TCID50to about 5.0×109TCID50. The dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight, and the nature of the formulation. The pharmaceutical compositions of the invention are administered in such an amount as will be therapeutically effective, immunogenic, and/or protective against a Plasmodium antigen. The dosage administered depends on the subject to be treated (e.g., the manner of administration and the age, body weight, capacity of the immune system, and general health of the subject being treated). The composition is administered in an amount to provide a sufficient level of expression that elicits an immune response without undue adverse physiological effects. Preferably, the composition of the invention is a heterologous viral vector that includes one or Plasmodium antigens or immunogenic fragments thereof and large matrix protein; and is administered at a dosage of, e.g., between 1.0×104and 9.9×1012TCID50of the viral vector, preferably between 1.0×105TCID50and 1.0×1011TCID50pfu, more preferably between 1.0×106and 1.0×1010TCID50pfu, or most preferably between 5.0×106and 5.0×109TCID50. The composition may include, e.g., at least 5.0×106TCID50of the viral vector (e.g., 1.0×108TCID50of the viral vector). A physician or researcher can decide the appropriate amount and dosage regimen. The composition of the method may include, e.g., between 1.0×104and 9.9×1012TCID50of the viral vector, preferably between 1.0×105TCID50and 1.0×1011TCID50pfu, more preferably between 1.0×106and 1.0×1010TCID50pfu, or most preferably between 5.0×106and 5.0×109TCID50. The composition may include, e.g., at least 5.0×106TCID50of the viral vector (e.g., 1.0×108TCID50of the viral vector). The method may include, e.g., administering the composition to the subject two or more times. The term “effective amount” is meant the amount of a composition administered to improve, inhibit, or ameliorate a condition of a subject, or a symptom of a disorder, in a clinically relevant manner (e.g., improve, inhibit, or ameliorate disease associated with malaria or provide an effective immune response to a Plasmodium antigen). Any improvement in the subject is considered sufficient to achieve treatment. Preferably, an amount sufficient to treat is an amount that prevents the occurrence or one or more symptoms of disease associated with malaria or is an amount that reduces the severity of, or the length of time during which a subject suffers from, one or more symptoms of malaria (e.g., by at least 10%, 20%, or 30%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 80%, 90%, 95%, 99%, or more, relative to a control subject that is not treated with a composition of the invention). A sufficient amount of the pharmaceutical composition used to practice the methods described herein (e.g., the prevention or treatment of malaria) varies depending upon the manner of administration and the age, body weight, and general health of the subject being treated. It is important to note that the value of the present invention may never be demonstrated in terms of actual clinical benefit. Instead, it is likely that the value of the invention will be demonstrated in terms of success against a surrogate marker for protection. For an indication such as malaria, in which it is impractical or unethical to attempt to measure clinical benefit of an intervention, the FDA's Accelerated Approval process allows approval of a new vaccine based on efficacy against a surrogate endpoint. Therefore, the value of the invention may lie in its ability to induce an immune response that constitutes a surrogate marker for protection. Similarly, FDA may allow approval of vaccines against Plasmodium antigens based on its Animal Rule. In this case, approval is achieved based on efficacy in animals. The composition of the method may include, e.g., between 1.0×104and 9.9×1012TCID50of the viral vector, preferably between 1.0×105TCID50and 1.0×1011TCID50pfu, more preferably between 1.0×106and 1.0×1010TCID50pfu, or most preferably between 5.0×106and 5.0×109TCID50. The composition may include, e.g., at least 5.0×106TCID50of the viral vector (e.g., 1.0×108TCID50of the viral vector). The method may include, e.g., administering the composition two or more times. In some instances it may be desirable to combine the Plasmodium antigen vaccines of the present invention with vaccines which induce protective responses to other agents, particularly other Plasmodium antigens. For example, the vaccine compositions of the present invention can be administered simultaneously, separately or sequentially with other genetic immunization vaccines such as those for influenza (Ulmer, J. B. et al., Science 259:1745-1749 (1993); Raz, E. et al., PNAS (USA) 91:9519-9523 (1994)), malaria (Doolan, D. L. et al., J. Exp. Med. 183:1739-1746 (1996); Sedegah, M. et al., PNAS (USA) 91:9866-9870 (1994)), and tuberculosis (Tascon, R. C. et al., Nat. Med. 2:888-892 (1996)). B. Administration As used herein, the term “administering” refers to a method of giving a dosage of a pharmaceutical composition of the invention to a subject. The compositions utilized in the methods described herein can be administered by a route selected from, e.g., parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical administration, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intraarterial, intravascular, and intramuscular administration. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered, and the severity of the condition being treated). Administration of the pharmaceutical compositions (e.g., vaccines) of the present invention can be by any of the routes known to one of skill in the art. Administration may be by, e.g., intramuscular injection. The compositions utilized in the methods described herein can also be administered by a route selected from, e.g., parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical administration, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The preferred method of administration can vary depending on various factors, e.g., the components of the composition being administered, and the severity of the condition being treated. In addition, single or multiple administrations of the compositions of the present invention may be given to a subject. For example, subjects who are particularly susceptible to developing malaria may require multiple treatments to establish and/or maintain protection against a Plasmodium antigen. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, e.g., measuring amounts of neutralizing secretory and serum antibodies. The dosages may then be adjusted or repeated as necessary to maintain desired levels of protection against malaria or to reduce symptoms of malaria. Increased vaccination efficacy can be obtained by timing the administration of the vector. Any of the priming and boosting compositions described above are suitable for use with the methods described here. In one embodiment, MVA vectors are used for both priming and boosting purposes. Such protocols include but are not limited to MM, MMM, and MMMM. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more than ten MVA boosts are administered. Vectors can be administered alone (i.e., a plasmid can be administered on one or several occasions with or without an alternative type of vaccine formulation (e.g., with or without administration of protein or another type of vector, such as a viral vector) and, optionally, with an adjuvant or in conjunction with (e.g., prior to) an alternative booster immunization (e.g., a live-vectored vaccine such as a recombinant modified vaccinia Ankara vector (MVA)) comprising an insert that may be distinct from that of the “prime” portion of the immunization or may be a related vaccine insert(s). For example, GM-CSF or other adjuvants known to those of skill in the art. The adjuvant can be a “genetic adjuvant” (i.e., a protein delivered by way of a DNA sequence). In exemplary embodiments, the present invention is an immunization method comprising (i) administering a priming composition comprising a DNA plasmid comprising one or more sequences encoding a Plasmodium antigen or immunogenic fragment thereof; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding a Plasmodium antigen or immunogenic fragment thereof; and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose, more particularly between about 14 and about 18 weeks after the first dose, even more particularly, about 16 weeks after the first dose. In a particular embodiment, the Plasmodium antigens are the same in step (i)-(iii). Optionally, the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition). The claimed invention is further described by way of the following non-limiting examples. Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures. EXAMPLES Example 1. Generating an Immune Response to CSP using MVA Vectors Expressing CSP-VLPs Overview Swiss mice were vaccinated with one of four constructs (N=5 per group) that contained CSP with 21 NANP-repeats (CSP21R) or a truncated CSP that had no repeats (CSPNR):1. MVA-CSP21R2. MVA-CSPNR3. MVA-VP40m.CSPNR4. MVA-Gag.CSPNR Methods Sera were tested by ELISA against the following antigens: recombinant full length CSP (Genova), (NANP)15peptide (Life Tein), and truncated CSP lacking the central repeat region (N+C, in-house). Samples were tested in duplicate, and corrected for background reactivity. Results Sera from all vaccine groups (N=20) were tested between 1/100-1/64000 dilution for anti-CSP IgG. Only group 3 (MVA-CSP)21R had detectable reactivity against full length CSP, which was relatively comparable to control mice vaccinated with recombinant CSP+Alum (FIG.1). IgG-reactivity was measured to a peptide representative of the central NANP repeat region of CSP. Again, strong reactivity was only detected in mouse serum of MVA-CSP21R, and in control mice vaccinated with recombinant CSP. A truncated CSP construct was expressed that contains the flanking regions of CSP only, termed N+C. To confirm this construct did not contain the central repeat region, it was tested using mouse anti-NANP MAbs against CSP-N+C, as well as full 25 length CSP and NANP-peptide as controls (FIG.2). Two antibody clones (2H8 and 3C1) demonstrated strong IgG-reactivity to full-length CSP and a NANP repeat polymer, but not against CSP-N+C. Furthermore, rabbits were vaccinated with the CSP-N+C and tested the purified polyclonal rabbit anti-N+C IgG by ELISA. A strong signal was observed to full length CSP and CSP-N+C, but not to the NANP peptide. Taken together, these data confirm the truncated N+C construct does not contain the central NANP repeat region. Therefore group 3 was vaccinated with CSP21R. To confirm that the CSP21R construct generates high levels of antibodies, repeat immunizations of mice are performed with this construct together with a new construct, VP4O-CSP21R. Given that only the mice vaccinated with CSP21R had detectable IgG responses, this group was further tested for IgG subclass reactivity to CSP at 1/1000 dilution (FIG.4). There were high levels IgG1, and variable levels of IgG2a, IgG2b and IgG3. The MVA-vaccinated mice showed a more favourable IgG subclass profile with a greater ratio of IgG2a, 2b, and IgG3 compared to IgG1, whereas CSP+Alum was strongly IgG1 skewed. Sera were then tested for the ability to fix human complement using our recently optimised assays (Kurtovic L. et al, BMC Medicine 2018), and the ability to interact with human Fc-receptors, FcγRIIa and FcγRIIIa (FIG.4). Strong levels of C1q-fixation were observed, particularly by in sera from animal #25 that had low levels of IgG1 (which cannot activate complement) and the highest level of IgG2a, although low levels of IgG2b and IgG3. This was followed by sera from animal #24 that notably had very high levels of IgG2b and IgG3, and moderate IgG1. Strong FcγRIIa binding was only apparent in sera from animal #25, which also had the highest signal for FcγRIIIa binding although this was relatively low overall. It should be noted that human Fc-receptors were used in these assays, so the results should only be used to indicate potential activity. Further studies immunize rabbits to generate Abs for functional assays, as rabbit IgG is similar to human IgG1 and can fix human complement and bind human Fc-receptors. Additional CSP-Based Vaccine 1. Immunize mice with VP4O-CSP21R construct and compare with mice with initial VP40-CSP21R versus CSP21R construct2. Repeat evaluation of IgG subclasses and functional activity with all mice3. Evaluate Ab longevity (extend time-points)4. Immunize rabbits to enable detailed studies of functional Ab activity5. Perform prime-boost protocols with MVA and protein vaccines such as GSK CSP VLP or other VLPs or subunit proteins or fragments thereof6. Test protection in a mouse model using genetically modified parasites that express Pf-CSP. Example 2: Generating an immune response to Pfs-230 using MVA vectors expressing Pfs-230-VLPs Overview Swiss mice are vaccinated with one of four constructs (N=5 per group) that contained Pfs-230:1. MVA-Pfs2302. MVA-VP40m.Pfs230 Methods Sera are tested by ELISA against the following antigens: recombinant full length Pfs230. Samples are tested in duplicate and corrected for background reactivity. Results Sera from all vaccine groups (N=20) are tested between 1/100-1/64000 dilution for anti-Pfs230 IgG. Sera are then tested for the ability to fix human complement using recently optimised assays (Kurtovic L et al, BMC Medicine 2018), and the ability to interact with human Fc-receptors, FcγRIIa and FcγRIIIa (FIG.4). Immunization Studies 1. Immunize mice with VP40-Pfs230 vector construct2. Repeat evaluation of IgG subclasses and functional activity with all mice3. Evaluate Ab longevity (extend time-points)4. Immunize rabbits to enable detailed studies of functional Ab activity5. Perform prime-boost protocols with MVA and protein vaccines. MVA and protein (plus adjuvant) are administered at the same time in the same anatomical sites to generate an immune response in the same draining lymph nodes. Vector and or proteins are administered on Day 1 to prime and then on Day 28 to boost the primed immune response6. Test protection in a mouse model using genetically modified parasites that express Pfs230 The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. All references cited herein are incorporated by reference in their entirety. | 79,633 |
11857612 | DETAILED DESCRIPTION Embodiments of the invention are directed to inducing and/or enhancing an immune response to neoplastic diseases. BCG therapy of bladder cancer results in extensive activation of the immune system. Within hours of BCG instillation, a marked increase in the number of leukocytes in the urine can be detected. These leukocytes consist mainly of granulocytes and, to a lesser degree, of macrophages and lymphocytes (De Boer, E. C. et al. Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guerin.Cancer Immunol. Immunother.33, 411-416 (1991)). Similarly, an influx of immune cells can be found in the bladder wall after BCG therapy (Bohle, A. et al., Effects of local bacillus Calmette-Guerin therapy in patients with bladder carcinoma on immunocompetent cells of the bladder wall.J. Urol,144, 53-58 (1990)). Additional evidence of immune activation is the release of a wide variety of cytokines and chemokines into the urine following BCG therapy (Redelman-Sidi G. et al.,Nat Rev Urol.2014 March; 11(3):153-62). Histopathologically, post-treatment bladder biopsies in patients treated with BCG reveal erosion of the superficial epithelium, and submucosal granulomatous inflammation, with oedema and non caseating granulomas surrounded by a lymphoplasmacytic and eosinophilic infiltrate (Lage, J. M. et al. Histological parameters and pitfalls in the interpretation of bladder biopsies in bacillus Calmette-Guerin treatment of superficial bladder cancer.J. Urol.135, 916-919 (1986)). The majority of lymphocytes in the urine of patients treated with BCG are T cells, most of which are CD4+(De Boer, E. C. et al. Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guerin.Cancer Immunol. Immunother.33, 411-416 (1991)). T cells, again mostly CD4+, can also be found infiltrating the bladder mucosa for months after BCG therapy (Bohle, A. et al.,J. Urol.144, 53-58 (1990); Boccafoschi, C. et al. Immunophenotypic characterization of the bladder mucosa infiltrating lymphocytes after intravesical BCG treatment for superficial bladder carcinoma.Eur. Urol.21, 304-308 (1992)). Although CD4+lymphocytes predominate, both CD4+and CD8+lymphocytes seem to be required for effective BCG therapy. The cytotoxicity of BCG-specific NK cells, which have also been termed BCG-activated killer (BAK) cells (Brandau, S. & Bohle, A. Activation of natural killer cells by Bacillus Calmette-Guerin.Eur. Urol.39, 518-524 (2001)), can be enhanced by IL-12 and interferon (IFN)-γ, and is inhibited by IL-1 (Suttmann, H. et al. Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation.J. Urol.172, 1490-1495 (2004)). Killing of bladder cancer cells by BAK cells seems to involve perforin, which is a cytolytic protein that is released from granules and forms a pore in the plasma membrane of the target cell (Brandau, S. et al. Perforin-mediated lysis of tumor cells byMycobacterium bovis BacillusCalmette-Guerin-activated killer cells.Clin. Cancer Res.6, 3729-3738 (2000)). Dendritic cells have been postulated to initiate activation of T cells after BCG administration. Although some evidence from in vitro studies supports this hypothesis, a role for dendritic cells in response to BCG has not been clearly defined. Immature dendritic cells have been identified in the urine of patients with bladder cancer who were treated with BCG (Beatty, J. D. et al. Urine dendritic cells: a noninvasive probe for immune activity in bladder cancer?BJU Int.94, 1377-1383 (2004)), and in vitro, dendritic cells that were exposed to BCG can activate NK cells and γδ T cells, and induce their cytotoxicity against BCG-infected bladder cancer cells (Naoe, M. et al. Bacillus Calmette-Guerin-pulsed dendritic cells stimulate natural killer T cells and gamma delta T cells.Int. J. Urol.14, 532-538 (2007); Higuchi, T. et al. A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth.Cancer Immunol. Immunother.58, 1245-1255 (2009)). Similar to the finding for tumor-associated macrophages, patients with high levels of tumor-associated dendritic cells prior to BCG treatment were more likely to experience cancer recurrence after BCG therapy (Ayari, C. et al. Bladder tumor infiltrating mature dendritic cells and macrophages as predictors of response to Bacillus Calmette-Guerin immunotherapy.Eur. Urol.55, 1386-1395 (2009)). As is the case with tumor-associated macrophages, this finding might be explained by immunosuppression induced by specific subsets of tumor-associated dendritic cells (Hurwitz, A. A. & Watkins, S. K. Immune suppression in the tumor microenvironment: a role for dendritic cell-mediated tolerization of T cells.Cancer Immunol. Immunother.61, 289-293 (2012)). BCG therapy is followed by a massive release of cytokines into the urine of treated patients. These cytokines include IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (De Boer, E. C. et al.Cancer Immunol. Immunother.34, 306-312 (1992); Eto, M. et al. Importance of urinary interleukin-18 in intravesical immunotherapy with Bacillus Calmette-Guerin for superficial bladder tumors.Urol. Int.75, 114-118 (2005)), as well as the chemokines macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, MIP-la, and interferon-inducible protein (IP)-10 (Luo, Y., et al.Mycobacterium bovis bacillusCalmette-Guerin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo. Clin. Exp. Immunol. 147, 370-378 (2007)). Although the array of cytokines found in the urine of patients treated with BCG cannot be strictly categorized as corresponding to a TH1 or TH2 response, the presence of IL-2, IL-12, and IFN-γ, and the absence of IL-4, are more consistent with a TH1-like response. Another cytokine that has been evaluated in response to BCG is TRAIL, a member of the TNF family that is expressed by various immune cells, including cytotoxic lymphocytes, NK cells, and neutrophils. In addition to the local inflammatory response in the bladder wall, BCG therapy induces a systemic immune response. More than 40% of patients receiving intra-vesical BCG instillation experience conversion of a previously negative tuberculin skin test (Kelley, D. R. et al. Prognostic value of purified protein derivative skin test and granuloma formation in patients treated with intravesical bacillus Calmette-Guerin.J. Urol.135, 268-271 (1986)). Furthermore, patients treated with BCG have increased serum levels of IL-2 and IFN-γ, and peripheral blood mononuclear cells in patients who have received repeated instillations of BCG exhibit increased killing activity against an NK-cell resistant cancer cell line, compared with before BCG treatment (Taniguchi, K. et al. Systemic immune response after intravesical instillation of bacillus Calmette-Guerin (BCG) for superficial bladder cancer. Clin. Exp. Immunol. 115, 131-135 (1999)). The immune response to BCG is preceded by an interaction between BCG and urothelial cells, which is essential to achieving antitumor activity. The initial step is attachment of BCG to urothelial cells. This step is facilitated by fibronectin, a glycoprotein that is part of the extracellular matrix and that can also be found in urine in a soluble form. BCG attaches to fibronectin through its fibronectin attachment protein (FAP) (Zhao, W. et al. Role of a bacillus Calmette-Guerin fibronectin attachment protein in BCG-induced antitumor activity.Int. J. Cancer86, 83-88 (2000)). In turn, fibronectin is thought to attach to urothelial cells through integrin α5β1 (Coplen, D. E., et al. Characterization of fibronectin attachment by a human transitional cell carcinoma line, T24. J. Urol.145, 1312-1315 (1991)). In vitro, BCG attachment and internalization is enhanced by addition of exogenous fibronectin and inhibited by antibodies against fibronectin or integrins a5 or R31 (Kuroda, K., et al. Characterization of the internalization of bacillus Calmette-Guerin by human bladder tumor cells.J. Clin. Invest.91, 69-76 (1993)). Bladder cancer cells can directly secrete immune-activating effectors following internalization of BCG. The main cytokine studied in this context has been IL-6, which is released from bladder cancer cells exposed to BCG. Dendritic Cells Dendritic Cells (DC) are the most powerful antigen presenting cells of the immune system, capable of stimulating naïve and memory CD8+T-cells as well as B-cells and CD4+helper T-cells. In the immature state DC are present in blood and tissues, processing foreign antigens for presentation to the immune system. The uptake of presentable antigen stimulates maturation of DC and promotes DC migration to lymph nodes, where these cells can directly interact with immune effector cells. Mature DC are capable of stimulating T helper type-1 immune responses and antigen specific CD8+cytotoxic T-lymphocytes (CTL), but within the tumor microenvironment DC promote tumor tolerance, facilitating T helper type-2 responses. Therefore DC can exert both strong positive and negative influences on the acquisition of tumor specific cellular immune responses. DC vaccines have generally consisted of autologous monocytes that are matured in vitro and pulsed with antigen before injection. Each step of DC vaccine production, DC generation, antigen loading, in vitro maturation, and inoculation with or without adjuvant is an opportunity to enhance efficacy. DC vaccine research has therefore focused on expanding the available sources of DC and improving DC immunogenicity, optimizing the source and presentation of antigen, developing new immune adjuvants, and investigation of concomitant immunomodulation or chemotherapy. (Kalijn F. Bol et al. “Dendritic Cell-Based Immunotherapy: State of the Art and Beyond.”Clinical Cancer Research,2016; 22:1897-1906; Elster, Jennifer D et al. “Dendritic cell vaccines: A review of recent developments and their potential pediatric application.” Human Vaccines & Immunotherapeutics vol. 12, 9 ( ): 2232-9. doi:10.1080/21645515.2016.1179844). Interleukin-15 IL-15 is a pleiotropic cytokine that plays various roles in the innate and adaptive immune systems, including the development, activation, homing and survival of immune effector cells, especially NK, NK-T and CD8+T cells (Cooper, M. A., et al.,Blood,2001. 97(10): p. 3146-51). IL-15, a member of the common gamma chain (γc) cytokine family, binds to a receptor complex that consists of IL-15Rα, IL-2Rβ and the γc chain (Grabstein, K. H., et al.,Science,1994. 264(5161): p. 965-8; Giri, J. G., et al.,Embo J,1995. 14(15): p. 3654-63). Furthermore, IL-15 functions as a key regulator of development, homeostasis and activity of NK cells (Prlic, M., et al.,J Exp Med,2003. 197(8): p. 967-76; Carson, W. E., et al.,J Clin Invest,1997. 99(5): p. 937-43). IL-15 administration to normal mice or overexpression of IL-15 in the transgenic mouse model increases the number and percentage of NK cells in the spleen (Evans, R., et al.,Cell Immunol,1997. 179(1): p. 66-73; Marks-Konczalik, J., et al.,Proc Natl Acad Sci USA,2000. 97(21): p. 11445-50), the proliferation and survival of NK cells, as well as their cytolytic activity and cytokine secretion. IL-15 administration could also increase the NK cell number and function in recipients of stem cell transplantation (Katsanis, E., et al.,Transplantation,1996. 62(6): p. 872-5; Judge, A. D., et al.,J Exp Med,2002. 196(7): p. 935-46; Alpdogan, O., et al.,Blood,2005. 105(2): p. 865-73; Sauter, C. T., et al.,Bone Marrow Transplantation,2013. 48(9): p. 1237-42). The primary limitations in clinical development of recombinant human IL-15 (rhIL-15) are low production yields in standard mammalian cell expression systems and a short serum half-life (Ward, A., et al.,Protein Expr Purif2009. 68(1): p. 42-8; Bessard, A., et al.,Mol Cancer Ther,2009. 8(9): p. 2736-45). The formation of the IL-15:IL-15Rα complex, with both proteins co-expressed in the same cell can stimulate immune effector cells bearing the IL-2βγc receptor through a trans-presentation mechanism. In addition, when IL-15 is bound to IL-15Rα, it increased the affinity of the IL-15 to IL-2Rβ approximately 150-fold, when compared with free IL-15 (Ring, A. M., et al.,Nat Immunol,2012. 13(12): p. 1187-95). A superagonist mutant of IL-15 (IL-15N72D), which has increased IL-2Rβ binding ability (4-5 fold higher than native IL-15) has been identified for therapeutic usages (Zhu, X., et al., Novel human interleukin-15 agonists.J Immunol,2009. 183(6): p. 3598-607). The strong interaction of IL-15N72D and soluble IL-15Rα was exploited to create an IL-15 superagonist complex with IL-15N72D bound to IL-15RαSu/Fc. The soluble fusion protein, IL-15RαSu/Fc, was created by linking the human IL-15RαSu domain with human IgG1 containing the Fc domain. Studies on IL-15:IL-15Rα complexes show an advantage of increased intracellular stability of IL-15 (Bergamaschi, C., et al.,J Biol Chem,2008. 283(7): p. 4189-99; Duitman, E. H., et al.,Mol Cell Biol,2008. 28(15): p. 4851-61). Co-expression of both the IL-15N72D and IL-15RαSu/Fc proteins resulted in a soluble and stable complex with significantly longer serum half-life and increased biological activity, compared to native IL-15 (Han, K. P., et al.,Cytokine,2011. 56(3): p. 804-10). As indicated above, this IL-15N72D:IL-15RαSu/Fc complex (N-803) was >10-fold more active than free IL-15 in promoting in vitro proliferation of IL-15-dependent cells (Zhu, X., et al., Novel human interleukin-15 agonists.J Immunol,2009. 183(6): p. 3598-607). N-803 has potent anti-tumor activity in syngeneic murine models of multiple myeloma (Xu, W., et al.,Cancer Res,2013. 73(10): p. 3075-86). IL-15:IL-15Rα Complex As defined above, an IL-15:IL-15Rα fusion protein complex can refer to a complex having IL-15 non-covalently bound to the soluble IL-15Rα domain of the native IL-15Rα. In some cases, the soluble IL-15Rα is covalently linked to a biologically active polypeptide and/or to an IgG Fc domain. The IL-15 can be either IL-15 or IL-15 covalently linked to a second biologically active polypeptide. The crystal structure of the IL-15:IL-15Rα complex is shown in Chirifu et al., 2007Nat Immunol8, 1001-1007, incorporated herein by reference. In certain embodiments, the IL-15Rα comprises IL-15RαSushi (IL-15RαSu). In other embodiments, the IL-15 is a variant IL-15 (e.g., IL-15N72D). In certain embodiments of the soluble fusion protein complexes of the invention, the IL-15 polypeptide is an IL-15 variant having a different amino acid sequence than native IL-15 polypeptide. The human IL-15 polypeptide is referred to herein as huIL-15, hIL-15, huIL15, hIL15, IL-15 wild type (wt) and variants thereof are referred to using the native amino acid, its position in the mature sequence and the variant amino acid. For example, huIL15N72D refers to human IL-15 comprising a substitution of N to D at position 72. In certain embodiments, the IL-15 variant functions as an IL-15 agonist as demonstrated, e.g., by increased binding activity for the IL-15RβγC receptors compared to the native IL-15 polypeptide. In certain embodiments, the IL-15 variant functions as an IL-15 antagonist as demonstrated by e.g., decreased binding activity for the IL-15RβγC receptors compared to the native IL-15 polypeptide. In certain embodiments, the IL-15 variant has increased binding affinity or a decreased binding activity for the IL-15RβγC receptors compared to the native IL-15 polypeptide. In certain embodiments, the sequence of the IL-15 variant has at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid change compared to the native IL-15 sequence. The amino acid change can include one or more of an amino acid substitution or deletion in the domain of IL-15 that interacts with IL-15Rβ and/or IL-15RγC. In certain embodiments, the amino acid change is one or more amino acid substitutions or deletions at position 8, 61, 65, 72, 92, 101, 108, or 111 of the mature human IL-15 sequence. For example, the amino acid change is the substitution of D to N or A at position 8, D to A at position 61, N to A at position 65, N to R at position 72 or Q to A at position 108 of the mature human IL-15 sequence, or any combination of these substitutions. In certain embodiments, the amino acid change is the substitution of N to D at position 72 of the mature human TL-15 sequence. N-803 N-803 comprises an IL-15 mutant with increased ability to bind IL-2Rβγ and enhanced biological activity (U.S. Pat. No. 8,507,222, incorporated herein by reference). This superagonist mutant of IL-15 was described in a publication (J Immunol2009 183:3598) and a patent has been issued by the U.S. Patent & Trademark Office on the super agonist and several patents applications are pending (e.g., U.S. Ser. Nos. 12/151,980 and 13/238,925). This IL-15 superagonist in combination with a soluble IL-15a receptor fusion protein (IL-15RαSu/Fc) results in a protein complex with highly potent IL-15 activity in vitro and in vivo (Han et al., 2011, Cytokine,56: 804-810; Xu, et al., 2013Cancer Res.73:3075-86, Wong, et al., 2013, Oncolmmunology2:e26442). This IL-15 super agonist complex (IL-15N72D:IL-15RαSu/Fc) is referred to as N-803. Pharmacokinetic analysis indicated that the complex has a half-life of 25 hours following i.v. administration in mice. N-803 exhibits impressive anti-tumor activity against aggressive solid and hematological tumor models in immunocompetent mice. It can be administered as a monotherapy using a twice weekly or weekly i.v. dose regimen or as combinatorial therapy with an antibody. The N-803 anti-tumor response is also durable. Tumor-bearing mice that were cured after N-803 treatment were also highly resistant to re-challenge with the same tumor cells indicating that N-803 induces effective immunological memory responses against the re-introduced tumor cells. Fc Domain N-803 comprises an IL-15N72D:IL-15RαSu/Fc fusion complex. Fusion proteins that combine the Fc regions of IgG with the domains of another protein, such as various cytokines and soluble receptors have been reported (see, for example, Capon et al.,Nature,337:525-531, 1989; Chamow et al.,Trends Biotechnol.,14:52-60, 1996; U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked through cysteine residues in the hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule without the heavy chain variable and CH1domains and light chains. The dimeric nature of fusion proteins comprising the Fc domain may be advantageous in providing higher order interactions (i.e. bivalent or bispecific binding) with other molecules. Due to the structural homology, Fc fusion proteins exhibit an in vivo pharmacokinetic profile comparable to that of human IgG with a similar isotype. Immunoglobulins of the IgG class are among the most abundant proteins in human blood, and their circulation half-lives can reach as long as 21 days. To extend the circulating half-life of IL-15 or an IL-15 fusion protein and/or to increase its biological activity, fusion protein complexes containing the IL-15 domain non-covalently bound to IL-15RαSu covalently linked to the Fc portion of the human heavy chain IgG protein have been made (e.g., N-803). The term “Fc” refers to a non-antigen-binding fragment of an antibody. Such an “Fc” can be in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG 1 and IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982),Nucleic Acids Res.10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms. Fc domains containing binding sites for Protein A, Protein G, various Fc receptors and complement proteins. In some embodiments, the term “Fc variant” refers to a molecule or sequence that is modified from a native Fc, but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, in certain embodiments, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, (7) antibody-dependent cell-mediated cytotoxicity (ADCC), or (8) antibody dependent cellular phagocytosis (ADCP). Fc variants are described in further detail hereinafter. The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by recombinant gene expression or by other means. Linkers In some cases, the fusion protein complexes of the invention also include a flexible linker sequence interposed between the IL-15 or IL-15Rα domains. The linker sequence should allow effective positioning of the polypeptide with respect to the IL-15 or IL-15Rα domains to allow functional activity of both domains. In certain cases, the soluble fusion protein complex has a linker wherein the first polypeptide is covalently linked to IL-15 (or functional fragment thereof) by a polypeptide linker sequence. In other aspects, the soluble fusion protein complex as described herein has a linker wherein the second polypeptide is covalently linked to IL-15Rα polypeptide (or functional fragment thereof) by polypeptide linker sequence. The linker sequence is preferably encoded by a nucleotide sequence resulting in a peptide that can effectively position the binding groove of a TCR molecule for recognition of a presenting antigen or the binding domain of an antibody molecule for recognition of an antigen. As used herein, the phrase “effective positioning of the biologically active polypeptide with respect to the IL-15 or IL-15Rα domains”, or other similar phrase, is intended to mean the biologically active polypeptide linked to the IL-15 or IL-15Rα domains is positioned so that the IL-15 or IL-15Rα domains are capable of interacting with each other to form a protein complex. For example, the IL-15 or IL-15Rα domains are effectively positioned to allow interactions with immune cells to initiate or inhibit an immune reaction, or to inhibit or stimulate cell development. The fusion protein complexes of the invention preferably also include a flexible linker sequence interposed between the IL-15 or IL-15Rα domains and the immunoglobulin Fc domain. The linker sequence should allow effective positioning of the Fc domain, biologically active polypeptide and IL-15 or IL-15Rα domains to allow functional activity of each domain. For example, the Fc domains are effectively positioned to allow proper fusion protein complex formation and/or interactions with Fc receptors on immune cells or proteins of the complement system to stimulate Fc-mediated effects including opsonization, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other Fc receptor-dependent processes; activation of the complement pathway; and enhanced in vivo half-life of the fusion protein complex. Linker sequences can also be used to link two or more polypeptides of the biologically active polypeptide to generate a single-chain molecule with the desired functional activity. Preferably, the linker sequence comprises from about 7 to 20 amino acids, more preferably from about 10 to 20 amino acids. The linker sequence is preferably flexible so as not hold the biologically active polypeptide or effector molecule in a single undesired conformation. The linker sequence can be used, e.g., to space the recognition site from the fused molecule. Specifically, the peptide linker sequence can be positioned between the biologically active polypeptide and the effector molecule, e.g., to chemically cross-link same and to provide molecular flexibility. The linker preferably predominantly comprises amino acids with small side chains, such as glycine, alanine and serine, to provide for flexibility. Preferably, about 80 or 90 percent or greater of the linker sequence comprises glycine, alanine or serine residues, particularly glycine and serine residues. Different linker sequences could be used including any of a number of flexible linker designs that have been used successfully to join antibody variable regions together (see, Whitlow, M. et al., (1991) Methods: A Companion to Methods in Enzymology, 2:97-105). Fusions Protein Complexes The invention provides N-803, which is a protein complex between IL-15N72D and IL-15RαSu/Fc. An exemplary IL-15N72D nucleic acid sequence is provided below (with leader peptide) (SEQ ID NO: 1): (Leader peptide)atggagacagacacactcctgttatgggtactgctgctctgggttccaggttccaccggt-(IL-15N72D)aactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacgacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct(Stop codon)taa An exemplary IL-15N72D amino acid sequence is provided below (with leader peptide) (SEQ ID NO: 2): (Leader peptide)METDTLLLWVLLLWVPGSTG-(IL-15N72D)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS In some cases, the leader peptide is cleaved from the mature IL-15N72D polypeptide (SEQ ID NO: 3): (IL-15N72D)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS An exemplary IL-15RαSu/Fc nucleic acid sequence (with leader peptide) is provided below (SEQ ID NO: 4): (Leader peptide)atggacagacttacttcttcattcctgctcctgattgtccctgcgtacgtcttgtcc-(IL-15RaSu)atcacgtgccctccccccatgtccgtggaacacgcagacatctgggtcaacgagctacagcttgtactccagggagcggtacatttgtaacttggtttcaagcgtaaagccggcacgtccagcctgacggagtgcgtgttgaacaaggccacgaatgtcgcccactggacaacccccagtctcaaatgtattaga-(IgG1 CH2-CH3 (Fc domain))gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa-(Stop codon)taa An exemplary IL-15RαSu/Fc amino acid sequence (with leader peptide) is provided below (SEQ ID NO: 5): (Leader peptide)MDRLTSSFLLLIVPAYVLS-(IL-15RaSu)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR-(IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some cases, the mature IL-15RαSu/Fc protein lacks the leader sequence (SEQ ID NO: 6): (IL-15RaSu)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR-(IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Bacillus Calmette-Guerin (BCG) Bacillus Calmette-Guerin (BCG), a live attenuated strain ofMycobacterium bovis, is currently the only agent approved by the US Food and Drug Administration for primary therapy of carcinoma in situ (CIS) of the bladder. The original bacillus Calmette-Guérin (BCG) strain was developed at the Pasteur Institute from an attenuated strain ofMycobacterium bovis. T wo BCG products are commercially available in the United States. The Tice strain, which is a substrain of the original Pasteur product, is manufactured by Organon Pharmaceuticals. The TheraCys strain is made by Aventis/Pasteur. BCG supplanted cystectomy as the treatment of choice for CIS in the mid-1980s. BCG therapy also reduces the risk of recurrence, and ongoing maintenance therapy with BCG reduces the risk of progression in patients with high-grade non-muscle invasive bladder cancer. Bladder cancer is the only cancer in which BCG is commonly used. For BCG to be effective, all the following criteria should be met: The patient is immunocompetent, the tumor burden is small; BCG makes direct contact with the tumor, the dose is adequate to incite a reaction. BCG viability is an important consideration for the vaccine to be effective. This viability is measured in colony-forming units (CFUs). A vaccine that contains no or very few live organisms would be clinically ineffective. One dose, either an ampule or vial, may vary in weight from one product to another, but the CFU should be similar. Tice BCG has 1-8×10−8CFUs. TheraCys has 10.5+/−8.7×10−8CFUs. Typically, BCG is administered in either an induction (once weekly for 6 weeks) or maintenance (once weekly for 3 weeks) course. Another 6-week course may be administered if a repeat cystoscopy (see image above) reveals tumor persistence or recurrence. Induction therapy combined with maintenance therapy every 3-6 months for 1-3 years may provide more lasting results. Periodic bladder biopsies are usually necessary to assess response. Accordingly, the administration of BCG and/or N-803 can be determined based on the progress of the patient. The guidelines from the American Urological Association (AUA) and the Society of Urologic Oncology (SUO) (Chang S S, et al. Diagnosis and Treatment of Non-Muscle Invasive Bladder Cancer: AUA/SUO Guideline.J Urol.2016 October 196 (4):1021-9) provide further guidance on the administration of BCG. Formulation of Pharmaceutical Compositions The administration of compositions embodied herein, such as, immune effector cells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cells cultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG for the treatment of a neoplasia may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a neoplasia, e.g. bladder cancer. The N-803 and BCG may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intravesicularly or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice or nonhuman primates, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 0.1 μg compound/kg body weight to about 5000 μg compound/kg body weight; or from about 1 μg/kg body weight to about 4000 μg/kg body weight or from about 10 μg/kg body weight to about 3000 μg/kg body weight. In other embodiments this dose may be about 0.1, 0.3, 0.5, 1, 3, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 μg/kg body weight. In other embodiments, it is envisaged that doses may be in the range of about 0.5 μg compound/kg body weight to about 20 μg compound/kg body weight. In other embodiments the doses may be about 0.5, 1, 3, 6, 10, or 20 mg/kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient. In particular embodiments, N-803 are formulated in an excipient suitable for parenteral or intravesical administration. In particular embodiments, N-803 is administered at 0.5 μg/kg-about 15 μg/kg (e.g., 0.5, 1, 3, 5, 10, or 15 μg/kg). For the treatment of bladder cancer, N-803 is administered by instillation into the bladder. Methods of instillation are known. See, for example, Lawrencia, et al.,Gene Ther8, 760-8 (2001); Nogawa, et al.,J Clin Invest115, 978-85 (2005); Ng, et al.,Methods Enzymol391, 304-13 2005; Tyagi, et al.,J Urol171, 483-9 (2004); Trevisani, et al.,J Pharmacol Exp Ther309, 1167-73 (2004); Trevisani, et al.,Nat Neurosci5, 546-51 (2002)). In certain embodiments, it is envisioned that the N-803 dosage for instillation may vary from between about 5 and 1000 μg/dose. In other embodiments the intravesical doses may be about 25, 50, 100, 200, or 400 μg/dose. In other embodiments, N-803 is administered by instillation into the bladder in combination with standard therapies, including mitomycin C or BCG. Pharmaceutical compositions are formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. Parenteral Compositions The pharmaceutical compositions embodied herein, such as, immune effector cells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cells cultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intravesicularly, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra. Compositions embodied herein, such as, immune effector cells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cells cultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG for intravesical or parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules, syringes or bags), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neoplasia, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents. As indicated above, the pharmaceutical compositions comprising N-803 and/or BCG may be in a form suitable for sterile injection. To prepare such a composition, the suitable active antineoplastic therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. The present invention provides methods of treating neoplastic and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a composition embodied herein, such as, immune effector cells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cells cultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a neoplastic or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a composition embodied herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated. The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a neoplastic disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). N-803 may be used in the treatment of any other disorders in which an increase in an immune response is desired. In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with neoplasia in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. Combination Therapies The compositions embodied herein, such as, immune effector cells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cells cultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG, can be administered in combination with an anti-neoplasia such as a chemotherapeutic agent, e.g. mitomycin C, an antibody, e.g., a tumor-specific antibody or an immune-checkpoint inhibitor. The compositions may be administered simultaneously or sequentially. In some embodiments, the chemotherapeutic treatment is an established therapy for the disease indication and addition of, for example, dendritic cells isolated from a subject, the treatment improves the therapeutic benefit to the patients. Such improvement could be measured as increased responses on a per patient basis or increased responses in the patient population. Combination therapy could also provide improved responses at lower or less frequent doses of chemotherapeutic agent resulting in a better tolerated treatment regimen. If desired, the immune effector cells, e.g. dendritic cells obtained from subjects having been administered BCG and cultured with N-803, are administered in combination with any conventional therapy, including but not limited to, surgery, radiation therapy, chemotherapy, protein-based therapy or biological therapy. Chemotherapeutic drugs include alkylating agents (e.g., platinum-based drugs, tetrazines, aziridines, nitrosoureas, nitrogen mustards), anti-metabolites (e.g., anti-folates, fluoropyrimidines, deoxynucleoside analogues, thiopurines), anti-microtubule agents (e.g., vinca alkaloids, taxanes), topoisomerase inhibitors (e.g., topoisomerase I and II inhibitors), cytotoxic antibiotics (e.g., anthracyclines) and immunomodulatory drugs (e.g., thalidomide and analogs). Anti-Cancer Therapeutic Agents The methods of the invention may include administration of second therapeutic agent or treatment with a second therapy (e.g., a therapeutic agent or therapy that is standard in the art). Exemplary therapeutic agents include chemotherapeutic agents. A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include mitomycin C, Erlotinib (TARCEVA™, Genentech/OSI Pharm.), Bortezomib (VELCADE™, Millennium Pharm.), Fulvestrant (FASLODEX™, Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA™, Novartis), Imatinib mesylate (GLEEVEC™, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin™, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE™, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA™, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozcicsin, carzcicsin and bizcicsin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin 71 and calicheamicin omega 1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX™ (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON™ (toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane), formestanie, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole), and ARIMIDEX™ (anastrozole); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME™ (ribozyme)) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™ topoisomerase 1 inhibitor; ABARELIX™ rmRH; (x) anti-angiogenic agents such as bevacizumab (AVASTIN™, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above. While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments. All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, applicants do not admit any particular reference is “prior art” to their invention. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. | 51,788 |
11857613 | DETAILED DESCRIPTION Necrotic enteritis (NE) is caused by type A strains of the bacteriumClostridium perfringens. The total global economic losses to the poultry industry due to NE is estimated to be over 2 billion dollars annually.C. perfringensproduces two toxins, alpha-toxin and NetB. The NetB toxin is responsible for the symptoms associated with NE and anti-NetB antibodies are protective. Immune responses against alpha-toxin are partially protective despite the fact that it does not play a direct role in NE. We describe a single fusion protein combining immunogenic and non-toxic components of alpha-toxin and NetB that can be used to immunize poultry against NE. The fusion protein is produced in plants which can be purified and used as an injectable preparation or fed directly to poultry to elicit a protective immune response. The use of plants for the production and oral delivery of a necrotic enteritis vaccine is novel. Moreover, the PlcC-NetB fusion protein described here is novel and is strongly expressed in plants, which lack cholesterol and thus may be immune to the toxic effects of NetB, thus permitting the observed high expression level. This novel fusion protein combines the two most potent protective antigens against necrotic enteritis, NetB and alpha-toxin, into a single antigenic protein. NetB is the toxin responsible for necrotic enteritis symptoms. The alpha-toxin (Plc) may also contribute to disease, and, in addition, antibodies against alpha-toxin are targeted to the surface ofC. perfringens, inhibiting its growth. However, strains lacking alpha-toxin can cause disease, such that a vaccine relying only on immune responses against alpha-toxin will not provide protection against these strains. Combining both alpha-toxin and NetB epitopes will provide robust protection against disease. The PlcC-NetB protein can be purified from plants, in either a glycosylated or non-glycosylated form, and used as an injectable vaccine to protect birds directly. Injection into hens will protect their offspring in the first two weeks of life via maternal antibodies passed on in the egg. When this protein is produced in a food plant, such as corn, with or without an LT adjuvant, the resulting recombinant plant can be applied directly to the feed, resulting in a potentially low cost, oral vaccine to protect chickens against necrotic enteritis. The use of plants for the production and oral delivery of a necrotic enteritis vaccine is novel. Moreover, the PlcC-NetB fusion protein described here is novel and is strongly expressed in plants, which lack cholesterol and thus may be immune to the toxic effects of NetB, thus permitting the observed high expression level. C. perfringenstype A strains produce alpha-toxin, a membrane-damaging phospholipase C enzyme. The toxin is hemolytic, necrotizing and lethal. It is the toxin that responsible forC. perfringens-mediated gas gangrene. Many of the symptoms of NE can be reproduced with culture-free supernatants ofC. perfringens. Since these supernatants were known to contain alpha-toxin, it was assumed that alpha-toxin was responsible. More recent studies have identified a novel toxin linked to necrotic enteritis, designated NetB toxin. It was first identified in a virulentC. perfringenstype A strain isolated in Australia and it has been detected in the vast majority of NE-associatedC. perfringensstrains throughout the world. Thus, it is now considered to be the most critical virulence factor for the development of NE in broilers. NetB is a pore-forming toxin encoded on a large conjugative plasmid (approximately 85 kb) within a 42 kilobase (kb) pathogenicity locus (NELoc-1), showing similarity toC. perfringensβ-toxin (38% identity). The presence of netB gene is highly correlated with necrotic enteritis strains. NetB is also a protective antigen, particularly in combination with other immunogenic components. One study showed that the levels of serum antibodies against both alpha-toxin and NetB toxin were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE, suggesting that these antitoxin antibodies play a role in protection. The large clostridial cytotoxin TpeL (predicted molecular mass=191 kDa), first identified in type C strains, is also produced by some type A strains and has been linked to increased virulence, particularly in strains producing netB. However, it should be noted that a recent study of historical NE strains collected >15 years ago in Alabama revealed a low prevalence of the netB gene, indicating that netB may be dispensable for some NE for some strains or in some situations. Nevertheless, it is clear that the overwhelming majority of current necrotic enteritis strains produce this toxin. Toxins have traditionally been targeted as antigens of interest for controlling clostridial infections. TheC. perfringensalpha-toxin (Plc) is the major virulence determinant for gas gangrene and antibodies toC. perfringensalpha-toxin prevent gas gangrene in mice. TheC. perfringensgene encoding alpha-toxin is plc (for phospholipase C). The protein is divided into two domains, the amino-terminal domain encodes the catalytic site responsible for phospholipase activity, while the carboxy-terminal domain is involved in interactions with phospholipids, targeting the enzyme to host cell membranes. The alpha-toxin carboxy-terminal fragment (amino acids 247-370) is non-toxic and immunization with this fragment confers protection against alpha-toxin andC. perfringensin a gas gangrene mouse model. Immune responses against the C-terminal domain, PlcC, can provide protection against subsequent challenge withC. perfringens. NetB binds to cholesterol in membranes, forming heptameric pores. A number of single amino acid substitutions in the rim loop region can significantly reduce its ability to bind to cells and its toxicity. These include Y191A, R200A, W257A and W262A, S254L, R230Q and W287R. Some of these were shown to retain the ability to generate protective immune responses, including W262A and S254L. A number of studies have demonstrated the potential of vaccination to control NE. A vaccine utilizing detoxified alpha-toxin can induce some protection against experimental infection. Since alpha-toxin is not required in order forC. perfringensto cause NE in chickens, it is not clear why alpha-toxoids are protective. One likely explanation is based on data showing that anti-alpha-toxin (anti-Plc) antibodies bind to the surface of Plc+C. perfringensstrains and that these antibodies can also inhibitC. perfringensgrowth. Thus, it is possible that the reason anti-Plc antibodies are protective is due to their growth inhibitory properties and not directly due to detoxification. NetB is also a protective antigen, which could provide significant protection against NE challenge, especially in combination with other immunogenic components. Both alpha-toxin (C-fragment) and NetB (W262A) toxoids were combined (30 g of each) in Quil A adjuvant and used to subcutaneously inject broiler birds 3 times, on days 3, 9 and 15. Birds injected with only one of the proteins were also included. The immunized birds were partially protected against a mild challenge (gavage only), but not against a more severe, in feed challenge. In some studies, hens were infected with NetB toxoid and antibodies against NetB were transferred from immunized hens to their progeny, providing protection to the chicks againstC. perfringenschallenge. In another study, immunization with both NetB and alpha-toxin toxoids using a liveSalmonelladelivery vector induced mucosal antibodies against both toxins and elicited a protective response.S. Typhimuriumvaccine trains engineered to deliver both toxoids provided significantly better protection than strains delivering each toxin alone. Recently an injectable alpha-toxoid preparation produced by Intervet, called Netvax, has come on the market for use in broiler breeders to increase protection in chicks during the first few weeks of life. However, there is no commercial vaccine that includes a NetB immunogenic component. Several vaccine antigens have been stably expressed in corn and rice, which are convenient for use in feed products. Despite concerns regarding oral tolerance, feeding animals plant-based vaccines has been shown to be effective in agricultural animals, including poultry. Proteins In certain embodiments, the present invention provides an antigenic protein comprising a PlcC protein unit that is operably linked to a peptide linker that is operably linked to a NetB protein unit. In certain embodiments, the PlcC protein unit, the peptide linker and the NetB protein unit each have an N-terminus and a C-terminus, and wherein the C-terminus of the PlcC protein unit is linked to the N-terminus of the peptide linker, and the C-terminus of the peptide linker is operably linked to the N-terminus of the NetB protein unit. In certain embodiments, the PlcC protein unit has at least 95% sequence identity to SEQ ID NO: 3. The PlcC protein unit of SEQ ID NO: 3 is amino acids 248-370 of alpha toxin (GenBank accession AAP-15462.1). In certain embodiments, the PlcC protein unit has 100% sequence identity to SEQ ID NO: 3. In certain embodiments, the NetB protein unit has at least 95% sequence identity to SEQ ID NO: 5. The NetB protein unit of SEQ ID NO: 5 is amino acids 31-322 of (GenBank accession ACN73257.1). In certain embodiments, the NetB protein unit has one or more amino acid substitutions at Y191A, R200A, W257A and W262A, S254L, R230Q or W287R of SEQ ID NO: 5. In certain embodiments, the NetB protein unit has 100% sequence identity to SEQ ID NO: 5. In certain embodiments, the peptide linker has at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the peptide linker has 100% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigenic protein further comprises a 6Hist tag having an N-terminus and a C-terminus, wherein the C-terminus of the 6Hist tag is operably linked to the N-terminus of the PlcC protein unit. In certain embodiments, the 6His tag has 100% identity to SEQ ID NO: 2. In certain embodiments, the antigenic protein further comprises a plant signal peptide having an N-terminus and a C-terminus, wherein the C-terminus of the plant signal peptide is operably linked to the N-terminus of the 6Hist tag. In certain embodiments, the plant signal peptide has at least 95% sequence identity to SEQ ID NO: 1. In certain embodiments, the plant signal peptide has 100% sequence identity to SEQ ID NO: 1. The term “amino acid” includes the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g., phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also includes peptides with reduced peptide bonds, which will prevent proteolytic degradation of the peptide. Also, the term includes the amino acid analog α-amino-isobutyric acid. The term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (C1-C6)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene,Protecting Groups In Organic Synthesis; Wiley: New York, 1981, and references cited therein). A “variant” of one of the proteins that one that is not completely identical to a native protein. Such variant protein can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acid. The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A “conserved substitution” is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Aline is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. The amino acid changes are achieved by changing the codons of the corresponding nucleic acid sequence. It is known that such polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that results in increased activity. Alternatively, amino acid substitutions in certain polypeptides may be used to provide residues, which may then be linked to other molecules to provide peptide-molecule conjugates which retain sufficient properties of the starting polypeptide to be useful for other purposes. One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated is intended for use in immunological embodiments. The greatest local average hydrophilicity of a “protein”, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to conduct substitutions of amino acids where these values are ±2, with ±1 being particularly preferred, and those with in ±0.5 being the most preferred substitutions. The variant protein has at least 80%, at least about 90%, or even at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native protein. A variant may include amino acid residues not present in the corresponding native protein or deletions relative to the corresponding native protein. A variant may also be a truncated “fragment” as compared to the corresponding native protein, i.e., only a portion of a full-length protein. Protein variants also include peptides having at least one D-amino acid. The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein. Amino acid sequence of 6H-plcC-netB fusion protein: (SEQ ID NO: 13)mankhlslslflvllglsaslasgHHHHHHgsDPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIKggsggsggpsggsggsELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQARGTNKLSSTSEYNEFMFKINWQDHKIEYYL Coding of Regions:Lower case, regular type=ER signal peptide from barley alpha amylase geneUpper case, Italics=6-His metal affinity tagLower case, italics=linker sequencesUpper case, regular type=plcCUpper case, Bold type=netB (W262A mutation underlined) In certain embodiments, e.g., for cytosolic instead of ER targeting, the N-terminal signal peptide (Lower case, regular type) is omitted. Nucleic Acid In certain embodiments, the present invention provides a nucleic acid encoding the antigenic protein described herein. In certain embodiments, the nucleic acid has been plant-codon optimized for plant expression. In certain embodiments, the nucleic acid has been plant-codon optimized for expression inNicotiana benthamianaorArabidopsis. In certain embodiments, the nucleic acid has at least 95% sequence identity to SEQ ID NO: 6. In certain embodiments, the nucleic acid has 100% sequence identity to SEQ ID NO: 6. The fusion protein gene for the 6H-plcC-netB fusion protein (SEQ ID NO: 13 (amino acid sequence)) was codon optimized for expression inNicotiana benthamiana. Each codon was assessed for its preference of use in highly expressed genes ofN. benthamiana, N. tabacum, andArabidopsis thaliana, using coding sequences obtained from Genbank accessions (Geyer, B. C., Kannan, L., Cherni, I., Woods, R. R., Soreq, H., and Mor, T. S. (2010) Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase. Plant Biotechnol J. 8(8):873-86). Use of a particular codon was avoided if it represented less than 50% of the frequency of the most preferred synonymous codon in the reference gene set. The remaining codons were used at frequencies proportional to their frequencies of use in the reference gene set. Furthermore, A-rich sequences that could function as a polyadenylation near-upstream element were avoided, including AATAAA, AATGGA, AATGAA, TATAAA, AATAAT, AATAAG, AATATT, GATAAA, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, CATAAA, ACTAAA, and AAAAAA. Sequences that could function as 5′ intron splice recognition sequences were avoided, including GTAACA, GTGCTC, GTTAGT, GTAAAT, GTAAAG, GTCTGT, GTAAGG, GTGAGT, GTAAAA, GTAAGT, GTAAGC, GTACGT, GTAACT, GTAAGA, GTTAAA, GTAATA, and GTACAT. As much as possible, sequences that could function as 3′ intron splice recognition sequences were avoided, including GCAGG, CCAGG, TCAGG, ATAGG, GTAGG, TTAGG, ACAGG, and AAAGG. Potential RNA destabilizing sequences were avoided, including ATTTA, TAGATY, ATAGAT, and TTTTTT. Potential termination signals for RNA polymerase II were avoided as much as possible, including CA(N7-9)AGTNNA. The potential DNA C-methylation signal CCGG was avoided as much as possible. The nucleotide sequence of pBYR2eK2M-6HplcCnetB is the following (FIG.6): (SEQ ID NO: 14)CGATCGGTCGATTCATAGAAGATTAGATTTTTCATAGTATTTTTTTAAAGTAAACCTTTAACTACGGTTAGGAGACTTTTAAGTTAAATTTAATTTGAACCCTTAAATTAATTTTTAAAATAGATAAATATCAATCATCCTGATATGCTTTTGAAAAAATGAATGAGAAAGATGATTCAATTAAGGCCACATTTTAATCATGACTAAAATAATATACAGTATAATTTCATATATATTTGCTTTAAAAAAAAATTGACAATCCATTCGTTTCTAGCAATAAATTTCTTCAACCACAAATATATTAAAGATAACTACGGCATAGAAACAAAAATCTATGAAGAATTTTTGTATACTTCATATGAAATTAAAAAAAACTTCATTGAACATCAAAATAATAATAATAATCATAAACTCCTCAATATTTATATTCCTAGCTTCTTGAATTAAATTGTTTACATATTCAACGATGTAAAAAATTATTTCTCTATCTATTTTCCTTATATCATGCATGGTTTCACATATATCAAAGGATAAAAGCAATCTATGTAAATTATCTCACTTTATTAAGTTTTCTATCTGAATTATTGAGAACGTAGATTTCTTTTTGCACTATCCCCCAATAATTAGCAAAACACACCTAGACTAGATTTGTTTTGCTAACCCAATTGATATTAATTATATATGATTAATATTTATATGTATATGGAATTGGTTAATAAAATGCATCTGGTTCATCAAAGAATTATAAAGACACGTGACATTCATTTAGGATAAGAAATATGGATGATCTCTTTCTCTTATTCAGATAATTAGTAATTAGACATAACACACAACTTTGATGCCCACATTATAGTGATTAGCATGTCACTATGTGTGCATCCTTTTATTTCATACATTAATTAACTTGGCCAATCCAGAAGATGGACAAGTCTAGGGTCACATTGCAGGGTACTCTAGCTTACTCGCCTTCTTTTTCGAAGGTTTGAGTACCTTCAGGGCATCCTCTTGATACATTACTTTCCACTTCGATTGGGGCAAGCTGTAGCAGTTCTTGCTTAGACCGAATTGCCATCTCACAGAGATGCTGAAGAGTTCGCGACCCTCCAGAAACGGTGATACTAACTCCTCGAAACCGAATACTATAGGTACATCCGATCTGGTCGAAACCGAAAAATCGAGATGCTGCATAGTTAACCGAATCTCCCGTCCAAGATCCAAGGACTCTGTGCAGTGAAGCTTCCGTCCTGTCGTATCTGAGATATCTCTTAAATACAACTTTCCCGAAACCCCAGCTTTCCTTGAAACCAAGGGGATTATCTTGATTCGAATTCGTCTCATCGTTATGTAGCCGCCACTCAGTCCAACTCGGACTTTCGTCAGGAAGTTTGAAGGGAGAAGTTGTACCTCCTGATCCTCCATCCCAACGTTCACTGTTAGCTTGTTCCCTAGCGTCGTTTCCTTGTATAGCTCGTTCCATGGATTGTAAATAGTAATTGTAATGTTGTTTGTTGTTTGTTGTTGTTGGTAATTGTTGTAAAAATACGCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCAGCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAAGCTGGCGCGCCGCTCTAGCAGAAGGCATGTTGTTGTGACTCCGAGGGGTTGCCTCAAACTCTATCTTATAACCGGCGTGGAGGCATGGAGGCAAGGGCATTTTGGTAATTTAAGTAGTTAGTGGAAAATGACGTCATTTACTTAAAGACGAAGTCTTGCGACAAGGGGGGCCCACGCCGAATTTTAATATTACCGGCGTGGCCCCACCTTATCGCGAGTGCTTTAGCACGAGCGGTCCAGATTTAAAGTAGAAAAGTTCCCGCCCACTAGGGTTAAAGGTGTTCACACTATAAAAGCATATACGATGTGATGGTATTTGATAAAGCGTATATTGTATGAGGTATTTCCGTCGGATACGAATTATTCGTACGACCCTCCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTAGTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGAGAAACAAACAAAATCAACAAATATAGAAAATAACGCATTTCCAATTCTTTGAAATTTCTGCAACATCTAGAACAATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTTTAAGAGCTCGAAGTGACATCACAAAGTTGAAGGTAATAAAGCCAAATTAATTAAGACATTTTCATAATGATGTCAAGAATGCAAAGCAAATTGCATAACTGCCTTTATGCAAAACATTAATATAATATAAATTATAAAGAACTGCGCTCTCTGCTTCTTATTTTCTTAGCTTCATTTATTAGTCACTAGCTGTTCAGAATTTTCAGTATCTTTTGATATTACTAAGAACCTAATCACACAATGTATATTCTTATGCAGGAAAAGCAGAATGCTGAGCTAAAAGAAAGGCTTTTTCCATTTTCGAGAGACAATGAGAAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAAGAGTAAATAATAAAGCCCCACAGGAGGCGAAGTTCTTGTAGCTCCATGTTATCTAAGTTATTGATATTGTTTGCCCTATATTTTATTTCTGTCATTGTGTATGTTTTGTTCAGTTTCGATCTCCTTGCAAAATGCAGAGATTATGAGATGAATAAACTAAGTTATATTATTATACGTGTTAATATTCTCCTCCTCTCTCTAGCTAGCCTTTTGTTTTCTCTTTTTCTTATTTGATTTTCTTTAAATCAATCCATTTTAGGAGAGGGCCAGGGAGTGATCCAGCAAAACATGAAGATTAGAAGAAACTTCCCTCTTTTTTTTCCTGAAAACAATTTAACGTCGAGATTTATCTCTTTTTGTAATGGAATCATTTCTACAGTTATGACGAATTCTCGATTAAAAATCCCAATTATATTTGGTCTAATTTAGTTTGGTATTGAGTAAAACAAATTCGAACCAAACCAAAATATAAATATATAGTTTTTATATATATGCCTTTAAGACTTTTTATAGAATTTTCTTTAAAAAATATCTAGAAATATTTGCGACTCTTCTGGCATGTAATATTTCGTTAAATATGAAGTGCTCCATTTTTATTAACTTTAAATAATTGGTTGTACGATCACTTTCTTATCAAGTGTTACTAAAATGCGTCAATCTCTTTGTTCTTCCATATTCATATGTCAAAATCTATCAAAATTCTTATATATCTTTTTCGAATTTGAAGTGAAATTTCGATAATTTAAAATTAAATAGAACATATCATTATTTAGGTATCATATTGATTTTTATACTTAATTACTAAATTTGGTTAACTTTGAAAGTGTACATCAACGAAAAATTAGTCAAACGACTAAAATAAATAAATATCATGTGTTATTAAGAAAATTCTCCTATAAGAATATTTTAATAGATCATATGTTTGTAAAAAAAATTAATTTTTACTAACACATATATTTACTTATCAAAAATTTGACAAAGTAAGATTAAAATAATATTCATCTAACAAAAAAAAAACCAGAAAATGCTGAAAACCCGGCAAAACCGAACCAATCCAAACCGATATAGTTGGTTTGGTTTGATTTTGATATAAACCGAACCAACTCGGTCCATTTGCACCCCTAATCATAATAGCTTTAATATTTCAAGATATTATTAAGTTAACGTTGTCAATATCCTGGAAATTTTGCAAAATGAATCAAGCCTATATGGCTGTAATATGAATTTAAAAGCAGCTCGATGTGGTGGTAATATGTAATTTACTTGATTCTAAAAAAATATCCCAAGTATTAATAATTTCTGCTAGGAAGAAGGTTAGCTACGATTTACAGCAAAGCCAGAATACAAAGAACCATAAAGTGATTGAAGCTCGAAATATACGAAGGAACAAATATTTTTAAAAAAATACGCAATGACTTGGAACAAAAGAAAGTGATATATTTTTTGTTCTTAAACAAGCATCCCCTCTAAAGAATGGCAGTTTTCCTTTGCATGTAACTATTATGCTCCCTTCGTTACAAAAATTTTGGACTACTATTGGGAACTTCTTCTGAAAATAGTGGTACCGAGTGTACTTCAAGTCAGTTGGAAATCAATAAAATGATTATTTTATGAATATATTTCATTGTGCAAGTAGATAGAAATTAGATATGTTAGATAACACACGAAATAAACAAAAAAACACAATCCAAAACAAACACCCCAAACAAAATAACACTATATATATCCTCGTATGAGGAGAGGCACGTTCAGTGACTCGACGATTCCCGAGCAAAAAAAGTCTCCCCGTCACACATATAGTGGGTGACGCAATTATCTTCAAAGTAATCCTTCTGTTGACTTGTCATTGATAACATCCAGTCTTCGTCAGGATTGCAAAGAATTATAGAAGGGATCCCACCTTTTATTTTCTTCTTTTTTCCATATTTAGGGTTGACAGTGAAATCAGACTGGCAACCTATTAATTGCTTCCACAATGGGACGAACTTGAAGGGGATGTCGTCGATGATATTATAGGTGGCGTGTTCATCGTAGTTGGTGAAGTCGATGGTCCCGTTCCAGTAGTTGTGTCGCCCGAGACTTCTAGCCCAGGTGGTCTTTCCGGTACGAGTTGGTCCGCAGATGTAGAGGCTGGGGTGTCTGACCCCAGTCCTTCCCTCATCCTGGTTAGATCGGCCATCCACTCAAGGTCAGATTGTGCTTGATCGTAGGAGACAGGATGTATGAAAGTGTAGGCATCGATGCTTACATGATATAGGTGCGTCTCTCTCCAGTTGTGCAGATCTTCGTGGCAGCGGAGATCTGATTCTGTGAAGGGCGACACGTACTGCTCAGGTTGTGGAGGAAATAATTTGTTGGCTGAATATTCCAGCCATTGAAGCTTTGTTGCCCATTCATGAGGGAACTCTTCTTTGATCATGTCAAGATACTCCTCCTTAGACGTTGCAGTCTGGATAATAGTTCGCCATCGTGCGTCAGATTTGCGAGGAGACACCTTATGATCTCGGAAATCTCCTCTGGTTTTAATATCTCCGTCCTTTGATATGTAATCAAGGACTTGTTTAGAGTTTCTAGCTGGCTGGATATTAGGGTGATTTCCTTCAAAATCGAAAAAAGAAGGATCCCTAATACAAGGTTTTTTATCAAGCTGGATAAGAGCATGATAGTGGGTAGTGCCATCTTGATGAAGCTCAGAAGCAACACCAAGGAAGAAAATAAGAAAAGGTGTGAGTTTCTCCCAGAGAAACTGGAATAAATCATCTCTTTGAGATGAGCACTTGGGGTAGGTAAGGAAAACATATTTAGATTGGAGTCTGAAGTTCTTGCTAGCAGAAGGCATGTTGTTGTGACTCCGAGGGGTTGCCTCAAACTCTATCTTATAACCGGCGTGGAGGCATGGAGGCAAGGGCATTTTGGTAATTTAAGTAGTTAGTGGAAAATGACGTCATTTACTTAAAGACGAAGTCTTGCGACAAGGGGGGCCCACGCCGAATTTTAATATTACCGGCGTGGCCCCACCTTATCGCGAGTGCTTTAGCACGAGCGGTCCAGATTTAAAGTAGAAAAGTTCCCGCCCACTAGGGTTAAAGGTGTTCACACTATAAAAGCATATACGATGTGATGGTATTTGATGGAGCGTATATTGTATCAGGTATTTCCGTCGGATACGAATTATTCGTACGGCCGGACCGGTCCCCTAGGCCGGCCAATTCGAGATCGGCCGCGGCTGAGTGGCTCCTTCAATCGTTGCGGTTCTGTCAGTTCCAAACGTAAAACGGCTTGTCCCGCGTCATCGGCGGGGGTCATAACGTGACTCCCTTAATTCTCCGCTCATGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAATCGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCAGATCTGGCGCCGGCCAGCGAGACGAGCAAGATTGGCCGCCGCCCGAAACGATCCGACAGCGCGCCCAGCACAGGTGCGCAGGCAAATTGCACCAACGCATACAGCGCCAGCAGAATGCCATAGTGGGCGGTGACGTCGTTCGAGTGAACCAGATCGCGCAGGAGGCCCGGCAGCACCGGCATAATCAGGCCGATGCCGACAGCGTCGAGCGCGACAGTGCTCAGAATTACGATCAGGGGTATGTTGGGTTTCACGTCTGGCCTCCGGAGACTGTCATACGCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGCAGTTGCCATGTTTTACGGCAGTGAGAGCAGAGATAGCGCTGATGTCCGGCGGTGCTTTTGCCGTTACGCACCACCCCGTCAGTAGCTGAACAGGAGGGACAGCTGATAGACACAGAAGCCACTGGAGCACCTCAAAAACACCATCATACACTAAATCAGTAAGTTGGCAGCATCACCCATAATTGTGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGATGTGGCGCAACGATGCCGGCGACAAGCAGGAGCGCACCGACTTCTTCCGCATCAAGTGTTTTGGCTCTCAGGCCGAGGCCCACGGCAAGTATTTGGGCAAGGGGTCGCTGGTATTCGTGCAGGGCAAGATTCGGAATACCAAGTACGAGAAGGACGGCCAGACGGTCTACGGGACCGACTTCATTGCCGATAAGGTGGATTATCTGGACACCAAGGCACCAGGCGGGTCAAATCAGGAATAAGGGCACATTGCCCCGGCGTGAGTCGGGGCAATCCCGCAAGGAGGGTGAATGAATCGGACGTTTGACCGGAAGGCATACAGGCAAGAACTGATCGACGCGGGGTTTTCCGCCGAGGATGCCGAAACCATCGCAAGCCGCACCGTCATGCGTGCGCCCCGCGAAACCTTCCAGTCCGTCGGCTCGATGGTCCAGCAAGCTACGGCCAAGATCGAGCGCGACAGCGTGCAACTGGCTCCCCCTGCCCTGCCCGCGCCATCGGCCGCCGTGGAGCGTTCGCGTCGTCTCGAACAGGAGGCGGCAGGTTTGGCGAAGTCGATGACCATCGACACGCGAGGAACTATGACGACCAAGAAGCGAAAAACCGCCGGCGAGGACCTGGCAAAACAGGTCAGCGAGGCCAAGCAGGCCGCGTTGCTGAAACACACGAAGCAGCAGATCAAGGAAATGCAGCTTTCCTTGTTCGATATTGCGCCGTGGCCGGACACGATGCGAGCGATGCCAAACGACACGGCCCGCTCTGCCCTGTTCACCACGCGCAACAAGAAAATCCCGCGCGAGGCGCTGCAAAACAAGGTCATTTTCCACGTCAACAAGGACGTGAAGATCACCTACACCGGCGTCGAGCTGCGGGCCGACGATGACGAACTGGTGTGGCAGCAGGTGTTGGAGTACGCGAAGCGCACCCCTATCGGCGAGCCGATCACCTTCACGTTCTACGAGCTTTGCCAGGACCTGGGCTGGTCGATCAATGGCCGGTATTACACGAAGGCCGAGGAATGCCTGTCGCGCCTACAGGCGACGGCGATGGGCTTCACGTCCGACCGCGTTGGGCACCTGGAATCGGTGTCGCTGCTGCACCGCTTCCGCGTCCTGGACCGTGGCAAGAAAACGTCCCGTTGCCAGGTCCTGATCGACGAGGAAATCGTCGTGCTGTTTGCTGGCGACCACTACACGAAATTCATATGGGAGAAGTACCGCAAGCTGTCGCCGACGGCCCGACGGATGTTCGACTATTTCAGCTCGCACCGGGAGCCGTACCCGCTCAAGCTGGAAACCTTCCGCCTCATGTGCGGATCGGATTCCACCCGCGTGAAGAAGTGGCGCGAGCAGGTCGGCGAAGCCTGCGAAGAGTTGCGAGGCAGCGGCCTGGTGGAACACGCCTGGGTCAATGATGACCTGGTGCATTGCAAACGCTAGGGCCTTGTGGGGTCAGTTCCGGCTGGGGGTTCAGCAGCCAGCGCTTTACTGGCATTTCAGGAACAAGCGGGCACTGCTCGACGCACTTGCTTCGCTCAGTATCGCTCGGGACGCACGGCGCGCTCTACGAACTGCCGATAAACAGAGGATTAAAATTGACAATTCAATGGCAAGGACTGCCAGCGCTGCCATTTTTGGGGTGAGGCCGTTCGCGGCCGAGGGGCGCAGCCCCTGGGGGGATGGGAGGCCCGCGTTAGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGCCCGGTGAGCGTCGCAAAGGCGCTCGGTCTTGCCTTGCTCGTCGAGATCTGGGGTCGATCAGCCGGGGATGCATCAGGCCGACAGTCGGAACTTCGGGTCCCCGACCTGTACCATTCGGTGAGCAATGGATAGGGGAGTTGATATCGTCAACGTTCACTTCTAAAGAAATAGCGCCACTCAGCTTCCTCAGCGGCTTTATCCAGCGATTTCCTATTATGTCGGCATAGTTCTCAAGATCGACAGCCTGTCACGGTTAAGCGAGAAATGAATAAGAAGGCTGATAATTCGGATCTCTGCGAGGGAGATGATATTTGATGACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGAGGCTTAGACAACTTAATAACACATTGCGGAGGTTTTTAATGTACTGGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG The sequence indicated in bold above is the portion that encodes the fusion protein: (SEQ ID NO: 6)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTAGACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTAGAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTTTAA The proteins of the present invention may be expressed from an isolated DNA sequence encoding the protein. “Recombinant” is defined as a peptide or nucleic acid produced by the processes of genetic engineering. It should be noted that it is well-known in the art that, due to the redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon, and still result in an identical amino acid sequence. The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term “nucleotide sequence” refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene. The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an “isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell or bacteriophage. For example, an “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By “fragment” or “portion” is meant a full length or less than full length of the nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein. The term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. “Naturally occurring” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring. The term “chimeric” refers to any gene or DNA that contains 1) DNA sequences, including regulatory and coding sequences that are not found together in nature or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. A “transgene” refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, DNA that is either heterologous or homologous to the DNA of a particular cell to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer. A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid which encodes a polypeptide is implicit in each described sequence. “Recombinant DNA molecule” is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook and Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (3rdedition, 2001). The terms “heterologous DNA sequence,” “exogenous DNA segment” or “heterologous nucleic acid,” each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced. “Wild-type” refers to the normal gene, or organism found in nature without any known mutation. Expression Cassettes In certain embodiments, the present invention provides an expression cassette comprising the nucleic acid described herein and a promoter. In certain embodiments, the promoter is a plant promoter. In certain embodiments, the plant promoter is operable in corn or rice. In certain embodiments, the plant promoter is operable in seed tissue. In certain embodiments, the seed tissue is embryo or endosperm tissue. “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. “Coding sequence” refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an “uninterrupted coding sequence”, i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An “intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein. “Regulatory sequences” and “suitable regulatory sequences” each refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive promoters, tissue-specific promoters, development-specific promoters, inducible promoters and viral promoters. “5′ non-coding sequence” refers to a nucleotide sequence located 5′ (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. “3′ non-coding sequence” refers to nucleotide sequences located 3′ (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The term “translation leader sequence” refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5′) of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. The term “mature” protein refers to a post-translationally processed polypeptide without its signal peptide. “Precursor” protein refers to the primary product of translation of an mRNA. “Signal peptide” refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway. The term “signal sequence” refers to a nucleotide sequence that encodes the signal peptide. “Promoter” refers to a nucleotide sequence, usually upstream (5′) to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. The “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3′ direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as “minimal or core promoters.” In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator. “Constitutive expression” refers to expression using a constitutive or regulated promoter. “Conditional” and “regulated expression” refer to expression controlled by a regulated promoter. “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. “Expression” refers to the transcription and/or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein. “Transcription stop fragment” refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of terminating transcription. Examples of transcription stop fragments are known to the art. “Translation stop fragment” refers to nucleotide sequences that contain one or more regulatory signals, such as one or more termination codons in all three frames, capable of terminating translation. Insertion of a translation stop fragment adjacent to or near the initiation codon at the 5′ end of the coding sequence will result in no translation or improper translation. Excision of the translation stop fragment by site-specific recombination will leave a site-specific sequence in the coding sequence that does not interfere with proper translation using the initiation codon. The terms “cis-acting sequence” and “cis-acting element” refer to DNA or RNA sequences whose functions require them to be on the same molecule. The terms “trans-acting sequence” and “trans-acting element” refer to DNA or RNA sequences whose function does not require them to be on the same molecule. “Chromosomally-integrated” refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not “chromosomally integrated” they may be “transiently expressed.” Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.” (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a known mathematical algorithm. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (available on the world wide web at ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See the world-wide-web at ncbi.nlm.nih.gov. Alignment may also be performed manually by visual inspection. For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California). (d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. (e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, and at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. (e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence. “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tmcan be approximated from the equation of Meinkoth and Wahl: Tm81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tmis reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tmcan be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tmfor the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the Tm. Using the equation, hybridization and wash compositions, and desired temperature, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a temperature of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. Generally, highly stringent hybridization and wash conditions are selected to be about SEC lower than the Tmfor the specific sequence at a defined ionic strength and pH. An example of highly stringent wash conditions is 0.15 M NaCl at 72EC for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65EC for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45EC for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40EC for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30EC and at least about 60° C. for long probes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2×(or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. Very stringent conditions are selected to be equal to the Tmfor a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the polypeptides of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activity. The deletions, insertions, and substitutions of the polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also “conservatively modified variations.” In certain embodiments, the nucleic acid sequences are the following: Fusion protein cds, with signal peptide:(SEQ ID NO: 7)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGAGATCCAGAACATGTGGATTAGGAAACGTAAGTAGACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTTFusion protein cds, without signal peptide:(SEQ ID NO: 8)ATGGCTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT6His-plcC, with signal peptide:(SEQ ID NO: 9)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTAGACCGCCTTCCCAGACGCTTAGAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAA6His-plcC, without signal peptide:(SEQ ID NO: 10)ATGGCTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAA6His-netB, with signal peptide:(SEQ ID NO: 11)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTAGAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT6His-netB, without signal peptide:(SEQ ID NO: 12)ATGGCTCACCATCACCATCATCACGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTAGAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTAGTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTAGAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT Vectors In certain embodiments, the present invention provides a recombinant vector comprising the expression cassette described herein and a vector. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a bean yellow dwarf virus replicon. In certain embodiments, the vector is pBYR2eK2M-6HplcCnetB. (SEQ ID NO: 14 andFIG.6). A “vector” is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). “Cloning vectors” typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance. Plant Cells and Animal Feed In certain embodiments, the present invention provides a plant cell comprising the antigenic protein described herein, the nucleic acid described herein, the expression cassette described herein or the recombinant vector described herein. In certain embodiments, the plant is a corn or rice cell. In certain embodiments, the plant cell further comprises anE. coliheat-labile enterotoxin (LT) and/or a cholera toxin (CT). In certain embodiments, the present invention provides animal feed comprising the plant cell described herein. The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”. “Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. For example, “transformed,” “transformant,” and “transgenic” cells have been through the transformation process and contain a foreign gene integrated into their chromosome. The term “untransformed” refers to normal cells that have not been through the transformation process. A “transgenic” organism is an organism having one or more cells that contain an expression vector. Vaccines In certain embodiments, the present invention provides a vaccine comprising the antigenic protein described herein, the nucleic acid described herein, the expression cassette described herein, the recombinant vector described herein, the plant cell described herein, or the animal feed described herein. The fusion antigen was readily purified using metal affinity chromatography, and used for chicken immunization experiments. The data indicate that the plant-made fusion protein was immunogenic and protective. Evidence was observed on western blots that the PlcC-NetB accumulated in several glycosylated forms. A search of the PlcC-NetB amino acid sequence for consensus Asn-linked glycosylation sites (Asn-X-Ser/Thr) showed one site in the PlcC and four sites in the NetB domain. Mapping of these sites on the 3-dimensional structures of Plc and NetB showed that they mostly occur in surface loops, and thus probably would not interfere with correct folding of the proteins or impair the antigen structure of protective epitopes. In some cases, such eukaryotic glycosylation was shown to be either neutral in effect or enhance immunogenicity of plant-made antigens. However, it is difficult to predict the effects of glycosylation on the immunogenicity of PlcC-NetB. The preliminary study showed it is immunogenic in chickens, it is possible that a non-glycosylated protein will be even more potent. Thus, a new expression vector was constructed that lacks the N-terminal signal sequence, which resulted in cytosolic accumulation and thus unglycosylated antigen. The glycosylated and unglycosylated antigens are used in further studies to test immunogenicity and protection in chickens. Several mutant forms of NetB have been studied and showed reduced toxicity and may retain protective immunogenicity. Single amino acid substitutions in the rim loop region that significantly reduce its toxicity include Y191A, R200A, W257A, W262A S254L, R230Q and W287R. Some of these were shown to retain the ability to generate protective immune responses, including W262A and S254L. Thus it is reasonable to contemplate the use of multiple different mutations in the NetB component of the PlcCNetB fusion protein, in order to maximize its safety. For production of the fusion protein in seeds of corn or rice, stable transgenic lines must be developed. The expression construct would use an appropriate promoter that will drive strong expression in a seed tissue, such as embryo or endosperm tissues.Agrobacterium-mediated delivery of DNA to embryogenic cell cultures enables creation of stably transformed whole plants that transmit the transgenes to sexual progeny. One may consider the co-delivery of a mucosal adjuvant to enhance immunogenicity of the PlcC-NetB antigens. TheE. coliheat-labile enterotoxin (LT) and related cholera toxin (CT) are potent stimulators of mucosal immunity. LT and mutants thereof (including LTA S63K and A72R have been expressed in transgenic tobacco cells, and were well tolerated and immunogenic in chickens by oral or parenteral delivery. Orally immunogenic LT-B was expressed in transgenic corn; and CT-B was expressed in transgenic rice. Methods for milling and formulating corn and rice for oral delivery are well developed and convenient. Methods of Administration In certain embodiments, the present invention provides a method of protecting an avian species fromC. perfringensinfections comprising administering the vaccine described herein. In certain embodiments, the avian species is chicken, turkey, duck or ostrich. In certain embodiments, the avian is a chicken or turkey. The present invention also provides a method of protecting poultry by administering to the poultry an immunologically protective amount of a vaccine of the present invention. As used herein, the term “immunologically protective” means that the vaccine is effective in inducing a protective immune response. An immunological response to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the protein or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cell, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest. The fusion can be purified and used to inject birds. Injections can be given to hens prior to lay, to enhance immunity of chicks during the first 2-3 weeks of life by passive transfer of antibodies against the fusion protein. In certain embodiments, a suitable adjuvant is used. For example, saponin adjuvant such as Quil A, various oil emulsion adjuvants such as water in oil or water in oil in water formulations are used. The agents of the invention are preferably administered so as to result in a reduction in at least one symptom associated with a disease. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems, which are well known to the art. Administration of therapeutic agents may be accomplished through the administration of the therapeutic agent, such as a fusion protein. Pharmaceutical formulations, dosages and routes of administration for peptide are generally known. The present invention envisions treating uveitis in a mammal by the administration of an agent, e.g., a fusion protein. Administration of the therapeutic agents in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. One or more suitable unit dosage forms having the therapeutic agent(s) of the invention, which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. When the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules, as a solution, a suspension or an emulsion. Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intraocular routes. The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension. Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0 saline solutions and water. As used herein, the term “therapeutic agent” refers to a fusion protein agent or material containing the fusion protein that has a beneficial effect on the mammalian recipient. “Treating” as used herein refers to preventing infection ofC. perfringensinfection. The present invention also provides a method of protecting poultry by administering a vaccine that is effective in inducing cellular and humoral immunity and that contains a biological agent or microbial component that is effective in stimulating a protective cellular and humoral immune response toC. perfringens. The purified protein can also be used for in ovo vaccination. Again, a suitable adjuvant may be used to enhance immunogenicity, as discussed above. The vaccine of the present invention can be administered via conventional modes of administration or in ovo. Methods of in ovo immunization are set forth, for example, in U.S. Pat. No. 6,048,535. Vaccination can be performed at any age. For in ovo vaccination, vaccination would be done in the last quarter of embryonal development but may be done at any time during embryonation. The vaccines according to the invention can, for example, be administered intramuscularly, subcutaneously, orally, intraocularly, intratracheally, intranasally, in ovo, in drinking water, in the form of sprays or by contact spread. Preferably, chickens are given the first vaccine in ovo or at one day of age. Subsequent vaccinations are done according to need. Breeder chickens can be vaccinated before and during the lay cycle (several inoculations). In certain embodiments, the vaccine is administered in poultry feed. In certain embodiments, the vaccine is administered by injection. In certain embodiments, the vaccine is administered in ovo. Adjuvants Vaccines are often formulated and inoculated with various adjuvants. The adjuvants aid in attaining a more durable and higher level of immunity using small amounts of antigen or fewer doses than if the immunogen were administered alone. The mechanism of adjuvant action is complex, and may involve the stimulation of cytokine production, phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen. Suitable adjuvants include but are not limited to surfactants, e.g., hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N′—N-bis(2-hydroxyethyl-propane di-amine), methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g., muramyl dipeptide, aimethylglycine, tuftsin, oil emulsions, alum, and mixtures thereof. Other potential adjuvants include the B peptide subunits ofE. coliheat labile toxin or of the cholera toxin, and mutant forms of complete toxin in which mutations have been introduced into the A subunit ofE. coliheat labile toxin or cholera toxin that attenuate its toxicity while retaining its adjuvant properties. McGhee, J. R., et al., “On vaccine development,”Sem. Hematol.,30:3-15 (1993). Finally, the immunogenic product may be incorporated into liposomes for use in a vaccine formulation, or may be conjugated to proteins such as keyhole limpet hemocyanin (KLH) or human serum albumin (HSA) or other polymers. In certain embodiments, a saponin adjuvant such as Quil A, various oil emulsion adjuvants such as water in oil or water in oil in water formulations are used. The invention will now be illustrated by the following non-limiting Example. Example 1 Introduction Clostridium perfringens(C. perfringens) induced necrotic enteritis (NE) is becoming an economically significant problem for the broiler industry. The acute form of the disease leads to increased mortality in broiler flocks, which can account for high losses of up to 1% per day, reaching mortality rates up to 10-40%. In the subclinical form, fibrin deposits and other damage to the intestinal mucosa caused byC. perfringens(FIG.1) leads to poor productivity (reduced growth, reduced feed efficiency) without mortality.C. perfringens-infected poultry also constitutes a risk for transmission to humans through the food chain. Historically,C. perfringensoutbreaks in the broiler industry were avoided by the use of growth-promoting antimicrobials in the diet. However, concerns regarding antibiotic resistance led to restrictions on the use of antibiotics. This, coupled with high-density living conditions and the reuse of litter materials, has culminated in a resurgence ofC. perfringensinfections, estimated to cause a global economic loss of over $US2 billion annually. C. perfringensis a Gram-positive anaerobic spore-forming bacterium. At least 17 exotoxins and enzymes responsible for the associated lesions and disease symptoms have been identified.C. perfringensstrains are classified into five types (A, B, C, D and E), based on their ability to produce different combinations of four major toxins (α, β, εand ι). NE and the subclinical form ofC. perfringensinfection in poultry are caused byC. perfringenstype A strains. For many years, the chromosome-encoded alpha-toxin, a membrane active phospholipase, was considered to be the major toxin associated with NE. Alpha-toxin is composed of two domains, which are associated with phospholipase C activity (N-domain, 1-250 residues) and membrane recognition (C-domain, 251-370 residues), respectively. The C-terminal domain contributes to maintaining the active form of the toxin and mediates interactions with membrane phospholipids in a calcium-dependent manner. Individually these domains are non-toxic but immunogenic in mice resulting in the generation of antibody that reacts with the holotoxin, however, only immune responses against the C-domain provided protection against a subsequent challenge, possibly due to the blocking effects on the initial membrane-binding event. Therefore, the C-terminal domain of the alpha-toxin has been studied extensively as a vaccine againstC. perfringensinfection, delivered as a purified protein or by live attenuated bacteria. Currently the only commercially available vaccine for necrotic enteritis, Netvax®, is composed of an alpha toxoid derived from aC. perfringenstype A strain. Recent studies have identified a β-like toxin linked to necrotic enteritis, designated NetB toxin. It was identified in an AustralianC. perfringenstype A strain and has been proposed to be the most critical virulence factor for the development of NE in broilers. NetB is a pore-forming toxin encoded on a large conjugative plasmid (approximately 85 kb) within a 42 kilobase (kb) pathogenicity locus (NELoc-1), showing similarity toC. perfringensβ-toxin (38% identity). Several studies have screened for the presence of the netB gene withinC. perfringensisolates and found that the presence of netB gene is highly correlated with necrotic enteritis strains. NetB is also a protective antigen, which could provide protection againstC. perfringenschallenge, especially in combination with other immunogenic components. Results consistent with a protective role for immune responses to NetB were obtained in a study that examined serum antibody levels againstC. perfringensalpha-toxin and NetB toxin in commercial birds from field outbreaks of NE. The results showed that the levels of serum antibodies against both alpha-toxin and NetB toxin were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE, suggesting that these antitoxin antibodies may play a role in protection against NE. Their results indicate a correlation between the presence of antitoxin antibodies in the serum and protective immunity against NE. In one study, purified α-toxin C-fragment and NetB (W262A) toxoids were mixed (30 μg of each) in Quil A adjuvant and used to subcutaneously inject broiler birds 3 times, on days 3, 9 and 15. Birds injected with only one of the proteins were also included. The immunized birds were partially protected against a mild gavage challenge, but not against a more severe, in feed challenge. In some studies, hens were infected with NetB toxoid and antibodies against NetB were transferred from immunized hens to progeny, providing protection againstC. perfringenschallenge. In another study, immunization with both NetB and α-toxin toxoids using a liveSalmonelladelivery vector induced mucosal antibodies against both toxins and elicited a protective response. Strains engineered to deliver both toxoids provided significantly better protection than strains delivering each toxin alone. In the current study, the immunogenicity of a novel PlcC-NetB fusion protein was examined in broiler birds. Materials and Methods Growth ofC. perfringens. C. perfringensCP4 was cultured in cooked meat medium (CMM; Difco) and fluid thioglycollate medium (FTG; Difco). Purification of PlcC, NetB and PlcC-NetB proteins. His-tagged PlcC (Zekarias, B., H. Mo, and R. Curtiss, III. 2008. Recombinant attenuatedSalmonella entericaserovarTyphimuriumexpressing the carboxy-terminal domain of alpha toxin fromClostridium perfringensinduces protective responses against necrotic enteritis in chickens. Clin Vaccine Immunol 15:805-816) and GST-NetB (Jiang, Y., H. Mo, C. Willingham, S. Wang, J. Y. Park, W. Kong, K. L. Roland, and R. Curtiss, 3rd. 2015. Protection Against Necrotic Enteritis in Broiler Chickens by Regulated Delayed LysisSalmonellaVaccines. Avian diseases 59:475-485) proteins were prepared fromE. colias described. A fusion protein PlcC-NetB was designed comprising the following components. The PlcC component represents aa 248-370 of alpha toxin (GenBank accession AAP15462.1) (SEQ ID NO: 3). The full-length, mature (i.e., after processing) Alpha toxin (GenBank accession AAP15462.1) is the following (SEQ ID NO: 16): WDGKIDGTGTHAMIVTQGVSILENDMSKNEPESVRKNLEILKDNMHELQLGSTYPDYDKNAYDLYQDHFWDPDTNNNFSKDNSWYLAYSIPDTGESQIRKFSALARYEWQRGNYKQATFYLGEAMHYFGDIDTPYHPANVTAVDSAGHVKFETFAEERKEQYKINTVGCKTNEDFYADILKNKDFNAWSKEYARGFAKTGKSIYYSHASMSHSWDDWDYAAKVTLANSQKGTAGYIYRFLHDVSEGNDPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIK The PlcC component, which is aa 248-370 of alpha toxin (GenBank accession AAP15462.1) (SEQ ID NO: 3) is the following: DPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDEMAGSKDTYTEKLKDENLKIDDIONMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIK The NetB component represents amino acids 31-322 of NetB (GenBank accession ACN73257.1) (SEQ ID NO: 5). The full-length NetB (GenBank accession ACN73257.1) is the following (SEQ ID NO: 17): MKRLKIISITLVLTSVISTSLFSTQTQVFASELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL The NetB component, which is amino acids 31-322 of NetB (GenBank accession ACN73257.1) (SEQ ID NO: 5) is the following: SELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL The PlcC component is linked to the NetB component by the peptide linker “GGSGGSGGPSGGSGG” (SEQ ID NO: 4), with NetB on the C-terminal side. A 6His tag (HHHHHH, SEQ ID NO:2) and linker “HHHHHHGS” (SEQ ID NO: 15) is fused to the N-terminus of PlcC (FIG.2). Because the toxins are naturally secreted inC. perfringensvia a processed N-terminal signal peptide, we directed the expressed fusion protein to the endoplasmic reticulum (ER) of plant cells, reasoning that correct protein folding may be enhanced by the chaperones present in the ER. In order to target the fusion protein to the ER of plant cells, the plant signal peptide from barley alpha amylase “MANKHLSLSLFLVLLGLSASLASG” (SEQ ID NO:1) is fused to the N-terminus of the 6His tag. Examination of the sequence using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and selecting “Eukaryotes” indicates that signal peptidase cleavage is likely to occur between positions 24 and 25: ASG-HH. A plant codon-optimized coding sequence was designed to enable high expression in a tobacco relative,Nicotiana benthamiana. Codons were selected that are more frequently used in highly expressed genes of tobacco andArabidopsis(Geyer, B. C., L. Kannan, I. Cherni, R. R. Woods, H. Soreq, and T. S. Mor. 2010. Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase. Plant Biotechnol J 8:873-886). Sequences were eliminated that could specify RNA processing (splicing, polyadenylation) or destabilization. A commercial service was used for gene synthesis and cloned the fragment via XbaI at 5′ and SacI at 3′ into an expression vector based on a bean yellow dwarf virus replicon, pBYR2eK2M (Diamos, A. G., S. H. Rosenthal, and H. S. Mason. 2016. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons inNicotiana benthamianaLeaves. Front Plant Sci 7:200). The resulting construct pBYR2eK2M-6HplcCnetB was verified by DNA sequencing and transformed into the disarmedAgrobacterium tumefaciensstrain EHA105. Transient expression in leaves We performed byAgrobacterium-mediated DNA delivery. Briefly,Agrobacteriumcells were grown overnight in LB media with 50 μg/ml kanamycin and 1 μg/ml rifampicin, and then cells were collected and resuspended in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 and 10 mM MgSO4to OD600=0.2. The resulting bacterial suspensions were injected into leaves through a small puncture using a syringe without needle (Huang, Z., and H. S. Mason. 2004. Conformational analysis of hepatitis B surface antigen fusions in anAgrobacterium-mediated transient expression system. Plant Biotechnol J 2:241-249). The plants were cultured in a growth room under moderate light at 25° C. for 4 days before leaves were harvested and weighed. The leaves were extracted using a blender in 3-fold mass of buffer (phosphate buffered saline pH 7.5 (PBS), 50 mM sodium ascorbate, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100), and insoluble debris was removed by centrifugation (10,000×g, 4° C., 15 min). The supernatant was collected and 1 M phosphoric acid was added while stirring at 4° C. until the pH=4.8, and then 1 M Tris base was added until the supernatant reached pH=7.5. Precipitated material was removed by centrifugation (10,000×g, 4° C., 15 min), and the supernatant containing recombinant PlcC-NetB was subjected to metal affinity chromatography, using Talon® affinity resin (http://www.clontech.com). Bound protein was eluted by washing the column with 150 mM imidazole, and fractions were assayed by absorbance at 280 nm. Combined fractions with the highest protein content were dialyzed against PBS, pH 7.5, and the A280was measured. Protein concentration was calculated using the theoretical extinction coefficient based on the amino acid sequence of the fusion protein. The ER-targeted construct resulted in high expression and accumulation of soluble PlcC-NetB fusion protein, which was verified by western blotting using anti-PlcC and anti-NetB antisera (data not shown). The fusion antigen was readily purified using metal affinity chromatography. Detection of Antibody Response by Enzyme-Linked Immunosorbent Assay (ELISA) ELISAs were performed in triplicate as described (Jiang, Y., Q. Kong, K. L. Roland, and R. Curtiss, 3rd. 2014. Membrane vesicles ofClostridium perfringenstype A strains induce innate and adaptive immunity. International journal of medical microbiology: IJMM 304:431-443) to determine the titer of IgY r against PlcC, NetB and PlcC-NetB in chicken sera. Nunc Immunoplate Maxisorb F96 plates (Nalge Nunc, Rochester, NY) were coated overnight at 4° C. with purified proteins at 100 ng/well suspended in sodium carbonate-bicarbonate buffer (pH 9.6). The plates were blocked with Sea Block blocking buffer (Fisher). Sera from individual birds were serially diluted in 2-fold steps from an initial dilution of 1:10 in PBS, respectively. After 1 h incubation at 37° C., wells were washed three times with PBS-0.05% Tween-20. The plates were incubated with biotinylated IgY (Southern Biotech) antibodies diluted 1:10,000 for 1 h at 37° C. Then streptavidin horseradish peroxidase conjugate (Southern Biotech) was added at a 1:4,000 dilution. 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, KPL, Inc) was then added to develop the reaction. Color development (absorbance) was recorded at 405 nm using a SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices, LLC). Endpoint titers were expressed as the reciprocal log 2 values as the last sample dilution with an absorbance of 0.1 OD unit above that for the negative controls. Chicken Experiments. All animal experiments were conducted in compliance with the Arizona State University Institutional Animal Care and Use Committee and the Animal Welfare Act. Any chickens that had reached a pre-determined severity of clinical illness prior to the end of the experiment were humanely euthanized and necropsied. One-day-old Cornish×Rock broiler chickens were purchased from Murray McMurray Hatchery (Webster City, Iowa) and typically arrived at our facility at 2 days of age. Birds were randomly sorted and placed in pens with pine shavings on the floor. Food and water was supplied ad libitum. Experiment 1. One week old broiler birds were vaccinated subcutaneously three times at weekly intervals with 50 μg of purified PlcC-NetB fusion protein plus 50 μg of Quil A as adjuvant. The first immunization was at 1 week of age. Control birds mock-vaccinated with Quil A only. The volume was 100 μl for all inoculations. Experiment 2. Broiler birds were vaccinated subcutaneously three times at weekly intervals with 100 μg of purified PlcC-NetB fusion protein plus 50 μg of Quil A as adjuvant. Control birds mock-vaccinated with Quil A only. The volume was 100 μl for 1stand 2ndinoculations and 200 μl for 3rdinoculation due to the lower concentration of protein in that preparation. In Experiment 1, serum was taken one week after the final immunization and assayed for IgY antibodies against PlcC, NetB and PlcC-NetB fusion protein. Challenge procedure. The in-feed challenge performed as described previously (Jiang, Y., H. Mo, C. Willingham, S. Wang, J. Y. Park, W. Kong, K. L. Roland, and R. Curtiss, 3rd. 2015. Protection Against Necrotic Enteritis in Broiler Chickens by Regulated Delayed LysisSalmonellaVaccines. Avian diseases 59:475-485; Shojadoost, B., A. R. Vince, and J. F. Prescott. 2012. The successful experimental induction of necrotic enteritis in chickens byClostridium perfringens: a critical review. Vet Res 43:74). Three weeks after the first immunization, birds were challenged in-feed for 5 days withC. perfringensCP4, a virulent strain isolated from a necrotic enteritis outbreak. The day after the final challenge birds were euthanized and necropsies performed. At necropsy, intestinal tracts were examined and scored for lesions typical of necrotic enteritis. The person performing the scoring was blinded to the treatment regimen each bird received. Intestinal lesions are scored as follows: 0=no gross lesions; 1=thin or friable wall or diffuse superficial but removable fibrin; 2=focal necrosis or ulceration, or non-removable fibrin deposit, 1 to 5 foci; 3=focal necrosis or ulceration, or non-removable fibrin deposit, 6 to 15 foci; 4=focal necrosis or ulceration, or non-removable fibrin deposit, 16 or more foci; 5=patches of necrosis 2 to 3 cm long; 6=diffuse necrosis typical of field cases. Results PlcC-NetB protein production inNicotiana benthamiana. A codon-optimized gene was designed (FIG.2) for expression of PlcC-NetB inNicotiana benthamiana, and it was cloned in an expression vector based on a bean yellow dwarf virus replicon (Diamos, A. G., S. H. Rosenthal, and H. S. Mason. 2016. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons inNicotiana benthamianaLeaves. Front Plant Sci 7:200). The system uses transient expression in leaves, with amplified DNA and greatly enhanced protein expression only four days afterAgrobacterium-mediated DNA delivery. Because the toxins are naturally secreted inC. perfringensvia a processed N-terminal signal peptide, the expressed fusion protein was directed to the ER of plant cells using a barley alpha-amylase signal peptide, reasoning that correct protein folding may be enhanced by the chaperones present in the ER. The construct resulted in high expression and accumulation of soluble PlcC-NetB fusion protein, which was readily purified using metal affinity chromatography, and used for a preliminary chicken immunization experiment (see below). The data indicate that the plant-made fusion protein was immunogenic. Evidence was observed on western blots that the PlcC-NetB accumulated in several glycosylated forms (FIG.3). A search of the PlcC-NetB amino acid sequence for consensus Asn-linked glycosylation sites (Asn-X-Ser/Thr) showed one site in the PlcC and four sites in the NetB domain. Mapping of these sites on the 3-dimensional structures of Plc and NetB showed that they mostly occur in surface loops, and thus probably would not interfere with correct folding of the proteins or impair the antigen structure of protective epitopes. In some cases, such eukaryotic glycosylation was shown to be either neutral in effect or enhance immunogenicity of plant-made antigens (Boes, A., H. Spiegel, G. Edgue, S. Kapelski, M. Scheuermayer, R. Fendel, E. Remarque, F. Altmann, D. Maresch, A. Reimann, G. Pradel, S. Schillberg, and R. Fischer. 2015. Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced inNicotiana benthamianaand analysis of growth inhibitory responses in rabbits. Plant Biotechnol J 13:222-234.; Joensuu, J. J., M. Kotiaho, T. H. Teeri, L. Valmu, A. M. Nuutila, K. M. Oksman-Caldentey, and V. Niklander-Teeri. 2006. Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice. Transgenic Res 15:359-373; Yuki, Y., M. Mejima, S. Kurokawa, T. Hiroiwa, Y. Takahashi, D. Tokuhara, T. Nochi, Y. Katakai, M. Kuroda, N. Takeyama, K. Kashima, M. Abe, Y. Chen, U. Nakanishi, T. Masumura, Y. Takeuchi, H. Kozuka-Hata, H. Shibata, M. Oyama, K. Tanaka, and H. Kiyono. 2013. Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification. Plant Biotechnol J 11:799-808). However, it is difficult to predict the effects of glycosylation on the immunogenicity of PlcC-NetB. Although the preliminary study showed it is immunogenic in chickens, it is possible that a non-glycosylated protein will be even more potent. Thus, a new expression vector was constructed that lacks the N-terminal signal sequence, which resulted in cytosolic accumulation and thus unglycosylated antigen. The glycosylated and unglycosylated antigens are used in further studies to test immunogenicity and protection in chickens. Serum antibody responses to the PlcC-NetB fusion protein. Serum IgY responses against the PlcC-NetB protein were significantly higher in immunized birds compared to non-vaccinated controls (FIG.4A), indicating that PlcC-NetB is highly immunogenic. However, the protein is glycosylated (FIG.3) and some of the reacting antibodies could be against the carbohydrate moieties, which are not present in the corresponding proteins produced byC. perfringens. To examine the responses against the proteinaceous epitopes, PlcC and NetB proteins purified fromE. coliwere used as the coating antigen. Although the titers were somewhat lower, they remained significantly higher than titers from control animals, indicating that protein epitopes in the PlcC (FIG.4B) and NetB (FIG.4C) were being recognized. Protection AgainstC. perfringensChallenge. The results from both challenge experiments are summarized below in Table 1 and graphically inFIG.4. The challenge in Experiment 1 was milder than in Experiment 2, based on the fact that in Experiment 1, none of the birds in the control group received a lesion score of 5. This was due to the fact that different subclones of CP4 were used in each experiment. Interestingly, the vaccinated birds in Experiment 2 had overall healthier intestinal tracts than in Experiment 1. In Experiment 1 in which the birds received three doses of 50 μg of PlcC-NetB, after challenge, the intestines of most of the vaccinated birds displayed friability, even in the absence of fibrin. In Experiment 2, where the birds received three doses of 100 μg of PlcC-NetB, there was little friability and only scattered, removable fibrin. This is remarkable considering that the challenge was stronger in Experiment 2. These results demonstrate that the PlcC-NetB protein is highly immunogenic and protective against an in-feed challenge with a highly virulentC. perfringensstrain. TABLE 1Lesion scores in immunized and non-immunized birdsLesion ScoreAverageGroup0123456Lesion ScoreExp. 1PlcC-NetB32200000.9*Mock00343003.0Exp. 2PlcC-NetB49000000.7*Mock00134203.7*Different from controls, P = 0.0004 by Mann-Whitney test**Different from controls, P < 0.001 by Mann-Whitney testExperiment 1: n = 7, PlcC-NetB group; n = 10, mock vaccinated groupExperiment 2: n = 13, PlcC-NetB group; n = 10, mock vaccinated group Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto. All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. | 120,737 |
11857614 | DISCLOSURE OF THE INVENTION Flagellin Flagellin, a principal component of bacterial flagella, stimulates host defence in a variety of organisms, including mammals, insects, and plants. The structure and composition of bacterial flagellin has been comprehensively studied in model organisms such asS. typhimuriumandE. coli. However, there is very limited data available describing flagellin from the genusEnterococcusand in particular the speciesEnterococcus gallinarum. Flagellin can exist either as a monomeric protein or can be polymerised to form a flagellum filament. Bacterial flagellum filaments inSalmonellaare composed of approximately 20,000 flagellin protein units. Flagellum filaments form part of a fully assembled bacterial flagellum. Flagellin proteins generally contain 3-4 domains (D0, D1, D2 and D3), with the D0 and D1 domains consisting of a fold over from the N- and C-termini [31]. During flagellum filament assembly, the D0 and D1 domains are buried within the flagellum filament with the D2 and/or D3 domains facing outwards (FIG.1). Amino acid motifs within the conserved domain D1 have been shown to be required for TLR5 recognition and interaction [32-33]. The D0 domain has also been shown to affect TLR5 signalling [34]. The TLR5 recognition site is generally not accessible in the flagellar filament. Flagellin interacts more effectively with TLR5 in its monomeric form, as the TLR5 binding sites are not accessible when assembled in a filament [30]. The central region of the flagellin protein displays sequence variability and has not been studied extensively but is considered to be an antigenic and immunogenic region of the protein. The D0 and D1 domains are highly conserved, while the D2 and D3 domains are more variable [35].FIG.2is a sequence alignment of flagellin proteins from the genusEnterococcuswith the extensively studied flagella protein fromS. typhimurium. The majority of the sequence variation between the flagellin protein fromS. typhimuriumand the flagellin proteins from the genusEnterococcuslies within the central region (D2-D3 domain). The majority of the sequence variation between differentEnterococcus gallinarumstrains is also observed in this region, for example between flagellin proteins from the strains MRx0518 and DSM100110 (FIG.3). The flagellum filament is a component of the bacterial flagellum. The genes known to be involved in bacterial flagellar assembly are summarised inFIG.4. Those shaded in grey are present inEnterococcus gallinarum. The flagellin proteins are encoded by FliC, which is also interchangeably referred to herein as FlaA. The invention provides a flagellin polypeptide from the genusEnterococcusfor use in therapy. In certain embodiments, the invention provides a flagellin polypeptide from the speciesEnterococcus gallinarumfor use in therapy. In preferred embodiments, the invention provides a flagellin polypeptide from the strain MRx0518 for use in therapy. The examples demonstrate that flagellin polypeptides from the genusEnterococcuscan activate a strong TLR5 response, which shows flagellin polypeptides may be useful in therapy, and in particular useful in treating cancer. The invention provides a flagellin polypeptide from the genusEnterococcusfor use in a method of treating or preventing cancer. In certain embodiments, the invention provides a flagellin polypeptide from the speciesEnterococcus gallinarumfor use in a method of treating or preventing cancer. In preferred embodiments, the invention provides a flagellin polypeptide from the strain MRx0518 for use in a method of treating or preventing cancer. The examples demonstrate that flagellin polypeptides from the genusEnterococcusare able to induce strong TLR5 responses, which is useful in treating and preventing cancer. In particular, the examples show that flagellin polypeptides from the speciesEnterococcus gallinarumand especially strain MRx0518 produce a very high TLR5 response. In preferred embodiments, the invention provides a flagellin polypeptide from the speciesEnterococcus gallinarumor the speciesEnterococcuscasseliflavus, for use in therapy, in particular for treating or preventing cancer. In certain embodiments, the flagellin polypeptide is from the speciesEnterococcuscasseliflavus. In the most preferred embodiments, the flagellin polypeptide is from the speciesEnterococcus gallinarum. Flagellin polypeptides from the strain MRx0518 were tested in the examples. MRx0518 is a strain ofEnterococcus gallinarumbacterium deposited under accession number NCIMB 42488. Strain MRx0518 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16 Nov. 2015 as “Enterococcussp” and was assigned accession number NCIMB 42488. All microorganism deposits were made under the terms of the Budapest Treaty and thus viability of the deposit is assured. Maintenance of a viable culture is assured for 30 years from the date of deposit. During the pendency of the application, access to the deposit will be afforded to one determined by the Commissioner of the United States Patent and Trademark Office to be entitled thereto. All restrictions on the availability to the public of the deposited microorganisms will be irrevocably removed upon the granting of a patent for this application. The deposit will be maintained for a term of at least thirty (30) years from the date of the deposit or for the enforceable life of the patent or for a period of at least five (5) years after the most recent request for the furnishing of a sample of the deposited material, whichever is longest. The deposit will be replaced should it become necessary due to inviability, contamination or loss of capability to function in the manner described in the specification. In certain embodiments, the invention provides a flagellin polypeptide with at least 75% sequence identity to the flagellin polypeptide from the strain MRx0518, and use of such polypeptides in therapy, and in particular in treating or preventing cancer. The examples show that such flagellin polypeptides are especially effective. In particular, the invention provides a flagellin polypeptide having a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:1. Preferably, the flagellin polypeptide of the invention comprises or consists of SEQ ID NO:1. In other embodiments, the flagellin polypeptide has a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:2. The flagellin polypeptide of the invention may comprise or consist of SEQ ID NO:2. The examples also demonstrate that such polypeptides are useful. In certain embodiments, the invention provides a flagellin polypeptide with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO:3-42. In preferred embodiments, the flagellin polypeptide has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO:3-16. In preferred embodiments, the flagellin polypeptide is from the speciesEnterococcus gallinarum. In preferred embodiments, the flagellin of the invention binds TLR5, for example with a KDvalue of at least 10−2, at least 10−3, at least 10−4, at least 10−5, at least 10−6, at least 10−7, at least 10−8, at least 10−9, at least 10−10, or at least 10−11. Binding to TLR5 may be measured by any appropriate method known in the art, including electrochemical impedance spectroscopy (EIS), scanning electrochemical microscopy (SECM) [36], or native PAGE and gel-filtration chromatography analyses using a recombinant TLR5 protein [37]. The flagellin polypeptides of the invention can contain four domains, D0, D1, D2 and D3. In preferred embodiments, the flagellin polypeptide does not comprise a D3 domain. In certain embodiments the flagellin polypeptide of the invention consists of three domains, D0, D1, D2. The location of the D0, D1 and D2 domains inE. gallinarumflagellin polypeptide are shown inFIG.3. The predicted shape of the flagellin polypeptide from the strain MRx0518 (FliCMRx0518) is shown inFIG.5. The predicted structure of FliCMRx0518was generated using the Phyre2 tool which predicts protein structure based on homology with publicly available structures [38] and the graphic was generated with the UCSF Chimera package [39]. The predicted lack of D3 domain leads to a conformation change in the protein structure compared to flagellin polypeptide fromS. typhimuriumSL1344 (GenBank Accession No. CBW17983). The predicted lack of a D3 domain may lead to the TLR5 recognition site being more exposed, which leads to an increase in the ability of the flagellin polypeptide to activate a TLR5 response. The predicted lack of a D3 domain may also affect the ability of flagellin polypeptide polymerise into a flagellum filament. Flagellin polypeptide activates TLR5 responses more effectively in the monomeric form, therefore preventing or reducing the polymerisation of flagellin polypeptides is beneficial. The flagellin polypeptides of the invention may contain a D1 domain, which may be required for TLR5 recognition and interaction. In certain embodiments, the invention provides a flagellin polypeptide with a D1 domain that has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to residues 43-164 and 273-317 in SEQ ID NO:1. In certain embodiments, the invention provides a flagellin polypeptide with a TLR5 recognition site in the D1 domain. The TLR recognition site in flagellin polypeptides from the speciesEnterococcus gallinarumis located at position 87-96 and 290-295 in SEQ ID NO:1. In preferred embodiments, the TLR5 recognition site of the flagellin polypeptide has at least 99%, 99.5% or 99.9% identity to residues 87-96 and 290-295 in SEQ ID NO:1. In further preferred embodiments, the flagellin polypeptide comprises the TLR5 recognition site of residues 87-96 and 290-295 in SEQ ID NO:1. The flagellin polypeptide of the invention may also contains a D0 domain, which can affect TLR5 signalling. In certain embodiments, the invention provides a flagellin polypeptide with at D0 domain that has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to residues 2-32 and 234-358 in SEQ ID NO:1. The flagellin polypeptide of the invention may also contain a D2 domain. The majority of the sequence variation between the flagella polypeptides fromS. typhimuriumand the genusEnterococcuslie within the central region (D2-D3 domain). The examples show that flagellin polypeptides from the speciesEnterococcus gallinarumare able to produce stronger TLR5 response at a lower dosage compared to flagellin polypeptide fromS. typhimurium. The sequence variation in the D2 or D3 domain may contribute to the increased ability of flagellin polypeptides from the speciesEnterococcus gallinarumto activate a TLR5 response. The flagellin polypeptide of the invention also contains a D0 domain, which can affect TLR5 signalling. In certain embodiments, the invention provides a flagellin polypeptide with at D2 domain that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to residues 165-272 in SEQ ID NO:1. Flagellin polypeptide can exist as a monomer or in a polymerised form as a flagellum filament. This filament is part of the bacterial flagellum. In certain embodiments, the flagellin polypeptide of the invention is arranged in flagellum filament. The invention also relates to flagellin polypeptides not arranged in flagellum filament. In preferred embodiments, the flagellin polypeptide is in its monomeric form. The monomeric form of the flagellin polypeptide is advantageous as it can interact more strongly with TLR5. In embodiments, the flagellin of the invention is not glycosylated, to aid its shedding and disaggregation into the monomeric form. In certain embodiments, the flagellin polypeptide is part of a bacterial flagellum. In certain embodiments, the flagellin polypeptide is part of a bacterial flagellum that contains one or more proteins selected from FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS. In preferred embodiments, the bacterial flagellar assembly contains FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS. In certain embodiments, the bacterial flagellar assembly contains the proteins FliL and FlaG. FliL is flagellar basal body-associated protein, which controls the rotational direction of the flagella during chemotaxis and FlaG regulates flagellin assembly along with FlaF. FliL and FlaG proteins are present in MRx0518. The examples show that flagellin polypeptide from MRx0518 can activate a stronger TLR5 response compared to flagellin polypeptide from anotherEnterococcus gallinarumspecies. The flagellin polypeptides of the invention may be recombinantly expressed, or they may be isolated fromEnterococcuscells. In certain embodiments, the flagellin polypeptide of the invention is not attached to anEnterococcuscell. In certain embodiments, the flagellin polypeptide of the invention is in a composition that is substantially free of bacterial cells. In certain embodiments, the flagellin polypeptides of the invention have been heat treated, and optionally denatured. The examples demonstrate that such flagellin polypeptides are still potently effective. In certain embodiments, the flagellin polypeptides of the invention are heat-stable to 80° C., for example can maintain activity following heating to 80° C. for, for example, 40 mins. In certain embodiments, the flagellin polypeptides are digested by trypsin. In other words, in certain embodiments, the flagellin polypeptides contain trypsin cleavage sites. The MRX518 flagellin is predicted to contain 36 trypsin cleavage sites. A flagellin polypeptide according to the invention does not have to function as a normal flagellin polypeptide in order to be considered a flagellin polypeptide. A flagellin polypeptide according to the invention may contain mutations or deletions that ablate certain activities. Generally, a flagellin polypeptide of the invention is a TLR5 agonist. In certain embodiments, the flagellin polypeptides of the invention are not able to polymerise into a flagellum filament. In other embodiments, the flagellin polypeptides of the invention are not able to form part of the bacterial flagellar assembly. In certain embodiments, the polypeptides of the invention are not flagellin polypeptides. In preferred embodiments, the polypeptides have sequence identity to SEQ ID NO:1, as set out above, or comprise or consist of SEQ ID NO:1. The invention also provides compositions comprising a flagellin polypeptide from the genusEnterococcusfor use in therapy. In certain embodiments, the composition comprises a flagellin polypeptide from the speciesEnterococcus gallinarumfor use in therapy. In preferred embodiments, the composition comprises a flagellin polypeptide from the strain MRx0518 for use in therapy. In alternative aspects of every embodiment of the invention, the flagellin polypeptide of the invention is of the speciesEnterococcus caselliflavus. Enterococcus caselliflavusis highly similar toEnterococcus gallinarumand is also flagellated. Fragments Flagellin polypeptides with sequence identity to those tested in the examples are expected to also be useful in therapy and are encompassed by the invention, as set out in the preceding section. In addition, fragments of flagellin polypeptides of the invention are also expected to be useful. Preferably, any fragment is at least 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or 350 amino acids in length. Preferred fragments comprise one or more of the following stretches of amino acids of SEQ ID NO:1: 2-32, 87-96, 165-272, 234-358, 290-295, or comprise a sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity to one of said fragments. Preferred fragments comprise a sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity to amino acids 165-272 of SEQ ID NO:1. Further preferred fragments comprise a sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity to amino acids 87-96, 165-272 or 290-295 of SEQ ID NO:1, or preferably to amino acids 87-96, 165-272 and 290-295 of SEQ ID NO:1. A fragment for use according to the invention will usually (i) have at least w % sequence identity to SEQ ID NO: 1 and/or (ii) comprise of a fragment of at least x contiguous amino acids from SEQ ID NO: 1. The value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more). The value of x is at least 7 (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300) and the fragment will usually include an epitope from SEQ ID NO: 1. The fragment will usually be able to bind to TLR5. Other fragments for use according to the invention will usually (i) have at least w % sequence identity to SEQ ID NO: 2 and/or (ii) comprise of a fragment of at least x contiguous amino acids from SEQ ID NO: 2. The value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more). The value of x is at least 7 (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300) and the fragment will usually include an epitope from SEQ ID NO: 2. The fragment will usually be able to bind to TLR5. Other fragments for use according to the invention will usually (i) have at least w % sequence identity to one of SEQ ID NO: 3-42 and/or (ii) comprise of a fragment of at least x contiguous amino acids from one of SEQ ID NO: 3-42. The value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more). The value of x is at least 7 (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300) and the fragment will usually include an epitope from one of SEQ ID NO: 3-42. The fragment will usually be able to bind to TLR5. Fusions Fusions comprising the flagellin polypeptides of the invention are also expected to be useful in therapy. Therefore, in certain embodiments, the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above, optionally for use in therapy, and in particular for use in treating cancer. Preferred fusion partners include targeting moieties and moieties intended to prolong half-life. In certain embodiments the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above and a polypeptide selected from the group consisting of an Fc region, transferrin, albumin, recombinant PEG, a homoamino acid polymer, a proline-alanine-serine polymer, an elastin-like peptide, or carboxy-terminal peptide [CTP; of chorionic gonadotropin(CG) b-chain]. The flagellin polypeptides of the invention may also be fused or conjugated to non-polypeptide moieties in order to improve their characteristics and therapeutic utility. For example, in certain embodiments, the invention provides a flagellin polypeptide as described above covalently linked to PEG or hyaluronic acid. The flagellin polypeptides of the invention may be fused or conjugated to an antigen, for example, for use as a vaccine. Presenting an antigen in close proximity to the flagellin of the invention may maximise their immunostimulatory activities and further enhance the protective immune response generated against the antigen. In addition, manufacturing and delivering therapeutics comprising an antigen and a flagellin of the invention may be more efficient and effective when they are fused or conjugated. Therefore, in certain embodiments, the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above and an antigen. In further embodiments, the invention provides a flagellin polypeptide as described above conjugated to an antigen. Preferred antigens for including in the fusions and conjugates of the invention are pathogen antigens and tumour antigens. An antigen will elicit an immune response specific for the antigen that will be effective for protecting against infection with the pathogen or attacking the tumour. Antigens may be, for example, peptides or polysaccharides. Exemplary antigens for use with the fusions and conjugates of the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. Further antigens for use with the fusions and conjugates of the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or opiates. Fusion polypeptides of the invention may include a linker sequence between the flagellin and the antigen. Preferred linker sequences include peptides that comprise (Gly4)n motif(s) (SEQ ID NO: 50), a (Gly4Ser)n motif(s) (SEQ ID NO: 51), and Ser(Gly4Ser)n motif(s) SEQ ID NO: 52). Linker sequences may be 1-50, 1-30, 1-20, 5-20, 5-10 or 10-20 amino acids in length. Linker sequences will allow the flagellin and the fusion partner, for example the antigen, to perform their roles and, for example, be accessed by the immune system. Therefore, in certain embodiments, the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above, a linker sequence, and an antigen, such as an antigen derived from a pathogen, or a tumour antigen. Flagellin polypeptides of the invention may be particularly useful when conjugated to non-polypeptide moieties such as polysaccharide antigens. Suitable conjugation methods include using carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, CDAP or TSTU. Polynucleotides The invention also provides a polynucleotide sequence that encodes a flagellin polypeptide from the genusEnterococcusfor use in therapy. In certain embodiments, the polynucleotide sequence encodes a flagellin polypeptide from the speciesEnterococcus gallinarumfor use in therapy. In preferred embodiments, the polynucleotide sequence encodes a flagellin polypeptide from the strain MRx0518 for use in therapy. The examples demonstrate that flagellin polypeptides from the genusEnterococcuscan activate a strong TLR5 response, so polynucleotides encoding such flagellin polypeptides may be useful in therapy, and in particular useful in treating cancer. The invention also provides a polynucleotide sequence that encodes a flagellin polypeptide from the genusEnterococcusfor use in a method of treating or preventing cancer. In certain embodiments, the invention provides a polynucleotide sequence that encodes a flagellin polypeptide from the speciesEnterococcus gallinarumfor use in a method of treating or preventing cancer. In preferred embodiments, the invention provides a polynucleotide sequence that encodes a flagellin polypeptide from the strain MRx0518 for use in a method of treating or preventing cancer. The examples demonstrate that flagellin polypeptide from the genusEnterococcusare able to induce strong TLR5 responses, which is useful in treating and preventing cancer. In particular, the examples show that flagellin polypeptides from the speciesEnterococcus gallinarumand especially strain MRx0518 produce a very high TLR5 response. In preferred embodiments, the polynucleotide of the invention for use in therapy encodes SEQ ID NO:1, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:1. In further preferred embodiments, the polynucleotide of the invention encodes a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful. In other embodiments, the polynucleotide of the invention for use in therapy encodes SEQ ID NO:2, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:2. In further preferred embodiments, the polynucleotide of the invention encodes a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful. In other embodiments, the polynucleotide of the invention for use in therapy encodes one of SEQ ID NO: 3-42, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO: 3-42. In further preferred embodiments, the polynucleotide of the invention encodes a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful. Nucleic acid according to the invention can take various forms (e.g. single stranded, double stranded, vectors etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2′,3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. Nucleic acids may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc. The nucleic acids of the invention comprise a sequence which encodes at least one polypeptide of the invention, such as a flagellin polypeptide of the invention. Typically, the nucleic acids of the invention will be in recombinant form, i.e. a form which does not occur in nature. For example, the nucleic acid may comprise one or more heterologous nucleic acid sequences (e.g. a sequence encoding another antigen and/or a control sequence such as a promoter or an internal ribosome entry site) in addition to the sequence encoding at least flagellin polypeptide. In such embodiments, the nucleic acid of the invention encodes a flagellin polypeptide as discussed above and an antigen, such as a pathogen antigen or a tumour antigen. Optionally, the nucleic acid also encodes a linker sequence. Host Cells The invention also provides host cells expressing a flagellin polypeptide of the invention, for use in therapy. The examples demonstrate thatEnterococcusstrains such as MRx0518, which expressEnterococcusflagellin of the invention, can activate TLR5 and therefore may have potent therapeutic effects. Host cells expressing flagellin polypeptides of the invention may be useful for producing flagellin polypeptide for inclusion in a therapeutic composition, or such host cells may be useful for administering to a patient, wherein they express the flagellin polypeptide to exert a direct therapeutic effect. In preferred embodiments, the host cell of the invention expresses the flagellin polypeptide of the invention recombinantly. In other words, the host cell of the invention comprises a heterologous polynucleotide sequence that encodes the flagellin polypeptide of the invention. In certain embodiments, the host cell is not of the genusEnterococcus, is not of the speciesEnterococcus gallinarum, or is not of the strain MRx0518. In preferred embodiments, the host cell for expressing recombinantEnterococcusflagellin polypeptides isE. coli. In alternative embodiments, wherein for example the host cell is for administration to a patient, the host cell is a probiotic strain, such as aLactobacillusorBifidobacteriumstrain. In certain embodiments, the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the genusEnterococcus. In certain embodiments, the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the speciesEnterococcus gallinarum. In preferred embodiments, the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the strain MRx0518. In certain embodiments, the host cell comprises a vector, wherein the vector comprises a polynucleotide sequence that encodes a flagellin polypeptide from the genusEnterococcus. In certain embodiments, the host cell comprises a polynucleotide sequence that encodes one or more of, preferably all of, the bacterial flagellar assembly genes FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS. In certain embodiments, the host cell of the invention expresses one or more of, preferably all of, the polypeptides FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS. In preferred embodiments, the host cell comprises a polynucleotide sequence that encodes a bacterial flagellar assembly comprising FliL and FlaG. In preferred embodiments, the host cell of the invention expresses the polypeptides FliL and FlaG. Preferably, any cell expressing a flagellin polypeptide of the invention, and in particular any cell for administration to a patient, is effective at shedding flagellin polypeptide. The examples demonstrate that such cells induce a stronger TLR5 response. Flagellin can be “actively” shed during flagellum assembly, through leakage of its monomeric units. However, in most cases monomeric flagellin arise from the passive degradation of detached flagella. Flagellar detachment can occur following mechanical strain or as a voluntary process. In some bacterial species flagellin is glycosylated to enhance filament stability and prevent uncontrolled shedding, for example inShewanella oneidensis[40]. In preferred embodiments, the flagellin expressed by the host cell is not glycosylated, to aid its shedding and disaggregation into the monomeric form. In certain embodiments, the flagellin polypeptide and/or the bacterial flagellar assembly of the invention is easily shed from a host cell. The ease of shedding of flagellin polypeptides and/or flagellar assemblies can be determined by separately growing i) cells expressing the flagellin of the invention and ii) one or more cells (e.g. 1, 2, 3, 4, 5, 10 or more than 10) expressing reference flagellins derived from the same or different strains belonging to the same bacterial species as the flagellin of the invention a) in a stationary phase for 18 hours using a 1% inoculum of the respective cells, then b) a late-log phase in which 10% inoculum of cells from the stationary phase are grown for 3 hours. 10 ml cultures of the cells expressing the flagellin of the invention and of the cells expressing reference flagellins can then be centrifuged at 5000×g for 5 minutes at room temperature to remove the bacterial pellet. The resulting supernatants can then be filtered (0.22 um) under sterile conditions. In embodiments in which the flagellin polypeptide and/or the bacterial flagellar assembly of the invention is a good shedder, the concentration of the flagellin protein in the supernatant derived from cells expressing the flagellin of the invention will be higher (e.g. 1.5× or higher, 2× or higher, 3× or higher, 4× or higher, 5× or higher, 7× or higher or 10× or higher) than the concentration of flagellin protein in the supernatants derived from the cells expressing reference flagellins. In preferred embodiments, the host cell of the invention releases flagellin polypeptides and/or flagellar assemblies into its environment with requiring chemical or physical disruption. In preferred embodiments, culturing a host cell of the invention under conditions suitable for expression of the flagellin polypeptide results in flagellin polypeptide being released from the cell into its environment. The examples demonstrate that cells expressing flagellin polypeptides of the invention induce a stronger TLR5 response. In other embodiments, the host cells of the invention are good shedders of the bacterial flagellar assembly. In certain embodiments, the host cells of the invention are good shedders of bacterial flagellum filaments and in particular good shedders of flagellin polypeptides. In certain embodiments, the bacterial flagellar assembly of the invention actively sheds monomeric flagellin. In certain embodiments, the flagellum filament of the invention actively sheds monomeric flagellin. Monomeric flagellin is more effective at inducing TLR5 responses. In certain embodiments, the invention provides a method for obtaining a flagellin polypeptide or flagellar assembly of the invention comprising culturing a cell expressing a flagellin polypeptide or flagellar assembly of the invention and culturing said flagellin polypeptide or flagellar assembly, without performing any step to remove separate flagellin polypeptide or flagellar assembly from the cell surface. The process of flagellum (or flagellin) shedding is not currently associated with the expression of a specific subset genes and is likely to be heavily influenced by environmental conditions. However, a number of genes have been shown to be involved in flagellum rigidity and stability. For example inSalmonella, fliF and fliL mutants have been shown to shed flagella more easily when grown in viscous media [41,42]. In both cases, flagella broke near the distal rod [43]. Therefore, in preferred embodiments, the host cell does not express fliF and/or fliL. In other embodiments, the host cell does express fliL. In certain embodiments, the host cells of the invention have been heat treated. The examples demonstrate that cells expressing flagellin polypeptides of the invention are still potently effective after heat treatment. In certain embodiments, the host cells of the invention are heat-stable to 80° C., for example can maintain activity following heating to 80° C. for, for example, 40 mins. The invention further provides methods of producing the flagellin polypeptides of the invention by culturing the host cells under conditions permitting production of the flagellin polypeptides, and recovering the flagellin polypeptides so produced. In certain embodiments, the host cell of the invention additionally expresses one or more antigens. Generally the antigen will be expressed recombinantly and will be heterologous to the host cell. The antigen may be part of a fusion polypeptide expressed with the flagellin polypeptide of the invention, or the host cell may separately express the flagellin and the antigen from different open reading frames. Therefore, the invention provides a host cell that expresses a flagellin polypeptide of the invention and a heterologous antigen. Exemplary antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. Further antigens for expressing in a host cell with a flagellin of the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or opiates. Vectors, Plasmids and Other Nucleic Acids The nucleic acid of the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or “viral vectors” which are designed to result in the production of a recombinant virus or virus-like particle. Alternatively, or in addition, the sequence or chemical structure of the nucleic acid may be modified compared to a naturally-occurring sequence which encodes a flagellin polypeptide. The sequence of the nucleic acid molecule may be modified, e.g. to increase the efficacy of expression or replication of the nucleic acid, or to provide additional stability or resistance to degradation. For example, the sequence of the nucleic acid molecule may be codon optimized for expression in a desired host, such as a mammalian (e.g. human) cell. Such modification with respect to codon usage may increase translation efficacy and half-life of the nucleic acid. A poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3′ end of the RNA to increase its half-life. The 5′ end of the RNA may be capped with a modified ribonucleotide with the structure m7G (5′) ppp (5′) N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5′ cap of the RNA molecule may be further modified by a 2′-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2′-O]N), which may further increases translation efficacy. The nucleic acids may comprise one or more nucleotide analogs or modified nucleotides. As used herein, “nucleotide analog” or “modified nucleotide” refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)). A nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate. The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, e.g. from U.S. Pat. Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, 5,700,642, and many modified nucleosides and modified nucleotides are commercially available. The invention further provides recombinant expression vectors capable of expressing a flagellin polypeptide. For example, the invention provides recombinant expression vectors comprising any of the nucleic acid molecules mentioned above. The invention further provides host cells into which any of the vectors mentioned above have been introduced. The invention further provides methods of producing the flagellin polypeptides of the invention by culturing the host cells under conditions permitting production of the flagellin polypeptides, and recovering the flagellin polypeptides so produced. A composition as disclosed herein comprising a nucleic acid sequence which encodes a flagellin polypeptides may be a nucleic acid-based therapeutic. The nucleic acid may, for example, be RNA (i.e. an RNA-based therapeutic) or DNA (i.e. a DNA-based therapeutic, such as a plasmid DNA therapeutic). In certain embodiments, the nucleic acid-based therapeutic is an RNA-based therapeutic. In certain embodiments, the RNA-based therapeutic comprises a self-replicating RNA molecule. The self-replicating RNA molecule may be an alphavirus-derived RNA replicon. Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is typically a + strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded antigen (i.e. a flagellin polypeptide), or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells. One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon. These replicons are + stranded RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell. The replicase is translated as a polyprotein which auto cleaves to provide a replication complex which creates genomic − strand copies of the + strand delivered RNA. These − strand transcripts can themselves be transcribed to give further copies of the + stranded parent RNA and also to give a subgenomic transcript which encodes the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons. In certain embodiments, the self-replicating RNA molecule described herein encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) a flagellin polypeptide. The polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4. The nucleic acid-based therapeutic may comprise a viral or a non-viral delivery system. The delivery system (also referred to herein as a delivery vehicle) may have adjuvant effects which enhance the immunogenicity of the encoded flagellin polypeptide. For example, the nucleic acid molecule may be encapsulated in liposomes, non-toxic biodegradable polymeric microparticles or viral replicon particles (VRPs), or complexed with particles of a cationic oil-in-water emulsion. In some embodiments, the nucleic acid-based therapeutic comprises a cationic nano-emulsion (CNE) delivery system or a lipid nanoparticle (LNP) delivery system. Alternatively, the nucleic acid-based therapeutic may comprise viral replicon particles. In other embodiments, the nucleic acid-based therapeutic may comprise a naked nucleic acid, such as naked RNA (e.g. mRNA), but delivery via LNPs is preferred. In certain embodiments, the nucleic acid-based therapeutic comprises a cationic nano-emulsion (CNE) delivery system. CNE delivery systems and methods for their preparation are described in the art. In a CNE delivery system, the nucleic acid molecule (e.g. RNA) which encodes the antigen is complexed with a particle of a cationic oil-in-water emulsion. Cationic oil-in-water emulsions can be used to deliver negatively charged molecules, such as an RNA molecule to cells. The emulsion particles comprise an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule thereby anchoring the molecule to the emulsion particles. Further details of useful CNEs can be found in the art. Thus, in a nucleic acid-based therapeutic of the invention, an RNA molecule encoding a flagellin polypeptide may be complexed with a particle of a cationic oil-in-water emulsion. The particles typically comprise an oil core (e.g. a plant oil or squalene) that is in liquid phase at 25 C, a cationic lipid (e.g. phospholipid) and, optionally, a surfactant (e.g. sorbitan trioleate, polysorbate 80); polyethylene glycol can also be included. In some embodiments, the CNE comprises squalene and a cationic lipid, such as 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP). In some preferred embodiments, the delivery system is a non-viral delivery system, such as CNE, and the nucleic acid-based therapeutic comprises a self-replicating RNA (mRNA). This may be particularly effective in eliciting humoral and cellular immune responses. Advantages also include the absence of a limiting anti-vector immune response and a lack of risk of genomic integration. LNP delivery systems and non-toxic biodegradable polymeric microparticles, and methods for their preparation are described in the art. LNPs are non virion liposome particles in which a nucleic acid molecule (e.g. RNA) can be encapsulated. The particles can include some external RNA (e.g. on the surface of the particles), but at least half of the RNA (and ideally all of it) is encapsulated. Liposomal particles can, for example, be formed of a mixture of zwitterionic, cationic and anionic lipids which can be saturated or unsaturated, for example; DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated). Preferred LNPs for use with the invention include an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.). A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is particularly effective. Other useful LNPs are disclosed in the art. In some embodiments, the LNPs are RV01 liposomes. NF-κB TLR signalling pathways culminate in the activation of the transcription factor nuclear factor-kappaB (NF-kB). NF-kB controls the expression of an array of inflammatory cytokine genes, including TNF-α. Flagellin induces the dimerization of TLR5, which subsequently recruits MyD88 and activates protein kinases, including IRAK1, IRAK2, IRAK4 and IRAK-M. The activation of these kinases leads to the nuclear localization of NF-kB, which is a proinflammatory cytokine [44]. As demonstrated in the examples (FIG.18), compositions of the invention lead to an increase in expression of NF-κB. Since administration of the compositions of the invention increase the expression of the proinflammatory cytokine NF-κB, compositions of the invention may be useful in stimulating the immune response. In addition, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and/or diseases treatable by an increased immune response. In one embodiment, the compositions of the invention are for use as an immune stimulant by increasing the level and/or activity of NF-κB. In one embodiment, the compositions of the invention are for use in treating diseases characterised by reduced immune activation by increasing the level and/or activity of NF-κB. In one embodiment, the compositions of the invention are for use in treating diseases treatable by an increased immune response by increasing the level and/or activity of NF-κB. In particular, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression and/or activation of NF-κB. In one embodiment, the compositions of the invention are for use in treating diseases characterised by a decrease in expression and/or activation of NF-κB The activation of NF-kB is important for eliciting innate immune responses and the subsequent development of adaptive immune responses. Thus, agonists of TLRs, such as flagellin, are likely to be useful as adjuvants to treat infectious diseases, allergies and tumours by promoting both innate and adaptive immune responses [44]. In one embodiment, the compositions of the invention are for use in treating infectious diseases, allergies and/or tumours. In one embodiment, the compositions of the invention are for use in treating infectious diseases, allergies and/or tumours by increasing the level and/or activity of NF-κB. Treating Cancer Toll-like receptors (TLRs) are membrane-bound receptors primarily expressed on innate immune cells that play key roles in both the innate and adaptive immune systems. TLRs respond to specific microbial pathogen-associated molecular patterns (PAMPs). TLRs sense bacterial cell wall components and TLR5 is known to recognise bacterial flagellin. Flagellin is the only known ligand for TLR5, a receptor protein expressed on the surface of a range of human cells, including epithelial cells, endothelial cells, macrophages, dendritic cells (DCs) and T cells [45-47]. Activation of TLR5 by bacterial flagellin leads to the activation of a variety of proinflammatory and immune response genes [15]. Activation of TLR5 byS. typhimuriumflagellin is known to suppress cell proliferation and tumour growth [48]. AS. typhimuriumstrain engineered to secreteVibrio vulnificusflagellin B (FlaB) has been shown to effectively suppress tumour growth and metastasis in mouse models and prolonged survival, through a two-step activation of the TLR4 and TLR5 signalling pathways [49]. The efficacy of flagellin polypeptides from other species to treat cancer is unknown. Assaying the ability of a compound to activate a TLR5 response shows that the compound may be effective at treating cancer. Compositions comprisingEnterococcus gallinarumstrains, and in particular the strain MRx0518, are effective at treating and preventing cancer [50]. The inventors have surprisingly shown that flagellin polypeptides from the speciesEnterococcus gallinarumare able to stimulate a strong TLR5 response, which may contribute to the anti-cancer activity ofEnterococcusstrains. Flagellin polypeptides fromEnterococcus gallinarumhave large sequence dissimilarity with flagellin polypeptides fromS. typhimurium. The examples show that flagellin polypeptides from the speciesEnterococcus gallinarumare able to produce a stronger TLR5 response than flagellin polypeptides fromS. typhimurium. Thus, the inventors have shown that flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarumare useful in therapy, and particularly effective in treating and preventing cancer. TLR5 has been shown to be expressed by a number of different cancers, including breast, colorectal and gastric cancer. The examples show that flagellin polypeptides from the genusEnterococcusare able to produce a strong TLR5 response. Therefore, in certain embodiments the invention provides flagellin polypeptides from the genusEnterococcusfor use in treating or preventing breast cancer, colorectal cancer or gastric cancer. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating or preventing breast, colorectal or gastric cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in breast, colorectal or gastric cancer. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating breast cancer. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating colorectal cancer. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating gastric cancer. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating or preventing TLR5 associated cancers. The invention provides flagellin polypeptides from the genusEnterococcusfor use in treating or preventing cancer. The invention also provides flagellin polypeptides the speciesEnterococcus gallinarumfor use in treating or preventing cancer. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO:1, for use in treating or preventing cancer. The invention also provides compositions comprising the flagellin polypeptides of the invention. The invention also provides compositions comprising a polynucleotide sequence encoding a flagellin polypeptide of the invention. The invention also provides a host cell that expresses a flagellin polypeptide of the invention. In addition, the invention also provides compositions comprising a host cell, wherein the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide of the invention. In certain embodiments, treatment with the compositions of the invention results in a reduction in tumour size or a reduction in tumour growth. In certain embodiments, the compositions of the invention are for use in reducing tumour size or reducing tumour growth. The compositions of the invention may be effective for reducing tumour size or growth. In certain embodiments, the compositions of the invention are for use in treating patients with solid tumours. In certain embodiments, the compositions of the invention are for use in reducing or preventing angiogenesis in the treatment of cancer. The compositions of the invention may have an effect on the immune or inflammatory systems, which have central roles in angiogenesis. In certain embodiments, the compositions of the invention are for use in preventing metastasis. In certain embodiments, the compositions of the invention are for use in treating or preventing breast cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against breast cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against breast cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer. In certain embodiments, the compositions of the invention are for use in treating or preventing lung cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against lung cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against lung cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer. In preferred embodiments the cancer is lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating or preventing liver cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against liver cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against liver cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of liver cancer. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma). In certain embodiments, the compositions of the invention are for use in treating or preventing colorectal cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against colorectal cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against colorectal cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colorectal cancer. In preferred embodiments the cancer is colorectal adenocarcinoma. In certain embodiments, the compositions of the invention are for use in treating or preventing colon cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against colon cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against colon cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colon cancer. In preferred embodiments the cancer is colon adenocarcinoma. In certain embodiments, the compositions of the invention are for use in treating or preventing rectal cancer. Reference [50] demonstrates thatEnterococcusstrains have a potent effect against rectal cancer, so following the data in the present application, flagellin polypeptides of the invention may be particularly effective against rectal cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of rectal cancer. In preferred embodiments the cancer is rectal adenocarcinoma. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating rectal cancer. In certain embodiments, the compositions of the invention are for use in treating or preventing gastric cancer. Gastric carcinomas have been show to express high levels of TLR5 and that treatment with flagellin elicits a potent anti-tumour activity [51]. The examples show that the flagellin polypeptides of the invention are able to produce a strong TLR5 response, therefore flagellin polypeptides of the invention may be particularly effective against gastric cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of gastric cancer. In preferred embodiments the cancer is gastric adenocarcinoma. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating gastric cancer. In certain embodiments, the compositions of the invention are for use in treating or preventing melanoma. Flagellin fromS. typhimurium, in combination with the major histocompatibility complex class II-restricted P10 peptide, has been shown to reduce the number of lung metastasis in a murine melanoma model [52]. The examples show that the flagellin polypeptides of the invention are able to produce a stronger TLR5 response thanS. typhimurium, therefore flagellin polypeptides of the invention may be particularly effective against melanoma. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of melanoma. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating melanoma. In certain embodiments, the compositions of the invention are for use in treating or preventing neuroblastoma. Activation of TLR5 byS. typhimuriumflagellin is known to suppress cell proliferation and tumour growth [53]. The examples show that the flagellin polypeptides of the invention are able to produce a stronger TLR5 response thanS. typhimurium, therefore flagellin polypeptides of the invention may be particularly effective against neuroblastoma. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of neuroblastoma. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating neuroblastoma. In some embodiments, the cancer is of the intestine. In some embodiments, the cancer is of a part of the body which is not the intestine. In some embodiments, the cancer is not cancer of the intestine. In some embodiments, the cancer is not colorectal cancer. In some embodiments, the cancer is not cancer of the small intestine. In some embodiments, the treating or preventing occurs at a site other than at the intestine. In some embodiments, the treating or preventing occurs at the intestine and also at a site other than at the intestine. Expression of TLR5 has been detected in haematological cancer cell lines, including multiple myeloma [54] and acute myeloid leukaemia [55]. The inventors have demonstrated in the examples that flagellin polypeptides from the genusEnterococcusare particularly effective at activating a strong TLR5 response. Flagellin polypeptides from the genusEnterococcusmay therefore be useful at treating or preventing haematological cancers, such as multiple myeloma and acute myeloid leukaemia, which express TLR5. Therefore, in certain embodiments the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating or preventing haematological malignancies, such as multiple myeloma, acute and chronic leukemias such as acute myeloid leukaemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and acute monocytic leukemia, lymphomas, such as Hodgkin's lymphomas and Non-Hodgkin's lymphomas, and myelodysplastic syndromes. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating acute myeloid leukaemia. In certain embodiments the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating or preventing multiple myeloma. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating or preventing acute myeloid leukaemia. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating multiple myeloma. Various TLR agonists have been tested in a wide array of tumour models and clinical trials [56]. For example, the TLR5 agonist CBLB502 has been shown to promote tumour clearance in mice models of T and B cell lymphomas [57]. Systemic administration of the TLR5 agonist entolimod (a pharmacologically optimized flagellin derivative) suppressed liver metastasis of colorectal cells in mice models. The liver shows the strongest TLR5 activation response following the administration of the TLR5 agonist entolimod [58]. The inventors have demonstrated in the examples that flagellin polypeptides from the genusEnterococcusact as strong TLR5 agonists. Therefore, flagellin polypeptides from the genusEnterococcusmay be particularly effective at treating or preventing T and B cell lymphomas, colorectal and liver cancer. In certain embodiments, the compositions of the invention are for use in treating or preventing T and B cell lymphomas. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating T and B cell lymphomas. In other embodiments, the compositions of the invention may be useful in treating or preventing metastasis. In other embodiments, the compositions of the invention may be useful in treating metastatic cancer. In other embodiments, the compositions of the invention may be useful in treating advanced cancer. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genusEnterococcusfor use in treating, reducing or preventing metastasis. In certain embodiments, the compositions of the invention may decrease or prevent metastatic growth. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating or preventing metastasis. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating or preventing metastatic cancer. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarum, for use in treating or preventing advanced cancer. In certain embodiments, the compositions of the invention are for use in treating or preventing carcinoma. Vibrio vulnificusflagellin B (FlaB) has been shown to effectively suppress tumour growth through a two-step activation of the TLR4 and TLR5 signalling pathways. In certain embodiments, the flagellin polypeptides of the invention can activate the TLR4 and TLR5 signalling pathways. In other embodiments the flagellin polypeptides of the invention only activate the TLR5 signalling pathway. In certain embodiments, the flagellin polypeptides of the invention can activate NFκB signalling pathways. In certain embodiments, host cells of the invention can activate the TLR9 signalling pathways. Reference [59] shows that synergy between flagellin and the TLR9 agonist CpG-containing oligodeoxynucleotides leads to tumour suppression. In certain embodiments, the host cells of the invention of the invention can activate the TLR5 and TLR9 signalling pathways. In certain such embodiments, the composition of the invention comprises CpG-containing oligodeoxynucleotides or is administered in combination with CpG-containing oligodeoxynucleotides. The therapeutic effects of the compositions of the invention on cancer may be mediated by a pro-inflammatory mechanism. Activation of TLR5 by flagellin initiates a proinflammatory signal cascade. Reference [59] shows that the interaction ofS. typhimuriumflagellin with highly immunogenic tumours induces a Th1 response and suppression of Tregs, resulting in the inhibition of tumour growth. In contrast, the growth rate of weakly immunogenic tumours was not affected by flagellin administration. Therefore, in certain embodiments, the flagellin polypeptides and compositions of the invention are for use in treating immunogenic tumours, in particular highly immunogenic tumours. In certain embodiments, the compositions of the invention are for use in promoting inflammation in the treatment of cancer. In preferred embodiments, the compositions of the invention are for use in promoting Th1 inflammation in the treatment of cancer. Th1 cells produce IFNγ and have potent anti-cancer effects [60]. In certain embodiments, the compositions of the invention are for use in treating an early-stage cancer, such as a cancer that has not metastasized, or a stage 0 or stage 1 cancer. Promoting inflammation may be more effective against early-stage cancers [60]. Reference [59] shows that administration of flagellin after tumour transplantation significantly inhibited growth of antigenic tumours. The differing effects of flagellin on tumour growth are correlated with the immune response that is induced. Flagellin administration after transplantation was associated with an increased IFN-γ:IL-4 ratio and the decreased frequency of CD4+CD25+T regulatory cells. Therefore, in certain embodiments, the flagellin polypeptides and compositions of the invention increase the IFN-γ:IL-4 ratio. In certain embodiments, the flagellin polypeptides and compositions of the invention decrease the frequency of CD4+CD25+T regulatory cells. In other embodiments, the flagellin polypeptides and compositions of the invention do not decrease the IFN-γ:IL-4 ratio and increase CD4+CD25+ T cell frequency. Inflammation can have a cancer-suppressive effect [60] and pro-inflammatory cytokines such as TNFα are being investigated as cancer therapies [61]. The up-regulation of genes such as TNF may indicate that the compositions of the invention may be useful for treating cancer via a similar mechanism. The up-regulation of CXCR3 ligands (CXCL9, CXCL10) and IFNγ-inducible genes (IL-32) may indicate that the compositions of the invention elicit an IFNγ-type response. IFNγ is a potent macrophage-activating factor that can stimulate tumirocidal activity [62], and CXCL9 and CXCL10, for example, also have anti-cancer effects [63-65]. In certain embodiments, the compositions of the invention are for use in promoting inflammation to enhance the effect of a second anti-cancer agent. In certain embodiments, the treatment or prevention of cancer comprises increasing the level of expression of one or more cytokines. For example, in certain embodiments, the treatment or prevention of cancer comprises increasing the level of expression of one or more of IL-1β, IL-6 and TNF-α, for example, IL-1β and IL-6, IL-1β and TNF-α, IL-6 and TNF-α or all three of IL-1β, IL-6 and TNF-α. Increases in levels of expression of any of IL-1β, IL-6 and TNF-α are known to be indicative of efficacy in treatment of cancer. In certain embodiments, the compositions of the invention are for use in treating a patient that has previously received chemotherapy. In certain embodiments, the compositions of the invention are for use in treating a patient that has not tolerated a chemotherapy treatment. The compositions of the invention may be particularly suitable for such patients. In certain embodiments, the compositions of the invention are for preventing relapse. The compositions of the invention may be suitable for long-term administration. In certain embodiments, the compositions of the invention are for use in preventing progression of cancer. In certain embodiments, the compositions of the invention are for use in treating non-small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating squamous-cell carcinoma. In certain embodiments, the compositions of the invention are for use in treating adenocarcinoma. In certain embodiments, the compositions of the invention are for use in treating glandular tumors, carcinoid tumors, or undifferentiated carcinomas. In certain embodiments, the compositions of the invention are for use in treating hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection. In certain embodiments, the compositions of the invention are for use in treating invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma. In further embodiments, the compositions of the invention are for use in treating or preventing acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer. The compositions of the invention may be particularly effective when used in combination with further therapeutic agents. The immune-modulatory effects of the compositions of the invention may be effective when combined with more direct anti-cancer agents. Therefore, in certain embodiments, the invention provides a composition comprising a flagellin polypeptide from the genusEnterococcus(or encoding polynucleotide or expressing host cell) and an anticancer agent. In preferred embodiments the anticancer agent is an immune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostatic drug. Preferably the anti-cancer agent is Keytruda (pembrolizumab, Merck). In further preferred embodiments, the composition comprises an anti-cancer agent selected from the group consisting of: Yervoy (ipilimumab, BMS); Opdivo (nivolumab, BMS); MEDI4736 (AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/MedImmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; CellDex Therapeutics); TRX-518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (Jounce Therapeutics); ARGX-115 (arGEN-X); NLG-9189 (indoximod, NewLink Genetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ); NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (Eli Lilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics); Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women's University, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre); Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto (blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva (obinutuzumab, Roche/Biogen); Kadcyla (ado-trastuzumab emtansine, Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris (brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK); Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-I131, GSK); Zevalin (ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab, Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab (Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Elotuzumab (Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C (Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38 (Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affimed); AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS-22ME (Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab, Alexion); ALT-836 (Altor Bioscience); ALT-801 (Altor Bioscience); ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232 (adecatumumab, Amgen); AMG211 (Amgen/MedImmune); BAY20-10112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen); MEDI575 (AZ/MedImmune); MEDI3617 (AZ/MedImmune); MEDI6383 (AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox (AZ/MedImmune); MEDI565 (AZ/MedImmune); MEDI0639 (AZ/MedImmune); MEDI0680 (AZ/MedImmune); MEDI562 (AZ/MedImmune); AV-380 (AVEO); AV203 (AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer); vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (Boehringer Ingleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioClin); BIIB015 (Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206 (Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-010t (Biosceptre); MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB); CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (Daiichi Sankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo); nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (Daiichi Sankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai); MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358 (Eli Lilly); LY2812176 (Eli Lilly); LY3012217 (Eli Lilly); LY2495655 (Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806 (Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IMC-TR1 (Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab (Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five Prime Therapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five Prime Therapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed); IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745 (Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab (GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen); IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox (Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics); IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab (Immunomedics); Clivatuzumab (Immunomedics); IPH41 (Innate Immunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab, Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios); mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science); Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab (Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646 (dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono); DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574 (Morphosys); BPC-1C (ensituximab, Precision Biologics); TAS266 (Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031 (Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355 (Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK); OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumab ozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer); PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab, Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221, RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636, RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160 (imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab), RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi); SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212 (Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (Seattle Genetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A (Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (Seattle Genetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab (Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845 (Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YM Biosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10 TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics); KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (Bellicum Pharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (Unum Therapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101 (Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences); Colo-Adl (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102 (Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, Jennerex Biotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene); HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienX010 (OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001 (Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin, oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide, 5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer), Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma), Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib (Chugai), LDK378 (Novartis), Genetespib (Synta Pharma), Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib (ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer); Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene); Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMIVIU-132 (Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis); Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (Taiho Pharmaceuticals); and TS-1 (Taiho Pharmaceuticals). In further preferred embodiments, flagellin polypeptides are administered in combination (contemporaneously or sequentially) with CpG-containing oligodeoxynucleotides, which have shown useful effects with other flagellins [59]. In some embodiments, a flagellin polypeptide from the genusEnterococcusis the only therapeutically active agent(s) in a composition of the invention. Protection Against Toxic Chemical, Pathogen or Radiation Exposure In certain embodiments, the invention provides a flagellin polypeptide from the genusEnterococcus, preferably a flagellin polypeptide from the speciesEnterococcus gallinarum, preferably a flagellin polypeptide from the strain MRx0518, for use in preventing or treating symptoms associated with toxic chemical, pathogen or radiation exposure. The flagellin of the invention may be administered to subjects at risk of toxic chemical, pathogen or radiation exposure, or subjects recently exposed to toxic chemicals, pathogens or radiation, for example subjects exposed 1, 2, 3, 4, or 5 hours previously. Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. One strategy to protect human populations is to activate innate host defence pathways, particularly those that rapidly induce cytoprotective and/or antimicrobial gene expression. A lot of work in this area has focused on the TLR4 agonist LPS/endotoxin, however LPS administration can lead to severe inflammatory pathologies, including sepsis or severe lung injury. In addition, LPS is a poor stimulator of epithelial cells, which are the first cells that interact with the pathogen or chemical challenge. Flagellin stimulates host defence in a variety of organisms, including mammals, insects, and plants. In contrast to LPS, flagellin is a potent activator of innate immune signalling pathways in epithelial cells but has generally observed to be a poor activator of hemopoietic cells, such as macrophages and dendritic cells.S. typhimuriumflagellin has been observed to not stimulate significant levels of “master” proinflammatory cytokines, such as TNF-α, that mediate the adverse effects associated with LPS.S. typhimuriumflagellin has been observed to activate TLR5 mediated innate immunity in mice, protecting the mice against chemical, pathogen or radiation exposure [66]. Administration ofS. typhimuriumflagellin has been shown to protect against radiation exposure via a mechanism requiring TLR5 [66]. Optimal protection was observed when flagellin was administered prophylactically 2 hours before radiation, but a significant degree of protection was still observed if the flagellin was administered up to 4 h following irradiation. The examples show that flagellin polypeptides from the genusEnterococcusare able to stimulate a stronger TLR5 response thanS. typhimurium. Thus, the inventors have shown that flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarummay be useful as radioprotective agents. The invention provides flagellin polypeptides from the genusEnterococcusfor use as radioprotective agents. The invention also provides flagellin polypeptides the speciesEnterococcus gallinarumfor use as radioprotective agents. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO:1, for use as radioprotective agents. Treatment with the TLR5 agonist Entolimod has also been shown to reduce radiation-induced damage in all three of the major types of radiosensitive tissues: hemato-poietic, gastrointestinal and skin. It has also been shown to mitigate radiation-induced epithelial damage in a mouse model of head and neck radiation [67]. The examples show that flagellin polypeptides from the genusEnterococcusact as a potent TLR5 agonist. Thus, the inventors have shown that flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarummay be useful at reducing radiation-induced damage. In certain embodiments, compositions comprising flagellin polypeptides from the genusEnterococcusare for use in reducing radiation-induced damage in hemato-poietic, gastrointestinal or skin tissues. In certain embodiments, compositions comprising flagellin polypeptides from the genusEnterococcusare for use in reducing radiation-induced epithelial damage. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the speciesEnterococcus gallinarumfor use in reducing radiation-induced damage. In certain embodiments, the composition of the invention may be administered, preferably orally, prior to radiotherapy. In certain embodiments, the composition of the invention may be administered, preferably orally, shortly after radiotherapy. In certain embodiments, the compositions of the invention are to be administered to a patient that has recently undergone radiotherapy, or that is scheduled to undergo radiotherapy. In preferred embodiments, the flagellin polypeptides from the genusEnterococcusare administered before the irradiation exposure. In other embodiments, the flagellin polypeptides from the genusEnterococcusare administered after the radiation exposure, preferably within 4 hours following exposure. Treating Bacterial Infections Administration ofS. typhimuriumflagellin has been shown stimulate immune responses to bacterial infection. Reference [68] shows that the prophylactic intranasal administration ofS. typhimuriumflagellin with an antibiotic protects mice against respiratoryStreptococcus pneumoniaebacteria, while reference [66] shows that oral administration ofS. typhimuriumflagellin 2 hours prior to infection withS. typhimuriumreduced mortality. Flagellin induced these responses by activating the TLR5 innate immune response. The examples show that flagellin polypeptides from the genusEnterococcusare able to stimulate a stronger TLR5 response thanS. typhimurium. Thus, the inventors have shown that flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarumcan be useful in the treatment or prevention of bacterial infections. The invention provides flagellin polypeptides from the genusEnterococcusfor use in treating or preventing bacterial infections. The invention also provides flagellin polypeptides the speciesEnterococcus gallinarumfor use in treating or preventing bacterial infections. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO:1, for use in treating or preventing bacterial infections. In certain embodiments, the flagellin polypeptides from the genusEnterococcusare used in combination with an antibiotic for use in treating bacterial infections. In certain embodiments, the flagellin polypeptides from the genusEnterococcusare used the only therapeutic agent in a composition for use in treating bacterial infections. Vaccine Adjuvants Microbial components can be used as adjuvants to enhance the immune responses of poorly immunogenic vaccines. Flagellin has been shown to act as adjuvants in vaccines for bacterial, viral and parasitic infections, however, most of these studies used flagellin fromS. typhimuriumandVibrio vulnificus[15]. The efficacy of flagellin polypeptides from other species to act as vaccine adjuvants, and in particularEnterococcusspp., is unknown. Flagellin acts as an adjuvant by stimulating the innate immune response through the TLR5 receptor. The inventors have surprisingly shown that flagellin polypeptides from the genusEnterococcusare able to stimulate a stronger TLR5 response thanS. typhimurium. Thus, the inventors have shown that flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarumcan be useful as vaccine adjuvants. Use of a flagellin polypeptide as a vaccine adjuvant means that the flagellin polypeptide is used, for example, to provide protection against an infective agent or prevent infection by an infective agent, generally by enhancing the immune response to a separate antigen. The invention provides flagellin polypeptides from the genusEnterococcusfor use as vaccine adjuvants. The invention also provides flagellin polypeptides the speciesEnterococcus gallinarumfor use as vaccine adjuvants. More preferably, the invention provides flagellin polypeptide from strain MRx0518, in particular with the sequence SEQ ID NO:1, for use as vaccine adjuvants. The invention also provides vaccine compositions comprising flagellin polypeptides of the invention as an adjuvant. In certain embodiments, the vaccine compositions are for use in treating or preventing bacterial infections. In certain embodiments, the bacterial pathogen to be treated or prevented isY. pestis, tetanus,Streptococcus pneumoniae, Escherichia coliorM. tuberculosis. In certain embodiments, the vaccine compositions are for use in treating or preventing viral infections. In certain embodiments, the viral pathogen to be treated or prevented is influenza, HIV, rabies or foot and mouth virus. In certain embodiments, the vaccine compositions are for use in treating or preventing parasitic infections. In certain embodiments, the parasite isPlasmodium vivax, Plasmodium yoeliiorEimeria tenella. In any such embodiments, the composition comprises at least one antigen from the relevant pathogen. In preferred such embodiments, the composition comprises the flagellin of the invention fused or conjugated to at least one antigen from the relevant pathogen. In such embodiments, the invention provides a fusion polypeptide comprising a flagellin of the invention, an optional linker, and an antigen from a pathogen or parasite. Administration through a mucosal route is an attractive vaccine administration route. Flagellin is a potent activator of innate immune signalling pathways in epithelial cells, which are the first major cell type that encounter agents administered by a mucosal route.S. typhimuriumandVibrio vulnificusflagellin proteins have been shown to potent adjuvant activity in mucosal vaccines against influenza, West Nile virus,Escherichia coli, Yersinia pestis, Clostridium tetani, C. jejuni, Streptococcusspp. andPlasmodium falciparum[15]. The efficacy of flagellin polypeptides from other species to act as mucosal vaccines adjuvants is unknown. Flagellin acts as an adjuvant by stimulating the innate immune response through the TLR5 receptor. The inventors have surprisingly shown that flagellin polypeptides from the genusEnterococcusare able to stimulate a stronger TLR5 response thanS. typhimurium. Thus, the invention provides flagellin polypeptides from the genusEnterococcusfor use as a mucosal vaccine adjuvant. In preferred embodiments the invention provides flagellin polypeptides from the speciesEnterococcus gallinarumfor use as mucosal vaccine adjuvants. More preferably, the invention provides flagellin polypeptide from strain MRx0518, in particular with the sequence SEQ ID NO:1, for use mucosal as vaccine adjuvants. In certain embodiments, flagellin polypeptides from the genusEnterococcusare for use in mucosal vaccine for use in treating or preventing bacterial infections. In preferred embodiments, the bacterial infection isEscherichia coli, Yersinia pestis, Clostridium tetani, C. jejuni, Streptococcusspp. orPlasmodium falciparum. In certain embodiments, flagellin polypeptides from the genusEnterococcusare for use in mucosal vaccines for use in treating or preventing viral infections. In preferred embodiments, the viral infection is influenza or the West Nile virus. Administration of the compositions of the invention may lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-α). TNF-α is known to be important for vaccine responses. For example, TNF-α has been shown to be required for an efficient vaccine response in a flu vaccination of the elderly population [69]. Since administration of the compositions of the invention may increase TNF-α expression, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of TNF-α. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in influenza therapy. In certain embodiments, the compositions of the invention are for use in enhancing an immune response against an antigen. In certain embodiments, the invention provides a composition to be administered in combination with an antigen. In such embodiments of the invention, the flagellin of the invention may be fused or conjugated to the antigen. In certain embodiments, the compositions of the invention are for administration to a patient shortly prior to or after vaccination. Enterococcus gallinarumand in particular strain MRX518 is flagellated and flagellins can be TLR5 agonists. TLR agonists are in development as vaccine adjuvants across a range of antigen types, particularly in the elderly population [70]. Also, MRX518 is a TLR5 agonist. Therefore, the compositions of the invention may be useful as vaccine adjuvants, in particular for vaccine administered to elderly patients (e.g. over 40, 50, 60, 70 or 80 years of age), who may have reduced immune system activity. TLR5 signalling also plays a key role in age-associated innate immune responses [71]. In certain embodiments, the compositions are for use in enhancing an innate immune response. Although TLR5 agonists are in development as vaccine adjuvants, these are all from known pathogens and/or synthetic. In contrast, the compositions of the invention comprise commensal bacteria. Administration of the compositions of the invention may lead to an increase in expression of IL-6. Increased 11-6 expression has been associated with vaccine responses for many diseases. For example, IL-6 was produced by CD14+CD16-inflammatory monocytes after adults were administered an influenza vaccine [72], and higher levels of IL-6 were associated with achieving a vaccine response to an influenza vaccine [73]. Furthermore, 11-6 was produced after injection of the AS03 adjuvant system [74] and downregulation of IL-6 in mice was shown to reduce the helper T cell response after administration of a tuberculosis vaccine [75]. Since administration of the compositions of the invention may increase IL-6 expression, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in tuberculosis therapy. Furthermore, IL-6 and TNF-α expression have been shown to be correlated with the efficacy of a therapeutic HIV vaccine [Huang et al] a tuberculosis vaccine and achlamydiavaccine [76]. Su et al. [77] showed that co-inoculation of IL-6 or TNF-α with the FMDV DNA vaccine resulted in increased IFN-γ expression by CD4+ and CD8+ T cells, higher expression of IL-4 in CD4+ T cells and a higher antigen-specific cytotoxic response. Since administration of the compositions of the invention may increase IL-6 and TNF-α expression, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF-α. In one embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of IL-6. In a particular embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF-α and IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in HIV therapy. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant inchlamydiatherapy. Administration of the compositions of the invention may lead to an increase in expression of IL-1β. Li et al. [78] showed that the adjuvant aluminium hydroxide activated the secretion of IL-1β, and suggested that IL-β itself can act as an adjuvant. Since administration of the compositions of the invention may increase IL-1β expression, compositions of the invention may be useful as a vaccine adjuvant. The examples show that administration of the compositions of the invention can increase the ratio of CD8+ T cells to Tregs. Adjuvants have been shown to stimulate CD8+ T cells [79] and since administration of the compositions of the invention were shown to increase the ratio of CD8+ T cells to Tregs, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the ratio of CD8+ T cells to Tregs. Administration of the compositions of the invention may lead to an increase in expression or levels of CXCR3 ligands CXCL9 and CXCL10. Known adjuvants such as ASO3, CpG, GLA-SE, αGalCer all increase CXCL9 and 10 [80,81], which suggests the compositions of the invention will be effective as adjuvants. Also, CXCL9 and 10 are associated with IFNγ/Th1 responses and promote antibody responses [82]. In certain embodiments, the compositions of the invention are for use in promoting an antibody response against an antigen, in particular a pathogenic or cancer antigen. Also, CXCL9 is a more sensitive measure than IFN-γ of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines [83]. In certain embodiments, the compositions of the invention are for use in promoting an T-cell response against an antigen, in particular a pathogenic or cancer antigen. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of CXCL9 and CXCL10. In certain embodiments, the compositions are for use in protecting against malaria. Administration of the compositions of the invention can lead to an increase in expression or levels of IL-12p70. This effect has been associated with vaccine adjuvant efficiency and IL-12 has been proposed as an adjuvant itself [84], which suggests the compositions of the invention will be effective as adjuvants. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-12p70. Generally, when used as a vaccine adjuvant, the compositions of the invention will be administered on their own to provide an adjuvant effect for an antigen that has been separately administered to the patient. In certain embodiments, the composition of the invention is administered orally, whilst the antigen is injected parenterally. In alternative embodiments, the flagellin of the invention may be fused or conjugated to the antigen. The compositions of the invention may be used for enhancing an immune response to any useful antigen. Exemplary antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. The invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus,Staphylococcus aureus, chlamydia, SARS coronavirus, varicella zoster virus,Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, human papillomavirus, etc. In certain embodiments, the flagellin of the invention is fused or conjugated to one or more of these antigens. Further antigens for use with the flagellins of the invention include glycoprotein and lipoglycan antigens. In certain embodiments, the antigen is an archaea antigen. Exemplary tumour-associated antigens include melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens and oncoproteins. Flagellins of the invention may also be useful for enhancing the response to vaccines against non-communicable diseases such as Alzheimer's Disease and other neurodegenerative disorders, in which case the antigen for use with the invention may be amyloid-beta or Tau. Other such antigens for non-communicable diseases include PCSK9 (for the treatment of elevated cholesterol). Flagellins of the invention may also be useful for enhancing the response to vaccines against habit forming substances, for example nicotine, alcohol or opiates. The invention also provides the use of: (i) an aqueous preparation of an antigen; and (ii) a composition comprising a flagellin polypeptide from the speciesEnterococcus gallinarum, in the manufacture of a medicament for raising an immune response in a patient. The immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response. Stimulating the Immune System The compositions of the invention can lead to immune stimulation. Since administration of the compositions of the invention may have an immunostimulatory effect, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an increased immune response. In certain embodiments, the compositions of the invention are for use in stimulating the immune system. In certain embodiments, the compositions of the invention are for use in treating disease by stimulating the immune system. In certain embodiments, the compositions of the invention are for use in promoting an immune response. Compositions of the invention may be useful in the treatment of diseases characterised by an increase in the percentage of Tregs in a cell population. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of Tregs in a cell population. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of CD4+CD25+CD127− cells in a cell population. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by decreasing the percentage of Tregs in cell populations. In one embodiment, compositions of the invention are for use in reducing suppression of the immune response by Tregs. In one embodiment, compositions of the invention are for use in stimulating the immune response by the selective reduction of Tregs. In one embodiment, compositions of the invention are for use in immunostimulation, wherein the compositions of the invention reduce the number or percentage of Tregs. Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the ratio of CD8/Treg and/or activated CD8/Treg cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of CD8/Treg cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of activated CD8/Treg cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the ratio of CD8/Treg cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the ratio of activated CD8/Treg cells. Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of B cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of B cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD19+CD3− cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of B cells in cell populations, wherein the increase in number or percentage of B cells results in immune stimulation. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of B cells. Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8 T-cytotoxic cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8 T-cytotoxic cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8 T-cytotoxic cells in cell populations, wherein the increase in number or percentage of CD8 T-cytotoxic cells results in immune stimulation. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8 T-cytotoxic cells. Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8+ activated cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8+ activated cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8+ activated cells in cell populations, wherein the increase in number or percentage of CD8+ activated cells results in immune stimulation. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8+ activated cells. Administration of the compositions of the invention may lead to an increase in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines. Examples of pro-inflammatory molecules that may show an increase in expression levels upon administration of compositions of the invention include IL-8, IL-12p70, IL-23, TNF-α, IL-1β, and IL-6. Since administration of the compositions of the invention may increase the expression of pro-inflammatory molecules, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of pro-inflammatory molecules, in particular diseases characterised by a decrease in the expression and/or activity of pro-inflammatory cytokines. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-8, IL-12p70, IL-23, TNF-α, IL-1β, and/or IL-6. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23, TNF-α, IL-1β, and/or IL-6. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-8, IL-12p70, IL-23, TNF-α, IL-1β, and/or IL-6. Administration of the compositions of the invention may lead to an increase in expression of IL-1β. IL-1β is a pro-inflammatory cytokine [85]. The production and secretion of IL-1β is regulated by the inflammasome, a protein complex which is associated with activation of the inflammatory response [86]. Since administration of the compositions of the invention may increase the expression of IL-1β, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-1β. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-1β. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-1β. Administration of the compositions of the invention may lead to an increase in expression of IL-23. IL-23 has been linked to inflammation [87,88]. The proposed functions of IL-23 in the immune response include promoting the proliferation of CD4+ memory T cells and promoting the secretion of IFN-γ by dendritic cells (DCs) [89]. Since administration of the compositions of the invention may increase the expression of IL-23, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-23. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-23. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-23. Administration of the compositions of the invention may lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-α). TNF-α is a pro-inflammatory cytokine which is known to be involved in various signalling pathways to promote cell death. TNF-α initiates apoptosis by binding to its cognate receptor, TNFR-1, which leads to a cascade of cleavage events in the apoptotic pathway [90]. TNF-α can also trigger necrosis via a RIP kinase-dependent mechanism [91]. Since administration of the compositions of the invention may increase TNF-α expression, compositions of the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF-α. In one embodiment, the compositions of the invention are for use in treating diseases characterised by decreased TNF-α expression. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of TNF-α. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of TNF-α. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of TNF-α. Administration of the compositions of the invention may lead to an increase in expression of IL-6. IL-6 a pro-inflammatory cytokine that is produced during inflammation, and promotes the differentiation of naïve CD4+ T cells and the differentiation of CD8+ T cells into cytotoxic T cells [92]. Since administration of the compositions of the invention may increase the expression of IL-6, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-6. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-6. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-6. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-6. Bettelli et al. [93] reported that IL-6 inhibits the production of Tregs. Compositions of the invention may increase the expression of IL-6, so compositions of the invention may selectively decrease the number or percentage of Tregs by increasing the expression of IL-6. In one embodiment, compositions of the invention are for use in immunostimulation by increasing the expression of IL-6. In another embodiment, compositions of the invention are for use in immunostimulation by decreasing the number or percentage of Tregs. The examples also demonstrate that the compositions of the invention promote the differentiation of T-helper cells and cytotoxic T lymphocytes. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the differentiation of T-helper cells and/or cytotoxic T lymphocytes. Cell Therapies Chimeric Antigen Receptor T Cell (CAR-T) Therapy Administration of the compositions of the invention may lead to an increase in expression of IL-6. Increased 11-6 expression has been correlated with response to CD19 CAR-T therapy of chronic lymphocyte leukaemia. An increase in serum IL-6 was associated with CAR-T cell expansion, whereas inhibition of IL-6 was associated with inhibition of CAR-T cell proliferation [94]. Since administration of the compositions of the invention may increase IL-6 expression, compositions of the invention may be useful in cell therapy, in particular CAR-T cell therapy. In one embodiment, the compositions of the invention are for use in cell therapy. In one embodiment, the compositions of the invention are for use in CAR-T cell therapy. In one embodiment, compositions of the invention are for use in the treatment of chronic lymphocyte leukaemia. Selective depletion of Tregs has been shown to enhance the efficacy of cytotoxic lymphocytes [95]. CAR-T cells are a subset of cytotoxic lymphocytes, and therefore it is thought that selective depletion of Tregs is effective in CAR-T cell therapy. Since administration of the compositions of the invention may deplete Tregs, compositions of the invention may be useful in cell therapy, in particular CAR-T cell therapy. Therefore, the compositions of the invention may be useful in cell therapy, in particular in enhancing the response to a cell therapy. Mesynchymal Stem Cell (MSC) Therapy Mesynchymal stem cell (MSC) therapy has been reported to have immunostimulatory properties. When MSCs are treated with LPS, they upregulate pro-inflammatory cytokines IL-6 and IL-8 which causes increased B cell proliferation [96]. Therefore, since compositions of the invention may increase the expression of IL-6, they may be useful in combination with MSC cell therapy. Stem Cell Transplantation Therapy It has been reported that, instead of using undifferentiated stem cells in stem cell transplantation therapy, it may be beneficial to differentiate stem cells to some extent prior to transplantation. For example, Heng et al. [97] reported that cardiomyogenic differentiation of stem cells may be beneficial by having a higher engraftment efficiency, enhanced regeneration of myocytes and increased restoration of heart function. Since administration of the compositions of the invention may initiate neuronal differentiation in undifferentiated neuroblastoma cells, compositions of the invention may be useful for stem cell differentiation in stem cell transplantation therapy. Hematopoietic Stem Cell Transplantation Hematopoietic stem cell transplantation is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. Colonisation of the gut with Enterococci (Enterococcus gallinarumandEnterococcus casseliflavus) prior to allogenic hematopoietic stem cell transplantation has been shown to lead to a significantly improved the 2-year survival of patients after due to decreased nonrelapse mortality [98]. Therefore, the immunomodulatory effect shown in the examples may be useful in hematopoietic stem cell transplantation therapy. In certain embodiments, the compositions of the invention may be useful in improving survival after hematopoietic stem cell transplantation and in particular after allogenic hematopoietic stem cell transplantation. The compositions of the invention may be useful in combination with allogenic hematopoietic stem cell transplantation. The compositions of the invention may be effecting in boosting successful patient response to allogenic hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are administered prior to hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are for administration to a patient scheduled to receive hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are administered following hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are for administration to a patient that has received hematopoietic stem cell transplantation. Immunosenescence Fulop et al. [99] identified that an increase in Treg cell number and a decrease in B cell number are associated with aging in the adaptive immune system. Therefore, compositions of the invention may be used to prevent or delay immunosenescence. In one embodiment, compositions of the invention are for use in preventing immunosenescence. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by a decrease in B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number and a decrease in B cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by increasing B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number and increasing B cell number. In one embodiment, compositions of the invention are for use in treating diseases caused by immunosenescence. In one embodiment, compositions of the invention are for use in treating aging-related diseases by delaying and/or preventing immunosenescence. Furthermore, it has been proposed that vaccine adjuvants may overcome immunosenescence [100]. Since the compositions of the invention are suitable for use as a vaccine adjuvant, compositions of the invention may be useful for preventing or delaying immunosenescence. In another embodiment, compositions of the invention are for use in delaying and/or preventing immunosenescence as a vaccine adjuvant. In another embodiment, compositions of the invention are for use as a vaccine adjuvant, wherein the compositions delay and/or prevent immunosenescence. Diseases that are associated with immunosenescence include cardiovascular disease, neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, cancer, diabetes mellitus type 2 [101] and autoimmune disorders [102]. Modes of Administration Preferably, the compositions of the invention are administered by injection, preferably subcutaneously or alternatively intravenously, or intraperitoneally. Further details regarding compositions suitable for administered by injection are provided in the next section. In alternative embodiments, the compositions of the invention are to be administered to the gastrointestinal tract. The immune system is known to be modulated by the presence of bacteria and bacterial proteins in the gastrointestinal tract. The compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes. In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a gel. In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for example in the form of atheobromaoil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition. In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports. The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily. In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the patient's gut microbiota. The compositions of the invention may be administered to a patient that has been diagnosed with cancer, or that has been identified as being at risk of a cancer. The compositions may also be administered as a prophylactic measure to prevent the development of cancer in a healthy patient. The compositions of the invention may be administered to a patient that has been identified as having an abnormal gut microbiota. For example, the patient may have reduced or absent colonisation byEnterococcusspp, in particularEnterococcus gallinarum. Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used. Compositions Generally, the composition of the invention comprises a flagellin polypeptide from the genusEnterococcusand in particular from the speciesEnterococcus gallinarum. In preferred embodiments, the composition of the invention is encapsulated to enable delivery of flagellin to the intestine. Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Compositions may thus be pharmaceutically acceptable. They will usually include components in addition to the flagellin polypeptide (or nucleic acid) e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). Compositions may be administered to a human in aqueous form. In such embodiments, prior to administration, however, the composition may have been in a non-aqueous form. For instance, although some therapeutics are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other therapeutics are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use. Thus a composition of the invention may be dried, such as a lyophilised formulation. The composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the therapeutics should be substantially free from (i.e. less than 5 μg/ml) mercurial material e.g. thiomersal-free. Therapeutics containing no mercury are more typical. Preservative-free therapeutics are particularly favoured. To improve thermal stability, a composition may include a temperature protective agent. Further details of such agents are provided below. To control tonicity, it is typical to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is generally used, which may be present at between 1 and 20 mg/ml e.g. about 10±2 mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc. Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, more often between 240-360 mOsm/kg, and will more typically fall within the range of 290-310 mOsm/kg. Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range. The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8. The composition is typically sterile. The composition is also typically non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, for example <0.1 EU per dose. The composition is often gluten free. The composition may include material for a single administration, or may include material for multiple administration (i.e. a ‘multidose’ kit). The inclusion of a preservative is typical in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Human protein therapeutics are typically administered in a dosage volume of about 0.5 ml, although a half dose (i.e. about 0.25 ml) may be administered to children. Compositions of the invention may also comprise one or more immunoregulatory agents. Often, one or more of the immunoregulatory agents include one or more adjuvants. The adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below. The compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a mammal. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens. In certain embodiments, the flagellin of the invention present in the composition may be fused or conjugated to one or more antigens. Where a composition is to be prepared extemporaneously prior to use (e.g. where a component is presented in lyophilised form) and is presented as a kit, the kit may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection. Compositions used as therapeutics comprise an immunologically effective amount of polypeptide or encoding nucleic acid, as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Where more than one antigen is included in a composition then two antigens may be present at the same dose as each other or at different doses. As mentioned above, a composition may include a temperature protective agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminium salt). As described in reference 103, a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0° C. Thus the composition can be stored below 0° C., but above its freezing point, to inhibit thermal breakdown. The temperature protective agent also permits freezing of the composition while protecting mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40° C. A starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1-80% by volume of the final mixture. Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components (e.g. antigen and adjuvant) in the composition. Examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG). Suitable PEGS may have an average molecular weight ranging from 200-20,000 Da. In one embodiment, the polyethylene glycol can have an average molecular weight of about 300 Da (PEG-300′). The invention provides a composition comprising: (i) one or more flagellin polypeptides(s); and (ii) a temperature protective agent. This composition may be formed by mixing (i) an aqueous composition comprising one or more antigen(s), with (ii) a temperature protective agent. The mixture may then be stored e.g. below 0° C., from 0-20° C., from 20-35° C., from 35-55° C., or higher. It may be stored in liquid or frozen form. The mixture may be lyophilised. The composition may alternatively be formed by mixing (i) a dried composition comprising one or more antigen(s), with (ii) a liquid composition comprising the temperature protective agent. Thus component (ii) can be used to reconstitute component (i). The composition may be administered orally and may be in the form of a tablet, capsule or powder. Other ingredients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and/or partial or total colonisation and survival in vivo. A composition of the invention includes a therapeutically effective amount of a flagellin polypeptide of the invention. A therapeutically effective amount of a flagellin polypeptide is sufficient to exert a beneficial effect upon a patient. The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [104]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [105]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. In some embodiments, the composition comprises more than one flagellin polypeptide from the genusEnterococcusfor use in treating or preventing cancer. In certain embodiments, the composition comprises a flagellin polypeptide with SEQ ID NO:1 and at least one further flagellin polypeptide from the genusEnterococcus. In certain embodiments, the composition comprises a flagellin polypeptide with SEQ ID NO:1 and at least one flagellin polypeptide from the speciesEnterococcus gallinarum. In certain embodiments, the composition comprises a flagellin polypeptide with SEQ ID NO:1 and at least one flagellin polypeptide selected from a group consisting of SEQ ID NO 2-42. In some embodiments, the composition can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or 45 flagellin polypeptides from the genusEnterococcusand in particular from the speciesEnterococcus gallinarum. In some embodiments, the compositions of the invention comprise less than 50 flagellin polypeptides from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain flagellin polypeptides from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 flagellin polypeptides from within the same species and, optionally, do not contain flagellin polypeptides from any other species. In some embodiments in which the composition of the invention comprises more than one flagellin polypeptide, the individual flagellin polypeptides may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of the flagellin polypeptides, or the flagellin polypeptides may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one flagellin polypeptides are stored separately but are mixed together prior to use. The compositions for use in accordance with the invention may or may not require marketing approval. The compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is lung cancer. In preferred embodiments the cancer is lung carcinoma. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is liver cancer. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma). In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is colon cancer. In preferred embodiments the cancer is colorectal adenocarcinoma. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is carcinoma. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is a non-immunogenic cancer. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is a immunogenic cancer. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of non-small-cell lung carcinoma, small-cell lung carcinoma, squamous-cell carcinoma, adenocarcinoma, glandular tumors, carcinoid tumors undifferentiated carcinomas. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection. In certain embodiments, the invention provides a pharmaceutical composition comprising: a flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma. In certain embodiments, the invention provides a pharmaceutical composition comprising: flagellin polypeptide of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer. A dose of a nucleic acid (e.g. a nucleic acid-based therapeutic) may have ≤100 μg nucleic acid; e.g. from 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg, but expression can be seen at much lower levels; e.g. using ≤1 μg/dose, ≤100 ng/dose, ≤10 ng/dose, ≤1 ng/dose, etc. Similarly, a dose of a protein antigen may have ≤100 μg protein; e.g. from 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg. The polypeptide may be administered in a dose of about 0.1-10 mg/kg body weight. In certain embodiments, the pharmaceutical compositions of the invention may be administered in a single dose or in more preferably in repeated doses. Preferably repeat doses are administered about every 2, 5, 10, 7, 10 or 15 days. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 100 μg/kg, 200 μg/kg, 300 μg/kg, 400 μg/kg, 500 μg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg or 500 mg/kg of subjects body weight. Suitable unit doses include 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1000 mg, 100-500 mg, 500-1000 mg, 200-400 mg, 5-00-750 mg, 750-1000 mg. In certain embodiments, the invention provides the above pharmaceutical composition comprising host cells of the invention. A suitable daily dose of the host cells, for example for an adult human, may be from about 1×103to about 1×1011colony forming units (CFU); for example, from about 1×107to about 1×1010CFU; in another example from about 1×106to about 1×1010CFU. In certain embodiments, the composition contains the host cell in an amount of from about 1×106to about 1×1011CFU/g, respect to the weight of the composition; for example, from about 1×108to about 1×1010CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g. In some embodiments, the composition comprises a mixture of live host cells and host cells that have been killed. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, sublingual, subcutaneous, intravenous, and intramuscular. In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol. In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water. In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride. In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer. In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4.0 or about 25.0 and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years. In some embodiments, the composition of the invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein. The composition of the present invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gelatine capsule (“gel-cap”). In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth. Pharmaceutical formulations suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches. In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastro-resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions. In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or an enteric-coated capsule, or the like. The enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery. The formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer. In some embodiments, the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [106]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel). In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gelatine or starch. Gelatine-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process. General The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [107] and [108-114], etc. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y. The term “about” in relation to a numerical value x is optional and means, for example, x±10%. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. The term “flagellin polypeptides from the genusEnterococcus” encompasses wild type flagellin polypeptides and mutated flagellin polypeptides that exhibit immunostimulatory properties, for example which can effectively activate TLR5 responses. The term “flagellin polypeptides” also encompasses fragments of wild type flagellin polypeptides and mutated flagellin polypeptides that exhibit immunostimulatory properties, for example which can effectively activate TLR5 responses. References to a percentage sequence identity between two protein sequences means that, when aligned, that percentage of amino acid residues are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in ref. [115]. A preferred method uses MUSCLE alignment of protein sequences and can be performed with Geneious (78.02% pairwise identity), Blossom62 score matrix, threshold=1. References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [116]. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [117]. Generally, unless specified otherwise (for example in relation to fragments), sequence identity is calculated over the length of the reference sequence (for example SEQ ID NO:1) and not over the length of the query sequence. Therefore, short and irrelevant sequences are not considered to have high sequence identity to the sequences of the invention. Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate. Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive). MODES FOR CARRYING OUT THE INVENTION Example 1—Activation of TLR5 Reporter Cells by MRx0518 Summary This study tested the efficacy of the bacterial strain MRx0518 to activate TLR5 reporter cells. TLR5 Activation Assay HEK-Blue TLR5 cells are HEK293 cells co-transfected with the human TLR5 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. The SEAP gene is placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Stimulation with a TLR5 ligand activates NF-κB and AP-1 which induce the production of SEAP. The level of alkaline phosphatase thus relates to the TLR5 activity. Materials Reporter cell line: HEK-Blue hTLR5 (Invivogen, hkb-htlr5) Cell growth media: DMEM (Sigma, D6171) supplemented with 10% (v/v) FBS (Sigma, F9665), 4 mM L-glutamine (Sigma, G7513), 100 U/ml penicillin and 100 ug/ml streptomiycin (Sigma, P4333), 100 ug/ml normocin (Invivogen, ant-nr-1) and selective antibiotics blasticidin (30 ug/ml, Invivogen, ant-bl-05) and zeocin (100 ug/ml, Invivogen, ant-zn-1) Antibiotic-free media: DMEM supplemented with 10% (v/v) FBS and 4 mM L-glutamine Bacterial Growth Conditions Bacterial growth conditions: AllEnterococcus gallinarumMRx0518 cultures were grown in yeast extract-casein hydrolysate-fatty acids (YCFA) broth (E&O Laboratories, UK) at 37° C., under anaerobic conditions. Stationary phase MRx0518 cells: 16-18 hour cultures of strain MRx0518 were grown under the conditions described above using a 1% inoculum of MRx0518 cells, stored frozen as working cell banks at −80° C. Late-log phase MRx0518 cells: 3 hour (approx.) cultures of strain MRx0518 were grown under the conditions described above using a 10% inoculum of stationary phase MRx0518 cells. Procedure The TLR5 activation assay was performed with the following treatments at two dosages—10:1 (3×105bacteria) or 100:1 (3×106bacteria):Live MRx0518 bacteriaHeat killed MRx0518 bacteriaCFS from MRx0518 A positive control was performed with ultrapure FLA-ST at a concentration of 20 ng/ml and 5 ng/ml A negative control were performed with antibiotic-free media containing no reporter cells to control for the production of alkaline phosphatase by anything other than the reporter cells. Two additional controls were performed with YCFA and water. For all treatments three replicates were performed. Preparation of Live and Cell-Free Supernatant (CFS) MRx0518 Live and CFS treatments were both prepared from one 10 ml culture of late-log phase MRx0518 cells following centrifugation of the cells at 5000×g for 5 mins at room temperature. The bacterial pellet was washed once in PBS (Sigma, D8537) and resuspended in antibiotic-free media to an estimated density of 1.4×108CFU/ml (2-fold dilution) and 1.4×107CFU/ml (20-fold dilution). The bacterial supernatant was collected, filtered (0.22 um) under sterile conditions and was diluted 10-fold and 20-fold using H2O (Sigma, W3500) to provide equivalents for the bacteria suspensions outlined above. The CFS contains MRx0518 flagella shed into the supernatant. Preparation of Heat Killed (HK) Cells Separately, HK cells were generated by incubating a 10 ml culture of late-log phase MRx0518 cells at 80° C. for 40 mins. Following centrifugation (conditions described above) the culture supernatant was discarded and the cell pellet was washed once in PBS (Sigma, D8537) and resuspended in antibiotic-free media to an estimated density of 1.4×108CFU/ml (2-fold dilution) and 1.4×107CFU/ml (20-fold dilution). Once prepared, all treatments were stored on ice prior to use. Viable cell counts per ml were determined for all live and heat-killed MRx0518 cell preparations. Preparation of the Positive Control A known agonist of TLR5, theS. typhimuriumflagellin (ultrapure, FLA-ST ultrapure from Invitrogen, tlrl-epsitfla) was prepared at 200 ng/ml or 50 ng/ml in H2O. TLR5 Assay Conditions 20 μl of the appropriate treatment (bacterial cells, cell-free supernatant, positive control ligands, recombinant flagellin, negative controls e.g. YCFA, antibiotic-free media, H2O, PBS) were plated in duplicate or triplicate in 96-well cell culture plates (Sigma, CLS3596). 180 μl of reporter cell suspension was added to each well to give a final volume of 200 ul and a final concentration HEK-blue TLR5 cells of 3×104. The assays were incubated at 37° C., 5% CO2for 22 h. Following the incubation, 20 μl of the assay was added to 180 μl of Quantiblue (Invivogen, rep-qbl) substrate in a new 96-well plate and incubated at 37° C., 5% CO2for 2 h. The Quantiblue-containing plates were imaged on a microplate reader (Bio-Rad, iMark) taking 655 nm optical density readings. Results were averaged from technical replicates and then independent experiments averaged thereafter to provide data for final graphs. Conclusions Treatment with live and heat inactivated MRx0518 led to intermediate and high levels of TLR5 responses. These data show that the MRx0518 strain is able to induce a TLR5 response and that this response is not eliminated by heat inactivation. These data suggest flagellin from the genusEnterococcusand in particular MRx0518 flagellin can elicit a TLR5 response and that the flagellin is heat stable. The highest level of TLR5 activity was observed after treatment with MRx0518-CFS and was even higher than the known TLR5 agonist FLA-ST (FIG.6). The CFS contains MRx0518 flagella shed into the supernatant. These data show that flagellin from the genusEnterococcusand in particular from MRx0518 produce a very strong TLR5 response, and so may be useful in therapy, in particular in the treatment of cancer. These data also show that MRx0518 is effective at shedding its flagellin into the supernatant. Example 2—TLR5 Activation by the CFS is Particularly Strong for MRx0518 Summary This study tested whether TLR5 activation using the CFS varies between strains. Both MRx0518 and DSM100110 are flagellatedEnterococcus gallinarumstrains. A study was conducted to observe if the CFS from both these strains leads to a TLR5 response. TLR5 Activation Assay A TLR5 assay as described in Example 1 was performed. Procedure The TLR5 activation assay was performed with the following treatments at two dosages—10:1 and 100:1:MRx0518-CFSDSM100110-CFS Three negative controls were performed with antibiotic-free media, YCFA and water. A positive control was performed with ultrapure FLA-ST at a concentration of 20 ng/ml. For all treatments three replicates were performed. Conclusions MRx0518-CFS was able to stimulate a high TLR5 response. DSM100110-CFS also produced a TLR5 response at an MOI of 100:1, although it was reduced relative to MRx0518-CFS. These data show that while supernatant from both strains elicited an immunostimulatory response, this varies between strains. These data are surprising as strains MRx0518 and DSM100110 are both flagellatedEnterococcus gallinarumstrains. The MRx0518-CFS produces a higher TLR5 response compared to DSM100110-CFS, potentially because MRx0518 has an improved ability to shed its flagellin into the supernatant compared to otherEnterococcus gallinarumstrains, which may make it particularly useful in therapy. These data suggest that flagellin polypeptides from the genusEnterococcusmay be effective for use in treating or preventing cancer, in particular cancers that are associated with TLR5, such as breast, colorectal and gastric cancer. Example 3—TLR5 Activation after Trypsin Treatment Summary This study tested whether MRx0518-CFS activation of TLR5 is trypsin-dependent. TLR5 Activation Assay A TLR5 assay as described in Example 1 was performed. Procedure The TLR5 activation assay was performed with the following treatments at two dosages—10:1 and 100:1 alone or in combination with trypsin:MRx0518-CFSDSM100110-CFSYCFA For all treatments three replicates were performed. CFSs were digested with 500 μg/ml trypsin (0.22 um filtered, Sigma, T3924), or an equivalent volume of trypsin vehicle (Hank's balanced salt solution, Fisher Scientific, 1175129) as a mock digest control, for 1 hr at 37° C., 5% CO2. After incubation digested and mock digested CFS samples were supplemented with FBS to a final concentration of 10% (v/v) to inhibit trypsin activity. Three negative controls were performed with antibiotic-free media, HMS and water. A positive control was performed with ultrapure FLA-ST at a concentration of 20 ng/ml. Conclusions TLR5 activation by MRx0518-CFS was abolished by treatment with trypsin (FIG.8). These data show that the TLR5 response is activated by proteins in the MRx0518-CFS. Degradation of these proteins by trypsin abolishes the TLR5 response. The MRx0518 flagellin protein (FlaAMRx0518) contains 36 trypsin cleavage sites, some of which are located in the TLR5 interaction domains. Example 4—TLR5 Activation with Purified Flagellin Proteins Summary This study tested the stimulatory profiles of purified MRx0518 and DSM100110 flagellin. Production of Recombinant Flagellin Proteins An overview of the cloning strategy is shown ifFIG.9a. The full length genes for MRx0518 and DSM100110 flagellin were cloned into the recombinant expression construct pQE-30 (which has an N-terminal 6×His tag (SEQ ID NO: 49) using a restriction enzyme digest to produce pQE-30-FlaAMRx0518and pQE-30-FlaADSM100110constructs. Positive colonies were identified using colony PCR, restriction enzymes digests and DNA sequencing techniques well-known in the art. Recombinant flagellin proteins, FlaAMRx0518and FlaADSM100110, were expressed inE. colicells and purified using an Immobilized Metal Affinity Chromatography and imidazole elution, followed by endotoxin removal (see table below). This results in high purity preparations with minimal endotoxin levels. The size and purity of the proteins was confirmed using an SDS-PAGE gel (FIG.9b). FlaAMRX0518FlaADSM100110VehicleVolume (ml)51010Protein amount (mg)3.136.260Protection0.6250.6260concentration (mg/ml)Endotoxin0.880.990BufferPBSPBSPBSSDS-PAGEMinorMinorNocontaminantscontaminantsband Dose Response to Purified Recombinant FlaAMRx0518and FlaADSM100110Proteins A TLR5 assay as described in Example 1 was performed. Procedure The TLR5 activation assay was performed with the following treatments:Recombinant purified FlaAMRx0518at concentrations of 5, 1, 0.2, 0.04 and 0.008 ng/mlRecombinant purified FlaADSM100110at concentrations of 5, 1, 0.2, 0.04 and 0.008 ng/ml A negative control was performed with a FLA vehicle at a concentration 5 ng/ml and a positive control was performed with ultrapure FLA-ST at a concentration of 20 ng/ml. For each treatment three replicates were performed. Conclusions Both recombinant FlaAMRx0518and FlaADSM100110can activate TLR5 responses.FIG.10compares the TLR5 activation with different dosages of recombinant FlaAMRx0518and FlaADSM100110with the treatments described in examples 3 and 4. At a concentration of 5 ng/ml, both recombinant flagellin proteins can produce a TLR5 response that is higher than produced by the well-known TLR5 agonist FLA-ST being used a higher concentration (20 ng/ml). These data show that flagellin polypeptides from the speciesEnterococcus gallinarumare more effective at activating a TLR5 response thanS. typhimurium. Flagellin polypeptides fromS. typhimuriumare known to suppress tumour growth through TLR5 activation. These data therefore suggest that flagellin polypeptides fromEnterococcus gallinarumare more effective than flagellin polypeptides fromS. typhimuriumat treating or preventing cancer. FlaAMRx0518and FlaADSM100110have different immunostimulatory profiles. At concentrations of 1 ng/ml and 0.2 ng/ml FlaAMRx0518produces approximately five times more potent TLR5 response than FlaADSM100110. Both MRx0518 and DSM100110 are highly related strains. Both are flagellatedEnterococcus gallinarumstrains, however they express fundamentally different flagellin that have different immunostimulatory profiles. The surprising finding that FlaAMRx0518is strong TLR5 activator shows that this flagellin polypeptide is particularly effective at treating or preventing cancer. These examples demonstrate that flagellin polypeptides from the genusEnterococcusare agonists for TLR5. TLR5 agonists have been implicated in treating T and B cell lymphomas. Therefore, flagellin polypeptides from the genusEnterococcusmay be particularly effective at treating or preventing T and B cell lymphomas. TLR5 activation has been shown to ameliorate radiation induced tissue damage. The examples show that flagellin polypeptides from the genusEnterococcusare strong activators of the TLR5 response. Therefore, flagellin polypeptides from the genusEnterococcus, and in particular the speciesEnterococcus gallinarum, may be particularly effective at ameliorating radiation induced tissue damage. Example 5—Production of MRx0518 flaA−Mutant Summary An insertion mutant of MRx0518 was created which disrupted the flagellin gene. Experimental Conditions An internal fragment of flaA was cloned into vector pORI19 (repA). The resulting vectors flaAMRx0518int-pORI19 was transformed into MRx0518 with and the insert integrated into the bacterial genome using homologous recombination (seeFIG.13). Positive colonies were screened for the presence of the em gene and using PCR and further confirmed using sequencing. Phenotypic analysis of the mutants using motility assays also confirmed the insertion mutant MRx0518 flaA−. The insert contains erythromycin resistance from the pORI19 vector.FIG.11shows that MRx0518 flaA−mutant is non-motile on BBL motility agar supplemented with 20 μg/ml erythromycin compared to wild type. Example 6—Activation of TLR5 is Eliminated in the MRx0518 Mutant Summary The ability of CFS from the MRx0518 flaA−mutant to activate a TLR5 response was tested. TLR5 Activation Assay A TLR5 assay as described in Example 1 was performed. Procedure The TLR5 activation assay was performed with the following treatments at two dosages—10:1 and 100:1:MRx0518-CFSMRx0518 flaA−-CFS Positive controls were performed with ultrapure FLA-ST at a concentrations of 5 ng/ml and 20 ng/ml. A negative control was performed with YFCA at two dosages—10:1 and 100:1. For all treatments three replicates were performed. Conclusions The ability of MRx0518-CFS to stimulate a TLR5 response was eliminated in the MRx0518 flaA−mutant (FIG.12). These data show flagellin polypeptide is essential for the activation of the TLR5 response by MRx0518-CFS. Example 7—Characterisation of MRx0518 Summary Bacteria morphology such as size, presence or absence of fimbrae/flagella/pilli, or the presence of extracellular matrix influence both motility and adherence and are therefore important in the characterization of bacteria. MRx0518 cultures were examined at the electron microscope level (Scanning and Transmission) to allow visualization of bacteria structure at a higher resolution and magnification than that allowed by conventional light microscopy methods. Methodology Transmission Electron Microscopy An aliquot of MRx0518 bacterial culture in the exponential phase was received directly from an anaerobic hood in a sealed Eppendorf. Cultures were fixed in freshly made ice cold 2.5% Glutaraldehyde 0.1M Na Cacodylate pH 7.2. The Eppendorf was inverted gently a number of times before being placed on ice and left to fix for 3-5 minutes. Following fixation the bacteria were centrifuged briefly to pellet and resuspended in milli pure water. Using a glass pipette one drop of the fixed culture was applied to Formvar coated 300 mesh Cu grids. The bacteria were allowed to settle and adsorb for approximately 1-2 minutes and the excess solution removed from the grid using Whatman No 3 filter paper. 10 μl 2% Uranyl acetate was applied to the grid for a few seconds and removed by capillary action using filter paper as above. The grids were allowed to dry completely before being examined in a Philips CM100 TEM at varying kV. Scanning Electron Microscopy An aliquot from the same culture as above was taken for SEM analysis. The bacteria were fixed as above but not pelleted. SPI PORE FILTERs (25 mm diameter, 0.2 um pore size) were pretreated with 0.01% poly-lysine immediately before the fixed bacteria were pushed through using a Luer-Lok syringe onto the filter. This was followed by gently pushing 1 ml milli pure water through the filter. The filters were cut to size and dehydrated through a series of ethanol solutions (50%, 70% 90% and 3×100%) The filters were then immersed sequentially through increasing concentrations of hexamethyldisilazane (HMDS 25%, 50%, 75% in ethanol) with the final dehydration step carried out using 100% HMDS. This was allowed to evaporate overnight. The filters were critical point dried and coated with 10 nm gold palladium before being examined in a Zeiss EVO MA10 Scanning Electron Microscope. Conclusions Both methods of visualization at the electron microscope level showed MRx0518 to have flagella (FIG.14). Both TEM and SEM analysis showed MRx0518 to have flagella (solid white arrows). The cell size ranged between 1-2 μm. The presence of outer membrane vesicles was only apparent in the SEM images of MRx0518. The SEM image shows what appears to be outer membrane vesicles (white dashed arrow). Example 8—Activation of Murine TLR5 by Recombinant Flagellin Proteins fromEnterococcus gallinarumStrains MRx0518 and DSM 100110 Summary MRx0518 is a bacterial strain isolated from a human sample, whereas DSM 100110 is of murine origin. The ability of the recombinant flagellin proteins from theEnterococcus gallinarumstrains MRx0518 and DSM 100110 to activate murine TLR5 was tested. Methodology Recombinant purified MRx0518 (FliCMRx0518) and DSM 100110 (FliCDSM100110) were produced as described in Example 4. A TLR5 assay as described in Example 1 was performed. The TLR5 activation assay was performed with the following treatments:Recombinant purified FliCMRx0518at concentrations of 5, 1, 0.2, 0.04 and 0.008 ng/mlRecombinant purified FliCDSM100110at concentrations of 5, 1, 0.2, 0.04 and 0.008 ng/ml Three independent replicates were performed. Conclusions Both FliCMRx0518and FliCDSM100110activated murine TLR5 and there was no difference in the activation levels between FliCMRx0518and FliCDSM100110at any dose (FIG.15). This effect is different to the results observed for the activation of human TLR5 by FliCMRx0518and FliCDSM100110. As shown in Example 4,FIG.10, FliCMRx0518can stimulate human TLR5 to greater levels than FliCDSM100110at concentrations of 1 ng/ml and lower. Without being bound by any particular theory, the different activation profiles of human and murine TLR5 by recombinant flagellin proteins from differentEnterococcus gallinarumstrains may be due to sequence differences between FliCMRx0518and FliCDSM100110, as well as binding site variability between human and murine TLR5. As shown inFIG.3, the inventors have noted that the majority of the sequence variation between different flagellin polypeptides is observed in the D2-D3 region. A sequence alignment of FliCMRx0518and FliCDSM100110shows a large variation in the D2-D3 domain (FIG.16). The alignment was performed using CLUTAL OMEGA v1.2.4 multiple sequence alignment software. Example 9—Immunostimulatory Capability of MotileE. gallinarumandE. casseliflavusStrains Derived from Humans Summary The ability of motileE. gallinarumandE. casseliflavusstrains derived from humans to activate human TLR5 in vitro was assessed. Methodology Bacterial Strains Tested Strain NameSpeciesMRx0518E. gallinarumTest 1E. gallinarumTest 2E. gallinarumTest 3E. gallinarumMRx0554E. gallinarumMRx0556E. gallinarumMRx1548E. gallinarumMRx1649E. gallinarumMRx1650E. gallinarumMRx1763E. gallinarumMRx1766E. gallinarumMRx1775E. gallinarumTest 4E. casseliflavusTest 5E. casseliflavusTest 6E. casseliflavusDSM25781E. casseliflavus The MRX and test strains were isolated from one of four donors from the 4D Pharma culture collection (donor numbers FOO, F14, F19 and F22). TLR5 Assay Conditions The bacterial strains were cultured in YCFA broth (E&O Laboratories, Bonnybridge, Scotland, UK) until they reached stationary growth phase. Cells and supernatants were separated by centrifugation at 5,000×g for 5 mins. Supernatants were passed through a 0.22 μm filter and diluted in water. HEK-Blue™-hTLR5 cells (InvivoGen, San Diego, CA, USA) were routinely cultured in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 4.5 mg/ml glucose, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™ (InvivoGen), 30 μg/ml blastocidin (InvivoGen) and 100 μg/ml zeocin (InvivoGen) to 90% density. Cell lines were cultured at 37° C. and 5% CO2. All reagents were supplied by Sigma-Aldrich, Gillingham, England, UK unless otherwise stated. For co-cultures, cells grown to 90% density were washed once with phosphate-buffered saline (PBS) (Sigma-Aldrich) and resuspended in growth media without antibiotic at a density of 140,000 cells/ml. Supernatants were added to cells at a multiplicity of infection (MOI) equivalent of 100:1. The assay positive control,Salmonella Typhimuriumflagellin (FLA-ST) (InvivoGen), was used at 20 ng/ml. Cells were incubated with supernatants at 37° C. in a 5% CO2atmosphere for 22 h. QUANTI-Blue™ (InvivoGen) was added, plates were incubated for a further 2 h and optical density at 655 nm was recorded. Three independent biological replicates were carried out for all strains. The data inFIG.18represents three independent replicates. Conclusions All of the supernatants of theE. gallinarumandE. casseliflavusstrains tested were able to potently activate a TLR5 response compared to the untreated control. Of the threeE. casseliflavusstrains tested, Test 6 was found to strongly activate a TLR5 response, while Test 5 and DSM25781 elicit less potent TLR5 responses. Example 10—Activation of NF-κB by MRx518 The ability of the bacterial strain MRx0518 to activate NF-κB was investigated. The results are presented inFIG.18. MRx0518 supernatant was the most potent activator of NF-κB. The activation of NF-κB was eliminated after treatment with trypsin. These data show that flagellin from the genusEnterococcusand in particular from MRx0518 produce a very strong NF-κB response, and so may be useful in therapy. Example 11—T Cell Differentiation The ability of MRx0518 to induce T-cell differentiation was explored in vitro on peripheral blood mononuclear cells (PBMCs, Stemcell, Cat: 70025). Methodology PBMCs were plated in 96-well plates plated with anti-CD3 (Ebioscience, CD3 monoclonal antibody (OKT3 clone), functional grade, cat. No. 16-0037-81) at 400,000/well in 50 μl cRPMI medium per well (cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2 mM final conc. Stock 200 mM.; 10% HI FBS (Gibco life technologies, 10082-147); 50 μm mercaptoethanol (Gibco life technologies, 21985-023); and 1% pen/strep (P4333, 10 mg/ml). MRx518 supernatant was then added to each well, 4,000,000 in 100 μl/well. Supernatants were passed through a 0.22 μm filter and diluted appropriately in co-culture. Following 3 days in a 37° C. incubator, the cells were removed and re-suspended in a medium containing PMA—(Sigma, Cat no. P8139), Ionomycin (Sigma, Cat no. I3909) and GolgiSTOP (BD, Cat no 554724) for 5 hours. PMA stock was 1 mg/ml in DMSO which was further diluted in 100 ug/ml (each sample required 50 ng/ml in cRPMI), Ionomycin stock was 1 mM in DMSO (1 μM in cRPMI was used) and GolgiStop concentration was used at 4 μl/6 ml. The cells were then subjected to a flow cytometry staining: After washing, the cells were incubated with Viobility 405/520 Fixable Dye from Miltenyi biotec (1 μl/sample)+human Fc block, cat. 564219 (1 μl/sample) in PBS for 10 mins in the dark at room temperature. The surface antibodies (2 μl of each) were then added directly to the wells for 10 mins in the dark at room temperature—CD3-APC-Vio 770 (Miltenyi, cat. No. 130-113-136), CD4-VioBlue (Miltenyi, cat. No. 130-114-534) and CD25-VioBright FITC (Miltenyi, cat. No. 130-113-283). The cells were then washed twice in PBS and spun down at 300 g/5 min/RT. The eBioscience FoxP3 transcription factor staining buffer was then used to fix and permeabilise the cells (cat. No. 00-5523). Following the eBioscience protocol, a perm/fix buffer was prepared using 1× concentrate and 3 diluent. The cells were fixed for 1 h at RT and then washed 2× in 1× Perm wash and spun down at 300 g/5 min/RT. The following intracellular staining or transcription factor antibodies were added to the samples in perm wash (1×) for 45 mis/dark/RT or in the fridge overnight (up to 18 h), followed by washing the antibodies 2× using Perm wash (300 μl) and re-suspension in PBS (250 μl) to acquire on the cytometer: Intracellular markersTranscription factors2 ul IL10-PE5.5 ul FoxP3-PE-Cy72 ul IFNy-PE Vio7709 ul Tbet-APC10 ul IL17a-APC9 ul RoRyt-PEAnti IFNγ-PE Vio770 human antibodies (Miltenyi, cat. No. 130-114-025)Anti IL10-PE human antibodies (Miltenyi, cat. No. 130-112-728)Anti IL17a-APC human antibodies (Miltenyi, cat. No. 130-099-202)Anti RoRyt-PE human antibodies (Miltenyi, cat. No. 130-103-837)Anti Tbet-APC human antibodies (Miltenyi, cat. No. 130-098-655Foxp3 monoclonal antibody (236A/E7), Pe cy7 (ebioscience) cat. No. 25-4777-41 Conclusions As can be seen inFIG.19, MRx518 supernatant (SP 518) can induce the differentiation of T helper cells and cytotoxic T cells. Example 12—Generation of anE. gallinarumMRx0518 Flagellin Gene Insertion Mutant This example provides an alternative strategy for inactivating the flagellin gene compared to Example 5. The fliC gene was disrupted by homology-driven insertion of the suicide plasmid pORI19 (seeFIG.20). Insertion of pORI19 within the fliC gene was confirmed by DNA sequencing and the non-motile phenotype of the resulting mutant strain was confirmed in vitro. Methodology The flagellin insertion mutant was created using the non-replicative plasmid pORI19 (Emr repA− Ori+; cloning vector [118]). An internal fragment ofE. gallinarumMRx0518 fliC gene was amplified using primers DC020 (SEQ ID NO: 43: CCCGGGGGATCCGCGGTAAATGTTGCTAAAGCATCATCG) and DC021 (SEQ ID NO: 44: ACGACGGTCGACCCACAGCATCTTAGGGCGTATGCG) and cloned into pORI19. Restriction enzymes and Quick Ligase (New England Biolabs, Ipswich, MA, USA) were used according to the manufacturer's instructions. This construct was propagated inE. coliEC101 by chemical transformation [118] and isolated using the Genopure Plasmid Maxi Kit (Roche Diagnostics, Basel, Switzerland) from a 500-ml culture. Isolated plasmid DNA was concentrated using 0.3 M sodium acetate pH 5.2 and ethanol down to 20 μl. Electrocompetent cells were successfully generated by growing bacterial cultures in sub-inhibitory concentrations of glycine followed by mutanolysin and lysozyme treatments to further weaken the cell wall peptidoglycan layer. A protocol was developed to prepareE. gallinarumMRx0518 electrocompetent cells, which was adapted from a previously published method [119]. In brief,E. gallinarumMRx0518 was grown for 18 h in GM17 broth, supplemented with 0.5 M sucrose and 3% (w/v) glycine (Sigma-Aldrich). Cells were then washed twice with 0.5 M sucrose and 10% (v/v) glycerol and treated with 10 μg/ml lysozyme and 10 U/ml mutanolysin (Sigma-Aldrich) for 30 min at 37° C.E. gallinarumMRx0518 cells were then transformed by electroporation with 10 μg of plasmid DNA and recovered in BHI broth before plating on selective BHI agar. Positive colonies were screened for the presence of the em gene using primers DC047 (SEQ ID NO:45: CCAAATTAAAGAGGGTTATAATGAACGAG) and DC048 (SEQ ID NO:46: GATGCAGTTTATGCATCCCTTAAC). Plasmid insertion was confirmed for successful transformants by PCR amplification and sequencing (GATC Biotech, Konstanz, Germany) using primers DC013 (SEQ ID NO:47: CCGATAAATAGTAGCAGAGGGAAACC) and DC014 (SEQ ID NO:48: GGCTGAATATCCATCAGAGCTTCCTC). In vitro motility of the flagellin insertion mutant was assessed using BBL™ Motility Test Medium supplemented with 0.005% (w/v) 2,3,5-triphenyltetrazolium chloride (BD, Sparks, MD, USA). In brief, a fresh colony was stab-inoculated in 20 ml equilibrated media and incubated for 48 h at 37° C. in anaerobic conditions. All assays were performed in triplicate. Sequences SEQ ID NO:1—Flagellin polypeptide fromEnterococcus gallinarumMRx0518 SEQ ID NO:2—Flagellin polypeptide fromEnterococcus gallinarumDSM100110 SEQ ID NO:3—Flagellin polypeptide fromEnterococcus gallinarumMRx0554 SEQ ID NO:4—Flagellin polypeptide fromEnterococcus gallinarumMRx0556 SEQ ID NO:5—Flagellin polypeptide fromEnterococcus gallinarumMRx1548 SEQ ID NO:6—Flagellin polypeptide fromEnterococcus gallinarumMRx1650 SEQ ID NO:7—Flagellin polypeptide fromEnterococcus gallinarumMRx1763 SEQ ID NO:8—Flagellin polypeptide fromEnterococcus gallinarumMRx1766 SEQ ID NO:9—Flagellin polypeptide fromEnterococcus gallinarumMRx1775 SEQ ID NO:10—Flagellin polypeptide fromEnterococcus gallinarumMRx1886 SEQ ID NO:11—Flagellin polypeptide fromEnterococcus gallinarum2A8 SEQ ID NO:12—Flagellin polypeptide fromEnterococcus gallinarum9402 SEQ ID NO:13—Flagellin polypeptide fromEnterococcus gallinarumA6981 SEQ ID NO:14—Flagellin polypeptide fromEnterococcus gallinarumMRx1649 SEQ ID NO:15—Flagellin polypeptide fromEnterococcus gallinarumEG2 SEQ ID NO:16—Flagellin polypeptide fromEnterococcus gallinarumSKF1 SEQ ID NO:17—Flagellin polypeptide fromEnterococcus casseliflavusDSM 7370 SEQ ID NO:18—Flagellin polypeptide fromEnterococcus casseliflavus1a6A SEQ ID NO:19—Flagellin polypeptide fromEnterococcus casseliflavus3h10B SEQ ID NO:20—Flagellin polypeptide fromEnterococcus casseliflavus14-MB-W-14 SEQ ID NO:21—Flagellin polypeptide fromEnterococcus casseliflavusATCC12755 SEQ ID NO:22—Flagellin polypeptide fromEnterococcus casseliflavusDSM4841 SEQ ID NO:23—Flagellin polypeptide fromEnterococcus casseliflavusDSM20680 SEQ ID NO:24—Flagellin polypeptide fromEnterococcus casseliflavusEC10 SEQ ID NO:25—Flagellin polypeptide fromEnterococcus casseliflavusEC20 SEQ ID NO:26—Flagellin polypeptide fromEnterococcus casseliflavusEC30 SEQ ID NO:27—Flagellin polypeptide fromEnterococcus casseliflavusF1129 SEQ ID NO:28—Flagellin polypeptide fromEnterococcus casseliflavusF1129F46 SEQ ID NO:29—Flagellin polypeptide fromEnterococcus casseliflavusNBRC 100478 SEQ ID NO:30—Flagellin polypeptide fromEnterococcus casseliflavusPAVET15 SEQ ID NO:31—Flagellin polypeptide fromEnterococcus casseliflavusNLAE-z1-G268 SEQ ID NO:32—Flagellin polypeptide fromEnterococcus casseliflavusNLAE-z1-C414 SEQ ID NO:33—Flagellin polypeptide fromEnterococcus gallinarumFDAARGOS-163 SEQ ID NO:34—Flagellin polypeptide fromEnterococcus casseliflavusMRx0858 SEQ ID NO:35—Flagellin polypeptide fromEnterococcus casseliflavusDSM25781 SEQ ID NO:36—Flagellin polypeptide fromEnterococcus gallinarumDSM28564 SEQ ID NO:37—Flagellin polypeptide fromEnterococcus gallinarumDSM28565 SEQ ID NO:38—Flagellin polypeptide fromEnterococcus gallinarumF1213F 228 SEQ ID NO:39—Flagellin polypeptide fromEnterococcus gallinarumDSM20718 SEQ ID NO:40—Flagellin polypeptide fromEnterococcus gallinarumDSM20628 SEQ ID NO:41—Flagellin polypeptide fromEnterococcus gallinarumNBRC100675 SEQ ID NO:42—Flagellin polypeptide fromEnterococcus gallinarumDSM24841 REFERENCES [1] Spor et al. 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11857615 | DETAILED DISCLOSURE OF THE INVENTION Definitions The term “polypeptide” is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Further-more, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide (s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups. The term “subsequence” means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively The term “amino acid sequence” s the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The term “adjuvant” has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone. “Sequence identity” is in the context of the present invention determined by comparing 2 optimally aligned sequences of equal length (e.g. DNA, RNA or amino acid) according to the following formula: (Nref−Ndif)·100/Nref, wherein Nrefis the number of residues in one of the 2 sequences and Ndifis the number of residues which are non-identical in the two sequences when they are aligned over their entire lengths and in the same direction. So, two sequences 5′-ATTCGGAACC-3′ (SEQ ID No. 91) and 5′-ATACGGGACC-3′ (SEQ ID NO. 92) will provide the sequence identity 80% (Nref=10 and Ndif=2). An “assembly of amino acids” means two or more amino acids bound together by physical or chemical means. The “3D conformation” is the 3 dimensional structure of a biomolecule such as a protein. In monomeric polypeptides/proteins, the 3D conformation is also termed “the tertiary structure” and denotes the relative locations in 3 dimensional space of the amino acid residues forming the polypeptide. “An immunogenic carrier” is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen/hapten. Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen/hapten, which is not sufficiently immunogenic in its own right—typically, the immunogenic carrier is capable of eliciting a strong T-helper lymphocyte response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen by B-lymphocytes and cytotoxic lymphocytes. More recently, the large carrier molecules have to a certain extent been substituted by so-called promiscuous T-helper epitopes, i.e. shorter peptides that are recognized by a large fraction of HLA haplotypes in a population, and which elicit T-helper lymphocyte responses. A “T-helper lymphocyte response” is an immune response elicited on the basis of a peptide, which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell and which stimulates T-helper lymphocytes in an animal species as a consequence of T-cell receptor recognition of the complex between the peptide and the MHC Class II molecule present. An “immunogen” is a substance of matter which is capable of inducing an adaptive immune response in a host, whose immune system is confronted with the immunogen. As such, immunogens are a subset of the larger genus “antigens”, which are substances that can be recognized specifically by the immune system (e.g. when bound by antibodies or, alternatively, when fragments of the are antigens bound to MHC molecules are being recognized by T-cell receptors) but which are not necessarily capable of inducing immunity—an antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against the antigen. A “hapten” is a small molecule, which can neither induce or elicit an immune response, but if conjugated to an immunogenic carrier, antibodies or TCRs that recognize the hapten can be induced upon confrontation of the immune system with the hapten carrier conjugate. An “adaptive immune response” is an immune response in response to confrontation with an antigen or immunogen, where the immune response is specific for antigene determinants of the antigen/immunogen—examples of adaptive immune responses are induction of antigen specific antibody production or antigen specific induction/activation of T helper lymphocytes or cytotoxic lymphocytes. A “protective, adaptive immune response” is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen or a pathology-related agent that includes the antigen. Typically, prophylactic vaccination aims at establishing a protective adaptive immune response against one or several pathogens. “Stimulation of the immune system” means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased “alertness” of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen. Hybridization under “stringent conditions” is herein defined as hybridization performed under conditions by which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to a probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Generally, stringent wash temperature conditions are selected to be about 5° C. to about 2° C. lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow temperature range and represents the disruption of the double helix into its complementary single strands. The process is described by the temperature of the midpoint of transition, Tm, which is also called the melting temperature. Formulas are available in the art for the determination of melting temperatures. The term “animal” is in the present context in general intended to denote an animal species (preferably mammalian), such asHomo sapiens, Canis domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention substantially all will mount an immune response against the immunogen of the present invention. As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies. “Specific binding” denotes binding between two substances which goes beyond binding of either substance to randomly chosen substances and also goes beyond simple association between substances that tend to aggregate because they share the same overall hydrophobicity or hydrophilicity. As such, specific binding usually involves a combination of electrostatic and other interactions between two conformationally complementary areas on the two substances, meaning that the substances can “recognize” each other in a complex mixture. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. The term further denotes certain biological vehicles useful for the same purpose, e.g. viral vectors and phage—both these infectious agents are capable of introducing a heterologous nucleic acid sequence The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide. SPECIFIC EMBODIMENTS OF THE INVENTION The Polypeptides of the Invention In some embodiments the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may preferably constitute at least or exactly or at most 6, such as at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, at least or exactly or at most 35 and at least or exactly or at most 36, at least or exactly or at most 37, at least or exactly or at most 38, at least or exactly or at most 39, at least or exactly or at most 40, at least or exactly or at most 41, at least or exactly or at most 42, at least or exactly or at most 43, at least or exactly or at most 44, at least or exactly or at most 45, at least or exactly or at most 46, at least or exactly or at most 47, at least or exactly or at most 48, at least or exactly or at most 49, at least or exactly or at most 50, at least or exactly or at most 51, at least or exactly or at most 52, at least or exactly or at most 53, at least or exactly or at most 54, at least or exactly or at most 55 and at least or exactly or at most 56, at least or exactly or at most 57, at least or exactly or at most 58, at least or exactly or at most 59, or at least or exactly or at most 60 contiguous amino acid residues. The number may, where applicable, be higher. Another way to phrase this is that for each of SEQ ID NOs: 1-30, the number of the contiguous amino acid residues is at least N−n, where N is the length of the sequence ID in question and n is any integer between 6 and N−1; that is, the at least 5 contiguous amino acids can be at least any number between 5 and the length of the reference sequence minus one, in increments of one. Consequently: Insofar as embodiment b relates to SEQ ID NO: 2-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 61, at least or exactly or at most 62, at least or exactly or at most 63, at least or exactly or at most 64, at least or exactly or at most 65, at least or exactly or at most 66 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 4-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 67, at least or exactly or at most 68, at least or exactly or at most 69, at least or exactly or at most 70, at least or exactly or at most 71, at least or exactly or at most 72, at least or exactly or at most 73 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 5-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 74, at least or exactly or at most 75, at least or exactly or at most 76, at least or exactly or at most 77, at least or exactly or at most 78, at least or exactly or at most 79, at least or exactly or at most 80, at least or exactly or at most 81, at least or exactly or at most 82, at least or exactly or at most 83, at least or exactly or at most 84, at least or exactly or at most 85, at least or exactly or at most 86, at least or exactly or at most 87, at least or exactly or at most 88, at least or exactly or at most 89, at least or exactly or at most 90, at least or exactly or at most 91, at least or exactly or at most 92, at least or exactly or at most 93, at least or exactly or at most 94, at least or exactly or at most 95, at least or exactly or at most 96, at least or exactly or at most 97, at least or exactly or at most 98, at least or exactly or at most 99, at least or exactly or at most 100, at least or exactly or at most 101, at least or exactly or at most 102, at least or exactly or at most 103, at least or exactly or at most 104, at least or exactly or at most 105, at least or exactly or at most 106, at least or exactly or at most 107, at least or exactly or at most 108 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 6-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 109, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least or exactly or at most 113, at least or exactly or at most 114 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 7-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 115, at least or exactly or at most 116, at least or exactly or at most 117, at least or exactly or at most 118, at least or exactly or at most 119, at least or exactly or at most 120, at least or exactly or at most 121, at least or exactly or at most 122, at least or exactly or at most 123, at least or exactly or at most 124, at least or exactly or at most 125, at least or exactly or at most 126 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 8-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 127, at least or exactly or at most 128, at least or exactly or at most 129, at least or exactly or at most 130, at least or exactly or at most 131, at least or exactly or at most 132, at least or exactly or at most 133, at least or exactly or at most 134, at least or exactly or at most 135, at least or exactly or at most 136, at least or exactly or at most 137, at least or exactly or at most 138, at least or exactly or at most 139 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 9-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 140, at least or exactly or at most 141, at least or exactly or at most 142, at least or exactly or at most 143, at least or exactly or at most 144, at least or exactly or at most 145, at least or exactly or at most 146, at least or exactly or at most 147, at least or exactly or at most 148, at least or exactly or at most 149, at least or exactly or at most 150, at least or exactly or at most 151, at least or exactly or at most 152, at least or exactly or at most 153, at least or exactly or at most 154, at least or exactly or at most 155, at least or exactly or at most 156, at least or exactly or at most 157, at least or exactly or at most 158, at least or exactly or at most 159 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 10-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 160, at least or exactly or at most 161, at least or exactly or at most 162 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 11-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 163, at least or exactly or at most 164, at least or exactly or at most 165, at least or exactly or at most 166 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 12-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 167, at least or exactly or at most 168, at least or exactly or at most 169, at least or exactly or at most 170, at least or exactly or at most 171, at least or exactly or at most 172, at least or exactly or at most 173, at least or exactly or at most 174, at least or exactly or at most 175, at least or exactly or at most 176, at least or exactly or at most 177, at least or exactly or at most 178, at least or exactly or at most 179, at least or exactly or at most 180, at least or exactly or at most 181, at least or exactly or at most 182, at least or exactly or at most 183, at least or exactly or at most 184, at least or exactly or at most 185, at least or exactly or at most 186, at least or exactly or at most 187, at least or exactly or at most 188, at least or exactly or at most 189, at least or exactly or at most 190, at least or exactly or at most 191, at least or exactly or at most 192, at least or exactly or at most 193, at least or exactly or at most 194, at least or exactly or at most 195, at least or exactly or at most 196, at least or exactly or at most 197, at least or exactly or at most 198, at least or exactly or at most 199, at least or exactly or at most 200, at least or exactly or at most 201, at least or exactly or at most 202, at least or exactly or at most 203, at least or exactly or at most 204, at least or exactly or at most 205, at least or exactly or at most 206, at least or exactly or at most 207, at least or exactly or at most 208, at least or exactly or at most 209, at least or exactly or at most 210, at least or exactly or at most 211, at least or exactly or at most 212, at least or exactly or at most 213, at least or exactly or at most 214, at least or exactly or at most 215, at least or exactly or at most 216, at least or exactly or at most 217, at least or exactly or at most 218, at least or exactly or at most 219, at least or exactly or at most 220, at least or exactly or at most 221, at least or exactly or at most 222, at least or exactly or at most 223, at least or exactly or at most 224, at least or exactly or at most 225, at least or exactly or at most 226, at least or exactly or at most 227, at least or exactly or at most 228, at least or exactly or at most 229, at least or exactly or at most 230 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 13-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 231, at least or exactly or at most 232, at least or exactly or at most 233, at least or exactly or at most 234, at least or exactly or at most 235, at least or exactly or at most 236, at least or exactly or at most 237, at least or exactly or at most 238 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 14-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 239, at least or exactly or at most 240, at least or exactly or at most 241, at least or exactly or at most 242, at least or exactly or at most 243, at least or exactly or at most 244, at least or exactly or at most 245, at least or exactly or at most 246, at least or exactly or at most 247, at least or exactly or at most 248, at least or exactly or at most 249, at least or exactly or at most 250, at least or exactly or at most 251, at least or exactly or at most 252, at least or exactly or at most 253 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 15-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 254, at least or exactly or at most 255, at least or exactly or at most 256, at least or exactly or at most 257, at least or exactly or at most 258, at least or exactly or at most 259, at least or exactly or at most 260, at least or exactly or at most 261, at least or exactly or at most 262, at least or exactly or at most 263, at least or exactly or at most 264, at least or exactly or at most 265, at least or exactly or at most 266, at least or exactly or at most 267, at least or exactly or at most 268, at least or exactly or at most 269, at least or exactly or at most 270, at least or exactly or at most 271, at least or exactly or at most 272, at least or exactly or at most 273, at least or exactly or at most 274, at least or exactly or at most 275, at least or exactly or at most 276, at least or exactly or at most 277, at least or exactly or at most 278, at least or exactly or at most 279, at least or exactly or at most 280, at least or exactly or at most 281, at least or exactly or at most 282, at least or exactly or at most 283, at least or exactly or at most 284, at least or exactly or at most 285, at least or exactly or at most 286, at least or exactly or at most 287, at least or exactly or at most 288, at least or exactly or at most 289, at least or exactly or at most 290, at least or exactly or at most 291, at least or exactly or at most 292, at least or exactly or at most 293, at least or exactly or at most 294 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 16-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 295, at least or exactly or at most 296, at least or exactly or at most 297, at least or exactly or at most 298, at least or exactly or at most 299, at least or exactly or at most 300, at least or exactly or at most 301, at least or exactly or at most 302, at least or exactly or at most 303, at least or exactly or at most 304, at least or exactly or at most 305, at least or exactly or at most 306, at least or exactly or at most 307, at least or exactly or at most 308, at least or exactly or at most 309, at least or exactly or at most 310, at least or exactly or at most 311, at least or exactly or at most 312, at least or exactly or at most 313, at least or exactly or at most 314, at least or exactly or at most 315, at least or exactly or at most 316, at least or exactly or at most 317, at least or exactly or at most 318, at least or exactly or at most 319, at least or exactly or at most 320, at least or exactly or at most 321, at least or exactly or at most 322, at least or exactly or at most 323, at least or exactly or at most 324, at least or exactly or at most 325, at least or exactly or at most 326, at least or exactly or at most 327, at least or exactly or at most 328, at least or exactly or at most 329, at least or exactly or at most 330, at least or exactly or at most 331, at least or exactly or at most 332, at least or exactly or at most 333, at least or exactly or at most 334, at least or exactly or at most 335, at least or exactly or at most 336, at least or exactly or at most 337, at least or exactly or at most 338, at least or exactly or at most 339, at least or exactly or at most 340, at least or exactly or at most 341, at least or exactly or at most 342, at least or exactly or at most 343, at least or exactly or at most 344, at least or exactly or at most 345, at least or exactly or at most 346, at least or exactly or at most 347, at least or exactly or at most 348, at least or exactly or at most 349, at least or exactly or at most 350, at least or exactly or at most 351, at least or exactly or at most 352, at least or exactly or at most 353, at least or exactly or at most 354, at least or exactly or at most 355, at least or exactly or at most 356 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 17-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 357, at least or exactly or at most 358, at least or exactly or at most 359, at least or exactly or at most 360, at least or exactly or at most 361, at least or exactly or at most 362, at least or exactly or at most 363, at least or exactly or at most 364, at least or exactly or at most 365, at least or exactly or at most 366, at least or exactly or at most 367 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 18-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 368, at least or exactly or at most 369, at least or exactly or at most 370, at least or exactly or at most 371, at least or exactly or at most 372, at least or exactly or at most 373, at least or exactly or at most 374, at least or exactly or at most 375, at least or exactly or at most 376, at least or exactly or at most 377, at least or exactly or at most 378, at least or exactly or at most 379, at least or exactly or at most 380, at least or exactly or at most 381, at least or exactly or at most 382, at least or exactly or at most 383, at least or exactly or at most 384, at least or exactly or at most 385, at least or exactly or at most 386, at least or exactly or at most 387, at least or exactly or at most 388, at least or exactly or at most 389, at least or exactly or at most 390, at least or exactly or at most 391, at least or exactly or at most 392, at least or exactly or at most 393, at least or exactly or at most 394, at least or exactly or at most 395, at least or exactly or at most 396, at least or exactly or at most 397, at least or exactly or at most 398, at least or exactly or at most 399, at least or exactly or at most 400, at least or exactly or at most 401, at least or exactly or at most 402, at least or exactly or at most 403, at least or exactly or at most 404, at least or exactly or at most 405, at least or exactly or at most 406, at least or exactly or at most 407, at least or exactly or at most 408, at least or exactly or at most 409, at least or exactly or at most 410, at least or exactly or at most 411, at least or exactly or at most 412, at least or exactly or at most 413, at least or exactly or at most 414, at least or exactly or at most 415, at least or exactly or at most 416, at least or exactly or at most 417, at least or exactly or at most 418, at least or exactly or at most 419, at least or exactly or at most 420 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 19-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 421, at least or exactly or at most 422, at least or exactly or at most 423, at least or exactly or at most 424, at least or exactly or at most 425, at least or exactly or at most 426, at least or exactly or at most 427, at least or exactly or at most 428, at least or exactly or at most 429, at least or exactly or at most 430, at least or exactly or at most 431, at least or exactly or at most 432, at least or exactly or at most 433, at least or exactly or at most 434, at least or exactly or at most 435, at least or exactly or at most 436, at least or exactly or at most 437, at least or exactly or at most 438, at least or exactly or at most 439, at least or exactly or at most 440, at least or exactly or at most 441, at least or exactly or at most 442, at least or exactly or at most 443, at least or exactly or at most 444, at least or exactly or at most 445 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 20-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 446, at least or exactly or at most 447, at least or exactly or at most 448, at least or exactly or at most 449, at least or exactly or at most 450, at least or exactly or at most 451, at least or exactly or at most 452, at least or exactly or at most 453, at least or exactly or at most 454, at least or exactly or at most 455, at least or exactly or at most 456, at least or exactly or at most 457, at least or exactly or at most 458, at least or exactly or at most 459, at least or exactly or at most 460, at least or exactly or at most 461, at least or exactly or at most 462, at least or exactly or at most 463, at least or exactly or at most 464, at least or exactly or at most 465, at least or exactly or at most 466, at least or exactly or at most 467, at least or exactly or at most 468, at least or exactly or at most 469, at least or exactly or at most 470, at least or exactly or at most 471, at least or exactly or at most 472, at least or exactly or at most 473, at least or exactly or at most 474, at least or exactly or at most 475, at least or exactly or at most 476, at least or exactly or at most 477, at least or exactly or at most 478, at least or exactly or at most 479, at least or exactly or at most 480, at least or exactly or at most 481, at least or exactly or at most 482, at least or exactly or at most 483, at least or exactly or at most 484, at least or exactly or at most 485, at least or exactly or at most 486, at least or exactly or at most 487, at least or exactly or at most 488, at least or exactly or at most 489, at least or exactly or at most 490, at least or exactly or at most 491, at least or exactly or at most 492, at least or exactly or at most 493, at least or exactly or at most 494, at least or exactly or at most 495, at least or exactly or at most 496, at least or exactly or at most 497, at least or exactly or at most 498, at least or exactly or at most 499, at least or exactly or at most 500, at least or exactly or at most 501, at least or exactly or at most 502, at least or exactly or at most 503, at least or exactly or at most 504, at least or exactly or at most 505, at least or exactly or at most 506, at least or exactly or at most 507, at least or exactly or at most 508, at least or exactly or at most 509, at least or exactly or at most 510, at least or exactly or at most 511, at least or exactly or at most 512, at least or exactly or at most 513, at least or exactly or at most 514, at least or exactly or at most 515, at least or exactly or at most 516, at least or exactly or at most 517, at least or exactly or at most 518, at least or exactly or at most 519, at least or exactly or at most 520, at least or exactly or at most 521, at least or exactly or at most 522, at least or exactly or at most 523, at least or exactly or at most 524, at least or exactly or at most 525, at least or exactly or at most 526, at least or exactly or at most 527, at least or exactly or at most 528, at least or exactly or at most 529, at least or exactly or at most 530, at least or exactly or at most 531, at least or exactly or at most 532, at least or exactly or at most 533, at least or exactly or at most 534, at least or exactly or at most 535, at least or exactly or at most 536, at least or exactly or at most 537, at least or exactly or at most 538, at least or exactly or at most 539, at least or exactly or at most 540, at least or exactly or at most 541, at least or exactly or at most 542, at least or exactly or at most 543, at least or exactly or at most 544, at least or exactly or at most 545, at least or exactly or at most 546, at least or exactly or at most 547, at least or exactly or at most 548, at least or exactly or at most 549, at least or exactly or at most 550, at least or exactly or at most 551, at least or exactly or at most 552, at least or exactly or at most 553, at least or exactly or at most 554, at least or exactly or at most 555, at least or exactly or at most 556, at least or exactly or at most 557, at least or exactly or at most 558, at least or exactly or at most 559, at least or exactly or at most 560, at least or exactly or at most 561, at least or exactly or at most 562, at least or exactly or at most 563, at least or exactly or at most 564, at least or exactly or at most 565, at least or exactly or at most 566, at least or exactly or at most 567, at least or exactly or at most 568, at least or exactly or at most 569, at least or exactly or at most 570, at least or exactly or at most 571, at least or exactly or at most 572, at least or exactly or at most 573, at least or exactly or at most 574, at least or exactly or at most 575, at least or exactly or at most 576, at least or exactly or at most 577, at least or exactly or at most 578, at least or exactly or at most 579, at least or exactly or at most 580, at least or exactly or at most 581, at least or exactly or at most 582, at least or exactly or at most 583, at least or exactly or at most 584, at least or exactly or at most 585, at least or exactly or at most 586, at least or exactly or at most 587, at least or exactly or at most 588, at least or exactly or at most 589, at least or exactly or at most 590, at least or exactly or at most 591, at least or exactly or at most 592, at least or exactly or at most 593, at least or exactly or at most 594, at least or exactly or at most 595, at least or exactly or at most 596, at least or exactly or at most 597, at least or exactly or at most 598, at least or exactly or at most 599, at least or exactly or at most 600, at least or exactly or at most 601, at least or exactly or at most 602, at least or exactly or at most 603, at least or exactly or at most 604, at least or exactly or at most 605, at least or exactly or at most 606, at least or exactly or at most 607, at least or exactly or at most 608, at least or exactly or at most 609, at least or exactly or at most 610, at least or exactly or at most 611, at least or exactly or at most 612, at least or exactly or at most 613, at least or exactly or at most 614, at least or exactly or at most 615, at least or exactly or at most 616, at least or exactly or at most 617, at least or exactly or at most 618, at least or exactly or at most 619, at least or exactly or at most 620, at least or exactly or at most 621, at least or exactly or at most 622, at least or exactly or at most 623, at least or exactly or at most 624, at least or exactly or at most 625, at least or exactly or at most 626, at least or exactly or at most 627, at least or exactly or at most 628, at least or exactly or at most 629, at least or exactly or at most 630, at least or exactly or at most 631, at least or exactly or at most 632, at least or exactly or at most 633, at least or exactly or at most 634, at least or exactly or at most 635, at least or exactly or at most 636, at least or exactly or at most 637, at least or exactly or at most 638, at least or exactly or at most 639, at least or exactly or at most 640, at least or exactly or at most 641, at least or exactly or at most 642, at least or exactly or at most 643, at least or exactly or at most 644, at least or exactly or at most 645, at least or exactly or at most 646, at least or exactly or at most 647, at least or exactly or at most 648, at least or exactly or at most 649, at least or exactly or at most 650, at least or exactly or at most 651, at least or exactly or at most 652, at least or exactly or at most 653, at least or exactly or at most 654, at least or exactly or at most 655, at least or exactly or at most 656, at least or exactly or at most 657, at least or exactly or at most 658, at least or exactly or at most 659, at least or exactly or at most 660, at least or exactly or at most 661, at least or exactly or at most 662, at least or exactly or at most 663, at least or exactly or at most 664, at least or exactly or at most 665, at least or exactly or at most 666, at least or exactly or at most 667, at least or exactly or at most 668, at least or exactly or at most 669, at least or exactly or at most 670, at least or exactly or at most 671, at least or exactly or at most 672, at least or exactly or at most 673, at least or exactly or at most 674, at least or exactly or at most 675, at least or exactly or at most 676, at least or exactly or at most 677, at least or exactly or at most 678, at least or exactly or at most 679, at least or exactly or at most 680, at least or exactly or at most 681, at least or exactly or at most 682, at least or exactly or at most 683, at least or exactly or at most 684, at least or exactly or at most 685, at least or exactly or at most 686, at least or exactly or at most 687, at least or exactly or at most 688, at least or exactly or at most 689, at least or exactly or at most 690, at least or exactly or at most 691, at least or exactly or at most 692, at least or exactly or at most 693, at least or exactly or at most 694, at least or exactly or at most 695, at least or exactly or at most 696, at least or exactly or at most 697, at least or exactly or at most 698, at least or exactly or at most 699, at least or exactly or at most 700, at least or exactly or at most 701, at least or exactly or at most 702, at least or exactly or at most 703, at least or exactly or at most 704, at least or exactly or at most 705, at least or exactly or at most 706, at least or exactly or at most 707, at least or exactly or at most 708, at least or exactly or at most 709, at least or exactly or at most 710, at least or exactly or at most 711 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 21-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 712, at least or exactly or at most 713, at least or exactly or at most 714, at least or exactly or at most 715, at least or exactly or at most 716, at least or exactly or at most 717, at least or exactly or at most 718, at least or exactly or at most 719, at least or exactly or at most 720, at least or exactly or at most 721, at least or exactly or at most 722, at least or exactly or at most 723, at least or exactly or at most 724, at least or exactly or at most 725, at least or exactly or at most 726, at least or exactly or at most 727 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 22-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 728 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 23-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 729, at least or exactly or at most 730, at least or exactly or at most 731, at least or exactly or at most 732, at least or exactly or at most 733, at least or exactly or at most 734, at least or exactly or at most 735, at least or exactly or at most 736, at least or exactly or at most 737, at least or exactly or at most 738, at least or exactly or at most 739, at least or exactly or at most 740, at least or exactly or at most 741, at least or exactly or at most 742, at least or exactly or at most 743, at least or exactly or at most 744, at least or exactly or at most 745, at least or exactly or at most 746, at least or exactly or at most 747, at least or exactly or at most 748, at least or exactly or at most 749, at least or exactly or at most 750, at least or exactly or at most 751, at least or exactly or at most 752, at least or exactly or at most 753, at least or exactly or at most 754 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 24-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 755, at least or exactly or at most 756, at least or exactly or at most 757, at least or exactly or at most 758, at least or exactly or at most 759, at least or exactly or at most 760, at least or exactly or at most 761, at least or exactly or at most 762, at least or exactly or at most 763, at least or exactly or at most 764, at least or exactly or at most 765, at least or exactly or at most 766, at least or exactly or at most 767, at least or exactly or at most 768, at least or exactly or at most 769, at least or exactly or at most 770, at least or exactly or at most 771, at least or exactly or at most 772, at least or exactly or at most 773, at least or exactly or at most 774, at least or exactly or at most 775, at least or exactly or at most 776, at least or exactly or at most 777, at least or exactly or at most 778, at least or exactly or at most 779, at least or exactly or at most 780, at least or exactly or at most 781, at least or exactly or at most 782, at least or exactly or at most 783, at least or exactly or at most 784, at least or exactly or at most 785, at least or exactly or at most 786, at least or exactly or at most 787, at least or exactly or at most 788, at least or exactly or at most 789, at least or exactly or at most 790, at least or exactly or at most 791, at least or exactly or at most 792, at least or exactly or at most 793, at least or exactly or at most 794, at least or exactly or at most 795, at least or exactly or at most 796, at least or exactly or at most 797, at least or exactly or at most 798, at least or exactly or at most 799, at least or exactly or at most 800, at least or exactly or at most 801, at least or exactly or at most 802, at least or exactly or at most 803, at least or exactly or at most 804, at least or exactly or at most 805, at least or exactly or at most 806, at least or exactly or at most 807, at least or exactly or at most 808, at least or exactly or at most 809, at least or exactly or at most 810, at least or exactly or at most 811, at least or exactly or at most 812, at least or exactly or at most 813, at least or exactly or at most 814, at least or exactly or at most 815, at least or exactly or at most 816, at least or exactly or at most 817, at least or exactly or at most 818, at least or exactly or at most 819, at least or exactly or at most 820, at least or exactly or at most 821, at least or exactly or at most 822, at least or exactly or at most 823, at least or exactly or at most 824, at least or exactly or at most 825, at least or exactly or at most 826, at least or exactly or at most 827, at least or exactly or at most 828, at least or exactly or at most 829, at least or exactly or at most 830, at least or exactly or at most 831, at least or exactly or at most 832, at least or exactly or at most 833, at least or exactly or at most 834, at least or exactly or at most 835, at least or exactly or at most 836, at least or exactly or at most 837, at least or exactly or at most 838, at least or exactly or at most 839, at least or exactly or at most 840, at least or exactly or at most 841, at least or exactly or at most 842, at least or exactly or at most 843, at least or exactly or at most 844, at least or exactly or at most 845, at least or exactly or at most 846, at least or exactly or at most 847, at least or exactly or at most 848, at least or exactly or at most 849, at least or exactly or at most 850, at least or exactly or at most 851, at least or exactly or at most 852, at least or exactly or at most 853, at least or exactly or at most 854, at least or exactly or at most 855, at least or exactly or at most 856, at least or exactly or at most 857, at least or exactly or at most 858, at least or exactly or at most 859, at least or exactly or at most 860, at least or exactly or at most 861, at least or exactly or at most 862, at least or exactly or at most 863, at least or exactly or at most 864, at least or exactly or at most 865, at least or exactly or at most 866, at least or exactly or at most 867, at least or exactly or at most 868, at least or exactly or at most 869, at least or exactly or at most 870, at least or exactly or at most 871, at least or exactly or at most 872, at least or exactly or at most 873, at least or exactly or at most 874, at least or exactly or at most 875, at least or exactly or at most 876, at least or exactly or at most 877, at least or exactly or at most 878, at least or exactly or at most 879, at least or exactly or at most 880, at least or exactly or at most 881, at least or exactly or at most 882, at least or exactly or at most 883, at least or exactly or at most 884, at least or exactly or at most 885, at least or exactly or at most 886, at least or exactly or at most 887, at least or exactly or at most 888, at least or exactly or at most 889, at least or exactly or at most 890, at least or exactly or at most 891, at least or exactly or at most 892, at least or exactly or at most 893, at least or exactly or at most 894, at least or exactly or at most 895, at least or exactly or at most 896, at least or exactly or at most 897, at least or exactly or at most 898, at least or exactly or at most 899, at least or exactly or at most 900, at least or exactly or at most 901, at least or exactly or at most 902, at least or exactly or at most 903, at least or exactly or at most 904, at least or exactly or at most 905, at least or exactly or at most 906 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 25-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 907, at least or exactly or at most 908, at least or exactly or at most 909, at least or exactly or at most 910, at least or exactly or at most 911, at least or exactly or at most 912, at least or exactly or at most 913, at least or exactly or at most 914, at least or exactly or at most 915, at least or exactly or at most 916, at least or exactly or at most 917, at least or exactly or at most 918, at least or exactly or at most 919, at least or exactly or at most 920, at least or exactly or at most 921, at least or exactly or at most 922, at least or exactly or at most 923, at least or exactly or at most 924, at least or exactly or at most 925, at least or exactly or at most 926, at least or exactly or at most 927, at least or exactly or at most 928, at least or exactly or at most 929, at least or exactly or at most 930, at least or exactly or at most 931, at least or exactly or at most 932, at least or exactly or at most 933, at least or exactly or at most 934, at least or exactly or at most 935, at least or exactly or at most 936, at least or exactly or at most 937, at least or exactly or at most 938, at least or exactly or at most 939, at least or exactly or at most 940, at least or exactly or at most 941, at least or exactly or at most 942, at least or exactly or at most 943, at least or exactly or at most 944, at least or exactly or at most 945, at least or exactly or at most 946, at least or exactly or at most 947, at least or exactly or at most 948, at least or exactly or at most 949, at least or exactly or at most 950, at least or exactly or at most 951, at least or exactly or at most 952, at least or exactly or at most 953, at least or exactly or at most 954, at least or exactly or at most 955, at least or exactly or at most 956, at least or exactly or at most 957, at least or exactly or at most 958, at least or exactly or at most 959, at least or exactly or at most 960, at least or exactly or at most 961, at least or exactly or at most 962, at least or exactly or at most 963, at least or exactly or at most 964, at least or exactly or at most 965, at least or exactly or at most 966, at least or exactly or at most 967, at least or exactly or at most 968, at least or exactly or at most 969, at least or exactly or at most 970, at least or exactly or at most 971, at least or exactly or at most 972, at least or exactly or at most 973, at least or exactly or at most 974 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 26-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 975, at least or exactly or at most 976, at least or exactly or at most 977, at least or exactly or at most 978, at least or exactly or at most 979, at least or exactly or at most 980, at least or exactly or at most 981, at least or exactly or at most 982, at least or exactly or at most 983, at least or exactly or at most 984, at least or exactly or at most 985, at least or exactly or at most 986, at least or exactly or at most 987, at least or exactly or at most 988, at least or exactly or at most 989, at least or exactly or at most 990, at least or exactly or at most 991, at least or exactly or at most 992, at least or exactly or at most 993, at least or exactly or at most 994, at least or exactly or at most 995, at least or exactly or at most 996, at least or exactly or at most 997, at least or exactly or at most 998, at least or exactly or at most 999, at least or exactly or at most 1000, at least or exactly or at most 1001, at least or exactly or at most 1002, at least or exactly or at most 1003, at least or exactly or at most 1004, at least or exactly or at most 1005, at least or exactly or at most 1006, at least or exactly or at most 1007, at least or exactly or at most 1008, at least or exactly or at most 1009, at least or exactly or at most 1010, at least or exactly or at most 1011, at least or exactly or at most 1012, at least or exactly or at most 1013, at least or exactly or at most 1014, at least or exactly or at most 1015, at least or exactly or at most 1016, at least or exactly or at most 1017, at least or exactly or at most 1018, at least or exactly or at most 1019, at least or exactly or at most 1020, at least or exactly or at most 1021, at least or exactly or at most 1022, at least or exactly or at most 1023, at least or exactly or at most 1024, at least or exactly or at most 1025, at least or exactly or at most 1026, at least or exactly or at most 1027, at least or exactly or at most 1028, at least or exactly or at most 1029, at least or exactly or at most 1030, at least or exactly or at most 1031, at least or exactly or at most 1032, at least or exactly or at most 1033, at least or exactly or at most 1034, at least or exactly or at most 1035, at least or exactly or at most 1036, at least or exactly or at most 1037, at least or exactly or at most 1038, at least or exactly or at most 1039, at least or exactly or at most 1040, at least or exactly or at most 1041, at least or exactly or at most 1042, at least or exactly or at most 1043, at least or exactly or at most 1044, at least or exactly or at most 1045, at least or exactly or at most 1046, at least or exactly or at most 1047, at least or exactly or at most 1048, at least or exactly or at most 1049, at least or exactly or at most 1050, at least or exactly or at most 1051, at least or exactly or at most 1052, at least or exactly or at most 1053, at least or exactly or at most 1054, at least or exactly or at most 1055, at least or exactly or at most 1056, at least or exactly or at most 1057, at least or exactly or at most 1058, at least or exactly or at most 1059, at least or exactly or at most 1060, at least or exactly or at most 1061, at least or exactly or at most 1062, at least or exactly or at most 1063, at least or exactly or at most 1064, at least or exactly or at most 1065, at least or exactly or at most 1066, at least or exactly or at most 1067, at least or exactly or at most 1068, at least or exactly or at most 1069, at least or exactly or at most 1070, at least or exactly or at most 1071 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 27-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 1072, at least or exactly or at most 1073, at least or exactly or at most 1074, at least or exactly or at most 1075, at least or exactly or at most 1076, at least or exactly or at most 1077, at least or exactly or at most 1078, at least or exactly or at most 1079, at least or exactly or at most 1080, at least or exactly or at most 1081, at least or exactly or at most 1082, at least or exactly or at most 1083, at least or exactly or at most 1084, at least or exactly or at most 1085, at least or exactly or at most 1086, at least or exactly or at most 1087, at least or exactly or at most 1088, at least or exactly or at most 1089, at least or exactly or at most 1090, at least or exactly or at most 1091, at least or exactly or at most 1092, at least or exactly or at most 1093, at least or exactly or at most 1094, at least or exactly or at most 1095, at least or exactly or at most 1096, at least or exactly or at most 1097, at least or exactly or at most 1098, at least or exactly or at most 1099, at least or exactly or at most 1100, at least or exactly or at most 1101, at least or exactly or at most 1102, at least or exactly or at most 1103, at least or exactly or at most 1104, at least or exactly or at most 1105, at least or exactly or at most 1106, at least or exactly or at most 1107, at least or exactly or at most 1108, at least or exactly or at most 1109, at least or exactly or at most 1110 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 28-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 1111, at least or exactly or at most 1112, at least or exactly or at most 1113, at least or exactly or at most 1114, at least or exactly or at most 1115, at least or exactly or at most 1116, at least or exactly or at most 1117, at least or exactly or at most 1118, at least or exactly or at most 1119, at least or exactly or at most 1120, at least or exactly or at most 1121, at least or exactly or at most 1122, at least or exactly or at most 1123, at least or exactly or at most 1124, at least or exactly or at most 1125, at least or exactly or at most 1126, at least or exactly or at most 1127, at least or exactly or at most 1128, at least or exactly or at most 1129, at least or exactly or at most 1130, at least or exactly or at most 1131, at least or exactly or at most 1132, at least or exactly or at most 1133, at least or exactly or at most 1134, at least or exactly or at most 1135, at least or exactly or at most 1136, at least or exactly or at most 1137, at least or exactly or at most 1138, at least or exactly or at most 1139, at least or exactly or at most 1140, at least or exactly or at most 1141, at least or exactly or at most 1142, at least or exactly or at most 1143, at least or exactly or at most 1144, at least or exactly or at most 1145, at least or exactly or at most 1146, at least or exactly or at most 1147, at least or exactly or at most 1148, at least or exactly or at most 1149, at least or exactly or at most 1150, at least or exactly or at most 1151, at least or exactly or at most 1152, at least or exactly or at most 1153, at least or exactly or at most 1154, at least or exactly or at most 1155, at least or exactly or at most 1156, at least or exactly or at most 1157, at least or exactly or at most 1158, at least or exactly or at most 1159, at least or exactly or at most 1160, at least or exactly or at most 1161, at least or exactly or at most 1162, at least or exactly or at most 1163, at least or exactly or at most 1164, at least or exactly or at most 1165, at least or exactly or at most 1166, at least or exactly or at most 1167, at least or exactly or at most 1168, at least or exactly or at most 1169, at least or exactly or at most 1170, at least or exactly or at most 1171, at least or exactly or at most 1172, at least or exactly or at most 1173, at least or exactly or at most 1174, at least or exactly or at most 1175, at least or exactly or at most 1176, at least or exactly or at most 1177, at least or exactly or at most 1178, at least or exactly or at most 1179, at least or exactly or at most 1180, at least or exactly or at most 1181, at least or exactly or at most 1182, at least or exactly or at most 1183, at least or exactly or at most 1184, at least or exactly or at most 1185, at least or exactly or at most 1186, at least or exactly or at most 1187, at least or exactly or at most 1188, at least or exactly or at most 1189, at least or exactly or at most 1190, at least or exactly or at most 1191, at least or exactly or at most 1192, at least or exactly or at most 1193, at least or exactly or at most 1194, at least or exactly or at most 1195, at least or exactly or at most 1196, at least or exactly or at most 1197, at least or exactly or at most 1198, at least or exactly or at most 1199, at least or exactly or at most 1200, at least or exactly or at most 1201, at least or exactly or at most 1202, at least or exactly or at most 1203, at least or exactly or at most 1204, at least or exactly or at most 1205, at least or exactly or at most 1206, at least or exactly or at most 1207, at least or exactly or at most 1208, at least or exactly or at most 1209, at least or exactly or at most 1210, at least or exactly or at most 1211, at least or exactly or at most 1212, at least or exactly or at most 1213, at least or exactly or at most 1214, at least or exactly or at most 1215, at least or exactly or at most 1216, at least or exactly or at most 1217, at least or exactly or at most 1218, at least or exactly or at most 1219, at least or exactly or at most 1220, at least or exactly or at most 1221, at least or exactly or at most 1222, at least or exactly or at most 1223, at least or exactly or at most 1224, at least or exactly or at most 1225, at least or exactly or at most 1226, at least or exactly or at most 1227, at least or exactly or at most 1228 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 29-30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 1229, at least or exactly or at most 1230, at least or exactly or at most 1231, at least or exactly or at most 1232, at least or exactly or at most 1233, at least or exactly or at most 1234, at least or exactly or at most 1235, at least or exactly or at most 1236, at least or exactly or at most 1237, at least or exactly or at most 1238, at least or exactly or at most 1239, at least or exactly or at most 1240, at least or exactly or at most 1241, at least or exactly or at most 1242, at least or exactly or at most 1243, at least or exactly or at most 1244, at least or exactly or at most 1245, at least or exactly or at most 1246, at least or exactly or at most 1247, at least or exactly or at most 1248, at least or exactly or at most 1249, at least or exactly or at most 1250, at least or exactly or at most 1251, at least or exactly or at most 1252, at least or exactly or at most 1253, at least or exactly or at most 1254, at least or exactly or at most 1255, at least or exactly or at most 1256, at least or exactly or at most 1257, at least or exactly or at most 1258, at least or exactly or at most 1259, at least or exactly or at most 1260, at least or exactly or at most 1261, at least or exactly or at most 1262, at least or exactly or at most 1263, at least or exactly or at most 1264, at least or exactly or at most 1265, at least or exactly or at most 1266, at least or exactly or at most 1267, at least or exactly or at most 1268, at least or exactly or at most 1269, at least or exactly or at most 1270, at least or exactly or at most 1271, at least or exactly or at most 1272, at least or exactly or at most 1273, at least or exactly or at most 1274, at least or exactly or at most 1275, at least or exactly or at most 1276, at least or exactly or at most 1277, at least or exactly or at most 1278, at least or exactly or at most 1279, at least or exactly or at most 1280, at least or exactly or at most 1281, at least or exactly or at most 1282, at least or exactly or at most 1283, at least or exactly or at most 1284 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NO: 30, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention will preferably constitute at least or exactly or at most 1285, at least or exactly or at most 1286, at least or exactly or at most 1287, at least or exactly or at most 1288, at least or exactly or at most 1289, at least or exactly or at most 1290, at least or exactly or at most 1291, at least or exactly or at most 1292, at least or exactly or at most 1293, at least or exactly or at most 1294, at least or exactly or at most 1295, at least or exactly or at most 1296, at least or exactly or at most 1297, at least or exactly or at most 1298, at least or exactly or at most 1299, at least or exactly or at most 1300, at least or exactly or at most 1301, at least or exactly or at most 1302, at least or exactly or at most 1303, at least or exactly or at most 1304, at least or exactly or at most 1305, at least or exactly or at most 1306, at least or exactly or at most 1307, at least or exactly or at most 1308, at least or exactly or at most 1309, at least or exactly or at most 1310, at least or exactly or at most 1311, at least or exactly or at most 1312, at least or exactly or at most 1313, at least or exactly or at most 1314, at least or exactly or at most 1315, at least or exactly or at most 1316, at least or exactly or at most 1317, at least or exactly or at most 1318, at least or exactly or at most 1319, at least or exactly or at most 1320, at least or exactly or at most 1321, at least or exactly or at most 1322, at least or exactly or at most 1323, at least or exactly or at most 1324, at least or exactly or at most 1325, at least or exactly or at most 1326, at least or exactly or at most 1327, at least or exactly or at most 1328, at least or exactly or at most 1329, at least or exactly or at most 1330, at least or exactly or at most 1331, at least or exactly or at most 1332, at least or exactly or at most 1333, at least or exactly or at most 1334, at least or exactly or at most 1335, at least or exactly or at most 1336, at least or exactly or at most 1337, at least or exactly or at most 1338, at least or exactly or at most 1339, at least or exactly or at most 1340, at least or exactly or at most 1341, at least or exactly or at most 1342, at least or exactly or at most 1343, at least or exactly or at most 1344, at least or exactly or at most 1345, at least or exactly or at most 1346, at least or exactly or at most 1347, at least or exactly or at most 1348, at least or exactly or at most 1349, at least or exactly or at most 1350, at least or exactly or at most 1351, at least or exactly or at most 1352, at least or exactly or at most 1353, at least or exactly or at most 1354, at least or exactly or at most 1355, at least or exactly or at most 1356, at least or exactly or at most 1357, at least or exactly or at most 1358, at least or exactly or at most 1359, at least or exactly or at most 1360, at least or exactly or at most 1361, at least or exactly or at most 1362, at least or exactly or at most 1363, at least or exactly or at most 1364, at least or exactly or at most 1365, at least or exactly or at most 1366, at least or exactly or at most 1367, at least or exactly or at most 1368, at least or exactly or at most 1369, at least or exactly or at most 1370, at least or exactly or at most 1371, at least or exactly or at most 1372, at least or exactly or at most 1373, at least or exactly or at most 1374, at least or exactly or at most 1375, at least or exactly or at most 1376, at least or exactly or at most 1377, at least or exactly or at most 1378, at least or exactly or at most 1379, at least or exactly or at most 1380, at least or exactly or at most 1381, at least or exactly or at most 1382, at least or exactly or at most 1383, at least or exactly or at most 1384, at least or exactly or at most 1385, at least or exactly or at most 1386, at least or exactly or at most 1387, at least or exactly or at most 1388, at least or exactly or at most 1389, at least or exactly or at most 1390, at least or exactly or at most 1391, at least or exactly or at most 1392, at least or exactly or at most 1393, at least or exactly or at most 1394, at least or exactly or at most 1395, at least or exactly or at most 1396, at least or exactly or at most 1397, at least or exactly or at most 1398, at least or exactly or at most 1399, at least or exactly or at most 1400, at least or exactly or at most 1401, at least or exactly or at most 1402, at least or exactly or at most 1403, at least or exactly or at most 1404, at least or exactly or at most 1405, at least or exactly or at most 1406, at least or exactly or at most 1407, at least or exactly or at most 1408, at least or exactly or at most 1409, at least or exactly or at most 1410, at least or exactly or at most 1411, at least or exactly or at most 1412, at least or exactly or at most 1413, at least or exactly or at most 1414, at least or exactly or at most 1415, at least or exactly or at most 1416, at least or exactly or at most 1417, at least or exactly or at most 1418, at least or exactly or at most 1419, at least or exactly or at most 1420, at least or exactly or at most 1421, at least or exactly or at most 1422, at least or exactly or at most 1423, at least or exactly or at most 1424, at least or exactly or at most 1425, at least or exactly or at most 1426, at least or exactly or at most 1427, at least or exactly or at most 1428, at least or exactly or at most 1429, at least or exactly or at most 1430, at least or exactly or at most 1431, at least or exactly or at most 1432, at least or exactly or at most 1433, at least or exactly or at most 1434, at least or exactly or at most 1435, at least or exactly or at most 1436, at least or exactly or at most 1437, at least or exactly or at most 1438, at least or exactly or at most 1439, at least or exactly or at most 1440, at least or exactly or at most 1441, at least or exactly or at most 1442, at least or exactly or at most 1443, at least or exactly or at most 1444, at least or exactly or at most 1445, at least or exactly or at most 1446, at least or exactly or at most 1447, at least or exactly or at most 1448, at least or exactly or at most 1449, at least or exactly or at most 1450, at least or exactly or at most 1451, at least or exactly or at most 1452, at least or exactly or at most 1453, at least or exactly or at most 1454, at least or exactly or at most 1455, at least or exactly or at most 1456, at least or exactly or at most 1457, at least or exactly or at most 1458, at least or exactly or at most 1459, at least or exactly or at most 1460, at least or exactly or at most 1461, at least or exactly or at most 1462, at least or exactly or at most 1463, at least or exactly or at most 1464, at least or exactly or at most 1465, at least or exactly or at most 1466, at least or exactly or at most 1467, at least or exactly or at most 1468, at least or exactly or at most 1469, at least or exactly or at most 1470, at least or exactly or at most 1471, at least or exactly or at most 1472, at least or exactly or at most 1473, at least or exactly or at most 1474, at least or exactly or at most 1475, at least or exactly or at most 1476, at least or exactly or at most 1477, at least or exactly or at most 1478, at least or exactly or at most 1479, at least or exactly or at most 1480, at least or exactly or at most 1481, at least or exactly or at most 1482, at least or exactly or at most 1483, at least or exactly or at most 1484, at least or exactly or at most 1485, at least or exactly or at most 1486, at least or exactly or at most 1487, at least or exactly or at most 1488, at least or exactly or at most 1489, at least or exactly or at most 1490, at least or exactly or at most 1491, at least or exactly or at most 1492, at least or exactly or at most 1493, at least or exactly or at most 1494, at least or exactly or at most 1495, at least or exactly or at most 1496, at least or exactly or at most 1497, at least or exactly or at most 1498, at least or exactly or at most 1499, at least or exactly or at most 1500, at least or exactly or at most 1501, at least or exactly or at most 1502, at least or exactly or at most 1503, at least or exactly or at most 1504, at least or exactly or at most 1505, at least or exactly or at most 1506, at least or exactly or at most 1507, at least or exactly or at most 1508, at least or exactly or at most 1509, at least or exactly or at most 1510, at least or exactly or at most 1511, at least or exactly or at most 1512, at least or exactly or at most 1513, at least or exactly or at most 1514, at least or exactly or at most 1515, at least or exactly or at most 1516, at least or exactly or at most 1517, at least or exactly or at most 1518, at least or exactly or at most 1519, at least or exactly or at most 1520, at least or exactly or at most 1521, at least or exactly or at 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most 1846, at least or exactly or at most 1847, at least or exactly or at most 1848, at least or exactly or at most 1849, at least or exactly or at most 1850, at least or exactly or at most 1851, at least or exactly or at most 1852, at least or exactly or at most 1853, at least or exactly or at most 1854, at least or exactly or at most 1855, at least or exactly or at most 1856, at least or exactly or at most 1857, at least or exactly or at most 1858, at least or exactly or at most 1859, at least or exactly or at most 1860, at least or exactly or at most 1861, at least or exactly or at most 1862, at least or exactly or at most 1863, at least or exactly or at most 1864, at least or exactly or at most 1865, at least or exactly or at most 1866, at least or exactly or at most 1867, at least or exactly or at most 1868, at least or exactly or at most 1869, at least or exactly or at most 1870, at least or exactly or at most 1871, at least or exactly or at most 1872, at least or exactly or at most 1873, at least or exactly or at most 1874, at least or exactly or at most 1875, at least or exactly or at most 1876, at least or exactly or at most 1877, at least or exactly or at most 1878, at least or exactly or at most 1879, at least or exactly or at most 1880, at least or exactly or at most 1881, at least or exactly or at most 1882, at least or exactly or at most 1883, at least or exactly or at most 1884, at least or exactly or at most 1885, at least or exactly or at most 1886, at least or exactly or at most 1887, at least or exactly or at most 1888, at least or exactly or at most 1889, at least or exactly or at most 1890, at least or exactly or at most 1891, at least or exactly or at most 1892, at least or exactly or at most 1893, at least or exactly or at most 1894, at least or exactly or at most 1895, at least or exactly or at most 1896, at least or exactly or at most 1897, at least or exactly or at most 1898, at least or exactly or at most 1899, at least or exactly or at most 1900, at least or exactly or at most 1901, at least or exactly or at most 1902, at least or exactly or at most 1903, at least or exactly or at most 1904, at least or exactly or at most 1905, at least or exactly or at most 1906, at least or exactly or at most 1907, at least or exactly or at most 1908, at least or exactly or at most 1909, at least or exactly or at most 1910, at least or exactly or at most 1911, at least or exactly or at most 1912, at least or exactly or at most 1913, at least or exactly or at most 1914, at least or exactly or at most 1915, at least or exactly or at most 1916, at least or exactly or at most 1917, at least or exactly or at most 1918, at least or exactly or at most 1919, at least or exactly or at most 1920, at least or exactly or at most 1921, at least or exactly or at most 1922, at least or exactly or at most 1923, at least or exactly or at most 1924, at least or exactly or at most 1925, at least or exactly or at most 1926, at least or exactly or at most 1927, at least or exactly or at most 1928, at least or exactly or at most 1929, at least or exactly or at most 1930, at least or exactly or at most 1931, at least or exactly or at most 1932, at least or exactly or at most 1933, at least or exactly or at most 1934, at least or exactly or at most 1935, at least or exactly or at most 1936, at least or exactly or at most 1937, at least or exactly or at most 1938, at least or exactly or at most 1939, at least or exactly or at most 1940, at least or exactly or at most 1941, at least or exactly or at most 1942, at least or exactly or at most 1943, at least or exactly or at most 1944, at least or exactly or at most 1945, at least or exactly or at most 1946, at least or exactly or at most 1947, at least or exactly or at most 1948, at least or exactly or at most 1949, at least or exactly or at most 1950, at least or exactly or at most 1951, at least or exactly or at most 1952, at least or exactly or at most 1953, at least or exactly or at most 1954, at least or exactly or at most 1955, at least or exactly or at most 1956, at least or exactly or at most 1957, at least or exactly or at most 1958, at least or exactly or at most 1959, at least or exactly or at most 1960, at least or exactly or at most 1961, at least or exactly or at most 1962, at least or exactly or at most 1963, at least or exactly or at most 1964, at least or exactly or at most 1965, at least or exactly or at most 1966, at least or exactly or at most 1967, at least or exactly or at most 1968, at least or exactly or at most 1969, at least or exactly or at most 1970, at least or exactly or at most 1971, at least or exactly or at most 1972, at least or exactly or at most 1973, at least or exactly or at most 1974, at least or exactly or at most 1975, at least or exactly or at most 1976, at least or exactly or at most 1977, at least or exactly or at most 1978, at least or exactly or at most 1979, at least or exactly or at most 1980, at least or exactly or at most 1981, at least or exactly or at most 1982, at least or exactly or at most 1983, at least or exactly or at most 1984, at least or exactly or at most 1985, at least or exactly or at most 1986, at least or exactly or at most 1987, at least or exactly or at most 1988, at least or exactly or at most 1989, at least or exactly or at most 1990, at least or exactly or at most 1991, at least or exactly or at most 1992, at least or exactly or at most 1993, at least or exactly or at most 1994, at least or exactly or at most 1995, at least or exactly or at most 1996, at least or exactly or at most 1997, at least or exactly or at most 1998, at least or exactly or at most 1999, at least or exactly or at most 2000, at least or exactly or at most 2001, at least or exactly or at most 2002, at least or exactly or at most 2003, at least or exactly or at most 2004, at least or exactly or at most 2005, at least or exactly or at most 2006, at least or exactly or at most 2007, at least or exactly or at most 2008, at least or exactly or at most 2009, at least or exactly or at most 2010, at least or exactly or at most 2011, at least or exactly or at most 2012, at least or exactly or at most 2013, at least or exactly or at most 2014, at least or exactly or at most 2015, at least or exactly or at most 2016, at least or exactly or at most 2017, at least or exactly or at most 2018, at least or exactly or at most 2019, at least or exactly or at most 2020, at least or exactly or at most 2021, at least or exactly or at most 2022, at least or exactly or at most 2023, at least or exactly or at most 2024, at least or exactly or at most 2025, at least or exactly or at most 2026, at least or exactly or at most 2027, at least or exactly or at most 2028, at least or exactly or at most 2029, at least or exactly or at most 2030, at least or exactly or at most 2031, at least or exactly or at most 2032, at least or exactly or at most 2033, at least or exactly or at most 2034, at least or exactly or at 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most 2224, at least or exactly or at most 2225, at least or exactly or at most 2226, at least or exactly or at most 2227, at least or exactly or at most 2228, at least or exactly or at most 2229, at least or exactly or at most 2230, at least or exactly or at most 2231, at least or exactly or at most 2232, at least or exactly or at most 2233, at least or exactly or at most 2234, at least or exactly or at most 2235, at least or exactly or at most 2236, at least or exactly or at most 2237, at least or exactly or at most 2238, at least or exactly or at most 2239, at least or exactly or at most 2240, at least or exactly or at most 2241, at least or exactly or at most 2242, at least or exactly or at most 2243, at least or exactly or at most 2244, at least or exactly or at most 2245, at least or exactly or at most 2246, at least or exactly or at most 2247, at least or exactly or at most 2248, at least or exactly or at most 2249, at least or exactly or at most 2250, at least or exactly or at most 2251, at least or exactly or at most 2252, at least or exactly or at most 2253, at least or exactly or at most 2254, at least or exactly or at most 2255, at least or exactly or at most 2256, at least or exactly or at most 2257, at least or exactly or at most 2258, at least or exactly or at most 2259, at least or exactly or at most 2260, at least or exactly or at most 2261, at least or exactly or at most 2262, at least or exactly or at most 2263, at least or exactly or at most 2264, at least or exactly or at most 2265, at least or exactly or at most 2266, at least or exactly or at most 2267, at least or exactly or at most 2268, at least or exactly or at most 2269, at least or exactly or at most 2270, at least or exactly or at most 2271, at least or exactly or at most 2272, at least or exactly or at most 2273, at least or exactly or at most 2274, at least or exactly or at most 2275, at least or exactly or at most 2276, at least or exactly or at most 2277, at least or exactly or at most 2278, at least or exactly or at most 2279, at least or exactly or at most 2280, at least or exactly or at most 2281, at least or exactly or at most 2282, at least or exactly or at most 2283, at least or exactly or at most 2284, at least or exactly or at most 2285, at least or exactly or at most 2286, at least or exactly or at most 2287, at least or exactly or at most 2288, at least or exactly or at most 2289, at least or exactly or at most 2290, at least or exactly or at most 2291, at least or exactly or at most 2292, at least or exactly or at most 2293, at least or exactly or at most 2294, at least or exactly or at most 2295, at least or exactly or at most 2296, at least or exactly or at most 2297, at least or exactly or at most 2298, at least or exactly or at most 2299, at least or exactly or at most 2300, at least or exactly or at most 2301, at least or exactly or at most 2302, at least or exactly or at most 2303, at least or exactly or at most 2304, at least or exactly or at most 2305, at least or exactly or at most 2306, at least or exactly or at most 2307, at least or exactly or at most 2308, at least or exactly or at most 2309, at least or exactly or at most 2310, at least or exactly or at most 2311, at least or exactly or at most 2312, at least or exactly or at most 2313, at least or exactly or at most 2314, at least or exactly or at most 2315, at least or exactly or at most 2316, at least or exactly or at most 2317, at least or exactly or at most 2318, at least or exactly or at most 2319, at least or exactly or at most 2320, at least or exactly or at most 2321, at least or exactly or at most 2322, at least or exactly or at most 2323, at least or exactly or at most 2324, at least or exactly or at most 2325, at least or exactly or at most 2326, at least or exactly or at most 2327, at least or exactly or at most 2328, at least or exactly or at most 2329, at least or exactly or at most 2330, at least or exactly or at most 2331, at least or exactly or at 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most 2386, at least or exactly or at most 2387, at least or exactly or at most 2388, at least or exactly or at most 2389, at least or exactly or at most 2390, at least or exactly or at most 2391, at least or exactly or at most 2392, at least or exactly or at most 2393, at least or exactly or at most 2394, at least or exactly or at most 2395, at least or exactly or at most 2396, at least or exactly or at most 2397, at least or exactly or at most 2398, at least or exactly or at most 2399, at least or exactly or at most 2400, at least or exactly or at most 2401, at least or exactly or at most 2402, at least or exactly or at most 2403, at least or exactly or at most 2404, at least or exactly or at most 2405, at least or exactly or at most 2406, at least or exactly or at most 2407, at least or exactly or at most 2408, at least or exactly or at most 2409, at least or exactly or at most 2410, at least or exactly or at most 2411, at least or exactly or at most 2412, at least or exactly or at 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most 2440, at least or exactly or at most 2441, at least or exactly or at most 2442, at least or exactly or at most 2443, at least or exactly or at most 2444, at least or exactly or at most 2445, at least or exactly or at most 2446, at least or exactly or at most 2447, at least or exactly or at most 2448, at least or exactly or at most 2449, at least or exactly or at most 2450, at least or exactly or at most 2451, at least or exactly or at most 2452, at least or exactly or at most 2453, at least or exactly or at most 2454, at least or exactly or at most 2455, at least or exactly or at most 2456, at least or exactly or at most 2457, at least or exactly or at most 2458, at least or exactly or at most 2459, at least or exactly or at most 2460, at least or exactly or at most 2461, at least or exactly or at most 2462, at least or exactly or at most 2463, at least or exactly or at most 2464, at least or exactly or at most 2465, at least or exactly or at most 2466, at least or exactly or at 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most 2494, at least or exactly or at most 2495, at least or exactly or at most 2496, at least or exactly or at most 2497, at least or exactly or at most 2498, at least or exactly or at most 2499, at least or exactly or at most 2500, at least or exactly or at most 2501, at least or exactly or at most 2502, at least or exactly or at most 2503, at least or exactly or at most 2504, at least or exactly or at most 2505, at least or exactly or at most 2506, at least or exactly or at most 2507, at least or exactly or at most 2508, at least or exactly or at most 2509, at least or exactly or at most 2510, at least or exactly or at most 2511, at least or exactly or at most 2512, at least or exactly or at most 2513, at least or exactly or at most 2514, at least or exactly or at most 2515, at least or exactly or at most 2516, at least or exactly or at most 2517, at least or exactly or at most 2518, at least or exactly or at most 2519, at least or exactly or at most 2520, at least or exactly or at 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most 2548, at least or exactly or at most 2549, at least or exactly or at most 2550, at least or exactly or at most 2551, at least or exactly or at most 2552, at least or exactly or at most 2553, at least or exactly or at most 2554, at least or exactly or at most 2555, at least or exactly or at most 2556, at least or exactly or at most 2557, at least or exactly or at most 2558, at least or exactly or at most 2559, at least or exactly or at most 2560, at least or exactly or at most 2561, at least or exactly or at most 2562, at least or exactly or at most 2563, at least or exactly or at most 2564, at least or exactly or at most 2565, at least or exactly or at most 2566, at least or exactly or at most 2567, at least or exactly or at most 2568, at least or exactly or at most 2569, at least or exactly or at most 2570, at least or exactly or at most 2571, at least or exactly or at most 2572, at least or exactly or at most 2573, at least or exactly or at most 2574, at least or exactly or at most 2575, at least or exactly or at most 2576, at least or exactly or at most 2577, at least or exactly or at most 2578, at least or exactly or at most 2579, at least or exactly or at most 2580, at least or exactly or at most 2581, at least or exactly or at most 2582, at least or exactly or at most 2583, at least or exactly or at most 2584, at least or exactly or at most 2585, at least or exactly or at most 2586, at least or exactly or at most 2587, at least or exactly or at most 2588, at least or exactly or at most 2589, at least or exactly or at most 2590, at least or exactly or at most 2591, at least or exactly or at most 2592, at least or exactly or at most 2593, at least or exactly or at most 2594, at least or exactly or at most 2595, at least or exactly or at most 2596, at least or exactly or at most 2597, at least or exactly or at most 2598, at least or exactly or at most 2599, at least or exactly or at most 2600, at least or exactly or at most 2601, at least or exactly or at 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most 2629, at least or exactly or at most 2630, at least or exactly or at most 2631, at least or exactly or at most 2632, at least or exactly or at most 2633, at least or exactly or at most 2634, at least or exactly or at most 2635, at least or exactly or at most 2636, at least or exactly or at most 2637, at least or exactly or at most 2638, at least or exactly or at most 2639, at least or exactly or at most 2640, at least or exactly or at most 2641, at least or exactly or at most 2642, at least or exactly or at most 2643, at least or exactly or at most 2644, at least or exactly or at most 2645, at least or exactly or at most 2646, at least or exactly or at most 2647, at least or exactly or at most 2648, at least or exactly or at most 2649, at least or exactly or at most 2650, at least or exactly or at most 2651, at least or exactly or at most 2652, at least or exactly or at most 2653, at least or exactly or at most 2654, at least or exactly or at most 2655, at least or exactly or at most 2656, at least or exactly or at most 2657, at least or exactly or at most 2658, at least or exactly or at most 2659, at least or exactly or at most 2660, at least or exactly or at most 2661, at least or exactly or at most 2662, at least or exactly or at most 2663, at least or exactly or at most 2664, at least or exactly or at most 2665, at least or exactly or at most 2666, at least or exactly or at most 2667, at least or exactly or at most 2668, at least or exactly or at most 2669, at least or exactly or at most 2670, at least or exactly or at most 2671, at least or exactly or at most 2672, at least or exactly or at most 2673, at least or exactly or at most 2674, at least or exactly or at most 2675, at least or exactly or at most 2676, at least or exactly or at most 2677, at least or exactly or at most 2678, at least or exactly or at most 2679, at least or exactly or at most 2680, at least or exactly or at most 2681, at least or exactly or at most 2682, at least or exactly or at most 2683, at least or exactly or at most 2684, at least or exactly or at most 2685, at least or exactly or at most 2686, at least or exactly or at most 2687, at least or exactly or at most 2688, at least or exactly or at most 2689, at least or exactly or at most 2690, at least or exactly or at most 2691, at least or exactly or at most 2692, at least or exactly or at most 2693, at least or exactly or at most 2694, at least or exactly or at most 2695, at least or exactly or at most 2696, at least or exactly or at most 2697, at least or exactly or at most 2698, at least or exactly or at most 2699, at least or exactly or at most 2700, at least or exactly or at most 2701, at least or exactly or at most 2702, at least or exactly or at most 2703, at least or exactly or at most 2704, at least or exactly or at most 2705, at least or exactly or at most 2706, at least or exactly or at most 2707, at least or exactly or at most 2708, at least or exactly or at most 2709, at least or exactly or at most 2710, at least or exactly or at most 2711, at least or exactly or at most 2712, at least or exactly or at most 2713, at least or exactly or at most 2714, at least or exactly or at most 2715, at least or exactly or at most 2716, at least or exactly or at most 2717, at least or exactly or at most 2718, at least or exactly or at most 2719, at least or exactly or at most 2720, at least or exactly or at most 2721, at least or exactly or at most 2722, at least or exactly or at most 2723, at least or exactly or at most 2724, at least or exactly or at most 2725, at least or exactly or at most 2726, at least or exactly or at most 2727, at least or exactly or at most 2728, at least or exactly or at most 2729, at least or exactly or at most 2730, at least or exactly or at most 2731, at least or exactly or at most 2732, at least or exactly or at most 2733, at least or exactly or at most 2734, at least or exactly or at most 2735, at least or exactly or at most 2736, at least or exactly or at most 2737, at least or exactly or at most 2738, at least or exactly or at most 2739, at least or exactly or at most 2740, at least or exactly or at most 2741, at least or exactly or at most 2742, at least or exactly or at most 2743, at least or exactly or at most 2744, at least or exactly or at most 2745, at least or exactly or at most 2746, at least or exactly or at most 2747, at least or exactly or at most 2748, at least or exactly or at most 2749, at least or exactly or at most 2750, at least or exactly or at most 2751, at least or exactly or at most 2752, at least or exactly or at most 2753, at least or exactly or at most 2754, at least or exactly or at most 2755, at least or exactly or at most 2756, at least or exactly or at most 2757, at least or exactly or at most 2758, at least or exactly or at most 2759, at least or exactly or at most 2760, at least or exactly or at most 2761, at least or exactly or at most 2762, at least or exactly or at most 2763, at least or exactly or at most 2764, at least or exactly or at most 2765, at least or exactly or at most 2766, at least or exactly or at most 2767, at least or exactly or at most 2768, at least or exactly or at most 2769, at least or exactly or at most 2770, at least or exactly or at most 2771, at least or exactly or at most 2772, at least or exactly or at most 2773, at least or exactly or at most 2774, at least or exactly or at most 2775, at least or exactly or at most 2776, at least or exactly or at most 2777, at least or exactly or at most 2778, at least or exactly or at most 2779, at least or exactly or at most 2780, at least or exactly or at most 2781, at least or exactly or at most 2782, at least or exactly or at most 2783, at least or exactly or at most 2784, at least or exactly or at most 2785, at least or exactly or at most 2786, at least or exactly or at most 2787, at least or exactly or at most 2788, at least or exactly or at most 2789, at least or exactly or at most 2790, at least or exactly or at most 2791, at least or exactly or at most 2792, at least or exactly or at most 2793, at least or exactly or at most 2794, at least or exactly or at most 2795, at least or exactly or at most 2796, at least or exactly or at most 2797, at least or exactly or at most 2798, at least or exactly or at most 2799, at least or exactly or at most 2800, at least or exactly or at most 2801, at least or exactly or at most 2802, at least or exactly or at most 2803, at least or exactly or at most 2804, at least or exactly or at most 2805, at least or exactly or at most 2806, at least or exactly or at most 2807, at least or exactly or at most 2808, at least or exactly or at most 2809, at least or exactly or at most 2810, at least or exactly or at most 2811, at least or exactly or at most 2812, at least or exactly or at most 2813, at least or exactly or at most 2814, at least or exactly or at most 2815, at least or exactly or at most 2816, at least or exactly or at most 2817, at least or exactly or at most 2818, at least or exactly or at most 2819, at least or exactly or at most 2820, at least or exactly or at most 2821, at least or exactly or at most 2822, at least or exactly or at most 2823, at least or exactly or at most 2824, at least or exactly or at most 2825, at least or exactly or at most 2826, at least or exactly or at most 2827, at least or exactly or at most 2828, at least or exactly or at most 2829, at least or exactly or at most 2830, at least or exactly or at most 2831, at least or exactly or at most 2832, at least or exactly or at most 2833, at least or exactly or at most 2834, at least or exactly or at most 2835, at least or exactly or at most 2836, at least or exactly or at most 2837, at least or exactly or at most 2838, at least or exactly or at most 2839, at least or exactly or at most 2840, at least or exactly or at most 2841, at least or exactly or at most 2842, at least or exactly or at most 2843, at least or exactly or at most 2844, at least or exactly or at most 2845, at least or exactly or at most 2846, at least or exactly or at most 2847, at least or exactly or at most 2848, at least or exactly or at most 2849, at least or exactly or at most 2850, at least or exactly or at most 2851, at least or exactly or at most 2852, at least or exactly or at most 2853, at least or exactly or at most 2854, at least or exactly or at most 2855, at least or exactly or at most 2856, at least or exactly or at most 2857, at least or exactly or at most 2858, at least or exactly or at most 2859, at least or exactly or at most 2860, at least or exactly or at most 2861, at least or exactly or at most 2862, at least or exactly or at most 2863, at least or exactly or at most 2864, at least or exactly or at most 2865, at least or exactly or at most 2866, at least or exactly or at most 2867, at least or exactly or at most 2868, at least or exactly or at most 2869, at least or exactly or at most 2870, at least or exactly or at most 2871, at least or exactly or at most 2872, at least or exactly or at most 2873, at least or exactly or at most 2874, at least or exactly or at most 2875, at least or exactly or at most 2876, at least or exactly or at most 2877, at least or exactly or at most 2878, at least or exactly or at most 2879, at least or exactly or at most 2880, at least or exactly or at most 2881, at least or exactly or at most 2882, at least or exactly or at most 2883, at least or exactly or at most 2884, at least or exactly or at most 2885, at least or exactly or at most 2886, at least or exactly or at most 2887, at least or exactly or at most 2888, at least or exactly or at most 2889, at least or exactly or at most 2890, at least or exactly or at most 2891, at least or exactly or at most 2892, at least or exactly or at most 2893, at least or exactly or at most 2894, at least or exactly or at most 2895, at least or exactly or at most 2896, at least or exactly or at most 2897, at least or exactly or at most 2898, at least or exactly or at 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most 2953, at least or exactly or at most 2954, at least or exactly or at most 2955, at least or exactly or at most 2956, at least or exactly or at most 2957, at least or exactly or at most 2958, at least or exactly or at most 2959, at least or exactly or at most 2960, at least or exactly or at most 2961, at least or exactly or at most 2962, at least or exactly or at most 2963, at least or exactly or at most 2964, at least or exactly or at most 2965, at least or exactly or at most 2966, at least or exactly or at most 2967, at least or exactly or at most 2968, at least or exactly or at most 2969, at least or exactly or at most 2970, at least or exactly or at most 2971, at least or exactly or at most 2972, at least or exactly or at most 2973, at least or exactly or at most 2974, at least or exactly or at most 2975, at least or exactly or at most 2976, at least or exactly or at most 2977, at least or exactly or at most 2978, at least or exactly or at most 2979, at least or exactly or at most 2980, at least or exactly or at most 2981, at least or exactly or at most 2982, at least or exactly or at most 2983, at least or exactly or at most 2984, at least or exactly or at most 2985, at least or exactly or at most 2986, at least or exactly or at most 2987, at least or exactly or at most 2988, at least or exactly or at most 2989, at least or exactly or at most 2990, at least or exactly or at most 2991, at least or exactly or at most 2992, at least or exactly or at most 2993, at least or exactly or at most 2994, at least or exactly or at most 2995, at least or exactly or at most 2996, at least or exactly or at most 2997, at least or exactly or at most 2998, at least or exactly or at most 2999, at least or exactly or at most 3000, at least or exactly or at most 3001, at least or exactly or at most 3002, at least or exactly or at most 3003, at least or exactly or at most 3004, at least or exactly or at most 3005, at least or exactly or at most 3006, at least or exactly or at most 3007, at least or exactly or at most 3008, at least or exactly or at most 3009, at least or exactly or at most 3010, at least or exactly or at most 3011, at least or exactly or at most 3012, at least or exactly or at most 3013, at least or exactly or at most 3014, at least or exactly or at most 3015, at least or exactly or at most 3016, at least or exactly or at most 3017, at least or exactly or at most 3018, at least or exactly or at most 3019, at least or exactly or at most 3020, at least or exactly or at most 3021, at least or exactly or at most 3022, at least or exactly or at most 3023, at least or exactly or at most 3024, at least or exactly or at most 3025, at least or exactly or at most 3026, at least or exactly or at most 3027, at least or exactly or at most 3028, at least or exactly or at most 3029, at least or exactly or at most 3030, at least or exactly or at most 3031, at least or exactly or at most 3032, at least or exactly or at most 3033, at least or exactly or at most 3034, at least or exactly or at most 3035, at least or exactly or at most 3036, at least or exactly or at most 3037, at least or exactly or at most 3038, at least or exactly or at most 3039, at least or exactly or at most 3040, at least or exactly or at most 3041, at least or exactly or at most 3042, at least or exactly or at most 3043, at least or exactly or at most 3044, at least or exactly or at most 3045, at least or exactly or at most 3046, at least or exactly or at most 3047 contiguous amino acid residues. In some embodiments, the polypeptide of the invention also has a sequence identity with the amino acid sequence of a) defined above of at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. Similarly, the polypeptide of the invention in some embodiments also has a sequence identity with the amino acid sequence of b) defined above of at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, and 56 in any one of SEQ ID NOs: 1-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 57, 58, 59, 60, 61 and 62 in any on of SEQ ID NOs: 2-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 63, 64, 65, 66, 67, 68, and 69 in any one of SEQ ID NOs: 4-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, and 104 in any one of SEQ ID NOs: 5-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 105, 106, 107, 108, 109, 110, and 110 in any one of SEQ ID NOs: 6-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122 in any one of SEQ ID NOs: 7-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, and 135 in any one of SEQ ID NOs: 8-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, and 155 in any one of SEQ ID NOs: 9-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 156, 157, and 158 in any one of SEQ ID NOs: 10-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 159, 160, 161, and 162 in any one of SEQ ID NOs: 11-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, and 226 in any one of SEQ ID NOs: 12-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 227, 228, 229, 230, 231, 232, 233, and 234 in any one of SEQ ID NOs: 13-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, and 249 in SEQ ID NOs: 14-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, and 290 in any one of SEQ ID NOs: 15-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, and 352 in any one of SEQ ID NOs: 16-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, and 363 in any one of SEQ ID NOs: 17-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, and 416 in any one of SEQ ID NOs: 18-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, and 441 in any one of SEQ ID NOs: 19-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, and 707 in any one of SEQ ID NOs: 20-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722 and 723 in any one of SEQ ID NOs: 21-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to amino acid residue 724 in any one of SEQ ID NOs: 22-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, and 750 in any one of SEQ ID NOs: 23-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, and 902 in any one of SEQ ID NOs: 24-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, and 970 in any one of SEQ ID NOs: 25-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, and 1067 in any one of SEQ ID NOs: 26-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, and 1106 in any one of SEQ ID NOs: 27-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, and 1224 in any one of SEQ ID NOs: 28-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, and 1280 in any one of SEQ ID NOs: 29-30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944, 2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956, 2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968, 2969, 2970, 2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980, 2981, 2982, 2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992, 2993, 2994, 2995, 2996, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3042, and 3043 in any one of SEQ ID NOs: 30, if the length of the at least 5 amino acid residues so permit—if the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N−L+1, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b. The polypeptide of the invention is in certain embodiments also fused or conjugated to an immunogenic carrier molecule; or, phrased otherwise, the polypeptide of the invention also includes such an immunogenic carrier molecule in addition to the material derived from SEQ ID NOs. 1-30. The immunogenic carrier molecule is a typically polypeptide that induces T-helper lymphocyte responses in a majority of humans, such as immunogenic carrier proteins selected from the group consisting of keyhole limpet hemocyanino or a fragment thereof, tetanus toxoid or a fragment thereof, dipththeria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra. One further fusion partner, which is preferably incorporated is a “His tag”, i.e. a stretch of amino acids, which is rich or only consists of histidinyl residues so as to facilitate protein purification. In preferred embodiments, the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a human being. Preferably, the adaptive immune response is a protective adaptive immune response against infection withA baumanii. The polypeptide may in these cases induce a humeral and/or a cellular immune response. Epitopes SEQ ID NOs: 1-30 include antigenic determinants (epitopes) that are as such recognized by antibodies and/or when bound to MHC molecules by T-cell receptors. For the purposes of the present invention, B-cell epitopes (i.e. antibody binding epitopes) are of particular relevance. It is relatively uncomplicated to identify linear B-cell epitopes—one very simple approach entails that antibodies raised againstA. baumanniiorA. baumanniiderived proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from any one of SEQ ID NO: 1-30. Thereby, the regions of theA. baumanniipolypeptide which are responsible for or contribute to binding to the antibodies can be identified. Alternatively, or additionally, one can produce mutated versions of the polypeptides of the invention, e.g. version where each single non-alanine residue in SEQ ID NOs.: 1-30 are point mutated to alanine—this method also assists in identifying complex assembled B-cell epitopes; this is the case when binding of the same antibody is modified by exchanging amino acids in different areas of the full-length polypeptide. Also, in silico methods for B-cell epitope prediction can be employed: useful state-of-the-art systems for β-turn prediction is provided in Petersen B et al. (November 2010),Plos One5(11): e15079; prediction of linear B-cell epitopes, cf: Larsen 3 E P et al. (April 2006), Immunome Research, 2:2; prediction of solvent exposed amino acids: Petersen B et al (July 2009), BMC Structural Biology, 9:51. The Nucleic Acid Fragments of the Invention The nucleic acid fragment of the invention referred to above is preferably is a DNA fragment (such as SEQ ID NOs: 31-60) or an RNA fragment (such as SEQ ID NOs 61-90). The nucleic acid fragment of the invention typically consists of at least or exactly or at most 11, such as at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17 at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27, at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, at least or exactly or at most 35, at least or exactly or at most 36, at least or exactly or at most 37, at least or exactly or at most 38, at least or exactly or at most 39, at least or exactly or at most 40, at least or exactly or at most 41, at least or exactly or at most 42, at least or exactly or at most 43, at least or exactly or at most 44, at least or exactly or at most 45, at least or exactly or at most 46, at least or exactly or at most 47, at least or exactly or at most 48, at least or exactly or at most 49, at least or exactly or at most 50, at least or exactly or at most 51, at least or exactly or at most 52, at least or exactly or at most 53, at least or exactly or at most 54, at least or exactly or at most 55, at least or exactly or at most 56, at least or exactly or at most 57, at least or exactly or at most 58, at least or exactly or at most 59, at least or exactly or at most 60, at least or exactly or at most 61, at least or exactly or at most 62, at least or exactly or at most 63, at least or exactly or at most 64, at least or exactly or at most 65, at least or exactly or at most 66, at least or exactly or at most 67, at least or exactly or at most 68, at least or exactly or at most 69, at least or exactly or at most 70, at least or exactly or at most 71, at least or exactly or at most 72, at least or exactly or at most 73, at least or exactly or at most 74, at least or exactly or at most 75, at least or exactly or at most 76, at least or exactly or at most 77, at least or exactly or at most 78, at least or exactly or at most 79, at least or exactly or at most 80, at least or exactly or at most 81, at least or exactly or at most 82, at least or exactly or at most 83, at least or exactly or at most 84, at least or exactly or at most 85, at least or exactly or at most 86, at least or exactly or at most 87, at least or exactly or at most 88, at least or exactly or at most 89, at least or exactly or at most 90, at least or exactly or at most 91, at least or exactly or at most 92, at least or exactly or at most 93, at least or exactly or at most 94, at least or exactly or at most 95, at least or exactly or at most 96, at least or exactly or at most 97, at least or exactly or at most 98, at least or exactly or at most 99, at least or exactly or at most 100, at least or exactly or at most 101, at least or exactly or at most 102, at least or exactly or at most 103, at least or exactly or at most 104, at least or exactly or at most 105, at least or exactly or at most 106, at least or exactly or at most 107, at least or exactly or at most 108, at least or exactly or at most 109, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least or exactly or at most 113, at least or exactly or at most 114, at least or exactly or at most 115, at least or exactly or at most 116, at least or exactly or at most 117, at least or exactly or at most 118, at least or exactly or at most 119, at least or exactly or at most 120, at least or exactly or at most 121, at least or exactly or at most 122, at least or exactly or at most 123, at least or exactly or at most 124, at least or exactly or at most 125, at least or exactly or at most 126, at least or exactly or at most 127, at least or exactly or at most 128, at least or exactly or at most 129, at least or exactly or at most 130, at least or exactly or at most 131, at least or exactly or at most 132, at least or exactly or at most 133, at least or exactly or at most 134, at least or exactly or at most 135, at least or exactly or at most 136, at least or exactly or at most 137, at least or exactly or at most 138, at least or exactly or at most 139, at least or exactly or at most 140, at least or exactly or at most 141, at least or exactly or at most 142, at least or exactly or at most 143, at least or exactly or at most 144, at least or exactly or at most 145, at least or exactly or at most 146, at least or exactly or at most 147, at least or exactly or at most 148, at least or exactly or at most 149, at least or exactly or at most 150, at least or exactly or at most 151, at least or exactly or at most 152, at least or exactly or at most 153, at least or exactly or at most 154, at least or exactly or at most 155, at least or exactly or at most 156, at least or exactly or at most 157, at least or exactly or at most 158, at least or exactly or at most 159, at least or exactly or at most 160, at least or exactly or at most 161, at least or exactly or at most 162, at least or exactly or at most 163, at least or exactly or at most 164, at least or exactly or at most 165, at least or exactly or at most 166, at least or exactly or at most 167, at least or exactly or at most 168, at least or exactly or at most 169, at least or exactly or at most 170, at least or exactly or at most 171, at least or exactly or at most 172, at least or exactly or at most 173, at least or exactly or at most 174, at least or exactly or at most 175, at least or exactly or at most 176, at least or exactly or at most 177, at least or exactly or at most 178, at least or exactly or at most 179, at least or exactly or at most 180, at least or exactly or at most 181, at least or exactly or at most 182 and at least or exactly or at most 183 consecutive nucleotides in any one of SEQ ID NOs: 31-90. Longer fragments are contemplated, i.e. fragments having at least or exactly or at most 200, at least or exactly or at most 300 at least or exactly or at most 400, at least or exactly or at most 500, at least or exactly or at most 600, at least or exactly or at most 700, at least or exactly or at most 800, at least or exactly or at most 900, at least or exactly or at most 1000, at least or exactly or at most 1500, at least or exactly or at most 2000, at least or exactly or at most 2500, at least or exactly or at most 3000, at least or exactly or at most 3500, and at least or exactly or at most 4000 nucleotides from those of SEQ ID NOs: 31-90 that encompass fragments of such lengths. Particularly preferred nucleic acid fragments (DNA or RNA) are those fragments of any one of SEQ ID NOs 31-90, which encode a polypeptide of the present invention discussed supra. The nucleic acid fragment of the invention discussed above typically has a sequence identity with the nucleotide sequence defined for i) or ii) above, which is at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. The nucleic acid fragment of the invention discussed above may also have a sequence identity with the nucleotide sequence defined for iii) above, which is at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. The Vectors of the Invention Vectors of the invention fall into several categories discussed infra. One preferred vector of the invention comprises in operable linkage and in the 5′-3′ direction, an expression control region comprising an enhancer/promoter for driving expression of the nucleic acid fragment defined for option i) above, optionally a signal peptide coding sequence, a nucleotide sequence defined for option i), and optionally a terminator. Hence, such a vector constitutes an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are bacterial of origin, recombinant production is conveniently effected in bacterial host cells, so here it is preferred that the expression control region drives expression in prokaryotic cell such as a bacterium, e.g. in E coll. However, if the vector is to drive expression in mammalian cell (as would be the case for a DNA vaccine vector), the expression control region should be adapted to this particular use. At any rate, certain vectors of the invention are capable of autonomous replication. Also, the vector of the invention may be one that is capable of being integrated into the genome of a host cell—this is particularly useful if the vector is use in the production of stably transformed cells, where the progeny will also include the genetic information introduced via the vector. Alternatively, vectors incapable of being integrated into the genome of a mammalian host cell are useful in e.g. DNA vaccination. Typically, the vector of the invention is selected from the group consisting of a virus, such as a attenuated virus (which may in itself be useful as a vaccine agent), a bacteriophage, a plasmid, a minichromosome, and a cosmid. A more detailed discussion of vectors of the invention is provided in the following: Polypeptides of the invention may be encoded by a nucleic acid molecule comprised in a vector. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found. Vectors include naked DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al, 2001; Ausubel et al, 1996, both incorporated herein by reference). In addition to encoding the polypeptides of this invention, a vector of the present invention may encode polypeptide sequences such as a tag or immunogenicity enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof). Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Vectors of the invention may be used in a host cell to produce a polypeptide of the invention that may subsequently be purified for administration to a subject or the vector may be purified for direct administration to a subject for expression of the protein in the subject (as is the case when administering a nucleic acid vaccine). Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202, 5,928,906, each incorporated herein by reference). Naturally, it may be important to employ a promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides. Examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchinson et al, 1986, 1987; toiler et al, 1987; Weinberger et al, 1984; Kiledjian et al, 1988; Porton et al; 1990), Immunoglobulin Light Chain (Queen et al, 1983; Picard et al, 1984), T Cell Receptor (Luria et al, 1987; Winoto et al, 1989; Redondo et al; 1990), HLA DQα and/or DQβ (Sullivan et al, 1987), 13-Interferon (Goodbourn et al, 1986; Fujita et al, 1987; Goodbourn et al, 1988), Interleukin-2 (Greene et al, 1989), Interleukin-2 Receptor (Greene et al, 1989; Lin et al, 1990), MHC Class II 5 (Koch et al, 1989), MHC Class II HLA-DRα (Sherman et al, 1989), β-Actin (Kawamoto et al, 1988; Ng et al; 1989), Muscle Creatine Kinase (MCK) (Jaynes et al, 1988; Horlick et al, 1989; Johnson et al, 1989), Prealbumin (Transthyretin) (Costa et al, 1988), Elastase I (Omitz et al, 1987), Metallothionein (MTII) (Karin et al, 1987; Culotta et al, 1989), Collagenase (Pinkert et al, 1987; Angel et al, 1987), Albumin (Pinkert et al, 1987; Tranche et al, 1989, 1990), α-Fetoprotein (Godbout et al, 1988; Campere et al, 1989), γ-Globin (Bodine et al, 1987; Perez-Stable et al, 1990), β-Globin (Trudel et al, 1987), c-fos (Cohen et al, 1987), c-HA-ras (Triesman, 1986; Deschamps et al, 1985), Insulin (Edlund et al, 1985), Neural Cell Adhesion Molecule (NCAM) (Hirsh et al, 1990), αl-Antitrypain (Larimer et al, 1990), H2B (TH2B) Histone (Hwang et al, 1990), Mouse and/or Type I Collagen (Ripe et al, 1989), Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al, 1989), Rat Growth Hormone (Larsen et al, 1986), Human Serum Amyloid A (SAA) (Edbrooke et al, 1989), Troponin I (TN I) (Yutzey et al, 1989), Platelet-Derived Growth Factor (PDGF) (Pech et al, 1989), Duchenne Muscular Dystrophy (Klamut et al, 1990), SV40 (Banerji et al, 1981; Moreau et al, 1981; Sleigh et al, 1985; Firak et al, 1986; Herr et al, 1986; Imbra et al, 1986; Kadesch et al, 1986; Wang et al, 1986; Ondek et al, 1987; Kuhl et al, 1987; Schaffner et al, 1988), Polyoma (Swartzendruber et al, 1975; Vasseur et al, 1980; Katinka et al, 1980, 1981; Tyndell et al, 1981; Dandolo et al, 1983; de Villiers et al, 1984; Hen et al, 1986; Satake et al, 1988; Campbell et al, 1988), Retroviruses (Kriegler et al, 1982, 1983; Levinson et al, 1982; Kriegler et al, 1983, 1984a, b, 1988; Bosze et al, 1986; Miksicek et al, 1986; Celander et al, 1987; Thiesen et al, 1988; Celander et al, 1988; Choi et al, 1988; Reisman et al, 1989), Papilloma Virus (Campo et al, 1983; Lusky et al, 1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky et al, 1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987; Stephens et al, 1987), Hepatitis B Virus (Bulla et al, 1986; Jameel et al, 1986; Shaul et al, 1987; Spandau et al, 1988; Vannice et al, 1988), Human Immunodeficiency Virus (Muesing et al, 1987; Hauber et al, 1988; Jakobovits et al, 1988; Feng et al, 1988; Takebe et al, 1988; Rosen et al, 1988; Berkhout et al, 1989; Laspia et al, 1989; Sharp et al, 1989; Braddock et al, 1989), Cytomegalovirus (CMV) IE (Weber et al, 1984; Boshart et al, 1985; Foecking et al, 1986), Gibbon Ape Leukemia Virus (Holbrook et al, 1987; Quinn et al, 1989). Inducible Elements include, but are not limited to MT II—Phorbol Ester (TFA)/Heavy metals (Palmiter et al, 1982; Haslinger et al, 1985; Searle et al, 1985; Stuart et al, 1985; Imagawa et al, 1987, Karin et al, 1987; Angel et al, 1987b; McNeall et al, 1989); MMTV (mouse mammary tumor virus)—Glucocorticoids (Huang et al, 1981; Lee et al, 1981; Majors et al, 1983; Chandler et al, 1983; Lee et al, 1984; Ponta et al, 1985; Sakai et al, 1988); β-Interferon—poly(rl)x/poly(rc) (Tavernier et al, 1983); Adenovirus 5 E2—EIA (Imperiale et al, 1984); Collagenase—Phorbol Ester (TPA) (Angel et al, 1987a); Stromelysin—Phorbol Ester (TPA) (Angel et al, 1987b); SV40—Phorbol Ester (TPA) (Angel et al, 1987b); Murine MX Gene—Interferon, Newcastle Disease Virus (Hug et al, 1988); GRP78 Gene—A23187 (Resendez et al, 1988); α-2-Macroglobulin—IL-6 (Kunz et al, 1989); Vimentin—Serum (Rittling et al, 1989); MHC Class I Gene H-2κb—Interferon (Blanar et al, 1989); HSP70—E1A/SV40 Large T Antigen (Taylor et al, 1989, 1990a, 1990b); Proliferin—Phorbol Ester/TPA (Mordacq et al, 1989); Tumor Necrosis Factor—PMA (Hensel et al, 1989); and Thyroid Stimulating Hormonea Gene—Thyroid Hormone (Chatterjee et al, 1989). Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest. The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention. The use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well. In embodiments in which a vector is administered to a subject for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down-regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/polypeptide of the current invention in a subject to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, a promoter that is up-regulated in the presence of cytokines is employed. The MHC I promoter increases expression in the presence of IFN-γ. Tissue specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Initiation Signals and Internal Ribosome Binding Sites (IRES) A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements. In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, herein incorporated by reference). 2. Multiple Cloning Sites Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference.) Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. 3. Splicing Sites Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al, 1997, incorporated herein by reference.) 4. Termination Signals The vectors or constructs of the present invention will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. 5. Polyadenylation Signals In expression, particularly eukaryotic expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. 6. Origins of Replication In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “on”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. 7. Selectable and Screenable Markers In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein of the invention. Further examples of selectable and screenable markers are well known to one of skill in the art. The Transformed Cells of the Invention Transformed cells of the invention are useful as organisms for producing the polypeptide of the invention, but also as simple “containers” of nucleic acids and vectors of the invention. Certain transformed cells of the invention are capable of replicating the nucleic acid fragment defined for option i) of the second aspect of the invention. Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment defined for option i). For recombinant production it is convenient, but not a prerequisite that the transformed cell according is prokaryotic, such as a bacterium, but generally both prokaryotic cells and eukaryotic cells may be used. Suitable prokaryotic cells are bacterial cells selected from the group consisting ofEscherichia(such asE. coli.),Bacillus[e.g.Bacillus subtilis], Salmonella, andMycobacterium[preferably non-pathogenic, e.g.M. bovisBCG]. Eukaryotic cells can be in the form of yeasts (such asSaccharomyces cerevisiae) and protozoans. Alternatively, the transformed eukaryotic cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. For production purposes, it is advantageous that the transformed cell of the invention is stably transformed by having the nucleic acid defined above for option i) stably integrated into its genome, and in certain embodiments it is also preferred that the transformed cell secretes or carries on its surface the polypeptide of the invention, since this facilitates recovery of the polypeptides produced. A particular version of this embodiment is one where the transformed cell is a bacterium and secretion of the polypeptide of the invention is into the periplasmic space. As noted above, stably transformed cells are preferred—these i.a. allows that cell lines comprised of transformed cells as defined herein may be established—such cell lines are particularly preferred aspects of the invention. Further details on cells and cell lines are presented in the following: Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotes and eukaryotes. Examples of prokaryotic cells includeE. coli; members of theStaphylococcusgenus, such asS. epidermidis; members of theLactobacillusgenus, such asL. plantarum; members of theLactococcusgenus, such asL. lactis; members of theBacillusgenus, such asB. subtilis; members of theCorynebacteriumgenus such asC. glutamicum; and members of thePseudomonasgenus such asPseudomonas fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells such as members of theSaccharomycesgenus (e.g.S. cerevisiae), members of thePichiagenus (e.g.P. pastoris), members of theHansenulagenus (e.g.H. polymorpha), members of theKluyveromycesgenus (e.g.K. lactisorK. fragilis) and members of theSchizosaccharomycesgenus (e.g.S. pombe). Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org) or from other depository institutions such as Deutsche Sammlung vor Micrroorganismen and Zellkulturen (DSM). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors or expression of encoded proteins. Bacterial cells used as host cells for vector replication and/or expression includeStaphylococcusstrains, DH5a, JMI 09, and KCB, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOP ACK™ Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such asE. coliLE392 could be used as host cells for phage viruses. Appropriate yeast cells includeSaccharomyces cerevisiae, Saccharomyces pombe, andPichia pastoris. Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides. Expression Systems Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available. The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ Baculovirus expression system from CLONTECH® In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, anE. coliexpression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called thePichia methanolicaExpression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeastPichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide. Amplification of Nucleic Acids Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 2001). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid. The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA. The term “primer,” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred. Pairs of primers designed to selectively hybridize to nucleic acids corresponding to sequences of genes identified herein are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced. The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Bellus, 1994). A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each of which is incorporated herein by reference in their entirety. Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety. Methods of Gene Transfer Suitable methods for nucleic acid delivery to effect expression of compositions of the present invention are believed to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al, 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al, 1989; Kato et al, 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacteriummediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al, 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al, 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed. The Antibodies of the Invention—and their Production/Isolation Antibodies directed against the proteins of the invention are useful for affinity chromatography, immunoassays, and for distinguishing/identifyingstaphylococcusproteins as well as for passive immunisation and therapy. Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antiserum is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25 C for one hour, followed by incubating at 4 C for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits. Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature(1975) 256: 495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective I aedium (elg. hypexanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies, which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32p and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 1151 may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with, 125I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention. According to the invention, the isolated monoclonal antibody or antibody analogue is preferably a monoclonal antibody selected from a multi-domain antibody such as a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and single-domain antibody of a llama or a camel, or which is an antibody analogue selected from a fragment of an antibody such as an Fab or an F(ab′)2, an scFV; cf. also the definition of the term “antibody” presented above. Compositions of the Invention; Vaccines Pharmaceutical compositions, in particular vaccines, according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie, to treat disease after infection). Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with “pharmaceutically acceptable carriers”, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera,H. pylori, etc. pathogen, cf. the description of immunogenic carriers supra. The pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following: Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ adjuvants are preferred. As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2″-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc. The immunogenic compositions (eg. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers. Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. However, for the purposes of protein vaccination, the amount administered per immunization is typically in the range between 0.5 μg and 500 mg (however, often not higher than 5,000 μg), and very often in the range between 10 and 200 μg. The immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO 98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intraarterial routes may be applicable. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. As an alternative to protein-based vaccines, DNA vaccination (also termed nucleic acid vaccination or gene vaccination) may be used [eg. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Annu Rev Immunol 15: 617-648; later herein]. A further aspect of the invention is the recognition that combination vaccines can be provided, wherein 2 or more antigens disclosed herein are combined to enhance the immune response by the vaccinated animal, including to optimize initial immune response and duration of immunity. For the purposes of this aspect of the invention, multiple antigenic fragments derived from the same, longer protein can also be used, such as the use a combination of different lengths of polypeptide sequence fragments from one protein. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 1 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 2 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 3 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 4 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 5 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 6 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 7 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 8 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 9 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 10 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 11 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 12 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 13 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 14 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 15 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 16 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 17 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 18 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 19 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 20 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 21 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 22 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 23 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 24 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 25 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 26 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 27 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 28 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 29 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Thus, embodiments of the invention relate to a composition (or the use as a vaccine of) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 30 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, or a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Treatment Methods of the Invention The method of the sixth aspect of the invention generally relates to induction of immunity and as such also entails method that relate to treatment, prophylaxis and amelioration of disease. When immunization methods entail that a polypeptide of the invention or a composition comprising such a polypeptide is administered the animal (e.g. the human) typically receives between 0.5 and 5,000 μg of the polypeptide of the invention per administration. In preferred embodiments of the sixth aspect, the immunization scheme includes that the animal (e.g. the human) receives a priming administration and one or more booster administrations. Preferred embodiments of the 6thaspect of the invention comprise that the administration is for the purpose of inducing protective immunity againstA. baumannii. In this embodiment it is particularly preferred that the protective immunity is effective in reducing the risk of attracting infection withA. baumanniior is effective in treating or ameliorating infection withA. baumannii. As mentioned herein, the preferred vaccines of the invention induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific forA. baumanniiand wherein said antibodies or B-lymphocytes producing said antibodies are subsequently recovered from the animal. But, as also mentioned the method of the 6thaspect may also be useful in antibody production, so in other embodiments the administration is for the purpose of inducing antibodies specific forA. baumanniiand wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies. Pharmaceutical compositions can as mentioned above comprise polypeptides, antibodies, or nucleic acids of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount thereof. The term “therapeutically effective amount” or “prophylactically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. Reference is however made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA or RNA constructs in the individual to which it is administered. A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991). Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. As is apparent from the claims, the invention also relates to related embodiments to the treatment and prophylaxis disclosed herein: the invention also includes embodiments wherethe polypeptide of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection withA. baumannii;the nucleic acid fragment of the invention or the vector of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection withA. baumannii;the transformed cell of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection withA. baumannii.the antibody, antibody fragment or antibody analogue of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection withA. baumannii. Example 1 Immunization Study Experimental 16 individual proteins derived fromA. baumanii(where 1 protein is a positive control) were tested. 16 mice were included in each group. The study was divided into 4 separate experiments comprising testing of 5 groups, that is 4 protein groups and 1 control group (adjuvant only). The 4 experiments are run in parallel, that is staggered with 7-14 days between each immunization. Hence the studies had a duration of 70-98 days (˜2.3-3.3 months): Challenge strain:A. baumaniiAB5075. Mouse strain: C57BL/6 (inbred). Dose: 25 μg protein in each immunization. Immunization route and interval: 3× subcutaneous with 14 days intervals. Inoculation route (challenge): intranasal. End point: Lethal challenge. Adjuvant: Priming immunization: Alum+incomplete Freund's adjuvant (IFA). 1st and second boost: Alum. Bled and ELISA: Mice were bled 4 days prior to challenge and ELISAs were run. Mouse Tag: Each mouse was tagged, hence making it possible to track the mouse-specific antibody titer with survival. Trial type: Double blinded. Number of mice per group: 16 mice. Monitoring period: 7-10 days. Experiment 1 Group 1 was immunized with the protein AB57_3582-22-253, i.e. SEQ ID NO: 14, amino acid residues 22-253. Study Group 3 was immunized with the protein AB57_1088-22-159, i.e. SEQ ID NO: 9, amino acid residues 22-159. Group 5 (the negative control) received phosphate buffered saline (PBS). Groups 2 and 4 were immunized with proteins irrelevant for the present invention and did not provide protection. Data are hence not shown. Experiment 2 Group 1 was immunized with a protein consisting of amino acids 2-346 of the protein having ATCC accession number 17978. Group 2 was immunized with a cocktail of AB57_2465-45-550, AB57_2465-551-906, AB57_1136-35-420, and AB57_1136-421-1071, i.e. 2 fragments of SEQ ID NO: 24 (amino acid residues 45 to 550 and 551 to 906, respectively) and 2 fragments of SEQ ID NO: 25 (amino acid residues 35 to 420 and 421 to 1071, respectively). Group 3 was immunized with a cocktail of the proteins AB57_1893-26-711, AB57_1893-48-176, AB57_1893-478-711, and AB57_1893-26-477, i.e. 4 fragments of SEQ ID NO: 20 (amino acid residues 26-711, 48-176, 478-711, and 26-477, respectively). Group 4 was immunized with the protein AB57_2233-22-162, i.e. a fragment of SEQ ID NO: 10 (amino acids 22-162). Group 5 (the negative control) received phosphate buffered saline (PBS). Experiment 3 Group 1 was immunized with the protein (AB57_3370-27-356), i.e. a fragment of SEQ ID NO: 16 (amino acid reissues 27-356) Group 2 was immunized with a cocktail of the proteins AB57_1059-1-754+AB57_1059-25-754+AB57_1059-25-466+AB57_1059-58-177, i.e. the complete protein having SEQ ID NO: 23, as well a 3 fragments thereof (amino acid residues 25-754, 25-466, and 58-177, respectively). Group 3 was immunized with the protein AB57_1621-1-367, i.e. SEQ ID NO: 17. Group 4 was immunized with a cocktail of the proteins AB57_3396-23-691+AB57_3396-306-691+AB57_3396-23-305, i.e. 3 fragments of a positive control (the protein having accession number ACJ 41966). Group 5 (the negative control) received phosphate buffered saline (PBS). Experiment 4 Group 1 was immunized with a cocktail of the 4 proteins AB57_0478-1-550, AB57_0478-551-1110, AB57_2309-22-294, and AB57_2309-1-294, i.e. 2 fragments of SEQ ID NO: 27 (amino acids 1-550 and 551-1110, respectively) and a fragment (amino acids 22-294) and the full-length version of SEQ ID NO: 15. Group 2 was immunized with a cocktail of the 4 proteins AB57_0053-19-108, AB57_1336-19-114, AB57_0830-1-166, and AB57_3389-22-126, i.e. amino acids 19-108 of SEQ ID NO: 5, amino acids 19-114 of SEQ ID NO: 6, the entire sequence SEQ ID NO: 11, and amino acids 22-126 of SEQ ID NO: 7. Group 3 was immunized with a cocktail of the 3 proteins AB57_3081-2651-3047, AB57_0596-29-580, and AB57_0596-581-727, i.e. amino acids 2651-3047 of SEQ ID NO: 30, and 2 fragments of SEQ ID NO: 21 (amino acids 29-580 and 581-727, respectively). Group 4 was immunized with a cocktail of the 2 proteins AB57_3778-23-230 and AB57_1830-35-238, i.e. amino acids 22-230 of SEQ ID NO: 12 and amino acids 35-238 of SEQ ID NO: 13. Group 5 (the negative control) received phosphate buffered saline (PBS). Results GroupSurvivalDay 0Day 1Day 2Day 3Day 4Day 5Day 6Day 7Experiment 11# Mice alive1313222222% SurvivalN/A100.015.415.415.415.415.415.43# Mice alive1515000000% SurvivalN/A1000000005# Mice alive1212000000% SurvivalN/A100.0000000Experiment 21# Mice alive1212322222% SurvivalN/A100.025.016.716.716.716.716.72# Mice alive1010544443% SurvivalN/A1005040404040303# Mice alive1313000000% SurvivalN/A1000000004# Mice alive99000000% SurvivalN/A1000000005# Mice alive1313211111% Survival100.015.47.77.77.77.77.7Experiment 31# Mice alive1511333333% SurvivalN/A73.320.020.020.020.020.020.02# Mice alive1615887777% SurvivalN/A93.850.050.043.843.843.843.83# Mice alive16130% SurvivalN/A81.30.04# Mice alive1615666666% SurvivalN/A93.837.537.537.537.537.537.55# Mice alive151310% Survival86.76.70.0Experiment 41# Mice alive1212976666% SurvivalN/A1007558505050502# Mice alive1313522222% SurvivalN/A1003815151515153# Mice alive1010522222% SurvivalN/A1005020202020204# Mice alive1313400000% SurvivalN/A10031000005# Mice alive1313111111% Survival100.07.77.77.77.77.77.7 The above observations has let the inventors to the following conclusions: Proteins tested that provide significant protection against challenge:1: AB57_1059-1-754, AB57_1059-25-754, AB57_1059-25-466, and AB57_1059-58-177;2: AB57_3396-23-691 (positive control), AB57_3396-306-691 (positive controls, and AB_3396-23-305 (positive control);3: AB57_0478-1-550 and AB57_0478-551-1110; 4: AB57_2309-22-294 and AB57_2309-22-294. A number of proteins tested are found to possibly provide protection against challenge infection:1: AB57_3582-22-253;2: AB57_2465-45-550 and AB57_2465-551-906;3: AB57_1136-35-420 and AB57_1136-421-1071;4. AB573370-27-356. Amino Acid Sequences of the Proteins of the Invention: In the present specification and claims, the amino acid sequences of the proteins are identified via the SEQ ID NOs. set forth in the sequence listing and the table below. However, alternative designations are used in the examples, according to the following table: Designation:SEQ ID NO:AB57_17591AB57_10092AB57_19023AB57_16144AB57_00535AB57_13366AB57_33897AB57_35788AB57_10889AB57_223310AB57_083011AB57_377812AB57_183013AB57_358214AB57_230915AB57_337016AB57_162117AB57_050418AB57_061919AB57_189320AB57_059621AB57_287022AB57_105923AB57_246524AB57_079125AB57_113626AB57_047827AB57_104828AB57_362129AB57_308130 When designating a fragment of one of these proteins, this is done using the nomenclature AB57_XXXX-A-p1-p2, where XXXX is any of the 4 digit numbers following “AB57” in the table above, and p1 and p2 are the start and end amino acids relative to the entire sequence of the protein. For instance AB57_3081-50-200 is the fragment of AB57_3081 that has the amino acid sequence defined by residues 50 to 200 of AB57_3081. SEQ ID NO: 1MTQLINKGGFRERANRSRKYQQSENKQVALPSKKYQPQTKLQDNQSEMIQAKAGTAETSDSEQ ID NO: 2MKLAKTLLATTLALTAASTFAASKEDQAHNTAGEEKVVVSTQEQANTANAASDAVGSASEAAPATRSEQ ID NO: 3MIDEEKPLNFEDDDEPLDFEDEEFIDDKKEDEMYNSITKDGSSVDPADDGTRHIRPEDGDPIEIDESEQ ID NO: 4MSTTNNQANQRNNQQQQQQQNDNRNQQQHGNQQQNDQQQQNNQQQQQNDNRGQQQGSNQKDSGQQNSNNNQQRSEQ ID NO: 5MSAKLVVTLLATSLLTVGCVAYTDDPYYRGGYGYHDHDDDRYDRNDGRRYSEWERKRWEERKRLYEQQRKDIREQQKDRREWEKRHREWEKKRLEDRDHDHRDYRHDDSEQ ID NO: 6MNKLLVALGLAATVALVGCNKDKAPETGATTGEHLENAAQQATADIKSAGDQAASDIATATDNASAKIDAAADHAADATAKAAAETEATARKATADTAQAVENAAADVKKDAQHSEQ ID NO: 7MKMTAKIALFSAAIVTMGSLAACQSTTQPPKPEHGMMQDGPRDGHHHRMKHREFTPEQKAAWEQHRAERKARFEQIQKACEGKVVGQTVNVQVGDKTLEGTCNLRFEPKRPQPPVNAPAPVATQAKSEQ ID NO: 8MKAIKILCITSSILVSSSLFAETPQPQQVNEATSKTMPYGDNPSLGRVLLYKTGKGIQNLGDSIQGASEKTSNKISEKWKDTKEFTAEKAEVVQQKADTAKVFTEQKIEQAKQNITSSRNGENIPIEQGELSKSSTTANSEQ ID NO: 9MKKSLLAIALMSTLLVACNKHENKTETTSDASTPVQTAQSNNNEAVDTAHTAENSLDWDGKYKGTLPCADCEGIKTELELKDDKTYELTETYLGKGDANPFETHGKFTFDKDNTSVITLDDKAQNRKFFIGENTATALDMEGKKVEGSLAEHYVLKKEDSEQ ID NO: 10MANKKLLICAAIAAGLLLTACVKKETPKEEEQDKVETAVSEPQPQKPAKFESLESVDTQEAQVQEQPQVEVHREETANTTTEIRRETRPARSDESSQTQVAEQPKSETPKVEPKPEKKPEPKAEPKPEKAQSKPAAKATEPANTEDDAVAAAIAAATPALKNSEQ ID NO: 11MTTENKLDELKANAADAKVQGEKALDDLKENVKEKQTAGKEAIADKVDELKTKAADAKVQGEKALEDLKENVKEKQAAAKEAVEDKASDLKGKLDDAQHSLQDKFDHLRTEAAHKLDDAKAKAAELKEEAATKFDELKTQATAKFDELKKTATEKLNKLKNHDSAESEQ ID NO: 12MHTRRILLAFSLAASAASVAFADYQNINQSTDSDRLEQLSKTLSQGSYTHPDDLDLPASAKVSVTLREKTVELNNDSLAKKYGTTTAKNSFKTSSSNPYSWLVSHPLPDTVRVSSNFGGRTMGGRAEHHGGLDMAAPSGTPIYATGPGIVTKSGWGTGYGQYVEINHGNGYLTRYAHASRLMVRVGDQVSAGDHIANVGCTGRCTGPHLHYEVVKDGQRKNPSTYLAMLPSEQ ID NO: 13MGMTFTDIENKSAKRLIGIAAVIFLHLLVAYILMSGLANNIQKPAEKPVELQIIQDIKPPPPPKPEEPKPKEKPPEPPKMVEKVAKVPEPPKEVEKVATPVQKTTPVAQTTKVATPAPAAPSTPSPSPVAAPAPVAAAAPALKPAGVTRGVSEGSAGCEKPEYPREALMNEEQGTVRIRVLVDTSGKVIDAKVKKSSGSKTLDKAATKAYSLCTFKPAMKDGVPQQDWYEIEYPFVIESEQ ID NO: 14MKMMKTAIVTASVLASASIFAQSAGVNAGASAQVNVQPGGLVSGVANTVKNTAHTVGNTAKHAGHVAADTTVKATKKTTGKVTELSSKAATGTKEVASEAVTGTKHFATEAATGTKNLATKAATGTKNLAVEAKADTKAHLDAVKTKVAEKQADQKEFTAEKQADAQARVDAVKARVAQNQAEQKEFVADTKADAQAKLNTAQPAHGVNAQTGVNVGVNVAGINANANVNAGAQASTQKGEKKSFIKGLFGTNSEQ ID NO: 15MQMKKHSLLFIALMSTTSLYANIPIESRGLSQNDGSASNTSSSNISVPTNLNWELMQKNQQLENDIRTLRGQLEEQANDIEQLKKDLANRYTDLDQRLELLHQKVDPDSATQDDSSNATSDNTTPASAPAPQTTESNKVAAVPATQTSEQQPSAPTTTTQPAPAAAQNQSNSLELEKAAYTVALDAYKQGGAKKAIAPMQNFIKNHPNSIYTGNAYFWLAEFHLATDPVNYNEAKKNYNVVANQYPNSSKAPRALYQLYSIAKDVDKNTVSANQYKNKLLSQYPKSEEAKFFNKSEQ ID NO: 16MSMNNKQRWMGGVVLLGGGVLLAALLLKGNEEIKQVDVQPQTSTSPKLQAKPKQSAQEGQMVQLQPLAVDVETEKRLLEEQRRSREKAVAEQEARAAEFLAMQQQAEADAARKAAAEYAAINARRAAAQESSDNIPPEVAGSENKAKGQQTDTKKSVDLAKADADKKAAEAKRLAEADKKAAEAKRQAEADKKAAEAKRQAEADKKAAEAKRQAEADKKAAEAKRQAEADKKAAEAKRQAEADKKAAEAKRKAEAEKKAEAEKARELLENGDKKWMVQVALAANQANADAVVSKLRAKGYKVTTSPTSKGIRIMVGPAKDRDTADTTRKKITSDASLNMKSAWVIDWVPLDQRKSDSEQ ID NO: 17MANTRYEDDNNSSGTSNRGFASMDPERVREIASKGGRAAHASGNAHEFTSEEAREAGRAAHASGNAHEFTSEEAREAGALSHKNDDRNGRGRSRYDDDEDDDRGRSSGRGRGRSRYDDDDEDDDRGRSGGRGRGRGRDDDDEDDDRGRSGGRGRGRSRDDDDEDDDRGRSGGRGRGRSRRDDDDEDDDRGRSGGRGRGRSRRDDDDEDDDRGRSGGRGRGRSRYDDDDEDDDRGRSGGRGRGRSRADDDDEDDDRGRSGGRGRGRSRYDDDDEDDDRGRSGGRGRGRSRRDDDDEDDDRGRSGGRGRGRSRYDDDDEDDDRGRSGGRGRGRSRRDDDDDDDDRRGRSDGRGQNSRNQKRDAYGRFTSSEQ ID NO: 18MLYVIPFIILLVVAVILKKRENSQKQEATSPKNINRKSGKKASAKSSKSSREKIKAKVIEENIPAIPQSNPVPEALRHNIQQLIQEKQFSAAEAQVNQALKKDNTQHELYLLLLEIHIAQKDEFAIQQLISHIRSLGLNEIAAQAETRQKEYESSSQPDAIDFPQAQTYEEPKNTDTTAQFDELTTSSSEASFDDLQKDYTPVKQEPAIEIEPLEFNFSFEQNSATENTNQPAQQPELSSTQETNELADLEFSFDLAPLHETEEKSQAVEVKADQENSINALDFNFDLNPSSSETKSVQQAPSLDEIKLIEQAPLEATSIAPLEFSLDEPALVPAPELETQNHIDVVNEAATQTQIEDPLLEAFPELKQINENELDLKLAEQYIKFGANQAARNLLQGDEQKFNTEQQQHAKNLLNRIASSEQ ID NO: 19MPKIKPIKLVIIVVCIAIIAVLAWKFLKPKQQQPQYITAEVTRGDIENNVLATGTLDATKLISVGAQVSGQVKKMYVQLGDQVKQGQLIAQIDSTTQENSLKTSDANIKNLEAQRLQQIASLNEKQLEYRRQQQMYAQDATPRADLESAEAAYKTAQAQVKALDAQIESAKITRSTAQTNIGYTRIVAPTDGTVVAIVTEEGQTVNANQSAPTIVKIAKLQNMTIKAQVSEADIMKVEKGQQVYFTTLGDETKRYATLRQIEPAPDSISSESNSTTSSTTSSAVYYNALFDVPNTDGKLRIDMTAQVYIVLNSAKNALLVPSSALSSKQFSGQRKQGQSADKASSTPSAERKHQGNGVRLERLNLTPEQKQLIEQGKATLSVVRVLQADGTTKPTQILVGINNRVNAQVLAGLKQGDQVVIADSSENSAASANSGNNRRRGPMGMSEQ ID NO: 20MNIPPRPFKLSVIACAICYANLTYAQDAQVQALQTIQVKASNAEQSSEQTKAYNVKNSSSATKLNIEAKETPQTINVVTRQQIEDFGLTSTRDVLRNTPGVTVSNQETERTTYMARGFEISNILTDGVGFPLSGYNYNNTNPDTYFYDRVEVVKGADSLTNAFGDPSATINNIRKRPTQEFQASGGVSYGSWDTQRYEADVSGSILPSGKVRGRIMGYEQTGDSYLDRYSAEKNGFAGIVEADLTDSTLLTAGYSQEQNKPNANNWGALPLLDANGKQISYDRSYNPNPDWAHWDNETQNAFVELKQKLNDQWNAKLTYNYLDTKHNSRLLYYYGYPKADGSGVSLTPWGGQEHQEKHAVDFNLEGTYKLENREHEATLGYSYVRNHQQDKQSTGTINDSNVIKSTTTDWASWTPQSITWSDFTEAANYKQNINSIYAATRLHLNEDLKLLLGANYVQAESKGESYSSPMSYSESKVSPYVGLTYNFTPEYTGYMSYTSIFRPQTGIDKDTNQALKPIEGKSYEMGVKSSWLDDRLTGTLSVFKTEQNNYPLRNSDGNPLNRKVPTSDLESQGVEVGLSGQITDNVNLSEGYAQFSIKDTKNGGEARTYNPNQTLNLLTTYTPPVLPKLKVGAGLQWQDGIKLYDSNVNGTIKQDAYALVNLMASYEVNDHITLQANGNNIFDKKYLNSFPDGQAFYGAPANYTVAVKFKYSEQ ID NO: 21MKLQTIACAVAIATGGLFFSHTMNEARAATNTAAVSQSIQPTQEQALVARQLATLVDRQHYLNMRLDANTSNRILDMYLDSLDPDHSLFLDAEVQNYKKLYGSNFGASLKAGNLTGPFAIHQQYRERLKQFYEFMLAELKKPQNLKQPNTFIEVDREKAPYFKTSAEQQNHWRKMLVSQLINLTISREEEQAKQKALKENPSLADGQDLTGPEDLTPAQTLTKRYTRQLERISRVKSDDVLDKTLNAMLATYDPHSNYYPPIDAIELNRQTTLQLEGIGVSIRPERGNEDYTKIETIVEGGPASKSGQVKSGDRIVGVAQEGGKMIDVVGWSSSEIVGLIRGKRGTKVTLKLLGAGASMSQARNVTLVRDVIQEEDAGVRSRTVEVTRDGKKHLLGVIEIPSFYFDYRSRRAGQQYRSVSEDTANAFEALKAKKVEGIIIDLRNDPGGSLEEVARMLGQVIKSGPVVQIRDGNGNVSVFEDNDGGQQIYTGPLAVLVNLASASASEIYSAAIQDYERGIIIGSTTTGKGTAQVQLDTLAYGQATLTQRKFYRVTGGSTQNKGVVPDIKLVDIYNEEFGERKSKNALKWDTIPTAPFKREGSVQPYVAKLSQLSEQRVAVDPQFKYLNKRTAIAKVTSDQKQVVLDIDKRRAELLSLEKQTLDAENERRIATGQKPFPNWESYQASLDALAESRAKMKANQRPALPEEETEVNEAANVLMDYAKLQNRSEQ ID NO: 22MTRIIVASKEGLDVLQDGQLNKVVLNQPTIIQIGVSQKDIASMEKQGGSLVIHLKNGETIVLENFFNEATNTTEHSLVFPTEQGKFVEAQFDAQGKVIDYRGLNHVTDLAYTSTSPSAATMAVDNDPSFSMGNVLKAGLAVLAAEGLYLWAFDKDDKDDSPSTPDLIAPAAPTATLADDTVTVTGKTEANAKIYIKDAAGNTVASGVADASGNYTIKLDKPLVNGDKLNVIAQDAAGNNSKVTVVTGTKDTIAPDVPQAQLSDDGSLLTGKAEANAKITVYDATGKVLGTVFANKDGIYSLKLTPPLTSEAGGKVVAEDAAGNKSEEVKIIAGKDTIPPASPFVEVNKEGSVIHGKTEANAKVQIKDADGKVIGSGTADAQGEFQITLSPALKEAQKGTVVVEDAAGNVSKPVEITPGFDSIAPDKPTVQINTDGTSVTGTAEANAKIEIKDTTGKVIGSGTADANGKFTISISPALTDNKHASVSAIDNAGNKSEVVDIVGTKDTTPPAKPILNSVDDDVGAVKGAITAGSETDDARPKLTGSGEANATLTIYDNGVAIGVVTVTSGRSWSFTFDKDLALGKHTITLTQTDAAGLTSEASSPFTFYVVAPKAASLSETSVDILSTEGPSLADSVGLHTLKVAQNTTTETNNPQKSVPLDDLLKSSTASESDPIAKLLSSTALKTTQASEPIEVNASVGQTTSNPNHPLPDTTSSVLQNLLDQTYPVVSEQ ID NO: 23MSKRIIQSVLSVSVLASMMSMAFAAQNEQEQAEQTLEKPAEPVKLETIFVTAEEQVKQSLGVSVITKEDLEKLPVRNDISDYVRRMPGVNLTGNSATGQRGNNRQIDIRGMGPENTLILVDGKPINSRNSVRYGWKGERDTRGDSNWVPAEAIESIEVLRGPAAARYGSGAAGGVVNIITKKVTNETHGSVEFYTSQPEDSKEGSSNRVGENVSGPLIKDVLSYRLYGNYNKTEADDVDINKSIGSTAAGREGVKNKDISGRLAWQATDQQTVLLDISSSKQGNIYSGDSQLNANAEADAILSQLIGKETNTMYRDSYALTHEGDWSWGKSKLVAQYDKTHNKRLPEGLAGSVEGKINNLDDKATSRLETLRFNGEANIPFEYYLPQVLTVGTEWVEDREKDNVSTTQGKDSSGSGYGDQLAKGDRSKMESRIASAYIEDNLKVTDSTDVVLGLRFDDHSKSGSNWSPSLNITQKLNDNFTLKGGVAKAYKAPNMYQNAEGYLLSTNGNGCPANIESRCLLQGNGDLKPETSVNKELGIQFQRDIVNASLTWFRNDYKDKIVAGTHVVGTVDGSSTNANTGAVTNTKWNILRWENTPKALIQGFEGSLGLDFGDIRWTNNFTYMMDSKDKQTGNPLSLVPIYTINSIFDYDITDQLDVNFVFTQYGRQKSRQFAENRLESGIGSGGANSALKPSTVKSYSTAGINVGYKFSDQISTRVGVSNLFDKQILRDSNSISQTYNEPGRAYYASLKYSFSEQ ID NO: 24MPSKIKFKQSTLSHSMHLILKMQSIPKLICSSLLLSLCVTPCYAQSSAETVIPEANQTVTDSLVQQTNTNNPSDVPITDVATLVTQAQQQQDSLAILQQQEQFPNQIEEFKPITLDNLEDLPVMPVDQNMANEIYRVAEEAKNEAQNFQNGTQKQPEMVVSDASQAELHEINQAPVNIDQLMHEIQSDSKIVVEANETGKTLPELTAAVEEPPEEKGFFRRIFNKIRPPRVIPMEQIPRITAEVTGAPDDLAKNIKGKLSTFTQESFEDFNAALPQLRSLSNQAAQAVGYYNAEFRFEKLSASRVRVNVTPNEPVRINEQNIEFTGAGAKQPQFQVIRLVPDQDVGDIFNHGLYETTKSRIVDAASDNGYFDAYWRLHDVKVSQPENKADINLKYETGERYKLGKVEFRMSDPSKPLPLNMNILESMAPWKEGDDYAFWRVNVLANNLINSRYFNYTLVDSIKPDPIEKPLELPPDLQALVDQQNVDIDESKLLPLEQQQLAKARQLASSSKEVTQNVVDEKQFAGTESVQAAPASLKAATVQHEEQESEQDRLQAQAREEKRIPVIVTLNADKLNSLETGIGYGTDTGARLRSQYRRSIVNKYGHSFDANLELSQIRQSIDGRYSIPYKHPLNDYFNIVGGYERETRDDIGPDVSLLTESAVLGGERVIKKPLGNWQHTIGVRYRLDRLTQKGNVDISELPDAFKTAASEQEALLFSYETSKTSSNTRLNPTKAFKQTYKLELGSESLLSDANMAIATAGWRFIYSLGENDDHQFVGRSDFSYIFTDEFDKVPYNLRFFTGGDQTIRGFDYKSLSPEDNGYKIGGQALAVGSLEYNYQFKEGWRAAVFSDFGNAYDKSFSNPTAYSVGVGIRWKSPIGPIRLDVASGISDDNHPIRLHFFIGPQLSEQ ID NO: 25MFIKSILSSITSIIPLPENSNTSSNLGNGSGDGLLNGISSGNGEHNYGIGNGIADDASITAPITIPLNLSGNSITLIGNSSSSSVNSSPTTTSNNVNDNDVTNNGNGSTIGSGTGNGSGDGLLNGAASGNGEHNYGIGNGIADDASITAPLSIPINLAGNSITLIGDSSSSSVNNSATNTSNTVNDNDTTYNGNGSGGGNGSGDGLLNGIGSGNGEQNYGIGNGIADDASITAPITLPINLSGNSITLIGNSSASSVNSSPTTTSNTVNDNDTTYNGNGTGDSGVSALGGSGNGSGDGAGNGIASGNGEHNYGIGNGNGDDVDITAPITGVLNISGNSFTLIGNSSSSSVNTAPTTTSNTVNDNDTIDNGNSGGTGSGSGNGSGDGLLNGAASGNGEHNYGIGNGNGDDVDITAPITGVFNFSGNSFSIIGNSSSSSINTAPTTTTNTVNDNDVTDNGNDGGGLVGGSSGNGSGDGLLNGAASGNGEHNYGIGNGNGDDADFTFPLTGVLNFSGNSLSGFGSSSSDSVNVAPTTATNTVNDNDTIDNANTGGLGDGSGNGSGDGLLNGAASGNGEHNYGIGNGNGDDADFTLPFTGGLNILGNALSGIGGSSTDSINISPTTTSNTVNDNDTTNNGNTSGGVIGSGDSGNGSGDGLLNGISSGNGEHNYGIGNGNGDDVDVVAPITTPLNVLGNSFSFIGGEGTGDILGPITGIIGGIGGDGDILSPITGIIGGIGGDGDILSPITGIIGSIGGIGGDLGDNPLTGIIQSGIDVLQNLESLKTGLINTGIDTIAGTIIGVFPDAEHPVGDFADLGKLLFETSRDSVNGTLEAISDLAGADLEGASGSITGVIDTLITNGSTASTIIQHIVGDDLVTENGGLLGSITTIIGGVDSGDGGLLGGLDGLISINYGDSDNSNSIDVEDILGNILGSVGSNQGIAVGEPDPTGGSLIHTISLNTVNQLTDQLLHALPTVSEQ ID NO: 26MYKPTTFVWQPSAASLFKITVLSSALAALGITTGCSSTPQSAKTSKTKQVSGAGYLDASSLDSLEDLLSATDMRAVEGDRLLILKHGDVWKRMAVGFKMDLNHWDPRIEAQRSWFISRQPYLDRLSARASRYLYHTVKEAERRGLPTELALLPVIESSYDPAATSSAAAAGLWQFIPSTGRIYGLQQTGMYDGRRDVVESTRAAYEFLGSLYNQFGSWELALAAYNAGPGRIQQAINRNQAAGLPTDYWSLKLPQETMNYVPRFLAVAQIIKNPRAYGVSLPPIANRPHFREVTLSAPLSLNEIASVTGLSRAELYALNPGYRGETVDPASPMRILIPADISPSVDNKLKGMKAGGSSGWWASVTSPSKPTTTTSTSVTVRTTPSTPAQPVRPSTPAKTSSSSVTVKTTTPRGSDALAAFAASADVPSAPRIPVAVTPAANIKPVRTEPPISATEREKILAAVRAEGEKETVDQALEPQATQAEKDQVVAELKALAPQGTEIVDPYDGKIKLTAIQTSQSVAEQQGKEVSKGFAYPKTLAEDATLANSEDAQRNKDKPYIKTDTDVVVVQPKGKRSTYTVQPGDTLAVIAMKNGVNWRDVAKWNQIDPEKTLFVGTSLYLYDAKPQEAETTAKSAAKPDVYVVQANDSLTGVANQFNLSVKQLAEYNDLSVTDGLFVGQKLQLKEPKGNRAAKVEPKAIQASTRRIATKSYTVKAGEYLKLIADRYALSNQELADLTPGLSAGSNLIVGQKINVPAKEITVDEVDDSKASGKYEKLAAGPSYKTESYKVQRGDTLSSIATKSKISLAELAELNNLKANSHVQLGQTLKVPAGASVPDQYVVQSGDSLNAIAAKYNLQTSYLADLNGLSRTAGLRAGQRLKLTGEVETTSKVSAKNTKEETPETYTVKSGDSLGNIANRYHLQLDYLAALNGLSRNSNVRVGQRLKLTGDLPTVETAKTDTAKSSPKAVVAGKNTEKYTVKAGESLNAIASRAGISVRELAEMNALKANANLQRGQNIVIPKTVVEYKVKRGDTLIGLASKYGLETTLLAELNNLTPSTQLRIGDIIKVPNLSEQ ID NO: 27MKRMLINATHAEEVRVALITGNRLYDFDLENRTREQKKSNIYKGHVTRVEPSLEAVFVEYGAGRQGFLSMREIANSYFQADPRQTSNIRELITEGTELLVQVEKEERGNKGAALSTFISLAGRYLVLMPNNPKGGGISRQISGSVREELKEILASLNVPRGMSVIVRTAGIGRTQEELQLDLQHLLDLWAQIQGTASSGPSPMLVHQEAGVVTRAIRDYLRDDVAEILIDSEQAYNEAYNFVKAVMPRQLDKLKTYTLNEPLFAHFGIESQIQTAYEREVKLPSGGSIVIDQTEALVSIDINSAKSTRGHDVEETALNTNLEAAEEIARQLRLRDIGGLVVIDFIDMTKERNQRMVEAKLREATQSDRARIQFGQLSRFGLMEMSRQRLRPSLEEATGYVCPRCHGTGMVRDLRSLSLSIMRKVEEIALRERHGEVQVEVPVEIAAFLLNEKRHSLVYLEQTSGVRVTVLPHPHLETPHYEIAYNPDGFAPSSYERTEATRSSEKELGYESSEWHLEEADHGHAHVTATASTHAAAQKKANHATQPVAQPSAQKAASPCAWLENLFVQKQAQTVDQSRSAQNAAAAIEQMVNTGAVSRGQFGQVAVPAVAEVAPVQSNNAYISQSPVKQDVRERVEKDDKSQQQRQNNKKRKHKEQREQHHQSHEQQHQVHEEVVQLSRQEQRELKRQQKRQQQQDQQHQNNDVQHTENAVPRRDRNNQQRPNRPNRHRDPSVLNENQNTLVVVDEKQIKVDVIDAPKEDVMNTALIINVDQGQSEIVALTPERRHVERVETTSTEVAQEPTPAPVVAEKAAVVETKEEAQPSQEAAQPQIKRASNDPRMRRRQQREAKHAKAATPSIAPSQIPTLAQHTIGSLIRHVYGEDCTVLIEQFGLVPTFNRALQKFAEQYASTLVVEVTAETEEKKPVTRDAELPSHKPAEEAEPAPVLPLTPPQAPAPRVANDPRERRRLAKLAAEQAFEQVKQQHSAQEEVATPAPVAEETVAAPTAETQATVEPAQQPLELNQSTEVVQPEAAPAEEKATEETVAEAPAAKEPAPSKAASKAKAAAEETVAPTEATTDAESEDVKADKDKPSRPRRPRGRPPKKANPVAESEQ ID NO: 28MSTLATLKALLAKRILIIDGAMGTMIQRHKLEEADYRGERFADWAHDLKGNNDLLVLTQPQIIQGIHEAYLDAGADIIETNSFNGTRVSMSDYHMEDLVPEINREAARLAKAACEKYSTPDKPRFVAGVLGPTSRTCSISPDVNNPAFRNISFDELKENYIEATHALIEGGADIILIETVFDTLNCKAAIFAVKEVFKQIGRELPIMISGTITDASGRTLTGQTAEAFWNSVRHGDLLSIGFNCALGADAMRPHVKTISDVADTFVSAHPNAGLPNAFGEYDETPEQTAAFLKEFAESGLINITGGCCGTTPDHIRAIANAVKDIAPRQVPETVPACRLSGLEPFNIYDDSLFVNVGERTNVTGSKKFLRLIREENFAEALEVAQQQVEAGAQIIDINMDEGMLDSQNAMVHFLNLVASEPDISRVPIMIDSSKWEIIEAGLKCVQGKPVVNSISLKEGYDEFVEKARLCRQYGAAIIVMAFDEVGQADTAERKREICKRSYDILVNEVGFPAEDIIFDPNVFAVATGIEEHNNYAVDFIEATGWIKQNLPHAMISGGVSNVSFSFRGNEPVREAIHSVFLYHAIKQGMTMGIVNAGQMAIYDDIPTELKEAVEDVILNQNQGESGQAATEKLLEVAEKYRGQGGATKEAENLEWRNESVEKRLEYALVKGITTYIDQDTEEARLKSKRPLDVIEGPLMDGMNVVGDLFGSGKMFLPQVVKSARVMKQAVAWLNPYIEAEKTEGQSKGKVLMATVKGDVEDIGKNIVGVVLGCNGYDIVDLGVMVPCEKILQTAIDEKCDIIGLSGLITPSLDEMVFVAKEMQRKGFNIPLLIGGATTSKAHTAVKIDPQYQNDAVIYVADASRAVGVATTLLSKEMRGAFIEEHRAEYAKIRERLANKQPKAAKLTYKESVENGFKIDESYVPPKPNLLGTQVLKNYPLATLVDYFDWTPFFISWSLTGKFPKILEDEVVGEAATDLYNQAQAMLKDIIDNNRFDARAVFGMFPAQRTDADTVSVFDEAGQNVTHTFEHLRQQSDKVTGKPNLSLADYIRADREQQDYLGGFTVSIFGAEELANEYKAKGDDYSAILVQSLADRFAEAFAEHLHERIRKEFWGYKADEQLSNEELIKEKYVGIRPAPGYPACPEHSEKAVLFDWLGSTDKIGTKLTEHFAMMPPSSVSGFYYSHPQSEYFNVGKISQDQLEDYAKRKGWTLDEAKRWLAPNLDDSIVSEQ ID NO: 29MKLKLKNFKPNNLWYAVCSSSMIFTWLMTSSVVQASDLQIYASPTAGKKTIVMMLDTSGSMTNNSYGENRLAMLKNGMNAFLASNNPVLNDTRVGLGNESANGDSRSGQILVAAAPLGDASTLNTVGSQRYKLKQAVANLTAGGSTPSAHAYAEAAAYLMGTTTYSETNYAIRKDSYIKRVRRSDNRTEYSYCTNYRDSQIDTANLWQPCRSNSYWSSWSTNNPGVDNATAYDTSSDWTYYYTYYYTTFNYAVANADSGIPKSKSNDTASNPNIVVDRNATNSNAVYQSPLPAVANRQSCDGQGIYELSDGEPNNTTNTRSASVMSTALGSTFGADFNCSGGLSNTTADSGWACMGEFAKELFDKTKNPAGVSIQTAFVGFGSDFSSLNSSDVKNACRLSSRTQSDRKGDDACSPNQSTNAVAAPGYGNGGFFPTQSSQGVTDSVIAFINNLDKVPLEPLTTGAISVPYDALNPKNLQEYGYLRAFEPNPANTYLTWRGNLKKYHVVLSGANAGAFEANSGGLVYNASGAFRTGTKDYWNSSTYTDGGKVFLGGSYANVPLPIAGQPETRDAEGNITKYYYAVQSKIRNLFTDVSAVAADGSLTKISTSGTNLLKIPAAPPEETNPFDTVANTASYVLGKFDPSTGQNILKAFPISLKLKILNYLGYSTDINATTLPSSLVTSNEPYLSMGGSIHSLPVQLTYNGTLDDNGNLTSAREQSILYGTMEGGLHIVDASSGIEQMVFVPADILNDSVASKALVVGQSDASAPAEGMDGAWVSDPAYNITTVGSGSSAVSKVTAKQMNIYGGMRMGGSSYYGLDVLSPTSPKLLFRIGADQNDYSRMGQSWSKPVLANIRYNGSIRRVLIVGGGYDQCYEKPNITLTDACFTNGKAKGNAVYIIDAKTGQRLWWTSDTGSNTDNANMKHSIVSRISTLDRDADGLVDHLYFGDLGGQIFRVDLNNNQTKTNSTYSSFGVRVVRLANLATNDSTYDGTNDYTGGNAPRFYEPPTVTIHDYGIHTFITVGIASGDRSTPLDVYPLTGREGMTPASALSGRPVNNVYGIIDRDFVKKNLMSLTDNQLETKDITRTGLRKNPQILRTGETRVAQIFFPTTGVGKGGWYRSLSSTSDGTEKANNSFRIKGGLKAFEEPMAITGNLIILVYDPQGTGIVAADPCLPRVVGETDRQTYCLPFGACLNSDGSIDQNKENHSGFETQTGTNCPVGASECNKNVIGSGIRSVTFVPTEDNPPTTNSCGKLKLSGNEQGTGQWQCTSHLVPTRWYERYRSEQ ID NO: 30MTDAAGNTSEQAVQKVVVDTTAPQAGELTLSDLSDTGISATDQITQDKNFNLKLEGQESGSRVTYLVSTDEGKTWQETTIAQKDLTDGVYQYKAVVTDAAGNTSETAVQKVVVDTTTPQAGELTLSDLNDTGVSVTDQITQDKNFNLKLEGQETGSRVTYLVSTDEGKTWQETTIAQKDLADGVYKYKAVVTDAAGNTSETAVQKVVVDTTAPQAGKLTLSDLNDTGVSATDQITQDNSFTLKLAQPIVIGEQAALLDHYEVSKDEGKTWQETTADQKDLADGIYQYKAIVTDLAGNISESAIQKVVVDNSLNVESTTVIVKPITEDNTISLVEKDQVISIRLEIANLPTDLNSSLTSVNTTLGNVTYNFHFDEVTQEWVTEIPAEFLWSVEPQTNISIEISLTDQAGNTAIIKHTQNYNVDHTPNSPTLDSLTFNNIDGAIISGSAYKGSKVDIYNKNGDWLASTITNEEGKFTLQDLSINSNQEVYAVATYNGYSSENSSIGLVTEVPAISITRISPEGVISGYATEGSHFIVKDQNGNILQEFNSNVFDSSGITPFSVMALGEVRPFILSLDQPLEEGAQIIISIDKDNISGHPQYITADYTPAVFLETPQFDISGETLSVHVNEPNSFIRAFSGEGNLIATGFTDEQGFASLQVFQFLKEGETVSVQVVDKNQNTSETLIEVPNFAYIPHVERITQEGLISGVAEDNSTVIVRDADGNELGKVTLGDDNSWSDFSHFSLSVNRPLIDGEKISVQIIDNKGLMSPEQNIIVDLTPPPAPTELNENDAGDLVYGHAEPFSEILVKDGQGNILNKWFWNNWTDESGSFSIELGTFLTNAETVYVTATDVNGNVSLAAQIQAPNYAFAPYVDSFTSDGVISGQAENNSTLVVKDAKGDVVAEIKVGEDNGWNGSSYFKLQLDRPLVDGEQFFLSIKDARGQVSADTVITADTVAPTPASNLVFSEDGSYLTGVAELNTTIQVFDHNGQLVNIWNNTINSDGTFTIYLGSNNLHGEAFTVTVKDQAGNVSEAISINAPLDDIAPNPIKNILLDANGQNFTAQAEANSQIEVFDSLGNQTGWGSTDSAGNVSGSFNQTYLHGEELTFVVIDRAGNRSIEFKQNALIDTIAPNPIANIIFNEDGQSFTAQAEAGSSIDVLDQTGNKIGFGYTDSSGNVSGYFQQVYLHGEELTFVVIDRAGNRSAEVKQSALNDDVVPNPIENIVLDLNGQNFTAQAEANSQIEIKNNNGDVVGYGSADSAGNVSGYLYQVHLHGEELTFIVVDRAGNRSTEVKQNALIDDIAPNPIENIVLDINGQNFTAQAEANTQIEVKNAVGEIVGLGYVDGAGNVSGYLYQVYLHGEELTFVVVDRAGNRSTEVKQNALIDDIAPNPIENIVLDINGQNFTAQAEANTQIEVKNAVGEIVGLGYVDGAGNVSGYLYQVYLHGEELTFVVVDRAGNRSTEVKQNALIDDIAPNPIENILLDANGQNFTAQAEANTQIEVKNTAGEVIGSGSTDSMGNVSGYFYQVYLHGEELTFVVVDRAGNRSTEVKQNALIDDIAPNAIENIIFNENGQNFTAQAEANSKVEVKNAAGFVVGSGYVDSVGNVSGYLNQVYLKGEELTFVVIDQAGNRSIEVKQTAFLDNTAPENATNLVFSEDGSYLSGMAFPNATIQIFDQYGQLLNQWNNNVNWDGTFNIYLNSNYMHGEVFKVVVVDHAGNLSGEVTVKAPLDDIAPVAASDLVFNEDGSSLSGVAEPNTFIQIFDQNGQQMNTWSQSVNADGTFTIFFGTYNLHGEEFTVIVKDLAGNVSEAVSVKAPLDDIAPKPIKNIVFDANGQSFTAQAEANSQIEIFDSFGSQIGWGSTDSTGSVTGYFYQVYLHGEELTFVVIDRVGNRSDEMKLNALMDTIAPKPIENIIFNENGQNFTAQAEANSFISVKNAAGEFVGYGYVDSTGNVSGHENQVYLKGEELTFIVIDKAGNQSIEYKQNALTDDIAPNPIENIVLNKNGQNFTAQAEADSQIEVKNTAGEVVGSGYVDSIGNVSGSFNQVYLHGEELTFVVVDRAGNRSTFVKQNALIDDIAPNQIENIVFDVNGQYFTGHAEADTRIEVLDQEGNRAGWGYVDSQGNVIGYFNQVYLHGEELTFIVVDIAGNRSVEVKQNALIDNVAPPAAANITLTSDGLLFGEAEPNSTVEIIDQYGAVITTTYVWYDGTFNQWINLSQYQTQNLSIVVKDQAGNRSEVVHELVPVFTNSPIAATELKLDIDGHILTGKATVGMSVVVTSTDGQTINGGWNNAVNEDGSFAIQLNDYYLQGQTLQVRVYDQNTNQYSLISEIIAPLDNIAPVINEVVINNDGYGITGQTDSKAIIQVMDADGDLRAEFQADETGYFNASIYPPILRGEQLFITAIDLAKNISKPFNITFNADTNAPPSAEHIVVSENGFFIEGTAVAISTVHIFDVHSNHVATNVADEAGNFNIQLYPPLASGQILRIVVEYNGYQSAYTEITAPIDTVAPNAATQLLLFDGNVLSGQAFAYSIVNIFDANNNLVGQTNVGSDGAFLTHLWYEYWHGETLTVKVVDANQNVSVGTTIVAINDTVVPDVVTQLAIDEWGSLTGRVESYATVELTYHFTDQPLSVTSTTALANGMFFIYLDRNATSLDLTVIDRAGNRSETISQIISDLPTVIIDHFKGDATDNTYNIDTIDDFVQFYIVEPYAIYKDVWIDNSYMYSDWVIEGHYEQIWFVDGYYESQWATSGYSTVQNIYQNQNGITYIDNGTADSDYSRYEQQYYDEVNGQWQEGYELTYIRSEEGWVDTSHYFDVYIDTSHYFEVWVDTSHYQDINVENSYWESQLVESGRRDVDLGGHDKIISSVNYSLVGLYQTVNDPTTVDSFLESGRYVEDLELVGSAHLNATGNALDNLLTGNSGNNVLNGRFGNDTYITNEGTDTIVFQLLNSQDATGGNGHDTVLDFTLGDIRTNLQADKIDLSFLLIDYSKDVSALAKFITVEQDAGNTTISLDRDGEGTMFNSVSLLTLNQVNTTLDELLNNQQIIV | 274,614 |
11857616 | EXAMPLES Generation of Vaccine Candidates Previous experiences with different flaviviruses (dengue, West Nile, Japanese encephalitis, tick-borne encephalitis) widely demonstrated that the flaviviral surface envelope (E) proteins are able to elicit protective neutralizing antibodies that allow reducing virus infectivity. The ZIKV genome consists of a single-stranded positive sense RNA molecule of ˜10800 kb of length with 2 flanking non-coding regions (5′ and 3′ NCR) and a single long open reading frame encoding a polyprotein that is cleaved into three structural proteins (capsid (C), precursor of membrane (prM), envelope (E)) and seven non-structural proteins (NS) (FIG.1). The E protein (53 kDa) is the major virion surface protein involved in various aspects of the viral cycle, mediating binding to target cells and membrane fusion. The inventors therefore chose to express the Zika virus E protein. Several forms of E protein were selected in order to express either soluble secreted proteins or anchored proteins onto the surface of VLPs. The following Zika virus antigens were cloned and expressed from a mammalian expression plasmid in human cells: prM-E and different forms of E with or without the stem or anchor region. These proteins contain either the original signal peptide sequence of Zika virus E or a heterologous signal peptide sequence from JEV or MV fusion protein. These proteins contain the signalase cleavage site located between the prM and the E sequences (FIGS.3A,3B,3C,3D). Antigens Selection and Design The Zika antigens were selected based on previous works concurring into suggesting that envelope antigens of flaviviruses may be able to elicit neutralizing antibodies and T cell responses. Selecting a suitable antigen should however take into consideration the evolution of the virus over time and the variety of existing virus strains. To this end, the inventors reconstructed the phylogeny of representative members of the flavivirus family, including Zika virus, using only the amino acid region of the flavivirus polyprotein corresponding to the envelope (E) gene. Unlike phylogenetic analyses based on the full genome, or the polymerase (NS5) of flaviviruses, where the closest relative of Zika virus are neurotropic viruses such as Saint-Louis Encephalitis virus, the inventors noticed that Zika E appeared closer to DENV E (FIG.2) (Barba-Spaeth, et al. Nature 2016, 536, 48-53). The inventors then proceeded to identify the different domains of Zika membrane (M), its precursor (prM) and E proteins through structural homology modelling based on available data on DENV (Ekins et al. Illustrating and homology modeling the proteins of the Zika virus, F1000Research 2016, 5:275). The inventors also identified the signal peptides at the end of the Capsid (C) gene, just upstream of prM, using again homology modelling with dengue virus as a reference, as well as publicly available algorithms to predict signal peptide sequences (sigpep. services. came. sbg. ac. at/sidnalblast. html; cbs. dtu. dk/services/SignalP/; predisi. de/). The inventors chose to include the signal peptide sequence to induce the export and secretion of the candidate antigen, either the full-length prM-E, or the E only, outside the cells. For the E antigen, the inventors also predicted the signal peptide at the end of M, just upstream of E, and designed versions of the antigen using this native signal (FIG.3A). In addition, the inventors also designed chimeric antigens where the native signal peptide of Zika virus was replaced with the signal peptide present at the end of JEV C (FIG.3B), or the signal peptide present at the N-terminal of the fusion protein (F) of MV (FIG.3C), hypothesizing that these sequences would provide enhanced export of the candidate antigens. The inventors designed an additional version of the chimeric antigen including the signal peptide of F from MV, where two amino acids corresponding to the junction between the end of the signal peptide of F and the beginning of F itself were removed (FIG.3D). Secondly, the inventors also designed shorter variations of the antigens by removing C terminal fragments of the E protein corresponding to the predicted stem and/or anchor domains, including the intermediate region between the stem and anchor (as predicted by comparison to DENV). The aim of these modifications that reduced antigens size was to generate antigens that were able to form VLPs. For a third variant, the inventors removed the anchor, the intermediate domain between the anchor and the stem, as well as a fragment of the second helix that composed the stem, this time in homology modelling with WNV (variant Ed445). Finally, the inventors designed chimeric prM-E and E antigens using the signal peptide from MV F protein, and replacing Zika E anchor by the transmembrane (TM) and intracytoplasmic tail of MV F protein (FIGS.3C and3D). For the selection sequence of the antigen itself, the inventors analyzed all publicly available sequences of Zika virus (both Asian and African lineages), as well as unpublished sequences generated by the inventors, from the epidemic in South America and Pacific. Based on the epidemiological data reporting an association of congenital syndromes and neurological afflictions in adults with only the Asian lineage, the inventors designed an antigen using the consensus amino acid sequence of Zika viruses as observed circulating from 2015 and onward, notably to include the S139N change that generated a novel potential N glycosylation site in prM that was absent from the African lineage, and the V763M in E. The sequences were codon-optimized forHomo sapiensexpression and adapted to measles vector cloning and to the “rule of six” (total number of nucleotides divisible by 6). Regions very rich (>80%) or very poor (<30%) in GC were avoided to increase RNA stability, a high CAI value (0.97) was obtained to increase translation efficacy, the following CIS active sequences were avoided: internal TATA-boxes, chi-sites, ribosomal entry sites, AT- or GC-rich sequence stretches, ARE, INS, CRS elements, repeat sequences, RNA secondary structures, cryptic splice donor and acceptor sites, branch points. The following measles virus editing sequences were avoided where possible: AAAGGG, AAAAGG, GGGAAA, GGGGAA, TTAAA, AAAA, and also their complementary sequences on the same strand: TTCCCC, TTTCCC, CCTTTT, CCCTT, TTTAA, TTTT. The enzyme restriction sites BssHII, BsiWI were avoided internally and inserted at both ends for cloning purpose. Antigen Expression in Mammalian Cells The optimized antigen sequences were cloned into pcDNA5 mammalian expression plasmid and transfected into HEK293 cells. The size and level of expression of each antigen were characterized after western blotting using appropriate antibodies for detection. Antigen Expression in Measles Vector The optimized Zika antigen sequences were inserted into the MV vector in different additional transcription units, according to the desired level of expression. After sequencing of the measles vector plasmids expressing the different Zika antigens, the replicating recombinant vectors were generated by reverse genetics using a cell-based system previously developed (Combredet, C. et al., 2003, J Virol,77(21): 11546-11554), and the rescued viruses were amplified and titrated on Vero cells. The recombinant viruses were grown on Vero cells to document the expression of Zika proteins detected both in supernatants and in cells by using Western Blot and indirect immunofluorescence staining with appropriate antibodies. The presence of Zika virus VLPs (in prM/E expressing vectors) was identified after ultracentrifugation of culture medium and western blot (FIG.7). The correct processing of antigens in infected cells was checked by Western Blot. The vectors with the best expression capacity of Zika antigens were isolated by serial dilution and single plaque cloning before amplification on Vero cells. Growth Capacity of Recombinant Vaccine Virus The growth capacity of selected vaccine viruses was compared with standard MV Schwarz. Growth curve analysis was performed in Vero cell culture by using different multiplicity of infection then titration. Stability of Recombinant Vaccine Virus The best vaccine vectors selected were tested for their genetic stability by serial passaging over 10 cell culture passages on Vero cell culture followed by western blot for antigen expression and full sequencing analysis. Preclinical Evaluation of First MV-Zika Recombinant in Mice Single Immunization The two recombinant vectors MV-prMEd404 (native sequence, insert 4) and MV-ssEd445 (native sequence, insert 5) were evaluated in CD46/IFNAR mice susceptible to measles infection. Mice were immunized with one or two intraperitoneal injections with defined infectious units of vaccine virus and functional antibodies and cell-mediated immune responses were analysed using both standard and specifically developed assays. Binding antibodies to Zika virus were determined with ELISA and neutralizing antibodies with specific plaque reduction neutralization test (PRNT). The T cell responses were analysed by Elispot assay using Zika virus-specific peptides for ex vivo stimulation of splenic cells. The vaccine vectors were then tested for protective efficacy: immunized mice were challenged with a lethal dose of Zika virus. A dose-response challenge was previously established in CD46/IFNAR mice showing that doses between 102and 106focus forming unit (ffu) of Zika virus African strain HD78788 (adapted to mouse) efficiently kill these mice. In a first experiment 6 mice per group were immunized with a single intraperitoneal injection of 106TCID50 of MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5) or empty MVSchw as a control. Blood was taken before immunization and at day 30 after immunization, and Zika virus ELISA titers were determined (FIG.5A). The immunized mice were then challenged at day 30 by intraperitoneal injection of 106ffu of Zika virus African strain HD78788 (mouse adapted). Morbidity and mortality were controlled during 12 days (FIG.5B) and Zika virus viremia was determined in serum by qRT-PCR (FIG.5C). To determine T-cell response to the vaccine, another group of CD46/IFNAR mice were immunized by MV-prMEd404 (insert 4) or empty MVSchw and spleens were collected at 8 days after immunization. Elispot assay was performed on freshly extracted splenocytes using MVSchw or Zika virus to re-stimulate T-cells or concanavalin A as a control (FIG.5D). Prime-Boost Immunization In a second set of experiments, groups of CD46/IFNAR mice were immunized with two successive intraperitoneal injections of 106TCID50 of MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5) or empty MVSchw as a control. Blood was taken before immunization and at day 30, 45 and 55 after immunizations and Zika virus ELISA titers were determined (FIG.6A). Neutralizing antibodies were determined in sera collected at day 50 using a specific neutralization test of Zika virus (FIG.6B). The immunized mice were then challenged at day 60 by intraperitoneal injection of 106ffu of Zika virus African strain HD78788 (mouse adapted). Morbidity and mortality were controlled during 12 days (FIG.6C) and Zika virus viremia was determined in serum by qRT-PCR at days 2, 4 and 6 post infection (FIG.6D). Preclinical Evaluation in Non-Human Primates (NHP) Validation of the ZIKV Strain Used in the NHP Challenge Study Because little is known about the physiopathology of ZIKV in cynomolgus macaque (Macaca fascicularis), two animals were inoculated in a preliminary assay with three doses of Zika wild-type virus (104, 105and 106pfu) to assess the viral stock and associated clinics in macaques. These two animals were submitted to the same follow-up than vaccinated and challenged animals but for a 6-month period. The following points were addressed: Virology (qRT-PCR; clinics (Rash, Fever); Blood cell count (Lymphocytes, Monocytes, Granulocytes, platelettes); Biochemistry (ASAT, ALAT, CRP); Non-specific (innate and inflammatory) and specific immune response: Cytokines/chemokines by luminex, NK, B and T cell profile (14 colors flow cytometry), Antibodies (neutralizing, binding) on serial sera samples, T cells functional response and memory cells (ELISpot, ICS). Shedding of the virus in biological fluid (saliva, tears, genital fluids) was assessed by qRT-PCR and/or isolation methods at various time-points. Vaccine Immunogenicity Study in NHP Macaques were immunized with one or two subcutaneous injections at 3 months interval of defined infectious units of vaccine virus. Humoral and cell-mediated immune responses were determined at different times post immunization. Macaques were then challenged with infectious doses of ZIKV. Infectious viremia and clinical signs were determined. For this task, twenty-one adult cynomolgus macaques were selected to be negative for anti-flaviviruses and anti-measles antibodies; Two groups of 7 animals were vaccinated with a single dose or a prime boost regiment with the best MV-ZIKV recombinant virus (MV-prMEd404 native) selected. Immunity (Humoral and cell associated) was explored and virology was followed up to 1 month post vaccination. Clinics and biological parameters are assessed in parallel to a third group of 7 animals vaccinated with the control empty MVSchw strain following the prime boost schedule. Antibody neutralization titer was determined. Vaccine Efficacy Study in NHP Immunized NHP were challenged with ZIKV two months after immunization. ZIKV viremia level (qRT-PCR) was analyzed in blood, saliva and tears. Inflammation and immune response was assessed in plasma (neutralizing Ab, cytokines). Expression Assays The expression assays performed for all constructs generated (FIG.8) showed a strong expression for several of them. Signal was detected in the ultracentrifugated fraction, which was compatible with the generation of virus-like particles, in varying amounts for some candidate antigens, notably A1 and A12. These two antigens were thus further cloned into the measles vector and demonstrated high-level expression as shown by immunofluorescence (FIG.9A). The recombinant MV-ZIKV-A1 vector replicated similarly to standard MV Schwarz virus, although with a lower final titer (FIG.9B). Tested for their immunogenicity in CD46/IFNAR mice, MV-ZIKV-A1 and MV-ZIKV-A12 vectors elicited strong immune responses following a prime and boost regimen with 1-month interval, comparable to MV-prMEd404 and MV-ssEd445 vectors, as detected by ELISA (FIG.10). However, different amounts of neutralizing antibodies were induced (FIG.11). Only the candidate MV-ZIKV-A1 induced a strong neutralizing response (2 log stronger). This correlated with the complete protection conferred to mice by immunization with MV-ZIKV-A1 (FIG.12) against viremia, as well as protection from a lethal challenge (FIG.13). In conclusion, this study demonstrated that the A1 full-length Zika antigen expressed in MV vector was able to provide sterile protection from infectious and lethal challenge of immunized animals, correlating with strong neutralizing antibody induction. | 15,288 |
11857617 | DETAILED DESCRIPTION In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection in an individual in need thereof, including humans and animals, by administration of an antibiotic and/or an anti-viral drugs and a vaccine directed to a causative agent of the infection, and/or an inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection. In alternative embodiments, the infection (or causative agent of the infection) is parasitic, bacterial or viral. In alternative embodiments, the viral infection is a coronavirus infection such a Covid-19 infection. In alternative embodiments, methods as provide herein prevent or decrease the prevalence or severity of “vaccine breakthrough infections” after vaccination, where external mutants of the infection's causative agent develop and infect or re-infect patients in spite of the fact that they have undergone immunization, for example, to prevent a Covid-19 infection. Thus, in alternative embodiments, methods as provided herein comprise combining an effective anti-microbial (for example anti-viral) treatment (for example, a drug or a mixture of drugs or other therapeutics) with an anti-microbial vaccine to prevent in vivo mutations (and thus also prevent a vaccine breakthrough infection) of an infectious agent such as virus, for example, a coronavirus such as COVID-19 or variant thereof. In alternative embodiments, methods as provide herein provide a solution to the problem of imperfect vaccine disease prevention, where a bigger and/or better vaccine or multiple vaccinations are ineffective because science will never keep up with the continuing viral mutations. In alternative embodiments, methods as provide herein prevent replication of newly-inhaled mutants in the already vaccinated or ‘about to be vaccinated’ population. In alternative embodiments, methods as provide herein comprise an added anti-replication method of treatment in addition to vaccination to protect the immunized population from mutants entering, replicating, and further mutating in the immunized population. In alternative embodiments, methods as provide herein prevent replication and thus prevent an ongoing viral mutation, a method utilized by the mutant to escape neutralizing antibodies and destruction. No replication, no mutation. In alternative embodiments, methods as provide herein comprise combining an effective anti-microbial (for example anti-viral) treatment (for example, a drug or a mixture of drugs or other therapeutics) with an anti-microbial vaccine such as a DNA vaccine such as an adenovirus-based vaccine, an mRNA vaccine, a peptide-based vaccine, an inactivated pathogen-based vaccine, and/or an vaccine manufactured by:Sanofi (optionally VAT00002 or VAT00008),GlaxoSmithKline,Takeda Pharmaceutical (optionally TAK-019),Pfizer (optionally tozinamera or COMIRNATY™),Moderna (optionally elasomeran or SPIKEVAX™)Novavax (optionally vaccine to SARS VLPs S protein and influenza M1 protein),CanSino Biologics,Inovio,Sinovac,BioNTech,Johnson and Johnson,Valneva (France) and Dynavax Technologies (optionally VLA2001 and VLA2101),Sinopharm (or China National Pharmaceutical Group Corporation),Emergent BioSolutions (optionally human polyclonal hyperimmune serum with antibodies to SARS-CoV-2),Bharat Biotech (optionally COVAXIN®), The Rockefeller University (optionally vaccine toMVA S alone, or MVA-S prime and Ad5-S boost),Helmholtz Centre for Infection Research; Technical University Munich; German Center for Environmental Health (optionally vaccine to NC protein add-mixed with MALP-2 by intranasal route and boosting with MVA-NC by intramuscular route),University of Manitoba; University of Pennsylvania School of Medicine; Southern Research Institute; Fox Chase Cancer Institute (optionally vaccine to Heterologous Adenoviral prime boost AdHu5 s AdC7-nS),University of North Carolina at Chapel Hill, USA (optionally vaccine to VEEV replicon particles expressing the SARS-CoV S),National Institute of Infectious Diseases, Japan (optionally vaccine to recombinant D1 expressing S protein),Beijing Institute of Genomics, China (optionally vaccine to Recombinant trunctuated S—N fusion protein),Saitama Medical University; Josai University; Nippon Oil and Fat Corporation; National Institute of Infectious Diseases, Japan (optionally vaccine to recombinant peptide N223 on liposomes),Chinese Center for Disease Control and Prevention; Canadian Science Centre for Human and Animal Health (optionally vaccine to Recombinant TM-truncated S protein),HKU-Pasteur Research Centre; The University of Hong Kong; National Institutes of Health; Centers for Disease (optionally vaccine to Trimeric Spike protein),Sun Yat-sen University, China (optionally vaccine to SARS S DNA prime and HLAA*0201 restricted peptides boost vaccine),State Key Laboratory of Virology; Graduate University of Chinese Academy of Sciences (optionally vaccine to or as a 3a DNA vaccine),Institute of ImmunoBiology, Shanghai Medical College of Fudan University, China (optionally vaccine to DNA prime—protein S437-459 and M1-20),CNB-CSIC; University of Iowa (optionally vaccine to rSARSCoV-E),International Vaccine Institute (IVI) (optionally vaccine to recombinant adenovirus expressing truncated S protein (rADV-S)),University Health Network, Canada, and United States Center for Disease Control and Prevention (CDC) (optionally vaccine to recombinant measles virus spike protein),Institut Pasteur (optionally vaccine to MV-SARS recombinant measles virus vaccine expressing SARS CoV antigen),Baylor College Medicine; Sabin; New York Blood Center (NYBC); University of Texas Medical Branch (UTMB); Walter Reed Army Institute ofResearch (WRAIR); National Institute of Allergy and Infectious Diseases (NIAID) (optionally vaccine to receptor binding domain (RBD) of the SARS-CoV spike (S) protein),Vaxine Pty Ltd, Australia (optionally vaccine to SARS recombinant spike protein plus delta inulin),Gamaleya Research Institute (optionally SPUTNIK V™ or Gam-COVID-Vac), and/orOxford-AstraZeneca (optionally AZD1222 or COVISHIELD™ or VAXZEVRIA™)and others, including anti-COVID-19 vaccines. Hence, methods as provided herein that combine antibiotic, anti-parasitic and/or anti-viral treatment with an inactivated or attenuated causative agent of an infection, or a vaccine or a live, viable or infectious causative agent of the infection (for example, a live or attenuated virus) administration, can treat, ameliorate or prevent a vaccine-breakthrough infection, as well as eradicating the infection if present pre-vaccination enhance protection and eradicate infection. In alternative embodiments, methods as provide herein comprise administering in coordination with (for example, before, during and/or after) an anti-causative agent vaccination, or an anti-viral vaccination, and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (such as a live or attenuated virus) administration, any one or combination of anti-viral, anti-parasitic or anti-bacterial therapies, for example, one or more anti-COVID-19 infection medications or drugs, including for example, the drug ivermectin, or the combination of ivermectin and an antibiotic with anti-viral properties such as doxycycline or azithromycin, for example the combination of ivermectin and doxycycline or azithromycin, or the combination ivermectin and doxycycline or azithromycin and zinc or any zinc salt (an anti-viral mineral, for example, and anti-COVID-19 mineral), which optionally also can be administered in conjunction or coordination with a vitamin or vitamins such as vitamin D and/or vitamin C. In alternative embodiments, a drug combination administered in coordination with a vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (optionally a live or attenuated virus) administration comprises the commencement, optionally orally or by inhalation, of the antiviral combination before, or just before, and/or the day (day zero) the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (such as a live or attenuated virus) administration is given. For example, in alternative embodiments, the patient (or individual in need thereof) is given a pre-vaccine drug or anti-viral treatment for between about 1 to 10 days, or between about 2 to 21 days, depending on dosing and conditions. If the patient is already infected but asymptomatic, because of this pre-vaccination (or pre-administration of the attenuated and/or the live infectious causative agent) treatment the patient will be free, or substantially free, of the infection but not yet endowed with complete or partial immunity. In other words, because of this pre-vaccination treatment there will be no virus, or substantially no virus, to replicate in vivo after the anti-viral treatment. After administration of the anti-microbial drug or treatment (optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days after a first anti-microbial drug or treatment is first administered) the vaccine is given (depending on the type of vaccine, this may be the first of a two or three injection process). In alternative embodiments, after the first dose of the vaccine or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is given, the patient is treated at day 14 (or, optionally, on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) with an antibiotic or antiviral, for example, with a single preventative ivermectin and antibiotic drug combination (for example, using an antibiotic with anti-viral activity such as doxycycline or azithromycin), or ivermectin and doxycycline drug combination, or ivermectin and doxycycline and zinc drug combination, or ivermectin and azithromycin and zinc or any zinc salt, or any of these combinations with additional drugs or agent or adjuncts such as one or more vitamins, for example, vitamin B, C and/or D. In alternative embodiments, the drug combination administration is repeated every 14 days (or, optionally, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 14 or more days) for several weeks until plasma ivermectin is detectable over the 14 days. In alternative embodiments, later, the drug combination administration is repeated every 1, 2, 3, 4, 5 or 6 weeks or more. In alternative embodiments, a second dose of the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, and optionally subsequent boosters, are carried out between the intermittent (for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or up to 28 days) anti-microbial (for example, anti-viral) doses. In alternative embodiments, combining vaccine or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection with initial then intermittent anti-microbial (for example, anti-viral) administrations as provided herein achieves:virtual 100%, or substantial (for example, 95% or more) infectious agent (for example, COVID-19) abolition or in vivo clearance, which can be achieved by the anti-microbial (for example, anti-viral) pre-vaccination treatment arm of methods as provided herein;prolonged immunity, which can be achieved by administration of combined vaccine and intermittent anti-microbial (for example, anti-viral) treatments as provided herein;lack of novel virus replication in the patient, which can be achieved by the anti-microbial (for example, anti-viral) pre-vaccination treatment arm of methods as provided herein;no in vivo infectious agent (for example, virus) mutation in treated patients, as there is no or substantially no (for example, 90% to 99% reduction in) replication;no ‘Long Covid Syndrome’, because if there is no infectious agent (or substantially no infectious agent) remaining in the patient in vivo, then can be no “Long Covid Syndrome”;no or minimal hospitalization, and no or substantially decreased number of deaths from Covid-19, because if there is no active in vivo infection there can be no progression to morbidity or mortality;inability or substantial decrease in risk for patients treated using the combination methods as provided herein catch or be re-infected with any strain nor any mutant strain, where patients administered methods as provided herein are induced to have combined immunity (by administration of a vaccine) and an anti-viral response induced by administration of a drug or drugs which are viral mutant agonists;eradication of primary viral (for example, COVID-19) infection, because patients administered methods as provided herein receive anti-microbial (for example, anti-viral) treatment at the beginning of therapy; and/or,ideal long-term preventative therapy for the elderly with senescent immunity by supporting waning antibody levels in the elderly patient with anti-microbial (for example, anti-viral) treatment as provided herein; and also in embodiments where ivermectin is administered, having the added benefit of rosacea improvement and prevention of scabies in aged-care facilities. Increasing the dose of the intermittent ivermectin combination increases its anti-Covid-19 preventative power. In alternative embodiments, the dose is raised from between about 12 mg to about 36 mg, about 48 mg or about 60 mg, or the dose is raised progressively to 120 mg with few if any adverse effects. This will create a more prolonged circulating level. This is expected to be close to 100% at 4 weeks, but when combined with the vaccine could well prevent for up to 6 weeks or more. Hence, creating the possibility of reducing dosing to ×7/year. Given the use of accompanying anti-viral drugs, even if the vaccine results in lower circulation of neutralizing antibody levels and so immunity, the risk of vaccine breakthrough infection will be minimal if at all possible. Hence, this combination of anti-viral treatment together with the vaccine would be ideal therapy for prevention of infection in the elderly population with senescent immune systems. In alternative embodiments, any vaccine will benefit from practicing methods as provided herein, particularly the mRNA vaccines, which will benefit profoundly when combined with an effective anti-viral treatment. In alternative embodiments, methods as provided herein comprise 1 to 10 days of treatment with an ivermectin-based (or ivermectin-comprising) combination, followed by (the first dose of) vaccination, and then every 1, 2, 3, 4, 5, 6 or 7 days, and later every 8, 9, 10, 11, 12, 13 or 14 days (for example, every 7 days, then every 14 days), and later to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days (for example, every 7 days, then every 14 days, and later every 28 days), administer a higher dose of ivermectin, for example, 60 mg ivermectin for 4 weeks together with zinc or a zinc salt, doxycycline, vitamin D and vitamin C or other appropriate combinations. As long as one continues the anti-viral treatment based on the 60 mg ivermectin regimen, a vaccinated patient has both circulating antibodies for many months and cannot catch mutated virus (for example, COVID-19 agents), and therefore “vaccine breakthrough” will be prevented or substantially decreased and super infection with mutants will be prevented or substantially decreased. In alternative embodiments, methods as provided herein, including for example the ivermectin, zinc or a zinc salt and doxycycline and optionally also an adjunct therapy (such as for example administering a vitamin such as vitamin C or D) is mutant agnostic. In alternative embodiments, because methods as provided herein, including for example the ivermectin combination therapy, functions and works using a different mechanism by prevention of replication within a cell, no mutants can affect its activity as has been shown by us in clinical practice in California, United States. Hence, the combination of an anti-viral with a vaccine as provided herein may be the best method of terminating the Covid-19 pandemic. In alternative embodiments, the vaccination or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection administration may need to be repeated, for example, repeated at 6 or 12 month (or between about 1 (monthly) to 12 month) intervals, but it is of no great importance whether it is 6 months or 12 months because the second arm or the therapy as provided herein, the anti-viral arm, is on board to prevent any further infection and therefore any further mutation in vivo in the patient. In alternative embodiments, even if a mutant or variant strain becomes the predominant viral agent in a community in the future necessitating that the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection be adjusted (or changed) to take in (be specific for) that new mutant or variant strain, the drug combination as provided herein, for example, the ivermectin, zinc or a zinc salt and doxycycline plus adjunct treatment, can remain the same as it is mutant agnostic. Hence, any vaccine produced by any institution can be supplemented with an anti-viral combination such that no individuals will catch any viral strains once the individual has begun this program (commenced receiving treatment regimens as provided herein. The concept of ‘redundancy’ of treatment is significant here; in medicine, redundant treatment is one where the medication carries extra power to cure the condition. In the event that the treatment had to be terminated early (for example, due to an allergy developing) the built-in redundancy still delivers near (substantially) 100% cure because it was designed to carry extra power. Hence, the combination of the anti-viral treatment and vaccination as provided herein carries a high level of redundancy and thus can achieve a close to 100% success rate (or a substantially completely successfully cure rate). Although alternative embodiments of methods as provided herein are best suited to prevent and treat a virus such as a coronavirus such as a Covid-19 infection, alternative embodiments have multiple other applications. For example, in alternative embodiments, with appropriate antivirals methods as provided herein are used to prevent influenza infections. In alternative embodiments, provided are methods comprising a treatment regimen of an influenza (or other viral) vaccine followed later by antiviral agents intermittently in once a week, 2 weekly, 3 weekly, 4 weekly or less frequently spaced intervals to prevent influenza mutants from re-infecting de novo susceptible elderly patients with senescent immune system where influenza infection causes the most mortality. Other exemplary antiviral combinations administered practicing methods as provided herein comprise use of hydroxychloroquine, for example, hydroxychloroquine, azithromycin and zinc or a zinc salt. In alternative embodiments, provided are methods comprising a treatment regimen for treating: dengue fever, Zika, HIV, hepatitis C, Ebola disease, SARS, MERS, polio, measles, chickenpox and other viral or retro-viral infections. Where current immunizations may not be adequately effective, the follow-on with intermittent antiviral therapy as provide by methods as provided herein gives extra power for the poor immune response combined with antivirals to have enough redundancy to make it clinically effective. In alternative embodiments, provided are methods comprising use of antiviral compounds used singly or in multiple combinations, for example, antiviral compounds are administered singly or in multiple combinations, for example, before, at the time of vaccination, and/or after vaccination: For example, in alternative embodiments, methods provided herein comprise administering in coordination with (optionally before, at the time of vaccination, and/or after vaccination of) an anti-microbial vaccine a single drug or a therapeutic combination of drugs, or a single drug, a pharmaceutical dosage form, a drug delivery device, or a product of manufacture, or the methods can comprise use of: one, two or more classes of antiviral drugs used against influenza, such as: M2 protein inhibitors (for example, amantadine and rimantadine); neuraminidase inhibitors (for example, oseltamivir, zanamivir, peramivir and laninamivir); favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™ Glenmark Pharmaceuticals); a 5- to 6-membered heterocyclic ring such as benzene, naphthalene, furan, benzofuran, pyrrole, pyridine, pyrazole, imidazole, benzimidazole, triazole, tetrazole, oxazole, oxadiazole, 1,3,5-triazine, thiazole, thiophene, benzothiophene, pyrazine, pyridazine, pyrimidine, indole, purine, quinoline or isoquinoline; amantadine; rimantadine; oseltamivir; zanamivir; peramivir; laninamivir; laninamivir octanoate hydrate; arbidol; ribavirin; stachyflin; ingavirin; fludase; a niclosamide compound; an emricasan compound; nitazoxanide; tizoxanide; and/or a compound selected from consisting of teriflunomide, hydroxocobalamin, ensulizole, tenonitrozole, isoliquiritigenin, nitazoxanide, febuxostat, leflunomide, fidofludimus SB-366791, emodin, diphenyl isophthalate, benzoylpas, fenobam, indobufen, 2-(2H-Benzotriazol-2-yl)-4-methylphenol, tiaprofenic acid, flufenamic acid, vitamin B12, cinanserin, 5-nitro-2-(3-phenylpropylamino)benzoic acid, veliflapon, thiabendazole, SIB 1893, anethole trithione, naringenin, phenazopyridine, fanetizole, terazosin, diacerein, CAY10505, hesperetin, suprofen, ketorolac tromethamine, piperine, pirarubicin, piraxostat, albendazole oxide, tyrphostin AG 494, genistin, fenbufen, apatinib, RITA, BF-170 hydrochloride, OSI-930, tribromsalan, pifexole, formononetin, ebselen, tranilast, benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, baicalein, nemorubicin, rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine (MPEP), 5,7-dihydroxyflavone, vitamin B12, pipofezine, flurbiprofen axetil, 2-Amino-6-nitrobenzothiazole, nalachite green oxalate, enfenamic acid, fenaminosulf, AS-252424, phenserine, epalrestat, alizarin, dalcetrapib, SN-38, echinomycin, (S)-(+)-camptothecin, BI-2536, 10-hydroxycamptothecin, topotecan, delanzomib, volasertib, ispinesib, paclitaxel, FK-506, emetine, AVN-944, digoxin, vincristine, idarubicin, thapsigargin, lexibulin, ixazomib, cephalomannine, mitoxantrone, MLN-2238, demecolcine, vinorelbine, bardoxolone methyl, cycloheximide, actinomycin D, AZD-7762, PF-184, CHIR-124, cyanein, triptolide, KX-01, PF-477736, epirubicin, mycophenolate (mycophenolic acid), daunorubicin, PIK-75, vindesine, torin-2, floxuridine, Go-6976, OSU-03012, and a prodrug, metabolite, or derivative of any of the foregoing. In alternative embodiments the following compound (or its isomer, or stereoisomer, or enantiomer, or deuterated version, or bioisostere) is used singly or in various combinations (for example, formulated with, or administered separately) with other drug such as anti-viral drugs before, during or after vaccination or administration of a causative agent of infection: (1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide administered orally or by inhalation (or nasally), for example, as liquid, solid, powder, mist or spray, which can target a protease (such as the 3CL protease in COVID-19) and optionally has the following structure and molecular weight: This protease inhibitor (PF-07321332, or PAXLOVID™) may be used alone before and after the vaccination and/or administration of the attenuated causative agent of infection, optionally administered with ritonavir (or NORVIR™) or lopinavir, or with any of the numerous antiviral agents as provided herein. In alternative embodiments the following compounds (or their isomers, or stereoisomers, or enantiomer, or bioisostere) can be used singly or in various combinations: These compounds (PF-07304814 and/or PF-00835231) (or its isomer, or stereoisomer, or enantiomer, or deuterated version, or bioisostere) may be used alone before and after the vaccination and/or administration of the attenuated causative agent of infection, optionally administered with ritonavir (or NORVIR™) or lopinavir, or with any of the numerous antiviral agents as provided herein. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered separately, or together (for example, formulated together) as a tablet, gel, geltab or capsule, as a powder, in a liquid, in a mist or a spray, or as a lozenge. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered before, at the same time as, and/or after the vaccination. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days before, and/or on the day of, a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days after a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered both before and after a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. Another agent which can be used singly or in combination before and accompanying vaccination is 2-deoxy-D-Glucose (2-DG). In alternative embodiments, methods as provided herein comprise (or further comprise) administering in coordination with (optionally before, at the time of vaccination, and/or after vaccination of) an anti-microbial vaccine (or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection) a therapeutic combination of drugs or a single drug, a pharmaceutical dosage form, a drug delivery device, or a product of manufacture, comprising:(a) a thiazolide class drug, optionally nitazoxanide (or ALINIA™, NIZONIDE™) or tizoxanide (or 2-Hydroxy-N-(5-nitro-2-thiazolyl)benzamide);(b) molnupiravir, optionally co-administered with and/or formulated with an avermectin class drug (optionally ivermectin), an antibiotic (optionally doxycycline or azithromycin) and/or zinc, or co-administered with and/or formulated with ivermectin, hydroxychloroquine, an antibiotic (optionally doxycycline or azithromycin) and/or zinc; (c) opaganib or YELIVA™, or opaganib or YELIVA™ and oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate, amodiaquine (or AMDAQUINE™, AMOBIN™) and/or hydroxychloroquine (optionally, PLAQUENIL™), wherein optionally each or both of the opaganib and the chloroquine (or ARALEN™) chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) are in or formulated as a formulation for inhalation, for example, formulated as an aerosol, spray, mist, liquid or powder, or each or both are formulated for oral, intramuscular or intravenous administration,wherein optionally the opaganib is administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc (optionally zinc sulfate, optionally at (50 mg daily, or any zinc salt);(d) lopinavir, ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(e) lopinavir combined (formulated) with ritonavir (or NORVIR™), or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™, and/or zanamivir (or RELENZA™), or lopinavir and ritonavir separately formulated;(f) lopinavir combined (formulated) with ritonavir (or NORVIR™) (or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™), or lopinavir and ritonavir (or NORVIR™), and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™), optionally also with inhaled or aerosol formulations or versions of chloroquine (or ARALEN™) amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(g) lopinavir, ritonavir (or NORVIR™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine and oseltamivir (or TAMIFLU™); wherein optionally the chloroquine comprises inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(H) lopinavir and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(i) ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(j) remdesivir (optionally, GS-5734™, Gilead Sciences) alone, or oseltamivir (optionally, TAMIFLU™) and remdesivir (optionally, GS-5734™, Gilead Sciences), and optionally the remdesivir is an oral formulation and/or an inhaled or aerosol remdesivir formulation;(k) oseltamivir (optionally, TAMIFLU™) and efavirenz (optionally, SUSTIVA™), and/or zanamivir (or RELENZA™);(l) oseltamivir (optionally, TAMIFLU™) and nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™);(m) oseltamivir (or TAMIFLU™) and amprenavir (optionally, AGENERASE™);(n) oseltamivir (optionally, TAMIFLU™) and nelfinavir (optionally, VIRACEPT™);(o) a thiazolide class drug, optionally nitazoxanide (optionally ALINIA™ NIZONIDE™) or tizoxanide (or 2-Hydroxy-N-(5-nitro-2-thiazolyl)benzamide), with or in combination with any of (a) to (nn), or any drug or drug combination as provided herein, optionally a thiazolide class drug, optionally nitazoxanide, with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; or a thiazolide class drug (optionally, nitazoxanide or tizoxanide) and oseltamivir (or TAMIFLU™),and optionally the thiazolide class drug (optionally, nitazoxanide or tizoxanide) is formulated or administered with ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), and an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(p) plitidepsin (also known as dehydrodidemnin B), or APLIDIN™ (PharmaMar, S. A.);(q) an inhibitor or S-phase kinase-associated protein 2 (SKP2), or dioscin, or niclosamide, or NICLOCIDE™, FENASAL™, or PHENASAL™(r) ritonavir (or NORVIR™), ribavirin or tribavirin (or COPEGUS™ REBETOL™, or VIRAZOLE™), interferon beta 1 b, or a combination of ribavirin and interferon beta, or a combination of lopinavir and ritonavir (or NORVIR™) and interferon-beta-1b;(s) a nucleoside analog reverse-transcriptase inhibitor (NRTI) (optionally abacavir, or ZIAGEN™), acyclovir or aciclovir (optionally ZOVIRAX™), adefovir (optionally HEPSERA™), amantadine(optionally GOCOVRI™, SYMADINE™, SYMMETREL™), rintatolimod (or AMPLIGEN™), amprenavir (optionally, AGENERASE™), aprepitant (or EMEND™), umifenovir (or ARBIDOL™) atazanavir (or REYATAZ™), atazanavir (or REYATAZ™), tenofovir, a combination of efavirenz and emtricitabine and tenofovir (or ATRIPLA™), balavir, baloxavir marboxil (XOFLUZA™), bepotastine, bevirimat, bictegravir, a combination of bictegravir and emtricitabine and tenofovir alafenamide (or BIKTARVY™), brilacidin, bivalirudin (or BIVALITROBAN™), cidofovir, caspofungin, lamivudine and zidovudine (optionally, COMBVIR™), cobicstat, colisitin, cocaine, darunavir, delavirdine, descovy, didanosine, docosanol, dolutegravir, ecoliever, edoxudine, efavirenz (optionally, SUSTIVA™), elvitegravir, emtricitabine, enfuvirtide, foscarnet, fosfonet, galidesivir, ibacitabine, icatibant, idoxuridine, ifenprodil, imiquimod, imunovir, indinavir, inosine, an interferon (optionally interferon type I, interferon type II and/or interferon type III), lamivudine (or EPIVIR™, ZEFFIX™) lopinavir, loviride, ledipasvir, leronlimab, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide (optionally ALINIA™, NIZONIDE™), norvir, a nucleoside analogue or derivative (optionally brincidofovir (or TEMBEXA™), didanosine (or VIDEX™), favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™ Glenmark Pharmaceuticals), vidarabine, galidesivir (optionally, BCX4430, IMMUCILLIN-A™), remdesivir (optionally, GS-5734™, Gilead Sciences), cytarabine, gemcitabine, emtricitabine, lamivudine, zalcitabine, entecavir, stavudine, telbivudine, zidovudine, idoxuridine and/or trifluridine or any combination thereof), oseltamivir (or TAMIFLU™), peginterferon alfa-2a, penciclovir, peramivir (optionally, RAPIVAB™), perfenazine, pleconaril, plurifloxacin, podophyllotoxin, pyramidine, raltegravir, rifampicin, ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), rilpivirine, rimantadine, ritonavir (or NORVIR™) saquinavir, sofosbuvir, stavudine, telaprevir, tegobuv, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadinc, truvada, valaciclovir (optionally, VALTREX™), valganciclovir, valrubicin, vapreotide, vicriviroc, vidarabine, viramidine, velpatasvir, vivecon, zalcitabine, zanamivir (optionally, RELENZA™), zidovudine, an immunosuppressive drug (optionally tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™) or any combination thereof;(t) an mucolytic therapy or drug, optionally acetylcysteine, ambroxol, bromhexine (or BISOLVON™), carbocisteine, erdosteine, mecysteine or dornase alfa, or an expectorant, optionally guaifenesin;(u) a viral, or a coronavirus or a COVID-19, protease inhibitor, optionally ASC09 (CAS registry no. 1000287-05-7) (Janssen Research and Development, LLC), ritonavir (or NORVIR™) or ASC09 and ritonavir (or NORVIR™), or a JAK1/2 inhibitor (optionally baricitinib), optionally compound 11r(University of Lubeck, Germany, see optionally, Zhang et al J. Med Chem 2020, Feb. 11, 2020), or darunavir, cobicistat or darunavir and cobicistat;(v) an angiotensin-converting enzyme 2 (ACE2) inhibitor, optionally to block the site of viral spike protein interaction for anti-SARS-CoV-2 infection control;(w) an anti-vascular endothelial growth factor (VEGF) (optionally VEGF-A) drug or antibody, optionally bevacizumab;(x) a protease inhibitor, optionally danoprevir, optionally a serine protease inhibitor, optionally camostat or narlaprevir (optionally ARLANSA™);(y) anti-PD-1 checkpoint inhibitor, optionally camrelizumab;(z) a compound or antibody capable of binding complement factor C5 and blocking membrane attack complex formation, optionally eculizumab;(aa) a cathepsin inhibitor, optionally a cathepsin K, B or L inhibitor, optionally relacatib;(bb) thalidomide, or thalidomide and glucocorticoid (optionally ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™)) (optionally low-dose glucocorticoid), or and thalidomide and celecoxib; (cc) an antibacterial antibiotic or a macrolide drug,wherein optionally the macrolide drug comprises azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended- or delayed-release formulation of azithromycin, or ZMAX™), clarithromycin (optionally, BIAXIN™), erythromycin (optionally, ERYTHROCIN™), or fidaxomicin (optionally, DIFICID™ or DIFICLIR™), troleandomycin (optionally, TEKMISIN™), tylosin (optionally, TYLOCINE™ or TYLAN™), solithromycin (optionally, SOLITHERA™), oleandomycin (or SIGMAMYCINE™), midecamycin, roxithromycin, kitasamycin or turimycin, josamycin, carbomycin or magnamycin, and/or spiramycin,and optionally the antibacterial antibiotic comprises a tetracycline class drug, a glycylcycline or a fluorocycline class drug, or an analogue thereof, and optionally the tetracycline, glycylcycline or fluorocycline drug or analogue thereof comprises or is: tetracycline or SUMYCIN™; chlortetracycline or AUREOMYCIN™; oxytetracycline; demeclocycline or DECLOMYCIN™, DECLOSTATIN™, LEDERMYCIN™, BIOTERCICLIN™, DEGANOL™, DETECLO™, DETRAVIS™, MECICLIN™, MEXOCINE™, CLORTETRIN™; lymecycline; meclocycline; metacycline; minocycline or MINOCIN™; rolitetracycline; doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™; tigecycline or TYGACIL™; eravacycline or XERAVA™; sarecycline or SEYSARA™; omadacycline or NUZYRA™; or any combination thereof,and optionally the antibacterial antibiotic or macrolide drug, optionally azithromycin (or ZMAX™), is administered in combination with, and/or is combined with, chloroquine (or ARALEN™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), and the combination is administered commencing on the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth day of therapy, or is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days, or for between about 1 to 21 days or longer, or is administered until within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days of ending the therapy for treating, preventing, ameliorating, slowing the progress of, decreasing the severity of or preventing the coronavirus infection,and optionally the chloroquine (or ARALEN™), chloroquine phosphate, amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered the entire length of the treatment but the azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended-release formulation of azithromycin, or ZMAX™) administration is halted or ceased after two, three, four, five or six days after treatment is commenced, and optionally the azithromycin administration is replaced by a tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™ administration,and optionally the antibacterial antibiotic, optionally azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day,and optionally an oral extended-release formulation of azithromycin, or ZMAX™), is administered or formulated with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or cholecalciferol (vitamin D3) or calcifediol,and optionally the antibacterial antibiotic comprises an antimycobacterial drug, and optionally the antimycobacterial drug comprises clofazimine (optionally LAMPRENE™);(dd) an avermectin class drug such as ivermectin (optionally STROMECTOL™, SOOLANTRA™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally dosaged and/or administered at about 5 microgram/kg to about 1 gram (g) per day, optionally formulated or administered at about 1 to 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200, 220 or 240 mg per day, or between about 1 to 240 mg per day, or between about 3 to 240 mg per day,optionally formulated or administered with an antibiotic (optionally azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline, and optionally the doxycycline is at between about 25 to 600 mg per dose or per day, or at about 100 mg per dose or per day, and optionally the azithromycin is at between about 50 mg to 2000 mg per dose or per day), optionally as a single or a divided dose, and optionally formulated and administered as an inhalant or a mist (optionally using a nebulizer, nasal spray or equivalent), optionally formulated as an aerosol, spray, mist, liquid or powder, optionally formulated as an aerosol, spray, mist, liquid or powder,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated with and/or administered with chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) with or without zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt), and optionally this combination is administered weekly, or every two week, or one every 5 to 28 days, as a prophylactic,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone in the morning (AM), and an antibiotic (optionally doxycycline) and/or a chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered in the afternoon and/or evening,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days, followed by administration of an antibiotic (optionally doxycycline) for a corresponding period of days, and optionally repeating the cycle of dosaging,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated or administered with:(i) at least one antibiotic (wherein optionally the antibiotic is doxycycline(optionally, DORYX™, DOXYHEXA™, DOXYLIN™) (optionally formulated or administered at a dosage of between about 25 mg to 600 mg per dose or per day), or azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day, optionally an oral extended-release formulation of azithromycin, or ZMAX™) (optionally formulated or administered at a dosage of between an about 50 mg to 2000 mg);(ii) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) (optionally formulated or administered at a dosage of between an about 10 mg to 2000 mg per day);(iii) a zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally formulated or administered at a dosage of between about 1 mg to 250 mg; and/or(iv) at least one vitamin, and optionally the at least one vitamin comprises: vitamin C optionally formulated or administered at a dosage of between about 500 to 5000 units (U) per dose, and/or Vitamin D (or cholecalciferol) optionally formulated or administered at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered or formulated alone or in combination with any of the above (i) to (iv) (for example, at least one antibiotic, chloroquine (or ARALEN™) chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), zinc or any zinc salt and/or at least one vitamin are formulated (and administered) as oral formulations (for example, as tablets, capsules, gels or geltabs), injectable formulations, powders (for example, for inhalation or for addition to an ingestible liquid) or liquids (for example, for ingestion, infusion or injection);(ee) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) alone or with (or formulated with) or in combination with any of (a) to (bb), or chloroquine, chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and oseltamivir (or TAMIFLU™);(ff) chloroquine (optionally, ARALEN™), chloroquine phosphate, alone or with:(i) an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally at a dosage of between about 3 to 340 mg per day, or about 6 mg to 60 mg, or about 10 mg to 80 mg dosages, or about 12 to 50 mg dosages;(ii) vitamin D, vitamin D2 (or ergocalciferol), vitamin D3 (or cholecalciferol) optionally at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day, and/or(iii) with (i) and (ii) and zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally at a dosage of between about 1 mg to 250 mg, or (iv) the combination of (iii) also with a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™, optionally dosages at between about 25 mg to 600 mg per day or per dose, optionally between about 100 mg to 500 mg, or a between about 200 mg to 400 mg per dose or per day;(gg) colchicine, or COLCRYS™, MITIGARE™, optionally administered or dosaged at between about 0.5 mg to 20 mg, or about 1 mg to 15 mg, or about 3 mg to 10 mg, or about 4 mg to 6 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(hh) a corticosteroid or glucocorticoid class drug such as ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™), budesonide (optionally RHINOCORT™ or PULMICORT™), prednisolone (or ORAPRED™), methyl-prednisolone, prednisone (or DELTASONE™ or ORASONE™) or hydrocortisone (or CORTEF™), wherein optionally the corticosteroid or glucocorticoid class drug (optionally ciclesonide) is inhaled,or a selective estrogen receptor modulator (SERM), or toremifene (or FARESTON™), or clomifene or clomiphene (or CLOMID™, SEROPHENE™) wherein optionally the SERM is inhaled;and optionally the corticosteroid class drug (for example budesonide) is administered by inhalation, for example, in a nebulized form, for example, between about 1 mg to 12 mg per day of budesonide is administered by inhalation, or between about 6 to 80 mg per day of prednisolone is administered orally, or between about 6 to 100 mg per day of prednisone is administered orally, or between about 30 to 400 mg per day of hydrocortisone is administered orally,and optionally the corticosteroid class drug is formulated as a powder or for administration in an inhaler or by nasal spray, or for rectal administration,and optionally the corticosteroid class drug (for example, budesonide) is administered together with or in combination with 10 mg to 80 mg, an antibiotic (optionally azithromycin or a tetracycline class drug,wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™), zinc or any zinc salt and/or a vitamin (optionally vitamin D or calcifediol, D2 (or ergocalciferol), D3 (or cholecalciferol), C, E, B12, B6);(ii) an anti-androgen drug, and optionally the anti-androgen drug is bicalutamide, optionally CASODEX™, or dutasteride (or AVODART™),and optionally the anti-androgen drug is a nonsteroidal anti-androgen (NSAA) or an androgen receptor (AR) antagonist, and optionally the NSAA or AR antagonist comprises proxalutamide (or its developmental name GT-0918) (Suzhou Kintor Pharmaceuticals, Inc., a subsidiary of Kintor Pharmaceutical Limited), or flutamide (or niftolide, or EULEXIN™), or bicalutamide (or CASODEX™) or enzalutamide (or XTANDI™),and optionally the anti-androgen drug comprises a 5α-reductase inhibitor, and optionally the 5α-reductase inhibitor comprises finasteride (or PROSCAR™, PROPECIA™, or FINIDE™)and optionally the anti-androgen drug, or NSAA, or proxalutamide or bicalutamide, is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, eprinomectin or abamectin;and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered at dosages of about 50 to 100 mg optionally administered once, twice (BID), three times (TID) or four times a day, or is administered at dosages of about 50 to 100 mg per day,and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with an avermectin class drug, or ivermectin, optionally also administered with hydroxychloroquine, zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day) or vitamin C, B or A;and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(jj) a hydrocortisone or cortisol (optionally CORTEF™, SOLUCORTEF™), optionally hydrocortisone sodium succinate or hydrocortisone acetate or dexamethasome (optionally DEXTENZA™, OZURDEX™, NEOFORDEX™);(kk) an alpha-ketoamide (α-ketoamide), wherein optionally the alpha-ketoamide is a structure as described by Zhang et al, J. Med. Chem. 2020, 63, 9, 4562-4578, or Meng et al Chem. Sci. (2019) vol. 10, pg 5156 (optionally the structure KAM-2),and optionally the alpha-ketoamide is formulated or administered as an inhalant or a powder or mist, and optionally formulated or administered with (optionally as an inhalant): an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; an antibiotic (optionally azithromycin or a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™); chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™); zinc or any zinc salt; remdesivir (optionally, GS-5734™, Gilead Sciences); oseltamivir (or TAMIFLU™); and/or, hydrocortisone; or, any combination thereof;(ll) a compound, drug or formulation that decreases stomach acid production or decreases stomach pH, wherein optionally the compound, drug or formulation comprises famotidine, or PEPCID™, and optionally the famotidine is administered at a dosage of between about 10 to 60 mg per day, or between about 20 to 40 mg per day, and optionally the famotidine is administered is administered with: an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or a tetracycline tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™;(mm) a dendrimer, optionally astodrimer sodium (Starpharma, Melbourne, Australia);(nn) an antihistamine class drug such as azelastine, or ASTELIN™, OPTIVAR™, ALLERGODIL™, brompheniramine, fexofenadine or ALLEGRA™ pheniramine or AVIL™, or chlorpheniramine;(oo) a selective serotonin reuptake inhibitor (SSRI) class drug, optionally fluvoxamine, or LUVOX™, FAVERIN™, FLUVOXIN™;(pp) a nicotinic antagonist, a dopamine agonist or a noncompetitive N-Methyl-d-aspartic acid or N-Methyl-d-aspartate (NMDA) antagonist, wherein optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is 1-adamantylamine or amantadine, or GOCOVRI™, SYMADINE™, SYMMETREL™, optionally administered or dosaged at between about 50 mg to 150 mg, or about 100 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily), and optionally the amantadine is formulated or administered at 100 mg per day for the first two days of treatment, which optionally can then be elevated to 100 mg twice daily, optionally for the next 10 days;(qq) an immunosuppressive drug, wherein optionally the immunosuppressive drug comprises tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™, or a calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin), or NEORAL™, or SANDIMMUNE™, or tacrolimus, or PROTOPIC™, or PROGRAF™, and optionally the immunosuppressive drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally the calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin) is formulated combination of CNI (optionally cyclosporine) at a dose of 3 mg/kg (180 mg daily) together with 12 mg ivermectin once, and optionally also plus zinc 50 mg base and doxycycline 100 mg bid, optionally all for 10 days;(rr) a protein kinase inhibitor, wherein optionally the protein kinase inhibitor is a p38 mitogen-activated protein kinase inhibitor, or ralimetinib, and optionally the protein kinase inhibitor is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(ss) an anti-inflammatory therapy or at least one anti-inflammatory therapy drug, wherein optionally the anti-inflammatory therapy or drug comprises: a sphingosine kinase-2 (SK2) selective inhibitor (optionally, opaganib (optionally, YELIVA™), sirolimus, a JAK1/2/TYK2 inhibitor (optionally ruxolitinib), an anti-CD47 mAb (optionally meplazumab), a cyclooxygenase (COX) (optionally, COX2) inhibitor, a glucocorticoid (optionally a synthetic glucocorticoid, hydrocortisone, dexamethasone (or DEXTENZA™, OZURDEX™, or NEOFORDEX™) or cortisol, or CORTEF™), plitidepsin or dehydrodidemnin B, or APLIDIN™, or a nonsteroidal anti-inflammatory drug (NSAID), wherein optionally the NSAID comprises indomethacin (or indomethacin) or INDOCID™ or INDOCIN™, or naproxen, or NAPROSYN™ or ALEVE™, or a cyclooxygenase inhibitor, or a COX-1 or an COX-2 inhibitor, or aspirin, or ibuprofen or ADVIL™, MOTRIN™ or NUROFEN™, or celecoxib or CELEBREX™, or parecoxib or DYNASTAT™, or etoricoxib or ARCOXIA™,and optionally the anti-inflammatory therapy or anti-inflammatory therapy drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally opaganib, or YELIVA™, or opaganib, or YELIVA™ administered or formulated together with an oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™),and optionally the opaganib or YELIVA™ is formulated or administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(tt) a calcium channel blocker, or verapamil (or ISOPTIN™, CALAN™), or a voltage gated potassium (KCNH2) channel or a voltage gated calcium channel (CACNA2D2) blocker, or amiodarone (or CORDARONE™, NEXTERONE™);(uu) suramin, or ANTRYPOL™, BAYER 305™, or GERMANIN™;(vv) a peroxisome proliferator-activated receptor (PPAR) agonist, wherein optionally the PPAR agonist comprises fenofibrate, or TRICOR™, FENOBRAT™, FENOGLIDE™, or LIPOFEN™, or a combination of fenofibrate and simvastatin, or CHOLIB™, optionally the PPAR agonist comprises a combination of fenofibrate and pravastatin, or PRAVAFENIX™, or the PPAR agonist comprises bezafibrate, or BEZALIP™, or combination of bezafibrate and chenodeoxycholic acid, or HEPACONDA™, or aluminium clofibrate, or alfibrate, or ciprofibrate, or clinofibrate or LIPOCLIN™, or clofibrate or ATROMID-S™, or clofibride, or gemfibrozil or LOPID™, or ronifibrate, or simfibrate or CHOLESOLVIN™, or any combination thereof,(ww) a synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or a prodrug of N4-hydroxycytidine, optionally molnuvpiravir (Merck), or favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals),wherein the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is given as between about 10 mg to 3 gm per dose, or between about 10 mg to 3 gm per day, or can be dosed either as a single dose or given one, two, three or four times a day, or is administered at 200 to 800 mg twice daily, or 200, 400, 600 or 800 mg twice daily, or at 200 to 800 mg three times a day, or at 200, 400, 600 or 800 mg three times a day, or is administered at 200 to 800 mg three times a day for between about 2 to 15 days, or for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, and optionally when combined with other drugs a lower dosage is used, optionally administered at 100 or 200 mg three times a day for between about 5 to 15 days, or for about 7, 8, 9, 10, 11 or 12 days,and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin) with an antibiotic, and optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), and optionally the synthetic nucleoside analog or derivative, avermectin class drug, and antibiotic are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally molnuvpiravir, ivermectin and hydroxychloroquine are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally the synthetic nucleoside analog or derivative (optionally N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir), and antibiotic (optionally doxycycline or hydroxychloroquine) is administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an antibiotic (optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), optionally also administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D,and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with colchicine (or COLCRYS™, MITIGARE™), and optionally also zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day), or vitamin C, B or A),and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with an antibiotic (optionally azithromycin or doxycycline), and optionally also zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day), or vitamin C, B or A), and optionally also with hydroxychloroquine;(xx) an anti-malarial drug, wherein optionally the anti-malarial drug comprises mefloquine (or LARIAM™, MEPHAQUIN™, or MEFLIAM™);(yy) an antisera or an antibody or antibody or vaccine therapy for treating, preventing or ameliorating a microbial or a viral infection (optionally a coronavirus infection, optionally a COVID-19 infection) or a microbial infection (optionally a protozoan, helminthiasis, insect and/or parasitic infection), and optionally the antibody comprises a monoclonal antibody, and optionally the monoclonal antibody comprises sotrovimab (GlaxoSmithKline and Vir Biotechnology), or casirivimab, imdevimab or casirivimab and imdevimab (REGEN-COV™) (Regeneron), or bamlanivimab oretesevimab or bamlanivimab and etesevimab (Junshi Biosciences), or tocilizumab or ACTEMRA™ or ROACTEMRA™ (Hoffmann-La Roche), and optionally the vaccine comprises tozinamera or COMIRNATY™ (Pfizer), or elasomeran or SPIKEVAX™ (Moderna), or SPUTNIK V™ or Gam-COVID-Vac (Gamaleya Research Institute), or AZD1222 or COVISHIELD™ or VAXZEVRIA™ (Oxford-AstraZeneca),and optionally the antibody or antibody therapy comprises or is contained in a convalescent sera or plasma, and/or(zz) any combination of (a) to (yy), and optionally any of these combinations is administered very 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more days for between about 1 month and one year or more,and optionally any one or several or all of (a) to (zz) with an (or formulated with or formulated as an) inhaled or aerosol formulation such as a powder, spray or a mist or aerosol, and/or formulated with or formulated as an oral, intramuscular (IM) or intravenous (IV) formulation, wherein optionally both the inhaled (or aerosol) and the oral, IV and/or IM formulations are administered simultaneously or sequentially,and optionally the inhaled or aerosol formulation comprises chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) administered simultaneously or overlapping,and optionally the inhaled or aerosol formulation comprises an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally any one or several or all of (a) to (zz), or any therapeutic combination of drugs or a drug, or a pharmaceutical dosage form as provided herein, are administered orally, intramuscularly, subcutaneously, topically, by use of an enema, intravaginally, or intravenously, or administration is by subcutaneous administration, sublingual administration, inhalation or by aerosol (optionally by inhalation of a liquid, an aerosol, a spray, a mist or a powder), by absorbable patch, by use of an implant, or by use of an enema or a suppository. In alternative embodiments, the anti-viral drug or medication, or anti-microbial drug, is or comprises: molnupiravir, efavirenz (optionally, SUSTIVA™), tenofovir, emtricitabine and tenofovir, nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™), amprenavir (optionally, AGENERASE™), nelfinavir (optionally, VIRACEPT™) and/or remdesivir (optionally, GS-5734™, Gilead Sciences), a viral RNA-dependent RNA polymerase inhibitor, optionally galidesivir, a nucleoside analog reverse-transcriptase inhibitor (NRTI) (optionally abacavir, or ZIAGEN™),and optionally the anti-viral drug or medication is or comprises an anti-retroviral drug or drug combination, and optionally the anti-retroviral drug or drug combination comprises: darunavir and cobicistat (optionally, REZOLSTA™ or PREZCOBIX™); atazanavir and cobicistat (or EVOTAZ™); abacavir, lamivudine and dolutegravir (TRIUMEQ™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (optionally, STRIBILD™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (COMPLERA™ or EVIPLERA™); molnupiravir, efavirenz (optionally, SUSTIVA™), emtricitabine and tenofovir (ATRIPLA); lamivudine, nevirapine and stavudine (optionally, TRIOMUNE™); atazanavir and cobicistat (optionally, EVOTAZ™); lamivudine and raltegravir (optionally, DUTREBIS™); lamivudine and dolutegravir (or DOVATO™); doravirine, lamivudine and tenofovir (optionally, DELSTRIGO™); or lamivudine, zidovudine and nevirapine (optionally, CUOVIR-N™);and optionally the additional anti-viral drug or medication, or anti-microbial drug, is formulated with the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir or is formulated separately from the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir,and optionally the anti-viral drug or medication, or anti-microbial drug, or palliative agent comprises or further comprises: magnesium (Mg, optionally administer intravenously (IV) to maintain a blood concentration of between about 2.0 and 2.4 mmol/1); zinc or any zinc salt (optionally a zinc sulphate, acetate, gluconate or picolinate, optionally administered at about 75 to 100 mg/day or at a dosage of between about 1 mg to 250 mg); at least one vitamin, wherein optionally the at least one vitamin comprises vitamin K, vitamin D or calcifediol (optionally D2 (or ergocalciferol) or Vitamin D3 or cholecalciferol), optionally administered at about 1000 to 4000 ugm/day), vitamin B6 (or pyridoxine), vitamin B12, vitamin E, and/or vitamin C (optionally administered at 500 mg bid); a flavonoid, plant flavonol or quercetin optionally administered at between about 250 to 500 mg bid; atorvastatin, or LIPITOR™, SORTIS™ (optionally administered at between about 40 mg/day to 80 mg/day); or, melatonin, or CIRCADIN™, SLENYTO™ (optionally between about 6 to 12 mg a day, optionally, at night), any of which are optionally given enterally or parenterally. Anti-Clotting or Blood Thinning Agents In alternative embodiments for practicing methods as provided herein, to address the possibility of blood clotting, whether the blood clotting is caused by the infectious agent, the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, and/or the vaccine or for any another reason, an anti-clotting or anti-coagulant or blood thinning drug or agent is also administered, for example, before and/or at the commencement of the vaccination, and optionally is continued for between about 1 to 2 or 1 to 6 weeks after the vaccination, or for the duration of the anti-microbial drug treatment though administration of a second or booster vaccination, and/or for between about 1 to 2 weeks after administration of the second or booster vaccination. In alternative embodiments, the anti-clotting agent or anti-coagulant or blood thinning drug or agent comprises aspirin, for example between about 100 mg to 500 mg aspirin administered (for example, in the morning, or AM, or MANE) for 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days commencing on the day of the vaccination or commencing one or two days before the vaccination day. In alternative embodiments, antiplatelet drugs that can be used include clopidogrel (PLAVIX™), prasugrel (EFFIENT™) and ticagrelor (BRILINTA™). In alternative embodiments, the anti-clotting or anti-coagulant agent or blood thinning drug or agent comprises: heparin; warfarin (or COUMADIN™); a coumarin; phenprocoumon (or MARCUMAR™); rivaroxaban (XARELTO™); dabigatran (PRADAXA™); apixaban (ELIQUIS™); edoxaban (LIXIANA™) and/or betrixaban (BEVYXXA™). Vaccines In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection by administration of an antibiotic and/or an anti-viral drugs and a vaccine directed to a causative agent of the infection, and/or an inactivated or attenuated causative agent of the infection, or a live, viable or infectious causative agent of the infection. In alternative embodiments, vaccines used to practice methods as provided herein are directed to an exterior-expressed protein of a pathogen, for example, where the pathogen is a bacteria or a virus, for example, the exterior-expressed protein comprises a spike protein of a virus, for example, a spike protein of a coronavirus, for example, a Covid-19 spike protein. In alternative embodiments, vaccines used to practice methods as provided herein are formulated and administered using any formulations, protocols or techniques known in the art, for example, pharmaceutical formulations or vaccines as provided herein can be administered as peptides, or can be administered in the form of nucleic acids that encode the immunogenic peptides or proteins. In alternative embodiments, vaccines used to practice methods as provided herein comprise orally and intra-nasally administered vaccines. In alternative embodiments, vaccines used to practice methods as provided herein comprise administration of inactivated pathogen, for example, an inactivated virus (optionally an inactivated whole or entire pathogen (or virus) or substantially a whole or entire pathogen (or virus), for example, an inactivated coronavirus, for example, and inactivated COVID-19 virus, for example, as manufactured by Valneva, France), Sinopharm, or Bharat Biotech. In alternative embodiments, the pathogen (or virus) is inactivated using a chemical, for example, a beta-propiolactone (BPL) or equivalent, or any means used to inactivate a viruses for a vaccine. This type of inactivation can preserve the structure of the pathogen (for example, viral) proteins, as they would occur in nature. This means the immune system will be presented with something similar to what occurs naturally and mount a strong immune response. In alternative embodiments, after being inactivated, the vaccine (or, the inactivated pathogen, or virus) is highly purified. In alternative embodiments, an adjuvant (or any immune stimulant) is added or co-administered to induce a boosted or strong immune response. In alternative embodiments, vaccines used to practice methods as provided herein are DNA vaccine or RNA vaccines. For example, in alternative embodiments the immunogen-encoding nucleic acid can be a DNA encoding one or more immunogenic peptides or proteins, and the DNA can be carried in an expression vehicle such as a viral vector, for example an adenovirus vector such as an Ad5 or adeno-associated vector (AAV). In alternative embodiments, recombinant adenoviruses as used in vaccines as provided herein can be as described in U.S. patent application no. US 20200399323 A1, which describes for example recombinant adenoviruses including a deletion in or of the E1 region or any deletion that renders the virus replication-defective, for example, the replication-defective virus can include a deletion in one or more of the E1, E3, and/or E4 regions; or, can be as described in U.S. patent application no. US 20190382793 A1, which described how to make recombinant adenoviruses for gene therapy. In alternative embodiments, the immunogen-encoding nucleic acid can be an RNA, for example, mRNA, which can be formulated in a lipid formulation or a liposome and injected for example intramuscularly (IM), for example using formulations and methods as described in U.S. patent application no. US 20210046173 A1, which describes delivering to a subject (for example, via intramuscular administration) an immunogenic composition that comprises a RNA (for example, mRNA) that comprises an open reading frame (ORF) that comprises (or consists of, or consists essentially of) an immunogenic or antigenic sequence as provided herein; wherein optionally the RNA (or the DNA-carrying expression vehicle) is formulated in a liposome, or a lipid nanoparticle (LNP), or nanoliposome, that comprises: non-cationic lipids comprise a mixture of cholesterol and DSPC, or a PEG-lipid, or PEG-modified lipid, or LNP, or an ionizable cationic lipid; or a mixture of (13Z,16Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, cholesterol, DSPC, and PEG-2000 DMG. In alternative embodiments, the PEG-lipid is 1,2-Dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), or, the PEG-lipid is PEG coupled to dimyristoylglycerol (PEG-DMG). In alternative embodiments, the LNP comprises 20-99.8 mole % ionizable cationic lipids, 0.1-65 mole % non-cationic lipids, and 0.1-20 mole % PEG-lipid. In alternative embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of (2S)-1-({6-[(3))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9 Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing. In alternative embodiments, the PEG modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In alternative embodiments, the ionizable cationic lipid comprises: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy) heptadecanedioate (L319), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine. In one embodiment, the lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine or N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, each of which are described in PCT/US2011/052328, the entire contents of which are hereby incorporated by reference. In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. Attenuated, or Live, Viable or Infectious Causative Agent of the Infection In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection, comprising administering to a subject or an individual in need thereof:(a) at least one antibiotic and/or anti-viral drug capable of killing a causative agent of the infection, or completely or partially inhibiting the ability of the causative agent of the infection to replicate or become infectious or cause pathology in the subject or the individual in need thereof; and,(b) (i) at least one dose of a vaccine directed to the causative agent of the infection upon entry into the vaccinated subject or individual in need thereof,wherein the vaccine is capable of initiating an immune response in the individual that can substantially or partially kill or neutralize a causative agent of the infection, or the vaccine can completely, substantially or partially inhibit the ability of the causative agent of the infection to replicate, or be infectious, or cause pathology, in the subject or the individual in need thereof, and/or(ii) an inactivated or attenuated causative agent of the infection, or a live, viable or infectious causative agent of the infection, wherein optionally the live causative agent of the infection is a completely or partially attenuated version of the causative agent,wherein at least one dosage of the at least one antibiotic and/or anti-viral drug is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days before, or on the day of, a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection. In alternative embodiments, the causative agent of the infection is or comprises a bacteria, protozoan or a virus, orthe causative agent of the infection is or comprises the causative agent of:a viral infection, optionally a coronavirus, a virus that causes a common cold, an influenza virus (optionally an influenza A, B or C), a hepatitis virus, a respiratory syncytial virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus, a viral meningitis, a rhinovirus, a human immunodeficiency virus (HIV), a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EBV), an Adenovirus, a Hantavirus, a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection,a coronavirus infection, optionally a COVID-19 or a COVID-19 variant infection, or a Middle East respiratory syndrome virus (MERS-CoV) infection;malaria caused by a parasite of the genusPlasmodium(optionallyP. vivax, P. falciparum, P. malariae, P. ovale, orP. knowlesi);dengue fever or dengue shock syndrome caused by a virus of the Flaviviridae family or a dengue virus;a Flaviviridae family virus infection or a hepatitis or a hepatocellular carcinoma associated with viral hepatitis caused by a virus of the Flaviviridae family or a virus of the genus Hepacivirus or Hepacivirus C virus or hepatitis C;filariasis, leprosy or streptocerciasis or an infection caused by a parasite of the superfamily Filarioidea (optionallyBrugia malayi, Brugia timori, Wuchereria bancrofti, Loa loa, Mansonella streptocerca, Mansonella ozzardi, orMansonellaperstans);leprosy or an infection caused by a parasite of the genusMycobacterium(optionallyM. lepraeor M. lepromatosis);river blindness or onchocerciasis caused by a parasitic worm or a parasite of the genusOnchocerca(optionallyO. volvulus);a hookworm or a roundworm infection caused by a parasite of the genusAncylostoma(optionallyA. duodenaleorA. ceylanicum) or Necator (optionallyN. americanus);trichuriasis or a whipworm infection caused by a parasite of the genusTrichuris(optionallyT. trichiura); roundworm or anAscarisinfection that is caused byAscaris lumbricoides;scabies or a mite-carried infection caused by the parasite of the genusSarcoptes(optionallyS. scabiei);typhus or an infection caused by a lice or a parasite of the order Phthiraptera (optionallyPediculus humanuscapitis);enterobiasis or an infection caused by a pinworm or a parasite of the genusEnterobius(optionallyE. vermicularis); and/orpulicosis or an infection caused by a flea or an insect of the order Siphonaptera or of the genusPulex(optionallyP. irritans). In alternative embodiments, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is administered orally or by inhalation. Alternatively, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection can be administered by inclusion of the live, viable or infectious causative agent of the infection in a liquid (optionally to be administered as a drink or in drops such as nasal drops), a tablet, a lozenge, an aerosol, spray, or mist formulation that is inhaled or administered nasally or orally (optionally, by a puffer of a nebulizer), or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is formulated in a liquid (optionally the liquid is a sterile saline) solution which is ingested or gargled by the individual in need thereof. In alternative embodiments, the source of the inactivated or attenuated causative agent of the infection, or the administered live, viable or infectious causative agent of the infection can be from an infected individual, such as a human patient, a domesticated, wild or lab animal, or from a lab-grown culture. In alternative embodiments, the source of the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is from swab or sputum or other biological samples from an infected individual or patient. In alternative embodiments, the sputum or other biological sample from an infected individual or patient is diluted in a water or a saline prior to administrations. In alternative embodiments, the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is attenuated (in other words, its ability to cause pathogenesis is completely, substantially or partially abrogated or diminished, for example is genetically deleted or diminished by genomic engineering). In alternative embodiments, to generate an attenuated (e.g., completed inactivated) version of a causative agent of the infection to be administered, the causative agent of the infection is passaged multiple times in culture (or in vitro) or in an animal (or in vivo), where variants from each passage are selected for a phenotype and/or genotype that has diminished ability to cause pathogenesis. In alternative embodiments, to generate an attenuated (e.g., completed inactivated, completely non-infectious) version of a causative agent of the infection to be administered, the causative agent of the infection is treated with radiation and/or a chemical. For example, the chemical can be iodine (for example, povidone-iodine or PVP-I, also known as iodopovidone, or BETADINE™, WOKADINE™, PYODINE™), or any complex of polyvinylpyrrolidone and iodine, alcohol and/or formalin. In alternative embodiments, the causative agent of the infection is rendered inactive, or non-infectious, by exposing the causative agent of the infection to iodine or povidone-iodine or PVP-I (povidone is also known as polyvinylpyrrolidone (PVP), or 1-vinyl-2-pyrrolidinon-polymere), also known as iodopovidone, or BETADINE™, WOKADINE™, PYODINE™, as in the production of nasodine (Firebrick Pharma Pty Ltd, Australia). Povidone-iodine is a chemical complex of povidone, hydrogen iodide, and elemental iodine or triiodide (I3−); and it contains 10% povidone, with total iodine species equaling 10,000 ppm or 1% total titratable iodine, and it works by releasing iodine which results in the death of a range of microorganisms. In alternative embodiment, the causative agent of infection is mixed with PVP-I and water, ethyl alcohol, isopropyl alcohol, polyethylene glycol or glycerol. In alternative embodiments, the attenuated, or inactivated, causative agent, or live causative agent, is administered with an adjuvant, where the adjuvant can comprise: an inorganic compound such as alum (e.g., potassium alum), an aluminium salt or aluminium hydroxide, aluminium phosphate, or calcium phosphate; an oil such as paraffin oil, propolis or Adjuvant 65; a bacterial product such as killed bacteria of the genusBordetellaorMycobacteriumor of the speciesBordetella pertussisorMycobacterium bovis; a plant saponin or soybean extract; a cytokine such as interleukin-1 (IL-1), IL-2 or IL-12; Freund's complete adjuvant or Freund's incomplete adjuvant; and/or, an organic compound such as squalene. In alternative embodiments, the attenuated, or inactivated, causative agent, or live causative agent, with or without an adjuvant, is administered by nasal spray or nebulizer, or orally for example by lozenge, tablet, capsule or geltab, or by subcutaneous injection, or intramuscularly (IM), or by suppository, or via an implant. In alternative embodiments, attenuated viruses are made using a live attenuated codon-pair-deoptimized virus approach as described for example in Wang et al PNAS, Jul. 20, 2021, vol. 18 (29) e2102775118; or as described by Coleman et al. Science 320, 1784-1787 (2008), or Cheng et al J. Virol. 89, 3523-3533 (2015), or Gonsalves-Carneiro, mBio 12, e02238-20 (2021). For example, methods as provided herein comprise administration of the Wang et al, COVI-VAC™ attenuated virus, which was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. As described by Wang et al, synthetic highly attenuated live vaccine is generated by recoding portions of the WT SARS-CoV-2 genome according to the SAVE algorithm of codon-pair bias deoptimization. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. Attenuated viruses can be generated from viral genomes recover from WT SARS-CoV-2, strain USA-WA1/2020 (GenBank accession No. MN985325). In alternative embodiments, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, is administered in unit dosages of between about 10 to 50, or 1 to 20, trillion infectious units (or particles, if attenuated), or between about one infectious unit to 10, 20 or 30 billion infection units (or particles, if attenuated). Hand-Held or Portable Devices In alternative embodiments, provided are portable, for example, hand-held (or worn around the neck), medical devices, for example, an inhaler, ionizer, asthma puffer or nebulizer, capable of administering an inhalation product comprising a composition or formulation as provided herein or as described herein, for example, an inactivated or attenuated agent of the infection, or a live, viable or infectious causative agent of the infection with or without a vaccine or with or without an adjuvant, or with or without an antimicrobial drug, for example, as described herein. In alternative embodiments, a portable, for example, hand-held, medical device, for example, inhaler, asthma puffer or nebulizer, as provided herein can administer ionized air or air comprising generated electrons and/or negatively-charged oxygen ions and/or positively-charged ions. In alternative embodiments, a portable or hand-held medical device as provided herein comprises a cassette, packette, interchangeable disk (for example, for holding a powder) or reservoir (optionally a refillable reservoir) in or on the product of manufacture, or a removable cassette or packette, interchangeable disk (for example, for holding a powder) that can be inserted into a slot or port on the product of manufacture, or a separate reservoir or container operatively linked or joined to the product of manufacture, that comprises a vaccine or or live or attenuated causative agent of invention or a formulation or a medication, for inhalation as provided herein for delivery to a user. In alternative embodiments, provided is a modified hairdryer-type medical device capable of having an adjustable temperature, adjustable air intake; a provision (or receptacle) for insertion of a drug-containing cassette (optionally providing or delivering a combination of medications, or the live or attenuated causative agents and/or a vaccine as provided herein); and/or warm-to hot air availability (optionally with temperature control) to inhibit viral and bacterial growth. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection and/or a vaccine as provided herein drug or a medication or combinations thereof to a user is fabricated as a meter-dose inhaler (MDI) (either open or closed mouth MDI), which can comprise a pressurized canister of the drug or medication in a plastic case with a mouthpiece, and a holding chamber having a plastic tube with a mouthpiece, a valve to control mist delivery and a soft sealed end to hold the MDI; the holding chamber can assist delivery of the drug or medication to the nose and/or lungs, for example, as an AEROCHAMBER™ device. In alternative embodiments, the inhaler or nebulizer is breath activated, for example, as an REDIHALER™ device. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection and/or a vaccine or a drug or a medication or combinations thereof to a user is fabricated a dry powder inhaler (such as a dry powder disk inhaler, for example, as a DISKUS™ device), optionally having a dose counter window so user can see how many doses are left), for example, where the powder is dose dispensed by (using) a disposable, refillable or replaceable cassette, packette or disk; and the dry powder dispensing can be breath activated, for example, as an AEROLIZER™, FLEXHALER™, PRESSAIR™, DISKUS™, HANDIHALER™, TWISTHALER™, ELLIPTA™, NEOHALER™, RESPICLICK™, ROTAHALER™ or TUBUHALER™ device. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection, or a vaccine or a drug or a medication or combinations thereof, to a user is fabricated a nebulizer or soft mist inhaler, which can comprise a nebulizer delivery system comprising a nebulizer (for example, a small plastic bowl with a screw-top lid) and a source for compressed air to generate a mist comprising the drug or medication, which also can be dose dispensed using a disposable, refillable or replaceable cassette, packette or disk. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection, or a vaccine or drug or a medication or combinations thereof to a user is fabricated a dry powder inhaler (such as a dry powder disk inhaler, for example, as a DISKUS™ device), optionally having a dose counter window so user can see how many doses are left), for example, where the powder is dose dispensed by (using) a disposable, refillable or replaceable cassette, packette or disk; and the dry powder dispensing can be breath activated, for example, as an AEROLIZER™, FLEXHALER™, PRESSAIR™, DISKUS™, HANDIHALER™, TWISTHALER™, ELLIPTA™, NEOHALER™, RESPICLICK™, ROTAHALER™ or TUBUHALER™ device. Products of Manufacture and Kits Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein. Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections. As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court. Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. RELATED APPLICATIONS This U.S. Utility Patent application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. (USSN) 63/186,660, filed May 10, 2021; U.S. Ser. No. 63/188,311, May 13, 2021; U.S. Ser. No. 63/214,997, Jun. 25, 2021; U.S. Ser. No. 63/223,427, Jul. 19, 2021; U.S. Ser. No. 63/241,485 filed Sep. 7, 2021; U.S. Ser. No. 63/253,813 filed Oct. 8, 2021; and, U.S. Ser. No. 63/273,069, filed Oct. 28, 2021. The aforementioned applications are expressly incorporated herein by reference in its entirety and for all purposes. TECHNICAL FIELD This invention generally relates to medicine and the medical treatment of infections with vaccines, antibiotics and anti-viral drugs. In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection by administration of an antibiotic and/or an anti-viral drugs and a vaccine directed to a causative agent of the infection and/or an inactivated or attenuated causative agent of the infection, or a live, viable or infectious causative agent of the infection. In alternative embodiments, the infection is bacterial, parasitic or viral. In alternative embodiments, the viral infection is a coronavirus infection such a Covid-19 or Covid-19 variant infection. In alternative embodiments, methods as provided herein prevent or decrease the prevalence or severity of “vaccine breakthrough infections” after vaccination, where mutants of the infection's causative agent develop and infect patients in spite of the fact that they have undergone immunization, for example, to prevent a Covid-19 infection. In alternative embodiments, methods as provided herein are used to prevent in vivo mutations of an infectious agent to enhance to efficacy of an administered vaccination; in other word, methods as provided herein are used to prevent in vivo replication of a viral infectious agent, and thus also prevents mutations of the viral infectious agent because where there is no replication of infectious agent there is no mutation of infectious agent. In alternative embodiments, methods comprising administration of a combination of an antiviral medication, drug or treatment such as PF-07321332 or PAXLOVID™ and/or ritonavir and a vaccine prevents acquisition of externally mutated viruses infecting the vaccinated person, as well as preventing replication in those possessing less effective neutralising antibodies to the mutants. BACKGROUND The process of immunization attempts to create immunity to prevent acquisition of the new coronavirus or viruses. However, there is ongoing mutation occurring in the surrounding population because of ongoing replication of viruses. Hence, “vaccine breakthrough” infections are becoming reported, for example, see Hacisuleyman et al., New Eng J Med, Apr. 21, 2021. If the vaccine creates immunity to only the strains it was created for, it may permit ‘reinfection’ with Covid-19 of a mutant strain inhaled by the immunized person. This is termed “Vaccine breakthrough’. Within the immunized person replication of an external infectious mutant can take place because the vaccine alone is inadequate to control the mutant replicating and the mutant strain may further mutate in this seemingly safely immunized subject. With COVID-19, there has been a rush into development of vaccines that could prevent the disease. However, the nature of this infection, being an intracellular RNA type of virus, does not result in an easy development of a vaccine. There are vaccines which work, for example, small pox, tetanus, polio, or measles, and there are conditions where vaccines are of little use, for example, hepatitis C, human immunodeficiency virus (HIV) infection and tuberculosis (TB), and then there are vaccines which only give partial immunization and for a short period of time, such as for example influenza virus immunization and more recently malaria immunization attempts. Currently used mRNA vaccines as well as non-mRNA vaccines are manufactured by different companies such as Pfizer, AstraZeneca, Moderna, Johnson and Johnson (Janssen), Sputnik V, NovaVax, Sinovac, Sinopharm, Biological E, Valneva, EpiVac Corona, Convidiciae, Covaxin and others. All have been subject to various local side effects such as a swollen arm, systemic side effects such as fever, aches and pains, overwhelming feeling of doom, discomfort, profound tiredness and other symptoms. A small percentage of patients develop thrombocytopenia and clotting which is reminiscent of disseminated intravascular coagulation as described by some, as well as neurologic, dermatologic, cardiac and other adverse effects. The preventative success of mRNA vaccines has been reported to be up to 90% or more. However, in real life experience data in some countries has shown results of 50% to 90% efficacy. Some of these reduced efficacy levels may be due to mutants being present in that population. Unless vaccine manufacturers can predict the development of specific mutations, which is virtually impossible, the vaccine market will always suffer from such reduced efficacy in various regions of the world. Because of the ongoing mutations around the world of COVID-19, there are multiple strains, including a current India strain, which has caused super-infections in patients who have been immunized, i.e., the so-called vaccine breakthrough phenomenon. The B.1.617 variant of the Covid-19, known more commonly as the double mutant strain, was first detected in India in October 2020. As the name suggests, the strain involves two variants of the virus. The E484Q mutation has characteristics of a previously detected variant—the E484K—which was seen in the fast-spreading Brazilian and South African variants, making it highly transmissible. The L452R mutation, on the other hand, helps the virus evade the body's immune response. The double mutation strain was subsequently named B.1.617 Yet it is clear that the virus had to replicate to mutate. Hence the adage “if it cannot replicate it cannot mutate”. Hence, one simple solution to the problem of imperfect vaccine disease prevention is not a bigger and better vaccine or multiple vaccinations because we will never keep up with the mutations. The best solution may well be prevention of mutations. Yet it is clear that the virus had to replicate to mutate. And subsequently replicate in the immunized person. Hence the adage “if it cannot replicate it cannot mutate”. SUMMARY In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection, comprising administering to a subject or an individual in need thereof, wherein optionally the individual in need thereof is a human or an animal:(a) at least one antibiotic and/or anti-viral drug capable of killing a causative agent of the infection, or completely or partially inhibiting the ability of the causative agent of the infection to replicate or become infectious or cause pathology in the subject or the individual in need thereof; and,(b) (i) at least one dose of a vaccine directed to the causative agent of the infection upon entry into the vaccinated subject or individual in need thereof,wherein the vaccine is capable of initiating an immune response in the individual that can substantially or partially kill or neutralize a causative agent of the infection, or the vaccine can completely, substantially or partially inhibit the ability of the causative agent of the infection to replicate, or be infectious, or cause pathology, in the subject or the individual in need thereof, and/or(ii) an inactivated or attenuated agent of the infection, or a live, viable or infectious causative agent of the infection, wherein optionally the live causative agent of the infection is a completely or partially attenuated version of the causative agent,wherein at least one dosage of the at least one antibiotic and/or anti-viral drug is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days before, or on the day of, a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection. In alternative embodiments, the causative agent of the infection is or comprises a bacteria, protozoan or a virus, or the causative agent of the infection is or comprises the causative agent of:a viral infection, optionally a coronavirus, a virus that causes a common cold, an influenza virus (optionally an influenza A, B or C), a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus, a viral meningitis, a rhinovirus, a human immunodeficiency virus (HIV), a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EBV), an Adenovirus, a Hantavirus, a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection,a coronavirus infection, optionally a COVID-19 or a COVID-19 variant infection, or a Middle East respiratory syndrome virus (MERS-CoV) infection;malaria caused by a parasite of the genusPlasmodium(optionallyP. vivax, P. falciparum, P. malariae, P. ovale, orP. knowlesi);dengue fever or dengue shock syndrome caused by a virus of the Flaviviridae family or a dengue virus;a Flaviviridae family virus infection or a hepatitis or a hepatocellular carcinoma associated with viral hepatitis caused by a virus of the Flaviviridae family or a virus of the genus Hepacivirus or Hepacivirus C virus or hepatitis C;filariasis, leprosy or streptocerciasis or an infection caused by a parasite of the superfamily Filarioidea (optionallyBrugia malayi, Brugia timori, Wuchereria bancrofti, Loa loa, Mansonella streptocerca, Mansonella ozzardi, orMansonellaperstans);leprosy or an infection caused by a parasite of the genusMycobacterium(optionallyM. lepraeor M. lepromatosis);river blindness or onchocerciasis caused by a parasitic worm or a parasite of the genusOnchocerca(optionallyO. volvulus);a hookworm or a roundworm infection caused by a parasite of the genusAncylostoma(optionallyA. duodenaleorA. ceylanicum) or Necator (optionallyN. americanus);trichuriasis or a whipworm infection caused by a parasite of the genusTrichuris(optionallyT. trichiura); roundworm or anAscarisinfection that is caused byAscaris lumbricoides;scabies or a mite-carried infection caused by the parasite of the genusSarcoptes(optionallyS. scabiei);typhus or an infection caused by a lice or a parasite of the order Phthiraptera (optionallyPediculus humanuscapitis);enterobiasis or an infection caused by a pinworm or a parasite of the genusEnterobius(optionallyE. vermicularis); and/orpulicosis or an infection caused by a flea or an insect of the order Siphonaptera or of the genusPulex(optionallyP. irritans). In alternative embodiments, the virus is an influenza virus or a coronavirus, optionally a COVID-19 virus. In alternative embodiments, the at least one antibiotic and/or anti-viral drug comprises: an avermectin class drug (optionally ivermectin) alone; a combination of an avermectin class drug and an antibiotic, or a combination of an ivermectin and an antibiotic or an antiviral drug or therapeutic, optionally a combination of an avermectin class drug, an antibiotic and zinc or a zinc salt, or a combination of ivermectin and an antibiotic and zinc or a zinc salt. In alternative embodiments, the avermectin class drug comprises: ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin. In alternative embodiments, the avermectin class drug is administered with a synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or a prodrug of N4-hydroxycytidine, optionally molnuvpiravir (Merck), or favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals). In alternative embodiments, the antibiotic comprises an antibacterial antibiotic or a macrolide drug,wherein optionally the macrolide drug comprises azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended- or delayed-release formulation of azithromycin, or ZMAX™), clarithromycin (optionally, BIAXIN™), erythromycin (optionally, ERYTHROCIN™), or fidaxomicin (optionally, DIFICID™ or DIFICLIR™), troleandomycin (optionally, TEKMISIN™), tylosin (optionally, TYLOCINE™ or TYLAN™), solithromycin (optionally, SOLITHERA™), oleandomycin (or SIGMAMYCINE™), midecamycin, roxithromycin, kitasamycin or turimycin, josamycin, carbomycin or magnamycin, and/or spiramycin,and optionally the antibacterial antibiotic comprises a tetracycline class drug, a glycylcycline or a fluorocycline class drug, or an analogue thereof, and optionally the tetracycline, glycylcycline or fluorocycline drug or analogue thereof comprises or is: tetracycline or SUMYCIN™; chlortetracycline or AUREOMYCIN™; oxytetracycline; demeclocycline or DECLOMYCIN™, DECLOSTATIN™, LEDERMYCIN™, BIOTERCICLIN™, DEGANOL™, DETECLO™, DETRAVIS™, MECICLIN™, MEXOCINE™, CLORTETRIN™; lymecycline; meclocycline; metacycline; minocycline or MINOCIN™; rolitetracycline; doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™; tigecycline or TYGACIL™; eravacycline or XERAVA™; sarecycline or SEYSARA™; omadacycline or NUZYRA™; or any combination thereof. In alternative embodiments, the at least one antibiotic and/or anti-viral drug comprises a combination of ivermectin and doxycycline (optionally DORYX™, DOXYHEXA™, DOXYLIN™), optionally a combination of ivermectin, doxycycline and zinc or a zinc salt. In alternative embodiments, the at least one antibiotic and/or anti-viral drug comprises a combination of ivermectin and azithromycin ZITHROMAX™, or AZITHROCIN™, optionally an oral extended-release formulation of azithromycin, or ZMAX™), optionally a combination of ivermectin, azithromycin and zinc or a zinc salt. In alternative embodiments, the at least one antibiotic and/or anti-viral drug comprises a combination of hydroxychloroquine (optionally, PLAQUENIL™) and azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended-release formulation of azithromycin, or ZMAX™), optionally a combination of hydroxychloroquine, azithromycin and zinc or a zinc salt. In alternative embodiments, the at least one antibiotic and/or anti-viral drug further comprises administration of a vitamin, optionally vitamin D and/or vitamin C. In alternative embodiments, the zinc comprises: zinc sulphate, zinc acetate, zinc gluconate or zinc picolinate or a zinc salt. In alternative embodiments, on the day of administration of, or at least one day after the first dose of: (a) the attenuated and/or the live, viable or infectious causative agent of the infection, and/or (b) the vaccine, is given, the individual in need thereof is administered more of the at least one antibiotic and/or anti-viral drug, or a different combination of an least one antibiotic and/or anti-viral drug, or a different dosage of the at least one antibiotic and/or anti-viral drug. In alternative embodiments, the individual in need thereof is administered more of the at least one antibiotic and/or anti-viral drug, or a different combination of an least one antibiotic and/or anti-viral drug, or a different dosage of the at least one antibiotic and/or anti-viral drug on day zero (the day of administration), or day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 after administration of the first dosage of the vaccine and/or the attenuated and/or the live, viable or infectious causative agent of the infection. In alternative embodiments, a booster, or at least one second or follow-up administration of: at least one dosage of a vaccine and/or an attenuated and/or a live, viable or infectious causative agent of the infection, is given between about 1 week to one year (or between about two weeks to 9 months, or between about three weeks to 8 months, or between about one month to 7 months, or about 3, 4, 5, or 6 months) after the first administration of the at least one vaccine and/or the attenuated and/or the live, viable or infectious causative agent of the infection,and optionally, wherein on day zero, or at least one day, or about two days, after, administration of the second or additional or booster dose of the vaccine, and/or the attenuated and/or the live, viable or infectious causative agent of the infection, is given, the individual in need thereof is administered more of the at least one antibiotic and/or anti-viral drug, or a different combination of an least one antibiotic and/or anti-viral drug, or a different dosage of the at least one antibiotic and/or anti-viral drug. In alternative embodiments, the individual in need thereof is administered more of the at least one antibiotic and/or anti-viral drug, or a different combination of an least one antibiotic and/or anti-viral drug, or a different dosage of the at least one antibiotic and/or anti-viral drug on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 after administration of the second or additional or booster dose of the vaccine and/or the attenuated and/or the live, viable or infectious causative agent of the infection. In alternative embodiments, the at least one antibiotic and/or anti-viral drug is repeatedly administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days for one or several weeks (optionally, 2, 3, 4, 5, or 6 weeks) until plasma ivermectin is detectable. In alternative embodiments, the vaccine is: a nucleic acid-based vaccine, optionally an RNA vaccine or a DNA vaccine; a peptide or polypeptide-based vaccine; or, the vaccine comprises an inactivated virus. In alternative embodiments, the anti-bacterial and/or anti-viral drug or drug combination, or the attenuated and/or the live, viable or infectious causative agent of the infection, or the vaccine, is administered orally or by inhalation, or the the anti-bacterial and/or anti-viral drug or drug combination or the attenuated and/or the live, viable or infectious causative agent of the infection is administered by inclusion in a liquid (optionally to be administered as a drink or in drops such as nasal drops or in a mist), a tablet, a capsule, a gel, a geltab, a powder, a lozenge, an aerosol, spray, or mist formulation that is inhaled or administered nasally or orally (optionally, by a puffer or a nebulizer), or is formulated in a liquid (optionally the liquid is a sterile saline) solution which is ingested or gargled by the individual in need thereof. In alternative embodiments, the attenuated and/or the live, viable or infectious causative agent of the infection is administered in unit dosages of between about 10 to 50 trillion infectious units or particles, or between about one infectious unit or particle to 10, 20 or 30 billion infection units or particles. In alternative embodiments, after the first administration of (a) the attenuated and/or the live, viable or infectious causative agent of the infection, and/or (b) the vaccine, the IgM, IgG and/or IgA levels in the individual in need thereof is tested and measured (qualitatively and/or quantitatively), and optionally if levels of the measured IgM, IgG and/or IgA are low, a second or additional dosage or administration of the (a) the attenuated and/or the live, viable or infectious causative agent of the infection, and/or (b) the vaccine, is given,and optionally levels of the measured IgM, IgG and/or IgA are measured at between about 7 to 21 days, or at 14 and 20 days, after the first administration. In alternative embodiments, the individual in need thereof is a human or an animal, and optionally the animal is a domestic, farm or zoo animal. In alternative embodiments, the methods comprise administering in coordination with (optionally before, at the time of and/or after vaccination of and/or administration of the attenuated and/or the live, viable or infectious causative agent of the infection) the anti-microbial (optionally antiviral) vaccine and/or the attenuated and/or the live, viable or infectious causative agent of the infection, a therapeutic combination of drugs or a single drug, an antisera or an antibody, a pharmaceutical dosage form, a drug delivery device, or a product of manufacture, comprising:(a) a thiazolide class drug, optionally nitazoxanide (or ALINIA™ NIZONIDE™) or tizoxanide (or 2-Hydroxy-N-(5-nitro-2-thiazolyl)benzamide);(b) molnupiravir, optionally co-administered with and/or formulated with an avermectin class drug (optionally ivermectin), an antibiotic (optionally doxycycline or azithromycin) and/or zinc, or co-administered with and/or formulated with ivermectin, hydroxychloroquine, an antibiotic (optionally doxycycline or azithromycin) and/or zinc;(c) opaganib or YELIVA™, or opaganib or YELIVA™ and oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate, amodiaquine (or AMDAQUINE™, AMOBIN™) and/or hydroxychloroquine (optionally, PLAQUENIL™), wherein optionally each or both of the opaganib and the chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) are in or formulated as a formulation for inhalation, for example, formulated as an aerosol, spray, mist, liquid or powder, or each or both are formulated for oral, intramuscular or intravenous administration,wherein optionally the opaganib is administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc (optionally zinc sulfate, optionally at (50 mg daily, or any zinc salt);(d) lopinavir, ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(e) lopinavir combined (formulated) with ritonavir (or NORVIR™), or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™, and/or zanamivir (or RELENZA™), or lopinavir and ritonavir separately formulated;(f) lopinavir combined (formulated) with ritonavir (or NORVIR™) (or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™), or lopinavir and ritonavir (or NORVIR™), and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™), optionally also with inhaled or aerosol formulations or versions of chloroquine (or ARALEN™) amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(g) lopinavir, ritonavir (or NORVIR™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine and oseltamivir (or TAMIFLU™); wherein optionally the chloroquine comprises inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(h) lopinavir and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(i) ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(j) remdesivir (optionally, GS-5734™, Gilead Sciences) alone, or oseltamivir (optionally, TAMIFLU™) and remdesivir (optionally, GS-5734™, Gilead Sciences), and optionally the remdesivir is an oral formulation and/or an inhaled or aerosol remdesivir formulation;(k) oseltamivir (optionally, TAMIFLU™) and efavirenz (optionally, SUSTIVA™), and/or zanamivir (or RELENZA™);(l) oseltamivir (optionally, TAMIFLU™) and nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™);(m) oseltamivir (or TAMIFLU™) and amprenavir (optionally, AGENERASE™);(n) oseltamivir (optionally, TAMIFLU™) and nelfinavir (optionally, VIRACEPT™);(o) a thiazolide class drug, optionally nitazoxanide (optionally ALINIA™, NIZONIDE™) or tizoxanide (or 2-Hydroxy-N-(5-nitro-2-thiazolyl)benzamide), with or in combination with any of (a) to (hh), or any drug or drug combination as provided herein, optionally a thiazolide class drug, optionally nitazoxanide, with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; or a thiazolide class drug (optionally, nitazoxanide or tizoxanide) and oseltamivir (or TAMIFLU™),and optionally the thiazolide class drug (optionally, nitazoxanide or tizoxanide) is formulated or administered with ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), and an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(p) plitidepsin (also known as dehydrodidemnin B), or APLIDIN™ (PharmaMar, S. A.);(q) an inhibitor or S-phase kinase-associated protein 2 (SKP2), or dioscin, or niclosamide, or NICLOCIDE™, FENASAL™, or PHENASAL™;(r) ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™) interferon beta 1b, or a combination of ribavirin and interferon beta, or a combination of lopinavir and ritonavir (or NORVIR™) and interferon-beta-1b;(s) abacavir, acyclovir, or optionally ACICLOVIR™, adefovir, amantadine, ampligen, amprenavir (optionally, AGENERASE™), aprepitant, umifenovir (or ARBIDOL™), atazanavir, atripla, balavir, baloxavir marboxil (XOFLUZA™), bepotastine, bevirimat, bictegravir, a combination of bictegravir and emtricitabine and tenofovir alafenamide (or BIKTARVY™), brilacidin, bivalirudin (or BIVALITROBAN™), cidofovir, caspofungin, lamivudine and zidovudine (optionally, COMBVIR™), cobicstat, colisitin, cocaine, darunavir, delavirdine, descovy, didanosine, docosanol, dolutegravir, ecoliever, edoxudine, efavirenz (optionally, SUSTIVA™), elvitegravir, emtricitabine, enfuvirtide, entecavir, epirubicin, epoprostenol, etravirine, famciclovir, fomivirsen, fosamprenavi, foscarnet, fosfonet, galidesivir, ibacitabine, icatibant, idoxuridine, ifenprodil, imiquimod, imunovir, indinavir, inosine, an interferon (optionally interferon type I, interferon type II and/or interferon type III), lamivudine (or EPIVIR™, ZEFFIX™), lopinavir, loviride, ledipasvir, leronlimab, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide (optionally ALINIA™, NIZONIDE™), norvir, a nucleoside analogue or derivative (optionally brincidofovir (or TEMBEXA™), didanosine (or VIDEX™), favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals), vidarabine, galidesivir (optionally, BCX4430, IMMUCILLIN-A™), remdesivir (optionally, GS-5734™, Gilead Sciences), cytarabine, gemcitabine, emtricitabine, lamivudine, zalcitabine, abacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine and/or trifluridine or any combination thereof), oseltamivir (or TAMIFLU™), peginterferon alfa-2a, penciclovir, peramivir (optionally, RAPIVAB™), perfenazine, pleconaril, plurifloxacin, podophyllotoxin, pyramidine, raltegravir, rifampicin, ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), rilpivirine, rimantadine, ritonavir (or NORVIR™) saquinavir, sofosbuvir, stavudine, telaprevir, tegobuv, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir (optionally, VALTREX™), valganciclovir, valrubicin, vapreotide, vicriviroc, vidarabine, viramidine, velpatasvir, vivecon, zalcitabine, zanamivir (optionally, RELENZA™), zidovudine, an immunosuppressive drug (optionally tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™) or any combination thereof;(t) a mucolytic therapy or drug, optionally acetylcysteine, ambroxol, bromhexine (or BISOLVON™), carbocisteine, erdosteine, mecysteine or dornase alfa, or an expectorant, optionally guaifenesin;(u) a viral, or a coronavirus or a COVID-19, protease inhibitor, optionally ASC09 (CAS registry no. 1000287-05-7) (Janssen Research and Development, LLC), ritonavir (or NORVIR™) or ASC09 and ritonavir, or a JAK1/2 inhibitor (optionally baricitinib), optionally compound 11r (University of Lubeck, Germany, see optionally, Zhang et al J. Med Chem 2020, Feb. 11, 2020), or darunavir, cobicistat or darunavir and cobicistat;(v) an angiotensin-converting enzyme 2 (ACE2) inhibitor, optionally to block the site of viral spike protein interaction for anti-SARS-CoV-2 infection control;(w) an anti-vascular endothelial growth factor (VEGF) (optionally VEGF-A) drug or antibody, optionally bevacizumab;(x) a protease inhibitor, optionally danoprevir, optionally a serine protease inhibitor, optionally camostat or narlaprevir (optionally ARLANSA™);(y) anti-PD-1 checkpoint inhibitor, optionally camrelizumab;(z) a compound or antibody capable of binding complement factor C5 and blocking membrane attack complex formation, optionally eculizumab;(aa) a cathepsin inhibitor, optionally a cathepsin K, B or L inhibitor, optionally relacatib;(bb) thalidomide, or thalidomide and glucocorticoid (optionally low-dose glucocorticoid), or and thalidomide and celecoxib;(cc) an antibacterial antibiotic or a macrolide drug,wherein optionally the macrolide drug comprises azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended- or delayed-release formulation of azithromycin, or ZMAX™), clarithromycin (optionally, BIAXIN™), erythromycin (optionally, ERYTHROCIN™), or fidaxomicin (optionally, DIFICID™ or DIFICLIR™), troleandomycin (optionally, TEKMISIN™), tylosin (optionally, TYLOCINE™ or TYLAN™), solithromycin (optionally, SOLITHERA™), oleandomycin (or SIGMAMYCINE™), midecamycin, roxithromycin, kitasamycin or turimycin, josamycin, carbomycin or magnamycin, and/or spiramycin,and optionally the antibacterial antibiotic comprises a tetracycline class drug, a glycylcycline or a fluorocycline class drug, or an analogue thereof, and optionally the tetracycline, glycylcycline or fluorocycline drug or analogue thereof comprises or is: tetracycline or SUMYCIN™; chlortetracycline or AUREOMYCIN™; oxytetracycline; demeclocycline or DECLOMYCIN™, DECLOSTATIN™, LEDERMYCIN™, BIOTERCICLIN™, DEGANOL™, DETECLO™, DETRAVIS™, MECICLIN™, MEXOCINE™, CLORTETRIN™; lymecycline; meclocycline; metacycline; minocycline or MINOCIN™; rolitetracycline; doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™; tigecycline or TYGACIL™; eravacycline or XERAVA™; sarecycline or SEYSARA™; omadacycline or NUZYRA™; or any combination thereof,and optionally the antibacterial antibiotic or macrolide drug, optionally azithromycin (or ZMAX™), is administered in combination with, and/or is combined with, chloroquine (or ARALEN™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), and the combination is administered commencing on the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth day of therapy, or is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 20 or more days, or for between about 1 to 21 days or longer, or is administered until within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days of ending the therapy for treating, preventing, ameliorating, slowing the progress of, decreasing the severity of or preventing the coronavirus infection,and optionally the chloroquine (or ARALEN™), chloroquine phosphate, amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered the entire length of the treatment but the azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended-release formulation of azithromycin, or ZMAX™) administration is halted or ceased after two, three, four, five or six days after treatment is commenced, and optionally the azithromycin administration is replaced by a tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™ administration,and optionally the antibacterial antibiotic, optionally azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day,and optionally an oral extended-release formulation of azithromycin, or ZMAX™), is administered or formulated with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or cholecalciferol (vitamin D3) or calcifediol,and optionally the antibacterial antibiotic comprises an antimycobacterial drug, and optionally the antimycobacterial drug comprises clofazimine (optionally LAMPRENE™);(dd) an avermectin class drug such as ivermectin (optionally STROMECTOL™, SOOLANTRA™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally dosaged and/or administered at about 5 microgram/kg to about 1 gram (g) per day, optionally formulated or administered at about 1 to 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200, 220 or 240 mg per day, or between about 1 to 240 mg per day, or between about 3 to 240 mg per day,optionally formulated or administered with an antibiotic (optionally azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline, and optionally the doxycycline is at between about 25 to 600 mg per dose or per day, or at about 100 mg per dose or per day, and optionally the azithromycin is at between about 50 mg to 2000 mg per dose or per day), optionally as a single or a divided dose, and optionally formulated and administered as an inhalant or a mist (optionally using a nebulizer, nasal spray or equivalent), optionally formulated as an aerosol, spray, mist, liquid or powder, optionally formulated as an aerosol, spray, mist, liquid or powder,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated with and/or administered with chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) with or without zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt), and optionally this combination is administered weekly, or every two week, or one every 5 to 28 days, as a prophylactic,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone in the morning (AM), and an antibiotic (optionally doxycycline) and/or a chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered in the afternoon and/or evening,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days, followed by administration of an antibiotic (optionally doxycycline) for a corresponding period of days, and optionally repeating the cycle of dosaging,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated or administered with:(i) at least one antibiotic (wherein optionally the antibiotic is doxycycline(optionally, DORYX™, DOXYHEXA™, DOXYLIN™) (optionally formulated or administered at a dosage of between about 25 mg to 600 mg per dose or per day), or azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day, optionally an oral extended-release formulation of azithromycin, or ZMAX™) (optionally formulated or administered at a dosage of between an about 50 mg to 2000 mg);(ii) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) (optionally formulated or administered at a dosage of between an about 10 mg to 2000 mg per day);(iii) a zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally formulated or administered at a dosage of between about 1 mg to 250 mg; and/or(iv) at least one vitamin, and optionally the at least one vitamin comprises: vitamin C optionally formulated or administered at a dosage of between about 500 to 5000 units (U) per dose, and/or Vitamin D (or cholecalciferol) optionally formulated or administered at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered or formulated alone or in combination with any of the above (i) to (iv) (for example, at least one antibiotic, chloroquine (or ARALEN™) chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), zinc or any zinc salt and/or at least one vitamin are formulated (and administered) as oral formulations (for example, as tablets, capsules, powders, gels or geltabs), injectable formulations, powders (for example, for inhalation or for addition to an ingestible liquid) or liquids (for example, for ingestion, infusion or injection);(ee) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) alone or with (or formulated with) or in combination with any of (a) to (bb), or chloroquine, chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and oseltamivir (or TAMIFLU™);(ff) chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) alone or with:(i) an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally at a dosage of between about 3 to 340 mg per day, or about 6 mg to 60 mg, or about 10 mg to 80 mg dosages, or about 12 to 50 mg dosages;(ii) vitamin D, vitamin D2 (or ergocalciferol), vitamin D3 (or cholecalciferol) optionally at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day, and/or(iii) with (i) and (ii) and zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally at a dosage of between about 1 mg to 250 mg, or (iv) the combination of (iii) also with a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™, optionally dosages at between about 25 mg to 600 mg per day or per dose, optionally between about 100 mg to 500 mg, or a between about 200 mg to 400 mg per dose or per day;(gg) colchicine, or COLCRYS™, MITIGARE™, optionally administered or dosaged at between about 0.5 mg to 20 mg, or about 1 mg to 15 mg, or about 3 mg to 10 mg, or about 4 mg to 6 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(hh) a corticosteroid or glucocorticoid class drug such as ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™) budesonide (optionally RHINOCORT™ or PULMICORT™), prednisolone (or ORAPRED™), methyl-prednisolone, prednisone (or DELTASONE™ or ORASONE™) or hydrocortisone (or CORTEF™), or a selective estrogen receptor modulator (SERM), or toremifene (or FARESTON™), or clomifene or clomiphene (or CLOMID™, SEROPHENE™), wherein optionally the corticosteroid or glucocorticoid class drug (optionally ciclesonide) is inhaled;and optionally the corticosteroid class drug (for example budesonide) is administered by inhalation, for example, in a nebulized form, for example, between about 1 mg to 12 mg per day of budesonide is administered by inhalation, or between about 6 to 80 mg per day of prednisolone is administered orally, or between about 6 to 100 mg per day of prednisone is administered orally, or between about 30 to 400 mg per day of hydrocortisone is administered orally,and optionally the corticosteroid class drug is formulated as a powder or for administration in an inhaler or by nasal spray, or for rectal administration,and optionally the corticosteroid class drug (for example, budesonide) is administered together with or in combination with 10 mg to 80 mg, an antibiotic (optionally azithromycin or a tetracycline class drug,wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™), zinc or any zinc salt and/or a vitamin (optionally vitamin D or calcifediol, D2 (or ergocalciferol), D3 (or cholecalciferol), C, E, B12, B6);(ii) an anti-androgen drug, and optionally the anti-androgen drug is bicalutamide, optionally CASODEX™, or dutasteride (or AVODART™),and optionally the anti-androgen drug is a nonsteroidal anti-androgen (NSAA) or an androgen receptor (AR) antagonist, and optionally the NSAA or AR antagonist comprises proxalutamide (or its developmental name GT-0918) (Suzhou Kintor Pharmaceuticals, Inc., a subsidiary of Kintor Pharmaceutical Limited), or flutamide (or niftolide, or EULEXIN™), or bicalutamide (or CASODEX™) or enzalutamide (or XTANDI™),and optionally the anti-androgen drug comprises a 5α-reductase inhibitor, and optionally the 5α-reductase inhibitor comprises finasteride (or PROSCAR™, PROPECIA™, or FINIDE™)and optionally the anti-androgen drug, or NSAA, or proxalutamide or bicalutamide, is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered at dosages of about 50 to 100 mg optionally administered once, twice (BID), three times (TID) or four times a day, or is administered at dosages of about 50 to 100 mg per day,and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with an avermectin class drug, or ivermectin, optionally also administered with hydroxychloroquine, zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day) or vitamin C, B or A; and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(jj) a hydrocortisone or cortisol (optionally CORTEF™, SOLUCORTEF™), optionally hydrocortisone sodium succinate or hydrocortisone acetate or dexamethasome (optionally DEXTENZA™, OZURDEX™, NEOFORDEX™);(kk) an alpha-ketoamide (α-ketoamide), wherein optionally the alpha-ketoamide is a structure as described by Zhang et al, J. Med. Chem. 2020, 63, 9, 4562-4578, or Meng et al Chem. Sci. (2019) vol. 10, pg 5156 (optionally the structure KAM-2),and optionally the alpha-ketoamide is formulated or administered as an inhalant or a powder or mist, and optionally formulated or administered with (optionally as an inhalant): an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; an antibiotic (optionally azithromycin or a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™); chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™); zinc or any zinc salt; remdesivir (optionally, GS-5734™, Gilead Sciences); oseltamivir (or TAMIFLU™); and/or, hydrocortisone; or, any combination thereof;(ll) a compound, drug or formulation that decreases stomach acid production or decreases stomach pH, wherein optionally the compound, drug or formulation comprises famotidine, or PEPCID™, and optionally the famotidine is administered at a dosage of between about 10 to 60 mg per day, or between about 20 to 40 mg per day, and optionally the famotidine is administered is administered with: an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or a tetracycline tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™;(mm) a dendrimer, optionally astodrimer sodium (Starpharma, Melbourne, Australia);(nn) an antihistamine class drug such as azelastine, or ASTELIN™, OPTIVAR™, ALLERGODIL™, brompheniramine, fexofenadine or ALLEGRA™, pheniramine or AVIL™, or chlorpheniramine;(oo) a selective serotonin reuptake inhibitor (SSRI) class drug, optionally fluvoxamine, or LUVOX™, FAVERIN™, FLUVOXIN™;(pp) a nicotinic antagonist, a dopamine agonist or a noncompetitive N-Methyl-d-aspartic acid or N-Methyl-d-aspartate (NMDA) antagonist, wherein optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is 1-adamantylamine or amantadine, or GOCOVRI™, SYMADINE™, SYMMETREL™, optionally administered or dosaged at between about 50 mg to 150 mg, or about 100 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily), and optionally the amantadine is formulated or administered at 100 mg per day for the first two days of treatment, which optionally can then be elevated to 100 mg twice daily, optionally for the next 10 days;(qq) an immunosuppressive drug, wherein optionally the immunosuppressive drug comprises tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™, or a calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin), or NEORAL™, or SANDIMMUNE™, or tacrolimus, or PROTOPIC™, or PROGRAF™, and optionally the immunosuppressive drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally the calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin) is formulated combination of CNI (optionally cyclosporine) at a dose of 3 mg/kg (180 mg daily) together with 12 mg ivermectin once, and optionally also plus zinc 50 mg base and doxycycline 100 mg bid, optionally all for 10 days;(rr) a protein kinase inhibitor, wherein optionally the protein kinase inhibitor is a p38 mitogen-activated protein kinase inhibitor, or ralimetinib, and optionally the protein kinase inhibitor is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(ss) an anti-inflammatory therapy or at least one anti-inflammatory therapy drug, wherein optionally the anti-inflammatory therapy or drug comprises: a sphingosine kinase-2 (SK2) selective inhibitor (optionally, opaganib (optionally, YELIVA™), sirolimus, a JAK1/2/TYK2 inhibitor (optionally ruxolitinib), an anti-CD47 mAb (optionally meplazumab), a cyclooxygenase (COX) (optionally, COX2) inhibitor, a glucocorticoid (optionally a synthetic glucocorticoid, hydrocortisone, dexamethasone (or DEXTENZA™, OZURDEX™, or NEOFORDEX™) or cortisol, or CORTEF™) or ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™), plitidepsin or dehydrodidemnin B, or APLIDIN™, or a nonsteroidal anti-inflammatory drug (NSAID), wherein optionally the NSAID comprises indomethacin (or indomethacin) or INDOCID™ or INDOCIN™, or naproxen, or NAPROSYN™ or ALEVE™, or a cyclooxygenase inhibitor, or a COX-1 or an COX-2 inhibitor, or aspirin, or ibuprofen or ADVIL™ MOTRIN™ or NUROFEN™, or celecoxib or CELEBREX™, or parecoxib or DYNASTAT™, or etoricoxib or ARCOXIA™and optionally the anti-inflammatory therapy or anti-inflammatory therapy drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally opaganib, or YELIVA™, or opaganib, or YELIVA™ administered or formulated together with an oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™)and optionally the opaganib or YELIVA™ is formulated or administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(tt) a calcium channel blocker, or verapamil (or ISOPTIN™, CALAN™), or a voltage gated potassium (KCNH2) channel or a voltage gated calcium channel (CACNA2D2) blocker, or amiodarone (or CORDARONE™, NEXTERONE™);(uu) suramin, or ANTRYPOL™, BAYER 305™, or GERMANIN™;(vv) a peroxisome proliferator-activated receptor (PPAR) agonist, wherein optionally the PPAR agonist comprises fenofibrate, or TRICOR™, FENOBRAT™, FENOGLIDE™, or LIPOFEN™, or a combination of fenofibrate and simvastatin, or CHOLIB™, optionally the PPAR agonist comprises a combination of fenofibrate and pravastatin, or PRAVAFENIX™, or the PPAR agonist comprises bezafibrate, or BEZALIP™, or combination of bezafibrate and chenodeoxycholic acid, or HEPACONDA™, or aluminium clofibrate, or alfibrate, or ciprofibrate, or clinofibrate or LIPOCLIN™, or clofibrate or ATROMID-S™, or clofibride, or gemfibrozil or LOPID™, or ronifibrate, or simfibrate or CHOLESOLVIN™, or any combination thereof,(ww) a synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or a prodrug of N4-hydroxycytidine, optionally molnuvpiravir (Merck), or favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals),wherein the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is given as between about 10 mg to 3 gm per dose, or between about 10 mg to 3 gm per day, or can be dosed either as a single dose or given one, two, three or four times a day, or is administered at 200 to 800 mg twice daily, or 200, 400, 600 or 800 mg twice daily, or at 200 to 800 mg three times a day, or at 200, 400, 600 or 800 mg three times a day, or is administered at 200 to 800 mg three times a day for between about 2 to 15 days, or for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, and optionally when combined with other drugs a lower dosage is used, optionally administered at 100 or 200 mg three times a day for between about 5 to 15 days, or for about 7, 8, 9, 10, 11 or 12 days,and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin) with an antibiotic, and optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), and optionally the synthetic nucleoside analog or derivative, avermectin class drug, and antibiotic are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally molnuvpiravir, ivermectin and hydroxychloroquine are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally the synthetic nucleoside analog or derivative (optionally N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir), and antibiotic (optionally doxycycline or hydroxychloroquine) is administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an antibiotic (optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), optionally also administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D,and optionally any of these combinations is administered very 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more days for between about 1 month and one year or more;(xx) an antisera or an antibody or antibody or vaccine therapy for treating, preventing or ameliorating a microbial or a viral infection (optionally a coronavirus infection, optionally a COVID-19 infection) or a microbial infection (optionally a protozoan, helminthiasis, insect and/or parasitic infection), and optionally the antibody comprises a monoclonal antibody, and optionally the monoclonal antibody comprises sotrovimab (GlaxoSmithKline and Vir Biotechnology), or casirivimab, imdevimab or casirivimab and imdevimab (REGEN-COV™) (Regeneron), or bamlanivimab oretesevimab or bamlanivimab and etesevimab (Junshi Biosciences), or tocilizumab or ACTEMRA™ or ROACTEMRA™ (Hoffmann-La Roche), and optionally the vaccine comprises tozinamera or COMIRNATY™ (Pfizer), or elasomeran or SPIKEVAX™ (Moderna), or SPUTNIK V™ or Gam-COVID-Vac (Gamaleya Research Institute), or AZD1222 or COVISHIELD™ or VAXZEVRIA™ (Oxford-AstraZeneca),and optionally the antibody or antibody therapy comprises or is contained in a convalescent sera or plasma, and/or(yy) any combination of (a) to (xx),and optionally any one or several or all of (a) to (yy) with an (or formulated with or formulated as an) inhaled or aerosol formulation such as a powder or a mist or aerosol, and/or formulated with or formulated as an oral, intramuscular (IM) or intravenous (IV) formulation, wherein optionally both the inhaled (or aerosol) and the oral, IV and/or IM formulations are administered simultaneously or sequentially,and optionally the inhaled or aerosol formulation comprises chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) administered simultaneously or overlapping,and optionally the inhaled or aerosol formulation comprises an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally any one or several or all of (a) to (yy), or any therapeutic combination of drugs or a drug, or a pharmaceutical dosage form as provided herein, are administered orally, intramuscularly, subcutaneously, topically, by use of an enema, intravaginally, or intravenously, or administration is by subcutaneous administration, sublingual administration, inhalation or by aerosol (optionally by inhalation of a liquid, an aerosol, a spray, a mist or a powder), by absorbable patch, by use of an implant, or by use of an enema or a suppository. In alternative embodiments, the anti-viral drug or medication, or anti-microbial drug, is or comprises: molnupiravir, efavirenz (optionally, SUSTIVA™), tenofovir, emtricitabine and tenofovir, nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™), amprenavir (optionally, AGENERASE™), nelfinavir (optionally, VIRACEPT™) and/or remdesivir (optionally, GS-5734™, Gilead Sciences), a viral RNA-dependent RNA polymerase inhibitor, optionally galidesivir,and optionally the anti-viral drug or medication is or comprises an anti-retroviral drug or drug combination, and optionally the anti-retroviral drug or drug combination comprises: darunavir and cobicistat (optionally, REZOLSTA™ or PREZCOBIX™); atazanavir (or REYATAZ™) and cobicistat (or EVOTAZ™); a nucleoside analog reverse-transcriptase inhibitor (NRTI) (optionally abacavir, or ZIAGEN™), lamivudine and dolutegravir (TRIUMEQ™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (optionally, STRIBILD™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (COMPLERA™ or EVIPLERA™); molnupiravir, efavirenz (optionally, SUSTIVA™), emtricitabine and tenofovir (ATRIPLA); lamivudine, nevirapine and stavudine (optionally, TRIOMUNE™); atazanavir (or REYATAZ™) and cobicistat (optionally, EVOTAZ™); lamivudine and raltegravir (optionally, DUTREBIS™); lamivudine and dolutegravir (or DOVATO™); doravirine, lamivudine and tenofovir (optionally, DELSTRIGO™); or lamivudine, zidovudine and nevirapine (optionally, CUOVIR-N™);and optionally the additional anti-viral drug or medication, or anti-microbial drug, is formulated with the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir or is formulated separately from the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir,and optionally the anti-viral drug or medication, or anti-microbial drug, or palliative agent comprises or further comprises: magnesium (Mg, optionally administer intravenously (IV) to maintain a blood concentration of between about 2.0 and 2.4 mmol/1); zinc or any zinc salt (optionally a zinc sulphate, acetate, gluconate or picolinate, optionally administered at about 75 to 100 mg/day or at a dosage of between about 1 mg to 250 mg); at least one vitamin, wherein optionally the at least one vitamin comprises vitamin K, vitamin D or calcifediol (optionally D2 (or ergocalciferol) or Vitamin D3 or cholecalciferol), optionally administered at about 1000 to 4000 ugm/day), vitamin B6 (or pyridoxine), vitamin B12, vitamin E, and/or vitamin C (optionally administered at 500 mg bid); a flavonoid, plant flavonol or quercetin optionally administered at between about 250 to 500 mg bid; atorvastatin, or LIPITOR™, SORTIS™ (optionally administered at between about 40 mg/day to 80 mg/day); or, melatonin, or CIRCADIN™, SLENYTO™ (optionally between about 6 to 12 mg a day, optionally, at night), any of which are optionally given enterally or parenterally. In alternative embodiments, provided are kits comprising a vaccine and/or an attenuated and/or live causative agent of infection, and at least one antibiotic and/or anti-viral drug capable of killing a causative agent of the infection, or completely or partially inhibiting the ability of the causative agent of the infection to replicate or become infectious or cause pathology in an individual, as described herein or used in any method as provided herein, wherein optionally the kit comprises instructions for practicing a method as provided herein. In alternative embodiments of methods and kits as provided herein, the(i) 1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, or a compound having the following structure and molecular weight: or stereoisomer, or enantiomer, or deuterated version thereof, and(ii) ritonavir,are formulated together, or separately, and optionally are formulated together or separately in or as a liquid (optionally to be administered as a drink or in drops, optionally as nasal drops or in a mist), a tablet, a capsule, a gel, a geltab, a powder, a lozenge, an aerosol or spray. In alternative embodiments of methods and kits as provided herein, the anti-viral drug combination is formulated in or as a pharmaceutical dosage form, optionally formulated to be administered orally, intramuscularly, subcutaneously, topically, by use of an enema, intravaginally, or intravenously, or formulated for subcutaneous administration, sublingual administration, inhalation or by aerosol (optionally by inhalation of a liquid, an aerosol, a spray, a mist or a powder), by absorbable patch, by use of an implant, or by use of an enema or a suppository. In alternative embodiments of methods and kits as provided herein, the(a) 1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, or a compound having the following structure and molecular weight: or stereoisomer, or enantiomer, or deuterated version thereof, and/or(b) ritonavir,is or are administered:(a) at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage, or(b) at a dosage of between about 10 mg to 3 gm per dose, or between about 10 mg to 3 gm per day, or 12 mg or 3 mg/kg orally twice daily, or 125 mg orally twice daily or 520 mg/130 mg solution twice per day (optionally administered with efavirenz, fosamprenavir, nelfinavir, or nevirapine), or(c) is dosed either as a single dose or given one, two, three or four times a day, or(d) at 200 to 800 mg twice daily, or 200, 400, 600 or 800 mg twice daily, or at 200 to 800 mg three times a day, or at 200, 400, 600 or 800 mg three times a day, or is administered at 200 to 800 mg three times a day for between about 2 to 15 days, or for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days,(e) for pediatric patients dosage at 16 mg or 4 mg/kg orally twice daily, or(f) when combined with other drugs a lower dosage, optionally administered at 100 or 200 mg three times a day for between about 5 to 15 days, or for about 7, 8, 9, 10, 11 or 12 days. The details of one or more exemplary embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, and from the claims. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes. DETAILED DESCRIPTION In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection in an individual in need thereof, including humans and animals, by administration of an antibiotic and/or an anti-viral drugs and a vaccine directed to a causative agent of the infection, and/or an inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection. In alternative embodiments, the infection (or causative agent of the infection) is parasitic, bacterial or viral. In alternative embodiments, the viral infection is a coronavirus infection such a Covid-19 infection. In alternative embodiments, methods as provide herein prevent or decrease the prevalence or severity of “vaccine breakthrough infections” after vaccination, where external mutants of the infection's causative agent develop and infect or re-infect patients in spite of the fact that they have undergone immunization, for example, to prevent a Covid-19 infection. Thus, in alternative embodiments, methods as provided herein comprise combining an effective anti-microbial (for example anti-viral) treatment (for example, a drug or a mixture of drugs or other therapeutics) with an anti-microbial vaccine to prevent in vivo mutations (and thus also prevent a vaccine breakthrough infection) of an infectious agent such as virus, for example, a coronavirus such as COVID-19 or variant thereof. In alternative embodiments, methods as provide herein provide a solution to the problem of imperfect vaccine disease prevention, where a bigger and/or better vaccine or multiple vaccinations are ineffective because science will never keep up with the continuing viral mutations. In alternative embodiments, methods as provide herein prevent replication of newly-inhaled mutants in the already vaccinated or ‘about to be vaccinated’ population. In alternative embodiments, methods as provide herein comprise an added anti-replication method of treatment in addition to vaccination to protect the immunized population from mutants entering, replicating, and further mutating in the immunized population. In alternative embodiments, methods as provide herein prevent replication and thus prevent an ongoing viral mutation, a method utilized by the mutant to escape neutralizing antibodies and destruction. No replication, no mutation. In alternative embodiments, methods as provide herein comprise combining an effective anti-microbial (for example anti-viral) treatment (for example, a drug or a mixture of drugs or other therapeutics) with an anti-microbial vaccine such as a DNA vaccine such as an adenovirus-based vaccine, an mRNA vaccine, a peptide-based vaccine, an inactivated pathogen-based vaccine, and/or an vaccine manufactured by:Sanofi (optionally VAT00002 or VAT00008),GlaxoSmithKline,Takeda Pharmaceutical (optionally TAK-019),Pfizer (optionally tozinamera or COMIRNATY™),Moderna (optionally elasomeran or SPIKEVAX™)Novavax (optionally vaccine to SARS VLPs S protein and influenza M1 protein),CanSino Biologics,Inovio,Sinovac,BioNTech,Johnson and Johnson,Valneva (France) and Dynavax Technologies (optionally VLA2001 and VLA2101),Sinopharm (or China National Pharmaceutical Group Corporation),Emergent BioSolutions (optionally human polyclonal hyperimmune serum with antibodies to SARS-CoV-2),Bharat Biotech (optionally COVAXIN®),The Rockefeller University (optionally vaccine toMVA S alone, or MVA-S prime and Ad5-S boost),Helmholtz Centre for Infection Research; Technical University Munich; German Center for Environmental Health (optionally vaccine to NC protein add-mixed with MALP-2 by intranasal route and boosting with MVA-NC by intramuscular route),University of Manitoba; University of Pennsylvania School of Medicine; Southern Research Institute; Fox Chase Cancer Institute (optionally vaccine to Heterologous Adenoviral prime boost AdHu5 s AdC7-nS),University of North Carolina at Chapel Hill, USA (optionally vaccine to VEEV replicon particles expressing the SARS-CoV S),National Institute of Infectious Diseases, Japan (optionally vaccine to recombinant D1 expressing S protein),Beijing Institute of Genomics, China (optionally vaccine to Recombinant trunctuated S—N fusion protein),Saitama Medical University; Josai University; Nippon Oil and Fat Corporation; National Institute of Infectious Diseases, Japan (optionally vaccine to recombinant peptide N223 on liposomes),Chinese Center for Disease Control and Prevention; Canadian Science Centre for Human and Animal Health (optionally vaccine to Recombinant TM-truncated S protein),HKU-Pasteur Research Centre; The University of Hong Kong; National Institutes of Health; Centers for Disease (optionally vaccine to Trimeric Spike protein),Sun Yat-sen University, China (optionally vaccine to SARS S DNA prime and HLAA*0201 restricted peptides boost vaccine),State Key Laboratory of Virology; Graduate University of Chinese Academy of Sciences (optionally vaccine to or as a 3a DNA vaccine),Institute of ImmunoBiology, Shanghai Medical College of Fudan University, China (optionally vaccine to DNA prime—protein S437-459 and M1-20),CNB-CSIC; University of Iowa (optionally vaccine to rSARSCoV-E),International Vaccine Institute (IVI) (optionally vaccine to recombinant adenovirus expressing truncated S protein (rADV-S)),University Health Network, Canada, and United States Center for Disease Control and Prevention (CDC) (optionally vaccine to recombinant measles virus spike protein),Institut Pasteur (optionally vaccine to MV-SARS recombinant measles virus vaccine expressing SARS CoV antigen),Baylor College Medicine; Sabin; New York Blood Center (NYBC); University of Texas Medical Branch (UTMB); Walter Reed Army Institute of Research (WRAIR); National Institute of Allergy and Infectious Diseases (NIAID) (optionally vaccine to receptor binding domain (RBD) of the SARS-CoV spike (S) protein),Vaxine Pty Ltd, Australia (optionally vaccine to SARS recombinant spike protein plus delta inulin),Gamaleya Research Institute (optionally SPUTNIK V™ or Gam-COVID-Vac), and/orOxford-AstraZeneca (optionally AZD1222 or COVISHIELD™ or VAXZEVRIA™)and others, including anti-COVID-19 vaccines. Hence, methods as provided herein that combine antibiotic, anti-parasitic and/or anti-viral treatment with an inactivated or attenuated causative agent of an infection, or a vaccine or a live, viable or infectious causative agent of the infection (for example, a live or attenuated virus) administration, can treat, ameliorate or prevent a vaccine-breakthrough infection, as well as eradicating the infection if present pre-vaccination enhance protection and eradicate infection. In alternative embodiments, methods as provide herein comprise administering in coordination with (for example, before, during and/or after) an anti-causative agent vaccination, or an anti-viral vaccination, and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (such as a live or attenuated virus) administration, any one or combination of anti-viral, anti-parasitic or anti-bacterial therapies, for example, one or more anti-COVID-19 infection medications or drugs, including for example, the drug ivermectin, or the combination of ivermectin and an antibiotic with anti-viral properties such as doxycycline or azithromycin, for example the combination of ivermectin and doxycycline or azithromycin, or the combination ivermectin and doxycycline or azithromycin and zinc or any zinc salt (an anti-viral mineral, for example, and anti-COVID-19 mineral), which optionally also can be administered in conjunction or coordination with a vitamin or vitamins such as vitamin D and/or vitamin C. In alternative embodiments, a drug combination administered in coordination with a vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (optionally a live or attenuated virus) administration comprises the commencement, optionally orally or by inhalation, of the antiviral combination before, or just before, and/or the day (day zero) the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection (such as a live or attenuated virus) administration is given. For example, in alternative embodiments, the patient (or individual in need thereof) is given a pre-vaccine drug or anti-viral treatment for between about 1 to 10 days, or between about 2 to 21 days, depending on dosing and conditions. If the patient is already infected but asymptomatic, because of this pre-vaccination (or pre-administration of the attenuated and/or the live infectious causative agent) treatment the patient will be free, or substantially free, of the infection but not yet endowed with complete or partial immunity. In other words, because of this pre-vaccination treatment there will be no virus, or substantially no virus, to replicate in vivo after the anti-viral treatment. After administration of the anti-microbial drug or treatment (optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days after a first anti-microbial drug or treatment is first administered) the vaccine is given (depending on the type of vaccine, this may be the first of a two or three injection process). In alternative embodiments, after the first dose of the vaccine or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is given, the patient is treated at day 14 (or, optionally, on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) with an antibiotic or antiviral, for example, with a single preventative ivermectin and antibiotic drug combination (for example, using an antibiotic with anti-viral activity such as doxycycline or azithromycin), or ivermectin and doxycycline drug combination, or ivermectin and doxycycline and zinc drug combination, or ivermectin and azithromycin and zinc or any zinc salt, or any of these combinations with additional drugs or agent or adjuncts such as one or more vitamins, for example, vitamin B, C and/or D. In alternative embodiments, the drug combination administration is repeated every 14 days (or, optionally, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days) for several weeks until plasma ivermectin is detectable over the 14 days. In alternative embodiments, later, the drug combination administration is repeated every 1, 2, 3, 4, 5 or 6 weeks or more. In alternative embodiments, a second dose of the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, and optionally subsequent boosters, are carried out between the intermittent (for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or up to 28 days) anti-microbial (for example, anti-viral) doses. In alternative embodiments, combining vaccine or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection with initial then intermittent anti-microbial (for example, anti-viral) administrations as provided herein achieves:virtual 100%, or substantial (for example, 95% or more) infectious agent (for example, COVID-19) abolition or in vivo clearance, which can be achieved by the anti-microbial (for example, anti-viral) pre-vaccination treatment arm of methods as provided herein;prolonged immunity, which can be achieved by administration of combined vaccine and intermittent anti-microbial (for example, anti-viral) treatments as provided herein;lack of novel virus replication in the patient, which can be achieved by the anti-microbial (for example, anti-viral) pre-vaccination treatment arm of methods as provided herein;no in vivo infectious agent (for example, virus) mutation in treated patients, as there is no or substantially no (for example, 90% to 99% reduction in) replication;no ‘Long Covid Syndrome’, because if there is no infectious agent (or substantially no infectious agent) remaining in the patient in vivo, then can be no “Long Covid Syndrome”;no or minimal hospitalization, and no or substantially decreased number of deaths from Covid-19, because if there is no active in vivo infection there can be no progression to morbidity or mortality;inability or substantial decrease in risk for patients treated using the combination methods as provided herein catch or be re-infected with any strain nor any mutant strain, where patients administered methods as provided herein are induced to have combined immunity (by administration of a vaccine) and an anti-viral response induced by administration of a drug or drugs which are viral mutant agonists;eradication of primary viral (for example, COVID-19) infection, because patients administered methods as provided herein receive anti-microbial (for example, anti-viral) treatment at the beginning of therapy; and/or,ideal long-term preventative therapy for the elderly with senescent immunity by supporting waning antibody levels in the elderly patient with anti-microbial (for example, anti-viral) treatment as provided herein; and also in embodiments where ivermectin is administered, having the added benefit of rosacea improvement and prevention of scabies in aged-care facilities. Increasing the dose of the intermittent ivermectin combination increases its anti-Covid-19 preventative power. In alternative embodiments, the dose is raised from between about 12 mg to about 36 mg, about 48 mg or about 60 mg, or the dose is raised progressively to 120 mg with few if any adverse effects. This will create a more prolonged circulating level. This is expected to be close to 100% at 4 weeks, but when combined with the vaccine could well prevent for up to 6 weeks or more. Hence, creating the possibility of reducing dosing to ×7/year. Given the use of accompanying anti-viral drugs, even if the vaccine results in lower circulation of neutralizing antibody levels and so immunity, the risk of vaccine breakthrough infection will be minimal if at all possible. Hence, this combination of anti-viral treatment together with the vaccine would be ideal therapy for prevention of infection in the elderly population with senescent immune systems. In alternative embodiments, any vaccine will benefit from practicing methods as provided herein, particularly the mRNA vaccines, which will benefit profoundly when combined with an effective anti-viral treatment. In alternative embodiments, methods as provided herein comprise 1 to 10 days of treatment with an ivermectin-based (or ivermectin-comprising) combination, followed by (the first dose of) vaccination, and then every 1, 2, 3, 4, 5, 6 or 7 days, and later every 8, 9, 10, 11, 12, 13 or 14 days (for example, every 7 days, then every 14 days), and later to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days (for example, every 7 days, then every 14 days, and later every 28 days), administer a higher dose of ivermectin, for example, 60 mg ivermectin for 4 weeks together with zinc or a zinc salt, doxycycline, vitamin D and vitamin C or other appropriate combinations. As long as one continues the anti-viral treatment based on the 60 mg ivermectin regimen, a vaccinated patient has both circulating antibodies for many months and cannot catch mutated virus (for example, COVID-19 agents), and therefore “vaccine breakthrough” will be prevented or substantially decreased and super infection with mutants will be prevented or substantially decreased. In alternative embodiments, methods as provided herein, including for example the ivermectin, zinc or a zinc salt and doxycycline and optionally also an adjunct therapy (such as for example administering a vitamin such as vitamin C or D) is mutant agnostic. In alternative embodiments, because methods as provided herein, including for example the ivermectin combination therapy, functions and works using a different mechanism by prevention of replication within a cell, no mutants can affect its activity as has been shown by us in clinical practice in California, United States. Hence, the combination of an anti-viral with a vaccine as provided herein may be the best method of terminating the Covid-19 pandemic. In alternative embodiments, the vaccination or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection administration may need to be repeated, for example, repeated at 6 or 12 month (or between about 1 (monthly) to 12 month) intervals, but it is of no great importance whether it is 6 months or 12 months because the second arm or the therapy as provided herein, the anti-viral arm, is on board to prevent any further infection and therefore any further mutation in vivo in the patient. In alternative embodiments, even if a mutant or variant strain becomes the predominant viral agent in a community in the future necessitating that the vaccine and/or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection be adjusted (or changed) to take in (be specific for) that new mutant or variant strain, the drug combination as provided herein, for example, the ivermectin, zinc or a zinc salt and doxycycline plus adjunct treatment, can remain the same as it is mutant agnostic. Hence, any vaccine produced by any institution can be supplemented with an anti-viral combination such that no individuals will catch any viral strains once the individual has begun this program (commenced receiving treatment regimens as provided herein. The concept of ‘redundancy’ of treatment is significant here; in medicine, redundant treatment is one where the medication carries extra power to cure the condition. In the event that the treatment had to be terminated early (for example, due to an allergy developing) the built-in redundancy still delivers near (substantially) 100% cure because it was designed to carry extra power. Hence, the combination of the anti-viral treatment and vaccination as provided herein carries a high level of redundancy and thus can achieve a close to 100% success rate (or a substantially completely successfully cure rate). Although alternative embodiments of methods as provided herein are best suited to prevent and treat a virus such as a coronavirus such as a Covid-19 infection, alternative embodiments have multiple other applications. For example, in alternative embodiments, with appropriate antivirals methods as provided herein are used to prevent influenza infections. In alternative embodiments, provided are methods comprising a treatment regimen of an influenza (or other viral) vaccine followed later by antiviral agents intermittently in once a week, 2 weekly, 3 weekly, 4 weekly or less frequently spaced intervals to prevent influenza mutants from re-infecting de novo susceptible elderly patients with senescent immune system where influenza infection causes the most mortality. Other exemplary antiviral combinations administered practicing methods as provided herein comprise use of hydroxychloroquine, for example, hydroxychloroquine, azithromycin and zinc or a zinc salt. In alternative embodiments, provided are methods comprising a treatment regimen for treating: dengue fever, Zika, HIV, hepatitis C, Ebola disease, SARS, MERS, polio, measles, chickenpox and other viral or retro-viral infections. Where current immunizations may not be adequately effective, the follow-on with intermittent antiviral therapy as provide by methods as provided herein gives extra power for the poor immune response combined with antivirals to have enough redundancy to make it clinically effective. In alternative embodiments, provided are methods comprising use of antiviral compounds used singly or in multiple combinations, for example, antiviral compounds are administered singly or in multiple combinations, for example, before, at the time of vaccination, and/or after vaccination: For example, in alternative embodiments, methods provided herein comprise administering in coordination with (optionally before, at the time of vaccination, and/or after vaccination of) an anti-microbial vaccine a single drug or a therapeutic combination of drugs, or a single drug, a pharmaceutical dosage form, a drug delivery device, or a product of manufacture, or the methods can comprise use of: one, two or more classes of antiviral drugs used against influenza, such as: M2 protein inhibitors (for example, amantadine and rimantadine); neuraminidase inhibitors (for example, oseltamivir, zanamivir, peramivir and laninamivir); favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals); a 5- to 6-membered heterocyclic ring such as benzene, naphthalene, furan, benzofuran, pyrrole, pyridine, pyrazole, imidazole, benzimidazole, triazole, tetrazole, oxazole, oxadiazole, 1,3,5-triazine, thiazole, thiophene, benzothiophene, pyrazine, pyridazine, pyrimidine, indole, purine, quinoline or isoquinoline; amantadine; rimantadine; oseltamivir; zanamivir; peramivir; laninamivir; laninamivir octanoate hydrate; arbidol; ribavirin; stachyflin; ingavirin; fludase; a niclosamide compound; an emricasan compound; nitazoxanide; tizoxanide; and/or a compound selected from consisting of teriflunomide, hydroxocobalamin, ensulizole, tenonitrozole, isoliquiritigenin, nitazoxanide, febuxostat, leflunomide, fidofludimus SB-366791, emodin, diphenyl isophthalate, benzoylpas, fenobam, indobufen, 2-(2H-Benzotriazol-2-yl)-4-methylphenol, tiaprofenic acid, flufenamic acid, vitamin B12, cinanserin, 5-nitro-2-(3-phenylpropylamino)benzoic acid, veliflapon, thiabendazole, SIB 1893, anethole trithione, naringenin, phenazopyridine, fanetizole, terazosin, diacerein, CAY10505, hesperetin, suprofen, ketorolac tromethamine, piperine, pirarubicin, piraxostat, albendazole oxide, tyrphostin AG 494, genistin, fenbufen, apatinib, RITA, BF-170 hydrochloride, OSI-930, tribromsalan, pifexole, formononetin, ebselen, tranilast, benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, baicalein, nemorubicin, rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine (MPEP), 5,7-dihydroxyflavone, vitamin B12, pipofezine, flurbiprofen axetil, 2-Amino-6-nitrobenzothiazole, nalachite green oxalate, enfenamic acid, fenaminosulf, AS-252424, phenserine, epalrestat, alizarin, dalcetrapib, SN-38, echinomycin, (S)-(+)-camptothecin, BI-2536, 10-hydroxycamptothecin, topotecan, delanzomib, volasertib, ispinesib, paclitaxel, FK-506, emetine, AVN-944, digoxin, vincristine, idarubicin, thapsigargin, lexibulin, ixazomib, cephalomannine, mitoxantrone, MLN-2238, demecolcine, vinorelbine, bardoxolone methyl, cycloheximide, actinomycin D, AZD-7762, PF-184, CHIR-124, cyanein, triptolide, KX-01, PF-477736, epirubicin, mycophenolate (mycophenolic acid), daunorubicin, PIK-75, vindesine, torin-2, floxuridine, Go-6976, OSU-03012, and a prodrug, metabolite, or derivative of any of the foregoing. In alternative embodiments the following compound (or its isomer, or stereoisomer, or enantiomer, or deuterated version, or bioisostere) is used singly or in various combinations (for example, formulated with, or administered separately) with other drug such as anti-viral drugs before, during or after vaccination or administration of a causative agent of infection: (1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide administered orally or by inhalation (or nasally), for example, as liquid, solid, powder, mist or spray, which can target a protease (such as the 3CL protease in COVID-19) and optionally has the following structure and molecular weight: This protease inhibitor (PF-07321332, or PAXLOVID™) may be used alone before and after the vaccination and/or administration of the attenuated causative agent of infection, optionally administered with ritonavir (or NORVIR™) or lopinavir, or with any of the numerous antiviral agents as provided herein. In alternative embodiments the following compounds (or their isomers, or stereoisomers, or enantiomer, or bioisostere) can be used singly or in various combinations: These compounds (PF-07304814 and/or PF-00835231) (or its isomer, or stereoisomer, or enantiomer, or deuterated version, or bioisostere) may be used alone before and after the vaccination and/or administration of the attenuated causative agent of infection, optionally administered with ritonavir (or NORVIR™) or lopinavir, or with any of the numerous antiviral agents as provided herein. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered separately, or together (for example, formulated together) as a tablet, gel, geltab or capsule, as a powder, in a liquid, in a mist or a spray, or as a lozenge. In alternative embodiments, the PE-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered before, at the same time as, and/or after the vaccination. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days before, and/or on the day of, a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days after a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. In alternative embodiments, the PF-07321332 (or PAXLOVID™) and ritonavir (or NORVIR™) or lopinavir combination; or the PF-07304814 and/or PF-00835231 and ritonavir (or NORVIR™) or lopinavir combination; or the KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™ and/or zanamivir (or RELENZA™) combination; is administered both before and after a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection is administered. Another agent which can be used singly or in combination before and accompanying vaccination is 2-deoxy-D-Glucose (2-DG). In alternative embodiments, methods as provided herein comprise (or further comprise) administering in coordination with (optionally before, at the time of vaccination, and/or after vaccination of) an anti-microbial vaccine (or a dose of the inactivated, attenuated, or the live, viable or infectious causative agent of the infection) a therapeutic combination of drugs or a single drug, a pharmaceutical dosage form, a drug delivery device, or a product of manufacture, comprising:(a) a thiazolide class drug, optionally nitazoxanide (or ALINIA™, NIZONIDE™) or tizoxanide (or 2-Hydroxy-N-(5-nitro-2-thiazolyl)benzamide);(b) molnupiravir, optionally co-administered with and/or formulated with an avermectin class drug (optionally ivermectin), an antibiotic (optionally doxycycline or azithromycin) and/or zinc, or co-administered with and/or formulated with ivermectin, hydroxychloroquine, an antibiotic (optionally doxycycline or azithromycin) and/or zinc; (c) opaganib or YELIVA™, or opaganib or YELIVA™ and oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate, amodiaquine (or AMDAQUINE™, AMOBIN™) and/or hydroxychloroquine (optionally, PLAQUENIL™), wherein optionally each or both of the opaganib and the chloroquine (or ARALEN™) chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) are in or formulated as a formulation for inhalation, for example, formulated as an aerosol, spray, mist, liquid or powder, or each or both are formulated for oral, intramuscular or intravenous administration,wherein optionally the opaganib is administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc (optionally zinc sulfate, optionally at (50 mg daily, or any zinc salt);(d) lopinavir, ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(e) lopinavir combined (formulated) with ritonavir (or NORVIR™), or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™, and/or zanamivir (or RELENZA™), or lopinavir and ritonavir separately formulated;(f) lopinavir combined (formulated) with ritonavir (or NORVIR™) (or KALETRA™, ALTERA™, ALUVIA™, KALMELTREX, LOPIMUNE™ or LOPINAVIR™), or lopinavir and ritonavir (or NORVIR™), and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™), optionally also with inhaled or aerosol formulations or versions of chloroquine (or ARALEN™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(g) lopinavir, ritonavir (or NORVIR™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine and oseltamivir (or TAMIFLU™); wherein optionally the chloroquine comprises inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) simultaneously;(H) lopinavir and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(i) ritonavir (or NORVIR™) and oseltamivir (optionally, TAMIFLU™), and/or zanamivir (or RELENZA™);(j) remdesivir (optionally, GS-5734™, Gilead Sciences) alone, or oseltamivir (optionally, TAMIFLU™) and remdesivir (optionally, GS-5734™, Gilead Sciences), and optionally the remdesivir is an oral formulation and/or an inhaled or aerosol remdesivir formulation;(k) oseltamivir (optionally, TAMIFLU™) and efavirenz (optionally, SUSTIVA™), and/or zanamivir (or RELENZA™);(l) oseltamivir (optionally, TAMIFLU™) and nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™);(m) oseltamivir (or TAMIFLU™) and amprenavir (optionally, AGENERASE™);(n) oseltamivir (optionally, TAMIFLU™) and nelfinavir (optionally, VIRACEPT™);(o) a thiazolide class drug, optionally nitazoxanide (optionally ALINIA™, NIZONIDE™) or tizoxanide (or 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide), with or in combination with any of (a) to (nn), or any drug or drug combination as provided herein, optionally a thiazolide class drug, optionally nitazoxanide, with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; or a thiazolide class drug (optionally, nitazoxanide or tizoxanide) and oseltamivir (or TAMIFLU™),and optionally the thiazolide class drug (optionally, nitazoxanide or tizoxanide) is formulated or administered with ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), and an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(p) plitidepsin (also known as dehydrodidemnin B), or APLIDIN™ (PharmaMar, S. A.);(q) an inhibitor or S-phase kinase-associated protein 2 (SKP2), or dioscin, or niclosamide, or NICLOCIDE™, FENASAL™, or PHENASAL™;(r) ritonavir (or NORVIR™), ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), interferon beta 1b, or a combination of ribavirin and interferon beta, or a combination of lopinavir and ritonavir (or NORVIR™) and interferon-beta-1b;(s) a nucleoside analog reverse-transcriptase inhibitor (NRTI) (optionally abacavir, or ZIAGEN™), acyclovir or aciclovir (optionally ZOVIRAX™), adefovir (optionally HEPSERA™), amantadine(optionally GOCOVRI™, SYMADINE™, SYMMETREL™), rintatolimod (or AMPLIGEN™), amprenavir (optionally, AGENERASE™), aprepitant (or EMEND™), umifenovir (or ARBIDOL™), atazanavir (or REYATAZ™), atazanavir (or REYATAZ™), tenofovir, a combination of efavirenz and emtricitabine and tenofovir (or ATRIPLA™), balavir, baloxavir marboxil (XOFLUZA™), bepotastine, bevirimat, bictegravir, a combination of bictegravir and emtricitabine and tenofovir alafenamide (or BIKTARVY™), brilacidin, bivalirudin (or BIVALITROBAN™), cidofovir, caspofungin, lamivudine and zidovudine (optionally, COMBVIR™), cobicstat, colisitin, cocaine, darunavir, delavirdine, descovy, didanosine, docosanol, dolutegravir, ecoliever, edoxudine, efavirenz (optionally, SUSTIVA™), elvitegravir, emtricitabine, enfuvirtide, foscarnet, fosfonet, galidesivir, ibacitabine, icatibant, idoxuridine, ifenprodil, imiquimod, imunovir, indinavir, inosine, an interferon (optionally interferon type I, interferon type II and/or interferon type III), lamivudine (or EPIVIR™, ZEFFIX™), lopinavir, loviride, ledipasvir, leronlimab, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide (optionally ALINIA™, NIZONIDE™), norvir, a nucleoside analogue or derivative (optionally brincidofovir (or TEMBEXA™), didanosine (or VIDEX™), favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals), vidarabine, galidesivir (optionally, BCX4430, IMMUCILLIN-A™), remdesivir (optionally, GS-5734™, Gilead Sciences), cytarabine, gemcitabine, emtricitabine, lamivudine, zalcitabine, entecavir, stavudine, telbivudine, zidovudine, idoxuridine and/or trifluridine or any combination thereof), oseltamivir (or TAMIFLU™), peginterferon alfa-2a, penciclovir, peramivir (optionally, RAPIVAB™), perfenazine, pleconaril, plurifloxacin, podophyllotoxin, pyramidine, raltegravir, rifampicin, ribavirin or tribavirin (or COPEGUS™, REBETOL™, or VIRAZOLE™), rilpivirine, rimantadine, ritonavir (or NORVIR™), saquinavir, sofosbuvir, stavudine, telaprevir, tegobuv, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir (optionally, VALTREX™), valganciclovir, valrubicin, vapreotide, vicriviroc, vidarabine, viramidine, velpatasvir, vivecon, zalcitabine, zanamivir (optionally, RELENZA™), zidovudine, an immunosuppressive drug (optionally tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™) or any combination thereof;(t) an mucolytic therapy or drug, optionally acetylcysteine, ambroxol, bromhexine (or BISOLVON™), carbocisteine, erdosteine, mecysteine or dornase alfa, or an expectorant, optionally guaifenesin;(u) a viral, or a coronavirus or a COVID-19, protease inhibitor, optionally ASC09 (CAS registry no. 1000287-05-7) (Janssen Research and Development, LLC), ritonavir (or NORVIR™) or ASC09 and ritonavir (or NORVIR™), or a JAK1/2 inhibitor (optionally baricitinib), optionally compound 11r (University of Lubeck, Germany, see optionally, Zhang et al J. Med Chem 2020, Feb. 11, 2020), or darunavir, cobicistat or darunavir and cobicistat;(v) an angiotensin-converting enzyme 2 (ACE2) inhibitor, optionally to block the site of viral spike protein interaction for anti-SARS-CoV-2 infection control;(w) an anti-vascular endothelial growth factor (VEGF) (optionally VEGF-A) drug or antibody, optionally bevacizumab;(x) a protease inhibitor, optionally danoprevir, optionally a serine protease inhibitor, optionally camostat or narlaprevir (optionally ARLANSA™);(y) anti-PD-1 checkpoint inhibitor, optionally camrelizumab;(z) a compound or antibody capable of binding complement factor C5 and blocking membrane attack complex formation, optionally eculizumab;(aa) a cathepsin inhibitor, optionally a cathepsin K, B or L inhibitor, optionally relacatib;(bb) thalidomide, or thalidomide and glucocorticoid (optionally ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™)) (optionally low-dose glucocorticoid), or and thalidomide and celecoxib;(cc) an antibacterial antibiotic or a macrolide drug,wherein optionally the macrolide drug comprises azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended- or delayed-release formulation of azithromycin, or ZMAX™), clarithromycin (optionally, BIAXIN™), erythromycin (optionally, ERYTHROCIN™), or fidaxomicin (optionally, DIFICID™ or DIFICLIR™), troleandomycin (optionally, TEKMISIN™), tylosin (optionally, TYLOCINE™ or TYLAN™), solithromycin (optionally, SOLITHERA™), oleandomycin (or SIGMAMYCINE™), midecamycin, roxithromycin, kitasamycin or turimycin, josamycin, carbomycin or magnamycin, and/or spiramycin,and optionally the antibacterial antibiotic comprises a tetracycline class drug, a glycylcycline or a fluorocycline class drug, or an analogue thereof, and optionally the tetracycline, glycylcycline or fluorocycline drug or analogue thereof comprises or is: tetracycline or SUMYCIN™; chlortetracycline or AUREOMYCIN™; oxytetracycline; demeclocycline or DECLOMYCIN™, DECLOSTATIN™, LEDERMYCIN™, BIOTERCICLIN™, DEGANOL™, DETECLO™, DETRAVIS™, MECICLIN™, MEXOCINE™, CLORTETRIN™; lymecycline; meclocycline; metacycline; minocycline or MINOCIN™; rolitetracycline; doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™; tigecycline or TYGACIL™; eravacycline or XERAVA™; sarecycline or SEYSARA™, omadacycline or NUZYRA™; or any combination thereof, and optionally the antibacterial antibiotic or macrolide drug, optionally azithromycin (or ZMAX™), is administered in combination with, and/or is combined with, chloroquine (or ARALEN™), amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), and the combination is administered commencing on the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth day of therapy, or is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days, or for between about 1 to 21 days or longer, or is administered until within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days of ending the therapy for treating, preventing, ameliorating, slowing the progress of, decreasing the severity of or preventing the coronavirus infection,and optionally the chloroquine (or ARALEN™), chloroquine phosphate, amodiaquine (or AMDAQUINE™, AMOBIN™), chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered the entire length of the treatment but the azithromycin, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day (optionally, ZITHROMAX™, or AZITHROCIN™, optionally an oral extended-release formulation of azithromycin, or ZMAX™) administration is halted or ceased after two, three, four, five or six days after treatment is commenced, and optionally the azithromycin administration is replaced by a tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™ administration,and optionally the antibacterial antibiotic, optionally azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day,and optionally an oral extended-release formulation of azithromycin, or ZMAX™), is administered or formulated with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or cholecalciferol (vitamin D3) or calcifediol,and optionally the antibacterial antibiotic comprises an antimycobacterial drug, and optionally the antimycobacterial drug comprises clofazimine (optionally LAMPRENE™);(dd) an avermectin class drug such as ivermectin (optionally STROMECTOL™, SOOLANTRA™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally dosaged and/or administered at about 5 microgram/kg to about 1 gram (g) per day, optionally formulated or administered at about 1 to 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200, 220 or 240 mg per day, or between about 1 to 240 mg per day, or between about 3 to 240 mg per day,optionally formulated or administered with an antibiotic (optionally azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline, and optionally the doxycycline is at between about 25 to 600 mg per dose or per day, or at about 100 mg per dose or per day, and optionally the azithromycin is at between about 50 mg to 2000 mg per dose or per day), optionally as a single or a divided dose, and optionally formulated and administered as an inhalant or a mist (optionally using a nebulizer, nasal spray or equivalent), optionally formulated as an aerosol, spray, mist, liquid or powder, optionally formulated as an aerosol, spray, mist, liquid or powder,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated with and/or administered with chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) with or without zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt), and optionally this combination is administered weekly, or every two week, or one every 5 to 28 days, as a prophylactic,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone in the morning (AM), and an antibiotic (optionally doxycycline) and/or a chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) is administered in the afternoon and/or evening,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered alone for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 or more days, followed by administration of an antibiotic (optionally doxycycline) for a corresponding period of days, and optionally repeating the cycle of dosaging,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is formulated or administered with:(i) at least one antibiotic (wherein optionally the antibiotic is doxycycline(optionally, DORYX™, DOXYHEXA™, DOXYLIN™) (optionally formulated or administered at a dosage of between about 25 mg to 600 mg per dose or per day), or azithromycin (optionally, ZITHROMAX™, or AZITHROCIN™, optionally dosaged at between about 50 mg to about 2000 mg per dose or per day, optionally an oral extended-release formulation of azithromycin, or ZMAX™) (optionally formulated or administered at a dosage of between an about 50 mg to 2000 mg);(ii) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) (optionally formulated or administered at a dosage of between an about 10 mg to 2000 mg per day);(iii) a zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally formulated or administered at a dosage of between about 1 mg to 250 mg; and/or(iv) at least one vitamin, and optionally the at least one vitamin comprises: vitamin C optionally formulated or administered at a dosage of between about 500 to 5000 units (U) per dose, and/or Vitamin D (or cholecalciferol) optionally formulated or administered at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day,and optionally the avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin is administered or formulated alone or in combination with any of the above (i) to (iv) (for example, at least one antibiotic, chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™), zinc or any zinc salt and/or at least one vitamin are formulated (and administered) as oral formulations (for example, as tablets, capsules, gels or geltabs), injectable formulations, powders (for example, for inhalation or for addition to an ingestible liquid) or liquids (for example, for ingestion, infusion or injection);(ee) chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) alone or with (or formulated with) or in combination with any of (a) to (bb), or chloroquine, chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and oseltamivir (or TAMIFLU™);(ff) chloroquine (optionally, ARALEN™), chloroquine phosphate, alone or with:(i) an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, optionally at a dosage of between about 3 to 340 mg per day, or about 6 mg to 60 mg, or about 10 mg to 80 mg dosages, or about 12 to 50 mg dosages;(ii) vitamin D, vitamin D2 (or ergocalciferol), vitamin D3 (or cholecalciferol) optionally at a dosage of between about 3,000 to 100,000 units per day, or between about 10,000 to 50,000 units a day, and/or(iii) with (i) and (ii) and zinc (optionally a zinc sulphate, acetate, gluconate or picolinate or any zinc salt) optionally at a dosage of between about 1 mg to 250 mg, or (iv) the combination of (iii) also with a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™, optionally dosages at between about 25 mg to 600 mg per day or per dose, optionally between about 100 mg to 500 mg, or a between about 200 mg to 400 mg per dose or per day;(gg) colchicine, or COLCRYS™, MITIGARE™, optionally administered or dosaged at between about 0.5 mg to 20 mg, or about 1 mg to 15 mg, or about 3 mg to mg, or about 4 mg to 6 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(hh) a corticosteroid or glucocorticoid class drug such as ciclesonide (or ALVESCO™, OMNARIS™, OMNIAIR™, ZETONNA™ or ALVESCO™) budesonide (optionally RHINOCORT™ or PULMICORT™), prednisolone (or ORAPRED™), methyl-prednisolone, prednisone (or DELTASONE™ or ORASONE™) or hydrocortisone (or CORTEF™), wherein optionally the corticosteroid or glucocorticoid class drug (optionally ciclesonide) is inhaled,or a selective estrogen receptor modulator (SERM), or toremifene (or FARESTON™), or clomifene or clomiphene (or CLOMID™, SEROPHENE™) wherein optionally the SERM is inhaled;and optionally the corticosteroid class drug (for example budesonide) is administered by inhalation, for example, in a nebulized form, for example, between about 1 mg to 12 mg per day of budesonide is administered by inhalation, or between about 6 to 80 mg per day of prednisolone is administered orally, or between about 6 to 100 mg per day of prednisone is administered orally, or between about 30 to 400 mg per day of hydrocortisone is administered orally,and optionally the corticosteroid class drug is formulated as a powder or for administration in an inhaler or by nasal spray, or for rectal administration,and optionally the corticosteroid class drug (for example, budesonide) is administered together with or in combination with 10 mg to 80 mg, an antibiotic (optionally azithromycin or a tetracycline class drug,wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™), zinc or any zinc salt and/or a vitamin (optionally vitamin D or calcifediol, D2 (or ergocalciferol), D3 (or cholecalciferol), C, E, B12, B6);(ii) an anti-androgen drug, and optionally the anti-androgen drug is bicalutamide, optionally CASODEX™, or dutasteride (or AVODART™),and optionally the anti-androgen drug is a nonsteroidal anti-androgen (NSAA) or an androgen receptor (AR) antagonist, and optionally the NSAA or AR antagonist comprises proxalutamide (or its developmental name GT-0918) (Suzhou Kintor Pharmaceuticals, Inc., a subsidiary of Kintor Pharmaceutical Limited), or flutamide (or niftolide, or EULEXIN™), or bicalutamide (or CASODEX™) or enzalutamide (or XTANDI™),and optionally the anti-androgen drug comprises a 5α-reductase inhibitor, and optionally the 5α-reductase inhibitor comprises finasteride (or PROSCAR™, PROPECIA™, or FINIDE™)and optionally the anti-androgen drug, or NSAA, or proxalutamide or bicalutamide, is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, eprinomectin or abamectin;and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered at dosages of about 50 to 100 mg optionally administered once, twice (BID), three times (TID) or four times a day, or is administered at dosages of about 50 to 100 mg per day,and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with an avermectin class drug, or ivermectin, optionally also administered with hydroxychloroquine, zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day) or vitamin C, B or A;and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally bicalutamide is administered together with or in combination with an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin;(jj) a hydrocortisone or cortisol (optionally CORTEF™, SOLUCORTEF™), optionally hydrocortisone sodium succinate or hydrocortisone acetate or dexamethasome (optionally DEXTENZA™, OZURDEX™, NEOFORDEX™);(kk) an alpha-ketoamide (α-ketoamide), wherein optionally the alpha-ketoamide is a structure as described by Zhang et al, J. Med. Chem. 2020, 63, 9, 4562-4578, or Meng et al Chem. Sci. (2019) vol. 10, pg 5156 (optionally the structure KAM-2),and optionally the alpha-ketoamide is formulated or administered as an inhalant or a powder or mist, and optionally formulated or administered with (optionally as an inhalant): an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin; an antibiotic (optionally azithromycin or a tetracycline class drug, wherein optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™); chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™); zinc or any zinc salt; remdesivir (optionally, GS-5734™, Gilead Sciences); oseltamivir (or TAMIFLU™); and/or, hydrocortisone; or, any combination thereof;(ll) a compound, drug or formulation that decreases stomach acid production or decreases stomach pH, wherein optionally the compound, drug or formulation comprises famotidine, or PEPCID™, and optionally the famotidine is administered at a dosage of between about 10 to 60 mg per day, or between about 20 to 40 mg per day, and optionally the famotidine is administered is administered with: an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin, and/or a tetracycline tetracycline class drug, and optionally the tetracycline class drug comprises doxycycline, or DORYX™, DOXYHEXA™, DOXYLIN™;(mm) a dendrimer, optionally astodrimer sodium (Starpharma, Melbourne, Australia);(nn) an antihistamine class drug such as azelastine, or ASTELIN™, OPTIVAR™, ALLERGODIL™, brompheniramine, fexofenadine or ALLEGRA™ pheniramine or AVIL™, or chlorpheniramine;(oo) a selective serotonin reuptake inhibitor (SSRI) class drug, optionally fluvoxamine, or LUVOX™, FAVERIN™, FLUVOXIN™;(pp) a nicotinic antagonist, a dopamine agonist or a noncompetitive N-Methyl-d-aspartic acid or N-Methyl-d-aspartate (NMDA) antagonist, wherein optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is 1-adamantylamine or amantadine, or GOCOVRI™, SYMADINE™, SYMMETREL™, optionally administered or dosaged at between about 50 mg to 150 mg, or about 100 mg, per day for a period of between about 7 and 21 days, or about 14 days, and optionally the nicotinic antagonist, dopamine agonist or noncompetitive NMDA antagonist is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily), and optionally the amantadine is formulated or administered at 100 mg per day for the first two days of treatment, which optionally can then be elevated to 100 mg twice daily, optionally for the next 10 days;(qq) an immunosuppressive drug, wherein optionally the immunosuppressive drug comprises tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™, or a calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin), or NEORAL™, or SANDIMMUNE™, or tacrolimus, or PROTOPIC™, or PROGRAF™, and optionally the immunosuppressive drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally the calcineurin inhibitor (CNI), wherein the CNI comprises ciclosporin (or cyclosporine or cyclosporin) is formulated combination of CNI (optionally cyclosporine) at a dose of 3 mg/kg (180 mg daily) together with 12 mg ivermectin once, and optionally also plus zinc 50 mg base and doxycycline 100 mg bid, optionally all for 10 days;(rr) a protein kinase inhibitor, wherein optionally the protein kinase inhibitor is a p38 mitogen-activated protein kinase inhibitor, or ralimetinib, and optionally the protein kinase inhibitor is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(ss) an anti-inflammatory therapy or at least one anti-inflammatory therapy drug, wherein optionally the anti-inflammatory therapy or drug comprises: a sphingosine kinase-2 (SK2) selective inhibitor (optionally, opaganib (optionally, YELIVA™), sirolimus, a JAK1/2/TYK2 inhibitor (optionally ruxolitinib), an anti-CD47 mAb (optionally meplazumab), a cyclooxygenase (COX) (optionally, COX2) inhibitor, a glucocorticoid (optionally a synthetic glucocorticoid, hydrocortisone, dexamethasone (or DEXTENZA™, OZURDEX™, or NEOFORDEX™) or cortisol, or CORTEF™), plitidepsin or dehydrodidemnin B, or APLIDIN™, or a nonsteroidal anti-inflammatory drug (NSAID), wherein optionally the NSAID comprises indomethacin (or indomethacin) or INDOCID™ or INDOCIN™, or naproxen, or NAPROSYN™ or ALEVE™, or a cyclooxygenase inhibitor, or a COX-1 or an COX-2 inhibitor, or aspirin, or ibuprofen or ADVIL™, MOTRIN™ or NUROFEN™, or celecoxib or CELEBREX™, or parecoxib or DYNASTAT™, or etoricoxib or ARCOXIA™,and optionally the anti-inflammatory therapy or anti-inflammatory therapy drug is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin, hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily),and optionally opaganib, or YELIVA™, or opaganib, or YELIVA™ administered or formulated together with an oral and/or inhaled or aerosol chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™),and optionally the opaganib or YELIVA™ is formulated or administered at a dosage of QD (once a day), bid (twice a day) or tid (three times a day) at a dosage of between about 100 to 600 mg per day or per dosage, or at about 100, 200, 300, 400, 500 or 600 mg per day or per dosage,and optionally the opaganib, or YELIVA™ is also administered or formulated with an antibiotic (optionally azithromycin or doxycycline), ivermectin (optionally at 12 mg ivermectin, optionally administered on days 1, 3, 6 and 8), hydroxychloroquine (optionally, PLAQUENIL™) and/or zinc or any zinc salt (optionally zinc sulfate, optionally at (50 mg daily);(tt) a calcium channel blocker, or verapamil (or ISOPTIN™, CALAN™), or a voltage gated potassium (KCNH2) channel or a voltage gated calcium channel (CACNA2D2) blocker, or amiodarone (or CORDARONE™, NEXTERONE™);(uu) suramin, or ANTRYPOL™, BAYER 305™, or GERMANIN™(vv) a peroxisome proliferator-activated receptor (PPAR) agonist, wherein optionally the PPAR agonist comprises fenofibrate, or TRICOR™, FENOBRAT™, FENOGLIDE™, or LIPOFEN™, or a combination of fenofibrate and simvastatin, or CHOLIB™, optionally the PPAR agonist comprises a combination of fenofibrate and pravastatin, or PRAVAFENIX™, or the PPAR agonist comprises bezafibrate, or BEZALIP™, or combination of bezafibrate and chenodeoxycholic acid, or HEPACONDA™, or aluminium clofibrate, or alfibrate, or ciprofibrate, or clinofibrate or LIPOCLIN™, or clofibrate or ATROMID-S™, or clofibride, or gemfibrozil or LOPID™, or ronifibrate, or simfibrate or CHOLESOLVIN™, or any combination thereof,(ww) a synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or a prodrug of N4-hydroxycytidine, optionally molnuvpiravir (Merck), or favipiravir (also known as T-705 or AVIGAN™, or favilavir, Toyama Chemical, Fujifilm, Japan, or FABIFLU™, Glenmark Pharmaceuticals),wherein the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is given as between about 10 mg to 3 gm per dose, or between about 10 mg to 3 gm per day, or can be dosed either as a single dose or given one, two, three or four times a day, or is administered at 200 to 800 mg twice daily, or 200, 400, 600 or 800 mg twice daily, or at 200 to 800 mg three times a day, or at 200, 400, 600 or 800 mg three times a day, or is administered at 200 to 800 mg three times a day for between about 2 to 15 days, or for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, and optionally when combined with other drugs a lower dosage is used, optionally administered at 100 or 200 mg three times a day for between about 5 to 15 days, or for about 7, 8, 9, 10, 11 or 12 days,and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an avermectin class drug (optionally ivermectin) with an antibiotic, and optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), and optionally the synthetic nucleoside analog or derivative, avermectin class drug, and antibiotic are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally molnuvpiravir, ivermectin and hydroxychloroquine are administered together or as separate formulations, and optionally are administered every one, two, three, four or five weeks for between about one month and one year or more;and optionally the synthetic nucleoside analog or derivative (optionally N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir), and antibiotic (optionally doxycycline or hydroxychloroquine) is administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D),and optionally the synthetic nucleoside analog or derivative, or N4-hydroxycytidine, or the prodrug of N4-hydroxycytidine, optionally molnuvpiravir or favipiravir, is administered with an antibiotic (optionally the antibiotic comprises azithromycin, minocycline, amoxicillin, niclosamide, nitazoxanide, hydroxychloroquine or doxycycline), optionally also administered with zinc (optionally a zinc sulphate, acetate, gluconate or picolinate, or zinc oxide nanoparticles, optionally at a dosage of between about 1 mg to 250 mg, or about 50 mg per day) and/or a vitamin, optionally vitamin C or D,and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with colchicine (or COLCRYS™, MITIGARE™), and optionally also zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day), or vitamin C, B or A),and optionally the anti-androgen drug, or NSAA, or bicalutamide, proxalutamide, flutamide or niftolide, bicalutamide, enzalutamide or dutasteride, is administered with an antibiotic (optionally azithromycin or doxycycline), and optionally also zinc and/or a vitamin (optionally vitamin D (optionally vitamin D2, or ergocalciferol, or Vitamin D3 or cholecalciferol, optionally administered at about 1000 to 4000 ugm/day), or vitamin C, B or A), and optionally also with hydroxychloroquine;(xx) an anti-malarial drug, wherein optionally the anti-malarial drug comprises mefloquine (or LARIAM™, MEPHAQUIN™, or MEFLIAM™)(yy) an antisera or an antibody or antibody or vaccine therapy for treating, preventing or ameliorating a microbial or a viral infection (optionally a coronavirus infection, optionally a COVID-19 infection) or a microbial infection (optionally a protozoan, helminthiasis, insect and/or parasitic infection), and optionally the antibody comprises a monoclonal antibody, and optionally the monoclonal antibody comprises sotrovimab (GlaxoSmithKline and Vir Biotechnology), or casirivimab, imdevimab or casirivimab and imdevimab (REGEN-COV™) (Regeneron), or bamlanivimab oretesevimab or bamlanivimab and etesevimab (Junshi Biosciences), or tocilizumab or ACTEMRA™ or ROACTEMRA™ (Hoffmann-La Roche), and optionally the vaccine comprises tozinamera or COMIRNATY™ (Pfizer), or elasomeran or SPIKEVAX™ (Moderna), or SPUTNIK V™ or Gam-COVID-Vac (Gamaleya Research Institute), or AZD1222 or COVISHIELD™ or VAXZEVRIA™ (Oxford-AstraZeneca),and optionally the antibody or antibody therapy comprises or is contained in a convalescent sera or plasma, and/or(zz) any combination of (a) to (yy), and optionally any of these combinations is administered very 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more days for between about 1 month and one year or more,and optionally any one or several or all of (a) to (zz) with an (or formulated with or formulated as an) inhaled or aerosol formulation such as a powder, spray or a mist or aerosol, and/or formulated with or formulated as an oral, intramuscular (IM) or intravenous (IV) formulation, wherein optionally both the inhaled (or aerosol) and the oral, IV and/or IM formulations are administered simultaneously or sequentially,and optionally the inhaled or aerosol formulation comprises chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) and/or oral chloroquine (or ARALEN™), chloroquine phosphate, chloroquine diphosphate and/or hydroxychloroquine (optionally, PLAQUENIL™) administered simultaneously or overlapping,and optionally the inhaled or aerosol formulation comprises an avermectin class drug such as ivermectin (optionally STROMECTOL™), moxidectin (optionally CYDECTIN™, EQUEST™, QUEST™), selamectin (optionally STRONGHOLD™), a milbemycin (optionally milbemectin, milbemycin oxime, moxidectin or nemadectin), doramectin (optionally DECTOMAX™), eprinomectin or abamectin,and optionally any one or several or all of (a) to (zz), or any therapeutic combination of drugs or a drug, or a pharmaceutical dosage form as provided herein, are administered orally, intramuscularly, subcutaneously, topically, by use of an enema, intravaginally, or intravenously, or administration is by subcutaneous administration, sublingual administration, inhalation or by aerosol (optionally by inhalation of a liquid, an aerosol, a spray, a mist or a powder), by absorbable patch, by use of an implant, or by use of an enema or a suppository. In alternative embodiments, the anti-viral drug or medication, or anti-microbial drug, is or comprises: molnupiravir, efavirenz (optionally, SUSTIVA™), tenofovir, emtricitabine and tenofovir, nevirapine (or the combination efavirenz with emtricitabine and tenofovir, or ATRIPLA™), amprenavir (optionally, AGENERASE™), nelfinavir (optionally, VIRACEPT™) and/or remdesivir (optionally, GS-5734™, Gilead Sciences), a viral RNA-dependent RNA polymerase inhibitor, optionally galidesivir, a nucleoside analog reverse-transcriptase inhibitor (NRTI) (optionally abacavir, or ZIAGEN™)and optionally the anti-viral drug or medication is or comprises an anti-retroviral drug or drug combination, and optionally the anti-retroviral drug or drug combination comprises: darunavir and cobicistat (optionally, REZOLSTA™ or PREZCOBIX™); atazanavir and cobicistat (or EVOTAZ™); abacavir, lamivudine and dolutegravir (TRIUMEQ™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (optionally, STRIBILD™); tenofovir (or disoproxil or emtricitabine) and elvitegravir and cobicistat (COMPLERA™ or EVIPLERA™); molnupiravir, efavirenz (optionally, SUSTIVA™), emtricitabine and tenofovir (ATRIPLA); lamivudine, nevirapine and stavudine (optionally, TRIOMUNE™); atazanavir and cobicistat (optionally, EVOTAZ™); lamivudine and raltegravir (optionally, DUTREBIS™); lamivudine and dolutegravir (or DOVATO™); doravirine, lamivudine and tenofovir (optionally, DELSTRIGO™); or lamivudine, zidovudine and nevirapine (optionally, CUOVIR-N™);and optionally the additional anti-viral drug or medication, or anti-microbial drug, is formulated with the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir or is formulated separately from the chloroquine (optionally, ARALEN™), chloroquine phosphate, chloroquine diphosphate, hydroxychloroquine (optionally, PLAQUENIL™), lopinavir, ritonavir (or NORVIR™) and/or oseltamivir,and optionally the anti-viral drug or medication, or anti-microbial drug, or palliative agent comprises or further comprises: magnesium (Mg, optionally administer intravenously (IV) to maintain a blood concentration of between about 2.0 and 2.4 mmol/l); zinc or any zinc salt (optionally a zinc sulphate, acetate, gluconate or picolinate, optionally administered at about 75 to 100 mg/day or at a dosage of between about 1 mg to 250 mg); at least one vitamin, wherein optionally the at least one vitamin comprises vitamin K, vitamin D or calcifediol (optionally D2 (or ergocalciferol) or Vitamin D3 or cholecalciferol), optionally administered at about 1000 to 4000 ugm/day), vitamin B6 (or pyridoxine), vitamin B12, vitamin E, and/or vitamin C (optionally administered at 500 mg bid); a flavonoid, plant flavonol or quercetin optionally administered at between about 250 to 500 mg bid; atorvastatin, or LIPITOR™, SORTIS™ (optionally administered at between about 40 mg/day to 80 mg/day); or, melatonin, or CIRCADIN™, SLENYTO™ (optionally between about 6 to 12 mg a day, optionally, at night), any of which are optionally given enterally or parenterally. Anti-Clotting or Blood Thinning Agents In alternative embodiments for practicing methods as provided herein, to address the possibility of blood clotting, whether the blood clotting is caused by the infectious agent, the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, and/or the vaccine or for any another reason, an anti-clotting or anti-coagulant or blood thinning drug or agent is also administered, for example, before and/or at the commencement of the vaccination, and optionally is continued for between about 1 to 2 or 1 to 6 weeks after the vaccination, or for the duration of the anti-microbial drug treatment though administration of a second or booster vaccination, and/or for between about 1 to 2 weeks after administration of the second or booster vaccination. In alternative embodiments, the anti-clotting agent or anti-coagulant or blood thinning drug or agent comprises aspirin, for example between about 100 mg to 500 mg aspirin administered (for example, in the morning, or AM, or MANE) for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days commencing on the day of the vaccination or commencing one or two days before the vaccination day. In alternative embodiments, antiplatelet drugs that can be used include clopidogrel (PLAVIX™), prasugrel (EFFIENT™) and ticagrelor (BRILINTA™). In alternative embodiments, the anti-clotting or anti-coagulant agent or blood thinning drug or agent comprises: heparin; warfarin (or COUMADIN™); a coumarin; phenprocoumon (or MARCUMAR™); rivaroxaban (XARELTO™); dabigatran (PRADAXA™); apixaban (ELIQUIS™); edoxaban (LIXIANA™) and/or betrixaban (BEVYXXA™) Vaccines In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection by administration of an antibiotic and/or an anti-viral drugs and a vaccine directed to a causative agent of the infection, and/or an inactivated or attenuated causative agent of the infection, or a live, viable or infectious causative agent of the infection. In alternative embodiments, vaccines used to practice methods as provided herein are directed to an exterior-expressed protein of a pathogen, for example, where the pathogen is a bacteria or a virus, for example, the exterior-expressed protein comprises a spike protein of a virus, for example, a spike protein of a coronavirus, for example, a Covid-19 spike protein. In alternative embodiments, vaccines used to practice methods as provided herein are formulated and administered using any formulations, protocols or techniques known in the art, for example, pharmaceutical formulations or vaccines as provided herein can be administered as peptides, or can be administered in the form of nucleic acids that encode the immunogenic peptides or proteins. In alternative embodiments, vaccines used to practice methods as provided herein comprise orally and intra-nasally administered vaccines. In alternative embodiments, vaccines used to practice methods as provided herein comprise administration of inactivated pathogen, for example, an inactivated virus (optionally an inactivated whole or entire pathogen (or virus) or substantially a whole or entire pathogen (or virus), for example, an inactivated coronavirus, for example, and inactivated COVID-19 virus, for example, as manufactured by Valneva, France), Sinopharm, or Bharat Biotech. In alternative embodiments, the pathogen (or virus) is inactivated using a chemical, for example, a beta-propiolactone (BPL) or equivalent, or any means used to inactivate a viruses for a vaccine. This type of inactivation can preserve the structure of the pathogen (for example, viral) proteins, as they would occur in nature. This means the immune system will be presented with something similar to what occurs naturally and mount a strong immune response. In alternative embodiments, after being inactivated, the vaccine (or, the inactivated pathogen, or virus) is highly purified. In alternative embodiments, an adjuvant (or any immune stimulant) is added or co-administered to induce a boosted or strong immune response. In alternative embodiments, vaccines used to practice methods as provided herein are DNA vaccine or RNA vaccines. For example, in alternative embodiments the immunogen-encoding nucleic acid can be a DNA encoding one or more immunogenic peptides or proteins, and the DNA can be carried in an expression vehicle such as a viral vector, for example an adenovirus vector such as an Ad5 or adeno-associated vector (AAV). In alternative embodiments, recombinant adenoviruses as used in vaccines as provided herein can be as described in U.S. patent application no. US 20200399323 A1, which describes for example recombinant adenoviruses including a deletion in or of the E1 region or any deletion that renders the virus replication-defective, for example, the replication-defective virus can include a deletion in one or more of the E1, E3, and/or E4 regions; or, can be as described in U.S. patent application no. US 20190382793 A1, which described how to make recombinant adenoviruses for gene therapy. In alternative embodiments, the immunogen-encoding nucleic acid can be an RNA, for example, mRNA, which can be formulated in a lipid formulation or a liposome and injected for example intramuscularly (IM), for example using formulations and methods as described in U.S. patent application no. US 20210046173 A1, which describes delivering to a subject (for example, via intramuscular administration) an immunogenic composition that comprises a RNA (for example, mRNA) that comprises an open reading frame (ORF) that comprises (or consists of, or consists essentially of) an immunogenic or antigenic sequence as provided herein; wherein optionally the RNA (or the DNA-carrying expression vehicle) is formulated in a liposome, or a lipid nanoparticle (LNP), or nanoliposome, that comprises: non-cationic lipids comprise a mixture of cholesterol and DSPC, or a PEG-lipid, or PEG-modified lipid, or LNP, or an ionizable cationic lipid; or a mixture of (13Z,16Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, cholesterol, DSPC, and PEG-2000 DMG. In alternative embodiments, the PEG-lipid is 1,2-Dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), or, the PEG-lipid is PEG coupled to dimyristoylglycerol (PEG-DMG). In alternative embodiments, the LNP comprises 20-99.8 mole % ionizable cationic lipids, 0.1-65 mole % non-cationic lipids, and 0.1-20 mole % PEG-lipid. In alternative embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of (2S)-1-({6-[(3))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9 Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing. In alternative embodiments, the PEG modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In alternative embodiments, the ionizable cationic lipid comprises: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy) heptadecanedioate (L319), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine. In one embodiment, the lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine or N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, each of which are described in PCT/US2011/052328, the entire contents of which are hereby incorporated by reference. In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. Attenuated, or Live, Viable or Infectious Causative Agent of the Infection In alternative embodiments, provided are methods for treating, ameliorating, decreasing the chances of having any adverse effects from, decreasing the severity of adverse effects from, or preventing an infection, comprising administering to a subject or an individual in need thereof:(a) at least one antibiotic and/or anti-viral drug capable of killing a causative agent of the infection, or completely or partially inhibiting the ability of the causative agent of the infection to replicate or become infectious or cause pathology in the subject or the individual in need thereof; and,(b) (i) at least one dose of a vaccine directed to the causative agent of the infection upon entry into the vaccinated subject or individual in need thereof,wherein the vaccine is capable of initiating an immune response in the individual that can substantially or partially kill or neutralize a causative agent of the infection, or the vaccine can completely, substantially or partially inhibit the ability of the causative agent of the infection to replicate, or be infectious, or cause pathology, in the subject or the individual in need thereof, and/or(ii) an inactivated or attenuated causative agent of the infection, or a live, viable or infectious causative agent of the infection, wherein optionally the live causative agent of the infection is a completely or partially attenuated version of the causative agent,wherein at least one dosage of the at least one antibiotic and/or anti-viral drug is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more days before, or on the day of, a first dose of the at least one of a plurality of dosages of the vaccine is administered, or a dose of the inactivated, attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection. In alternative embodiments, the causative agent of the infection is or comprises a bacteria, protozoan or a virus, orthe causative agent of the infection is or comprises the causative agent of:a viral infection, optionally a coronavirus, a virus that causes a common cold, an influenza virus (optionally an influenza A, B or C), a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus, a viral meningitis, a rhinovirus, a human immunodeficiency virus (HIV), a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EBV), an Adenovirus, a Hantavirus, a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection,a coronavirus infection, optionally a COVID-19 or a COVID-19 variant infection, or a Middle East respiratory syndrome virus (MERS-CoV) infection;malaria caused by a parasite of the genusPlasmodium(optionallyP. vivax, P. falciparum, P. malariae, P. ovale, orP. knowlesi);dengue fever or dengue shock syndrome caused by a virus of the Flaviviridae family or a dengue virus;a Flaviviridae family virus infection or a hepatitis or a hepatocellular carcinoma associated with viral hepatitis caused by a virus of the Flaviviridae family or a virus of the genus Hepacivirus or Hepacivirus C virus or hepatitis C;filariasis, leprosy or streptocerciasis or an infection caused by a parasite of the superfamily Filarioidea (optionallyBrugia malayi, Brugia timori, Wuchereria bancrofti, Loa loa, Mansonella streptocerca, Mansonella ozzardi, orMansonellaperstans);leprosy or an infection caused by a parasite of the genusMycobacterium(optionallyM. lepraeor M. lepromatosis);river blindness or onchocerciasis caused by a parasitic worm or a parasite of the genusOnchocerca(optionallyO. volvulus);a hookworm or a roundworm infection caused by a parasite of the genusAncylostoma(optionallyA. duodenaleorA. ceylanicum) or Necator (optionallyN. americanus);trichuriasis or a whipworm infection caused by a parasite of the genusTrichuris(optionallyT. trichiura); roundworm or anAscarisinfection that is caused byAscaris lumbricoides;scabies or a mite-carried infection caused by the parasite of the genusSarcoptes(optionallyS. scabiei);typhus or an infection caused by a lice or a parasite of the order Phthiraptera (optionallyPediculus humanuscapitis);enterobiasis or an infection caused by a pinworm or a parasite of the genusEnterobius(optionallyE. vermicularis); and/orpulicosis or an infection caused by a flea or an insect of the order Siphonaptera or of the genusPulex(optionallyP. irritans). In alternative embodiments, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is administered orally or by inhalation. Alternatively, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection can be administered by inclusion of the live, viable or infectious causative agent of the infection in a liquid (optionally to be administered as a drink or in drops such as nasal drops), a tablet, a lozenge, an aerosol, spray, or mist formulation that is inhaled or administered nasally or orally (optionally, by a puffer of a nebulizer), or the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is formulated in a liquid (optionally the liquid is a sterile saline) solution which is ingested or gargled by the individual in need thereof. In alternative embodiments, the source of the inactivated or attenuated causative agent of the infection, or the administered live, viable or infectious causative agent of the infection can be from an infected individual, such as a human patient, a domesticated, wild or lab animal, or from a lab-grown culture. In alternative embodiments, the source of the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is from swab or sputum or other biological samples from an infected individual or patient. In alternative embodiments, the sputum or other biological sample from an infected individual or patient is diluted in a water or a saline prior to administrations. In alternative embodiments, the administered inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection is attenuated (in other words, its ability to cause pathogenesis is completely, substantially or partially abrogated or diminished, for example is genetically deleted or diminished by genomic engineering). In alternative embodiments, to generate an attenuated (e.g., completed inactivated) version of a causative agent of the infection to be administered, the causative agent of the infection is passaged multiple times in culture (or in vitro) or in an animal (or in vivo), where variants from each passage are selected for a phenotype and/or genotype that has diminished ability to cause pathogenesis. In alternative embodiments, to generate an attenuated (e.g., completed inactivated, completely non-infectious) version of a causative agent of the infection to be administered, the causative agent of the infection is treated with radiation and/or a chemical. For example, the chemical can be iodine (for example, povidone-iodine or PVP-1, also known as iodopovidone, or BETADINE™, WOKADINE™, PYODINE™), or any complex of polyvinylpyrrolidone and iodine, alcohol and/or formalin. In alternative embodiments, the causative agent of the infection is rendered inactive, or non-infectious, by exposing the causative agent of the infection to iodine or povidone-iodine or PVP-1 (povidone is also known as polyvinylpyrrolidone (PVP), or 1-vinyl-2-pyrrolidinon-polymere), also known as iodopovidone, or BETADINE™, WOKADINE™, PYODINE™, as in the production of nasodine (Firebrick Pharma Pty Ltd, Australia). Povidone-iodine is a chemical complex of povidone, hydrogen iodide, and elemental iodine or triiodide (I3−); and it contains 10% povidone, with total iodine species equaling 10,000 ppm or 1% total titratable iodine, and it works by releasing iodine which results in the death of a range of microorganisms. In alternative embodiment, the causative agent of infection is mixed with PVP-I and water, ethyl alcohol, isopropyl alcohol, polyethylene glycol or glycerol. In alternative embodiments, the attenuated, or inactivated, causative agent, or live causative agent, is administered with an adjuvant, where the adjuvant can comprise: an inorganic compound such as alum (e.g., potassium alum), an aluminium salt or aluminium hydroxide, aluminium phosphate, or calcium phosphate; an oil such as paraffin oil, propolis or Adjuvant 65; a bacterial product such as killed bacteria of the genusBordetellaorMycobacteriumor of the speciesBordetella pertussisorMycobacterium bovis; a plant saponin or soybean extract; a cytokine such as interleukin-1 (IL-1), IL-2 or IL-12; Freund's complete adjuvant or Freund's incomplete adjuvant; and/or, an organic compound such as squalene. In alternative embodiments, the attenuated, or inactivated, causative agent, or live causative agent, with or without an adjuvant, is administered by nasal spray or nebulizer, or orally for example by lozenge, tablet, capsule or geltab, or by subcutaneous injection, or intramuscularly (IM), or by suppository, or via an implant. In alternative embodiments, attenuated viruses are made using a live attenuated codon-pair-deoptimized virus approach as described for example in Wang et al PNAS, Jul. 20, 2021, vol. 18 (29) e2102775118; or as described by Coleman et al. Science 320, 1784-1787 (2008), or Cheng et al J. Virol. 89, 3523-3533 (2015), or Gonçalves-Carneiro, mBio 12, e02238-20 (2021). For example, methods as provided herein comprise administration of the Wang et al, COVI-VAC™ attenuated virus, which was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. As described by Wang et al, synthetic highly attenuated live vaccine is generated by recoding portions of the WT SARS-CoV-2 genome according to the SAVE algorithm of codon-pair bias deoptimization. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. Attenuated viruses can be generated from viral genomes recover from WT SARS-CoV-2, strain USA-WA1/2020 (GenBank accession No. MN985325). In alternative embodiments, the inactivated or attenuated causative agent of the infection, or the live, viable or infectious causative agent of the infection, is administered in unit dosages of between about 10 to 50, or 1 to 20, trillion infectious units (or particles, if attenuated), or between about one infectious unit to 10, 20 or 30 billion infection units (or particles, if attenuated). Hand-Held or Portable Devices In alternative embodiments, provided are portable, for example, hand-held (or worn around the neck), medical devices, for example, an inhaler, ionizer, asthma puffer or nebulizer, capable of administering an inhalation product comprising a composition or formulation as provided herein or as described herein, for example, an inactivated or attenuated agent of the infection, or a live, viable or infectious causative agent of the infection with or without a vaccine or with or without an adjuvant, or with or without an antimicrobial drug, for example, as described herein. In alternative embodiments, a portable, for example, hand-held, medical device, for example, inhaler, asthma puffer or nebulizer, as provided herein can administer ionized air or air comprising generated electrons and/or negatively-charged oxygen ions and/or positively-charged ions. In alternative embodiments, a portable or hand-held medical device as provided herein comprises a cassette, packette, interchangeable disk (for example, for holding a powder) or reservoir (optionally a refillable reservoir) in or on the product of manufacture, or a removable cassette or packette, interchangeable disk (for example, for holding a powder) that can be inserted into a slot or port on the product of manufacture, or a separate reservoir or container operatively linked or joined to the product of manufacture, that comprises a vaccine or or live or attenuated causative agent of invention or a formulation or a medication, for inhalation as provided herein for delivery to a user. In alternative embodiments, provided is a modified hairdryer-type medical device capable of having an adjustable temperature, adjustable air intake; a provision (or receptacle) for insertion of a drug-containing cassette (optionally providing or delivering a combination of medications, or the live or attenuated causative agents and/or a vaccine as provided herein); and/or warm-to hot air availability (optionally with temperature control) to inhibit viral and bacterial growth. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection and/or a vaccine as provided herein drug or a medication or combinations thereof to a user is fabricated as a meter-dose inhaler (MDI) (either open or closed mouth MDI), which can comprise a pressurized canister of the drug or medication in a plastic case with a mouthpiece, and a holding chamber having a plastic tube with a mouthpiece, a valve to control mist delivery and a soft sealed end to hold the MDI; the holding chamber can assist delivery of the drug or medication to the nose and/or lungs, for example, as an AEROCHAMBER™ device. In alternative embodiments, the inhaler or nebulizer is breath activated, for example, as an REDIHALER™ device. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection and/or a vaccine or a drug or a medication or combinations thereof to a user is fabricated a dry powder inhaler (such as a dry powder disk inhaler, for example, as a DISKUS™ device), optionally having a dose counter window so user can see how many doses are left), for example, where the powder is dose dispensed by (using) a disposable, refillable or replaceable cassette, packette or disk; and the dry powder dispensing can be breath activated, for example, as an AEROLIZER™, FLEXHALER™, PRESSAIR™, DISKUS™, HANDIHALER™, TWISTHALER™, ELLIPTA™, NEOHALER™, RESPICLICK™, ROTAHALER™ or TUBUHALER™ device. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection, or a vaccine or a drug or a medication or combinations thereof, to a user is fabricated a nebulizer or soft mist inhaler, which can comprise a nebulizer delivery system comprising a nebulizer (for example, a small plastic bowl with a screw-top lid) and a source for compressed air to generate a mist comprising the drug or medication, which also can be dose dispensed using a disposable, refillable or replaceable cassette, packette or disk. In alternative embodiments, a medical device as provided herein for inhalation delivery of a live or attenuated causative agent of infection, or a vaccine or drug or a medication or combinations thereof to a user is fabricated a dry powder inhaler (such as a dry powder disk inhaler, for example, as a DISKUS™ device), optionally having a dose counter window so user can see how many doses are left), for example, where the powder is dose dispensed by (using) a disposable, refillable or replaceable cassette, packette or disk; and the dry powder dispensing can be breath activated, for example, as an AEROLIZER™, FLEXHALER™, PRESSAIR™, DISKUS™, HANDIHALER™, TWISTHALER™, ELLIPTA™, NEOHALER™, RESPICLICK™, ROTAHALER™ or TUBUHALER™ device. Products of Manufacture and Kits Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein. Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections. As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court. Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. | 273,350 |
11857618 | DETAILED DESCRIPTION OF THE INVENTION Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. “Subject” refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. As used herein, the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof. As used herein, the term “intermixed with” when used to describe administration in combination with an additional treatment means that the agent may be administered “together with.” In certain embodiments, an “effective amount” in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a viral infection, disease or symptom associated therewith; (ii) reduce the duration of a viral infection, disease or symptom associated therewith; (iii) prevent the progression of a viral infection, disease or symptom associated therewith; (iv) cause regression of a viral infection, disease or symptom associated therewith; (v) prevent the development or onset of a viral infection, disease or symptom associated therewith; (vi) prevent the recurrence of a viral infection, disease or symptom associated therewith; (vii) reduce or prevent the spread of a viral from one cell to another cell, one tissue to another tissue, or one organ to another organ; (viii) prevent or reduce the spread of a viral from one subject to another subject; (ix) reduce organ failure associated with a viral infection; (x) reduce hospitalization of a subject; (xi) reduce hospitalization length; (xii) increase the survival of a subject with a viral infection or disease associated therewith; (xiii) eliminate a viral infection or disease associated therewith; (xiv) inhibit or reduce viral replication; (xv) inhibit or reduce the entry of an virus into a host cell(s); (xvi) inhibit or reduce replication of the virus genome; (xvii) inhibit or reduce synthesis of virus proteins; (xviii) inhibit or reduce assembly of virus particles; (xix) inhibit or reduce release of virus particles from a host cell(s); (xx) reduce virus titer; and/or (xxi) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. In certain embodiments, the effective amount does not result in complete protection from an influenza virus disease but results in a lower titer or reduced number of viruses compared to an untreated subject with a viral infection. In certain embodiments, the effective amount results in a 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reduction in titer of virus relative to an untreated subject with a viral infection. Benefits of a reduction in the titer, number or total burden of virus include, but are not limited to, less severe symptoms of the infection, fewer symptoms of the infection and a reduction in the length of the disease associated with the infection. “HA” and “hemagglutinin” refer to any hemagglutinin known to those of skill in the art. In certain embodiments, the hemagglutinin is influenza hemagglutinin, such as an influenza A hemagglutinin, an influenza B hemagglutinin, or an influenza C hemagglutinin. A typical hemagglutinin comprises domains known to those of skill in the art including a signal peptide (optional herein), a stem domain, a globular head domain, a luminal domain (optional herein), a transmembrane domain (optional herein) and a cytoplasmic domain (optional herein). “NA” and “neuraminidase” refer to any neuraminidase known to those of skill in the art. In certain embodiments, the neuraminidase is influenza neuraminidase, such as an influenza A neuraminidase, an influenza B neuraminidase, or an influenza C neuraminidase. As used herein, the terms “neuraminidase” and “NA” encompass neuraminidase polypeptides that are modified by post-translational processing such as disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation). As used herein, the terms “chimeric influenza virus hemagglutinin polypeptide,” “chimeric influenza virus HA polypeptide,” “chimeric hemagglutinin polypeptide,” “chimeric HA,” “chimeric hemagglutinin,” and “chimeric influenza hemagglutinin polypeptide” refer to an influenza hemagglutinin that comprises an influenza virus hemagglutinin stem domain and an influenza virus hemagglutinin head domain, wherein the influenza virus hemagglutinin head domain is heterologous to the influenza virus hemagglutinin stem domain. As used herein, the term “heterologous” in the context of a polypeptide, nucleic acid or virus refers to a polypeptide, nucleic acid or virus that is not normally found in nature or not normally associated in nature with a polypeptide, nucleic acid, or virus of interest. For example, a “heterologous polypeptide” may refer to a polypeptide derived from a different virus, e.g., a different influenza strain or subtype, or an unrelated virus or different species. In specific embodiments, when used in the context of a globular head domain of a chimeric influenza virus hemagglutinin described herein, the term heterologous refers to an influenza HA globular head domain that is associated with an influenza HA stem domain that it would not normally be found associated with (e.g., the head and stem domains of the HA would not be found together in nature). As used herein, the term “infection” means the invasion by, multiplication and/or presence of a virus in a cell or a subject. In one embodiment, an infection is an “active” infection, i.e., one in which the virus is replicating in a cell or a subject. Such an infection is characterized by the spread of the virus to other cells, tissues, and/or organs, from the cells, tissues, and/or organs initially infected by the virus. An infection may also be a latent infection, i.e., one in which the virus is not replicating. In certain embodiments, an infection refers to the pathological state resulting from the presence of the virus in a cell or a subject, or by the invasion of a cell or subject by the virus. Vaccines and Adjuvant Mixtures Viral vaccines are typically produced by injection of a desired viral strain into eggs, or other cells, and incubation for several days to allow the viruses to replicate. The fluid containing virus is harvested. For inactivated vaccines, virus nucleic acids are completely inactivated (or killed) with a chemical, for example, formalin or beta-propiolactone, or by physical means. After, the virus antigens are typically purified prior to use in the vaccine. Influenza viruses may be propagated in embryonated chicken eggs. The virus-containing fluids are harvested and inactivated with formaldehyde. Influenza virus may be concentrated and purified in a linear sucrose density gradient solution using a continuous flow centrifuge. The virus may be chemically disrupted using a nonionic surfactant, octoxinol-9, producing a “split virus.” The split virus may be further purified by chemical means and suspended in sodium phosphate-buffered isotonic sodium chloride solution. Attenuated vaccines are those created by passaging a virus in cultured cells. Virus strains are selectively and repeatedly exposed to, collected, and subsequently grown in non-human cells. Repeatedly selecting strains most capable of non-human cell infection and replication are eventually weakened in their ability to infect human cells, e.g., virus that are selected as superior at entering the chicken cells become less able to infect human cells. Viruses may also be attenuated by deleterious gene mutation, altered replication fidelity, codon deoptimization. Recombinant viral vaccines also may be created synthetically using recombinant techniques. A DNA plasmid encoding a viral antigen may be combined with a baculovirus. The role of the baculovirus is to help transport the DNA instructions for making the viral antigen and/or proteins that assemble into a virus like particle containing the viral antigen but lack intact viral nucleic acids. Once the recombinant virus enters a host cell line, the cells produce the viral antigens or particles containing the same. In certain embodiments, this disclosure relates to methods for inducing an immune response (e.g., an antibody response) against a virus, such as influenza virus, using a viral vaccine, e.g., an influenza viral vaccine, and an adjuvant mixture comprising a saponin and an agonist of the intracellular stimulator of interferon genes pathway such as cyclic dinucleotide. In certain embodiments, immunization regimens involve the intradermal administration of an effective amount of a hemagglutinin, chimeric hemagglutinin, a headless hemagglutinin or another influenza virus stem domain based construct (e.g., the hemagglutinin stem domain or a fragment thereof) in combination with saponins and an agonist of the intracellular stimulator of interferon genes pathway to a subject. In certain aspects, the immunization regimens also involve the administration of an influenza virus hemagglutinin (HA) and/or neuraminidase (NA) immunogen(s). Also provided herein are vaccine compositions for use in methods of immunizing against influenza virus in human subjects. A full-length influenza hemagglutinin typically comprises an HAI domain and an HA2 domain. In certain embodiments, a globular head domain is heterologous to the stem domain formed by the other segments of the HA1 domain and the HA2 domain. In some embodiments, the immunization/vaccinating regimens involve intradermally administering to the subject an immunogenic composition comprising an inactivated influenza virus in combination with saponins and an agonist of the intracellular stimulator of interferon genes pathway to a subject. In certain embodiments, the inactivated influenza virus comprises a hemagglutinin (HA) or chimeric HA and optionally a neuraminidase (NA). In certain embodiments, the chimeric HA comprises an influenza virus HA globular head domain and the HA stem domain, wherein the globular head domain is heterologous to the HA stem domain. In certain embodiments, the HA stem domain of the chimeric HAs are from one influenza virus subtype, and the HA globular head domains of the chimeric HAs are from other influenza virus subtypes. In certain embodiments, the HA stem domain of the chimeric HAs is from an influenza virus H1 or H3 subtype. In some embodiments, the influenza virus HA globular head domain is from an influenza A virus H4, H5, H7, H8, H11, H12, H14 or H15 subtype. In certain embodiments, the influenza virus HA globular head domain is the influenza virus HA globular head domain of an influenza virus of subtype H4, H6, H7, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18. In some embodiments, the influenza virus neuraminidase polypeptide is from an influenza virus of subtype N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and/or N11 In certain embodiments, provided herein is a method for immunizing against influenza virus in a human subject, comprising (a) administering to the subject a first vaccine comprising a chimeric HA, a headless HA or another influenza virus stem domain based construct (e.g., the HA stem domain or a fragment thereof), or an influenza virus hemagglutinin core polypeptide and/or an NA immunogen(s) or a vector comprising such a construct; and (b) a certain time after the administration of the first vaccine formulation, intradermally administering to the subject an inactivated influenza virus vaccine, or a vector comprising such a construct in combination with saponins and an agonist of the intracellular stimulator of interferon genes pathway. In certain embodiments, the second immunogenic composition is administered about 6 weeks, about 12 weeks, about 4 months, about 6 months, or about 9 months after the administration of the first immunogenic composition. In another specific embodiment, the second immunogenic composition is administered 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months after the administration of the first immunogenic composition. Viral polypeptides described herein can be incorporated into virus-like particle (VLP) vectors, e.g., purified/isolated VLPs. VLPs generally comprise viral polypeptide(s) derived from a structural protein(s) of a virus. In some embodiments, the VLPs are not capable of replicating. In certain embodiments, the VLPs may lack the complete genome of a virus or comprise a portion of the genome of a virus. In some embodiments, the VLPs are not capable of infecting a cell. In some embodiments, the VLPs express on their surface one or more of viral (e.g., virus surface glycoprotein) or non-viral (e.g., antibody or protein) targeting moieties known to one skilled in the art. In specific embodiments, VLPs, e.g., VLPs comprising an influenza hemagglutinin (HA) polypeptide and/or an influenza virus neuraminidase (NA) polypeptide, are expressed in cells (such as, e.g., mammalian cells (e.g., 293T cells) and insect cells (e.g., High Five cells and Sf9 cells). In certain embodiments, the VLPs are expressed in cells that express surface glycoproteins that comprise sialic acid. In accordance with such embodiments, the cells are cultured in the presence of neuraminidase. In certain embodiments, VLPs, e.g., VLPs comprising an influenza hemagglutinin (HA) polypeptide and/or an influenza virus neuraminidase polypeptide, are expressed in cells that do not express surface glycoproteins that comprise sialic acid. In certain embodiments, a viral polypeptide may be incorporated into a virosome. A virosome containing a viral polypeptide and/or an influenza virus polypeptide may be produced using techniques known to those skilled in the art. For example, a virosome may be produced by disrupting a purified virus, extracting the genome, and reassembling particles with the viral proteins (e.g., influenza virus polypeptide) and lipids to form lipid particles containing viral proteins. In certain embodiments, provided herein are subunit vaccines comprising a viral polypeptide in combination with a saponin and an agonist of the intracellular stimulator of interferon genes pathway. In certain embodiments, the subunit vaccine is prepared using influenza virus that is propagated in embryonated chicken eggs. In certain embodiments, provided herein are immunogenic compositions/vaccines comprising an inactivated virus containing a viral peptide (e.g., a chimeric influenza virus hemagglutinin polypeptide and/or an influenza virus neuraminidase polypeptide) in combination with a saponin and an agonist of the intracellular stimulator of interferon genes pathway. Compositions described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, conjunctival, and subcutaneous routes. In some embodiments, a composition is formulated for topical administration, for example, for application to the skin. In specific embodiments, the route of administration is nasal, e.g., as part of a nasal spray. In certain embodiments, a composition is formulated for intramuscular administration. In some embodiments, a composition is formulated for subcutaneous administration. In certain embodiments, a composition is not formulated for administration by injection. In certain embodiments, immunogenic compositions disclosed herein are administered intradermally. In certain embodiments, this disclosure contemplates administration using a transdermal patch for diffusion of the drug across the skin or by microneedle injection. In certain embodiments, it may be desirable to introduce the pharmaceutical compositions into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In some embodiments, cells stimulated with vaccine and adjuvant combinations disclosed herein in vitro may be introduced (or re-introduced) into a subject using techniques known to one of skill in the art. In some embodiments, the cells can be introduced into the dermis, under the dermis, or into the peripheral blood stream. In some embodiments, the cells introduced into a subject are preferably cells derived from that subject, to avoid an adverse immune response. In other embodiments, cells also can be used that are derived from a donor host having a similar immune background. Other cells also can be used, including those designed to avoid an adverse immunogenic response. In certain embodiments, provided herein are devices with a needle or an array of needles for intradermal administration wherein the needle(s) and used to administer compositions disclosed herein and/or the needles are coated with vaccine compositions and adjuvant mixtures disclosed herein. In certain embodiments, the vaccines comprise an inactivated virus, attenuated virus, virus protein, virus like particle, or virosome in combination with a saponin and an agonist of the intracellular stimulator of interferon genes pathway. In certain embodiments, the needles may be hollow or solid and made out of a biodegradable material. In certain embodiments, provided herein is a device comprising a substrate having an array of microneedles for intradermal administration wherein the needles are coated with a vaccine and an adjuvant composition comprising a saponin and an agonist of the intracellular stimulator of interferon genes pathway. In certain embodiments, the microneedle devices include a substrate; one or more microneedles; and, optionally, a reservoir for delivery of drugs, as well as pump(s), sensor(s), and/or microprocessor(s) to control the interaction of the foregoing. In certain embodiments, the microneedles are between 1 μm and 1 mm long, inclusive or are between 10 μm and 500 μm long, inclusive or are between 30 μm and 200 μm long, inclusive. In certain embodiments, the microneedles have a cross-sectional dimension between 10 nm and 1 mm, inclusive or have a cross-sectional dimension between 1 μm and 200 μm, inclusive, or have a cross-sectional dimension between 10 μm and 100 μm, inclusive, or have a circular cross section with an outer diameter between 10 μm and 100 μm, inclusive. The substrate includes the base to which the microneedles are attached or integrally formed. A reservoir may also be attached to the substrate. In certain embodiments, microneedles of the substrate can be constructed from a variety of materials, including metals, ceramics, semiconductors, organics, polymers, biodegradable polymers, and composites. Preferred materials of construction include pharmaceutical grade stainless steel, gold, titanium, nickel, iron, gold, tin, chromium, copper, alloys of these or other metals, silicon, silicon dioxide, and polymers. Representative biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone). Representative non-biodegradable polymers include polycarbonate, polymethacrylic acid, ethylenevinyl acetate, polytetrafluoroacetate (TEFLON™), and polyesters. Although viral vaccines with specified adjuvants are exemplified used herein, it is contemplated the one can apply methods and use compositions disclosed herein for vaccination against other infectious microbes such as bacteria, fungus, or other parasites. Agonists of the Intracellular Stimulator of Interferon Genes (STING) Pathway Stimulator of interferon genes (STING) is a protein that in humans is encoded by the TMEM173 gene. STING induces type I interferon production when cells are infected with intracellular pathogens. Cyclic di-nucleotides are agonists of the intracellular stimulator of interferon genes (STING) pathway. The signaling cascade triggered by activation of STING leads to production of IFN and other cytokines important for innate immunity. However, rather high amounts of cGAMP or other cyclic di-nucleotides have been required for adjuvant activity. Experiments were performed to determine whether combinations with other adjuvants increase adjuvant efficiency of the cGAMP in elderly subjects. In the presence of a membrane-active saponin-based adjuvant, the immunogenicity of an influenza subunit vaccine was assessed. In certain embodiments, this disclosure relates to vaccination methods comprising intradermally administering to a human subject an effective amount of a virus, attenuated virus, virus protein, virus like particle, or virosome in combination with a saponin and a cyclic dinucleotide or derivative. In certain embodiments, the cyclic dinucleotide or derivative of this disclosure is cyclic-di-AMP, cyclic-di-GMP, cyclic-di-IMP, cyclic-AMP-GMP, cyclic-AMP-IMP, cyclic-GMP-IMP, and cyclic-GMP-AMP (cGAMP). In certain embodiments, the cyclic dinucleotide or derivative of this disclosure has a fluoro substitution of one or both 2′-hydroxyls on cyclic-di-AMP, cyclic-di-GMP, cyclic-di-IMP, cyclic-AMP-GMP, cyclic-AMP-IMP, cyclic-GMP-IMP, In certain embodiments, the cyclic dinucleotide or derivative of this disclosure comprises bis-3′,5′ linkage between the two nucleotides or comprise one 2′,5′ linkage and one 3′,5′ linkage. In certain embodiments, the cyclic dinucleotide or derivative of this disclosure dinucleotide is a compound of Formula I or Formula II: or salt thereof, wherein each R1and R2is independently a purine; each R3and R4is independently H, OH or F, and each R5and R6is independently OH or SH. In certain embodiments, purines R1and R2are independently selected from the following structures: each R7or R11is independently —CR— or —N—; R8is —C(R)2—, —O—, or —NR—;each R9, R10, R12, R13, or R14is independently selected from the group consisting of hydrogen, halogen, —CN, —OR, —SR, —N(R)2, —C(O)R, —CO2R, —S(O)R, —S(O)2R, —C(O)N(R)2, —SO2N(R)2, —OC(O)R, —N(R)C(O)R, —N(R)N(R)2, —C═NOR, —N(R)C(O)N(R)2, —N(R)SO2N(R)2, —N(R)SO2R, —OC(O)N(R)2or an optionally substituted substituent selected from the group consisting of C1-12aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; wherein each R is independently an optionally substituted substituent selected from the group consisting of C1-12aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or two R groups on the same nitrogen are taken together with their intervening atoms to form an optionally substituted 3-7 membered saturated, partially unsaturated, or heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; wherein each C1-12aliphatic, phenyl, 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, 3-7 membered saturated or partially unsaturated heterocyclic ring, 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring, and 5-6 membered heteroaryl ring, or two R groups on the same nitrogen taken together to form 3-7 membered saturated, partially unsaturated, or heteroaryl ring is optionally substituted with 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 independently selected substituents selected from the group consisting of halogen, —CN, —NO2, —OH, ═O, —NH2, C1-6alkyl, C1-6alkoxy, C1-6alkylamino, and C1-6di-alkylamino. In certain embodiments, purines R1and R2are independently selected from adenine, guanine, isoguanine, hypoxanthine, or xanthine. In certain embodiments, the cyclic dinucleotide is 2′,3′-cGAMP (cyclic [G(2′,5′)pA(3′,5′)p]): derivative, ester, or salt thereof. Adjuvant Saponins Purified from an Aqueous Extract of the Bark of the South American Tree,Quillaia saponariaMolina “Quil-A,” refers to an adjuvant mixture of triterpenoid quillaic acids glycosidically linked to carbohydrate moieties isolated from the bark of the South American tree,Quillaja saponariaMolina. See U.S. Pat. No. 5,057,540. Quil-A veterinary applications induces humoral and cellular responses. However, Quil-A is typically considered unsuitable for human use due to its highly complex mixture nature. “QS-21” is a purified component of Quil-A useful as an adjuvant. See U.S. Pat. No. 6,231,859. For example, ASO1 adjuvant contains QS-21 and 3-O-desacyl-4′-monophosphoryl lipid A (MPL). Malaria vaccine studies with using ASO1 adjuvant showed enhanced immunogenicity in intramuscular-based vaccinations. See The RTS,S Clinical Trials Partnership reports results of a phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N Engl J Med, 2011, 365, 1863-1875. Saponins may be purified from an aqueous extract of the bark of the South American tree,Quillaja saponariaMolina. The predominant purifiedQuillaja saponinshave been identified as fractions QA-7, QA-17, QA-18, and QA-21. These saponins may be purified by high pressure liquid chromatography (HPLC) and low-pressure silica chromatography. In certain embodiments, QA-19 may be removed from the other components. Aqueous extracts of theQuillaja saponariaMolina bark may be dialyzed against water. The dialyzed extract may be lyophilized to dryness, extracted with methanol, and the methanol-soluble extract may be further fractionated on silica gel chromatography and by reverse phase high pressure liquid chromatography (RP-HPLC) as described in U.S. Pat. No. 5,057,540. Peaks (denominated QA-1 to QA-22) are reported to be separable. Each peak exhibited a single band on reverse phase thin layer chromatography. The individual components were identified by retention time on a Vydac C4 HPLC column as reported in U.S. Pat. No. 5,057,540. The substantially pure QA-7 saponin is characterized as having immune adjuvant activity, containing about 35% carbohydrate per dry weight, having a uv absorption maxima of 205-210 nm, a retention time of approximately 9-10 minutes on RP-HPLC on a Vydac C4 column having 5 μm particle size, 330 Å pore, 4.6 mm ID×25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 52-53% methanol from a Vydac C4 column having 5 μm particle size, 330 Å pore, 10 mM ID×25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of approximately 0.06% in water and 0.07% in phosphate buffered saline, causing no detectable hemolysis of sheep red blood cells at concentrations of 200 μg/ml or less, and containing the monosaccharide residues terminal rhamnose, terminal xylose, terminal glucose, terminal galactose, 3-xylose, 3,4-rhamnose, 2,3-fucose, and 2,3-glucuronic acid, and apiose. The substantially pure QA-17 saponin is characterized as having adjuvant activity, containing about 29% carbohydrate per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 35 minutes on RP-HPLC on a Vydac C4 column having 5 μm particle size, 330 Å pore, 4.6 mm ID×25 cm L in a solvent of 40 mM acetic acid in methanol-water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 63-64% methanol from a Vydac C4 column having 5 μm particle size, 330 Å pore, 10 mm ID×25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of 0.06% (w/v) in water and 0.03% (w/v) in phosphate buffered saline, causing hemolysis of sheep red blood cells at 25 μg/ml or greater, and containing the monosaccharide residues terminal rhamnose, terminal xylose, 2-fucose, 3-xylose, 3,4-rhamnose, 2,3-glucuronic acid, terminal glucose, 2-arabinose, terminal galactose and apiose. The substantially pure QA-18 saponin is characterized as having immune adjuvant activity, containing about 25-26% carbohydrate per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 38 minutes on RP-HPLC on a Vydac C4 column having 5 μm particle size, 330 Å pore, 4.6 mm ID×25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 64-65% methanol from a Vydac C4 column having 5 μm particle size, 330 Å pore, 10 mm ID×25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of 0.04% (w/v) in water and 0.02% (w/v) in phosphate buffered saline, causing hemolysis of sheep red blood cells at concentrations of 25 μg/ml or greater, and containing the monosaccharides terminal rhamnose, terminal arabinose, terminal apiose, terminal xylose, terminal glucose, terminal galactose, 2-fucose, 3-xylose, 3,4-rhamnose, and 2,3-glucuronic acid. The substantially pure QA-21 saponin is characterized as having immune adjuvant activity, containing about 22% carbohydrate per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 51 minutes on RP-HPLC on a Vydac C4 column having 5 μm particle size, 330 Å pore, 4.6 mm ID×25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 69 to 70% methanol from a Vydac C4 column having 5 μm particle size, 330 Å pore, 10 mm×ID 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, with a critical micellar concentration of about 0.03% (w/v) in water and 0.02% (w/v) in phosphate buffered saline, causing hemolysis of sheep red blood cells at concentrations of 25 μg/ml or greater, and containing the monosaccharides terminal rhamnose, terminal arabinose, terminal apiose, terminal xylose, 4-rhamnose, terminal glucose, terminal galactose, 2-fucose, 3-xylose, 3,4-rhamnose, and 2,3-glucuronic acid. EXAMPLES Methods Female BALB/c (AnNCrl) mice from Charles River Labs (Wilmington, MA) were used in all experiments. Mice were housed and subjected to a 12/12-h light/dark cycle until they reached 4 (adults) or 19 (aged) months of age. H1N1 Influenza A/California07/09 virus was obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA), grown in MDCK cells and used for hemagglutination inhibition (HAI) titration of sera. The virus was mouse-adapted by serial passage in the lungs of adult BALB/c mice and was used in challenge experiments. Influenza A (H1N1) 2009 A/California/07/09 H1N1 vaccine was obtained from BEI resources (NR-20347). The AddaVax™ formulation is an oil-in-water emulsion with about 160 nm in diameter (e.g., 140-170 nm or 130-180 nm in diameter). The stock solutions of Quil-A and cGAMP (2′,3′-cyclicGAMP) were prepared in 50 mM potassium phosphate buffer, pH 7.4. AddaVax™ (nanoemulsions produced from Span™ 85 (sorbitan trioleate 0.5%) in 5% squalene oil and 0.5% Tween™ 80 (Polyoxyethylene (80) sorbitan monooleate, 0.5%) in 10 mM sodium citrate buffer pH 6.5), 25 μl per dose, was mixed with the same volume of vaccine prior to immunization. Except for the high dose vaccine formulation, the amount of vaccine antigen was 1 μg in animal experiments. The immunogen was mixed with Quil-A in a vaccine/adjuvant ratio between 1:1 and 1:10 and with cGAMP between 1:1 and 1:5 (wt/wt, μg), as specified for each experiment. BALB/c mice were employed that were 19 months old at the time of vaccination and are classified as aged, as well as 4-month-old mature adult mice. Mice were immunized once intramuscularly (IM) by injection (0.05 ml volume, 30-gauge needle) either into the upper quadrant of the hind leg, or intradermally (ID) into depilated dorsal skin (bleb was observed) under xylazine/ketamine anesthesia. Blood samples were collected from the fascial vein on days 7, 14, and 28 post vaccination and analyzed for HAI titers and vaccine-specific immunoglobulins. HAI titers were converted into log 2 values for statistical analysis. For challenge studies, aged mice were infected with approximately 300 plaque forming units (pfu) of the mouse-adapted virus, and adult mice received a 10-fold higher dose which was equivalent to 70×LD50. Challenge was performed by intranasal installation of 30 μl of diluted virus under brief isofluorane anesthesia 5.5 weeks after single immunization. Mice were monitored for signs of infection for 2 weeks. The humane endpoint used for euthanasia was 25% loss of the initial body weight. HeLa cells and murine embryonic fibroblasts isolated from the wild type (STING+/+) or STING knockout (STING−/−) mice with C57BL/6J genetic background, were grown in 48-well plates in DMEM media supplemented with 1% FBS and Penn/Strep antibiotics. Confluent cells were treated with A/California/07/09 H1N1 vaccine and individual adjuvants or their combination for 1 h at 37° C., after which they were immediately collected on ice into reducing Laemmli sample buffer supplemented with protease inhibitors, phosphatase inhibitors and DNAse I. Cell lysates were analyzed by SDS-PAGE and western blot, and probed for pIRF3 and actin using antibodies and ECL detection and imager software for quantification. cGAMP and Quil-A as Individual Adjuvants in Aged Mice The effects of cGAMP or Quil-A administered were explored with 1 μg of purified hemagglutinin (HA) of A/California 07/09 (H1N1) virus as a vaccine to evaluate candidate adjuvants in aged mice. The unadjuvanted vaccine was not protective: only 22% of vaccinated animals survived the challenge. In experiments all aged mice immunized intradermally (ID) with the vaccine supplemented with 5 μg cGAMP succumbed to infection upon challenge (FIGS.1A,B). Quil-A alone, in a 5 μg dose, increased survival from 22 to 75% (FIG.1A) with about 14% maximal weight loss (FIG.1B). Compared with the unadjuvanted vaccine, the Quil-A supplemented formulation induced a significant 10 to 30-fold increase in vaccine-specific antibody levels, while cGAMP alone induced 3 to 4-fold increase in IgG1 and IgG/IgM by day 14 (FIGS.1C-F). The use of Quil-A as adjuvant elicited an increase in the IgG2a level by seven fold detected as soon as day 7 of vaccination (FIG.1E), but the changes in the IgG2a/IgG1 ratios were not statistically significant between groups of vaccinated mice (FIG.1G), and the HAI titers remained mostly below the level of detection in all groups (FIG.1H). These data indicate that in aged mice, Quil-A alone is more effective than cGAMP alone at the concentrations tested, but neither adjuvant ensured complete protection against live virus challenge. Effect of Quil-A+cGAMP Combination in Aged Mice Aged mice were immunized with the same vaccine adjuvanted with a combination of 5 μg of each compound by ID or IM injections. It was observed that survival of the ID-immunized animals increased from 22 to 80%, with a 12% average weight loss after challenge. When this formulation was delivered IM, a remarkable improvement was observed in survival from zero to 100%, and the average maximal weight loss was as low as 5% in this group (FIGS.2A,B). All isotypes of vaccine-induced antibodies increased to a greater extent than was observed with the individual adjuvants (compare inFIGS.1C-Eand2C-E). In particular, the levels of IgG2a isotype antibodies exhibited a 10-15-fold increase on day 7 post vaccination in the IM or ID groups, respectively, compared to the unadjuvanted vaccine delivered by the same route (insert onFIG.2E). The difference reached 93-fold in the ID group 1 week later. By day 28 the level of vaccine specific IgG2a rose slightly in the unadjuvanted groups, but it remained significantly higher in the adjuvanted groups (FIG.2E). A significant 10-fold increase in the vaccine-specific IgG2a/IgG1 ratio, indicative of a Th-1 shift in the immune response, was observed in the adjuvanted vs. non-adjuvanted ID group at day 7 of vaccination (p=0.003, Student two-tailed t-test) and an about 3-fold increase (p=0.051, Student two-tailed t-test) was detected between the corresponding IM groups (FIG.2G). Almost all aged mice in the Quil-A/cGAMP combination groups developed HAI titers of 10 or 20 by day 28 (FIG.2H). This substantial improvement in protection and functional antibody titers over non-adjuvanted vaccine exceeded the effects of the individual adjuvants, demonstrating a synergy between them. Comparison of Quil-A/cGAMP Combinations in Mature Adult vs. Aged Mice Groups of mature adult mice were challenged ID or IM for vaccination with a 10-fold higher infectious dose compared to the aged animals. The groups were ranked by rate of survival and average weight loss (FIG.3). In spite of the high infectious dose, even those adult mice that received an unadjuvanted vaccine were partially protected, with 60 and 80% survival rates observed in the ID and IM groups, respectively, and all adjuvants in the doses tested except for 1 μg cGAMP completely prevented mortality. Differences in protection in the Quil-A/cGAMP combination group (5 μg each) delivered ID or IM were not observe (FIG.3). In the adult mice, the maximal geometric mean HAI titer 45.9 was detected in the 5 μg Quil-A group, while in the aged mice this was detected in the Quil-A/cGAMP combination groups using 5 μg of each (FIG.2H). Quil-A alone (5 μg) increased vaccine-specific antibody levels as effectively as in combination with 1-5 μg cGAMP. A drop in the level of vaccine-specific IgM from day 7 to day 28 in mature adults (FIG.4A) was accompanied by a corresponding increase of vaccine-specific IgG (FIG.4C). The initial IgM response was 3-4 fold lower in the aged animals than in the adults (compareFIGS.4A,B) and a 1.6-fold increase of vaccine-specific IgM in the Quil-A/cGAMP group was observed between days 7 and 14 (p=0.04), but essentially remained at day 7 levels in the Quil-A group (FIG.4B). An increase in the level of vaccine-specific IgG was observed between days 7 and 14 in the aged animals (FIG.4D), but it was about 20-fold lower than observed in the adult mice by day 28 (FIG.4C). These data indicate that the adjuvant combination improved antibody class switching in the aged mice, but this process was significantly more efficient in the adult animals without use of an adjuvant. Mechanism of Potentiation of cGAMP Signaling by Quil-A Binding of cGAMP to the STING adaptor protein triggers phosphorylation of the downstream factor IRF3. We compared the effect of each adjuvant alone or in combination on IRF3 phosphorylation in HeLa cells, which are known to respond to cGAMP. The cells were incubated with adjuvants for 1 hour, followed by assay of phosphorylated IRF3 levels in cell lysates by western blot (FIG.5A). Comparison of the intensities of the pIRF3 band normalized to actin showed that the addition of vaccine or Quil-A did not change pIRF3 levels, while cGAMP increased them up to 3-fold in a concentration-dependent manner (FIG.5B). A combination of Quil-A and cGAMP yielded the highest increase, about 8-fold, in pIRF3 levels as compared to untreated control. Notably, in the presence of 5 μg/ml cGAMP the increase in concentration of Quil-A from 5 to 10 μg/ml increased phosphorylation of IRF3 in HeLa cells six-fold (FIG.5B). Same experiments carried out in MEFs provided similar results and confirmed that phosphorylation of IRF3 was due to STING activation because it only occurred in STING+/+ but not in STING−/− MEFs. These results support the conclusion that Quil-A enhances access of cGAMP to STING, and demonstrate that the combination of these compounds activates the IRF3 complex more effectively than cGAMP alone. Comparison with Current Approaches for Vaccination in Aged Humans Experiments were performed to determine whether a cGAMP/QuilA combination was more effective than the two currently used approaches for boosting the human immune response in aged patients. Aged mice were administered a single dose of 1 μg vaccine alone or in combination with a squalene-based adjuvant, AddaVax™, which, according to the manufacturer instruction, is similar to the MF59 formulation used in humans. Addition of AddaVax™ increased survival after lethal challenge to 60% in both IM and ID groups (FIG.6A), but did not prevent high ˜19% average weight loss at day 7 post challenge (FIG.6B). Consistent with previously reported data for a similar squalene-based adjuvant, the levels of vaccine-specific immunoglobulins were significantly elevated in the AddaVax™ groups as compared to the vaccine only groups (FIGS.6C-E). The vaccine-specific IgG/IgM ratio in the AddaVax™ groups was also consistently higher than in non-adjuvanted groups (FIG.6F), indicating an increase in the efficiency of antibody class switch. Changes were not observed in the vaccine-specific IgG2a/IgG1 ratio (FIG.6G) indicating a change in the Th type of response. Although aged animals immunized IM with the adjuvanted formulation demonstrated slightly higher levels of vaccine-specific IgG and IgG1 and IgG/IgM ratio as compared to the ID-vaccinated mice, survival percentages were similar for both delivery routes. The aged mice developed very low HAI titers which were at or below the limit of detection in all groups in response to a single 1 μg vaccination dose (FIG.6H). In comparison, AddaVax′ effectively prevented mortality in vaccinated mature adult mice (4-month-old control). Thus, although very effective in adults, AddaVax′ did not effectively prevent mortality or lessen morbidity in aged animals. Experiments were performed to determine whether an ID vaccination with a 4-fold higher dose of an unadjuvanted antigen was protective in the aged mice. A significant amount (75%) of mice in the 4 μg dose group survived the challenge, compared to 22% survival in the 1 μg vaccine group (FIG.7A), but the maximal weight loss was as high as 16% (FIG.7B). No significant differences were observed in the levels of vaccine-specific IgG, IgG1, IgG2a, or in IgG/IgM and IgG2a/IgG1 ratios (FIGS.7C-G) that would correlate with better survival in the 4×antigen dose group, and HAI titers were mostly below the level of detection in both groups (FIG.7H). Thus, in aged mice, use of a 4-fold higher antigen dose yielded a comparable level of protection as that observed with the AddaVax′ adjuvant. In both cases, survival was improved, although not to 100%, but morbidity was not prevented as seen by significant weight loss observed in all groups after challenge. These data show that the current strategies used to vaccinate the aged population are also limited in their effectiveness in the aged mouse model. In particular, the improvement in protection and functional antibody titers over non-adjuvanted vaccine was reduced compared with the cGAMP/Quil-A combination, demonstrating the high potential of this adjuvant combination in overcoming the effects of immunosenescence. | 46,658 |
11857619 | DETAILED DESCRIPTION OF THE INVENTION Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising”, “containing”, “including”, and “having”, whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of” or “consisting essentially of” to vary scopes of the disclosure. As used herein, the term “about,” when used in conjunction with a number, refers to any number within ±10%, e.g. ±5%, or ±1%, of the referenced number. For example, a pH of about 5.0 means any pH from 4.5-5.5, inclusive. As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to who will be or has been administered a protein or vaccine according to embodiments of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human. The invention generally relates to a process of purifying a protein, preferably a glycosylated protein, more preferably a human immunodeficiency virus (HIV) envelope protein, such as the envelope protein of HIV-1 clade C or HIV-1 mosaic envelope protein, the process comprises:a. obtaining a cell sample, such as a cell supernatant, comprising the protein;b. adjusting the pH of the cell sample to about 5.0 to thereby precipitate host cell proteins (HCPs) in the cell sample;c. removing the precipitated HCPs from the cell sample by depth filtration to obtain a filtrate comprising the protein; andd. purifying the protein in the filtrate by chromatography. Preferably, the cell sample is a cell supernatant comprising the protein secreted by the cell. The cell sample can also be a cell lysate or a processed cell lysate comprising the protein produced by the cell. Such lysate can for instance be prepared by breaking down of the membrane of a cell. A cell sample useful for a process of the application can be obtained using methods known in the art in view of the present disclosure. For example, a cell supernatant can be obtained by applying a cell culture to centrifugation to remove cells. A cell lysate can be obtained by disrupting or lysing the cells and removing the cell debris by centrifugation. The cell supernatant or cell lysate can be used directly or it can be further processed before being used for a process of the application. Preferably, a continuous centrifugation is used to remove cells produced from a bioreactor to obtain a cell sample useful for a process of the application. In some embodiments, the host cells produce the protein in a fed-batch process in a bioreactor. In certain embodiments, the bioreactor has a volume of between about 1 L and about 20000 L, e.g. from about 10 L to about 16500 L, e.g. from about 100 L to about 15000 L. The pH of the cell sample, such as a cell supernatant, can be adjusted, for example, by adding a suitable amount of acid (e.g., 1M acetic acid) to the cell sample to precipitate host cell proteins (HCPs) in the cell sample. This process is sometimes also referred to as “low pH flocculation.” Preferably, the pH of the cell sample is adjusted to about 5, e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or any value in between, to precipitate host cell proteins (HCPs) in the cell sample, while a sufficient amount of the protein of interest (e.g., HIV gp140) in the cell sample is not precipitated. Other proteins in the cell sample, such as proteins in the culture medium for the cells, can also be precipitated at the pH of about 5. In some embodiments, during the process of low pH flocculation, the cell sample is incubated at about pH 5 for about 15 minutes to about 15 hours, e.g. for about 0.5-12 hours, e.g. about 1-3 hours, e.g. about 3 hours, preferably about 1 hour, to precipitate the HCPs. In some embodiments, the low pH flocculation can be performed after centrifugation. In preferred embodiments, the low pH flocculation is performed before centrifugation. The precipitated HCPs from the cell sample can be removed by depth filtration to obtain a filtrate comprising the protein. Depth filters with various media types (single layer or multiple layers of cellulose, polyacrylic fiber, diatomaceous earth, silica, activated carbon, etc.) and various grades can be used for depth filtration in a process of the application in view of the disclosure herein. Examples of the depth filters useful for the invention include, but are not limited to, depth filters available from commercial sources, such as the Millistak+® family and Clarisolve® depth filters from Millipore Sigma. In certain embodiments, the depth filtration uses a depth filter such as a Millistak+® C0HC, C0SP, CE35, CE50, D0HC, D0SP, DE, A1HC, B1HC, F0HC, X0HC, X0SP, etc. Suitable buffers can be used to equilibrate the depth filters prior to use and to chase the filters after the acid precipitated harvest (e.g., precipitated HCPs and other proteins) was filtered through the depth filter. Preferably, the buffer has a pH of about 5.0. Preferably, the depth filtrate is sterile filtered to remove any contaminating microbes, e.g., with a filter pore size of 0.45 μm or less, preferably 0.22 μm. In certain embodiments, an ultrafiltration and diafiltration (UFDF) step is used to remove HCPs and concentrate the protein of interest (e.g., gp140) prior to or in between of the chromatography steps. Ultrafiltration (UF) is a commonly used process for concentrating a dilute product stream. It separates molecules in solution based on the membrane pore size or molecular weight cutoff. Diafiltration (DF) is often used to exchange product into a desired buffer (e.g., from an elution buffer into a final formulation buffer). UF and DF typically use tangential flow filtration, where feed flows parallel to the membrane surface rather than perpendicular to the surface. Various UF/DF membranes can be used, including, e.g., membranes of cellulose acetate, polyvinylidene fluoride (PVDF), and polyethersulfone (PES). Depending on the need, the membranes used in UF/DF can have different molecular weight cut off (MWCO). For example, the MWCO for a UF/DF can be, e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 kDa. In some embodiments, the UF/DF is configured with one or more flat plate membranes are stacked together. UF/DF processes include, e.g., sanitization and pre-use testing, equilibration, concentration, diafiltration, product recovery, cleaning and post-use testing, and storage. The integrity of a UF/DF system can be confirmed using a diffusion test. Suitable UF and/or DF buffers can be used for the UF/DF process in view of the present disclosure. For example, the buffer can have a pH of 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 8.5. Various cross-flow rates and membrane load rate can be used depending on the need, in view of the present disclosure. In some embodiments, a process of the application uses three or more chromatographic column steps. Suitable columns can be used in the invention in view of the present disclosure. Examples of such columns include, but are not limited to, those described in the embodiments, and for instance illustrated inFIGS.3and4. In one embodiment, a process of the application comprises a capture chromatography step using a multimodal resin (also called mixed mode resin). Multimodal or mixed-mode chromatography resins are based on media that have been functionalized with ligands inherently capable of several different types of interaction: for example combinations of two or more of ion exchange, affinity, size exclusion, and hydrophobic. The ability to merge and take advantage of these modes of protein separations can enhance overall selectivity in a purification process. This enhanced selectivity can be used to remove process impurities in a single column step that would otherwise require multiple processing steps to remove. Preferably, the multimodal resin has hydrophobic interaction and cation exchange properties, which is more salt tolerant, enabling binding of the protein to the resin with minimal or no dilution. Resins useful for the invention can be in different formats, e.g. as beads, filters (membranes), cartridges, etc., all to be considered as ‘resin’ according to the invention, and in certain embodiments the resins are in the form of beads that can be used in columns, and that resins that can be used according to the invention can be commercially obtained from vendors, e.g. Cytiva (former GE Healthcare) and/or others. In some embodiment, a multimodal resin can be a resin that is prepared by directly or indirectly immobilizing two or more types of functional groups having different selectivity onto a base resin. For example, a multimodal resin can comprise a multimodal strong anion exchange chromatography material having a matrix of high-flow agarose and a multimodal strong anion exchanger as ligand, or a matrix of high-flow agarose and a multimodal weak cation exchanger as ligand. Specific examples of the multimodal resin can include, but are not limited to, Capto Adhere, Capto MMC, Capto Adhere ImpRes or Capto MMC ImpRes (which are manufactured by Cytiva, Capto is registered trademark), HEA HyperCel, PPA HyperCel, MEP HyperCel (which are manufactured by Pall Corp., HyperCel is trademark), TOYOPEARL (registered trademark) MX-Trp-650M (manufactured by TOSOH Corp.) or the like, but are not limited thereto. In certain embodiments, the multimodal capture chromatography is performed in the flow-through mode. In preferred embodiments, the multimodal capture chromatography is performed in the bind and elute mode. Preferably, the multimodal capture chromatography is performed in the bind and elute mode in order to remove host cell proteins and DNA. The protein of interest is loaded to the multimodal capture resin, e.g. column, at a certain salt concentration and pH and binds to the column, and then is eluted later by an elution solution to obtain a pooled elute. The protein of interest can be loaded to the column in any suitable buffer (such as acetate buffer, histidine buffer, HEPES buffer, phosphate buffer, or Tris buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising sodium acetate at about 15-100 mM (e.g., 25 mM) and sodium chloride at about 10-50 mM (e.g., 25 mM), at any suitable pH such as pH between about 4-6 (e.g., pH 4 or 5), and with any suitable conductivity such as conductivity between about 1-50 mS/cm, e.g. e.g. between about 3-50 mS/cm, e.g. between about 5-50 mS/cm, e.g. between about 3-40 mS/cm, e.g. between about 6-40 mS/cm, preferably between about 3-20 mS/cm, e.g. between about 10-20 mS/cm (e.g., about 5 mS/cm, or about 15 mS/cm). The elution solution can comprise any suitable buffer (such as 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising HEPES at about 20-80 mM (e.g., 50 mM) and sodium chloride at about 50-600 mM, e.g. about 100-600 mM (e.g. 400 mM), and at any suitable pH such as pH between about 4-8, e.g. between about 4.5-7.5 (e.g., pH 7), and with any suitable conductivity such as conductivity between about 10-60 mS/cm (e.g., 40 mS/cm). The protein of interest can also be eluted from the multimodal capture column by gradient elution. In another embodiment, a process of the application comprises a second chromatography comprising an anion exchange resin. The anion exchanger used in this step can be a strong anion exchanger or a week anion exchanger. Preferably, the anion exchanger comprises an anion exchange ligand such as quaternary ammonium, quaternary aminoethyl, diethylaminoethyl, trimethylaminoethyl, or dimethylaminoethyl. More preferably, the anion exchanger is selected from a weak anion exchange resin (e.g. Capto DEAE) or a strong anion exchange resin (e.g. POROS 50 HQ). Other examples of anion exchanger include, but are not limited to, DEAE Sepharose FF, Q-Sepharose (HP and FF), AEX Sepharose FF (low and high substituted), Capto Q, Q XP, Source 30 Q and 15 Q, most preferably Fractogel DEAE and MPHQ. In certain embodiments, the second chromatography is performed in the flow-through mode. In preferred embodiments, the second chromatography is performed in the bind and elute mode. Preferably, the second chromatography is performed in the bind and elute mode in order to further remove host cell proteins and DNA. The protein of interest is loaded to the anion exchange resin, e.g. column, at a certain salt concentration and pH and binds to the column, and then is eluted later by an elution solution to obtain a pooled elute. The protein of interest can be loaded to the column in any suitable buffer (such as Tris buffer or HEPES buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising Tris at about 15-75 mM (e.g., 25 mM), and sodium chloride at about 0-75 mM, e.g. about 25-75 mM (e.g., 50 mM, or e.g. 5 mM), at any suitable pH such as pH between about 6-8 (e.g., pH 7.5 or 8), and with any suitable conductivity such as conductivity between about 2-8 mS/cm (e.g., 5.5 mS/cm). The elution solution can comprise any suitable buffer (such as Tris buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising Tris at about 15-75 mM (e.g., 25 mM, or e.g. 50 mM) and sodium chloride at about 50-500 mM, e.g. about 100-300 mM (e.g. 185 mM, or 200 mM), at any suitable pH such as pH between about 6-9 (e.g., pH 7.5), and with any suitable conductivity such as conductivity between about 5-50 mS/cm, e.g. about 5-40 mS/cm (e.g., 20 mS/cm). The protein of interest can also be eluted from the second column by gradient elution. In another embodiment, a process of the application comprises a third chromatography using an affinity medium that binds to glycan. The affinity medium resin can comprise the ligand sulfate or dextran sulfate. Examples of affinity medium include, but are not limited to, the cellulose sulfate medium or the agarose sulfate medium such as Cellufine sulfate, Cellufine sulfate m, Cellufine sulfate c, Cellulofine sulfate m, Cellulofine sulfate c, Cellufine sulfate m or Cellufine sulfate c (which are manufactured by JNC Corp.), Cytiva CAPTO™ Core 700 or Capto DeVirS (manufactured by Cytiva) or the like. In certain embodiments, the third chromatography is performed in the flow-through mode. In preferred embodiments, the third chromatography is performed in the bind and elute mode. Preferably, the third chromatography is performed in the bind and elute mode in order to further remove host cell proteins and DNA. The protein of interest is loaded to the affinity medium resin, e.g. column, at a certain salt concentration and pH and binds to the column, and then is eluted later by an elution solution to obtain a pooled elute. The protein of interest can be loaded to the column in any suitable buffer (such as Tris buffer or HEPES buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising Tris at about 5-25 mM (e.g., 6 mM) or HEPES at about 5-50 mM (e.g., 20 mM), and sodium chloride at about 0-100 mM, e.g. at about 25-75 mM (e.g., 45 mM, or 50 mM), and at any suitable pH such as pH between about 4-8, e.g. between about 5-8 (e.g., pH 6.5), and with any suitable conductivity such as conductivity between about 1-15 mS/cm, e.g. about 1-10 mS/cm (e.g., 5 mS/cm). The elution solution can comprise any suitable buffer (such as Tris buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising Tris at about 10-100 mM, e.g. at about 15-75 mM (e.g., 25 mM) and sodium chloride at about 100-300 mM (e.g. 185 mM), and at any suitable pH such as pH between about 6-9 (e.g., pH 7.5), and with any suitable conductivity such as conductivity between about 10-30 mS/cm, e.g. about 15-25 mS/cm (e.g., 19 mS/cm). The protein of interest can also be eluted from the third column by gradient elution. In certain embodiments, a process of the application comprises a low pH viral inactivation step, e.g. holding for about 15 minutes to about 4 hours, e.g. about one hour at about pH 3-4, e,g. pH about 3.5 and subsequently filtering through a 0.45-0.2 micrometer filter. This step is performed after the second chromatography step in case the third chromatography using an affinity medium is not used, or after the third chromatography step when the third chromatography step is performed. The filtrate is then neutralized to a target pH, such as a pH of 5-7, prior to the next processing step. The low pH viral inactivation step can denature the proteins of virus contaminants, which then can be removed in the subsequent column chromatography. In another embodiment, a process of the application comprises a fourth chromatography using a multimodal resin, preferably a multimodal resin comprising anion exchange and hydrophobic interaction chromatography functionalities. Specific examples of the multimodal resin can include, but are not limited to, Capto Adhere, Capto MMC, Capto Adhere ImpRes or Capto MMC ImpRes (which are manufactured by Cytiva, Capto is registered trademark), HEA HyperCel, PPA HyperCel, MEP HyperCel (which are manufactured by Pall Corp., HyperCel is trademark), TOYOPEARL (registered trademark) MX-Trp-650M (manufactured by TOSOH Corp.) or the like, but are not limited thereto. A suitable multimodal resin, such as Capto Adhere, can be used in this step in view of the present disclosure. The multimodal chromatography is performed in the bind and elute mode or preferably in flow-through mode. The fourth chromatography can further reduce hexamer and host cell protein impurities in the product pool. Preferably, the fourth chromatography is performed in the flow-through mode in order to remove host cell proteins and nucleic acids. The protein of interest can be loaded to the resin, e.g. column, in any suitable buffer (such as sodium acetate buffer) and/or salt solution (such as sodium chloride solution), for instance a solution comprising sodium acetate at about 25-75 mM (e.g., 50 mM) and sodium chloride at about 50-800 mM, e.g. about 200-800 mM (e.g., 317 mM or 650 mM), and at any suitable pH such as pH between about 3-8, e.g. between about 3-5 (e.g., pH 3.5 or 4.5), and with any suitable conductivity such as conductivity between about 5-70 mS/cm. Preferably, the flow-through solution comprises the HIV Env protein, e.g. gp140 protein, while certain impurities remain bound to the column. In yet another embodiment, a process of the application comprises one or more of a nanofiltration (viral retentive filtration) step and a final UFDF step. A viral-retentive filtration operates on a size exclusion principle. For example, a virus filter having an effective pore size of maximum 75 nm can be used for the viral retentive filtration. Examples of the filters for the viral-retentive filtration include, but are not limited to, Virosart HC, Virosart® HF, a Planova 20N filter, etc. In yet another embodiment, a process of the application comprises a final formulation step, wherein the purified protein can be formulated into a final product, such as a vaccine or an immunogenic composition. Final products of the invention can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. Each of the chromatography steps can be performed under suitable conditions in view of the disclosure herein. In certain embodiments, the protein of interest, such as HIV envelope protein, is loaded at a certain salt concentration and pH, and eluted in purer form at an increased salt concentration and increased pH as compared to the loading conditions. It is an aspect of the invention to provide a process for purifying HIV-1 gp140 protein, comprising capturing the protein on a multimodal resin comprising hydrophobic interaction and cation exchange properties, and eluting a purified fraction from said resin, wherein the purity of the HIV-1 gp140 protein is substantially increased as compared to the protein in the mixture that was loaded on the resin during the capturing step. Such multimodal resins appear particularly suitable for purification of HIV-1 gp140 protein. In another aspect of the invention, a process for purifying HIV-1 gp140 protein is provided, the process comprising the steps of:i) providing a composition comprising HIV-1 gp140 protein and other, non-desired proteins, such as host cell proteins derived from the host cell in which HIV-1 gp140 protein was expressed;ii) capturing the HIV-1 gp140 protein on a multimodal resin comprising hydrophobic interaction and cation exchange properties, and eluting a purified fraction comprising the HIV-1 gp140 protein from said resin;iii) applying the purified fraction of step ii) to an anion exchange resin to bind the HIV-1 gp140 protein, and eluting a further purified fraction comprising the HIV-1 gp140 protein from said resin; andiv) subjecting the further purified fraction of step iii) to a mixed mode resin that has anion-exchange and hydrophobic functionalities, and eluting a further purified HIV-1 gp140 protein. In certain embodiments, the HIV-1 gp140 protein is clade C gp140 protein or mosaic gp140 protein, preferably mosaic gp140 protein. In certain embodiments, the process further comprises a step of applying the further purified fraction of HIV-1 gp140 protein of step iii) to a resin that comprises dextran sulfate, and eluting a further purified fraction comprising the HIV-1 gp140 protein from said resin, before subjecting this fraction to step iv) of this process. Preferably, the HIV-1 gp140 protein in these embodiments is clade C gp140 protein. According to the embodiments of the application, the inventive process can be used in both laboratory scale and commercial scale. For example, the process of protein purification can be used to provide purified HIV-1 gp140 proteins for the purpose of investigation study. The process of the application can also be used in commercial and large scale to provide large quantities of purified HIV-1 gp140 proteins, preferably in trimeric state. In particular, large scale of purification of clade C gp140 protein can be achieved using a process described inFIG.3, and large scale of purification of mosaic gp140 protein can be achieved using a process described inFIG.4. Human immunodeficiency virus (HIV) is a member of the genus Lentivirinae, which is part of the family of Retroviridae. Two species of HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strain of HIV virus, and is known to be more pathogenic than HIV-2. As used herein, the terms “human immunodeficiency virus” and “HIV” refer, but are not limited to, HIV-1 and HIV-2. HIV is categorized into multiple clades with a high degree of genetic divergence. As used herein, the term “HIV clade” or “HIV subtype” refers to related human immunodeficiency viruses classified according to their degree of genetic similarity. There are currently three groups of HIV-1 isolates: M, N and O. Group M (major strains) consists of at least ten clades, A through J. Group O (outer strains) can consist of a similar number of clades. Group N is a new HIV-1 isolate that has not been categorized in either group M or O. As used herein, the terms “HIV antigenic polypeptide,” “HIV antigenic protein,” “HIV antigen,” and “HIV immunogen” refer to a polypeptide capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against HIV in a subject. The antigenic polypeptide or antigen can be a protein of the HIV, a fragment or epitope thereof, or a combination of multiple HIV proteins or portions thereof that can induce an immune response or produce an immunity, e.g., protective immunity, against the HIV in a subject. Preferably, an antigenic polypeptide or antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity in (i.e., vaccinates) a subject against a viral disease or infection, that protects the subject against the viral disease or infection. For example, the antigenic polypeptide or antigen can comprise a protein or fragments thereof from Simian Immunodeficiency Virus (SIV) or an HIV, such as the HIV or SIV envelope gp160 protein, the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env gene products. An HIV antigenic polypeptide or antigen can be any HIV-1 or HIV-2 antigen or fragment thereof. Examples of HIV antigens include, but are not limited to gag, pol, and env gene products, which encode structural proteins and essential enzymes. Gag, pol, and env gene products are synthesized as polyproteins, which are further processed into multiple other protein products. The primary protein product of the gag gene is the viral structural protein gag polyprotein, which is further processed into MA, CA, SP1, NC, SP2, and P6 protein products. The pol gene encodes viral enzymes (Pol, polymerase), and the primary protein product is further processed into RT, RNase H, IN, and PR protein products. The env gene encodes structural proteins, specifically glycoproteins of the virion envelope. The primary protein product of the env gene is gp160, which is further processed into gp120 and gp41. Other examples of HIV antigens include gene regulatory proteins Tat and Rev; accessory proteins Nef, Vpr, Vif and Vpu; capsid proteins, nucleocapsid proteins, and p24 viral protein. In certain embodiments, the HIV antigenic polypeptide or antigen comprises an HIV Gag, Env, or Pol antigen, or any antigenic portion or epitope or combination thereof, preferably an HIV-1 Gag, Env, or Pol antigen or any antigenic portion or epitope or combination thereof. HIV antigenic polypeptides can also be mosaic HIV antigens. As used herein, “mosaic antigen” refers to a recombinant protein assembled from fragments of natural sequences. Mosaic antigens resemble natural antigens, but are optimized to maximize the coverage of potential T-cell epitopes found in the natural sequences, which improves the breadth and coverage of the immune response. Mosaic HIV antigens can for instance be mosaic Gag, Pol, and/or Env antigens, and more preferably a mosaic HIV-1 Env antigen. As used herein, “a mosaic HIV Gag, Pol, and/or Env antigen” specifically refers to a mosaic antigen comprising multiple epitopes derived from one or more of the Gag, Pol and/or Env polyprotein sequences of HIV. As used herein, each of the terms “HIV envelope protein,” “env protein,” and “Env” refers to a protein that is expressed on the envelope of an HIV virion and enables an HIV to target and attach to the plasma membrane of HIV infected cells, or a fragment or derivative thereof that can induce an immune response or produce an immunity against the HIV in a subject in need thereof. The HIV env gene encodes the precursor protein gp160, which is proteolytically cleaved into the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction is mediated by a host cell protease, furin, at a sequence highly conserved in retroviral envelope glycoprotein precursors. More specifically, gp160 trimerizes to (gp160)3and then undergoes cleavage into the two noncovalently associated gp120 and gp41. Viral entry is subsequently mediated by a trimer of gp120/gp41 heterodimers. Gp120 is the receptor binding fragment, and binds to the CD4 receptor on a target cell that has such a receptor, such as, e.g., a T-helper cell. Gp41, which is non-covalently bound to gp120, is the fusion fragment and provides the second step by which HIV enters the cell. Gp41 is originally buried within the viral envelope, but when gp120 binds to a CD4 receptor, gp120 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell. Gp140 is the uncleaved ectodomain of trimeric gp160, i.e., (gp160)3,that has been used as a surrogate for the native state of the cleaved, viral spike. According to embodiments of the invention, an “HIV envelope protein” can be a gp160, gp140, gp120, gp41 protein, combinations, fusions, truncations or derivatives thereof. For example, an “HIV envelope protein” can include a gp120 protein noncovalently associated with a gp41 protein. It can also include a stabilized trimeric gp140 protein that can have or can be modified to include a trimerization domain that stabilizes trimers of gp140. Examples of trimerization domains include, but are not limited to, the T4-fibritin “foldon” trimerization domain; the coiled-coil trimerization domain derived from GCN4; and the catalytic subunit ofE. coliaspartate transcarbamoylase as a trimer tag. An “HIV envelope protein” can also be a truncated HIV envelope protein including, but not limited to, envelope proteins comprising a C-terminal truncation in the ectodomain (i.e. the domain that extends into the extracellular space), a truncation in the gp41, such as a truncation in the transmembrane domain of gp41, or a truncation in the cytoplasmic domain of gp41. An “HIV envelope protein” can further be a derivative of a naturally occurring HIV envelope protein having sequence mutations, e.g., in the furin cleavage sites, and/or so-called SOSIP mutations. In preferred embodiments of the invention, HIV envelope protein is a gp140 protein, more preferably HIV-1 clade C gp140 protein or HIV-1 mosaic gp140 protein. As used herein, each of the terms “stabilized trimeric gp140 protein” and “stabilized trimer of gp140” refers to a trimer of gp140 polypeptides that includes a polypeptide sequence that increases the stability of the trimeric structure. The gp140 polypeptides can have or can be modified to include a trimerization domain that stabilizes trimers of gp140. Examples of trimerization domains include, but are not limited to, the T4-fibritin “foldon” trimerization domain; the coiled-coil trimerization domain derived from GCN4; and the catalytic subunit ofE. coliaspartate transcarbamoylase as a trimer tag. Examples of antigenic HIV envelope polypeptides are stabilized trimeric gp140 such as those described in Nkolola et al 2010,J. Virology84(7): 3270-3279; Kovacs et al,PNAS2012, 109(30):12111-6; WO 2010/042942 and WO 2014/107744, all of which are incorporated by reference in their entirety. In some embodiments of the invention, the “envelope polypeptide” or “envelope glycoprotein” is a mosaic envelope protein comprising multiple epitopes derived from one or more of Env polyprotein sequences of one or more HIV clades. For example, as used herein a “gp140 protein” can be a “mosaic gp140 protein” that contains multiple epitopes derived from one or more gp140 protein sequences of one or more HIV clades. Preferably, a mosaic gp140 protein is a stabilized trimeric gp140 protein. In a preferred embodiment, a mosaic gp140 protein is a stabilized trimer of mosaic gp140 protein comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments of the invention, the “envelope polypeptide” or “envelope glycoprotein” is an envelope protein derived from a particular HIV clade, such as HIV clade A, B, or C. For example, as used herein a “gp140 protein” can be a “clade C gp140 protein” that contains envelope protein sequence derived from HIV clade C. Preferably, a clade C gp140 protein is a stabilized trimeric clade C gp140 protein. In a preferred embodiment, a clade C gp140 protein is a stabilized trimer of clade C gp140 protein comprising the amino acid sequence of SEQ ID NO: 1. According to certain embodiments of the invention, a gp140 polypeptide, such as a stabilized trimeric gp140 protein can be administered together with viral expression vectors, e.g., adenovirus 26 (see e.g. WO 2016/049287, WO 2017/102929). In certain embodiments, two gp140 proteins are administered to the same subject, preferably a clade C gp140 having the amino acid sequence of SEQ ID NO: 1 and a mosaic gp140 having the amino acid sequence of SEQ ID NO: 2. These two gp140 proteins can be together in one pharmaceutical composition, preferably administered together with an adjuvant, such as aluminum phosphate adjuvant. A preferred dose for the total amount of gp140 for administration to humans is between about 125 and 350 μg, such as 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 μg, or any amount in between, preferably about 250 μg. If clade C gp140 and mosaic gp140 are both administered, a suitable dose would for instance be about 125 μg of each glycoprotein, to provide a total dose of 250 μg of gp140 glycoprotein for an administration to a human subject. As used herein, unless indicated otherwise, the amount of a gp140 polypeptide refers to the amount of the gp140 polypeptide measured as glycoprotein. An isolated gp140 protein can be co-delivered or administered in combination with an adenovirus (e.g., Ad26) expression vector or other expression vector such as MVA. According to a preferred embodiment, a gp140 protein and Ad26 or other expression vector are administered separately, as two distinct formulations. Alternatively, a gp140 protein can be administered with Ad26 or other expression vector together in a single formulation. Simultaneous administration or co-delivery can take place at the same time, within one hour, or within the same day. Furthermore, a gp140 protein can be administered in an adjuvanted formulation. Suitable adjuvants can be, for example, aluminum phosphate or a saponin-based adjuvant, preferably aluminum phosphate adjuvant. Antigenic polypeptides such as gp140 can be produced and isolated using any method known in the art in view of the present disclosure. For example, an antigenic polypeptide can be expressed from a host cell, preferably a recombinant host cell optimized for production of the antigenic polypeptide. According to an embodiment of the invention, a recombinant gene is used to express a gp140 protein containing mutations to eliminate cleavage and fusion activity, preferably an optimized gp140 protein with increased breadth, intensity, depth, or longevity of the antiviral immune response (e.g., cellular or humoral immune responses) generated upon immunization (e.g., when incorporated into a composition, e.g., vaccine) of a subject (e.g., a human). The optimized gp140 protein can also include cleavage site mutation(s), a factor Xa site, and/or a foldon trimerization domain. A leader/signal sequence can be operably linked to the N-terminal of an optimized gp140 protein for maximal protein expression. The leader/signal sequence is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Any leader/signal sequence suitable for a host cell of interest can be used. An exemplary leader/signal sequence comprises the amino acid sequence of SEQ ID NO: 3. Preferably, an “HIV envelope protein” is a “synthetic HIV envelope protein.” As used herein, the term “synthetic HIV envelope protein” refers to a non-naturally occurring HIV envelope protein that is optimized to induce an immune response or produce an immunity against one or more naturally occurring HIV strains in a subject in need thereof. Mosaic HIV Env proteins are examples of synthetic HIV Env proteins, and the invention provides synthetic HIV Env antigens, e.g. the ones comprising SEQ ID NO: 1 or SEQ ID NO: 2. A protein of interest to be purified by a process according to an embodiment of the application can be expressed by a host cell, preferably a recombinant host cell. In certain embodiments, the protein of interest, such as an HIV envelope protein, can be expressed with a signal sequence, and the signal sequence is cleaved from the nascent polypeptide chain during its transport into the lumen of the endoplasmic reticulum (ER). Any suitable signal sequence could be used. Preferably an HIV Env signal sequence or a variant thereof is used. Different signal sequences have been used in the art for HIV Env proteins (see e.g. WO 2014/107744). In a preferred embodiment, a protein of interest is recombinantly produced from a host cell transfected with an expression vector comprising nucleic acid sequence encoding the protein, such as an HIV envelope protein. Any suitable expression vectors can be used for recombinant protein expression, including, but not limited to, non-viral vectors, such as plasmids, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc., or viral vectors, such as adenoviral vectors, adeno-associated virus vectors, baculovirus vectors, poxvirus vectors, MVA vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc. The nucleic acid sequence encoding the synthetic HIV envelope protein can be operably linked to a promoter, meaning that the nucleic acid is under the control of a promoter. The promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). Non-limiting examples of suitable promoters for the adenoviral vectors include the cytomegalovirus (CMV) immediate early promoter and the Rous Sarcoma virus (RSV) promoter. Preferably, the promoter is located upstream of the nucleic acid within an expression cassette. A host cell is typically used to produce sufficient amounts of protein for use in the invention. According to a preferred embodiment, a cell of a suitable cell culture can be transformed or transfected with an expression vector. Any host cells, preferably eukaryotic host cells, more preferably mammalian host cells, can be used for recombinant protein expression, including but not limited to PER.C6, HEK293, CHO cells, etc. transfected with an expression vector encoding a protein of interest. The expression vector usually also contains a cassette comprising a marker and/or selection gene that facilitate the identification and isolation of the recombinant host cells expressing the protein of interest. However, a recombinant host cell can also be identified by PCR technology. In certain embodiments, the nucleic acid that encodes the recombinant protein, e.g. HIV envelope protein, is incorporated into the genome of the host cell. This allows production of the recombinant protein from a stable host cell line. In view of the degeneracy of the genetic code, the skilled person is well aware that several nucleic acid sequences can be designed that encode the same protein, according to methods entirely routine in the art. The nucleic acid encoding a protein of interest, such as an HIV envelope protein, can optionally be codon-optimized to ensure proper expression in the host cell. Codon-optimization is a technology widely applied in the art. Accordingly, a method of the invention can further comprise producing a protein of interest, such as an HIV antigenic polypeptide, from a recombinant host cell. Preferably, the method comprises transfecting a host cell with an expression vector comprising nucleic acid encoding the HIV antigenic polypeptide operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the synthetic HIV antigenic polypeptide, and isolating the synthetic HIV antigenic polypeptide from the cell using a process of the invention. Techniques used for recombinant protein expression are well known to one of ordinary skill in the art in view of the present disclosure. Another general aspect of the invention relates to a pharmaceutical composition, such as a vaccine or an immunogenic composition, comprising a protein purified by a process of the invention, and a carrier. A carrier can include one or more pharmaceutically acceptable excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives. Compositions of the invention can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, intra-arterial injection, subcutaneous injection, intramuscular injection, and intra-articular injection. Compositions of the invention can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal. According to certain embodiments of the invention, a composition comprises an immunogenically effective amount of a protein, such as an HIV envelope protein, purified by a method of the invention, and optionally one or more additional HIV antigens and/or adjuvants. Said compositions can be formulated as a vaccine (also referred to as an “immunogenic composition”) according to methods known in the art in view of the present disclosure. In general, when used with reference to a polypeptide, such as an isolated antigenic polypeptide, an immunogenically effective amount can range from, e.g. about 0.3 to about 3000 microgram (μg), e.g. 1-1000 μg, e.g. 10-500 μg, e.g. about 50 or 250 μg. In some embodiments, compositions of the invention can further optionally comprise an adjuvant to enhance immune responses. The terms “adjuvant” and “immune stimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the vectors encoding synthetic HIV envelope proteins of the invention and optionally one or more additional HIV antigens and/or HIV antigenic polypeptides used in combination with vectors encoding synthetic HIV envelope proteins of the invention and optionally one or more additional HIV antigens. Adjuvants suitable for use with the invention should be ones that are potentially safe, well tolerated and effective in people, such as for instance QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminum salts (e.g. AdjuPhos), Adjuplex, and MF59. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure. In a preferred embodiment, the adjuvant is an aluminum salt, such as aluminum hydroxide or aluminum phosphate, e.g. AdjuPhos. In certain embodiments, the aluminum phosphate is preferably present in or administered with a composition with isolated HIV antigenic polypeptide, such as gp140. The preparation and use of immunogenic compositions are well known to those of ordinary skill in the art. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can also be included. Alternatively, the vaccine shots can be prepared by stepwise, freeze-drying of the virus in a formulation. In certain embodiments, the formulation contains additional additives such as mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, or other additives, such as, including, but not limited to, antioxidants or inert gas, stabilizers or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration. The ampoule is then sealed and can be stored at a suitable temperature, for example, between 4° C. and room temperature for several months. However, as long as no need exists, the ampoule is stored preferably at temperatures below −20° C. In various embodiments involving vaccination or therapy, the lyophilisate is dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline or tris(hydroxymethyl)aminomethane (Tris) buffer, and administered either systemically or locally, i.e., by parenteral, subcutaneous, intravenous, intramuscular, intranasal, intradermal, or any other path of administration known to a skilled practitioner. Optimization of the mode of administration, dose, and number of administrations is within the skill and knowledge of one skilled in the art. In certain embodiments, the HIV envelope proteins such as gp140 proteins made by methods according to the invention are included into a composition comprising sorbitol (e.g. 2 to 15% (w/v), e.g. 5% or 12%), polysorbate 20 (e.g. 0.01 to 0.05% (w/v), e.g. 0.02%), and histidine buffer (e.g. 5 to 20 mM, pH 5.5 to 7.0, e.g. 10 mM at pH 6.5), see e.g. WO 2017/216288. Such compositions can optionally further comprise an adjuvant, e.g. aluminum phosphate (e.g. 0.7-4.0 mg/mL, e.g. 0.7-1 mg/mL, e.g. 085 mg/mL). The HIV envelope proteins can for instance be present at a concentration of about 0.05-5 mg/mL, e.g. 0.2 mg/mL or 1 mg/mL. Such compositions can be stored at for instance between about −80° to about 25° C., e.g. at about −80° C., −60° C., −20°, or preferably at about 2-8° C., which provides for stable liquid compositions that are directly usable for administration as vaccines. The invention also relates to a method of inducing an immune response against one or more HIV clades in a subject in need thereof using a pharmaceutical composition or vaccine of the invention. According to embodiments of the invention, “inducing an immune response” when used with reference to the methods and compositions described herein encompasses providing protective immunity and/or vaccinating a subject against an infection, such as a HIV infection, for prophylactic purposes, as well as causing a desired immune response or effect in a subject in need thereof against an infection, such as a HIV infection, for therapeutic purposes, i.e., therapeutic vaccination. “Inducing an immune response” also encompasses providing a therapeutic immunity for treating against a pathogenic agent, i.e., HIV. Typically, for prophylactic vaccination, compositions and vaccines are administered to subjects who have not been previously infected with HIV, whereas for therapeutic vaccination, compositions and vaccines are administered to a subject already infected with HIV. The immune response can be a cellular immune response and/or a humoral immune response. As used herein, the term “protective immunity” or “protective immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a “protective immune response” or “protective immunity” against a certain agent will not die as a result of the infection with said agent. As used herein, the term “therapeutic immunity” or “therapeutic immune response” means that the HIV infected vaccinated subject is able to control an infection with the pathogenic agent, i.e., HIV, against which the vaccination was done. In certain embodiments, the methods of inducing an immune response according to the invention are for therapeutic purposes, such as for therapeutic vaccination, in which the compositions and vaccines described herein are administered to a subject already infected with HIV. The terms “HIV infection” and “HIV-infected” as used herein refer to invasion of a human host by HIV. As used herein, “an HIV-infected subject” refers to a subject in whom HIV has invaded and subsequently replicated and propagated within the host, thus causing the host to be infected with HIV or have an HIV infection or symptoms thereof. In other embodiments, the proteins and compositions of the invention can be used for prophylactic vaccination, e.g. by administration to a subject, preferably a human subject, that is not HIV infected. Administration of an immunogenic compositions comprising an antigenic polypeptide is typically intramuscular, intradermal or subcutaneous. However, other modes of administration such as intravenous, rectal, cutaneous, oral, nasal, etc. can be envisaged as well. Intramuscular administration of the immunogenic compositions can be achieved by using a needle to inject a suspension of the antigenic polypeptides. An alternative is the use of a needleless injection device to administer the composition (using, e.g., Biojector™) or a freeze-dried powder containing the vaccine. For intramuscular, intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the isolated antigenic polypeptide will typically be in the form of a parenterally acceptable solution having a suitable pH, isotonicity, and stability. Those of ordinary skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, and Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required. A slow-release formulation can also be employed. Examples of suitable formulations for HIV gp140 proteins are provided in WO 2017/216288, incorporated by reference herein. An amount of a composition sufficient to induce a detectable immune response is defined to be an “immunogenically effective dose” or “immunogenically effective amount.” The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in generally available textbooks and manuals. EXAMPLES Upstream and downstream process for the production and purification of a recombinant protein (e.g., gp140 of Clade C HIV (SEQ ID NO:1) or mosaic HIV gp140 (SEQ ID NO: 2)) expressed by recombinant PER. C6 cell lines were studied. Multiple growth media were tested to improve gene expression and productivity of the recombinant host cell in a bioreactor. For example, the feed to the bioreactor was concentrated by 20% to allow for increased productivity. Various processes, conditions and columns were studied for purification of the protein of interest (e.g., gp140 of Clade C HIV or mosaic HIV gp140), with the goals to, e.g., minimize final host cell protein (HCP) levels, maintain product variant level and/or conformation, eliminate DNA and other contaminations, and with relatively high yield (e.g., at least about 10%, preferably at least about 15% overall yield). Using the processes of the invention, HCP levels in the final gp140 protein product were reduced below 5000 ppm, typically below 1000 ppm, and host cell DNA was below detection levels. Due to the high cell density and cell type of the recombinant host cells, it was difficult to filter harvests of the cells. Gravity based cell settling is not feasible for large scale production of the protein of interest. Thus, a continuous centrifugation was used to replace the gravity-settled step. It was noticed that precipitation occurred after the first ultrafiltration in preparation for loading onto the 1st purification column (being a mixed mode resin comprising hydrophobic interaction and cation exchange properties, which surprisingly was found to be the most suitable capture column from a wide variety of possibilities). Acid precipitation (or low pH flocculation) and depth filtration were used to remove cell debris and other precipitates while maintaining sufficient amount of the protein of interest (e.g., HIV gp140) in the filtrate, prior to the first ultrafiltration to avoid fouling of the filter membranes by the precipitates. It was shown that, the turbidity of the clarified harvest material adjusted to pH 5.0 +/−0.1 (e.g., with 1 M acetic acid) reached a plateau of about 70 nephelometric turbidity units (NTU) after about 3 hours incubation but reached >95% of the final NTU (see, e.g.,FIGS.1A and1B). Although large precipitation was visible during the acid precipitation, recovery yield of the recombinant protein was also high, e.g., about 100% for Clade C gp140, suggesting that no or minimal amount of Clade C gp140 precipitated out or lost during the acid precipitation. The acid precipitate materials were filtered through various depth filters for selection of filters and the turbidity was again measured after filtration. Suitable filters gave significant reduction in turbidity and removal of HCPs, but with no significant loss in the recombinant protein. Ultrafiltration (UF) and diafiltration (DF) are used for product concentration and buffer exchange before column separation (FIG.1A). The UF/DF prepared the product for the following chromatography stage. Columns are often operating under different pH or molarity conditions and the product needs to be primed for chromatography use beforehand by the UF and DF. Suitable UF/DF can be selected in view of the disclosure in the application. A process of the invention comprises three or more chromatographic column steps, which can be preceded by UF/DF of the product. Various resins were assessed through screening methods, in view of the unpredictability of a suitable combination of columns for a given specific protein, with the aim of fulfilling the requirements of sufficient yield and high purity during a large scale manufacturing process. Binding isotherms were generated to assess product impurity binding. Different columns were studied and compared. Columns with the greatest binding capacity were viewed as “capture” columns. Using a purification process illustrated inFIG.2A, desired low level of HCP was achieved (FIG.2B). Further improvement to the process was made to optimize the scale-up production. Development efforts focusing on facility fit and process robustness were conducted. Such efforts include, for example, feed variance, seed density, pH sensitivities, bioreactor temperature, pCO2 variation, reactor duration, etc. Pilot scale, scale down model and engineering principles were used to show readiness of the process for good manufacturing practice (GMP) production. Excellent results were found for purification of the clade C gp140 protein using a process described inFIG.3, and for purification of mosaic gp140 protein using a process described inFIG.4. These processes were found suitable for large scale manufacturing of pharmaceutical grade products. Example 1 Purification of Clade C gp140 Clade C gp140 was manufactured by a fed batch cell culture process. The expansion of cells and the production of Clade C gp140 occurred in the first 2 stages of the process, including Stage 1 (preculture and seed bioreactor) which uses a PER.C6 cell line that expresses Clade C gp140, and Stage 2 (production in a bioreactor with volume of 15,000 L to 16,500 L). The subsequent purification and manufacture of formulated bulk (FB) occurred in the remaining 11 stages. A flow diagram of the Clade C gp140 drug substance manufacturing process from preculture and expansion through drug substance (DS) is shown inFIG.3. The target run duration of the 15,000 L production process is 18 days. The contents of the 15,000 L production then undergo flocculation by adjustment with 25% acetic acid to a target of pH 4.8 (Stage 3, low pH flocculation). The flocculation is followed by clarification (Stage 4) through centrifugation and depth/polish filtration. The subsequent ultrafiltration and diafiltration (UFDF) step (Stage 5) was conducted in a solution containing 50 mM tris(hydroxymethyl)aminomethane (Tris) and 150 mM sodium chloride at pH 7.6 to obtain a pooled UFDF retentate. In Stage 6, the pH of the pooled UFDF retentate was adjusted with 1M acetic acid to 5.0. Then the retentate was loaded to a column of Capto MMC ImpRes, which was already equilibrated with a solution containing 50 mM sodium acetate at pH 5.0. This Capto MMC ImpRes column chromatography was performed in bind and elute mode in order to remove host cell proteins and potentially present DNA. The Clade C gp140 bound to the column and was eluted later by an elution solution containing 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) and 400 mM sodium chloride at pH 7.0 to obtain a pooled elute. In Stage 7, the pooled elute collected from the above Capto MMC ImpRes chromatography step was neutralized with 1M Tris (pH 9.0) to pH 7.5 and then diluted with water for injection (to a conductivity of less than 6 mS/cm). The resulting pH-adjusted and diluted elute was loaded to a column of POROS 50 HQ, which was already equilibrated with a solution containing 25 mM Tris at pH 7.5. This POROS 50 HQ column chromatography was performed in bind and elute mode to further remove host cell proteins and potentially DNA. The Clade C gp140 bound to the column and was eluted later by an elution solution containing 25 mM Tris and 185 mM sodium chloride at pH 7.5 (conductivity about 19 mS/cm) to obtain a pooled elute. In Stage 8, the pooled elute collected from the above POROS 50 HQ chromatography step was adjusted with 1M acetic acid to pH 6.5 and then diluted with water for injection (to a conductivity of less than 7 mS/cm). The resulting pH-adjusted and diluted elute was loaded to a column of Capto DeVirS, which was already equilibrated with a solution containing 20 mM HEPES and 50 mM sodium chloride at pH 6.5. This Capto DeVirS column chromatography was performed in bind and elute mode. The Clade C gp140 bound to the column and was eluted later by an elution solution containing 25 mM Tris and 185 mM sodium chloride at pH 7.5 (conductivity about 19 mS/cm) to obtain a pooled elute. In Stage 9, 5M sodium chloride was added to the pooled eluate collected from the above Capto DeVirS chromatography step to adjust its conductivity to be 62 mS/cm, adjusted with 1M acetic acid to pH 3.5 for viral inactivation, neutralized with 1M Tris (pH 9.0) to pH 4.5, and then diluted with water for injection to a conductivity of 30 mS/cm. In Stage 10, the diluted elute obtained from Stage 9 was loaded to a column of Capto Adhere, which was already equilibrated with a solution containing 50 mM sodium acetate and 317 mM sodium chloride at pH 4.5. This Capto Adhere column chromatography was performed in flow-through mode to remove potentially present nucleic acids and host cell proteins, and the flow-through solution contains the clade C gp140 protein in 50 mM sodium acetate and 317 mM sodium chloride at pH 4.5. In Stage 11, the eluate (actually being the flow-through) collected from the above Capto Adhere chromatography step was neutralized with 1M Tris (pH 9.0) to be pH 6.5, and then was processed through a Planova 20N viral filter for viral retentive filtration. The obtained filtrate was subjected to final ultrafiltration and diafiltration (UFDF) into the formulation buffer (Stage 12) and final formulation of the drug substance (Stage 13). The purification process also included the in-process control (IPC) tests performed during each process stage of the manufacturing process. The IPC tests were defined as tests, checks and measurements made during the course of manufacturing to monitor and, if necessary, adjust the process to ensure that the resulting API or finished product would comply with its specification. The remaining in-process tests were defined as Process Monitoring tests (PMT's) and are tests, checks, and measurements performed during the course of routine production to monitor the process to assure that the process remains in a state of control. Example 2 Purification of Mosaic gp140 A flow diagram of the Mosaic gp140 drug substance manufacturing process from preculture and expansion through drug substance (DS) is shown inFIG.4. The large scale manufacture of Mosaic gp140 includes Stage 1 (preculture and seed bioreactor), Stage 2 (2000 L production in single use bioreactor (SUB)), Stage 3 (pH 5 flocculation) and Stage 4 (clarification) processes. The preculture process uses a PER.C6 cell line that expresses mosaic gp140 and entails expansion from vial thaw through shake flasks, wave bags and the 500 L Seed Bioreactor. The maximum duration of Stage 1 is 40 days for preculture including the seed bioreactor. Then the batch is transferred to the 2000 L production SUB process (Stage 2) after inoculation. The target run duration of the 2000 L production SUB process is 19 days. The contents of the 2000 L production SUB then undergo flocculation by adjustment with 1M acetic acid to a target of pH 5.0 (Stage 3). The flocculation is followed by clarification (Stage 4) through centrifugation, depth filtration and polish filtration, or through depth filtration and polish filtration only. In Stage 5, the obtained filtrate was adjusted with 1M Tris (pH 9) and 5M sodium chloride to pH 5.25 with a conductivity of 15 mS/cm, and then loaded to a column of Capto MMC ImpRes, which was already equilibrated with 50 mM sodium acetate at pH 5.0. This Capto MMC ImpRes column chromatography was performed in bind and elute mode in order to remove host cell proteins and potentially present DNA. The mosaic gp140 bound to the column and was eluted later by an elution solution containing 50 mM HEPES and 400 mM sodium chloride at pH 7.0 to obtain a pooled elute. In Stage 6, the pooled elute collected from the above Capto MMC ImpRes chromatography step was neutralized with 1M Tris (pH 9.0) and then diluted with water for injection to obtain a pooled elute at pH 8.0 with a conductivity of 5.5 mS/cm. The resulting pH-adjusted and diluted elute was loaded to a column of POROS 50 HQ, which was already equilibrated with a solution containing 50 mM Tris at pH 8.0. This POROS 50 HQ column chromatography was performed in bind and elute mode to further remove host cell proteins and potentially present DNA. The mosaic gp140 bound to the column and was eluted later by gradient elution 6%-42% of buffer B with gradient length=11.0 CV, wherein buffer A was 50 mM Tris at pH 8.0, and buffer B was a mixture of 50 mM Tris and 500 mM sodium chloride at pH 8.0, and the conductivity was increased from about 6 to 20 mS/cm. In Stage 7, the pooled eluate collected from the above POROS 50 HQ chromatography step was added 5M sodium chloride to adjust the conductivity of the pooled eluate to be 56 mS/cm, and adjusted with 1M acetic acid to pH 3.5 for viral inactivation. In Stage 8, the elute obtained from Stage 7 was loaded to a column of Capto Adhere, which was already equilibrated with a solution containing 50 mM sodium acetate and 650 mM sodium chloride at pH 3.5. This Capto Adhere column chromatography was performed in flow-through mode to remove potentially present nucleic acids and host cell proteins. The pooled eluate was neutralized with 1M Tris (pH 9.0) to pH 6.5. In Stage 9, the neutralized elute was processed through a Planova 20N viral filter for viral retentive filtration. The obtained filtrate was subjected to final ultrafiltration and diafiltration (UFDF) into the formulation buffer (Stage 10) and final formulation of the drug substance (Stage 11). It is understood that the examples and embodiments described herein are for illustrative purposes only, and that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims. TABLE 1Sequences HIV-1 envelope proteinsSEQ ID NO: 1 clade C gp140 protein(679 amino acids)AENLWVGNMW VTVYYGVPVW TDAKTTLFCA SDTKAYDREVHNVWATHACV PTDPNPQEIV LENVTENFNM WKNDMVDQMHEDIISLWDQS LKPCVKLTPL CVTLHCTNAT FKNNVTNDMNKEIRNCSFNT TTEIRDKKQQ GYALFYRPDI VLLKENRNNSNNSEYILINC NASTITQACP KVNFDPIPIH YCAPAGYAILKCNNKTFSGK GPCNNVSTVQ CTHGIKPVVS TQLLLNGSLAEKEIIIRSEN LTDNVKTIIV HLNKSVEIVC TRPNNNTRKSMRIGPGQTFY ATGDIIGDIR QAYCNISGSK WNETLKRVKEKLQENYNNNK TIKFAPSSGG DLEITTHSFN CRGEFFYCNTTRLFNNNATE DETITLPCRI KQIINMWQGV GRAMYAPPIAGNITCKSNIT GLLLVRDGGE DNKTEEIFRP GGGNMKDNWRSELYKYKVIE LKPLGIAPTG AKERVVEREE RAVGIGAVFLGFLGAAGSTM GAASLTLTVQ ARQLLSSIVQ QQSNLLRAIEAQQHMLQLTV WGIKQLQTRV LAIERYLKDQ QLLGIWGCSGKLICTTNVPW NSSWSNKSQT DIWNNMTWME WDREISNYTDTIYRLLEDSQ TQQEKNEKDL LALDSWKNLW SWFDISNWLWYIKSRIEGRG SGGYIPEAPR DGQAYVRKDG EWVLLSTFLSEQ ID NO: 2 mosaic gp140 protein(695 amino acids)AGKLWVTVYY GVPVWKEATT TLFCASDAKA YDTEVHNVWATHACVPTDPN PQEVVLENVT ENFNMWKNNM VEQMHEDIISLWDQSLKPCV KLTPLCVTLN CTDDVRNVTN NATNTNSSWGEPMEKGEIKN CSFNITTSIR NKVQKQYALF YKLDVVPIDNDSNNTNYRLI SCNTSVITQA CPKVSFEPIP IHYCAPAGFAILKCNDKKFN GTGPCTNVST VQCTHGIRPV VSTQLLLNGSLAEEEVVIRS ENFTNNAKTI MVQLNVSVEI NCTRPNNNTRKSIHIGPGRA FYTAGDIIGD IRQAHCNISR ANWNNTLRQIVEKLGKQEGN NKTIVFNHSS GGDPEIVMHS FNCGGEFFYCNSTKLFNSTW TWNNSTWNNT KRSNDTEEHI TLPCRIKQIINMWQEVGKAM YAPPIRGQIR CSSNITGLLL TRDGGNDTSGTEIFRPGGGD MRDNWRSELY KYKVVKIEPL GVAPTKAKERVVQREERAVG IGAVFLGFLG AAGSTMGAAS MTLTVQARLLLSGIVQQQNN LLRAIEAQQH LLQLTVWGIK QLQARVLAVERYLKDQQLLG IWGCSGKLIC TTTVPWNASW SNKSLDKIWNNMTWMEWERE INNYTSLIYT LIEESQNQQE KNEQELLELDKWASLWNWFD ISNWLWYIKS RIEGRGSGGY IPEAPRDGQAYVRKDGEWVL LSTFLSEQ ID NO: 3 (exemplary leader sequence) -amino acidsMRVRGIQRNC QHLWRWGTLI LGMLMICSA | 69,655 |
11857620 | DETAILED DESCRIPTION Disclosed herein are recombinant viruses and yeasts. The viruses and yeasts disclosed herein may be useful for a variety of purposes, such as treating and/or preventing a coronavirus disease. In one aspect, disclosed herein is a replication defective adenovirus, wherein the adenovirus comprises an E1 gene region deletion; an E2b gene region deletion; an E3 gene region deletion, a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein CoV2 nucleocapsid protein fused to an endosomal targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike protein sequence optimized for cell surface expression (S— Fusion). In one embodiment, the N-ETSD may comprises a sequence with at least 80% identity to SEQ ID NO:2. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. It is further contemplated that the fusion protein contains a linker between the N-ETSD domain and the nucleocapsid protein. For example this linker may be a 16 amino acid linker having the sequence (GGGS)4. In certain embodiments, methods are disclosed herein for enhancing the immunogenicity of an intracellular antigen, the methods comprising tagging the antigen with ETSD and expressing the tagged antigen in an antigen-presenting cell (e.g., a dendritic cell). In some embodiments, the fusion protein comprising N-ETSD and CoV2 nucleocapsid protein may be encoded by a nucleic acid sequence having at least 80% identity to SEQ ID NO:3. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The CoV2 spike protein is contemplated to have at least 85% identity to SEQ ID NO:6. The nucleic acid encoding the CoV2 spike protein has at least 99% identity to SEQ ID NO:5 or SEQ ID NO:7. In a second aspect of this disclosure, provided herein is a recombinant yeast comprising a nucleic acid encoding a protein selected from the group consisting of coronavirus 2 (CoV2) nucleocapsid protein, CoV2 spike protein, and a combination thereof. Preferably, the recombinant yeast isSaccharomyces cerevisiae. In some embodiments of this second aspect, the CoV2 nucleocapsid protein comprises a sequence with at least 80% identity to SEQ ID NO:2 or SEQ ID NO:3. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. In some embodiment of this second aspect, the CoV2 spike protein comprises a sequence with at least 80% identity to SEQ ID NO:5. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. In some embodiments, the nucleic acid encoding the CoV2 spike protein comprises a sequence with at least 80% identity to SEQ ID NO:5 or SEQ ID NO:7. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The adenoviruses and yeasts disclosed herein may further comprise a nucleic acid encoding a trafficking sequence, a co-stimulatory molecule, and/or an immune stimulatory cytokine. The co-stimulatory molecule is selected from the group consisting of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3. The immune stimulatory cytokine may be selected from the group consisting of IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, and LMP1. Additionally or alternatively, the vaccines disclosed herein may also encode SARS-CoV-2 M protein, with or without an ETSD tag. In yet another embodiment, disclosed herein is a vaccine composition comprising the adenovirus or yeast as disclosed above, and wherein the composition is formulated for injection. The vaccine composition may be used for inducing immunity against CoV2 in a patient in need thereof, by administering to the patient the vaccine composition Also disclosed herein are methods for preventing and/or treating coronavirus diseases, and especially COVID-19. Preferably, the method includes using a viral or yeast vector that encodes the nucleocapsid protein and/or spike protein of the coronavirus in an immunogenic composition that is administered to a subject individual. The virus and/or yeast vaccine, thus administered, would infect the individual with CoV2 nucleocapsid or spike protein. With that in place, the individual would have an immune response against it, and be vaccinated. Notably, as the nucleocapsid protein and the spike protein are relatively conserved polypeptides, immune responses can be elicited for a variety of members of the coronavirus family. Where the recombinant vector is an adenovirus, the adenoviral vector may be modified to encode the nucleocapsid protein, and/or the spike protein. Similarly, in case of yeast, the yeast vector may also be modified to encode the nucleocapsid protein, and/or the spike protein. Positive responses were obtained on cell mediated immunity upon administration of immunogenic compositions comprising the viral and/or yeast vectors in patients in need thereof. Thus, in one embodiment, the present disclosure envision creating the coronaviral spikes to be expressed on the yeast surface. So, in this embodiment, the yeast is acting as an avatar coronavirus to stimulate the B cells. The stimulation of the B cells then results in humoral immunity. In another embodiment, disclosed herein is a next generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, comprising both an S sequence optimized for cell surface expression (S— Fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal subcellular compartment, with the potential to generate durable immune protection. As further described in this disclosure, this bivalent vaccine has been found to be is optimized for immunogenicity as evidenced by the following findings:The optimized S-Fusion displayed improved S receptor binding domain (RBD) cell surface expression compared to S-WT where little surface expression was detected;The expressed RBD from S-Fusion retained conformational integrity and recognition by ACE2-Fc;The viral N protein modified with an enhanced T-cell stimulation domain (ETSD) localized to endosomal/lysosomal subcellular compartments for MHC I/II presentation; andThese optimizations to S and N (S-Fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T-cell responses in antigen-naïve preclinical models. Both the T-cell and antibody immune responses to S and N demonstrated a T-helper 1 (Th1) bias. The antibody responses were neutralizing as demonstrated by two independent SARS-CoV-2 neutralization assays. Thus, in one embodiment, this next generation bivalent hAd5 S-Fusion+N-ETSD vaccine provides robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection. This vaccine construct may be administered orally, intranasally or sublingually. Thus, in one embodiment, the instant disclosure provides vaccine construct in oral, intranasal, and sublingual formulations to induce mucosal immunity in addition to cell-mediated and humoral immunity. In one embodiment, the COVID-19 vaccine disclosed herein generates long-term T and B cell memory. Coronaviruses and Vaccines Therefor Coronaviruses are found in avian and mammalian species. They resemble each other in morphology and chemical structure: for example, the coronaviruses of humans and cattle are antigenically related. There is no evidence, however, that human coronaviruses can be transmitted by animals. In animals, various coronaviruses invade many different tissues and cause a variety of diseases in humans. One such disease was Severe acute respiratory syndrome (SARS) coronavirus disease that spread to several countries in Asia, Europe and North America in late 2002/early 2003. Another such disease is the novel Coronvirus Disease of 2019 (COVID 19) that has spread to several countries in the world. In December of 2019, reports emerged from Wuhan, China concerning a new infectious respiratory disease with high morbidity and mortality 1-3 that displayed human-to-human transmission.4 The causative agent was rapidly identified as a novel coronavirus and was designated SARS-coronavirus 2 (SARS-CoV-2). The disease it causes is referred to as COVID-19 and has rapidly become a worldwide pandemic that has disrupted socioeconomic life and resulted in more than 32 million infections and more than 1,100,000 deaths worldwide as of late October 2020. COVID 19 usually begins with a fever greater than 38° C. Initial symptoms can also include cough, sore throat, malaise and mild respiratory symptoms. Within two days to a week, patients may have trouble breathing. Patients in more advanced stages of COVID 19 develop either pneumonia or respiratory distress syndrome. Public health interventions, such as surveillance, travel restrictions and quarantines, are being used to contain the spread of COVID 19. It is unknown, however, whether these draconian containment measures can be sustained with each appearance of the COVID 19 in humans. Furthermore, the potential of this new and sometimes lethal CoV as a bio-terrorism threat is obvious. Coronavirus virions are spherical to pleomorphic enveloped particles. The envelope is studded with projecting glycoproteins, and surrounds a core consisting of matrix protein enclosed within which is a single strand of positive-sense RNA (Mr 6×106) associated with nucleocapsid protein. In that regard, it should be noted that the terms “nucleocapsid protein,” “nucleoprotein,” and “nucleocapsid” are used interchangeably throughout this disclosure. The coronavirus nucleocapsid (N) is a structural protein found in all coronaviruses, including COVID 19. The nucleocapsid protein forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. Another protein found throughout all coronavirus virions is the viral spike(S) protein. Coronaviruses are large positive-stranded RNA viruses typically with a broad host range. Like other enveloped viruses, CoV enter target cells by fusion between the viral and cellular membranes, and that process is mediated by the viral spike (S) protein. SARS-CoV-2 is an enveloped positive sense, single-strand RNA β coronavirus primarily composed of four structural proteins—spike (S), nucleocapsid (N), membrane (M), and envelope—as well as the viral membrane and genomic RNA. Of these, S is the largest and N the most prevalent. The S glycoprotein is displayed as a trimer on the viral surface (FIG.8a), whereas N is located within the viral particle. A schematic of the S primary structure is shown inFIG.8b. The sequence of SARS-CoV-2 was published8 and compared to that of previous coronaviruses. This was soon followed by reports on the crystal structure of the S protein. The virus uses S protein to enter host cells by interaction of the S receptor binding domain (S RBD) with angiotensin-converting enzyme 2 (ACE2), an enzyme expressed broadly on a variety of cell types in the nose, mouth, gut and lungs as well as other organs, and importantly on the alveolar epithelial cells of the lung where infection is predominantly manifested. As represented inFIG.8b, the S RBD is found within the S1 region of spike. The methods and compositions disclosed herein target the nucleoprotein and the spike protein that is conserved in all types of coronaviruses. In one embodiment, the present disclosure provides a vaccine formulation comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2); and/or wherein the recombinant entity encodes a spike protein of CoV2. The vaccine formulation may be useful for treating a disease, such as a coronavirus mediated disease or infection. Thus, in another embodiment, disclosed is a method for treating a coronavirus disease, in a patient in need thereof, comprising: administering to the subject an immunotherapy composition comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2). The coronavirus contemplated herein may be coronavirus disease 2019 (COVID-19) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) The instant disclosure also provides a method for treating coronavirus disease 2019 (COVID-19) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), in a patient in need thereof, comprising: administering to the subject a first immunotherapy composition comprising a recombinant virus, wherein the recombinant virus comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2), administering to the subject a second immunotherapy composition comprising a recombinant yeast, wherein the recombinant yeast comprises a nucleic acid that encodes a spike protein of CoV2. The first and second immunotherapy compositions may be administered concurrently or sequentially to the patient. Viewed form a different perspective, contemplated herein is a viral vector (e.g., recombinant adenovirus genome, optionally with a deleted or non-functional E2b gene) that comprises a nucleic acid that encodes (a) at least a nucleocapsid protein; and (b) at least one spike protein. The viral vector may further comprise co-stimulatory molecule. Most typically, the nucleic acid will further include a trafficking signal to direct a peptide product encoded by the nucleic acid to the cytoplasm, the endosomal compartment, or the lysosomal compartment, and the peptide product will further comprise a sequence portion that enhances intracellular turnover of the peptide product. The majority of current SARS-CoV-2 vaccines under development target S because of the potential to neutralize the ability of the virus to bind host cells by production of antibodies against the RBD. Support for RBD as a key antigen was recently confirmed, and it was reported that in 44 hospitalized COVID-19 patients, RBD-specific IgG responses and neutralizing antibody titers are detectable in all patients by 6 days post-PCR confirmation of infection, and that the two are correlated. See Suthar, M. S. et al. Rapid generation of neutralizing antibody responses in COVID-19 patients. Cell Reports Medicine, 2020, which is incorporated by reference herein. They confirmed this finding in an additional 231 PCR-confirmed COVID-19 patient samples. In addition to humoral responses, S epitopes are also frequent targets of COVID-19 recovered patient T cells, providing further justification for inclusion of S in prophylactic immunization strategies. Despite the urgent need for rapid development of SARS-CoV-2 vaccines, reliance on any one antigen cargo or immunological pathway as occurring in the monovalent vaccines under development is not without risk. Evaluation of nearly 4000 SARS-CoV-2 genomic sequences has identified numerous mutations in S with the D614G variant emerging recently as a potentially more infectious strain six months after identification of the original virus. In designing the vaccine disclosed herein, to overcome the risk of the emergence of new strains of the virus with mutations in S and to provide additional antigens against which responses can be elicited, an optimized N sequence was added. The N protein is a highly conserved and antigenic SARS-CoV-2-associated protein that has been studied previously as an antigen in coronavirus vaccine design for SARS-CoV. N associates with viral RNA within the virus and has a role in viral RNA replication, virus particle assembly, and release. SARS-CoV-2 N is a highly antigenic protein and recent studies have shown that nearly all patients infected with SARS-CoV-2 have antibody responses to N. Furthermore, another study reported that most, if not all, COVID-19 survivors tested were shown to have N-specific CD4+ T-cell responses. Currently, there is keen focus on generation of humoral responses to vaccines with, arguably, less attention being paid to T-cell responses. The natural history of SARS-CoV-2 infection would suggest, however, that a robust T-cell response to vaccination is at least as important as the production of antibodies and should be a critical consideration for COVID-19 vaccine efficacy. First, the humoral and T-cell responses are highly correlated, with titers of neutralizing antibodies being proportional to T-cell levels, suggesting the T response is necessary for an effective humoral response. It is well established that the activation of CD4+T helper cells enhances B-cell production of antibodies. Second, virus-specific CD4+ and CD8+ T cells are not only widely detected in COVID-19 patients, based on findings from patients recovered from the closely-related SARS-CoV, but such T cells persist for at least 6-17 years, suggesting that T cells may be an important part of long-term immunity. These T-cell responses were predominantly to N, and it has been reported that in all 36 convalescent COVID-19 patients in their study, the presence of CD4+ and CD8+ T cells recognizing multiple regions of the N protein could be demonstrated. Examination of blood from 23 individuals who had recovered from SARS-CoV and found that the memory T cells acquired 17 years ago also recognized multiple proteins of SARS-CoV-2. These findings emphasize the importance of designing a vaccine with the highly conserved nucleocapsid present in both SARS-CoV and SARS-CoV-2. Third, recovered patients exposed to SARS-CoV-2 have been found without seroconversion, but with evidence of T-cell responses. The T-cell based responses become even more critical given the finding in at least one study that neutralizing antibody titers decline in some COVID-19 patients after about 3 months. In one embodiment, the vaccines disclosed herein results in the generation of T-cell in addition to humoral responses. A bivalent vaccine comprising many antigens— S RBD as displayed by inclusion of full-length S including SD1, S1 and S2 epitopes, along with N— would be more effective in eliciting both T-cell and antibody-based responses than a construct with either antigen alone by presenting both unique and conserved SARS-CoV-2 antigenic sites to the immune system. The importance of both S and N was highlighted by identifying that both S and N antigens as a priori potential B and T-cell epitopes for the SARS-CoV virus that shows close similarity to SARS-CoV-2 that are predicted to induce both T and B cell responses. An additional consideration for design of an effective vaccine is the likelihood of antigen presentation on the surface of the vectored-protein-expressing cell and in a conformation that recapitulates natural virus infection. First, because wild type N does not have a signaling domain that directs it to endosomal processing and ultimately MEW class II complex presentation to CD4+ T cells, the wild type N sequence is not optimal for induction of a vigorous CD4+ T-cell responses, a necessity for both cell-mediated and B cell memory. To overcome this limitation, we have designed an Enhanced T-cell Stimulation Domain (ETSD) to N to allow the necessary processing and presentation. Second, to display the highly antigenic RBD region of S on the cell surface, we have optimized the wild type S protein “S Fusion sequence”, to increase the likelihood of native folding, increased stability, and proper cell surface expression of RBD. Thus, in one embodiment, the vaccine construct design comprises an S-Fusion+N-ETSD sequence. The vaccine platform utilized here is a next-generation recombinant human adenovirus serotype 5 (hAd5) vector with deletions in the E1, E2b, and E3 gene regions (hAd5 [E1-, E2b-, E3-]). This hAd5 [E1-, E2b-, E3-] vector (FIG.8c) is primarily distinguished from other first-generation [E1-, E3-] recombinant Ad5 platforms by having additional deletions in the early gene 2b (E2b) region that remove the expression of the viral DNA polymerase (pol) and in pre terminal protein (pTP) genes, and its propagation in the E.C7 human cell line. Removal of these E2b regions confers advantageous immune properties by minimizing immune responses to Ad5 viral proteins such as viral fibers, 37 thereby eliciting potent immune responses to specific antigens in patients with pre-existing adenovirus (Ad) immunity. As a further benefit of these deletions, the vector has an expanded gene-carrying/cloning capacity compared to the first generation Ad5 [E1-, E3-] vectors. This next generation hAd5 [E1-, E2b-, E3-] vaccine platform, in contrast to Ad5 [E1-, E3-]-based platforms, does not promote activities that suppress innate immune signaling, thereby allowing for improved vaccine efficacy and a superior safety profile independent of previous Ad immunity. Since these deletions allow the hAd5 platform to be efficacious even in the presence of existing Ad immunity, this platform enables relatively long-term antigen expression without significant induction of anti-vector immunity. It is therefore also possible to use the same vector/construct for homologous prime-boost therapeutic regimens unlike first-generation Ad platforms which face the limitations of pre-existing and vaccine-induced Ad immunity. Importantly, this next generation Ad vector has demonstrated safety in over 125 patients with solid tumors. In these Phase I/II studies, CD4+ and CD8+antigen-specific T cells were successfully generated to multiple somatic antigens (CEA, MUC1, brachyury) even in the presence of pre-existing Ad immunity. The instant disclosure provides findings of confirmed enhanced cell-surface expression and physiologically-relevant folding of the expressed S RBD from S-Fusion by ACE2-Fc binding. The N-ETSD protein was successfully localized to the endosomal/lysosomal subcellular compartment for MEW presentation and consequently generated both CD4+ and CD8+ T-cell responses. Immunization of CD-1 mice with the hAd5 S Fusion+N-ETSD vaccine elicited both humoral and cell-mediated immune responses to vaccine antigens. CD8+ and CD4+ T-cell responses were noted for both S and N. Statistically significant IgG responses were seen for antibody generation against S and N. Potent neutralization of SARS-CoV-2 by sera from hAd5 S Fusion+N-ETSD-immunized mice was confirmed by two independent SARS-CoV-2 neutralization assays: the cPass assay measuring competitive inhibition of RBD binding to ACE2,44 and in the live SARS-CoV-2 virus assay with infected Vero E6 cells. Analysis of T-cell responses as well as humoral responses to S and N were skewed toward a Th1-specific response. Taken together, these findings illustrate that hAd5 S-Fusion+N-ETSD vaccine would be particularly effective against the SARS-CoV-2. Recombinant Viruses With respect to recombinant viruses it is contemplated that all known manners of making recombinant viruses are deemed suitable for use herein, however, especially preferred viruses are those already established in therapy, including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, etc. Among other appropriate choices, adenoviruses are particularly preferred. Moreover, it is further generally preferred that the virus is a replication deficient and non-immunogenic virus. For example, suitable viruses include genetically modified alphaviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc. However, adenoviruses are particularly preferred. For example, genetically modified replication defective adenoviruses are preferred that are suitable not only for multiple vaccinations but also vaccinations in individuals with preexisting immunity to the adenovirus (see e.g., WO 2009/006479 and WO 2014/031178, which are incorporated by reference in its entirety). In some embodiments, the replication defective adenovirus vector comprises a replication defective adenovirus 5 vector. In some embodiments, the replication defective adenovirus vector comprises a deletion in the E2b region. In some embodiments, the replication defective adenovirus vector further comprises a deletion in the E1 region. In that regard, it should be noted that deletion of the E2b gene and other late proteins in the genetically modified replication defective adenovirus to reduce immunogenicity. Moreover, due to these specific deletions, such genetically modified viruses were replication deficient and allowed for relatively large recombinant cargo. For example, WO 2014/031178 describes the use of such genetically modified viruses to express CEA (colorectal embryonic antigen) to provide an immune reaction against colon cancer. Moreover, relatively high titers of recombinant viruses can be achieved using genetically modified human 293 cells as has been reported (e.g., J Virol. 1998 February; 72(2): 926-933). E1-deleted adenovirus vectors Ad5 [E1-] are constructed such that a trans gene replaces only the E1 region of genes. Typically, about 90% of the wild-type Ad5 genome is retained in the vector. Ad5 [E1-] vectors have a decreased ability to replicate and cannot produce infectious virus after infection of cells not expressing the Ad5 E1 genes. The recombinant Ad5 [E1-] vectors are propagated in human cells allowing for Ad5 [E1-] vector replication and packaging. Ad5 [E1-] vectors have a number of positive attributes; one of the most important is their relative ease for scale up and cGMP production. Currently, well over 220 human clinical trials utilize Ad5 [E1-] vectors, with more than two thousand subjects given the virus sc, im, or iv. Additionally, Ad5 vectors do not integrate; their genomes remain episomal. Generally, for vectors that do not integrate into the host genome, the risk for insertional mutagenesis and/or germ-line transmission is extremely low if at all. Conventional Ad5 [E1-] vectors have a carrying capacity that approaches 7 kb. One obstacle to the use of first generation (E1-deleted) Ad5-based vectors is the high frequency of pre-existing anti-adeno virus type 5 neutralizing antibodies. Attempts to overcome this immunity is described in WO 2014/031178, which is incorporated by reference herein. Specifically, a novel recombinant Ad5 platform has been described with deletions in the early 1 (E1) gene region and additional deletions in the early 2b (E2b) gene region (Ad5 [E1-, E2b-]). Deletion of the E2b region (that encodes DNA polymerase and the pre-terminal protein) results in decreased viral DNA replication and late phase viral protein expression. E2b deleted adenovirus vectors provide an improved Ad-based vector that is safer, more effective, and more versatile than First Generation adenovirus vectors. In a further embodiment, the adenovirus vectors contemplated for use in the present disclosure include adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally, deletions in the E1, E3 and, also optionally, partial or complete removal of the E4 regions. In a further embodiment, the adenovirus vectors for use herein have the E1 and/or the preterminal protein functions of the E2b region deleted. In some cases, such vectors have no other deletions. In another embodiment, the adenovirus vectors for use herein have the E1, DNA polymerase and/or the preterminal protein functions deleted. The term “E2b deleted”, as used herein, refers to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one E2b gene product. Thus, in certain embodiments, “E2b deleted” is used in relation to a specific DNA sequence that is deleted (removed) from the Ad genome. E2b deleted or “containing a deletion within the E2b region” refers to a deletion of at least one base pair within the E2b region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, the deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E2b region of the Ad genome. An E2b deletion may be a deletion that prevents expression and/or function of at least one E2b gene product and therefore, encompasses deletions within exons of encoding portions of E2b-specific proteins as well as deletions within promoter and leader sequences. In certain embodiments, an E2b deletion is a deletion that prevents expression and/or function of one or both of the DNA polymerase and the preterminal protein of the E2b region. In a further embodiment, “E2b deleted” refers to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein. As noted before, the desired nucleic acid sequences (for expression from virus infected cells) are under the control of appropriate regulatory elements well known in the art. In view of the above, it should be appreciated that compositions and methods presented are not only suitable for directing virally expressed antigens specifically to one or another (or both) MHC systems, but will also provide increased stimulatory effect on the CD8+ and/or CD4+ cells via inclusion of various co-stimulatory molecules (e.g., ICAM-1 (CD54), ICOS-L, LFA-3 (CD58), and at least one of B7.1 (CD80) and B7.2 (CD86)), and via secretion or membrane bound presentation of checkpoint inhibitors. With respect to viral expression and vaccination systems it is contemplated that all therapeutic recombinant viral expression systems are deemed suitable for use herein so long as such viruses are capable to lead to expression of the recombinant payload in an infected cell. Regardless of the type of recombinant virus it is contemplated that the virus may be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus may be injected subcutaneously or intravenously, or may be administered intranasaly or via inhalation to so infect the patient's cells, and especially antigen presenting cells. Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) of the patient (or from an allogeneic source) may be infected in vitro and then transfused to the patient. Alternatively, immune therapy need not rely on a virus but may be effected with nucleic acid transfection or vaccination using RNA or DNA, or other recombinant vector that leads to the expression of the neoepitopes (e.g., as single peptides, tandem mini-gene, etc.) in desired cells, and especially immune competent cells. As noted above, the desired nucleic acid sequences (for expression from virus infected cells) are under the control of appropriate regulatory elements well known in the art. For example, suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoter), but inducible promoters are also deemed suitable for use herein, particularly where induction conditions are typical for a tumor microenvironment. For example, inducible promoters include those sensitive to hypoxia and promoters that are sensitive to TGF-β or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive elements promoter). In other examples, suitable inducible promoters include the tetracycline-inducible promoter, the myxovirus resistance 1 (Mx1) promoter, etc. The replication defective adenovirus comprising an E1 gene region deletion, an E2b gene region deletion, and a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein and/or a CoV2 spike protein, as disclosed herein may be administered to a patient in need for inducing immunity against CoV2. Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, may vary from individual to individual, and the severity of the disease, and may be readily established using standard techniques. In some embodiments, the administration comprises delivering 4.8-5.2×1011replication defective adenovirus particles, or 4.9-5.1×1011replication defective adenovirus particles, or 4.95-5.05×1011replication defective adenovirus particles, or 4.99-5.01×1011replication defective adenovirus particles. The administration of the virus particles can be through a variety of suitable paths for delivery. One preferred route contemplated herein is by injection, such as intracutaneous injection, intramuscular injection, intravenous injection or subcutaneous injection. In some embodiments, a subcutaneous delivery may be preferred. Recombinant Yeasts With respect to yeast expression and vaccination systems, it is contemplated that all known yeast strains are deemed suitable for use herein. However, it is preferred that the yeast is a recombinantSaccharomycesstrain that is genetically modified with a nucleic acid construct encoding a protein selected from the group consisting of coronavirus 2 (CoV2) nucleocapsid protein, CoV2 spike protein, and a combination thereof, to thereby initiate an immune response against the CoV2 viral disease. In one aspect of any of the embodiments of the disclosure described above or elsewhere herein, the yeast vehicle is a whole yeast. The whole yeast, in one aspect is killed. In one aspect, the whole yeast is heat-inactivated. In one preferred embodiment, the yeast is a whole, heat-inactivated yeast fromSaccharomyces cerevisiae. The use of a yeast based therapeutic compositions are disclosed in the art. For example, WO 2012/109404 discloses yeast compositions for treatment of chronic hepatitis b infections. It is noted that any yeast strain can be used to produce a yeast vehicle of the present disclosure. Yeasts are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes andFungi Imperfecti. One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In preferred embodiments, the yeast is a non-pathogenic strain such asSaccharomyces cerevisiaeas non-pathogenic yeast strains minimize any adverse effects to the individual to whom the yeast vehicle is administered. However, pathogenic yeast may also be used if the pathogenicity of the yeast can be negated using pharmaceutical intervention. For example, suitable genera of yeast strains includeSaccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, SchizosaccharomycesandYarrowia. In one aspect, yeast genera are selected fromSaccharomyces, Candida, Hansenula, PichiaorSchizosaccharomyces, and in a preferred aspect,Saccharomycesis used. Species of yeast strains that may be used includeSaccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianusvar.lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, andYarrowia lipolytica. It should further be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned species. In one aspect, yeast species used in the instant disclosure includeS. cerevisiae, C. albicans, H. polymorpha, P. pastorisandS. pombe. S. cerevisiaeis useful due to it being relatively easy to manipulate and being “Generally Recognized As Safe” or “GRAS” for use as food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997). Therefore, particularly contemplated herein is a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as aS. cerevisiaecir strain. TheS. cerevisiaestrain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation. Expression of contemplated peptides/proteins in yeast can be accomplished using techniques known to those skilled in the art. Most typically, a nucleic acid molecule encoding at least one protein is inserted into an expression vector such manner that the nucleic acid molecule is operatively linked to a transcription control sequence to be capable of effecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. As will be readily appreciated, nucleic acid molecules encoding one or more proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. Particularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the methods and compositions of the present disclosure and a variety of such promoters are known to those skilled in the art and have generally be discussed above. Promoters for expression inSaccharomyces cerevisiaeinclude promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translational elongation factor EF-1 alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uridyl-transferase (GALT), UDP-galactose epimerase (GAL10), cytochrome cl (CYC1), Sec7 protein (SECT) and acid phosphatase (PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10 promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiaeinclude the UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GALT and GAL10, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression inSaccharomyces cerevisiaeinclude the termination sequences of the alpha-factor, GAPDH, and CYC1 genes. Transcription control sequences to express genes in methyltrophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase. Likewise, transfection of a nucleic acid molecule into a yeast cell according to the present disclosure can be accomplished by any method by which a nucleic acid molecule administered into the cell and includes diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins. Further exemplary yeast expression systems, methods, and conditions suitable for use herein are described in US20100196411A1, US2017/0246276, or US 2017/0224794, and US 2012/0107347. So produced recombinant viruses and yeasts may then be individually or in combination used as a therapeutic vaccine in a pharmaceutical composition, typically formulated as a sterile injectable composition with a virus of between 104-1013virus or yeast particles per dosage unit, or more preferably between 109-1012virus or yeast particles per dosage unit. Alternatively, virus or yeast may be employed to infect patient cells ex vivo and the so infected cells are then transfused to the patient. However, alternative formulations are also deemed suitable for use herein, and all known routes and modes of administration are contemplated herein. Second Generation hAd5 [E1-, E2b-, E3-] Based Vaccines Disclosed Herein Overcome Pre-Existing Anti-Ad5 Immunity To avoid the Ad immunization barrier and circumvent the adverse conditions for first generation Ad5 [E1-E3-] vectors, an advanced 2nd generation human adenoviral (hAd5) vector was constructed having two (2) additional deletions in the E2b region, removing the DNA polymerase and the preterminal protein genes [E1-, E2b-, E3-]. (Former names of our adenovirus vector were Ad5, ETBX in literature) E2b-deleted hAd5 vectors have up to a 12-14 kb gene-carrying capacity as compared to the 7-kb capacity of first generation Ad5 [E1-] vectors, providing space for multiple genes if needed. hAd5 [E1-, E2b-, E3-] based recombinant vectors are produced using the human E.C7 cell line. Deletion of the E2b region also confers advantageous immune properties on these novel Ad vectors, eliciting potent immune responses to specific, non-viral antigens while minimizing the immune responses to Ad viral proteins. hAd5 [E1-, E2b-, E3-] vectors induce a potent cell mediated immune (CMI) response, as well as Abs against the vectored antigens even in the presence of Ad immunity. hAd5 [E1-, E2b-, E3-] vectors also have reduced adverse reactions as compared to Ad5 [E1-] vectors, in particular the appearance of hepatotoxicity and tissue damage. In one embodiment, the reduced inflammatory response against hAd5 [E1-, E2b-, E3-] vector viral proteins and the resulting evasion of pre-existing Ad immunity increases the capability for the hAd5 [E1-, E2b-, E3-] vectors to infect dendritic cells (DC), resulting in greater immunization of the vaccine. In addition, increased infection of other cell types provides high levels of antigen presentation needed for a potent CD8+ and CD4+ T cell responses, leading to memory T cell development. In one embodiment, hAd5 [E1-, E2b-, E3-] vectors are superior to Ad5 [E1-] vectors in immunogenicity and safety and will be the best platform to develop a COVID-19 vaccine in a rapid and efficient manner. In one embodiment, a prophylactic vaccine is tested against COVID-19 by taking advantage of this new hAd5 vector system that overcomes barriers found with other Ad5 systems and permits the immunization of people who have previously been exposed to Ad5. Track Record of Rapid Vaccine Development Utilizing Second Generation Human (hAd5) Adenovirus Platform During Pandemic Treats: H1N1 Experience in 2009 To address emerging pathogen threats, especially in times of pandemic, it is critical that modernized vaccine technologies be deployed. These technologies will utilize the power of genomic sequencing, rapid transfection in well-established vaccine vectors to rapidly identify constructs with high immunogenicity. Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (hAd5 [E1-, E2b-, E3-]) has been utilized to induce immune responses to various antigenic targets. This vector platform expressed hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The hAd5 [E1-, E2b-, E3-] has thus demonstrated the capability for the rapid development of effective vaccines against infectious diseases. hAd5 Vaccine Constructs and Results Disclosed herein are constructs that have been constructed and tested, a hAd5-COVID-19 vaccine construct E1-, E2b-, E3-hAd5 vector with SARS-CoV-2 (S/N) protein insert (FIG.1). This construct has been tested in preclinical experiments, including in vitro expression (FIG.2) and small animal immunogenicity. In addition, ImmunityBio has developed multiple COVID-19 constructs including RBD-alone, S1-alone, S1-fusion proteins, and combinations of RBD, S1 and S1 fusions with N. Preliminary in-vitro studies demonstrate that these constructs (FIG.3) recognize convalescent serum antibodies and could serve as alternative vaccines following analysis of the two (2) constructs above (FIG.1) which is intended to initiate in our first in human Phase 1b study. Rationale for Inclusion of Nucleocapsid (N) in hAd5 Constucts for COVID-19 The nucleocapsid (N) protein of SARS-CoV-2 is highly conserved and highly expressed. Previous research with the related coronavirus that causes SARS demonstrated that N protein is immunogenic (Gupta, 2006), when integrated with intracellular trafficking constructs. To date, vaccine strategies in development all involve developing immunogenicity against spike (S) protein. However, very recent evidence in patients who recovered from COVID-19 demonstrates Th1 immunity generated against the nucleocapsid (N) (Grifoni, 2020). A second report by Grifoni et al. further confirmed that in the predictive bioinformatics model, T and B cell epitopes were highest for both spike glycoprotein and nucleoprotein (Grifoni, 2020). The present disclosure confirms the potential that combining S with N, that long-term cell-mediated immunity with a Th1 phenotype can be induced. The potential exists for this combination vaccine to serve as a long-term “universal” COVID-19 vaccine in light of mutations undergoing in S and the finding that the structural N protein is highly conserved in the coronavirus family. The clinical trial is designed to compare S alone versus S+N, to demonstrate safety and to better inform the immunogenicity of S and S+N. A single construct having S & N would be selected to induce potent humoral and cell mediated immunity. Immunogenicity Studies (Small Animal Model): Homologous prime-boost immunogenicity in BALB-c mice. Mice have been treated with 1, 2 or 3 doses of the hAd5 COVID-19 vaccine and serum and splenocyte samples are being tested for SARS-CoV-2 antigen-specific immune responses. Serum is tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes is tested for spike- and nucleocapsid-specific cell mediated immune responses by ELISPOT and intracellular cytokine simulation assays. The results show promising immunogenic activity. In one embodiment, hAd5 [E1-,E2b-, E3-] N-ETSD, a vaccine containing SARS-CoV-2 nucleocapsid plus an enhanced T cell stimulation domain (ETSD), alters T cell responses to nucleocapsid. Mice were immunized subcutaneously (SC) with a dose of 1010 VP twice at 7-day intervals. Blood was collected at several time points and spleen was collected upon sacrifice in order to perform immunogenicity experiments. Splenocytes were isolated and tested for cell mediated immune (CMI) responses. The results showed that SARS-CoV-2 nucleocapsid antigen specific CMI responses were detected by ELISpot and flow cytometry analyses in the spleens of all the mice immunized with hAd5 [E1-, E2b-, E3-] N-ETSD vaccine but not vector control (hAd5 [E1-, E2b-, E3-] null) immunized mice. In addition, antibody responses were detected in all the mice immunized with hAd5 [E1-, E2b-, E3-]-N-ETSD vaccine but not vector control (Ad5 [E1-, E2b-, E3-]-null) immunized mice (FIG.4&FIG.5). Additional studies to confirm and extend these results are ongoing. Enhanced RBD Cell Surface Expression: Further evidence of the potential enhancing immunogenicity value of N when combined with S was the surprising finding of enhanced surface expression of the RBD protein in 293 cells transfected with the N-ETSD+S construct as seen inFIG.6. Expression and presentation of RBD appears to be highly important as evidenced by the recent report by Robbiani et al who showed that rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested who had recovered from COVID-19 infections (Robbiani 2020). This finding of enhanced expression of RBD when N is combined with S-Fusion was corroborated in studies using plasma from a patient recovered from COVID-19 infection (FIG.7). The alternative construct of RBD-ETSD could serve as alternative vaccines following analysis of the two (2) constructs above (FIG.1) which is intended to initiate in human Phase 1b studies. In summary, on the basis of enhanced expression and exposure of the RBD protein with S Fusion and S Fusion+N construct, both were tested in the hAd5 vector. Furthermore, on the basis of recent clinical data from patients recovered from COVID-19, as well as the corroborating preclinical data that the N construct induces long lasting CD4+ and Th1 cell-mediated immunity, this combination of S Fusion+N construct could provide long-lasting immunity beyond short term neutralizing antibodies. Immunogenicity Testing of Candidate COVID-19 Vaccine Constructs Two (2) Adenovirus-based COVID-19 vaccine constructs will be tested in preclinical experiments, including in vitro expression; small animal immunogenicity, and non-human primate immunogenicity and efficacy. Constructs description: ImmunityBio has generated two (2) second generation hAd5-based COVID-19 vaccine constructs for preclinical testing and clinical evaluation. First is a hAd5 vector with SARS-CoV-2 with spike protein insert (seeFIG.1). Second is E1-, E2b-, E3-hAd5 vector with SARS-CoV-2 wild type spike protein (S) insert and Nucleocapsid protein (N) insert containing an Endosomal-targeting domain sequence (ETSD) in the same vector backbone. Immunogenicity Studies: Homologous prime-boost immunogenicity in mice was examined by treating Mice with 1, 2 or 3 doses of the ImmunityBio adenovirus vaccine candidates listed inFIG.1and serum and splenocyte samples will be tested for SARS-CoV-2 antigen-specific immune responses. Serum is being tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes will be tested for spike- and nucleocapsid-specific cell mediated immune responses by ELISPOT and intracellular cytokine simulation assays. Data from these studies are disclosed throughout this disclosure. SARS-CoV-2 Virus Neutralization Studies: Serum from the mice immunized during the course of the immunogenicity studies described above is used will be sent to a third-party subcontractor for SARS-CoV-2 neutralization studies to be performed in their ABSL-3 facility. Serum will be tested for COVID 19 virus neutralizing activity by mixing various dilutions of serum with COVID 19 virus, incubating the mixture, and then exposing the mixture to Vero cells to detect cytopathic effect (CPE). The last dilution that prevents CPE will be considered the endpoint neutralizing titer. Immunogenicity and Efficacy Evaluation in Non-Human Primates (third-party subcontractor): Rhesus macaques will be treated with three doses of the ImmunityBio adenovirus vaccine candidates listed inFIG.1. SARS-CoV-2 antigen-specific immune responses will be monitored in serum and PBMCs by ELISA, ELISPOT and ICS throughout the course of the therapy. Four weeks after the final vaccination, animals will be challenged with SARS-CoV-2 and monitored for disease hallmarks and virus shedding. Phase 1b Clinical trial: ImmunityBio has submitted an IND for Phase 1b clinical trial testing of hAd5 [E1-, E2b-, E3-] CoV-2 vaccine. Study Design: This is a Phase 1b open-label study in adult healthy subjects. This clinical trial is designed to assess the safety, reactogenicity, and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines. The hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines are hAd5 [E1-, E2b-, E3-] vector-based targeting vaccines encoding the SARS-CoV-2 Spike (S) protein alone or together with the SARS-CoV-2 nucleocapsid (N) protein. The hAd5 [E1-, E2b-, E3-] vector is the platform technology for targeted vaccines that has demonstrated safety in over 125 patients with cancer to date at doses as high as 5×1011 virus particles per dose. Co-administration of three different hAd5 [E1-, E2b-, E3-] vector-based vaccines on the same day at 5×1011 virus particles per dose each (1.5×1012 total virus particles) has also been demonstrated to be safe. COVID-19 infection causes significant morbidity and mortality in a worldwide population. The hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines are designed to induce both a humoral and cellular response even in individuals with pre-existing adenoviral immunity. Thus, the potential exists for the hAd5-COVID-19-S and hAd5-COVID-19-S/N to induce anti-COVID-19 immunity and prevent or lessen the health impact of COVID-19 infection in healthy subjects. Phase 1b Safety Analysis: In the initial safety analysis of phase 1b, a total of 40 healthy subjects will be divided into 4 dosing cohorts (cohorts 1A, 1B, 2A, 2B; n=10 for each cohort):Cohort 1A—hAd5-COVID-19-S at 5×1010 viral particles (VP) per dose (n=10),Cohort 1B—hAd5-COVID-19-S at 1×1011 VP per dose (n=10),Cohort 2A—hAd5-COVID-19-S/N at 5×1010 VP per dose (n=10),Cohort 2B—hAd5-COVID-19-S/N at 1×1011 VP per dose (n=10). Each subject will receive a subcutaneous (SC) injection of hAd5-COVID-19-S or hAd5-COVID-19-S/N on Day 1 and Day 22 (ie, 2 doses). This dosing schedule is consistent with hAd5 [E1-, E2b-, E3-] vector-based vaccines currently in clinical trials. Cohorts 1-2 will enroll in parallel and may be opened at the same time or in a staggered manner depending upon investigational product supply. Subjects in cohorts 1A and 2A will complete the low-dose vaccination regimen first. After all subjects in cohorts 1A and 2A have completed at least a single dose and follow-up assessments during the toxicity assessment period through study day 8, enrollment will proceed if ImmunityBio Safety Review Committee (SRC) and at least one qualified infectious disease physician, independent of the Sponsor and trial, confirms absence of safety concerns. Subjects will then be enrolled in higher-dose cohorts 1B and 2B, and vaccinated. For all subjects, follow-up study visits will occur at days 8, 22, 29, 52, and at months 3, 6, and 12 following the final vaccination. Additional follow up for safety information will occur via telephone contact as noted in the Schedule of Events. The primary objectives of the initial safety phase 1b are to evaluate preliminary safety and reactogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines. The secondary objectives are to evaluate the extended safety and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines. Expanded Phase 1b: Safety and Immunogenicity for Construct Selection Phase 1b expansion will proceed if the SRC determines it is safe to do so based on a review of safety data from the phase 1b safety assessment. In phase 1b expansion, a total of 60 healthy subjects will be divided into 4 dosing cohorts (cohorts 1A, 1B, 2A, 2B; n=15 for each cohort):Cohort 1A—hAd5-COVID-19-S at 5×1010 VP per dose (n=15)Cohort 1B—hAd5-COVID-19-S at 1×1011 VP per dose (n=15)Cohort 2A—hAd5-COVID-19-S/N at 5×1010 VP per dose (n=15)Cohort 2B—hAd5-COVID-19-S/N at 1×1011 VP per dose (n=15) Each subject will receive a SC injection of hAd5-COVID-19-S or hAd5-COVID-19-S/N on Day 1 and Day 22 (ie, 2 doses). For all subjects, follow-up study visits will occur at days 8, 22, 29, 52, and at months 3, 6, and 12 following the final vaccination. Additional follow up for safety information will occur via telephone contact as noted in the Schedule of Events. The primary objective of the expanded phase 1b is to select the most immunogenic construct between hAd5-COVID-19-S and hAd5-COVID-19-S/N and dose level as determined by changes in humoral and cellular immunogenicity indexes. The secondary objectives are to assess safety and reactogenicity of hAd5-COVID-19-S and hAd5-COVID-19-S/N. As used herein, the term “administering” a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, wherein direct administration of the pharmaceutical composition or drug is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively, or additionally, antigen presenting cells may be isolated or grown from cells of the patient, infected in vitro, and then transfused to the patient. In one aspect of any of the embodiments described above or elsewhere herein, the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject. It is still further contemplated that the recombinant viruses and yeasts contemplated herein may further comprises a sequence that encodes at least one of a co-stimulatory molecule, an immune stimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition. For example, suitable co-stimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and/or LFA3, while suitable immune stimulatory cytokine include IL-2, IL-12, IL-15, IL-15 super agonist (N803), IL-21, IPS1, and/or LMP1, and/or suitable proteins that interfere include antibodies against or antagonists of CTLA-4, PD-1, TIM1 receptor, 2B4, and/or CD160. It should be appreciated that all of the above noted co-stimulatory genes are well known in the art, and sequence information of these genes, isoforms, and variants can be retrieved from various public resources, including sequence data bases accessible at the NCBI, EMBL, GenBank, RefSeq, etc. Moreover, while the above exemplary stimulating molecules are preferably expressed in full length form as expressed in human, modified and non-human forms are also deemed suitable so long as such forms assist in stimulating or activating T-cells. Therefore, muteins, truncated forms and chimeric forms are expressly contemplated herein. The immunotherapeutic compositions disclosed herein may be either “prophylactic” or “therapeutic”. When provided prophylactically, the compositions of the present disclosure are provided in advance of the development of, or the detection of the development of, a coronavirus disease, with the goal of preventing, inhibiting or delaying the development of the coronavirus disease; and/or generally preventing or inhibiting progression of the coronavirus disease in an individual. Therefore, prophylactic compositions can be administered to individuals that appear to be coronavirus disease free (healthy, or normal, individuals), or to individuals who has not yet been detected of coronavirus. Individuals who are at high risk for developing a coronavirus disease, may be treated prophylactically with a composition of the instant disclosure. When provided therapeutically, the immunotherapy compositions are provided to an individual who is diagnosed with a coronavirus disease, with the goal of ameliorating or curing the coronavirus disease; increasing survival of the individual; preventing, inhibiting, reversing or delaying development of coronavirus disease in the individual. The contemplated subject matter further includes methods for administering a vaccine to a patient by more than one route of administration to induce both local and systemic immune responses to the vaccine. The contemplated subject matter also includes compositions and methods for assaying the presence or absence of the relevant antibodies (e.g., anti-SARS-CoV2 antibodies) in a patient sample (e.g., saliva, nasal mucosa, alimentary mucosa, or serum). The antibody status in the patient's sample may be used to assess the need for an additional vaccine dose (e.g., a booster dose/shot). In addition to the coveted molecular epitopes presented in a vaccine, the route of administration of the vaccine as well as the regimen for administering additional (i.e., booster) doses of the vaccine, can also affect whether or not the patient's immune response is robust enough to establish protection. For an emerging virus such as the severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV2), the duration of immunity (both humoral and cell-mediated) in a patient recovered from a SARS-CoV2 infection is not yet completely known, and furthermore, a vaccine protocol has not yet been tested across a varied population. Considering the current SARS-CoV2 pandemic and the high rate of transmission for the SARS-CoV2 virus, there is a need for a robust vaccination protocol and effective testing for the virus or immunity to the virus (e.g., presence of anti-SARS-CoV2 antibodies). Vaccine Administration. The presently disclosed contemplated methods for inducing immunity in a patient include administering a vaccine by at least oral administration, and preferably by oral administration and by injection to the blood supply. Many vaccines are given via the intramuscular (IM) route to optimize immunogenicity with the direct delivery of the vaccine to the blood supply in the muscle to induce systemic immunity. The IM administration is typically preferred over subcutaneous (SC) injection which is more likely to have adverse reactions at the injection site than IM injections. In addition to IM injection, induction of mucosal immunity has been reported to be essential to stop person-to-person transmission of pathogenic microorganisms and to limit their multiplication within the mucosal tissue. Furthermore, for protective immunity against mucosal pathogens, (e.g., SARS coronaviruses) immune activation in mucosal tissues instead of the more common approach of tolerance to maintain mucosal homeostasis allows for enhanced mucosal immune responses and better local protection. For example, nasal vaccination (delivery of a vaccine by nasal administration) induces both mucosal immunity as well as systemic immunity. See, e.g., Fujkuyama et al., 2012, Expert Rev Vaccines, 11:367-379 and Birkhoff et al., 2009, Indian J. Pharm. Sci.,71:729-731. In order to induce both mucosal and systemic immunity in a patient, embodiments of the present disclosure include providing a vaccine to the patient by at least administration to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient. In some embodiments, the routes of administration include administering the vaccine to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient together with injection into the blood supply (e.g., intramuscular (IM), intravenous (IV), or subcutaneous (SC)). As used herein, oral administration of a vaccine composition includes nasal injection, nasal inhalation, ingestion by mouth, and administration (e.g., inhalation, ingestion, injection) to the alimentary mucosa. Preferably, the routes of administering the vaccine include oral administration selected from delivery to the alimentary mucosa, nasal injection, nasal inhalation, ingestion by mouth, or inhalation by mouth together with administration by intramuscular (IM) injection. Notably, the vaccine administered for inducing immunity in the mucosal tissue of a patient is a vaccine against SARS-CoV2. In exemplary embodiments, the vaccine a replication defective adenovirus construct, comprising an E1 gene region deletion and an E2b gene region deletion. In certain embodiments the adenovirus comprises a sequence (e.g. SEQ ID NO:11) encoding a SARS-CoV2 spike protein antigen with at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity to SEQ ID NO:10. In certain embodiments the adenovirus comprises a sequence (e.g. SEQ ID NO:13) encoding a SARS-CoV2 spike protein antigen with at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity to SEQ ID NO:12. In certain embodiments, the adenovirus includes a sequence encoding a soluble ACE2 protein coupled to an immunoglobulin Fc portion, forming an ACE2-Fc hybrid construct that may also include a J-chain portion, as disclosed in U.S. Ser. No. 16/880,804 and U.S. 63/016,048, the entire contents of both of which are herein incorporated by reference. In other exemplary embodiments, the SARS-CoV2 vaccine (e.g., an adenovirus construct) includes a mutant variant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 9), wherein the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that imparts an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV2 spike protein as disclosed in U.S. 63/022,146, the entire content of which is herein incorporated by reference. In another exemplary embodiment, the SARS-CoV2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosomal targeting sequence (N-ETSD), as disclosed in U.S. Ser. No. 16/883,263 and U.S. 63/009,960, the entire contents of both of which are herein incorporated by reference. Additionally or alternatively, the SARS-CoV2 vaccine includes modified yeast cells (e.g.,Saccharomyces cerevisiae) genetically engineered to express coronaviral spike proteins on the yeast cell surface thereby creating yeast presenting cells to stimulate B cells (e.g., humoral immunity) as disclosed in U.S. 63/010,010. In some embodiments, more than one vaccine composition as disclosed herein may be administered to a patient to induce immunity to SARS-CoV2. For example, a patient may be administered genetically modified yeast cells expressing corona viral spike proteins as a single type of vaccine, or the genetically modified yeast cells may be administered together or concurrently with one or more SARS-CoV2 adenovirus constructs as disclosed herein. Monitoring presence of antibodies. The contemplated subject matter also includes monitoring or assessing a patient's immune response either to a vaccine administered as disclosed herein (e.g., by oral administration and injection into the blood supply), or to infection by the virus. In particular, disclosed herein are compositions and methods for assessing the continued presence of antibodies in a patient's respiratory and digestive mucosa following infection with SARS-CoV2 or following inoculation against SARS-CoV2 with administration of a SAR coronavirus vaccine. For assaying a sample from a patient having received a vaccine against a pathogenic infection (e.g., targeting SARS-CoV2) and/or having been infected with a virus (e.g., SARS-CoV2), the presence of antibodies against the pathogen may be carried out using any one of many diagnostic tests. In some embodiments, the diagnostic test is a cell viability assay that allows for the detection of antibodies in the presence of antigen. Diagnostic tests using a cell viability assay for anti-SARS-CoV2 antibody detection are disclosed in U.S. 62/053,691, the entire contents of which are herein incorporated by reference. The cellular diagnostic assay relies on the expression of the target receptor for a given pathogen (e.g., ACE2 for SARS-CoV2 infection) on the surface of an immune effector cell line (e.g., killer T cells, natural killer cells, NK92® cells and derivatives thereof, etc.) and the expression of the pathogen ligand (e.g., Spike proteins for SARS-CoV2 infection) on the surface of a surrogate cell line (e.g., HEK293 cells or SUP-B15 cells). Additional diagnostic tests using recombinant protein variants of the ACE2 protein (the human receptor targeted by SARS-CoV2 spike protein) are disclosed in U.S. Ser. No. 16/880,804, the entire contents of which are herein incorporated by reference. Antibody testing in saliva samples. In order to more easily monitor a patient for the presence of anti-pathogen antibodies, assaying a saliva sample from the patient allows for expedited sample collection, increased patient participation, and may allow for the patient to obtain the sample themselves and either mail or transport the sample to the lab for testing. However, in order to assay saliva for the presence of neutralizing antibodies against SARS-CoV2, it may be necessary to stabilize proteins in the saliva against degradation during transport and storage after sample collection prior to testing. Upon collection of the saliva sample, the saliva is placed into a preservative solution to stabilize the components (e.g., anti-SARS CoV2 antibody or viral spike protein) therein. Preservatives for biological samples are disclosed, for example, in Cunningham & al. (2018) report (“Effective Long-term Preservation of Biological Evidence,” U.S. Department of Justice grant #2010-DN-BX-K193) and U.S. Pat. No. 6,133,036 to Putcha et al. For example, a stabilizing preservative solution for a patient's saliva sample may include any one of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof. In specific embodiments, saliva samples may be mixed with stabilizing preservative solutions of glutaraldehyde to achieve a final glutaraldehyde concentration between 0.1%(w/v) and 2.0%(w/v), for example about 0.2%(w/v), about 0.3%(w/v), about 0.4%(w/v), about 0.5%(w/v), about 0.6%(w/v), about 0.7%(w/v), about 0.8%(w/v), about 1.0%(w/v), about 1.1%(w/v), about 1.2%(w/v), about 1.3%(w/v), about 1.4%(w/v), about 1.5%(w/v), about 1.6%(w/v), about 1.7%(w/v), about 1.8%(w/v), or about 1.9%(w/v). In additionally or alternatively embodiments, saliva samples may be mixed with a stabilizing preservative solution of about 0.10% to about 1.00% sodium benzoate (weight/volume of sample) and/or about 0.025% to about 0.20% citric acid (weight/volume of sample). For example, the saliva sample may be mixed with 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, or 1.00% w/v sodium benzoate. In additional embodiments, the saliva sample is mixed a stabilizing preservative solution of at least 0.5 mg/mL (for example, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, at least 1 mg/mL, at least 1.5 mg/mL, at least 2 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, at least 4 mg/mL, at least 4.5 mg/mL, or even 5 mg/mL) of benzoic acid and/or at least 0.2 mg/mL (for example, at least 0.2 mg/mL, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.40 mg/mL, at least 0.50 mg/mL, at least 0.75 mg/mL, at least 1.0 mg/mL, at least 1.25 mg/mL, at least 1.5 mg/mL, at least 1.75 mg/mL, or even 2.0 mg/mL) of citric acid. As used herein, “benzoic acid” is interchangeable with benzoate salt (e.g., sodium benzoate) and “citric acid” is interchangeable with citrate salt (e.g., sodium citrate). The saliva samples with preservatives as described above are stable for storage at temperatures between 15° C. and 40° C. for at least one hour (e.g., at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 48 hours, or even 36 hours). Therefore, disclosed herein is a method of preserving a saliva sample for neutralizing antibody testing, the method including mixing the saliva sample with the stabilizing solution made of one or more of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and/or sodium azide and storing between 15° C. and 25° C. for at least one hour, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours. In some embodiments, the saliva sample is mixed with a glutaraldehyde concentration between 0.1% (w/v) and 2.0% (w/v), and the glutaraldehyde-saliva is stored between 15° C. and 25° C. In certain embodiments, the glutaraldehyde-saliva may further comprise citric acid and/or benzoic acid at a concentration of as disclosed herein. Aragonite. In some embodiments, any antibody proteins or any specific antibody protein may be captured from the saliva sample with oolitic aragonite particles. For example, the saliva preserving solution of glutaraldehyde, sodium benzoate and citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof as disclosed herein, may also include oolitic aragonite (calcium carbonate, CaCO3) particles. Use of aragonite particles for binding to proteins is disclosed, for example, in U.S. Ser. No. 16/858,548 and PCT/US20/29949, the entire contents of both of which are herein incorporated by reference. Accordingly, aragonite particles may be added to that have been modified to capture (e.g., bind to) any antibodies present in the saliva sample or specifically capture an antibody against a specific antigen. For example, aragonite may be functionalized with moieties capable of binding to an immunoglobulin (Ig) protein. Preferably, the Ig protein is an immunoglobulin A (IgA), immunoglobulin G (IgG), or immunoglobulin E (IgE) protein. More preferably, the aragonite is functionalized to bind to an IgA protein. Most preferably, the aragonite particles are functionalized with moieties capable of binding to specific antibodies. For example, the aragonite particles may be coupled with a moiety specific to anti-SARS-CoV2 antibodies. Preferably, the aragonite particle is coupled with a recombinant ACE2 protein as disclosed, for example, in U.S. Ser. No. 16/880,804, supra. In typical embodiments, the aragonite particle is coupled with a recombinant human ACE2 protein having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 8. In additional or alternative embodiments, the aragonite particle is functionalized (e.g., coupled to) a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 9). For more efficient capture or binding of an anti-SARS-CoV2 antibody or the spike protein of SARS CoV-2, the recombinant soluble ACE2 may be mutated to form ACE2 variants having higher binding affinities for SARS-CoV2 spike protein (e.g., the RBD domain of the spike protein). These ACE2 variant mutants of the recombinant soluble ACE2 protein include T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and/or D355L. As used herein, the term “functionalized” refers to coupling or binding of a moiety to the aragonite particle thereby imparting any function of the coupled moiety to the aragonite particle. For example, the aragonite particle may be functionalized with a protein moiety. Methods for preparing and using aragonite particle beads are disclosed in U.S. Ser. No. 16/858,548 and PCT/US20/29949. In some embodiments, the aragonite composition includes a plurality of aragonite particle beads. Preferably, the plurality of aragonite particle beads have an average particle size of between 100 nm to 1 mm, In some embodiments a protein moiety is coupled directly to the natural, untreated surface of aragonite particles. Aragonite particles approximately 2-3% amino acid content including aspartic acid and glutamic acid rendering the aragonite surface hydrophilic. Accordingly, in some embodiments, protein moieties may be directly coupled to the surface of the aragonite particles. In alternative embodiments, the aragonite particle surface may be treated to modify the binding surface. For example, treatment with stearic acid (i.e., octadecanoic acid) provides for a hydrophobic surface, as disclosed in U.S. Ser. No. 16/858,548 and PCT/US20/29949. For protein loading, treatment of the aragonite with phosphoric acid forms lamellar structures. Additional conjugation techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art as disclosed, for example, inBioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013. Monitoring of Vaccine Protocol. Patients who do not show sufficient titers of (e.g., presence of) neutralizing antibody in their saliva may be sent oral dosages of the respective vaccine (e.g., a SARS-CoV2 vaccine as disclosed herein). The patients inhale or ingest these vaccine dosages, and then two weeks later send another saliva sample—prepared and stored in the same manner as above—to the test facility to confirm that the oral vaccine dose has restored their anti-SARS-CoV2 antibody (e.g., IgA) titers. Accordingly, in additional embodiments, a kit for collecting a saliva sample from a patient includes a collection container with the saliva preservative solution as disclosed herein. For example, the kit includes a collection container with a solution of any of one or combination of glutaraldehyde, sodium benzoate and/or citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and sodium azide. The kit may also include adhesive packaging and/or mailing supplies in order to secure the collection container with the saliva sample for transport or mailing. In some embodiments, the kit may also include at least one dose of the vaccine for oral administration. Recited ranges of values herein are merely intended as a shorthand referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. EXAMPLES The advantageous features of the compositions and methods described herein are further illustrated (but not limited) by the following examples. Example 1. Two groups of Rhesus macaques (5 per group) were immunized subcutaneously on day 0 with an adenoviral anti-SARS-CoV2 vaccine as described above. Blood was drawn from each macaque before immunization. On day 14, one group of macaques (Group 1) received another subcutaneous booster injection of the same vaccine, while another group (Group 2) received an oral vaccine as described herein (E1-/E2b-Ad5 with SEQ ID NO:11 or SEQ ID NO:13). On day 28, both groups received an oral vaccine booster dose. Two macaques (Control) were vaccinated at the indicated time points with shams. Blood was drawn on days 14, 21, 28, 35, & 42. Serum samples drawn at the indicated time points from these macaques was then assessed by ELISA for anti-spike protein IgG and IgM seroreactivity. Briefly, 96 well EIA/RIA plates (ThermoFisher, Cat #07-200-642) were coated with 50 μL/well of 1 μg/mL solution of purified recombinant SARS-CoV-2-derived Spike protein (S-Fusion. ImmunityBio, Inc.) suspended in coating buffer (0.05 M Carbonate-Bicarbonate, pH 9.6) and incubated overnight at 4° C. Individual 96 well plates were prepared for each immunoglobulins type (IgG or IgM) by washing three times each per well with 150 μL of TPBS solution (PBS+0.05% Tween 20). 100 μL/well of blocking solution (2% non-fat milk in TPBS) was then added and incubated for 1 hour at room temperature (RT). Plasma and serum samples were heat-inactivated at 56° C. for 1 hour before use. Serial dilutions of plasma, serum or antibody samples were prepared in 1% non-fat milk in TPBS. Plates were washed as described above and 50 μL/well of each serial dilution were added to the plate and incubated at RT for 1 hour. Plates were washed three times with 200 μL of TPBS. Dilutions (1:6000) of each goat anti-Human IgG (H+L) Cross-Adsorbed, HRP, Polyclonal; or Goat anti-Human IgM (Heavy chain) Cross-Adsorbed Secondary Antibody, HRP (ThermoFisher, Cat #62-842-0 or A18841 respectively) were 1 prepared in 1% non-fat milk/TPBS and 50 μL/well of these secondary antibodies were added in separate reactions/plates per immunoglobulin type (IgG or IgM) and incubated for 1 hour at RT. Plates were washed three times with 200 μL of TPBS. One component (3,3′,5,5′-tetramethylbenzidine (TMB) substrate, 50 μL/well, VWR, Cat #100359-156) was added to each well and incubated at RT for 10 minutes and then the reaction was stopped by addition of 50 μL/well of IN Sulfuric acid (H2504). The optical density at 450 nm was measured with a Synergy 2 plate reader (BioTek Instruments, Inc). Data were analyzed using Prism 8 (GraphPad Software, LLC), and shown inFIG.19. Example 2. On day 56, the macaques were challenged with respiratory exposure to the SARS-CoV2 virus. Nasal swabs were collected daily from these macaques on days 56-63. Bronchoalveolar lavage (BAL) fluid was collected on days 57, 59, 61, & 63. The ability of serum to inhibit SARS-CoV2 infectivity from the samples collected is shown inFIG.22. As can be seen, the sera from both the Group 1 and Group 2 macaques inhibited infectivity, with later collected sera inhibited more powerfully than early collected sera. Sera from control macaques had no inhibitory effect at any time point tested. Viral load over time in the nasopharynx is shown inFIG.23. Viral load over time in the lungs is shown inFIG.24. Example 3. Serum samples from various human volunteers who have received various experimental anti-SARS-CoV2 vaccines were collected and assayed by ELISA as described above for IgG and IgM seroreactivity against SARS-CoV2 S protein. The results are shown inFIG.25. Example 4. Human volunteers were divided into three cohorts. Cohort 1 (10 individuals) was immunized by subcutaneous injection with 5×1010viral particles of a vaccine as described herein (E1-/E2b-Ad5 containing SEQ ID NO:11 or SEQ ID NO:13). Cohort 2 (10 individuals) was immunized by subcutaneous injection with 1011 viral particles of a vaccine as described herein. Cohort 3 (15 individuals) was immunized by subcutaneous injection with 1011viral particles of a vaccine as described herein (or 5×1010viral particles if safety concerns indicated a lower dose). Blood was drawn from each volunteer on the same day as the initial prime vaccination was administered. Blood was drawn again on days 8, 15, & 22. A booster injection of the same vaccine was administered on day 22. ELISpot tests were run on the blood collected on days 1 & 15 to assess cell-mediated immunity against SARS-CoV2. 400,000 viable PBMCs from each blood draw per well (Cellometer K2 w/AO/PI viability stain) were stimulated with empty medium, SARS-CoV2 S, SARS-CoV2 N, SARS-CoV2 M, CD3/CD28/CD2, and CEFT. After 48 hrs of stimulation, supernatants were frozen (−80° C.) for later testing. FIG.26shows the results of this test from Th1 N-responsive patients 3, 6, & 11.FIG.27shows results from patient 4 (N-unresponsive) and patient 10 (weakly Th1 N-responsive). None of these patients showed a Th2 response to N. Example 5. Human volunteers received 5×1010viral particles of vaccine by subcutaneous injection on day 1 of the study, and again on day 22. Blood was drawn from each subject on days 1 and 29. These blood samples were assayed for immune reactivity to the SARS-CoV2 S protein by the methods described in co-pending U.S. 63/124,979 (filed 14 Dec. 2020).FIG.28shows the results of these assays. As can be seen, subject #8 shown a level of immune response to the S protein above the level of detection already on the first day of the experiment, indicating that this particular individual had already been previously infected with SARS-CoV2. The course of immunization produced a notable increase in immune response relative to baseline. This result constitutes in vivo evidence that the vaccines described herein can serve as vaccine boosts even to individuals whose immunity derives from some other source than prior immunization with the vaccines described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The present disclosure, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest manner consistent with the context. Example 6: The hAd5 [E1-, E2b-, E3-] Platform and Constructs For studies here, the next generation hAd5 [E1-, E2b-, E3-] vector was used (FIG.1c) to create viral vaccine candidate constructs. As shown inFIG.8d-h, a variety of constructs were created: FIG.8(d): S WT: S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot PODTC2); FIG.8(e): S RBD-ETSD: S Receptor Binding Domain with an Enhanced T-cell Stimulation Domain (ETSD); FIG.8(f): S Fusion: S optimized to enhance surface expression and display of RBD; FIG.8(g): N-ETSD: The nucleocapsid (N) sequence with the ETSD; and FIG.8(h): Bivalent S-Fusion+N-ETSD; S-WT+N-ETSD and S RBD-ETSD+N-ETSD constructs were also produced, but are not shown. Example 7: Enhanced HEK 293T Cell-Surface Expression of RBD Following Transfection with Ad5 S— Fusion+N-ETSD As shown inFIG.9, anti-RBD-specific antibodies did not detect RBD on the surface of HEK 293T cells transfected with hAd5 S-WT (FIG.9a) or hAd5 S-WT+N-ETSD (FIG.9b) constructs, while hAd5 S-Fusion alone was slightly higher (FIG.9e). As expected, both constructs with RBD, hAd5 RBD-ETSD and RBD-ETSD+N-ETSD, showed high binding of anti-RBD antibody (FIGS.9candd). Notably, high cell-surface expression of RBD was detected after transfection with bivalent hAd5 S-Fusion+N-ETSD (FIG.9f). These findings support our proposition that an hAd5 S-Fusion+N-ETSD construct, containing a high number and variety of antigens provided by both full-length, optimized S with proper folding and N leads to enhanced expression and cell surface display of RBD in a vaccine construct. Example 8: Immunoblot Correlation of Enhanced S Expression with hAd5 S-Fusion+N-ETSD Immunoblot analysis of S expression correlated with enhanced S expression (FIG.10), showing again that the bivalent hAd5 S-Fusion+N-ETSD construct enhances expression of S compared to S-Fusion alone. Example 9: Confirmation of Native Folding of Enhanced Surface RBD Following hAd5 S-Fusion+N-ETSD Transfection Determination of the binding of recombinant ACE2-Fc was performed to confirm the native, physiologically-relevant folding of the S RBD after expression from the hAd5 S-Fusion+N-ETSD vaccine candidate. S RBD binds ACE2 during the course of SARS-CoV-2 infection and an effective neutralizing antibody prevents this interaction and thus infection. Such a neutralizing antibody is more likely to be effective if raised in response to S presented in the correct conformation. In addition to enhancement of cell surface expression, the optimized S allows for proper protein folding. It was found that compared to either hAd5 S-WT or hAd5 S-Fusion (FIGS.11aandb, respectively), ACE2-Fc binding to S RBD expressed from the hAd5 S-Fusion+N-ETSD was clearly enhanced (FIG.11c). Anti-RBD antibody binding studies (FIG.1if j) performed with the same experiment, confirmed the enhanced surface expression findings noted by ACE2-Fc binding. These findings of conformationally correct and enhanced S RBD expression, important for production of neutralizing antibodies, directed us to elect the hAd5 S-Fusion+N-ETSD vaccine candidate for clinical trials. Example 10: hAd5 N-ETSD Successfully Directs N to an Endosomal/Lysosomal Compartment The ETSD design successfully translocated N to the endosomal subcellular compartment. After infection of HeLa cells with N-ETSD, N co-localized with the endosomal marker 45 transferrin receptor (CD71), as shown inFIG.12c, and also co-localized with the lysosomal marker Lamp1 (FIG.12d), demonstrating that N-ETSD is translocated throughout the endosomal pathway to lysosomes, enabling processing for MHC II presentation. N-wild type (N-WT), compared to N-ETSD, shows diffuse cytoplasmic distribution and does not co-localize with the lysosomal marker (FIG.12e). These findings confirm the role of the ETSD in directing N to an endosomal/lysosomal compartment that will result in increased MHC II presentation and CD4+activation by N. Example 11: In Vivo hAd5 S-Fusion+N-ETSD Vaccine Immunogenicity Studies Based on the evidence that S-Fusion+N-ETSD resulted in enhanced expression of physiologically-relevant RBD and that N-ETSD successfully translocated to the endosomal/lysosomal compartment, the bivalent hAd5 S-Fusion+N-ETSD vaccine was chosen for inoculation of 7-week old female CD-1 mice. The unique properties of this construct would result in the generation of both CD8+ and CD4+ T-cell responses and neutralizing antibodies. As described in Methods, mice received an initial injection on Day 0 and a second injection on Day 21. Sera were collected on Day 0 and at the end of the study on Day 28 for antibody and neutralization analyses. Splenocytes were also collected on Day 28 for intracellular cytokine staining (ICS) and ELISpot analyses. All age- and gender-matched animals assigned to the study appeared normal with no site reactions and no loss of body weight throughout the dosing were seen, consistent with previous observations with the hAd5 [E1-, E2b-, E3-] platform Example 12: hAd5 S-Fusion+N-ETSD Generates Both CD8□+ and CD4+ T-Cell Responses CD8+activation by both S and N: CD80+splenocytes from hAd5 S-Fusion+N-ETSD vaccinated mice exposed to S peptide pool 1 (containing RBD and 51) show IFN-γ expression that is significantly higher compared to hAd5 null mice (FIG.13a); splenocytes from these mice also expressed intracellular IFN-γ in response to the N peptide pool. Evaluation of simultaneous IFN-γ/TNF-α expression from CD8β+splenocytes (FIG.13c) mirrored those for IFN-γ expression alone. These results indicate that both S and N activate CD8+ T cells. CD4+activation by N: Although CD8+ cytotoxic T cells mediate killing of virus infected cells, CD4+ T cells are required for sustained cytotoxic T lymphocyte (CTL) activity. Thus, CD4+ T cells in the vaccinated animals was evaluated. In contrast to CD8β+splenocytes, only the N peptide pool stimulated CD4+splenocytes from hAd5 S-Fusion+N-ETSD-inoculated mice to express IFN-γ (FIG.13b) or IFN-γ/TNF-α (FIG.13d) at levels that were substantially higher than hAd5 Null control. The contribution by N of CD4+ T-cell responses is vital to an effective immune response to the candidate vaccine. Example 13: hAd5 S-Fusion+N-ETSD Generates Antibody Responses to Both S and N Antigens The primary objective of coronavirus vaccines currently in development are neutralizing antibodies against spike, thus we examined antibody production in mice vaccinated with our bivalent vaccine. There was significant production of both anti-S(FIG.14a) and anti-N(FIG.14c) antibodies in the sera from CD-1 mice vaccinated with hAd5 S-Fusion+N-ETSD at Day 28 in the study. Compared to anti-S antibodies, anti-N antibodies were higher in sera, given the dilution factor for sera was 1:90 for anti-N antibody analysis and 1:30 for anti-S antibody analysis. A standard curve of IgG was generated, then absorbance values were converted into mass equivalents for both anti-S and anti-N antibodies (FIGS.14bandd). These values were used to calculate that hAd5 S-Fusion+NETSD vaccination generated a geometric mean value of 5.8 μg S-specific IgG and 42 μg N-specific IgG per mL of serum, therefore the relative μg amount of anti-N antibodies is higher than that for anti-S antibodies and reflects the strong contribution of N to anti-SARS-CoV-2 antibody production. Example 14: hAd5 S-Fusion+N-ETSD Vaccine Generates Potent Neutralizing Antibodies as Assessed by Both cPass and Live Virus Neutralization Assays Neutralizing antibody activity was evaluated using a cell free assay (cPass) as well as live virus infection in vitro. As seen inFIG.15a, the cPass assay showed inhibition of S RBD:ACE2 binding for all mice and −100% inhibition for two mice at both dilutions of 1:20 and 1:60. The Vero E6 neutralization assay results are shown for the four mice that showed S-specific antibodies by ELISA. The high persistent neutralization seen even at the high dilution factors suggests the intriguing possibility that the bivalent, multi-antigen, multi-epitope generation by hAd5 S-Fusion+N-ETSD vaccine, could result in synergies of neutralizing immune responses (FIG.15b); at epitopes in addition to those associated with RBD-ACE2 binding. As can be seen inFIG.15b, the value for 50% neutralization (IC50) is present at 1:10,000 serum dilution for the G4 pool of sera from mice that showed S-specific antibodies, ten times higher than the convalescent serum with a dilution of 1:1,000. The potent neutralization, confirmed by two assays, supports the predicted efficacy of the hAd5 S-Fusion+ETSD vaccine candidate and its advancement to clinical trials Example 15: hAd5 S-Fusion+N-ETSD Generates Th1 Dominant Responses Both in Humoral and T-Cell Immunity Antibody Th1 dominance in response to N and S: IgG2a, IgG2b, and IgG3 represent Th1 dominance; while IgG1 represents Th2 dominance. For both anti-S(FIG.16a) and anti-N(FIG.16c) antibodies in sera from hAd5 S-Fusion+N-ETSD vaccinated mice, IgG2a and IgG2b isotypes were predominant and significantly higher compared to the hAd5 Null control. These data show the Th1 dominance of antibody production in response to the hAd5 S-Fusion+N-ETSD vaccine T-cell Th1 dominance in response to N and S: IFN-γ production correlates with CTL activity 47 (Th1 dominance), whereas, IL-4 causes delayed viral clearance 48 (Th2 dominance). A ratio of IFN-γ to IL-4 of 1 is balanced and a ratio greater than 1 is demonstrative of Th1 dominance. Thus, we examined IFN-γ and IL-4 production in animals immunized with the bivalent S plus N vaccine. As determined by ELISpot, IFN-γ secretion was significantly higher for hAd5 S-Fusion+N-ETSD than for hAd5 Null splenocytes in response to both S peptide pool 1 and the N peptide pool (FIG.17a), but IL-4 was only secreted at significantly higher levels for hAd5 S-Fusion+N-ETSD in response to the N peptide pool (FIG.17b). The Th1-type predominance is also seen when the ratio of IFN-□to IL-4 based on spot forming units in response to the combined S peptide pools and the N peptide pool, is considered (FIG.18a). Th1 predominance was seen again in humoral responses, where the ratio based on ng equivalence of Th1 related antibodies (IgG2a, IgG2b, and IgG3) to Th2 related antibodies (IgG1) for both anti-S and anti-N antibodies is greater than 1 in all mice (FIG.18b). This Th1 dominant profile of the hAd5 S-Fusion+N-ETSD vaccine candidate provides further justification for hAd5 S-Fusion+N-ETSD to be our lead candidate for clinical testing The hAd5 S-Fusion+N-ETSD vaccine was designed to overcome the risks of an S-only vaccine and elicit both T-cell immunity and neutralizing antibodies, leveraging the vital role T cells play in generating long-lasting antibody responses and in directly killing infected cells. Both CD4+ and CD8+ T cells are multifunctional, and induction of such multifunctional T cells by vaccines correlated with better protection against infection. We posit that enhanced CD4+ T-cell responses and Th1 predominance resulting from expression of an S antigen optimized for surface display and an N antigen optimized for endosomal/lysosomal subcellular compartment localization and thus MHC I and II presentation, led to increased dendritic cell presentation, cross-presentation, B cell activation, and ultimately high neutralization capability. Furthermore, the potent neutralization capability at high dilution seen for the pooled sera from hAd5 S-Fusion+N-ETSD vaccinated mice, combined with Th1 dominance of antibodies generated in response to both S and N antigens, supports the objective of this vaccine design. Contemporaneous MHC I and MHC II presentation of an antigen by the antigen presenting cell activates CD4+ and CD8+ T cells simultaneously and is optimal for the generation of memory B and T cells. A key finding of our construct is that N-ETSD, which we show is directed to the endosomal/lysosomal compartment, elicits a CD4+response, a necessity for induction of memory T cells and helper cells for B cell antibody production. Others have also reported on the importance of lysosomal localization for eliciting the strongest T-cell IFN-γ and CTL responses, compared to natural N.50,51 The T-cell responses to the S and N antigens expressed by hAd5 S-Fusion+N-ETSD were polycytokine, including IFN-g and TNF-α, consistent with successful antimicrobial immunity in bacterial and viral infections. Post-vaccination polycytokine T-cell responses have been shown to correlate with vaccine efficacy, including those with a viral vector. Highly relevant here, polycytokine T-cell responses to SARS-CoV-2 N protein are consistent with recovered COVID-19 patients, suggesting that the bivalent hAd5 S-Fusion+N-ETSD vaccine will provide vaccine subjects with greater protection against SARS-CoV-2. In contrast to N, the S protein, here expressed as S-Fusion with confirmed enhanced RBD cell-surface expression and conformational integrity as evidenced by high ACE2-Fc binding, generated predominantly CD8+ T cells. Our results confirmed our vaccine design goal, showing that S-Fusion induced elevated levels of antigen-specific T-cell responses against S compared to S-WT. To ensure MHC presentation to both MHC I (for CD8+ T-cell activation) and MHC II (for CD4+ T-cell activation), it is necessary to vaccinate with both S and N antigens optimized to produce this coordinated response. The neutralization data with live SARS-CoV-2 virus demonstrated the potency of the antibody response generated following vaccination with hAd5 S-Fusion+N-ETSD, with evidence of high neutralization even at a high dilution factor. In addition, a striking synergistic effect of pooled sera was evident, with potent neutralization even greater than control convalescent serum at >1:1,000 dilution. The hAd5 S-Fusion+N-ETSD construct described above is delivered by a next generation hAd5 [E1-, E2b-, E3-] platform wherein the E2b deletion (pol) alone enables prolonged transgene production and allows homologous vaccination (prime and the boost formulation is the same) in the presence of pre-existing adenoviral immunity.38 In addition to the generation of cellular and humoral immunity by the subcutaneous injection of hAd5 S-Fusion+N-ETSD, we are also exploring the potential of inducing IgA mucosal immunity by utilizing the same vaccine in an oral or sublingual formulation in clinical trials. Example 16: Methods The hAd5 [E1-, E2b-, E3-] platform and constructs For studies herein, the 2nd generation hAd5 [E1-, E2b-, E3-] vector was used (FIG.1c) to create viral vaccine candidate constructs. hAd5 [E1-, E2b-, E3-] backbones containing SARS-CoV-2 antigen expressing inserts and virus particles were produced as previously described.37 In brief, high titer adenoviral stocks were generated by serial propagation in the E1- and E2b-expressing E.C7 packaging cell line, followed by CsCl2 purification, and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by ViraQuest Inc. (North Liberty, IA). Viral particle counts were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm and viral titers were determined using the Adeno-X™ Rapid Titer Kit (Takara Bio). The constructs created included:S-WT: S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot PODTC2);S RBD-ETSD: S Receptor Binding Domain (S RBD) with an ETSD;N-ETSD: Nucleocapsid (N) with ETSD;S-WT+N-ETSD: S-WT with an Enhanced T-cell Stimulation Domain (ETSD);S-RBD-ETSD+N-ETSD;S Fusion: S optimized to enhance surface expression and display of RBD; and Bivalent S-Fusion+N-ETSD; Transfection of HEK 293T Cells with hAd5 Constructs To determine surface expression of the RBD epitope by vaccine candidate constructs, we transfected HEK 293T cells with hAd5 construct DNA and quantified surface RBD by flow cytometric detection using anti-RBD antibodies. There were seven constructs tested: S-WT, S-WT+N-ETSD, S RBD-ETSD, S RBD-ETSD+N-ETSD, S-Fusion, S-Fusion+N-ETSD, and N-ETSD. HEK 293T cells (2.5×105cells/well in 24 well plates) were grown in DMEM (Gibco Cat #11995-065) with 10% FBS and 1X PSA (100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 ug/mL Amphotericin B) at 37° C. Cells were transfected with 0.5 ug of hAd5 plasmid DNA using a JetPrime transfection reagent (Polyplus Catalog #89129-924) according to the manufacturer's instructions. Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog #40150-D003) and F(ab′)2-Goat anti-Human IgG-Fc secondary antibody conjugated with R-phycoerythrin (ThermoFisher Catalog #H10104). Labeled cells were acquired using a Thermo-Fisher Attune NxT flow cytometer and analyzed using Flowjo Software. Immunocytochemical Labeling of hAd5 Infected HeLa Cells To determine subcellular localization of N after infection or transfection of HeLa cells with hAd5 N-wild type (WT) or hAd5 N-ETSD (each with a flag tag to allow labeling), 48 hours after infection or transfection cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X100, in PBS) for 15 min. at room temperature. To label N, cells were then incubated with an anti-flag monoclonal (Anti-Flag M2 produced in mouse, Sigma cat #F1804) antibody at 1:1000 in phosphate buffered saline with 3% BSA overnight at 4° C., followed by washes in PBS and a 1 hour incubation with a goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Life Technologies, Cat #A32727) at 1:500. For co-localization studies, cells were also incubated overnight at 4° C. with a sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems, Cat #IC7985G) at 1:10 or a rabbit anti-CD71 (transferrin receptor, endosomal marker) antibody (ThermoFisher Cat #PAS-83022) at 1:200. After removal of the primary antibody, two washes in PBS and three 3 washes in PBS with 3% BSA, cells were incubated with fluor-conjugated secondary antibodies when applicable at 1:500 (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Life technologies, A-11034) for 1 hour at room temperature. After brief washing, cells were mounted with Vectashield Antifade mounting medium with DAPI (Fisher Scientific, Cat #NC9524612) and immediately imaged using a Keyence all-in-one Fluorescence microscope camera and Keyence software. Immunoblot Analysis of S Antigen Expression HEK 293T cells transfected with hAd5 S-WT, S-Fusion, or S-Fusion+N-ETSD constructs were cultured and transfected as described in the main manuscript and harvested 3 days after transfection in 150 mL RIPA lysis buffer with 1× final Protease Inhibitor cocktail (Roche). After protein assay, equivalent amounts of total protein were loaded into and run on a 4 to 12% gradient polyacrylamide gel (type) and transferred to nitrocellulose membranes using semi-dry transfer apparatus. Anti-Spike S2 (SinoBiological Cat #40590-T62) was used as the primary antibody and IRDye® 800CW Goat anti-Rabbit IgG (H+L) (Li-Cor, 925-32211) as the secondary antibody using the Ibind Flex platform. Antibody-specific signals were detected with an infrared Licor Odyssey instrument. ACE2-IgG1Fc Binding to hAd5 Transfected HEK 293T Cells HEK 293T cells were cultured at 37° C. under conditions described above for transfection with hAd5 S-WT, S-Fusion, S-Fusion+N-ETSD, S RBD-ETSD, or S RBD-ETSD+N-ETSD and were incubated for 2 days and harvested for ACE2-Fc binding analysis. Recombinant ACE2-IgG1Fc protein was produced using Maxcyte transfection in CHO-S cells that were cultured for 14 days. ACE2-IgG1Fc was then purified using a Mab Select SuRe affinity column on AKTA Explorer. Purified ACE2-IgG1Fc was dialyzed into 10 mM HEPES, pH7.4, 150 mM NaCl and concentrated to 2.6 mg/mL. For binding studies, the ACE2-IgG1Fc was used at a concentration of 1 □g/mL for binding. Cells were incubated with ACE2-Fc for 20 minutes and, after a washing step, were then labeled with a PE conjugated F(ab′)2-goat anti-human IgG Fc secondary antibody at a 1:100 dilution, incubated for 20 minutes, washed and acquired on flow cytometer. Histograms are based on normalized mode (NM) of cell count—count of cells positive for signal in PE channel. Vaccination of CD-1 Mice with the hAd5 S-Fusion+N-ETSD Vaccine Candidate CD-1 female mice (Charles River Laboratories) 7 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA). After an initial blood draw, mice were injected with either hAd5 Null (a negative control) or vaccine candidate hAd5 S-Fusion+N-ETSD on Day 0 at a dose of 1×1010 viral particles (VP). There were 5 mice per group. Mice received a second vaccine dose on Day 21 and on Day 28, blood was collected via the submandibular vein from isoflurane-anesthetized mice for isolation of sera and then mice were euthanized for collection of spleen and other tissues. Splenocyte Collection and Intracellular Cytokine Staining (ICS) Spleens were removed from each mouse and placed in 5 mL of sterile medium of RPMI (Gibco Cat #22400105), HEPES (Hyclone Cat #SH30237.01), 1X Pen/Strep (Gibco Cat #15140122), and 10% FBS (Gibco Cat #16140-089). Splenocytes were isolated within 2 hours of collection. ICS for flow cytometric detection of CD8β+ and CD4+ T-cell-associated IFN-γ and IFN-γ/TNFα+production in response to stimulation by S and N peptide pools. Stimulation assays were performed using 106 live splenocytes per well in 96-well U-bottom plates. Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of peptide pools at 2 μg/mL/peptide for 6 h at 37° C. in 5% CO2, with protein transport inhibitor, GolgiStop (BD) added two hours after initiation of incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8□and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-γ and TNF-α. Fluorescent-conjugated antibodies against mouse CD8□antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software. ELISpot Assay ELISpot assays were used to detect cytokines secreted by splenocytes from inoculated mice. Fresh splenocytes were used on the same day, as were cryopreserved splenocytes containing lymphocytes. The cells (2-4×105 cells per well of a 96-well plate) were added to the ELISpot plate containing an immobilized primary antibodies to either IFN-γ or IL-4 (BD), and were exposed to various stimuli (e.g. control peptides, target peptide pools/proteins) comprising 2 μg/mL peptide pools or 10 μg/mL protein for 36-40 hours. After aspiration and washing to remove cells and media, extracellular cytokine was detected by a secondary antibody to cytokine conjugated to biotin (BD). A streptavidin/horseradish peroxidase conjugate was used detect the biotin-conjugated secondary antibody. The number of spots per well, or per 2-4×105 cells, was counted using an ELISpot plate reader. ELISA for Detection of Antibodies For antibody detection in sera from inoculated mice, ELISAs specific for spike and nucleocapsid antibodies, as well as for IgG subtype (IgG1, IgG2a, IgG2b, and IgG3) antibodies were used. A microtiter plate was coated overnight with 100 ng of either purified recombinant SARS-CoV-2 S-FTD (full-length S with fibritin trimerization domain, constructed and purified in-house by ImmunityBio), SARS-CoV-2 S RBD (Sino Biological, Beijing, China; Cat #401591-VO8B1-100) or purified recombinant SARS-CoV-2 nucleocapsid (N) protein (Sino Biological, Beijing, China; Cat #40588-VO8B) in 100 μL of coating buffer (0.05 M Carbonate Buffer, pH 9.6). The wells were washed three times with 250 μL PBS containing 1% Tween 20 (PBST) to remove unbound protein and the plate was blocked for 60 minutes at room temperature with 250 μL PBST. After blocking, the wells were washed with PBST, 100 μL of diluted serum samples were added to wells, and samples incubated for 60 minutes at room temperature. After incubation, the wells were washed with PBST and 100 μL of a 1/5000 dilution of anti-mouse IgG HRP (GE Health Care; Cat #NA9310V), or anti-mouse IgG1 HRP (Sigma; Cat #SAB3701171), or anti-mouse IgG2a HRP (Sigma; Cat #SAB3701178), or anti-mouse IgG2b HRP (Sigma; catalog#SAB3701185), or anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat #SAB3701192) was added to wells. For positive controls, a 100 μL of a 1/5000 dilution of rabbit anti-N IgG Ab or 100 μL of a 1/25 dilution of mouse anti-S serum (from mice immunized with purified S antigen in adjuvant) were added to appropriate wells. After incubation at room temperature for 1 hour, the wells were washed with PBS-T and incubated with 200 μL o-phenylenediamine-dihydrochloride (OPD substrate (Thermo Scientific Cat #A34006) until appropriate color development. The color reaction was stopped with addition of 50 μL 10% phosphoric acid solution (Fisher Cat #A260-500) in water and the absorbance at 490 nm was determined using a microplate reader (SoftMax® Pro, Molecular Devices). Calculation of Relative μg Amounts of Antibodies A standard curve of IgG was generated and absorbance values were converted into mass equivalents for both anti-S and anti-N antibodies. Using these values, we were able to calculate that hAd5 S-Fusion+N-ETSD vaccination generated a geometric mean value of 5.8 S-specific IgG and 42 μg N-specific IgG per milliliter of serum. cPassTM Neutralizing Antibody Detection The GenScript cPassTM (https://www.genscript.com/cpass-sars-cov-2-neutralization-antibody-detection-Kit.html) for detection of neutralizing antibodies was used according to the manufacturer's instructions.44 The kit detects circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the S RBD with the ACE2 cell surface receptor. It is suitable for all antibody isotypes and appropriate for use with in animal models without modification. Vero E6 Cell Neutralization Assay All aspects of the assay utilizing virus were performed in a BSL3 containment facility according to the ISMMS Conventional Biocontainment Facility SOPs for SARS-CoV-2 cell culture studies. Vero e6 kidney epithelial cells fromCercopithecus aethiops(ATCC CRL-1586) were plated at 20,000 cells/well in a 96-well format and 24 hours later, cells were incubated with antibodies or heat inactivated sera previously serially diluted in 3-fold steps in DMEM containing 2% FBS, 1% NEAAs, and 1% Pen-Strep; the diluted samples were mixed 1:1 with SARS-CoV-2 in DMEM containing 2% FBS, 1% NEAAs, and 1% Pen-Strep at 10,000 TCID 50/mL for 1 hr. at 37° C., 5% CO2. This incubation did not include cells to allow for neutralizing activity to occur prior to infection. The samples for testing included sera from the four mice that showed >20% inhibition of ACE2 binding in cPass, pooled sera from those four mice, sera from a COVID-19 convalescent patient, and media only. For detection of neutralization, 120 μL of the virus/sample mixture was transferred to the Vero E6 cells and incubated for 48 hours before fixation with 4% PFA. Each well received 60 μL of virus or an infectious dose of 600 TCID50. Control wells including 6 wells on each plate for no virus and virus-only controls were used. The percent neutralization was calculated as 100-((sample of interest-[average of “no virus”])/[average of “virus only”])*100) with a stain for CoV-2 Np imaged on a Celigo Imaging Cytometer (Nexcelom Bioscience). The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the disclosures herein, and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention. Many more modifications besides those already described are possible without departing from the concepts disclosed herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc. | 116,630 |
11857621 | DETAILED DESCRIPTION OF THE INVENTION One aspect of the invention is directed to a pDNA or pDNA vaccine that induces protective humoral and/or cellular responses against SARS-CoV-2 S protein epitopes and to methods using the pDNA to prevent or treat SARS-CoV-2 infection. Surprisingly, it has been found that particular modifications to DNA encoding full-length S protein or the S1 subunit of S protein, enhance vaccine efficacy and stability and specific pDNA vectors are disclosed herein that have been demonstrated to induce immune responses targeting SARS-CoV-2. SARS-CoV-2 Spike (S) protein. The total length of SARS-CoV-2 S is 1273 aa and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the S1 subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S1 subunit, there is an N-terminal domain (H-305 residues) and a receptor-binding domain (RBD, 319-541 residues); the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasmic tail (1237-1273 residues) comprise the S2 subunit. S protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle. Based on the structure of coronavirus S protein monomers, the S1 subunit forms the globular head while S2 subunits forms the stalk region.FIGS.7and8illustrate S protein and S protein mediated attachment and invasion of a host cell. S protein polynucleotide or polypeptide variants. In some embodiments the segment of plasmid DNA encoding a segment of S protein, including but not limited to, full-length S protein or the S1 subunit of S protein or an immunogenic segment of S protein, may comprise an polynucleotide sequence that is at least 95, 96, 97, 98, 99 or <100% identical to SEQ ID NO: 1 or 3 or have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, substitutions, or insertions of nucleotides to a sequence of SEQ ID NO: 1 or 3, and encode a protein that comprises at least one epitope of S protein. In some embodiments, the plasmid DNA may encode a variant full-length S protein or S1 protein that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue deletions, substitutions, or additions to the amino acid sequence encoded by SEQ ID NOS: 1, 2, 3, or 4 or that encodes an S protein or S1 protein that is at least 95, 96, 97, 98, 99 or <100% identical to that encoded by SEQ ID NOS: 1, 2, 3 or 4. BLASTN may be used to identify a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100% sequence identity to a reference polynucleotide such as a polynucleotide encoding an S protein, antigenic or immunogenic S protein fragment, or S1 subunit. A representative BLASTN setting modified to find highly similar sequences uses an Expect Threshold of 10 and a Wordsize of 28, max matches in query range of 0, match/mismatch scores of 1/−2, and linear gap cost. Low complexity regions may be filtered or masked. Default settings of a Standard Nucleotide BLAST are described by and incorporated by reference to ≤hypertext transfer protocol secure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blas tn&PAGE_TYPE=BlastSearch& LINK_LOC=blasthome≥(last accessed Mar. 23, 2021). BLASTP can be used to identify an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100% sequence identity, or similarity to a reference amino acid, such as an S protein, S1 subunit protein, or antigenic or immunogenic segment of S protein, amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at: ≤hypertext transfer protocol secure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp& PAGE_TYPE=BlastSearch&LINK_LOC=blasthome> (last accessed Mar. 23, 2021). The inventor made several modifications to the SARS-CoV-2 S protein and 51 protein nucleic acid sequences to attain these benefits. Codon Adaptation Index (CAI) is the most widespread technique for analyzing codon usage bias. As opposed to other measures of codon usage bias, such as the effective number of codons (Nc), which measure deviation from a uniform bias (null hypothesis), CAI measures the deviation of a given protein coding gene sequence with respect to a reference set of genes. CAI is used as a quantitative method of predicting the level of expression of a gene based on its codon sequence; see Sharp, Paul M. & Li, Wen-Hsiung,The codon adaptation index-α measure of directional synonymous codon usage bias, and its potential applications, NUCLEICACIDSRESEARCH, 1987, 15 (3): 1281-1295 (incorporated by reference). Software suitable for optimizing codon usage is known and may be used to optimize codon usage in a pDNA construct or a segment thereof, such as an immunogenic portion of the S protein of SARS-CoV-2; see Optimizer available at ≤hypertext transfer protocol://genomes._urv.cat/OPTIMIZER/≥(last accessed Mar. 17, 2021). Codon usage frequencies for various organisms are known and are also incorporated by reference to hypertext transfer protocol://genomes.urv.cat/OPTIMIZER/CU_human_nature.html or to the Codon Usage Database at worldwide web.kazusa.or.jp/codon/ (last accessed Mar. 17, 2021). A pDNA construct or its elements as described herein may have a CAI ranging from 0.8, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, preferably about 0.94. These CAI percentages are considered favorable in terms of level of gene expression from the pDNA. Codon Adaptation Index (CAI) is the most widespread technique for analyzing codon usage bias. As opposed to other measures of codon usage bias, such as the effective number of codons (Nc), which measure deviation from a uniform bias (null hypothesis), CAI measures the deviation of a given protein coding gene sequence with respect to a reference set of genes. CAI is used as a quantitative method of predicting the level of expression of a gene based on its codon sequence; see Sharp, Paul M. & Li, Wen-Hsiung,The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications, NUCLEICACIDSRESEARCH, 1987, 15 (3): 1281-1295 (incorporated by reference). Software suitable for optimizing codon usage is known and may be used to optimize codon usage in a pDNA construct or a segment thereof, such as an immunogenic portion of the S protein of SARS-CoV-2; see Optimizer available at hypertext transfer protocol://genomes_urv.cat/OPTIMIZER/(last accessed Mar. 17, 2021). Codon usage frequencies for various organisms are known and are also incorporated by reference to hypertext transfer protocol://genomes.urv.cat/OPTIMIZER/CU_human_nature.html or to the Codon Usage Database at worldwide web.kazusa.orjp/codon/(last accessed Mar. 17, 2021). A pDNA construct or its elements as described herein may have a CAI ranging from 0.8, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, preferably about 0.94. These CAI percentages are considered favorable in terms of level of gene expression from the pDNA. In some embodiments, the techniques described above are used to design the polynucleotide sequence of the pDNA or pDNA encoding S protein determinants. GC content. In molecular biology and genetics, GC-content (or guanine-cytosine content) is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). This measure indicates the proportion of G and C bases out of an implied four total bases, also including adenine and thymine in DNA and adenine and uracil in RNA. GC-content may be given for a certain fragment of DNA or RNA or for an entire genome. When it refers to a fragment, it may denote the GC-content of an individual gene or section of a gene (domain), a group of genes or gene clusters, a non-coding region, or a synthetic oligonucleotide such as a primer. While high GC content may stabilize a DNA construct, its effects on uptake of a pDNA vaccine, structural effects on transcribed mRNA, and expression level of a protein expressed by pDNA cannot be accurately predicted. A pDNA construct as described herein may have a GC content ranging from about 30 to 70%, for example, about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, preferably about 55-56%. CpG dinucleotide content. The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5′→3′ direction. CpG sites occur with high frequency in genomic regions called CpG islands (or CG islands). Nucleic acids containing CpG motifs can activate host innate and acquired immune responses. Further characterization of CpG sequences which may be used in conjunction with the pDNA vaccine disclosed herein are incorporated by reference to H. L. Davis, Dev Biol (Basel) 2000; 104:165-9. RNA stability/instability motifs (AU-rich elements, ARE). The presence of AU-rich elements in some mammalian mRNAs tends to destabilize those transcripts through the action of cellular proteins that bind these sequences and stimulate poly(A) tail removal. Adenylate-uridylate-rich elements (AU-rich elements; AREs) are found in the 3′ untranslated region (UTR) of many messenger RNAs (mRNAs) that code for proto-oncogenes, nuclear transcription factors, and cytokines. AREs are defined as a region with frequent adenine and uridine bases in a mRNA.FIG.1describes the modified sequence containing modifications described below. Cryptic splicing sites can be present at the mRNA level. A cryptic splice site is a mRNA sequence that has the potential for interacting with the spliceosome. Mutations, including splice site mutations, in the underlying DNA or errors during transcription can activate a cryptic splice site in part of the transcript that usually is not spliced. Premature polyA sites may occur in a sense strand encoding mRNA. These A-rich coding strands result in premature polyadenylation and aberrant mRNA splicing. Repeat sequences and Secondary mRNA structures such as hairpins, loops, and stems can cause interference with the translation of protein. In addition to modification of the S protein nucleic acid sequences, the inventor sought and found that particular vectors were suitable for expression of the modified S protein and S1 subunit nucleic acid sequences. Plasmid vectors. Description pcDNA™3.1(+) and pcDNA™3.1(−) are commercially available vectors derived from pcDNA 3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. In some embodiments, other vectors may be selected, for example, based on the promoters they contain or on other features contributing to their genetic stability when administered to a subject. Promoters which may be used to express or enhance expression of S protein determinants include SV40, RSV and CMV promoters. Additional modifications to improve expression rates include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences; see Alarcon, J. B., et al., Parasitology, 1999, 42, 343-410 which is incorporated by reference. Advantageously, it was found that the pDNA vaccine as disclosed herein was thermostable and did not require encapsulation in order to induce protective humoral and cellular responses against SARS-CoV-2./ Carriers, Excipients and Adjuvants. In a preferred embodiment, the pDNA is not encapsulated and it is not necessary to admix it with a polymer, a liposome, or particles (e.g. microparticles or nanoparticles). For example, it is not necessary to form particles of pDNA and cationic polymers such as polyamines (e.g., polyethyleneimine (PEI), polyhistidine, carboxymethylcellulose (CMC), putrescine, spermidine, or spermine), laminar or multilaminar liposomes, or particles of calcium phosphate, or other compositional forms such as an emulsion, a microcapsule, a microsphere, or a nanoparticle Compositions. Pharmaceutical compositions of the present disclosure comprise an effective amount of pDNA formulation disclosed herein, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical” or “pharmacologically acceptable” refers to molecular entities and compositions that produce no adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human. The preparation of a pharmaceutical composition that contains at least one compound or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 21stedition, 2005, incorporated herein by reference. Moreover, for animal, mammal or human administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. A composition disclosed herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. It was also found that the pDNA vaccine as disclosed herein could be easily and conveniently administered intramuscularly. Routes of administration. Preferably, the pDNA compositions disclosed herein are administered intramuscularly. Other modes for pDNA administration include electroporation and gene gun; see Wang, S. et al., DNA immunization, Curr. Protoc. Microbiol., 2013, 31, 18.3.1-18.3.24, incorporated by reference. Alternatively, the compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, subcutaneously, mucosally, in utero, orally, topically, locally, via inhalation (e.g., by aerosol inhalation, dry powder inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., in liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference). In certain embodiments, a composition herein and/or additional agents is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with edible carrier that can be assimilated. In further embodiments, a composition described herein may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. Solutions of the compositions disclosed herein as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. In some embodiments, agents such as EDTA or EGTA (e.g., Tris-EDTA buffer) are incorporated to prevent degradation of the pDNA by scavenging divalent cations. Other stabilizers including malic acid, ethanol, and Pluronic F-68 are known in the art and are incorporated by reference to Y. Zeng, et al., JOURNAL OFPHARMACEUTICALSCIENCES, March 2011, 100, 904-914. Dispersions of the pDNA may be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate or gelatin. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards. Sterile injectable solutions are prepared by incorporating the pDNA compositions in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent. In other embodiments, the compositions may be formulated for administration via various miscellaneous routes, for example, topical (e.g., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or via inhalation. Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture. Transdermal administration of the compositions may also comprise the use of a “patch.” For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time. In certain embodiments, the pDNA compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in t its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., JOURNAL OFCONTROLLEDRELEASE, 1998, 52, 81-87) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts and could be employed to deliver the compositions described herein. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated by reference), and could be employed to deliver the compositions described herein. It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms. Dosage. The actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight or surface area, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 or 1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. Dosing Regimen. One skilled in the medical and immunological arts may select an appropriate dosing regimen. To enhance the magnitude of antibody responses against SARS-CoV-2, preferably, a regimen comprises administering three separate doses of pDNA intramuscularly over a four week period, preferably at 2 week intervals (0, 2, 4 weeks). Alternatively, the pDNA vaccine may be administered less frequently, for example, a two dose of the pDNA may be given to healthy individuals intramuscularly. EXAMPLES Construct Modification and Vaccination Strategy. The S glycoprotein of SARS-CoV is composed of two subunits, S1 and S2. The S1 subunit consists of four domains, namely, the N-terminal domain (NTD), the C-terminal domain (CTD), and subdomains II and I. In addition, the S1 subunit contains the receptor binding domain (RBD), an essential component required for binding to the human (h)ACE2 receptor on the host cell (FIG.1A). The S2 subunit consists of the fusion peptide (FP) domain, heptad repeats (HR) 1 and 2, the transmembrane domain (TM), and the cytoplasmic tail (CT). These elements are necessary for the fusion of SARS-CoV-2 with the host cell membrane (FIG.1A). The S protein of coronaviruses is a trimeric type I transmembrane, and each monomer consists of S1 and S2 subunits (FIG.1B). Two vaccine constructs were tested in this study: pDNA S.opt.FL containing the full-length S gene and pDNA S1.opt including only the globular head, S1 subunit. The codons of S.FL gene were changed to mammalian codon preference (Homo sapiens) to enhance the gene expression in mammalian cells (FIGS.2A-C) and were subsequently synthesized and inserted into pcDNA 3.1(+). Furthermore, the S1.opt was generated from S.FL via mutagenesis study. Both sequences were tested for the correct gene size (FIG.2B). Mice were divided into seven groups (n=6 per group); the first group received pDNA S.opt.FL, the second group received pDNA S1.opt, and the third group receive done dose of pDNA S.opt.FL followed by two doses of pDNA S1.opt. These groups each received three doses of vaccine. Group four received pDNA S.opt.FL, group five received pDNA S1.opt, and group six received one dose of pDNA S.opt.FL followed by three doses of pDNA S1.opt; these groups each received four doses of vaccine. Group seven was the control group and received only phosphate-buffered saline (PBS) (FIG.3A). A mouse from the control group died prior to first immunization and another mouse from group 4 died after first immunization. Immunogenicity in Mice: Production of Binding Antibodies. All C57BL/6 mice were vaccinated intramuscularly (IM) at 6-8 weeks of age with the pDNA vaccines or with the PBS control; blood was collected at 2 week intervals. Total immunoglobulin G (IgG) antibodies against the S protein were measured in serum samples collected 2 weeks after the last immunization (FIG.3B). The results indicated that sera from all groups of immunized mice, except the PBS control group, contained detectable levels of binding antibodies at weeks 6 and 8 (FIGS.4A,4B). Comparisons among vaccine groups further revealed that mice vaccinated with S.opt.FL pDNA vaccine (groups 1 and 4) generated the highest levels of binding antibodies, with three and four doses of vaccine eliciting equivalent antibody responses (FIGS.4A,4B). Mouse groups immunized with the pDNA S1.opt vaccine produced the lowest levels of antibody responses, while the heterologous vaccine produced a moderate immune response (FIG.4A,4B). Immunogenicity in Mice: Production of Neutralizing Antibodies. To assess the immunological efficacy of the two pDNA vaccines, a surrogate virus-neutralizing assay was performed. This technique is based on the fact that neutralizing antibodies can block the interaction between the SARS-CoV-2 RBD and the ACE2 receptor. Neutralization assay results revealed that mice who received three immunization doses with pDNA S.opt.FL produced higher levels of neutralizing antibodies than mice vaccinated with three doses of pDNA S1.opt (FIG.5A). Mice immunized with S.opt.FL at weeks 6 and 8 produced similar levels of neutralizing antibodies. It was also found that an additional dose enhanced the levels of neutralizing antibodies; that is, mice who received S.opt.FL priming, followed by the three S1.opt booster doses, had higher antibody responses than those who received only two S1.opt booster doses (FIGS.5A,5B). Interestingly, mice immunized with four doses of S1.opt produced comparable levels of neutralizing antibody responses to immunization with three doses (FIGS.5A,5B). Immunogenicity in Mice: Production of IFN-γ. Recent studies highlighted the role of cell-mediated responses in controlling COVID-19. We, therefore, measured the serum levels of IFN− in mice immunized with our vaccine constructs, as an indicator of innate immunity/cellular immunity. It was found that consistent with the antibody data, mice immunized with S.opt.FL pDNA vaccine produced significantly higher serum levels of IFN−, relative to the other experimental vaccine groups (FIG.6). The pDNA platform is as an attractive strategy for vaccine development during pandemics. This technology is simple and highly scalable. Furthermore, unlike mRNA vaccines that are fragile and require encapsulation to protect from degradation, pDNA vaccines are thermally stable, which is particularly beneficial during vaccine shipment and storage. Limited data are available on the effect that multiple vaccine doses can have on eliciting potent neutralizing antibodies. The pDNA vaccines designed and produced by the inventor encode the full-length SARS-CoV-2 S gene and S1 as the antigens of interest. In addition, combining multiple gene inserts in a plasmid vector may interfere with expression of the proteins encoded by these gene inserts; hence, we tested combined administration of the different constructs (S.opt.FL and S1.opt genes) at different doses. Previous studies on pDNA vaccines against other viral pathogens determined that the optimal dosage required for effective immunity is dependent on the antigen/virus type and how these interact with the immune system. For example, one to two doses of pDNA vaccine are sufficient to produce effective neutralizing antibodies for influenza viruses; however, three to four doses are needed to elicit a sufficient protective immune response in HIV [17]. Neutralizing antibodies against SARS-CoV-2 target the spike RBD known to bind to ACE2 of host cell, thereby blocking viral entry. However, the number of pDNA vaccine doses needed to elicit optimal neutralizing antibody responses to SARS-CoV-2 remains unexplored. The inventor considered that multiple doses of a SARS-CoV-2 pDNA vaccine would be needed to generate an effective SARS-CoV-2 antibody-mediated immune response. Therefore, both a three and four dose regimen of each SARS-CoV-2 pDNA vaccine was used to determine which of these could elicit the most potent neutralizing antibody response. As shown herein, it was found that three doses of pDNA S.opt.FL vaccine induced the highest levels of neutralizing antibodies, with no added antibody production conferred by the fourth vaccine dose. In addition, the full-length S protein elicited the most potent immune response, as compared to the pDNA S1.opt vaccine or the S.opt.FL with an S1.opt booster, suggesting that multiple doses of full-length S are needed to elicit high-level immune responses. The inventor consider that non-RBD epitopes may have greater surface accessibility and thus be more immunogenic than some RBD epitopes. Consistent with this observation, mice vaccinated with S.opt.FL elicited higher IFN-production than mice vaccinated with the S1.opt or the combined vaccine. This result is also consistent with the identification of epitopes outside of the S1 domain; see Zheng, et al., Cell Mol. Immunol, 2020 17, 536-538. The data described herein was obtained following the procedures described below. Ethics Statement This preclinical study was registered under the Animal Study Registry 10.17590/asr.0000212. Animal protocols were approved by the Institutional Review Board (IRB NO-2020-333-IRMC) at Imam Abdulrahman Bin Faisal University (IAU), and experiments were done in compliance with the institution guidelines. pDNA Vaccine Constructs. Polynucleotide constructs encoding the full-length S protein (3840 bp) (YP_009724390.1) or its 51 subunit was codon-modified forHomo sapiens. SEQ ID NO: 1 describes the DNA sequence of the gene insert of S.opt.FL which is designated Almansour-I. SEQ ID NO: 2 describes the DNA sequence of the S.opt.FL construct. SEQ ID NO: 3 describes the DNA sequence of the gene insert of S1.opt SEQ ID NO: 4 describes the DNA sequence of the S1.opt construct which is designated Almansour-II. SEQ ID NO: 5 describes a Kozac sequence. SEQ ID NO: 6 describes a native DNA sequence encoding SARS CoV-2 Spike protein. This sequence as well as the amino acid sequence it encodes and other information are described by, and incorporated by reference to, NCBI Reference Sequence: NC_045512.2 and to ≤hypertext transfer protocol secure:// www.ncbi.nlm.nih.gov/nuccore/NC_045512.2?reportgenbank&from=21563&to=25384≥ (last accessed May 3, 2021). SEQ ID NO: 7 describes an amino acid sequence translated from the DNA sequence of SEQ ID NO: 6. Other modifications that were made include changes to GC % content, mRNA secondary structure, cryptic splicing sites, premature polyA sites, internal Chi sites, ribosomal-binding sites, and RNA stability motifs. For example, the entire S.FL sequence was codon-enhanced. To increase translation initiation a Kozac sequence (comprising SEQ ID NO: 5) was added downstream of the NheI restriction site in the constructs. A Shine-Dalgarno sequence is not required for eukaryotic expression but may be incorporated for use in prokaryotic expression systems. The designed sequences were chemically synthesized and BamHI and NheI sequences were incorporated upstream and downstream of the S.opt.FL sequence, respectively. The S.opt.FL sequence was inserted into pcDNA3.1 (+) and cloned to further increase the efficiency of translation in eukaryotes. The S1.opt sequence was synthesized by mutagenesis from the template S.opt.FL. A Kozac sequence was added upstream of the coding sequence: (GCCACC SEQ ID NO: 5. The S.opt.FL was de novo synthesized (GenScript, Piscataway, NJ, USA), and NheI and BamHI restriction sites were incorporated up- and downstream, respectively, of the coding sequence. The S.opt.FL insert was individually cloned into pcDNA 3.1(+). The nucleotide sequence of the S.opt.FL construct was confirmed by sequencing. The S1.opt construct (2043 bp) was synthesized by mutagenesis using the synthesized S.opt.FL as a template. Briefly, a mutagenesis oligo was synthesized and the S.opt.FL pcDNA 3.1(+) was amplified by PCR using the mutagenesis oligo. The mutagenesis construct was linearized by NheI and BamHI and subsequently ligated. The construct was transformed into competent cells and was incubated overnight in LB media with ampicillin at 37° C. A colony was picked and verified by colony PCR and sequencing. For pDNA vaccine production, each cloned vaccine construct was grown in LB media containing ampicillin and was incubated overnight at 37° C. A plasmid DNA purification kit (Cat #12163, QIAGEN®) was used to purify each vaccine construct. The purification levels for S.opt.FL and S1.opt, verified at absorbance 260/280, were 1.91 and 1.89, respectively. Construct lengths were checked by restriction analyses prior to immunization. Immunizations. C57BL/6 mice, 6-8 weeks of age, were provided by the King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. These were maintained by the animal house facility at Imam Abdulrahman Bin Faisal University. Mice were vaccinated intramuscularly (IM) into the tibialis anterior muscle. For each immunization, animals received 100 μg of pDNA in 200 μL of phosphate-buffered saline (PBS), pH 7.4, or the PBS control. Mice were administered vaccines at multiple sites. Serum samples were collected prior to the first immunization and 2 weeks after each immunization. Enzyme-Linked Immunosorbent Assay (ELISA). For ELISAs, 96-well plates (Cat #44-2404-21; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 10 ug/mL of the full-length S antigen (Cat #Z03483-1, Genscript) and incubated overnight at 4° C. Using a 96-well plate washer, plates were washed five times with 300 μL of 1×PBS. For blocking, 2004, of 5% non-fat dry milk in Tris-buffered saline (Blocker BLOTTO Cat #170-6404; Bio-Rad Laboratories, Hercules, CA, USA) was added to each well, and plates were incubated for 1 h at room temperature. Blocked plates were washed five times with 300 μL of 1×PBS, and 100 of serially diluted serum from vaccinated mice was added to each well, followed by incubation for 1 h at room temperature. After five washes with 300 μL of 1×PBS, 100 μL of goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Cat #31430; Invitrogen, Thermo Fisher Scientific) was added to each well, and plates were incubated for 1 h at room temperature. Plates were washed five times with 300 μL of 1×PBS, and 100_L of tetramethylbenzidine (TMB) substrate (Cat #1854050; Thermo Fisher Scientific) was added to all wells, according to the manufacturer's instructions. Lastly, 100 μL of 2 M sulfuric acid (2M H2SO4) was added to all wells to stop reactions; optical density (OD) values were read at 450 nm. Neutralization Assay. The test used to measure antibody neutralization was based on the surrogate virus neutralization test (Cat #L0084; GenScript), a robust assay for testing vaccine efficacy. Briefly, serum samples, as well as positive and negative controls, were serially diluted and incubated with an equal volume (1:1) of diluted HRP-conjugated receptor-binding domain (RBD) a 37° C. for 30 min. Mixtures were then added to plates coated with ACE2, which were covered and incubated at 37° C. for 15 min. After washing four times with 1×wash solution, 100 μL of TMB was added to each well, and plates were incubated in the dark at room temperature for 20 min. Lastly, 50 μL of stop solution was added to each well, and the absorbance was read immediately at 450 nm. Percentage neutralization was calculated based on the following formula: (1−sample absorbance/negative control absorbance)×100%, with a cutoff value of >20%. IFN-gγ Levels of secreted IFN-were measured by ELISA using the mouse IFN-(improved) ELISA Kit (Cat #KMC4021; Invitrogen), according to manufacturer instructions. Briefly, 100 μL of pre-diluted serum samples with standard diluent buffer were added to wells. Samples were incubated at room temperature for 2 h, and plates were washed four times with the provided wash buffer. Next, 100 μL of streptavidin-HRP solution was added to each well, and plates were incubated at room temperature for 30 min. After washing four times with wash buffer, 100 μL of stabilized chromogenic substrate was added to each well, and plates were incubated at room temperature for 30 min. Lastly, 100 μL of stopping solution was added to each well, and plates were read at 450 nm. The results disclosed above show that immunization with a codon-modified pDNA encoding the full-length or 51 subunit of the SARS-CoV-2 S generated potent and robust binding and neutralizing antibodies, as well as IFN-cytokine responses. Terminology. Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”. As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “substantially”, “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/−0.1% of the stated value (or range of values), +/−1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/−5% of the stated value (or range of values), +/−10% of the stated value (or range of values), +/−15% of the stated value (or range of values), +/−20% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all subranges subsumed therein. Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if parameter X is exemplified herein to have values in the range of 1-10 it also describes subranges for Parameter X including 1-9, 1-8, 1-7, 2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 as mere examples. A range encompasses its endpoints as well as values inside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2, 3, 4, <5 and 5. As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology. As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features. Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention. The description and specific examples, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested. All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears. The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references, and does not constitute an admission as to the accuracy of the content of such references. | 48,571 |
11857622 | SEQUENCE IDENTIFIERS SEQ ID NO: 1 sets forth an amino acid sequence derived from a native HCMV gB (strain Towne). SEQ ID NO: 2 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Q98C, G271C. SEQ ID NO: 3 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Q98C, I653C. SEQ ID NO: 4 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: G99C, A267C. SEQ ID NO: 5 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T100C, A267C. SEQ ID NO: 6 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T100C, S269C. SEQ ID NO: 7 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T100C, L651C. SEQ ID NO: 8 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: D217C, F584C. SEQ ID NO: 9 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Y218C, A585C. SEQ ID NO: 10 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: S219C, D654C. SEQ ID NO: 11 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: N220C, D652C. SEQ ID NO: 12 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T221C, D652C. SEQ ID NO: 13 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: W240C, G718C. SEQ ID NO: 14 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Y242C, K710C. SEQ ID NO: 15 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Y242C, D714C. SEQ ID NO: 16 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: S269C, I653C. SEQ ID NO: 17 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: G271C, P614C. SEQ ID NO: 18 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: S367C, L499C. SEQ ID NO: 19 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T372C, W506C. SEQ ID NO: 20 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: F541C, Q669C. SEQ ID NO: 21 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: L548C, A650C. SEQ ID NO: 22 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: A549C, I653C. SEQ ID NO: 23 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: S550C, D652C. SEQ ID NO: 24 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: G604C, F661C. SEQ ID NO: 25 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: N605C, E665C. SEQ ID NO: 26 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: R607C, S675C. SEQ ID NO: 27 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T608C, D679C. SEQ ID NO: 28 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: E609C, F678C. SEQ ID NO: 29 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: R673C, S674C. SEQ ID NO: 30 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: N676C, V677C. SEQ ID NO: 31 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: L680C, E681C. SEQ ID NO: 32 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: I683C, M684C. SEQ ID NO: 33 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: F687C, N688C. SEQ ID NO: 34 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: Y690C, K691C. SEQ ID NO: 35 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: K695C, K724C. SEQ ID NO: 36 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: T746C, F747C. SEQ ID NO: 37 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutations are included: K749C, N750C. SEQ ID NO: 38 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: K670L. SEQ ID NO: 39 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: K670F. SEQ ID NO: 40 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: R673L. SEQ ID NO: 41 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: R673F. SEQ ID NO: 42 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: K691L. SEQ ID NO: 43 sets forth the amino acid sequence of SEQ ID NO: 1, wherein the following mutation is included: K691F. SEQ ID NO: 44 sets forth the amino acid sequence for a native HCMV gB (AD169; PDB: 5CXF) that folds into a postfusion conformation when expressed. SEQ ID NO: 45 sets forth the amino acid sequence for an HCMV gB variant (gB705) that folds into a postfusion conformation when expressed. SEQ ID NO: 46 sets forth the amino acid sequence for a native HCMV gB (Merlin strain) that folds into a postfusion conformation when expressed. SEQ ID NO: 47 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: M96C and D660C. SEQ ID NO: 48 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Q98C and N658C. SEQ ID NO: 49 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: T100C and R258C. SEQ ID NO: 50 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: T100C and L656C. SEQ ID NO: 51 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: T100C and N658C. SEQ ID NO: 52 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: 1117C and T406C. SEQ ID NO: 53 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: 1117C and 5407C. SEQ ID NO: 54 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Y153C and L712C. SEQ ID NO: 55 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: L162C and M716C. SEQ ID NO: 56 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: D217C and S587C. SEQ ID NO: 57 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: D217C and Y589C. SEQ ID NO: 58 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S219C and F584C. SEQ ID NO: 59 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S219C and A585C. SEQ ID NO: 60 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S219C and N586C. SEQ ID NO: 61 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: N220C and T659C. SEQ ID NO: 62 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S223C and T659C. SEQ ID NO: 63 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: W240C and A732A. SEQ ID NO: 64 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: W240C and G735C. SEQ ID NO: 65 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Y242C and V728C. SEQ ID NO: 66 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Y242C and G731C. SEQ ID NO: 67 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: R258C and L656C. SEQ ID NO: 68 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S269C and L656C. SEQ ID NO: 69 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S269C and N658C. SEQ ID NO: 70 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: D272C and P614C. SEQ ID NO: 71 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: V273C and V629C. SEQ ID NO: 72 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: W349C and A650C. SEQ ID NO: 73 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S367C and A500C. SEQ ID NO: 74 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S367C and A503C. SEQ ID NO: 75 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: K370C and Q501C. SEQ ID NO: 76 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: K522C and I683C. SEQ ID NO: 77 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: I523C and I683C. SEQ ID NO: 78 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: I523C and M684C. SEQ ID NO: 79 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: N524C and M684C. SEQ ID NO: 80 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: P525C and E681C. SEQ ID NO: 81 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: R540C and L680C. SEQ ID NO: 82 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: F541C and L680C. SEQ ID NO: 83 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: L548C and P655C. SEQ ID NO: 84 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: A549C and N658C. SEQ ID NO: 85 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S550C and P655C. SEQ ID NO: 86 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: S550C and E657C. SEQ ID NO: 87 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Q591C and S668C. SEQ ID NO: 88 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: L603C and Y667C. SEQ ID NO: 89 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: G604C and L672C. SEQ ID NO: 90 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: R607C and N688C. SEQ ID NO: 91 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: T608C and Q692C. SEQ ID NO: 92 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: E609C and K691C. SEQ ID NO: 93 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: E610C and S674C. SEQ ID NO: 94 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: E610C and S675C. SEQ ID NO: 95 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: Q612C and V663C. SEQ ID NO: 96 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: V737C and F755C. SEQ ID NO: 97 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: V741C and A754C. SEQ ID NO: 98 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutations are included: V741C and F755C. SEQ ID NO: 99 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: D679S. SEQ ID NO: 100 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: D679N. SEQ ID NO: 101 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: E682S. SEQ ID NO: 102 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: E682Q. SEQ ID NO: 103 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: E686S. SEQ ID NO: 104 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: E686Q. SEQ ID NO: 105 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: N118P. SEQ ID NO: 106 sets forth the amino acid of SEQ ID NO: 1, wherein the following mutation is included: D646P. SEQ ID NO: 107 sets forth the amino acid sequence for >5CXF:A|PDBID|CHAIN|SEQUENCE, fromFIG.8. SEQ ID NO: 108 sets forth the amino acid sequence for >5CXF:B|PDBID|CHAIN|SEQUENCE, fromFIG.8. SEQ ID NO: 109 sets forth the amino acid sequence for >5CXF:C|PDBID|CHAIN|SEQUENCE, fromFIG.8. SEQ ID NO: 110 sets forth the amino acid sequence for a gB polypeptide from HAN13 gi|2423456141gb|GQ221973.1|:81988-84705 Human herpesvirus 5 strain HAN13, complete genome reverse complement, referenced in the description forFIG.9. SEQ ID NOs: 111 sets forth the amino acid sequence for a gB polypeptide from VR1814 gi|012703557591gb|GU179289.1|:81925-84642 Human herpesvirus 5 strain VR1814, complete genome reverse complement, referenced in the description forFIG.9. SEQ ID NOs: 112-140 sets forth the amino acid sequence for a gB polypeptide from various CMV gB strains described inFIG.9. SEQ ID NO: 141-SEQ ID NO: 210 set forth a polynucleotide sequence encoding a polypeptide derived from HCMV, such as for example, gH, gL, UL128, UL130, UL131, gB or pp65. SEQ ID NO: 211-SEQ ID NO: 223 set forth an amino acid sequence for a polypeptide derived from HCMV, such as for example, gH, gL, UL128, UL130, UL131, gB or pp65. SEQ ID NO: 224 sets forth an amino acid sequence for a polypolypeptide derived from HCMV. SEQ ID NO: 225-SEQ ID NO: 254 set forth a polynucleotide sequence encoding a polypolypeptide derived from HCMV. SEQ ID NO: 255 sets forth the amino acid sequence of CMV gB1666, residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, Y589C, and I675S. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 256 sets forth the nucleic acid sequence of CMV gB1666 encoding residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, Y589C, and I675S. Does not include nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 257 sets forth the amino acid sequence of CMV gB2457 (Prefusion, Full length), residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, M371C, W506C, Y589C, and I675S. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 258 sets forth the nucleic acid sequence of CMV gB2457 (Prefusion, Full length) encoding residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, M371C, W506C, Y589C, and I675S. Does not include nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 259 sets forth the amino acid sequence of CMV gB2459 (Prefusion, Full length), residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, N524C, Y589C, M684C, and I675S. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 260 sets forth the nucleic acid sequence of CMV gB2459 (Prefusion, Full length) encoding residues V23 to V907 of SEQ ID NO: 1 (Towne) having the following mutations: D217C, N524C, Y589C, M684C, and I675S. Does not include nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 261 sets forth the amino acid sequence of CMV gB2555 (Prefusion, including trimerization domain (GCN4 CC tri2)), residues V23 to V702 of SEQ ID NO: 1 (Towne) having the following mutations: YIH to GHR (155-157), D217C, W240A, M371C, C246S, W506C, Y589C, and I675S. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 262 sets forth the nucleic acid sequence of CMV gB2555 (Prefusion, including trimerization domain (GCN4 CC tri2)) encoding residues V23 to V702 of SEQ ID NO: 1 (Towne) having the following mutations: YIH to GHR (155-157), D217C, W240A, M371C, C246S, W506C, Y589C, and I675S. Does not include nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 263 sets forth the amino acid sequence of CMV gB2556 (Prefusion, including trimerization domain (GCN4 CC tri2)), residues V23 to V702 of SEQ ID NO: 1 (Towne) having the following mutations: YIH to GHR (155-157), D217C, W240A, C246S, N524C, Y589C, I675S, and M684C. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 264 sets forth the nucleic acid sequence of CMV gB2556 (Prefusion, including trimerization domain (GCN4 CC tri2)) encoding residues V23 to V702 of SEQ ID NO: 1 (Towne) having the following mutations: YIH to GHR (155-157), D217C, W240A, C246S, N524C, Y589C, I675S, and M684C. Does not include nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 265 sets forth the amino acid sequence of CMV gB2796 (Prefusion, ectodomain), residues V23 to D646 of SEQ ID NO:1 (Towne) having the following mutations: YIH to GHR (155-157), D217C, W240A, C246S, M371C, W506C, and Y589C. Does not include the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 266 sets forth the nucleic acid sequence of CMV gB2796 (Prefusion, ectodomain) encoding amino acids V23 to D646 of SEQ ID NO:1 (Towne) having the following amino acid mutations: YIH to GHR (155-157), D217C, W240A, C246S, M371C, W506C, and Y589C. Does not include the nucleotides encoding the signal sequence (residues M1-A22 of SEQ ID NO: 1). SEQ ID NO: 267 sets for the CMV gB ectodomain, V23 to P707 of SEQ ID NO:1 (Towne strain). SEQ ID NO: 268 sets forth the amino acid sequence of the signal sequence of wt HCMV gB (Towne). SEQ ID NO: 269 sets forth the amino acid sequence of GCN4 CC tri2 trimerization domain (see Table 9). SEQ ID NO: 270 sets forth the nucleic acid sequence encoding the GCN4 CC tri2 trimerization domain. SEQ ID NO: 271 sets forth the amino acid sequence of T4 fibritin foldon domain (see Table 9). SEQ ID NO: 272-273 sets forth the amino acid sequences of various GCN4 trimerization domains. SEQ ID NO: 274 sets forth the amino acid sequences of C-terminal fusion sequence described in Table 9. DETAILED DESCRIPTION As described herein, the inventors elucidated a three-dimensional structure of a HCMV glycoprotein B (gB) polypeptide in a conformation that differs from the postfusion conformation and which we refer to as a prefusion conformation. Mutations to stabilize the polypeptide in a prefusion conformation were also discovered. The structures may be used to generate HCMV neutralizing antibody responses greater than those achieved with prior HCMV gB-based immunogens. The polypeptides described herein, and the nucleic acids that encode the polypeptides, may be used, for example, as potential immunogens in a vaccine against HCMV and as diagnostic tools, among other uses. The inventors further discovered mutations that can be introduced into a cytomegalovirus (CMV) gB polypeptide, which can, among other things, greatly facilitate the production and subsequent purification of a gB antigen stabilized in the prefusion conformation; significantly improve the efficiency of production of a gB polypeptide in the prefusion conformation; alter the antigenicity of a gB polypeptide, as compared to the wild-type gB polypeptide; facilitate a focused immune response to prefusion gB; and reduce and/or eliminate steric occlusion of neutralizing epitopes of gB. Definitions As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “additional mutation” shall include, but not be limited to, an amino acid substitution that introduces electrostatic mutations, fill cavities, alter the packing of residues, introduce N-linked glycosylation sites, introduce inter-protomer disulfide bonds, and combinations thereof, including conservative substitutions thereof, as compared to a native HCMV gB. Examples of an “additional mutation” may be found throughout this description but most specifically are set forth in Tables 3 and 4 and the Examples. The term “adjuvant” refers to a substance capable of enhancing, accelerating, or prolonging the body's immune response to an immunogen or immunogenic composition, such as a vaccine (although it is not immunogenic by itself). An adjuvant may be included in the immunogenic composition, such as a vaccine, or may be administered separately from the immunogenic composition. The term “administration” refers to the introduction of a substance or composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intramuscular, the composition (such as a composition including a disclosed immunogen) is administered by introducing the composition into a muscle of the subject. The term “antigen” refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. The term “conservative substitution” refers to the substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The term “degenerate variant” of a reference polynucleotide refers to a polynucleotide that differs in the nucleotide sequence from the reference polynucleotide but encodes the same polypeptide sequence as encoded by the reference polynucleotide. There are 20 natural amino acids, most of which are specified by more than one codon. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified within a protein encoding sequence, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. The term “effective amount” refers to an amount of agent that is sufficient to generate a desired response. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection. The term “epitope” (or “antigenic determinant” or “antigenic site”) refers to the region of an antigen to which an antibody, B cell receptor, or T cell receptor binds or responds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by secondary, tertiary, or quaternary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by higher order folding are typically lost on treatment with denaturing solvents. The term “subject” refers to either a human or a non-human mammal. The term “mammal” refers to any animal species of the Mammalia class. Examples of mammals include: humans; non-human primates such as monkeys; laboratory animals such as rats, mice, guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like. The term “glycoprotein” refers to a protein that contains oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification known as glycosylation. The term “glycosylation site” refers to an amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan. An N-linked glycosylation site is triplet sequence of NX(S/T) in which N is asparagine, X is any residue except proline, and (S/T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. The term “host cells” refers to cells in which a vector can be propagated and its DNA or RNA expressed. The cell may be prokaryotic or eukaryotic. The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, through supplement 104, 2013). The term “immunogen” refers to a compound, composition, or substance that is immunogenic as defined herein below. The term “immunogenic” refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response against a particular antigen, in a subject, whether in the presence or absence of an adjuvant. The term “immune response” refers to any detectable response of a cell or cells of the immune system of a host mammal to a stimulus (such as an immunogen), including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). Examples of immune responses include an alteration (e.g., increase) in Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th2 or Th17 type cytokines) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody generation and/or secretion), binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte (“CTL”) response, induction of a B cell response (e.g., antibody production), and, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells and B cells), and increased processing and presentation of antigen by antigen presenting cells. The term “immune response” also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of the immune system of a vertebrate animal in vitro. The term ‘immunogenic composition” refers to a composition comprising an immunogen. The term “mutation” refers to deletion, addition, or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the substitution of an amino acid at one particular location in the protein sequence is referred to using a notation “(amino acid residue in wild type protein)(amino acid position)(amino acid residue in engineered protein)”. For example, a notation Y75A refers to a substitution of a tyrosine (Y) residue at the 75th position of the amino acid sequence of the reference protein by an alanine (A) residue (in a mutant of the reference protein). In cases where there is variation in the amino acid residue at the same position among different wild-type sequences, the amino acid code preceding the position number may be omitted in the notation, such as “75A.” The term “native” or “wild-type” protein, sequence, or polypeptide refers to a naturally existing protein, sequence, or polypeptide that has not been artificially modified by selective mutations. The term “pharmaceutically acceptable carriers” refers to a material or composition which, when combined with an active ingredient, is compatible with the active ingredient and does not cause toxic or otherwise unwanted reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffering agents, lubricating agents, emulsifiers, absorbants, dispersion media, coatings, and stabilizers. The term “pre-fusion-specific antibody” refers to an antibody that specifically binds to the CMV gB glycoprotein in a pre-fusion conformation, but does not bind to the CMV gB protein in a post-fusion conformation. The term “pre-fusion trimer-specific antibody” refers to an antibody that specifically binds to the CMV gB glycoprotein in a pre-fusion, trimeric conformation, but does not bind to the CMV gB protein in a post-fusion conformation or in a pre-fusion conformation that is not also trimeric. “Pre-fusion trimer-specific antibodies” are a subset of “pre-fusion-specific antibodies.” The term “prime-boost vaccination” refers to an immunotherapy regimen that includes administration of a first immunogenic composition (the primer vaccine) followed by administration of a second immunogenic composition (the booster vaccine) to a subject to induce an immune response. The primer vaccine and the booster vaccine typically contain the same immunogen and are presented in the same or similar format. However, they may also be presented in different formats, for example one in the form of a vector and the other in the form of a naked DNA plasmid. The skilled artisan will understand a suitable time interval between administration of the primer vaccine and the booster vaccine. Further, the primer vaccine, the booster vaccine, or both primer vaccine and the booster vaccine additionally include an adjuvant. The term “soluble protein” refers to a protein capable of dissolving in aqueous liquid and remaining dissolved. The solubility of a protein may change depending on the concentration of the protein in the water-based liquid, the buffering condition of the liquid, the concentration of other solutes in the liquid, for example salt and protein concentrations, and the temperature of the liquid. The term “specifically bind,” in the context of the binding of an antibody to a given target molecule, refers to the binding of the antibody with the target molecule with higher affinity than its binding with other tested substances. For example, an antibody that specifically binds to the CMV gB protein in pre-fusion conformation is an antibody that binds CMV gB protein in pre-fusion conformation with higher affinity than it binds to the CMV gB protein in the post-fusion conformation. The term “therapeutically effective amount” refers to the amount of agent that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of a disorder. The term “vaccine” refers to a pharmaceutical composition comprising an immunogen that is capable of eliciting a prophylactic or therapeutic immune response in a subject. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen. The term “vector” refers to a nucleic acid molecule capable of transporting or transferring a foreign nucleic acid molecule. The term encompasses both expression vectors and transcription vectors. The term “expression vector” refers to a vector capable of expressing the insert in the target cell, and generally contains control sequences, such as enhancer, promoter, and terminator sequences, that drive expression of the insert. The term “transcription vector” refers to a vector capable of being transcribed but not translated. Transcription vectors are used to amplify their insert. The foreign nucleic acid molecule is referred to as “insert” or “transgene.” A vector generally consists of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or origin of vectors, major types of vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors, and artificial chromosomes. Native HCMV gB Native HCMV gB is synthesized as a 906 or 907 amino acid polypeptide (depending upon the strain of CMV) that undergoes extensive posttranslational modification, including glycosylation at N- and O-linked sites and cleavage by ubiquitous cellular endoproteases into amino- and carboxy-terminal fragments. The N- and C-terminal fragments of gB, gp116 and gp55, respectively, are covalently connected by disulfide bonds, and the mature, glycosylated gB assumes a trimeric configuration. The gB polypeptide contains a large ectodomain (which is cleaved into gp116 and the ectodomain of gp55), a transmembrane domain (TM), and the intraviral (or cytoplasmic) domain (cytodomain). Native HCMV gBs from various strains are known. For example, at least sixty HCMV gB sequences from clinical and laboratory-adapted strains are available from NCBI's RefSeq database as described in Burke et al., “Crystal Structure of the Human Cytomegalovirus Glycoprotein B.”PLoS Pathog.2015 Oct. 20; 11(10):e1005227, see S4 Fig., which is hereby incorporated herein in its entirety. Accordingly, the term “CMV gB” polypeptide or “HCMV gB” polypeptide as used herein is to be understood as the native HCMV gB polypeptide from any human HCMV strain (not limited to the Towne strain). The actual residue position number may need to be adjusted for gBs from other human CMV strains depending on the actual sequence alignment. However, one of skill in the art will understand how to align sequences from different strains in order to identify the corresponding residue position from one strain to another. HCMV gB is encoded by the UL55 gene of HCMV genome. It is an envelope glycoprotein that mediates the fusion of the HCMV viral membrane with a host cell membrane. The protein undergoes a series of conformational changes from a prefusion to a postfusion form. The crystal structure of gB in its postfusion form is available (PDB accession code 5CXF), and the prefusion conformation is set forth herein. Conformations A HCMV gB postfusion conformation refers to a structural conformation adopted by HCMV gB subsequent to the fusion of the virus envelope with the host cellular membrane. The native HCMV gB may also assume the postfusion conformation outside the context of a fusion event, for example, under stress conditions such as exposure to heat, extraction from a membrane, expression as an ectodomain or storage. More specifically, the gB postfusion conformation is described, for example, in Burke et al., Crystal Structure of the Human Cytomegalovirus Glycoprotein B.PLoS Pathog.2015 Oct. 20; 11(10): e1005227. See also, Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB): 5CXF, Crystal structure of the extracellular domain of glycoprotein B from Human Cytomegalovirus, from Human cytomegalovirus (strain AD169), deposited 2015-07-28; DOI: 10.2210/pdb5CXF/pdb; and Burke et al.,PLoS Pathog.2015 Oct. 20; 11(10):e1005227. A sequence of a protein that when expressed, can fold into a postfusion conformation, is provided as SEQ ID NO: 44. Another example of a protein that when expressed folds into a postfusion conformation is provided as SEQ ID NO: 45. The postfusion conformation is about 165 Å tall and 65 Å wide. As used herein, a “prefusion conformation” refers to a structural conformation adopted by the polypeptide that differs from the HCMV gB postfusion conformation at least in terms of molecular dimensions or three-dimensional coordinates. The prefusion conformation refers to a structural conformation adopted by HCMV gB prior to triggering of the fusogenic event that leads to transition of gB to the postfusion conformation. Isolating HCMV gB in a stable prefusion conformation may be useful in informing and directing development of improved vaccines and immunogenic compositions to address the important public health problem of cytomegalovirus infections. In some embodiments, a prefusion conformation includes a conformation that can bind to a prefusion-specific antibody. In some embodiments, a prefusion conformation includes a conformation that is characterized by coordinates set forth in Table 1A, which is incorporated by reference herein in its entirety. In some embodiments, the polypeptide is characterized by structure coordinates comprising a root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed on backbone atoms described by structural coordinates set forth in Table 1A. In some embodiments, a prefusion conformation includes a conformation that is characterized by coordinates set forth in Table 1B, which is incorporated by reference herein in its entirety. In some embodiments, the polypeptide is characterized by structure coordinates comprising a root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed on backbone atoms described by structural coordinates set forth in Table 1B. In some embodiments, a polypeptide having a HCMV gB prefusion conformation refers to a polypeptide that includes a trimeric helix bundle, centered on the three-fold axis of the trimer and comprising residues L479 to K522 of each protomer, wherein the direction of the bundle from N-terminal to C-terminal along the three-fold axis (shown by the arrows inFIGS.4A and4B) is towards the point on the three-fold axis intersected by the plane defined by residue W240 of each protomer, which is in a fusion loop near the tip of each Domain I of the trimer. In some embodiments, the helix bundle comprises the residues between L479 and K522, according to the numbering of SEQ ID NO: 1. Polypeptides of the Invention The present invention relates to polypeptides that include amino acid mutations relative to the amino acid sequence of the corresponding wild-type HCMV gB. The amino acid mutations include amino acid substitutions, deletions, or additions relative to a wild-type HCMV gB. Accordingly, the polypeptides are mutants of wild-type HCMV gBs. In some embodiments, the polypeptides possess certain beneficial characteristics, such as being immunogenic. In some embodiments, the polypeptides possess increased immunogenic properties or improved stability in the prefusion conformation, as compared to the corresponding wild-type HCMV gB. Stability refers to the degree to which a transition of the HCMV gB conformation from prefusion to postfusion is hindered or prevented. In still other embodiments, the present disclosure provides polypeptides that display one or more introduced mutations as described herein, which may also result in improved stability in the prefusion conformation. The introduced amino acid mutations in the HCMV gB include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequences of the mutants are amino acid substitutions relative to a wild-type HCMV gB. Several modes of stabilizing the polypeptide conformation include amino acid substitutions that introduce disulfide bonds, introduce electrostatic mutations, fill cavities, alter the packing of residues, introduce N-linked glycosylation sites, and combinations thereof, as compared to a native HCMV gB. In one aspect, the invention relates to a polypeptide that exhibits a conformation that is not the postfusion conformation. That is, the polypeptide exhibits a prefusion conformation as described above and does not exhibit a postfusion conformation. See, for example, the prefusion conformation illustrated inFIG.3A, as compared to the postfusion conformation illustrated inFIG.3B;FIG.4A, as compared to the postfusion conformation illustrated inFIG.4B; andFIG.6A, as compared to the postfusion conformation illustrated inFIG.6C. In some embodiments, the polypeptide is characterized by structure coordinates comprising a root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed on backbone atoms described by structural coordinates of Table 1A. In some embodiments, the polypeptide is characterized by structure coordinates comprising a root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed on backbone atoms described by structural coordinates of Table 1B. In some embodiments, the polypeptides are isolated, i.e., separated from HCMV gB polypeptides having a postfusion conformation. Thus, the polypeptide may be, for example, at least 80% isolated, at least 90%, 95%, 98%, 99%, or even 99.9% isolated from HCMV gB polypeptides in a postfusion conformation. In one aspect, the invention relates to a polypeptide that specifically binds to an HCMV gB prefusion-specific antibody. It will be understood that a homogeneous population of polypeptides in a particular conformation can include variations (such as polypeptide modification variations, e.g., glycosylation state), that do not alter the conformational state of the polypeptide. In several embodiments, the population of polypeptides remains homogeneous over time. For example, in some embodiments, the polypeptide, when dissolved in aqueous solution, forms a population of polypeptides stabilized in the prefusion conformation for at least 12 hours, such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more. Without being bound by theory, the polypeptides disclosed herein are believed to facilitate a stabilized prefusion conformation of an HCMV gB polypeptide. The polypeptides include at least one mutation as compared to a corresponding native HCMV gB polypeptide. A person of ordinary skill in the art will appreciate that the polypeptides are useful to elicit immune responses in mammals to CMV. The native HCMV gB is conserved among the HCMV entry glycoproteins and is required for entry into all cell types. In view of the substantial conservation of HCMV gB sequences, the amino acid positions amongst different native HCMV gB sequences may be compared to identify corresponding HCMV gB amino acid positions among different HCMV strains. Thus, the conservation of native HCMV gB sequences across strains allows use of a reference HCMV gB sequence for comparison of amino acids at particular positions in the HCMV gB polypeptide. Accordingly, unless expressly indicated otherwise, the polypeptide amino acid positions provided herein refer to the reference sequence of the HCMV gB polypeptide set forth in SEQ ID NO: 1. However, it should be noted that different native HCMV gB sequences may have different numbering systems from SEQ ID NO: 1, for example, there may be additional amino acid residues added or removed as compared to SEQ ID NO: 1 in a native HCMV gB sequence derived from a strain other than Towne. As such, it is to be understood that when specific amino acid residues are referred to by their number, the description is not limited to only amino acids located at precisely that numbered position when counting from the beginning of a given amino acid sequence, but rather that the equivalent or corresponding amino acid residue in any and all HCMV gB sequences is intended even if that residue is not at the same precise numbered position, for example if the HCMV sequence is shorter (e.g., a fragment) or longer than SEQ ID NO: 1, or has insertions or deletions as compared to SEQ ID NO: 1. In some embodiments, the polypeptide is full-length, wherein the polypeptide includes the same number of amino acid residues as the mature full-length wild-type HCMV gB. In some embodiments, the polypeptide is a fragment, wherein the polypeptide includes less than the total number of amino acid residues as the mature full-length wild-type HCMV gB. As used herein the term “fragment” and “truncated” are interchangeable. In some embodiments, the truncated gB polypeptide includes only the ectodomain sequence. 1. Cysteine (C) Substitutions In some embodiments, the polypeptide includes cysteine substitutions that are introduced, as compared to a native HCMV gB. In some embodiments, the polypeptide includes any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine substitutions. Without being bound by theory or mechanism, the cysteine substitutions described herein are believed to facilitate stability of the polypeptide in a conformation that is not the HCMV gB postfusion conformation. The introduced cysteine substitutions may be introduced by protein engineering, for example, by including one or more substituted cysteine residues that form a disulfide bond. In several embodiments, the amino acid positions of the cysteines are within a sufficiently close distance for formation of a disulfide bond in the prefusion, and not postfusion, conformation of the HCMV gB. The cysteine residues that form a disulfide bond can be introduced into native HCMV gB sequence by two or more amino acid substitutions. For example, in some embodiments, two cysteine residues are introduced into a native HCMV gB sequence to form a disulfide bond. In some embodiments, the polypeptide includes a recombinant HCMV gB stabilized in a prefusion conformation by a disulfide bond between cysteines that are introduced into a pair of amino acid positions that are close to each other in the prefusion conformation and more distant in the postfusion conformation. Exemplary cysteine substitutions as compared to a native HCMV gB include any mutation selected from Table 2, the numbering of which based on the numbering of SEQ ID NO: 1. TABLE 2Exemplary cysteine pairs for disulfide bond stabilization(ii) HCMV gBresidue pairs for(iv) Exemplarycysteinesequencesubstitution,thataccording to the(iii) Substitutionsincludes the(i) Mutantnumbering ofcorrespondingmutations isRowIDSEQ ID NO: 1to SEQ ID NO: 1set forth in:1gB-001;98 and 271Q98C and G271CSEQ ID NO: 2pSB015822gB-00298 and 653Q98C and I653CSEQ ID NO: 33gB-003;99 and 267G99C and A267CSEQ ID NO: 4pSB015794gB-004;100 and 267T100C and A267CSEQ ID NO: 5pSB015805gB-005;100 and 269T100C and S269CSEQ ID NO: 6pSB015816gB-006100 and 651T100C and L651CSEQ ID NO: 77gB-007217 and 584D217C and F584CSEQ ID NO: 88gB-008218 and 585Y218C and A585CSEQ ID NO: 99gB-009219 and 654S219C and D654CSEQ ID NO: 1010gB-010220 and 652N220C and D652CSEQ ID NO: 1111gB-011221 and 652T221C and D652CSEQ ID NO: 1212gB-012240 and 718W240C and G718CSEQ ID NO: 1313gB-013242 and 710Y242C and K710CSEQ ID NO: 1414gB-014242 and 714Y242C and D714CSEQ ID NO: 1515gB-015269 and 653S269C and I653CSEQ ID NO: 1616gB-016271 and 614G271C and P614CSEQ ID NO: 1717gB-017367 and 499S367C and L499CSEQ ID NO: 1818gB-018372 and 506T372C and W506CSEQ ID NO: 1919gB-019541 and 669F541C and Q669CSEQ ID NO: 2020gB-020548 and 650L548C and A650CSEQ ID NO: 2121gB-021549 and 653A549C and I653CSEQ ID NO: 2222gB-022550 and 652S550C and D652CSEQ ID NO: 2323gB-023604 and 661G604C and F661CSEQ ID NO: 2424gB-024605 and 665N605C and E665CSEQ ID NO: 2525gB-025607 and 675R607C and S675CSEQ ID NO: 2626gB-026608 and 679T608C and D679CSEQ ID NO: 2727gB-027609 and 678E609C and F678CSEQ ID NO: 2828gB-028673 and 674R673C and S674CSEQ ID NO: 2929gB-029676 and 677N676C and V677CSEQ ID NO: 3030gB-030680 and 681L680C and E681CSEQ ID NO: 3131gB-031683 and 684I683C and M684CSEQ ID NO: 3232gB-032687 and 688F687C and N688CSEQ ID NO: 3333gB-033690 and 691Y690C and K691CSEQ ID NO: 3434gB-034695 and 724K695C and K724CSEQ ID NO: 3535gB-035746 and 747T746C and F747CSEQ ID NO: 3636gB-036749 and 750K749C and N750CSEQ ID NO: 3737gB-043;96 and 660M96C andSEQ ID NO: 47pSB01656D660C38gB-044;98 and 658Q98C and N658CSEQ ID NO: 48pSB0165739gB-045;100 and 258T100C andSEQ ID NO: 49pSB01658R258C40gB-046;100 and 656T100C and L656CSEQ ID NO: 50pSB0165941gB-047;100 and 658T100C andSEQ ID NO: 51pSB01660N658C42gB-048;117 and 406I117C and T406CSEQ ID NO: 52pSB0166143gB-049;117 and 407I117C and S407CSEQ ID NO: 53pSB0166244gB-050;153 and 712Y153C and L712CSEQ ID NO: 54pSB0166345gB-051;162 and 716L162C andSEQ ID NO: 55pSB01664M716C46gB-052;217 and 587D217C andSEQ ID NO: 56pSB01665S587C47gB-053;217 and 589D217C andSEQ ID NO: 57pSB01666Y589C48gB-054;219 and 584S219C and F584CSEQ ID NO: 58pSB0166749gB-055;219 and 585S219C andSEQ ID NO: 59pSB01668A585C50gB-056;219 and 586S219C andSEQ ID NO: 60pSB01669N586C51gB-057;220 and 659N220C andSEQ ID NO: 61pSB01670T659C52gB-058;223 and 659S223C andSEQ ID NO: 62pSB01671T659C53gB-059;240 and 732W240C andSEQ ID NO: 63pSB01672A732C54gB-060;240 and 735W240C andSEQ ID NO: 64pSB01673G735C55gB-061;242 and 728Y242C andSEQ ID NO: 65pSB01674V728C56gB-062;242 and 731Y242C andSEQ ID NO: 66pSB01675G731C57gB-063258 and 656R258C andSEQ ID NO: 67L656C58gB-064269 and 656S269C and L656CSEQ ID NO: 6859gB-065;269 and 658S269C andSEQ ID NO: 69pSB01678N658C60gB-066;272 and 614D272C andSEQ ID NO: 70pSB01679P614C61gB-067;273 and 629V273C andSEQ ID NO: 71pSB01680V629C62gB-068;349 and 650W349C andSEQ ID NO: 72pSB01681A650C63gB-069;367 and 500S367C andSEQ ID NO: 73pSB01682A500C64gB-070;367 and 503S367C andSEQ ID NO: 74pSB01683A503C65gB-071;370 and 501K370C andSEQ ID NO: 75pSB01684Q501C66gB-072;522 and 683K522C and I683CSEQ ID NO: 76pSB0168567gB-073;523 and 683I523C and I683CSEQ ID NO: 77pSB0168668gB-074;523 and 684I523C andSEQ ID NO: 78pSB01687M684C69gB-075;524 and 684N524C andSEQ ID NO: 79pSB01688M684C70gB-076525 and 681P525C andSEQ ID NO: 80E681C71gB-077540 and 680R540C andSEQ ID NO: 81L680C72gB-078;541 and 680F541C and L680CSEQ ID NO: 82pSB0169173gB-079;548 and 655L548C andSEQ ID NO: 83pSB01692P655C74gB-080;549 and 658A549C andSEQ ID NO: 84pSB01693N658C75gB-081;550 and 655S550C andSEQ ID NO: 85pSB01694P655C76gB-082;550 and 657S550C andSEQ ID NO: 86pSB01695E657C77gB-083;591 and 668Q591C andSEQ ID NO: 87pSB01696S668C78gB-084;603 and 667L603C and Y667CSEQ ID NO: 88pSB0169779gB-085;604 and 672G604C andSEQ ID NO: 89pSB01698L672C80gB-086;607 and 688R607C andSEQ ID NO: 90pSB01699N688C81gB-087;608 and 692T608C andSEQ ID NO: 91pSB01700Q692C82gB-088;609 and 691E609C andSEQ ID NO: 92pSB01701K691C83gB-089;610 and 674E610C andSEQ ID NO: 93pSB01702S674C84gB-090;610 and 675E610C andSEQ ID NO: 94pSB01703S675C85gB-091;612 and 663Q612C andSEQ ID NO: 95pSB01704V663C86gB-092;737 and 755V737C andSEQ ID NO: 96pSB01705F755C87gB-093;741 and 754V741C andSEQ ID NO: 97pSB01706A754C88gB-094;741 and 755V741C andSEQ ID NO: 98pSB01707F755C89356 and 500I356C and A500C90371 and 505M371C andA505C91371 and 506M371C andW506C92374 and 503T374C andA503C93160 and 708Y160C andY708C94221 and 657T221C andE657C95541 and 681F541C andE681C96605 and 670N605C andK670C97219 and 587S219C and S587C98219 and 588S219C and S588C99219 and 589S219C andY589C100217 and 586D217C andN586C101217 and 588D217C andS588C In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) cysteine substitutions at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101 of column (ii) of Table 2, wherein the resulting polypeptide does not exhibit an HCMV postfusion conformation. In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) cysteine substitutions at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 47, 69 or 91 of column (ii) of Table 2, wherein the resulting polypeptide does not exhibit an HCMV postfusion conformation. In some embodiments, the polypeptide includes two cysteine substitutions as listed at any one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101 of column (ii) of Table 2. In an embodiment, the resulting polypeptide does not exhibit an HCMV postfusion conformation. In an embodiment, the resulting polypeptide exhibits an HCMV prefusion conformation. In a preferred embodiment, the polypeptide includes cysteine substitutions at positions 98 and 653 (listed in row 2, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at positions 100 and 269 (listed in row 5, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at positions 217 and 584 (listed in row 7, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at positions 242 and 710 (listed in row 13, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at positions 242 and 714 (listed in row 14, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at positions 367 and 499 (listed in row 17, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at positions 372 and 506 (listed in row 18, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at positions 550 and 652 (listed in row 22, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at positions 608 and 679 (listed in row 26, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at positions 695 and 724 (listed in row 34, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) disulfide bonds between pairs of cysteine residues substituted at any one of the pairs of positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 and 101 of column (ii) of Table 2. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 98 and 653 (listed in row 2, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 100 and 269 (listed in row 5, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 217 and 584 (listed in row 7, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 242 and 710 (listed in row 13, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 242 and 714 (listed in row 14, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 367 and 499 (listed in row 17, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 372 and 506 (listed in row 18, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 550 and 652 (listed in row 22, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 608 and 679 (listed in row 26, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 695 and 724 (listed in row 34, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 217 and 589 (listed in row 47, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 524 and 684 (listed in row 69, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 371 and 506 (listed in row 91, column (ii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In further embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) disulfide bonds between pairs of cysteine residues that are introduced by cysteine amino acid substitutions at any one of the pairs of positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of column (iii) of Table 2, wherein the polypeptide does not exhibit an HCMV postfusion conformation. In further embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) disulfide bonds between pairs of cysteine residues that are introduced by cysteine amino acid substitutions at any one of the pairs of positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101 of column (iii) of Table 2, wherein the polypeptide does not exhibit an HCMV postfusion conformation. In some embodiments, the polypeptide includes a disulfide bond between pairs of cysteine residues substituted at any one of the pairs of positions listed at any one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101 of column (ii) of Table 2. In an embodiment, the resulting polypeptide does not exhibit an HCMV postfusion conformation. In an embodiment, the resulting polypeptide exhibits an HCMV prefusion conformation. In a preferred embodiment, the polypeptide includes cysteine substitutions at Q98C and I653C (listed in row 2, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at T100C and S269C (listed in row 5, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at D217C and F584C (listed in row 7, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at Y242C and K710C (listed in row 13, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at Y242C and D714C (listed in row 14, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at S367C and L499C (listed in row 17, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at T372C and W506C (listed in row 18, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes cysteine substitutions at S550C and D652C (listed in row 22, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes cysteine substitutions at T608C and D679C (listed in row 26, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at K695C and K724C (listed in row 34, column (iii) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at D217C and Y589C (listed in row 47, column (ili) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at N524C and M684C (listed in row 69, column (iil) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes cysteine substitutions at M371C and W506C (listed in row 91, column (ili) of Table 2) according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 96 and 660 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 98 and 658 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 100 and 258 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 100 and 656 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In another preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 100 and 658 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a further preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 117 and 406 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 117 and 407 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 153 and 712 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 162 and 716 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 217 and 587 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 217 and 589 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 219 and 584 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 219 and 585 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 219 and 586 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 220 and 659 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 223 and 659 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 240 and 732 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 240 and 735 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 242 and 728 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 242 and 731 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 258 and 656 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 269 and 656 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 269 and 658 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 272 and 614 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 273 and 629 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 349 and 650 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 367 and 500 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 367 and 503 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 370 and 501 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 522 and 683 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 523 and 683 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 523 and 684 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 524 and 684 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 525 and 681 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 540 and 680 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 541 and 680 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 548 and 655 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 549 and 658 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 550 and 655 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 550 and 657 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 591 and 668 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 603 and 667 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 604 and 672 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 607 and 688 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 608 and 692 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 609 and 691 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 610 and 674 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 610 and 675 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 612 and 663 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 737 and 755 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 741 and 754 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In a preferred embodiment, the polypeptide includes a disulfide bond between a pair of cysteine residues substituted at positions 741 and 755 according to the numbering of SEQ ID NO: 1, relative to the amino acid sequence of the wild-type HCMV gB. In some embodiments, the polypeptide includes a combination of two or more of the disulfide bonds between cysteine residues listed in Table 2. In some embodiments, the polypeptide includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37. In some embodiments, the polypeptide includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from: SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98. In some embodiments, the polypeptide includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably 99%, or 100% identity to any sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 60. In some embodiments, the polypeptide includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably 99%, or 100% identity to any sequence selected from SEQ ID NO: 51, SEQ ID NO: 73, SEQ ID NO: 70, and SEQ ID NO: 78. In some embodiments, the composition preferably does not include a polypeptide having the sequence set forth in any one of SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 71, SEQ ID NO: 52, SEQ ID NO: 96, and SEQ ID NO: 50. In additional embodiments, the polypeptide includes the amino acid sequence as set forth in any one of the SEQ ID NOs listed in column (iv) of Table 2. That is, an exemplary polypeptide includes a polypeptide having the amino acid sequence selected from any one of: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37. In some embodiments, the polypeptide has the amino acid sequence selected from any one of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98. In a preferred embodiment, the polypeptide includes the amino acid sequence as set forth in any one of SEQ ID NO: 3; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 23; SEQ ID NO: 27; and SEQ ID NO: 35. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 98% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.5% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.6% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.7% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.8% identity to amino acids 23 to 907 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23 to 907 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 98% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.5% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.6% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.7% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.8% identity to amino acids 23 to 707 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23 to 707 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 94% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 96% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 98% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.5% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.6% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.7% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.8% identity to amino acids 23 to 646 of SEQ ID NO: 1. In an embodiment, the polypeptide comprises an amino acid sequence having at least 99.85% identity to amino acids 23 to 646 of SEQ ID NO: 1. In some embodiments, amino acids can be inserted (or deleted) from the native HCMV gB sequence to adjust the alignment of residues in the polypeptide structure, such that particular residue pairs are within a sufficiently close distance to form a disulfide bond in the prefusion, but not postfusion, conformation. In several such embodiments, the polypeptide includes a disulfide bond between cysteine residues located at any of the pairs of positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101 of column (ii) of Table 2, in addition to including at least one amino acid insertion. In some embodiments, the polypeptide includes a phenylalanine substitution as compared to a native HCMV gB. In some embodiments, the polypeptide includes a leucine substitution as compared to a native HCMV gB. In some embodiments, the polypeptide may be stabilized by amino acid mutations (such as, for example, phenylalanine (F) and leucine (L) substitutions) that decrease ionic repulsion between resides that are proximate to each other in the folded structure of the polypeptide, as compared to a HCMV gB polypeptide in postfusion conformation. In some embodiments, the polypeptide may be stabilized by amino acid mutations that increase ionic attraction between residues that are proximate to each other in the folded structure of the polypeptide, as compared to a HCMV gB in postfusion conformation. Exemplary mutations include any mutation selected from Table 3, according to the numbering of SEQ ID NO: 1 as compared to a native HCMV gB: TABLE 3Exemplary Phenylalanine (F) and Leucine (L) Substitutions(ii) Mutated(iv) Exemplaryresidue position,sequence thataccording to the(iii) Substitutionsincludes the(i) Mutantnumbering ofcorresponding tomutation is setRowIDSEQ ID NO: 1SEQ ID NO: 1forth in:1gB-037670K670LSEQ ID NO: 382gB-038670K670FSEQ ID NO: 393gB-039673R673LSEQ ID NO: 404gB-040673R673FSEQ ID NO: 415gB-041691K691LSEQ ID NO: 426gB-042691K691FSEQ ID NO: 437pSB02041686E686F8686E686L9354R354F10573R573F11101 and 260D101L and K260L TABLE 4Additional substitutions(ii) Mutatedresidueposition,according to(iv) Exemplarythesequencenumbering(iii) Substitutionsthat includes(i) Mutantof SEQ IDcorrespondingthe mutationRowIDNO: 1to SEQ ID NO: 1is set forth in:1gB-095;679D679SSEQ ID NO: 99pSB017082gB-096;679D679NSEQ ID NO: 100pSB017093gB-097682E682SSEQ ID NO: 1014gB-098682E682QSEQ ID NO: 1025gB-099;686E686SSEQ ID NO: 103pSB017126gB-100;686E686QSEQ ID NO: 104pSB017137gB-101118N118PSEQ ID NO: 1058gB-102;646D646PSEQ ID NO: 106pSB017159240W240A10246C246S11675I675S12155-157Y155G I156HH157R13pSB02043686E686I14pSB02044686E686V15679D679A16696 and 697Y696C and V697C17703 and 704D703C and P704C18767 and 768I767C and I768C19217, 589,D217C, Y589C,703, and 704D703C, and P704C20217, 589,D217C, Y589C,696, and 697Y696C, and V697C21pSB2795217, 371,D217C, M371C,506,W506C, N524C524 and 589and Y589C22pSB2796217, 371,D217C, M371C,506, and 589W506C, and Y589C23217, 524,D217C, N524C,589, and 684Y589C, M684C24217, 524,D217C, N524C,589, 684,Y589C, M684C,703, and 704D703C, and P704C25655P655S26pSB2797;217, 371,D217C, M371C,pSB2968506, 524 589W506C, N524Cand 684Y589C and M684C27pSB2797;648 -653M648G I649SpSB2968A650G L651KD652D I653G28693R693V29685R685A30678F678S31680L680T In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) residues substituted at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of column (ii) of Table 3, wherein the polypeptide does not exhibit an HCMV gB postfusion conformation. In an embodiment, the resulting polypeptide exhibits an HCMV gB prefusion conformation. In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) residues substituted at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 of column (ii) of Table 4, wherein the polypeptide does not exhibit an HCMV gB postfusion conformation. In an embodiment, the resulting polypeptide exhibits an HCMV gB prefusion conformation. In some embodiments, the polypeptide includes a mutation at position 670 (listed in rows 1 and 2, column (ii) of Table 3) according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 673 (listed in rows 3 and 4, column (ii) of Table 3) according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 691 (listed in rows 5 and 6, column (ii) of Table 3) according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 670 according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 682 according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 686 according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 118 according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a mutation at position 646 according to the numbering of SEQ ID NO: 1. In further embodiments, the polypeptide includes an electrostatic mutation that is introduced by substitutions at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of column (iii) of Table 3, wherein the polypeptide does not exhibit an HCMV postfusion conformation. In a preferred embodiment, the polypeptide includes a substitution K670L (listed in row 1, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution K670F (listed in row 2, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution R673L (listed in row 3, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a preferred embodiment, the polypeptide includes a substitution R673F (listed in row 4, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution K691L (listed in row 5, column (iii) of) Table 3 according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 6, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 7, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 8, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 9, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 10, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In a further preferred embodiment, the polypeptide includes a substitution K691F (listed in row 11, column (iii) of Table 3) according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a combination of two or more of the phenylalanine (F) and leucine (L) substitutions listed in Table 3. In a preferred embodiment, the polypeptide includes a substitution D679S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution D679N according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution E682S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution E682Q according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution E686S according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution E686Q according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution N118P according to the numbering of SEQ ID NO: 1. In another preferred embodiment, the polypeptide includes a substitution D646P according to the numbering of SEQ ID NO: 1. In some embodiments, the polypeptide includes a combination of two or more of the phenylalanine (F) and leucine (L) substitutions listed in Table 3. In some embodiments, the polypeptide includes an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from: SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; and SEQ ID NO: 43. In some embodiments, the polypeptide includes an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any sequence selected from: SEQ ID NO: 99; SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106. In additional embodiments, the polypeptide includes the amino acid sequence as set forth in any one of the SEQ ID NOs listed in column (iv) of Table 3. That is, an exemplary polypeptide includes a polypeptide having the amino acid sequence selected from any one of: SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; and SEQ ID NO: 43. In some embodiments, the polypeptide has the amino acid sequence selected from any one of: SEQ ID NO: 99; SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106. In some embodiments, the polypeptide includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) residues substituted at any one of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 of column (iii) of Table 4, wherein the polypeptide does not exhibit an HCMV gB postfusion conformation. In an embodiment, the resulting polypeptide exhibits an HCMV gB prefusion conformation. In some embodiments, amino acids can be inserted (or deleted) from the native HCMV gB sequence to adjust the alignment of residues in the polypeptide structure, such that particular residue pairs are within a sufficiently close distance to form a desired electrostatic interaction in the prefusion, but not postfusion, conformation. In several such embodiments, the polypeptide includes a desired electrostatic interaction at any of the positions listed in one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of column (ii) of Table 3, wherein the polypeptide does not exhibit an HCMV postfusion conformation. This invention provides a mutant of a wild-type cytomegalovirus (CMV) glycoprotein B (gB) protein, which mutant comprises at least two amino acid mutations relative to the amino acid sequence of the wild-type CMV gB protein, and wherein the amino acid mutation is selected from the group consisting of: (1) an engineered disulfide bond mutation; (2) an additional mutation; and (3) a combination of at least one engineered disulfide mutation and at least one additional mutation. In one aspect, the amino acid mutations comprise a combination of at least two engineered disulfide mutations and at least one additional mutation. In another aspect, the mutant of a wild-type CMV gB protein is in the form of a trimer. In another aspect, the mutant of a wild-type CMV gB protein has increased stability in prefusion form as compared with the corresponding wild-type CMV gB protein, wherein the stability is measured by binding of a prefusion-specific antibody, thermal shift assay or EM imaging. In another aspect, the mutant of the wild-type CMV gB is Towne strain. In another aspect of the invention, the engineered disulfide mutation is selected from the group consisting of: D217C and Y589C; M371C and W506C; and N524C and M684C. In a further aspect of the invention, the additional mutation is selected from the group consisting of: (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; and (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648-653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) substitution of Y at position 696 and V at position 697 with C. In another aspect of the invention, the additional mutation is selected from the group consisting of: (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; and (4) substitution of I at position 675 with S. In another aspect of the invention, the amino acid mutation is a combination of at least two engineered disulfide mutations and at least one additional mutation, and wherein: (i) the engineered disulfide mutations are selected from the group consisting of: D217C and Y589C; M371C and W506C; and N524C and M684C; and (ii) the additional mutation is selected from the group consisting of:(1) substitution of YIH at positions 155-157 with GHR;(2) substitution of W at position 240 with A;(3) substitution of C at position 246 with S;(4) substitution of P at position 655 with S;(5) substitution of F at position 678 with S; and(6) substitution of L at position 680 with T;(7) substitution of R at position 685 with A;(8) substitution of MIALDI at positions 648-653 with GSGKDG;(9) substitution of R at position 693 with V;(10) substitution of I at position 675 with S;(11) substitution of I at positions 767 and 768 with C;(12) substitution of D at position 703 and P at position 704 with C; and(13) substitution of Y at position 696 and V at position 697 with C. In another aspect of the invention, the amino acid mutations are a combination of mutations selected from the group consisting of: (1) combination of D217C and Y589C, M371C and W506C, and I675S; (2) combination of D217C and Y589C, N524C and M684C, and I675S; (3) combination of D217C and Y589C, M371C and W506C, Y155G I156H H157R, W240A, C246S and I675S; and (4) combination of D217C and Y589C, N524C and M684C, Y155G I156H H157R, W240A, C246S and I675S. In another aspect of the invention, the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 371 (371C) and at position 506 (506C), and a serine (S) at position 675 (675S), and wherein the mutant is selected from the group consisting of: (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO:257; and (2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:257. In another aspect of the invention, the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 524 (524C) and at position 684 (684C), and a serine (S) at position 675 (675S), and wherein the mutant is selected from the group consisting of: (1) a mutant comprising the amino acid sequence of SEQ ID NO: 259; and (2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 259. In another aspect of the invention, the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 371 (371C) and at position 506 (506C), a serine (S) at position 675 (675S), a glycine (G) at position 155 (155G), a histidine at position 156 (156H), an arginine at position 157 (157R), an alanine at position 240 (240A), and a serine at position 246 (246S), and wherein the mutant is selected from the group consisting of: (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO:261; and (2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 261; (1) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 261; (3) a mutant comprising the amino acid sequence set forth in SEQ ID NO: 265; and (4) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:265. In another aspect of the invention, the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 524 (524C) and at position 684 (684C), a serine (S) at position 675 (675S), a glycine (G) at position 155 (155G), a histidine at position 156 (156H), an arginine at position 157 (157R), an alanine at position 240 (240A), and a serine at position 246 (246S), and wherein the mutant is selected from the group consisting of: (1) a mutant comprising the amino acid sequence of SEQ ID NO: 263; and (2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:263. In another aspect of the invention, the amino acid sequence of the wildtype CMV gB polypeptide is set forth in SEQ ID NO: 1. In another aspect of the invention, the amino acid sequence of the mutant does not comprise a signal sequence. In another aspect of the invention, the mutant comprises residues 23-907 of SEQ ID NO: 1. In another aspect of the invention, the amino acid sequence of the mutant does not comprise an MPR, TM or CT domain. In another aspect of the invention, the mutant comprises residues 23-707 of SEQ ID NO: 1. In another aspect of the invention, the amino acid sequence of the mutant comprises a truncated Domain V region. In another aspect of the invention, the mutant comprises residues 23-702 or 23-703 of SEQ ID NO: 1. In another aspect of the invention, the amino acid sequence of the mutant does not comprise a Domain V region. In another aspect of the invention, the mutant comprises residues 23-646 of SEQ ID NO: 1. In another aspect of the invention, the mutant further comprises a trimerization motif linked to the C terminus of the mutant. In another aspect of the invention, the trimerization motif is selected from the group consisting of: (i) an inter-protomer disulfide ring; (ii) GCN4; (iii) T4 fibritin foldon; and (iv) C-terminus fusion sequence. In another aspect of the invention, (i) the GCN4 comprises an amino acid sequence as set forth in SEQ ID NOs: 269, 272 or 273; (ii) the T4 fibritin foldon comprises an amino acid sequence as set forth in SEQ ID NO: 271; or (iii) the C-terminus fusion sequence comprises an amino acid sequence as set forth in SEQ ID NO: 274. In another aspect of the invention, the inter-protomer disulfide ring comprises at least two engineered cysteine mutations selected from: (i) 696C and 697C; (ii) 703C and 704C; or (iii) 767C and 768C. Several exogenous multimerization domains that promote formation of stable trimers of soluble proteins are known in the art. Examples of such multimerization domains that can be linked to a mutant provided by the present disclosure include, but are not limited to: (1) the GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407); (2) the trimerization motif from the lung surfactant protein (Hoppe et al. 1994 FEB S Lett 344: 191-195); (3) collagen (McAlinden et al. 2003 Biol Chem 278:42200-42207); and (4) the phage T4 fibritin foldon (Miroshnikov et al. 1998 Protein Eng 11:329-414). In some embodiments, a multimerization domain is linked to a CMV gB mutant at the C-terminus. In specific embodiments, the trimerization domain is set forth in SEQ ID NOs: 269-274. Methods for connecting the multimerization domain to the gB polypeptide are well known in the art. As used herein, “inter-protomer disulfide ring” shall mean a covalent ring formation between three helices achieved by three interhelical disulfide bonds formed by a ring system comprising pairs of adjacent cysteine residues, which establishes a functional topology and stabilization of the multimer (e.g. trimer). See, Stewart-Jones G B E, et al. (2015) A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus. PLoS ONE 10(6): e0128779. doi:10.1371/journal.pone.0128779. In another aspect of the invention, the mutant is secreted. In another aspect of the invention, the mutant is soluble. In another aspect of the invention, the wildtype CMV gB polypeptide sequence is selected from SEQ ID NOs: 107-140 or 224. In another aspect of the invention, the wildtype CMV gB polypeptide sequence is encoded by the polynucleotide sequences set forth in SEQ ID NOs: 225-254. This invention also provides a nucleic acid molecule comprising nucleotides that encode an amino acid sequence of a CMV gB protein mutant according to the embodiment and aspects described herein. In one aspect, the nucleic acid comprises nucleotides having a sequence set forth in SEQ ID NOs: 225-254. This invention also provides a pharmaceutical composition comprising (i) a CMV gB protein mutant according to the embodiments and aspects described herein and (ii) a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition is a vaccine. This invention also provides a method of reducing CMV infection in a subject comprising administering to the subject an effective amount of the vaccine set forth in the embodiments herein. This invention also provides a method of eliciting an immune response to CMV infection in a subject comprising administering to the subject an effective amount of the vaccine set forth in the embodiments herein. This invention also provides a method of preventing CMV infection in a subject comprising administering to the subject an effective amount of the vaccine set forth in the embodiments herein. In one aspect, the subject is a human. 2. Further Embodiments of the Polypeptide In some embodiments, the polypeptide does not include a mutation at any one of the following amino acid positions: 280, 281, 283, 284, 285, 286, 290, 292, 295, 297, 298, 299, or any combinations thereof, according to the numbering of reference sequence SEQ ID NO: 46. In some exemplary embodiments, the polypeptide does not include a substitution of any one of the following residues, according to the numbering of reference sequence SEQ ID NO: 46: Y280; N281; T283; N284; R285; N286; F290; E292; N293; F297; F298; 1299; F298; and any combinations thereof. Without being bound by theory or mechanism, residues important for neutralizing antibodies may include Y280/N284 and Y280/N293/D295. Accordingly, in a preferred embodiment, the polypeptide does not include mutations at Y280, N293, N284, and D295, as compared to reference sequence SEQ ID NO: 46. In some embodiments, the polypeptide does not include a mutation at any one of the following amino acid positions: R562, P577, S587, Y588, G592, G595, L601/H605, C610, L612, P613, Y625, Y627, F632, and K633, and any combinations thereof, according to the numbering of reference sequence SEQ ID NO: 44. In some embodiments, the polypeptide does not include any one of the following amino acid mutations: R562C, P577L, S587L, Y588C, G592S, G595D, L601P/H605N, C610Y, L612F, P613Y, Y625C, Y627C, F632L, and K633T, or any combinations thereof, according to the numbering of reference sequence SEQ ID NO: 44. Without being bound by theory or mechanism, P577 and Y627 are believed to be located next to each other within the domain IV core while C610 participates in a conserved disulfide bond. Thus, all three residues may help maintain the position of domain IV in the prefusion structure and, therefore, the stability of entire antigenic site AD-1. Moreover, without being bound by theory or mechanism, F632 and G595 are believed to be exposed on the surface of the prefusion form of gB. Accordingly, in a preferred embodiment, the polypeptide does not include a mutation at P577, Y627, C610, F632, and G595, or any combinations thereof, according to the numbering of reference sequence SEQ ID NO: 44. 3. Cavity Filling Mutations In still other embodiments, the polypeptide includes amino acid mutations that are one or more cavity filling mutations. Examples of amino acids that may be replaced with the goal of cavity filling include small aliphatic (e.g. Gly, Ala, and Val) or small polar amino acids (e.g. Ser and Thr) and amino acids that are buried in the pre-fusion conformation, but exposed to solvent in the post-fusion conformation. Examples of the replacement amino acids include large aliphatic amino acids (Ile, Leu and Met) or large aromatic amino acids (His, Phe, Tyr and Trp). 4. Combination of Mutations In another aspect, the present invention relates to a polypeptide that includes a combination of two or more different types of mutations selected from at least two engineered disulfide bond mutations, and at least one additional mutation such as a cavity filling mutation, an electrostatic mutation, an inter-protomer disulfide ring each as described herein. In some embodiments, the polypeptide includes at least two disulfide bond mutations and at least one electrostatic mutation. More specifically, in some embodiments, the polypeptide includes at least two cysteine substitutions and at least one phenylalanine substitution. In some embodiments, the polypeptide includes at least two cysteine substitutions and at least one leucine substitution. In some further embodiments, the polypeptide includes at least two mutations selected from any one of the mutations in Table 2 and at least one mutation selected from any one of the mutations in Table 3. In some further embodiments, the polypeptide includes at least two mutations selected from any one of the mutations in Table 2 and at least one mutation selected from any one of the mutations in Table 4. In some further embodiments, the polypeptide includes at least two mutations selected from any one of the mutations in Table 2, at least one mutation selected from any one of the mutations in Table 3 and at least one mutation selected from any one of the mutations in Table 4. Preparation of the Polypeptide The polypeptides described herein may be prepared by routine methods known in the art, such as by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and HIGH FIVE cells (a clonal isolate derived from the parentalTrichoplusia niBTI-TN-5B1-4 cell line). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g. ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from. Avian cell expression systems are also known to those of skill in the art. Similarly, bacterial and mammalian cell expression systems are also known in the art. A number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and/or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species). For example, for expression in insect cells a suitable baculovirus expression vector, such as PFASTBAC, is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used. The polypeptide can be purified using any suitable methods. For example, methods for purifying a polypeptide by immunoaffinity chromatography are known in the art. Suitable methods for purifying desired polypeptides including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the polypeptide may include a “tag” that facilitates purification, such as an epitope tag or a histidine tag. Such tagged polypeptides can be purified, for example from conditioned media, by chelating chromatography or affinity chromatography. Nucleic Acids Encoding Polypeptides In another aspect, the invention relates to nucleic acid molecules that encode a polypeptide described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules that encode only the ectodomain of the polypeptide are also encompassed by the invention. The nucleic acid molecule can be incorporated into a vector, such as an expression vector. In some embodiments, the nucleic acid includes a self-replicating RNA molecule. In some embodiments, the nucleic acid includes a modified RNA molecule. In another aspect, the invention relates to a composition including a nucleic acid according to any one of the embodiments described herein. Compound-Stabilized Polypeptide The inventors discovered a polypeptide stabilized in a prefusion conformation that can be identified by, for example, the binding of a bis(aryl)thiourea compound to an HCMV gB. Bis(aryl)thiourea compounds, as exemplified by structures 1a,b (Formula I), are highly potent and specific inhibitors of CMV. In one aspect, the invention relates to a polypeptide that is capable of binding to a bis(aryl)thiourea compound. In preferred embodiments, the compound does not bind to a postfusion conformation of the HCMV gB polypeptide. In a preferred embodiment, the compound is a bis(aryl)thiourea thioziole analog thereof. Most preferably, in some embodiments, the compound is N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide, having the following structure: In another embodiment, the compound has the following structure: In several embodiments, the polypeptide includes an HCMV gB prefusion epitope, which is not present in a native HCMV gB a postfusion conformation. In some embodiments, at least about 90% of the polypeptides (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% of the polypeptides in the homogeneous population are bound by a bis(aryl)thiourea compound (e.g., such as a thiazole analog of bis(aryl)thiourea compounds, more preferably N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl) amino]phenyl}-1,3-thiazole-4-carboxamide). In some embodiments, the polypeptide that can bind to the bis(aryl)thiourea compound does not have a postfusion conformation. Rather, the polypeptide has a prefusion conformation, such as an HCMV gB prefusion conformation. In another embodiment, the polypeptide can be at least 80% isolated, at least 90%, 95%, 98%, 99%, or preferably 99.9% isolated from HCMV gB polypeptides that are not specifically bound by a bis(aryl)thiourea compound. Compositions Including a Polypeptide and Methods of Use Thereof The invention relates to compositions and methods of using the polypeptide described herein, or a nucleic acid encoding such polypeptide described herein. For example, the polypeptide of the invention can be delivered directly as a component of an immunogenic composition. Alternatively, nucleic acids that encode the polypeptide of the invention can be administered to produce the polypeptide or immunogenic fragment in vivo. Certain preferred embodiments, such as protein formulations, recombinant nucleic acids (e.g., DNA, RNA, self-replicating RNA, or any variation thereof) and viral vectors (e.g., live, single-round, non-replicative assembled virions, or otherwise virus-like particles, or alphavirus VRP) that contain sequences encoding polypeptides are further described herein and may be included in the composition. In one aspect, the invention provides an immunogenic composition comprising the polypeptide described herein. The immunogenic composition can include additional CMV proteins, such as gO, gH, gL, pUL128, pUL130, pUL131, pp65, an immunogenic fragment thereof, or a combination thereof. For example, the polypeptide can be combined with CMV pentameric complex comprising: gH or a pentamer-forming fragment thereof, gL or a pentamer-forming fragment thereof, pUL128 or a pentamer-forming fragment thereof, pUL130 or a pentamer-forming fragment thereof, and pUL131 or a pentamer-forming fragment thereof. The polypeptide of the invention can also be combined with CMV trimeric complex comprising: gH or a trimer-forming fragment thereof, gL or a trimer-forming fragment thereof, and gO or a trimer-forming fragment thereof. In another aspect, the invention relates to a composition including a polynucleotide that may elicit an immune response in a mammal. The polynucleotide encodes at least one polypeptide of interest, e.g., an antigen. Antigens disclosed herein may be wild type (i.e., derived from the infectious agent) or preferably modified (e.g., engineered, designed or artificial). The nucleic acid molecules described herein, specifically polynucleotides, in some embodiments, encode one or more peptides or polypeptides of interest. Such peptides or polypeptides may serve as an antigen or antigenic molecule. The term “nucleic acid” includes any compound that includes a polymer of nucleotides. These polymers are referred to as “polynucleotides.” Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), including mRNA, and deoxyribonucleic acids (DNAs). In some embodiments, the composition includes DNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition includes RNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the composition includes an mRNA polynucleotide encoding a polypeptide or fragment thereof described herein. Such compositions may produce the appropriate protein conformation upon translation. In one aspect, the invention relates to a composition that includes at least one polynucleotide encoding a polypeptide including at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB. In one aspect, the invention relates to a composition that includes at least one DNA polynucleotide encoding a polypeptide including at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB. In one aspect, the invention relates to a composition that includes at least one RNA polynucleotide encoding a polypeptide including at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB. In some embodiments, the invention relates to a composition that includes at least one polynucleotide encoding at least one hCMV gB polypeptide or an immunogenic fragment or epitope thereof. In some embodiments, the composition includes at least one polynucleotide encoding two or more antigenic polypeptides or an immunogenic fragment or epitope thereof. In some embodiments, the composition includes two or more polynucleotides encoding two or more antigenic polypeptides or immunogenic fragments or epitopes thereof. The one or more antigenic polypeptides may be encoded on a single polynucleotide or may be encoded individually on multiple (e.g., two or more) polynucleotides. In another aspect, the invention relates to a composition that includes (a) a polynucleotide encoding a polypeptide including at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB); and (b) a polynucleotide encoding an additional polypeptide. In another aspect, the invention relates to a composition that includes (a) a polynucleotide encoding a polypeptide including at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB); and (b) a polynucleotide encoding an additional polypeptide, preferably an HCMV antigenic polypeptide. The additional polypeptide may be selected from HCMV gH, gL, gB, gO, gN, and gM and an immunogenic fragment or epitope thereof. In some embodiments, the additional polypeptide is HCMV pp65. In some embodiments, the additional polypeptide may be selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131A, and fragments thereof. In some embodiments, the additional polypeptide is HCMV gH polypeptide. In some embodiments, the additional polypeptide is an HCMV gL polypeptide. In some embodiments, the additional polypeptide is an HCMV gB polypeptide. In some embodiments, the additional polypeptide is an HCMV gO polypeptide. In some embodiments, the additional polypeptide is an HCMV gN polypeptide. In some embodiments, the additional polypeptide is an HCMV gM polypeptide. In some embodiments, the additional polypeptide is a variant gH polypeptide, a variant gL polypeptide, or a variant gB polypeptide. In some embodiments, the variant HCMV gH, gL, or gB polypeptide is a truncated polypeptide lacking one or more of the following domain sequences: (1) the hydrophobic membrane proximal domain, (2) the transmembrane domain, and (3) the cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide lacks the hydrophobic membrane proximal domain, the transmembrane domain, and the cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide includes only the ectodomain sequence. In some embodiments, an antigenic polypeptide is an HCMV protein selected from UL83, UL123, UL128, UL130 and UL131A or an immunogenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is an HCMV UL83 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL123 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL128 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL130 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL131 polypeptide. In another aspect, the invention relates to a composition that includes (a) a polynucleotide encoding a polypeptide including at least two introduced amino acid mutations relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB); and (b) a polynucleotide encoding an additional polypeptide having any one of the amino acid sequences set forth in SEQ ID NOs: 211-223. In another aspect, the invention relates to a composition that includes (a) a polynucleotide encoding a polypeptide including at least two introduced amino acid mutations relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB); and (b) a polynucleotide having any one of the sequences selected from SEQ ID NOs: 141-210. In another aspect, the invention relates to a composition that includes (a) a polynucleotide encoding a polypeptide including at least two introduced amino acid mutations relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB); and (b) an additional polypeptide having any one of the amino acid sequences selected from SEQ ID NOs: 211-223. In some embodiments, the polynucleotide encoding the additional polypeptide includes at least one nucleic acid sequence selected from any of SEQ ID NOs: 225-254. In some embodiments, the polynucleotide encoding the additional polypeptide includes at least one nucleic acid sequence selected from any of SEQ ID NOs: 141-147. In some embodiments, the polynucleotide encoding the additional polypeptide has at least one sequence selected from any of SEQ ID NOs: 220-223. In some embodiments, the antigenic polypeptide includes two or more HCMV proteins, fragments, or epitopes thereof. In some embodiments, the antigenic polypeptide includes two or more glycoproteins, fragments, or epitopes thereof. In some embodiments, the antigenic polypeptide includes at least one HCMV polypeptide, fragment or epitope thereof and at least one other HCMV protein, fragment or epitope thereof. In some embodiments, the two or more HCMV polypeptides are encoded by a single RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides are encoded by two or more RNA polynucleotides, for example, each HCMV polypeptide is encoded by a separate RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides can be any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins includes pp65 or immunogenic fragments or epitopes thereof; and any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of HCMV gB and one or more HCMV polypeptides selected from gH, gL, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of HCMV gL and one or more HCMV polypeptides selected from gB, gH, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are gB and gH. In some embodiments, the two or more HCMV polypeptides are gB and gL. In some embodiments, the two or more HCMV polypeptides are gH and gL. In some embodiments, the two or more HCMV polypeptides are gB, gL, and gH. In some embodiments, the two or more HCMV proteins can be any combination of HCMV UL83, UL123, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are UL123 and UL130. In some embodiments, the two or more HCMV polypeptides are UL123 and 131 A. In some embodiments, the two or more HCMV polypeptides are UL130 and 131 A. In some embodiments, the two or more HCMV polypeptides are UL 128, UL130 and 131 A. In some embodiments, the two or more HCMV proteins can be any combination of HCMV gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of HCMV gL and one or more HCMV polypeptides selected from gH, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are gL, gH, UL 128, UL130 and 131 A. In any of these embodiments in which the composition includes two or more HCMV proteins, the HCMV gH may be a variant gH, such as any of the variant HCMV gH glycoproteins disclosed herein, for example, any of the variant HCMV gH disclosed herein. In any of these embodiments in which the composition includes two or more HCMV proteins, the HCMV gB may be a variant gB, such as any of the variant HCMV gB glycoproteins disclosed herein, for example, any of the variant HCMV gB disclosed herein. In any of these embodiments in which the composition includes two or more HCMV gL proteins, the HCMV gL may be a variant gL, such as any of the variant HCMV gL glycoproteins disclosed herein, for example, any of the variant HCMV gL disclosed herein. In certain embodiments in which the composition includes two or more RNA polynucleotides encoding two or more HCMV antigenic polypeptides or an immunogenic fragment or epitope thereof (either encoded by a single RNA polynucleotide or encoded by two or more RNA polynucleotides, for example, each protein encoded by a separate RNA polynucleotide), the two or more HCMV proteins are a variant gB, for example, any of the variant gB polypeptides disclosed herein, and an HCMV protein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131 polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are a variant gH, for example, any of the variant gH polypeptides disclosed herein, and an HCMV protein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are a variant gH, for example, any of the variant gH polypeptides disclosed herein, and an HCMV protein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131 polypeptides or immunogenic fragments or epitopes thereof. In some embodiments in which the variant HCMV proteins are variant HCMV gB, variant HCMV gL, and variant HCMV gH, the variant HCMV polypeptide is a truncated polypeptide selected from the following truncated polypeptides: lacks the hydrophobic membrane proximal domain; lacks the transmembrane domain; lacks the cytoplasmic domain; lacks two or more of the hydrophobic membrane proximal, transmembrane, and cytoplasmic domains; and includes only the ectodomain. In some embodiments, the composition includes multimeric RNA polynucleotides encoding at least one HCMV antigenic polypeptide or an immunogenic fragment or epitope thereof. In some embodiments, the composition includes at least one RNA polynucleotide encoding at least one HCMV antigenic polypeptide or an immunogenic fragment or epitope thereof, wherein the 5′UTR of the RNA polynucleotide includes a patterned UTR. In some embodiments, the patterned UTR has a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, the 5′ UTR of the RNA polynucleotide (e.g., a first nucleic acid) has regions of complementarity with a UTR of another RNA polynucleotide (a second nucleic acid). For example, UTR nucleotide sequences of two polynucleotides sought to be joined (e.g., in a multimeric molecule) can be modified to include a region of complementarity such that the two UTRs hybridize to form a multimeric molecule. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV antigenic polypeptide is modified to allow the formation of a multimeric sequence. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV protein selected from UL128, UL130, UL131 is modified to allow the formation of a multimeric sequence. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV polypeptide is modified to allow the formation of a multimeric sequence. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV polypeptide selected from gH, gL, gB, gO, gM, and gN is modified to allow the formation of a multimeric sequence. In any of these embodiments, the multimer may be a dimer, a trimer, pentamer, hexamer, heptamer, octamer nonamer, or decamer. Thus, in some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131 is modified to allow the formation of a dimer. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A is modified to allow the formation of a trimer. In some embodiments, the 5′ UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131 is modified to allow the formation of a pentamer. In some embodiments, the composition includes at least one RNA polynucleotide having a single open reading frame encoding two or more (for example, two, three, four, five, or more) HCMV antigenic polypeptides or an immunogenic fragment or epitope thereof. In some embodiments, the composition includes at least one RNA polynucleotide having more than one open reading frame, for example, two, three, four, five or more open reading frames encoding two, three, four, five or more HCMV antigenic polypeptides. In either of these embodiments, the at least one RNA polynucleotide may encode two or more HCMV antigenic polypeptides selected from gH, gB, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In some embodiments, the at least one RNA polynucleotide encodes UL83 and UL123. In some embodiments, the at least one RNA polynucleotide encodes gH and gL. In some embodiments, the at least one RNA polynucleotide encodes UL128, UL130, and UL131. In some embodiments, the at least one RNA polynucleotide encodes gH, gL, UL128, UL130, and UL131. In some embodiments, in which the at least one RNA polynucleotide has a single open reading frame encoding two or more (for example, two, three, four, five, or more) HCMV antigenic polypeptides, the RNA polynucleotide further comprises additional sequence, for example, a linker sequence or a sequence that aids in the processing of the HCMV RNA transcripts or polypeptides, for example a cleavage site sequence. In some embodiments, the additional sequence may be a protease sequence, such as a furin sequence. In some embodiments, the additional sequence may be self-cleaving 2A peptide, such as a P2A, E2A, F2A, and T2A sequence. In some embodiments, the linker sequences and cleavage site sequences are interspersed between the sequences encoding HCMV polypeptides. In some embodiments, at least one RNA polynucleotide includes any nucleic acid sequence selected from any one of nucleic acid sequences disclosed herein, or homologs thereof having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity with a nucleic acid sequence disclosed herein. In some embodiments, the open reading frame is encoded is codon-optimized. Some embodiments include a composition that includes at least one RNA polynucleotide encoding at least one HCMV antigenic polypeptide or an immunogenic fragment thereof and at least one 5′ terminal cap. In some embodiments, a 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp. In some embodiments, the at least one polynucleotide includes a nucleic acid sequence selected from any one of SEQ ID NOs: 141-210. In some embodiments, the at least one polynucleotide encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 211-223. In some preferred embodiments, the composition does not include a polypeptide having the amino acid sequence SEQ ID NO: 216. In some preferred embodiments, the composition does not include a polynucleotide encoding the amino acid sequence SEQ ID NO: 216. In some preferred embodiments, the composition does not include a polynucleotide having the sequence SEQ ID NO: 152. In some embodiments, the composition includes at least one polynucleotide, wherein the at least one polynucleotide has at least one chemical modification. In some embodiments, the at least one polynucleotide further includes a second chemical modification. Preferably, the polynucleotide is RAN. In some embodiments, the at least one polynucleotide having at least one chemical modification has a 5′ terminal cap. In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-I-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In some embodiments, the composition includes at least one polynucleotide, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracil in the open reading frame has a chemical modification, optionally wherein the composition is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. In some embodiments, the additional polypeptides or immunogenic fragments encoded by the polynucleotide (e.g., in an mRNA composition) are selected from gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, pp65 and I E1 antigens. In some embodiments, a first composition and a second composition are administered to the mammal. In some embodiments, a first composition includes a polynucleotide encoding a polypeptide including at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB; and a second composition includes a polynucleotide encoding HCMV pp65 or an antigenic fragment or epitope thereof. In some embodiments, a first composition includes a polynucleotide encoding a polypeptide including at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB; and a second composition includes a polynucleotide encoding at least one polynucleotide encoding an additional polypeptide selected from HCMV gH, gL, UL128, UL130, and UL131, or antigenic fragments or epitopes thereof. In another aspect, the invention relates to methods of inducing an immune response in a mammal, including administering to the mammal a composition in an amount effective to induce an immune response, wherein the composition includes a polynucleotide encoding a polypeptide including at least two introduced amino acid mutations relative to the amino acid sequence of the wild-type HCMV gB. In some embodiments, the immune response includes a T cell response or a B cell response. In some embodiments, the immune response includes a T cell response and a B cell response. In some embodiments, the method involves a single administration of the composition. In some embodiments, a method further includes administering to the subject a booster dose of the composition. The composition including a polynucleotide disclosed herein may be formulated in an effective amount to produce an antigen specific immune response in a mammal. The immunogenic composition may include an adjuvant. Exemplary adjuvants to enhance effectiveness of the composition include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific adjuvants such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% TWEEN 80, and 0.5% Span 85 formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (3) saponin adjuvants, such as QS-21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), which may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as adjuvants to enhance the effectiveness of the composition. In a preferred embodiment, the adjuvant is a saponin adjuvant, namely QS-21. In some embodiments, the composition does not include an adjuvant. In some embodiments, the composition further includes a lipid nanoparticle. In some embodiments, the composition is formulated in a nanoparticle. In some embodiments, the composition further includes a cationic or polycationic compounds, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL). Each of the immunogenic compositions discussed herein may be used alone or in combination with one or more other antigens, the latter either from the same viral pathogen or from another pathogenic source or sources. These compositions may be used for prophylactic (to prevent infection) or therapeutic (to treat disease after infection) purposes. In one embodiment, the composition may include a “pharmaceutically acceptable carrier,” which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as adjuvants. Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera,H. pylori, and etc. pathogens. In one embodiment, the composition includes a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. The compositions described herein may include an immunologically effective amount of the polypeptide or polynucleotide, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount,” it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for eliciting an immune response. The immune response elicited may be sufficient, for example, for treatment and/or prevention and/or reduction in incidence of illness, infection or disease. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctors assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The composition may be administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. In some embodiments, the composition is administered to the mammal by intradermal or intramuscular injection. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, nasal formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. Dosage treatment may be a single dose schedule or a multiple dose schedule. The immunogenic composition may be administered in conjunction with other immunoregulatory agents. In another aspect, the invention provides a method of eliciting an immune response against cytomegalovirus, comprising administering to a subject in need thereof an immunologically effective amount of the polypeptide and/or an immunogenic composition described herein, which comprises the proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), or VRPs as described above. In certain embodiments, the immune response comprises the production of neutralizing antibodies against CMV. The immune response can comprise a humoral immune response, a cell-mediated immune response, or both. In some embodiments an immune response is induced against each delivered CMV protein. A cell-mediated immune response can comprise a Helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both. In some embodiments the immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral infection of cells. CMV infects epithelial cells and also fibroblast cells. In some embodiments the immune response reduces or prevents infection of both cell types. Neutralizing antibody responses can be complement-dependent or complement-independent. In some embodiments the neutralizing antibody response is complement-independent. In some embodiments the neutralizing antibody response is cross-neutralizing; i.e., an antibody generated against an administered composition neutralizes a CMV virus of a strain other than the strain used in the composition. The polypeptide and/or immunogenic composition described herein may also elicit an effective immune response to reduce the likelihood of a CMV infection of a non-infected mammal, or to reduce symptoms in an infected mammal, e.g., reduce the number of outbreaks, CMV shedding, and risk of spreading the virus to other mammals. In one aspect, the invention relates to a method for reducing CMV viral shedding in a mammal. In some embodiments, the invention relates to a method for reducing CMV viral shedding in urine in a mammal. In some embodiments, the invention relates to a method for reducing CMV viral shedding in saliva in a mammal. In another aspect, the invention relates to a method for reducing CMV viral titers in a mammal. In one aspect, the invention relates to a method for reducing CMV nucleic acids in serum in a mammal. The term “viral shedding” is used herein according to its plain ordinary meaning in medicine and virology and refers to the production and release of virus from an infected cell. In some embodiments, the virus is released from a cell of a mammal. In some embodiments, virus is released into the environment from an infected mammal. In some embodiments the virus is released from a cell within a mammal. In one aspect, the invention relates to a method for reducing CMV viral shedding in a mammal. The method includes administering the modified CMV gB polypeptide and/or immunogenic composition described herein to the mammal that is infected with or is at risk of a CMV infection. In one embodiment, the reduction in CMV viral shedding in a mammal is as compared to the viral shedding in mammals that were not administered the modified CMV gB. In another embodiment, the reduction in CMV viral shedding in a mammal is as compared to the viral shedding following an administration of a CMV pentamer alone or following an administration of a CMV pentamer in the absence of the polypeptide. In some embodiments, the mammal is a human. In some embodiments, the human is a child, such as an infant. In some other embodiments, the human is female, including an adolescent female, a female of childbearing age, a female who is planning pregnancy, a pregnant female, and females who recently gave birth. In some embodiments, the human is a transplant patient. In one embodiment, the challenge cytomegalovirus strain is a human CMV strain. In one embodiment, the challenge cytomegalovirus strain is homologous to the CMV strain from which the polypeptide is derived. In another embodiment, the challenge cytomegalovirus strain is homologous to the CMV strain VR1814. In another embodiment, the challenge cytomegalovirus strain is homologous to the CMV strain Towne. In one embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the CMV strain from which the modified CMV gB polypeptide is derived. In another embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the VR1814 CMV strain. In another embodiment, the challenge cytomegalovirus strain is the VR1814 CMV strain. In another embodiment, the challenge cytomegalovirus strain is a human CMV strain that is heterologous to the CMV strain Towne. In another embodiment, the challenge cytomegalovirus strain is the CMV strain Towne. In another embodiment, the challenge cytomegalovirus strain is a rhesus CMV strain homologous to the macacine herpesvirus 3 isolate 21252 CMV strain. In another embodiment, the challenge cytomegalovirus strain is the macacine herpesvirus 3 isolate 21252 CMV strain. A useful measure of antibody potency in the art is “50% neutralization titer.” Another useful measure of antibody potency is any one of the following: a “60% neutralization titer”; a “70% neutralization titer”; a “80% neutralization titer”; and a “90% neutralization titer.” To determine, for example, a 50% neutralizing titer, serum from immunized animals is diluted to assess how dilute serum can be yet retain the ability to block entry of 50% of infectious viruses into cells. For example, a titer of 700 means that serum retained the ability to neutralize 50% of infectious virus after being diluted 700-fold. Thus, higher titers indicate more potent neutralizing antibody responses. In some embodiments, this titer is in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The 50%, 60%, 70%, 80%, or 90% neutralization titer range can have an upper limit of about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 1 1000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000. For example, the 50% neutralization titer can be about 3000 to about 6500. “About” means plus or minus 10% of the recited value. Neutralization titer can be measured as described in the specific examples, below. An immune response can be stimulated by administering proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules or nucleoside modified RNA molecules), or VRPs to an individual, typically a mammal, including a human. In some embodiments the immune response induced is a protective immune response, i.e., the response reduces the risk or severity of or clinical consequences of a CMV infection. Stimulating a protective immune response is particularly desirable in some populations particularly at risk from CMV infection and disease. For example, at-risk populations include solid organ transplant (SOT) patients, bone marrow transplant patients, and hematopoietic stem cell transplant (HSCT) patients. VRPs can be administered to a transplant donor pre-transplant, or a transplant recipient pre- and/or post-transplant. Because vertical transmission from mother to child is a common source of infecting infants, administering VRPs to a woman who is pregnant or can become pregnant is particularly useful. Administration of the compositions provided by the present disclosure, such as pharmaceutical compositions, can be carried out using standard routes of administration. Any suitable route of administration can be used. For example, a composition can be administered intramuscularly, intraperitoneally, subcutaneously, or transdermally. Some embodiments will be administered through an intra-mucosal route such as intra-orally, intra-nasally, intra-vaginally, and intra-rectally. Compositions can be administered according to any suitable schedule. Also provided herein is a method of inhibiting cytomegalovirus entry into a cell, comprising contacting the cell with the immunogenic composition described herein. In one aspect, the invention relates to compositions that include a polypeptide described above. In another aspect, the invention relates to compositions that include a nucleic acid molecule or vector encoding such polypeptide. In a further aspect, the invention relates to compositions that include a polypeptide described above and a nucleic acid molecule or vector encoding such polypeptide. In some embodiments, the composition is an immunogenic composition capable of eliciting an immune response against CMV in a subject. In some particular embodiments, the immunogenic composition is a pharmaceutical composition, which includes a polypeptide provided by the present disclosure and a pharmaceutically acceptable carrier. In still other embodiments, the pharmaceutical composition is a vaccine. In some embodiments, a composition, such as an immunogenic composition or a vaccine, includes two or more different polypeptides described above. The two or more different polypeptides may include the same introduced amino acid mutations but may be derived from gB from different HCMV strains or subtypes. In another embodiment, the two or more different polypeptides may include amino acid mutations, as compared to a native HCMV gB, that differ from one another. In preferred embodiments, the polypeptide is soluble in aqueous solution. In some embodiments, the polypeptide is soluble in a solution that lacks detergent. Antibodies and Diagnostic Uses The polypeptides described above may be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, guinea pig, horse, etc.) is immunized with an immunogenic polypeptide bearing a CMV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a CMV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. Monoclonal antibodies directed against CMV epitopes can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against CMV epitopes can be screened for various properties; i.e., for isotype, epitope affinity, etc. Antibodies, both monoclonal and polyclonal, which are directed against CMV epitopes are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Both the polypeptides which react immunologically with serum containing CMV antibodies, and the antibodies raised against these polypeptides, may be useful in immunoassays to detect the presence of CMV antibodies, or the presence of the virus, in biological samples, including for example, blood or serum samples. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. For example, the immunoassay may utilize the polypeptide having the sequence set forth in any one of SEQ ID NOs: 2-106. Alternatively, the immunoassay may use a combination of viral antigens derived from the polypeptides described herein. It may use, for example, a monoclonal antibody directed towards at least one polypeptide described herein, a combination of monoclonal antibodies directed towards the polypeptides described herein, monoclonal antibodies directed towards different viral antigens, polyclonal antibodies directed towards the polypeptides described herein, or polyclonal antibodies directed towards different viral antigens. Protocols may be based, for example, upon competition, or direct reaction, or may be sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays. Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the polypeptides of the invention containing CMV epitopes or antibodies directed against epitopes in suitable containers, along with the remaining reagents and materials required for the conduct of the assay, as well as a suitable set of assay instructions. The polynucleotide probes can also be packaged into diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit. The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test. Some embodiments of the present disclosure provide a HCMV vaccine that includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or an immunogenic fragment thereof and at least one 5′ terminal cap. In some embodiments, a 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp. Some embodiments of the present disclosure provide a HCMV vaccine that includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or an immunogenic fragment thereof, wherein the at least one ribonucleic acid (RNA) polynucleotide has at least one chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide further comprises a second chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide having at least one chemical modification has a 5′ terminal cap. In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyl uridine. Some embodiments of the present disclosure provide a HCMV vaccine that includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or an immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracil in the open reading frame have a chemical modification, optionally wherein the vaccine is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. Some embodiments of the present disclosure provide a HCMV vaccine that is formulated within a cationic lipid nanoparticle, also referred to herein as ionizable cationic lipid nanoparticles, ionizable lipid nanoparticles and lipid nanoparticles, which are used interchangeably. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid nanoparticle has a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. In some embodiments, the nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, 80% of the uracil in the open reading frame have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine, N1-ethylpseudouridine. In some embodiments, the vaccine is formulated within a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject a HCMV RNA vaccine in an amount effective to produce an antigen specific immune response. In some embodiments, an antigen specific immune response comprises a T cell response or a B cell response. In some embodiments, an antigen specific immune response comprises a T cell response and a B cell response. In some embodiments, a method of producing an antigen specific immune response involves a single administration of the vaccine. In some embodiments, a method further includes administering to the subject a booster dose of the vaccine. In some embodiments, a vaccine is administered to the subject by intradermal or intramuscular injection. Also provided herein are HCMV RNA vaccines for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen specific immune response. Further provided herein are uses of HCMV RNA vaccines in the manufacture of a medicament for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen specific immune response. Further provided herein are methods of preventing or treating HCMV infection comprising administering to a subject the vaccine of the present disclosure. The HCMV vaccine disclosed herein may be formulated in an effective amount to produce an antigen specific immune response in a subject. The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a native or reference sequence. In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” is one which contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine. “Orthologs” refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes. “Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide. The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule. As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support. “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. In some embodiments, the mutant CMV gB polypeptide is a truncated polypeptide lacking one or more of the following domain sequences as compared to SEQ ID NO: 1: (1) Domain V (residues 124-344), (2) MPR domain (residues 705-750), (3) TM domain (residues 751-772), or (3) the CT domain (residues 773-907). As used herein the term “truncated” shall mean that a sequence is missing some or all of the residues comprising a domain as set forth herein. As described herein, CMV gB polypeptide comprises the following domains and residues (SEQ ID NO:1): (i) Domain I (residues 134-344), (ii) Domain II (residues 121-133 and 345-436), (iii) Domain III (residues 97-111, 475-539 and 640-648), (iv) Domain IV (residues 88-96, 540-639 and 551-641), (v) Domain V (residues 649-707), (vi) membrane-proximal region (MPR) (residues 705-750), (vii) transmembrane domain (TM) (residues 751-772), and (viii) cytoplasmic domain (CT) (residues 773-907). In another aspect, the ectodomain of CMV gB comprises residues 1-707 or 23-707 (without signal sequence) of SEQ ID NO: 1. In another aspect, the ectodomain is lacking Domain V and comprises residues 1-646 or 23-646 (without signal sequence). EXAMPLES The invention is further described by the following illustrative examples. The examples do not limit the invention in any way. They merely serve to clarify the invention. Example 1: Isolation and Purification of Crosslinked and Native HCMV gB (Towne Strain) with Fusion Inhibitor During the sample preparation the HCMV fusion inhibitor (compound 28 described in Bloom et al.,Bioorganic&Medicinal Chemistry Letters14 (2004) 3401-3406; see alsoFIG.5D) was added to each step during the virus concentration, processing, extraction and purification to inhibit conversion of gB to the postfusion form. Following crosslinking of the proteins on the virion surface with bis(sulfosuccinimidyl) glutarate (BS2G) and extraction of gB from the virion with detergent, the SM5-1 His/Strep-tagged Fab (Potzsch et al.,PLoS pathogens7(8):e1002172, 2011) was added to assist in purification and identification of gB by electron cryomicroscopy. The Fab-gB complexes were purified by an affinity column. These extracted and purified proteins were then analyzed by electron cryomicroscopy for the presence of prefusion gB and used to solve the structure of a prefusion form. Example 2: Electron Microscopy Graphene oxide film-supported electron microscopy grids were prepared. The gB sample solutions were vitrified using a Vitrobot (ThermoFisher). The frozen grids were transferred to a FEI Titan Krios transmission electron microscope that operates at 300 kV. Target positions were set up in the SerialEM program, and high magnification (18000×) images were automatically collected with the program using a K2 direct detector camera (Gatan) using super resolution movie mode. The unbinned pixel size was 0.638 Å and the beam intensity was ˜8e/unbin pixel/s. The total electron dose on the sample for each movie was ˜40e/Å2. A total of 7,771 movies, each with 28 frames, was collected in three sessions. Image Processing Drift correction was done using the MotionCor2 program (Zheng S et al.Nature Methods14, 331-332 (2017)), and the final micrographs were binned 2× and averaged from all frames. Contrast transfer function parameters were calculated with Gctf (Kai Zhang,Journal of Structural Biology193(1), 1-12 (2016)). For particle picking, the published structure of HCMV gB in postfusion conformation (PDB:5CXF) was used to generate a 30 Å density map using pdb2mrc (EMAN) (Ludtke, S. et al.Journal of Structural Biology128(1), 82-97 (1999)). Projection images from this density maps was generated with project3d (EMAN) (FIG.1) and used as a template for the automatic particle picking using Gautomatch program (Urnavicius L, et al.Science347(6229):1441-1446 (2015)). Relion v2.1-beta (Scheres, S. H.Journal of Structural Biology180(3): 519-530 (2012)) was used to extract the resulting ˜1.9 million particles and to carry out all subsequent image processing steps, including 2D classification, 3D classification, auto-refinement and post-processing. The 2D classes were put into three groups based on the image features: the first group consisted of the 2D classes that showed features that resemble the crystallographically determined postfusion gB structure (>50%); the second group contained 2D classes with well resolved protein features that do not resemble the structural features from postfusion gB (<10%); the third group contained 2D classes that did not contain clearly defined protein (˜40%) (FIG.1). The first and second groups were further processed with 3D classification, auto refinement and post processing procedures with Relion. Following this processing, a ˜3.5 Å resolution electron density map showing the postfusion conformation structure was reconstructed from the first group; a ˜3.6 Å resolution electron density map showing a prefusion conformation structure was reconstructed from the second group. Based on these density maps and the known HCMV gB amino acid sequence (Towne strain P13201, SEQ ID NO:1), atomic models were built with the Coot program (Emsley P. et alActa Crystallogr D Biol Crystallogr66(Pt 4): 486-501 (2010)) for the prefusion and postfusion conformation structures. The postfusion gB crystal structure (PDB accession code 5CXF) and a crystal structure of a complex between the SM5-1 fab and gB domain II (PDB accession code 40T1) were used as initial models for both structures. For the postfusion structure model, small adjustment was enough to obtain a good fit to the electron density. For the prefusion conformation model, domains I, II, III and IV from the reference PDB model could be docked as rigid bodies into the electron density map as a starting point. Then, adjustments of individual residues were made for optimal fitting. The model for domains V, MPR and TM were built de novo. The models were iteratively refined with the Phenix.real_space_refine tool (Afonine P V et al.Acta Crystallogr D Struct Biol74(Pt 6): 531-544 (2018)) followed by local manual adjusting for several rounds. Results Sample Screening by cryoEM The prefusion conformation of gB is unstable, with a propensity to rearrange to the postfusion state, including during sample handling. Therefore, the samples studied contained a mixture of gB conformers, complicating structure determination. In addition, there was no pre-existing reliable information on the arrangement of domains or the unique structural features of prefusion gB. We used direct visualization by electron microscopy and image processing to screen different sample preparation conditions. Image sorting by 2D and 3D classification permits multiple structures to be determined from heterogeneous samples. However, it requires a large data set so that enough particles for each structure can be combined to produce a class average with good signal. This was especially the case for the gB samples because prefusion gB was a small population in the mixtures. Therefore, we collected ˜1,000 movies for each condition, and decided whether to pursue image processing with more data from the same sample or switch to another at the 2D classification stage. The structure of antibody Fab-bound postfusion conformation gB was readily obtained from many datasets. The projection images from these Fab-bound postfusion conformation structures were used as a reference to avoid selecting images for the prefusion image reconstruction. We selected any good class average with protein features that did not resemble any of the postfusion gB projection images for further image processing. We screened dozens of conditions for sample preparation with this strategy and eventually found a sample that produced some alternative 2D classes as a minor species in the particle populations (FIG.1B, circled). Then a total of 7,771 movies were collected from that sample and used for determination of a prefusion gB structure. Projection images of the antibody Fab-bound postfusion gB structure are shown inFIG.1A. The 2D class averages from the dataset collected are shown inFIG.1B. Some classes that do not resemble any of the postfusion gB reference 2D projections are circled. Obtaining a Prefusion Conformation Structure Approximately 1.9 million raw particle images were automatically selected from the data set. After 2D classification, the images were grouped into a postfusion class (55% of the particle population) and a prefusion class (10% of the particle population). The two groups were further processed in 3D with C3 symmetry applied to yield a density map of SM5-1 Fab-bound postfusion gB at 3.5 Å resolution and a density map of SM5-1 Fab-bound prefusion gB at 3.6 Å resolution. The X-ray crystallography-based models of the SM5-1 Fab and of the ectodomain of postfusion gB were fit to the postfusion density map with rigid body docking. Except for the constant domain of the Fab (which is likely too flexible to produce strong electron density), the density map of the postfusion gB-Fab complex and the model agreed well with each other (FIG.3A). The membrane proximal region, transmembrane region and cytoplasmic domain were not resolved in our final postfusion gB density map, suggesting that these regions of postfusion gB are flexible either intrinsically or through detergent solubilization in the sample preparations (FIG.2, lower line). The interaction of the Fab and DII of postfusion gB in the electron cryomicroscopy-based model agrees well with the previously determined crystal structure of the complex (PDB accession code 40T1). To build a prefusion gB model, guided by the known Fab binding position, domains I, II, III and part of domain IV from the postfusion gB crystal structure were docked into the density map of the prefusion gB-Fab complex individually and individual residues were manually adjusted as necessary for optimal fit of the electron density. The rest of the prefusion gB structure was built de novo. The amino acids of gB that were modeled in the prefusion structure are indicated inFIG.2, the top line. The model of the prefusion gB-Fab complex fits most parts of the prefusion density map, and the presence of Fab density confirms the identity of gB in the novel structure (FIG.3B). The coordinates and structure factors for the model of the prefusion gB associated with the present Example are provided in Table 1A. The Structure of gB in a Prefusion Conformation and Comparison to Postfusion gB The electron density for the complex of prefusion gB and the SM5-1 Fab allowed the building of a prefusion gB model that includes the gB ectodomain, membrane proximal region (MPR—a helical region that is oriented parallel to the viral membrane), and single span transmembrane helix (TM) (FIG.3BandFIG.4B). The MPR and TM regions were not resolved in the structural data for postfusion gB or included in postfusion gB models. The overall dimensions of prefusion and postfusion gB are different (FIG.4Avs.FIG.4B). The postfusion gB trimer ectodomain has a rod shape, with an approximate height of 165 Å (the distance between planes formed by proline 570 of each protomer at the membrane distal end and tryptophan 240 of the each protomer at the membrane proximal end;FIG.4A). It has a width of approximately 65 Å (the distance between alanine 315 on adjacent protomers). The structures described here were derived from gB of HCMV strain Towne. Although there is some natural variations of gB amino acid sequence, the overall postfusion structure of Towne gB is almost identical to the postfusion structure of gB from the strain AD169 (PDB accession code 5CXF). Thus, the description of the postfusion gB structure applies to both strains with measurements from equivalent amino acids from sequence alignments. The prefusion gB trimer has a more squat shape than the postfusion gB trimer (FIG.4Avs.FIG.4B). The distance between the plane formed by W240 of each protomer and the most membrane distal modeled residue in the prefusion structure, Q483, is roughly 115 Å. The prefusion model is 95 Å in width (measured by the distance between any two A315 from different protomers). The individual subunit structures of domains I, II, III and IV are similar in the prefusion and postfusion conformations. However, the overall arrangement of these domains is very different in the two conformations (FIGS.4A-4BandFIGS.6A-6C). In the prefusion conformation, the fusion loops at the tip of DI and the C-termini of the central helix bundle in domain III all point in the same direction, toward the virion envelope, as identified by the position of the TM region (FIG.4AIandFIG.6A). In contrast, in the postfusion conformation, the fusion loops and the C-termini of the central helix bundle point in opposite directions (FIG.4BandFIG.6C). In the prefusion structure, the hydrophobic residues in the fusion loops (residues Y155, 1156, H157 and W240, L241) are in close proximity to the MPR and are likely surrounded by detergents (FIG.4AIandFIG.6A). In the transition from prefusion to post fusion, domain II shifts from a position mid-way up the domain III central coiled-coil to a position at the membrane proximate end of the coiled-coil and near end of domain I opposite the fusion loops (FIG.4AandFIG.4B). The structure of DIII (FIGS.4A-4BandFIGS.6A-6C) is very similar in the prefusion and postfusion conformations. The central helix in both conformations spans from L479 to P525, indicating a minimal rearrangement during the prefusion to postfusion transition. However, the other domains change their positions relative to the central helix of domain III, so that, as noted above, the direction of the DIII helix bundle (from N-terminal to C-terminal) points away from the fusion loops towards the distal end of the trimer in the postfusion conformation and toward the viral membrane, in the same direction as the fusion loops in the prefusion conformation. In the prefusion structure, domain IV (FIG.4AandFIG.6A) is buried at the interface between domain 1 on the exterior of the trimer and domains III and V at the center of the trimer. In contrast, in the postfusion structure, domain IV forms a highly exposed “crown” at the membrane-distal tip of the trimer. Domain V has different structures in prefusion gB (FIG.4AandFIG.6A) and postfusion gB (FIG.4BandFIG.6C). In prefusion gB, the N-terminal half of the domain (about residues 642-660) is sandwiched between domain 1 and domain IV of an adjacent protomer and is sequestered from solvent. The region between residue 683-704 of domain V forms a trimeric helix bundle with its counterpart in other protomers. This helix bundle is cuddled mostly inside of the pocket of the “crown” formed by domain IV. There is an additional short helix (approximately residues 710-719) linking the helix bundle from domain V to the MPR region. In contrast, in the postfusion conformation (FIG.4BandFIG.6C), domain V is solvent exposed and extends along the outside of domain III helix bundle and the groove formed by the interface between domain I from adjacent protomers. Comparison of the prefusion and postfusion gB structures suggests a progression of conformational changes that is familiar from other well-studied fusion proteins (Harrison, S. C.Virology0:498-507 (2015)). The comparison provides confidence that the structure described in this invention is, in fact, in a prefusion conformation. In the prefusion state (FIG.6A), the fusion loops of domain I are buried by interaction with the MPR and potentially with the viral membrane. In the prefusion structure of the distant gB homolog, the vesicular stomatitis virus G glycoprotein, the fusion loops also point toward the viral membrane (also the anticipated position of an MPR region, which is not seen in that structure)(Roche et al.Science315:843-8 (2007)). Based on analogy to other fusion proteins, it is likely that rearrangement proceeds with lengthening of the central helix as part of a transition to a proposed extended intermediate between the prefusion and postfusion states (FIG.6B). In the proposed extended intermediate state, the TM region would still be anchored in the viral membrane, and the fusion loops, now extended far from the viral membrane at the tips of a rotated and translocated domain I, would interact with a cellular membrane. The transition from the proposed extended intermediate to the postfusion conformation would involve a fold-back so that the transmembrane region and the fusion loops are again in proximity to each other at the same end of the molecule, this time both interacting with the fused viral and cellular membrane (FIG.6C). We speculate that, in prefusion gB, there may be dynamic changes in the length of the central helix, with the prefusion structure we have determined representing a “snapshot” of a “breathing” molecule, locked into the conformation we see in the electron density by the fusion inhibitor and by the cross-linking agent used to prepare the sample studied by electron cryomicroscopy. Stabilizing Factors for the Observed Prefusion Conformation After modeling the gB amino acids into the electron density map, a region of density that was not filled by amino acid residues remained between the MPR, domain V, and the tip of domain I that contains the fusion loops (FIG.5A). The size and shape of the unfilled density fits the chemical structure of the HCMV fusion inhibitor, N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide (FIG.5D), which had been present throughout the production of the sample studied by electron cryomicroscopy (FIG.5B). The compound adopted a pose with a kink between the trifluoromethyl phenyl moiety and the rest of the compound. The thiazole forms contacts with hydrophobic residues of L712, A738 and Y153, Y155 from an adjacent protomer. The phenyl is surrounded in a hydrophobic environment formed by residues of L715, the aliphatic hydrocarbon of D714 from domain V, G734 and I 730 from MPR, and F752 from the TM domain of an adjacent protomer. The trifluoromethyl phenyl resides in a hydrophobic environment near the hinge between MPR and TM helixes from another protomer. It may act as a hook to prevent the outward movement of MPR and TM domains. In addition to the interaction coordinated by the inhibitor compound, the W240, Y242 from other fusion loop are forming van der waals interactions with the hydrophobic patch from the MPR region and L715 in domain V respectively. (FIG.5C). These specific interactions around the fusion inhibitor would be expected to hold domain I, domain V, and the MPR together and restrict movements among domain I, domain V, and the MPR during the fusion process (FIGS.6A-6C). The effects of cross linking on the stability of the prefusion conformation were also tested. During the sample preparation steps, BS2G cross linking reagents either were or were not added. In the absence of the cross linker, the ratio of particles in prefusion versus postfusion conformations was 1:100, while the ratio was 1:4 in the sample that had been cross linked by the BS2G reagent. The cross linker was not identified in the electron density. The prefusion structure of CMV gB and color versions of the prefusion and postfusion structures set forth in the Figures described herein may also be found in Liu et al. Science Advances 7(10): eabf3178 (2021), which is hereby incorporated by reference herein in its entirety. Example 3: Expression and Purification of gB1666 For the production of gB1666, the PSB1666 construct was transiently transfected into Expi293F cells. The cell pellets were harvested 96 hours after transfection. The PSB1666 protein was purified in 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS, 3 μg/ml WAY-174865 (inhibitor, seeFIG.5D) through a series or processes of solubilization, affinity column and size exclusion chromatography. The protein was analyzed on SDS-PAGE and by EM with negative staining to ensure at least 50% of the proteins displaying prefusion conformation. The PSB1666 protein is expressed efficiently in transfection of Expi293F cells and 1L expression would generate ˜0.1 mg of purified PSB1666 in high quality. The polypeptide gB1666 (PSB1666) (SEQ ID NO: 57) includes a mutation in Domains I and IV. The polypeptide includes the following mutations, D217C and Y589C, relative to the corresponding wild-type gB (Towne) set forth in SEQ ID NO: 1. Example 4: DNA-Expressed gB1666 is Immunogenic in Balb/c Mice One of the proposed stabilized full length prefusion gB constructs, gB1666 (SEQ ID NO: 57), has been shown by EM to have an increased proportion of molecules in the prefusion conformation relative to wild type gB of the Towne strain after purification from transfected mammalian cells in the presence of a fusion inhibitor (WAY-174865; seeFIG.5D). To assess whether this molecule can elicit immune responses in vivo, the DNA sequence corresponding to gB1666 and wild type gB were cloned into an in-house mammalian expression vector. Ten Balb/c mice were electroporated with 100 ug of DNA encoding gB1666 twice at a three-week interval (DO and D21). An additional 10 mice were electroporated by the same protocol with DNA encoding wild type gB, and a third group was electroporated with a placebo, consisting of phosphate-buffered saline. Serum samples were collected at Day 28. ELISA was performed against recombinant gB protein produced from mammalian cells, based on the wild type sequence of Towne strain but with the transmembrane domain removed (Sino Biologicals) to determine the anti-gB IgG responses according to a standard protocol. Ten out of ten animals from the wild type gB DNA immunized mice and nine of ten gB1666 DNA immunized mice generated detectable anti-gB IgG titers (FIG.11, showing mean±SD, LLOQ=25). The study demonstrates that gB1666 is immunogenic in Balb/c mice. Example 5: Immunogenicity Study of Stabilized Prefusion gB1666 Protein Immunogenicity study of gB1666 in mice. To evaluate the antibody response in mice, the following immunization scheme will be followed. At week 8, mice will be exsanguinated and the neutralization titers from the immunized animal serum will be determined and compared with those immunized with gB705 (postfuion) and/or gB wild type proteins. TABLE 5Mouse immunogenicity study design with gB1666 proteinNo. ofDosingGroupMiceImmunogenAdjuvantRouteSchedule110gB705 (postfusion)—0.2 ml/SCWeeks 0, 3, 6(1.25 mcg/0.2 ml)210gB705 (postfusion)—0.2 ml/SCWeeks 0, 3, 6(0.25 mcg/0.2 ml)310gB1666 (in inhibitor-—0.2 ml/SCWeeks 0, 3, 6containing buffer)(1.25 mcg/0.2 ml)410gB1666 (in inhibitor-—0.2 ml/SCWeeks 0, 3, 6containing buffer)(0.25 mcg/0.2 ml)510gB wt (in inhibitor-—0.2 ml/SCWeeks 0, 3, 6containing buffer)(1.25 mcg/0.2 ml)610gB wt (in inhibitor-—0.2 ml/SCWeeks 0, 3, 6containing buffer)(0.25 mcg/0.2 ml)75Buffer (+Inhibitor)—0.2 ml/SCWeeks 0, 3, 685Buffer only—0.2 ml/SCWeeks 0, 3, 6 Example 6 In Example 2, we disclosed the electron cryomicroscopy (cryoEM) structure of prefusion human cytomegalovirus (HCMV) strain Towne glycoprotein B (gB) in complex with an antibody fragment. The gB used for structure determination was obtained by adding a small molecule fusion inhibitor, WAY-174865, to a fermentation of authentic HCMV in mammalian cell culture and maintaining the presence of the inhibitor throughout production and analysis of gB; purifying the virus; treating the virus with a chemical cross linker, bis(sulfosuccinimidyl) glutarate (BS2G; 7.7 Å spacer arm); extracting gB from the virus with detergent; binding gB on the virion with an affinity tagged antibody fragment; and purifying the gB by affinity and sizing columns. We also disclosed the use of the prefusion gB cryoEM structure to engineer mutations that stabilize gB in the prefusion state. Specifically, we disclosed the recombinant gB protein gB1666, in which two residues are mutated to cysteine (D217C, Y589C). The resulting formation of an engineered disulfide bond between C217 and C589 increases the conformational stability of the recombinant gB in the prefusion state. gB1666 maintained prefusion structural features when it was expressed in Expi293F cells and purified in the presence of a fusion inhibitor, compound WAY-174865. In the absence of the inhibitor, gB1666 tends to undergo a conformational change and lose its prefusion structural state. Loss of prefusion conformational stability in the absence of inhibitor is not a desirable characteristic for use of the recombinant glycoprotein as an antigen for immunization. Even if gB1666 were formulated with the inhibitor, there is a risk that, upon injection into a person or animal, the dilution of the inhibitor in vivo would lead to its dissociation from gB1666 and the loss of prefusion conformation of gB1666. Thus, it is desired that HCMV gB be stabilized sufficiently in the prefusion conformation to remain in the prefusion state in the absence of WAY-174865. It is also preferable that a prefusion gB immunogen includes a soluble ectodomain to improve manufacturability, improve solubility, improve homogeneity, and reduce or eliminate the need for formulation with a detergent or other excipient to prevent aggregation or precipitation mediated by the gB transmembrane region. We now report the invention, through a structure-based engineering approach, of new mutations in HCMV gB that confer these improved characteristics for use of prefusion gB as an immunogen. First, we determined the structure by cryoEM of gB1666, which was solubilized by anchoring in nanodiscs and stabilized in the prefusion conformation by the presence of WAY-174865 (FIG.12). Most of the new structure of the recombinant, D217C and Y589C mutant gB is similar to the structure of the virion-derived, chemically cross-linked and antibody fragment bound HCMV Towne prefusion gB that we determined previously, but there are subtle differences between the two structures in certain local regions. The difference in the structures could reflect several differences in the preparations: first, the presence of the engineered disulfide bond in gB1666, which should restrict the breathing motion of the glycoprotein; second, the anchoring of gB1666 in a nanodisc, which provides a more natural local lipid environment for the transmembrane domain than the detergents used to extract and maintain gB in solution for the previous structure determination; third, the absence of chemical cross-linking of gB1666; fourth, the higher resolution of the new structure at 3.3 Å, compared to the 3.6 Å resolution of the previous structure, allowing more accurate modeling of amino acid side chains. Based on the new structural information, we designed additional stabilizing mutations on the background of the full length gB construct pSB1666 (Table 6 and Table 7). We hypothesized that adding these additional mutations on the pSB1666 background would further stabilize the gB in a prefusion state (FIG.13). For example, cysteine mutations at residues M371 and W506 may introduce a disulfide bond between domains II and III; cysteine mutations at the pairs of (F541, E681) and (N524,M684) may introduce disulfide bonds between domains IV and V; mutations of residues E686, D679 to hydrophobic residues could remove a locally destabilizing same charge repulsion patch and increase protein stability. Recombinant glycoproteins with a selection of the new, added mutations were expressed and purified in the absence of fusion inhibitor and without chemical crosslinking. The electrophoretic mobility of the expressed glycoproteins by SDS-PAGE showed the expected apparent molecular weight and heterogeneity consistent with glycosylation (FIG.14). The samples were stored at 4° C., and aliquots were taken for negatively stained electron microscopy analysis on day 1 and day 7. In the 2D class averaged images, triangular shape features that resemble “top views” of the prefusion conformation of the gB were apparent. The ratio of particles in the population belonging to prefusion and postfusion classes were 5:1 on day 1 and 3:1 at day 7 (FIG.15). Based on the new structural information, we designed several soluble, detergent-free gB ectodomains (Table 8) with prefusion-stabilizing mutations as illustrated inFIGS.16A-16D. The purified ectodomain of HCMV gB, residues 1-707, formed rosette-like aggregates, in which gB proteins associated through their exposed fusion loops. To eliminate aggregation and increase protein secretion to the condition media, we replaced four exposed hydrophobic residues within the fusion loops with the corresponding more hydrophilic amino acids from herpes simplex virus-1 (HSV-1) gB, e.g. YIH (155-157)→GHR, W240→A. We also mutated the exposed Cys246 to Ser (C246→S) to prevent formation of spurious disulfide bonds. To further stabilize the prefusion trimeric state of the antigen, we either introduced cysteine residues capable of forming inter-protomer disulfide bonds or appended C-terminal trimerization motifs, e.g. GCN4 or foldon from T4-bacteriophage fibritin. Disulfide mutations, e.g. D217C-Y589C, M317C-W506C, N524C-M684C, were further introduced to lock the proteins into the prefusion state. Recombinant glycoproteins were expressed, secreted to the conditioned media and purified in the absence of fusion inhibitor and without chemical detergent. Notably, the recombinant variant, fused to a GCN4 trimerization motif, showed optimal size-exclusion chromatography profile (FIG.17). The negatively stained electron microscopy showed recombinant proteins, gB2555 and gB2556, as monodispersed proteins in the absence of inhibitor and detergents. Expected gB protein features are observed in the 2D class averaged images (FIG.18andFIG.19). These results confirm that these engineered constructs are suitable to be used as a framework to add more stabilizing mutations towards a prefusion form of gB in the absence of inhibitor and detergents if needed. The coordinates and structural factors for the model of the prefusion gB associated with the present Example are provided in Table 1B. TABLE 6Exemplary cysteine pair mutations for disulfide bond stabilizationRowMutations1D217C, Y589C2I356C, A500C3S367C, A500C4S367C, A503C5M371C, A505C6M371C, W506C7T374C, A503C8Y160C, Y708C9L162C, M716C10N524C, M684C11G99C, A267C12T100C, R258C13T221C, E657C14S223C, T659C15F541C, E681C16L603C, Y667C17N605C, K670C18R607C, N688C19E609C, K691C TABLE 7Exemplary charge mutations for stabilizationRowMutations1E686L2E686I3D679A4R354F5R573F6D101L, K260L TABLE 8Construct mutations.ConstructMutation 1Mutation 2Mutation 3Mutation 4pSB1688N524CM684CpSB2455M371CW506CpSB2456F541CE681CpSB2457D217CY589CM371CW506CpSB2459D217CY589CN524CM684CpSB1666D217CY589C The constructs set forth in Table 8 were made for the purpose of testing the presence of prefusion gB in the purified recombinant protein preparation under different conditions. TABLE 9Exemplary soluble, detergent-free gB ectodomain proteinsCodegB*MutationsC-Terminus Fusion SequencegB707gB (1-707)D217C, Y589CgB2264gB (1-707)D217C, Y589C,D703C, P704CgB2265gB (1-707)D217C, Y589C,Y696C, V697CgB2266gB (1-702)D217C, Y589CRIKQIEDKIEEILSKQYHIENEIARIKKLIG(SEQ ID NO: 272)gB2267gB (1-702)D217C, Y589CKIEEILSKQYHIENEIARIKKLIG (SEQID NO: 269)gB2268gB (1-702)D217C, Y589CKIYHIENEIARIKKLIG (SEQ ID NO: 273)gB2269gB (1-703)D217C, Y589CLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL(SEQ ID NO: 274)gB2555gB (1-702)D217C, M371C,KIEEILSKQYHIENEIARIKKLIG (SEQW506C, Y589CID NO: 269)gB2556gB (1-702)D217C, N524C,KIEEILSKQYHIENEIARIKKLIG (SEQY589C, M684CID NO: 269)gB2557gB (1-707)D217C, N524C,Y589C, M684C,D703C, P704C*Mutations, including YIH (155-157)→GHR, W240→A and C246→S, had been incorporated in gB to decrease aggregation and increase protein secretion. Example 7: Overall Strategies to Engineer a Stabilized Prefusion gB To engineer a stabilized prefusion gB, two strategies were used: i) the first strategy is to strengthen the interactions in the prefusion structure, which include engineering disulfide bonds between the residues that are in close proximity in the prefusion structure and grafting stable structure motifs to the C-terminal end without disrupting the structures from other parts of the molecule; and ii) the second strategy is to remove the energetically unfavorable local structures from the prefusion conformation. These include: (a) removal of surface exposed hydrophobic residues, (b) changes of proline to non-proline and hydrophilic residues in the loops regions, and (c) removal and reengineering Domain V, which undergoes large conformational changes during the fusion process. The combination of such designs was used to obtain enough stabilization for the prefusion gB. The proteins carrying such designed mutations were individually purified and screened by their properties in size exclusion chromatography (SEC), thermal melting assay (TM) and their features from the electron microscopy images (EM). A panel of mutant cysteine pair constructs was screened against the CMV gB full length post-fusion construct pSB1764. For the first round, each new construct included one mutant cysteine pair. The complete list of mutants that were screened are in Table 10. TABLE 10The list of one pair disulfide bond mutants that were screened onfull length CMV gBPostfusion gB Full length Construct pSB1764Construct Framework CMV gB HAss FLAG/V23 . . . V907(ACM48044.1) I675S/Thrombin His6StrepTagII ** in pcDNA3.1(+)ConstructnameMutation 1Mutation 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 It was determined from the screen that cysteine pair D217C-Y589C (pSB1666), in combination with inhibitor WAY-174865, stabilized CMV gB in the prefusion state. In the presence of the fusion inhibitor, the construct gB1666 (engineered disulfide by mutating D217C and Y589C) showed a right shift in the in SEC retention volume and a distinct transition temperature at ˜73° C. indicating a different conformation from that of the wild type gB (FIGS.22A and22B). gB1666 was further characterized by EM imaging experiments. From the cryoEM dataset 2D classification results, prefusion classes were observed as the dominant population and the 3D structure density map confirmed the prefusion gB structure (data not shown). However, in the absence of the inhibitor, the right shifted peak from SEC and the transition temperature peak at ˜73° C. are less distinct (FIG.23). This suggested a single disulfide bond has limited effect to retain gB1666 in a prefusion state. Thus, a second round of mutations were screened in frame of gB1666 to create further stability of the prefusion conformation in the absence of the inhibitor (Table 11). A new mutant panel was cloned and screened in which a second cysteine pair was introduced into construct pSB1666. The goal was to add another stabilizing disulfide bridge, in order to further stabilize CMV gB in the prefusion state. TABLE 11The list of two pairs of disulfide bonds mutants that were screenedon full length CMV gBFull length Prefusion construct pSB1666Construct Description CMV HAss FLAG/V23 . . . V907(ACM48044.1)[D217C-Y589C] I675S/ThrombinHis6 StrepTagII ** in pcDNA3.1(+).ConstructnameMutation 1Mutation 2pSB02145T100CN658CpSB02146S367CA500CpSB02147D272CP614CpSB02148S219CF584CpSB02149E686VpSB02191D217CF584CpSB02193D217CN586CpSB02194D217CS588CpSB02195S219CS587CpSB02196S219CS588CpSB02197S219CY589CpSB02455M371CW506CpSB02456F541CE681CpSB02457M371CW506CpSB02459N524CM684C It was determined from this screen that cysteine pair D217C-Y589C, in combination with either pair M371C-W506C (pSB2457) or pair N524C-M684C (pSB2459) stabilized CMV gB in the prefusion state, without the need for a stabilizing inhibitor. gB2457 and gB2459 proteins showed similar profiles in SEC and TM results as the prefusion form (gB1666+ inhibitor) (FIG.24A-24B). gB2457 had a more distinct transition at ˜73° C. in thermal stability assay (FIG.24B). In addition, both gB2457 and gB2459 showed the prefusion gB feature in the negative staining EM 2D class averaged images (data not shown). In the absence of a fusion inhibitor, gB2457 and gB2459 represent more stable versions of prefusion gB than their parent design, i.e. gB1666. Such improvement also confirms that the right combination of stabilizing mutation sites can synergistically contribute to the overall stability of the prefusion conformation. In comparison to the full length and membrane bound forms of prefusion gB, a soluble format prefusion gB does not require detergent which provides advantages in scalability of protein production and ease of purification. Based on the known structure, the membrane interaction domains from gB are the membrane proximal region (MPR), the transmembrane (TM) domain and fusion loops. The cytoplasmic tail domain may also interact with membrane from the cytosolic side. Thus, these membrane-interacting hydrophobic regions were either removed or converted to hydrophilic types in the designs for a soluble form prefusion gB. In addition, structurally stable trimerization tags (e.g. GCN4, cysteine rings, trimeric foldon) were added at the carboxy terminal of the protein that truncated at before the MPR domain. Several truncation designs were tested before the GCN4 tag (gB2267) was selected to be used as an ectodomain construct frame. The stabilizing mutation sites identified from full length frame were made on this ectodomain frame and screened (Table 12). In the resulting gB2555 and gB2556, which were well behaved proteins after purification (FIG.25A), the phase transition peak at a lower temperature ˜73° was not obvious (FIG.25B). But the 2D classification from negative staining EM images indicated that both prefusion and postfuiosn forms were present (data not shown) and further stabilization were still needed for the ectodomain prefusion gB. The third pair of disulfide bonds were screened on the pSB2556 background (Table 12), but not much improvement was observed. TABLE 12Ectodomain CMV gB mutants with triple disulfide bond pairsEctodomain construct pSB2556Construct Description pSB2556: CMV HAss FLAG/V23 . . .V702 gB CMV (ACM48044)YIH to GHR (155-157)[D217C-Y589C] [N524C-M684C]W240A C246S I675S/GCN4 CC tri2 ** in pcDNA3.1(+).constructmutationname1mutation 2pSB2558I356CA500CpSB2559S367CA500CpSB2560S367CA503CpSB2561M371CA505CpSB2562M371CW506CpSB2563T374CA503CpSB2564G99CA267CpSB2565T100CR258CpSB2567L603CY667CpSB2568N605CK670CpSB2570E609CK691CpSB2571T221CE657CpSB2572S223CT659C Since domain V undergoes large conformational changes and was expected to provide the energy to drive the membrane fusion between the viral and host cells, a new approach was to remove Domain V from the ectodomain gB. SB2562 was chosen as the starting construct (Table 13 and Table 14). TABLE 13Additional Ectodomain DesignspSB2795CMV HAss FLAG/V23 . . . D646 gB CMV (ACM48044) YIH to GHR (155-157) D217CW240A C246S M371C N524C W506C Y589C I675S ** in pcDNA3.1(+).pSB2797CMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) YIH to GHR (155-157) 648MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589C I675SM684C/GCN4 CC tri2 ** in pcDNA3.1(+).pSB2968CMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) PI116117GS YIH to GHR (155-157) 648 MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589CI675S M684C/GCN4 CC tri2 ** in pcDNA3.1(+).ACMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) YIH to GHR (155-157) 648MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589C I675SM684C R693V, last res L695 + KIKQIEDK IEEILSK IYHIENE IARIKKL IG ** inpcDNA3.1(+)BCMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) YIH to GHR (155-157) 648MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589C I675SM684C R685A R693V, last res L695 + KIKQIEDK IEEILSK IYHIENE IARIKKL IG **in pcDNA3.1(+)CCMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) YIH to GHR (155-157) 648MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589C I675SM684C F678S, L680T R693V, last res L695 + KIKQIEDK IEEILSK IYHIENEIARIKKL IG ** in pcDNA3.1(+)DCMV HAss FLAG/V23 . . . V702 gB CMV (ACM48044) YIH to GHR (155-157) 648MIALDI to GSGKDG D217C W240A C246S M371C N524C W506C Y589C I675SM684C P655S R693V, last res L695 + KIKQIEDK IEEILSK IYHIENE IARIKKL IG **in pcDNA3.1(+) One of the designs removed the entire Domain V in which residues after D646 were truncated. Since the disulfide pair could no longer form at N524C and M684C, the N524C was reverted back to its wildtype asparagine residue (N) in order to remove the free cysteine (pSB2796) (Table 14). This construct was named gB2796 and showed a well-behaved protein as a single peak in SEC profile with a distinct phase transition temperature at ˜70° C. (FIGS.26A and26B). From the cryoEM dataset, 2D class average showed clear expected protein densities that corresponded to the structural features from the model of ectodomain prefusion gB. The 3D reconstructed density envelope also corresponded to the expected ectodomain structure. (data not shown). TABLE 14Domain V truncated ectodomain CMV gB mutant with disulfide bond pairsCMV ectodomain gB prefusionpSB2796DescriptionCMV HAss FLAG/V23 . . . D646 gB CMV (ACM48044) YIHto GHR (155-157) [D217C-Y589C] [M371C-W506C]W240AC246S I675S ** in pcDNA3.1(+). Construct pSB2796 was redesigned into different N-term tagged versions for use in animal studies (Table 15). The Flag tag was swapped out for a 6×His tag, which is more amenable for purification scale up. An untagged version was also created. The HA signal sequence was also replaced with the IgK signal sequence because it is more compatible with the His tag. TABLE 15Construct Design for CMV gB ectodomain prefusion mouse studypSB3075CMV: IgKss 6xHis rTEV/V23 . . . D646 gB CMV (ACM48044) YIH toGHR (155-157) D217C W240A C246S M371C W506C Y589C I675S **in pcDNA3.1(+).pSB3076CMV: IgKss 6xHis PP/V23 . . . D646 gB CMV (ACM48044) YIH to GHR(155-157) D217C W240A C246S M371C W506C Y589C I675S **in pcDNA3.1(+).pSB3077CMV: IgKss 6xHis/V23 . . . D646 gB CMV (ACM48044) YIH to GHR(155-157) D217C W240A C246S M371C W506C Y589C I675S **in pcDNA3.1(+).pSB3078CMV: IgKss/V23 . . . D646 gB CMV (ACM48044) YIH to GHR (155-157)D217C W240A C246S M371C W506C Y589C I675S ** in pcDNA3.1(+). These data showed that the prefusion gB is metastable and stabilizing mutations at multiple sites are needed to achieve enough restraints to maintain its prefusion structure. The combination of disulfide bonds and the modifications of unstable Domain V provide examples of locking the prefusion conformation. The stabilized gB can be engineered in full length (gB1666, gB2457, gB2459) and ectodomain protein formats (gB2796), which are suitable for use as prefusion gB antigens. In addition, the SEC, the thermal shift assay and EM imaging approaches provided ways to evaluate the conformational state of the gB samples without the need for prefusion or postfusion specific antibodies. Methods Cysteine Mutants Construct Design and Cloning All constructs were cloned into the pcDNA3.1(+) vector backbone (ThermoFisher Scientific, Waltham, Mass.). Primers, each containing a cysteine point mutation, were ordered from IDT (Coralville, Iowa). Site directed mutagenesis was performed using a QuikChange Multi Site-Directed Mutagenesis Kit (Agilient, Santa Clara, Calif.). The mutagenized DNA was transformed into DH5 alpha cells. Colonies were selected and sent for sequencing. Positive transformants were verified by DNA sequencing analysis. Plasmid DNA was amplified using DNA preparation kits. Mammalian Cell Expression EXP1293 cells were grown to an OD of 3×106cells/mL. Plasmid DNA was diluted in OptiMem media and mixed 1:1 with PEI solution. The DNA-PEI mixture was then transfected into the EXP1293 cells at a concentration of 1 ug DNA/mL culture. Enhancers were added 24 hours post transfection. Cells were monitored and harvested by centrifugation 4 to 5 days post transfection. Protein Purification CMVgB proteins were purified through a series or processes of solubilization, affinity column and size exclusion chromatography (SEC). SEC is run on Superose6 increase column in buffer 25 mM HEPES pH 7.5, 250 mM NaCl, 0.02% DDM, 0.002% CHS. For experiments with inhibitor, 3 ug/ml WAY-174865 was added. Thermal Stability Assay As a complementary biophysical study of the gB mutants to help identify prefusion stable mutants, thermal stability of the purified gB proteins was analyzed on Tycho NT.6 with 20° C./min heating rate from 35° C. to 95° C. Negative Staining Grid Preparation Thin carbon supported grids were glow discharged by EZ-glow with −20 mA current for 30 seconds before use. Aliquots of 4 ul sample solution at the protein concentration of 0.02 mg/ml were applied to the carbon surface of the grids and let sit for 45 seconds. The sample solution was then blotted away with filter paper and same carbon surface was rinsed with filtered water and excess water was blotted away with filter paper. The grid was stained with 2% uranyl acetate solution and air dried before being loaded to a TF20 electron microscope for imaging. Cryo Grids Preparations Quantifoil grids were plasma cleaned with Argon/Oxygen. Graphene oxide stock solution diluted to 0.2 mg/ml concentration (Sigma) was applied to the surface of the treated grid and let sit for 2 minutes. The excess solution was blotted with filter paper and washed with one droplet of water to remove excess graphene oxide. The grid was dried overnight before use. Aliquots of sample solution were vitrified on graphene oxide film-supported grids using a Vitrobot (ThermoFisher). The grids were stored in liquid nitrogen until loaded in the microscope under cryo conditions for imaging. CryoEM Data Collection, Image Processing Data collections were done on a Thermo Fisher Titan Krios transmission electron microscope that operates at 300 kV with SerialEM program at a nominal magnification (165,000×) using a K2 direct detector camera (Gatan) with super resolution movie mode. The unbinned pixel size was 0.434 Å, and the beam intensity was ˜8e/unbin pixels. The total electron dose on the sample for each movie was ˜40e/A2. Both Relion and cisTEM programs were used for the data processing. Example 8: gB2796 Immunogenicity One of the designed prefusion stabilized ectodomain gB proteins (gB2796) containing amino acids 23-646 with mutations YIH to GHR (155-157), D217C, W240A, C246S, M371C, W506C and Y589C was purified from transfected mammalian via FLAG tag in the absence of fusion inhibitor. Briefly, the protein was purified by adding 20 mM Tris pH 7.5, 200 mM NaCl into the 3L cell media overexpressing SB2796. After incubation with 10 ml of M2 column at 4° C. with rotation for 4 hours, the supernatant was removed and the column was washed thoroughly with PBS. Then the protein was eluted with PBS containing 150 μg/ml FLAG peptide and subsequently concentrated. Size-exclusion chromatography was performed on Superose6 increase 10/300 in PBS at 0.5 ml/min, and the fractions were collected. The purified protein was analyzed on 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Biorad) in Tris/glycine/SDS buffer (FIG.20A) as well as by electronic microscopy with negative staining (FIG.20B). The EM image showed the triangular shape of the protein particles which is the typical prefusion conformation. To assess whether gB2796 can elicit a better immune response compared to postfusion gB, a secreted postfusion gB was produced based on the same strain of HCMV with transmembrane domain removed. The protein was named Sanofi gB since it is equivalent to the gB protein used in previous clinical trial by Sanofi (Pass, et al. 2009). Purification of this tagless gB was achieved using affinity chromatography on Lentil Lectin-Sepharose (GE Healthcare) column, followed by anion-exchange chromatography on Toyopearl GigaCap Q-650M (Tosoh), and size-exclusion chromatography on Superdex 200 (GE Healthcare) column. An in vivo immunogenicity study was conducted in mice with prefusion and postfusion gB described above, as shown in Table 16 below. TABLE 16Immunization dose and scheduleNo. ofDosingGroupMiceImmunogenAdjuvantRouteSchedule110gB2796—0.2 ml/SCWeeks 0, 3, 6(4 mcg/0.2 ml)210gB2796—0.2 ml/SCWeeks 0, 3, 6(1 mcg/0.2 ml)310gB2796—0.2 ml/SCWeeks 0, 3, 6(0.25 mcg/0.2 ml)610Sanofi gB—0.2 ml/SCWeeks 0, 3, 6(4 mcg/0.2 ml)710Sanofi gB—0.2 ml/SCWeeks 0, 3, 6(1 mcg/0.2 ml)810Sanofi gB—0.2 ml/SCWeeks 0, 3, 6(0.25 mcg/0.2 ml) At week 5 (2 weeks after second dose), mice sera samples were analyzed by ELISA to determine the IgG titers against gB2796. The results show dose-dependent IgG responses in both gB2796 and Sanofi gB immunized mice (FIG.21). Listing of Raw SequencesSEQ IDConstructSequence*NO:CMV gB1666: V23..V907 ofVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH255SEQ ID NO: 1 (Towne)GVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFER(ACM48044.1)NIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRY589C I675SRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVIVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIIIYLIYTRQRRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC256ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCTACATCCATACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACATGGCTGTATAGAGAGACCTGTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGATGACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGGTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCAACCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACAGTATGATCGCACTGGACATTGACCCTTTGGAAAACACCGATTTTCGCGTCCTTGAGCTGTACTCCCAGAAAGAACTGCGCTCTAGCAACGTCTTTGATCTTGAGGAGATCATGAGGGAGTTTAACTCTTATAAACAGAGGGTGAAGTATGTGGAGGATAAAGTGGTCGACCCTCTGCCACCCTACCTCAAAGGACTGGACGATCTGATGAGCGGACTTGGAGCTGCCGGCAAAGCCGTCGGGGTGGCTATCGGTGCCGTGGGCGGCGCCGTGGCTTCTGTGGTTGAGGGAGTGGCCACTTTTCTTAAAAATCCTTTCGGAGCTTTTACCATTATTCTGGTCGCCATCGCCGTGGTGATCATCATTTATCTGATCTACACCCGCCAGCGCCGCTTGTGCATGCAGCCACTTCAGAACCTGTTTCCCTATCTGGTCAGTGCTGACGGTACAACCGTGACCAGCGGCAACACAAAGGACACAAGCCTTCAGGCTCCTCCAAGTTATGAAGAGTCCGTGTATAATTCTGGGAGGAAGGGACCTGGTCCCCCCTCTTCCGACGCCTCAACAGCGGCACCCCCCTACACCAATGAGCAGGCATATCAGATGCTCCTGGCCCTTGTGCGGCTCGATGCCGAGCAACGCGCACAACAGAACGGGACGGATTCTCTCGACGGACAGACAGGCACTCAGGACAAAGGCCAGAAGCCCAACCTTCTGGATCGGTTGCGGCATAGAAAAAACGGCTATAGACACCTCAAGGACTCAGACGAAGAAGAGAACGTCCMV gB2457 (Prefusion, FullVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH257length) V23 to V907 of SEQGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERID NO: 1 (Towne)NIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRM371C W506C Y589CRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAI675SGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVIVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKCTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEACCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIIIYLIYTRQRRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC258ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCTACATCCATACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACATGGCTGTATAGAGAGACCTGTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGTGCACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGCTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCAACCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACAGTATGATCGCACTGGACATTGACCCTTTGGAAAACACCGATTTTCGCGTCCTTGAGCTGTACTCCCAGAAAGAACTGCGCTCTAGCAACGTCTTTGATCTTGAGGAGATCATGAGGGAGTTTAACTCTTATAAACAGAGGGTGAAGTATGTGGAGGATAAAGTGGTCGACCCTCTGCCACCCTACCTCAAAGGACTGGACGATCTGATGAGCGGACTTGGAGCTGCCGGCAAAGCCGTCGGGGTGGCTATCGGTGCCGTGGGCGGCGCCGTGGCTTCTGTGGTTGAGGGAGTGGCCACTTTTCTTAAAAATCCTTTCGGAGCTTTTACCATTATTCTGGTCGCCATCGCCGTGGTGATCATCATTTATCTGATCTACACCCGCCAGCGCCGCTTGTGCATGCAGCCACTTCAGAACCTGTTTCCCTATCTGGTCAGTGCTGACGGTACAACCGTGACCAGCGGCAACACAAAGGACACAAGCCTTCAGGCTCCTCCAAGTTATGAAGAGTCCGTGTATAATTCTGGGAGGAAGGGACCTGGTCCCCCCTCTTCCGACGCCTCAACAGCGGCACCCCCCTACACCAATGAGCAGGCATATCAGATGCTCCTGGCCCTTGTGCGGCTCGATGCCGAGCAACGCGCACAACAGAACGGGACGGATTCTCTCGACGGACAGACAGGCACTCAGGACAAAGGCCAGAAGCCCAACCTTCTGGATCGGTTGCGGCATAGAAAAAACGGCTATAGACACCTCAAGGACTCAGACGAAGAAGAGAACGTCCMV gB2459 (Prefusion, FullVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH259length) V23 to V907 of SEQGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERID NO: 1 (Towne)NIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRN524C Y589C M684CRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAI675SGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKICPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEICREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIIIYLIYTRQRRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC260ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCTACATCCATACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACATGGCTGTATAGAGAGACCTGTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGATGACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGGTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCTGTCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACAGTATGATCGCACTGGACATTGACCCTTTGGAAAACACCGATTTTCGCGTCCTTGAGCTGTACTCCCAGAAAGAACTGCGCTCTAGCAACGTCTTTGATCTTGAGGAGATCTGTAGGGAGTTTAACTCTTATAAACAGAGGGTGAAGTATGTGGAGGATAAAGTGGTCGACCCTCTGCCACCCTACCTCAAAGGACTGGACGATCTGATGAGCGGACTTGGAGCTGCCGGCAAAGCCGTCGGGGTGGCTATCGGTGCCGTGGGCGGCGCCGTGGCTTCTGTGGTTGAGGGAGTGGCCACTTTTCTTAAAAATCCTTTCGGAGCTTTTACCATTATTCTGGTCGCCATCGCCGTGGTGATCATCATTTATCTGATCTACACCCGCCAGCGCCGCTTGTGCATGCAGCCACTTCAGAACCTGTTTCCCTATCTGGTCAGTGCTGACGGTACAACCGTGACCAGCGGCAACACAAAGGACACAAGCCTTCAGGCTCCTCCAAGTTATGAAGAGTCCGTGTATAATTCTGGGAGGAAGGGACCTGGTCCCCCCTCTTCCGACGCCTCAACAGCGGCACCCCCCTACACCAATGAGCAGGCATATCAGATGCTCCTGGCCCTTGTGCGGCTCGATGCCGAGCAACGCGCACAACAGAACGGGACGGATTCTCTCGACGGACAGACAGGCACTCAGGACAAAGGCCAGAAGCCCAACCTTCTGGATCGGTTGCGGCATAGAAAAAACGGCTATAGACACCTCAAGGACTCAGACGAAGAAGAGAACGTCCMV gB2555: V23..V702 gBVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH261CMV (ACM48044) of SEQ IDGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNO: 1 (Towne) includingNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRtrimerization domain (GCN4RSYAGHRTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVICC tri2)AGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVTVKDQW[YIH to GHR (155-157) D217CHSRGSTALYRETSNLNCMVTITTARSKYPYHFFATSTGDVVDISW240A M371C C246S W506CPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLY589C I675S/GCN4 CC tri2]VAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKCTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEACCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVKIEEILSKQYHIENEIARIKKLIGGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC262ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCGGTCATCGTACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACAGCACTGTATAGAGAGACCTCTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGTGCACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGCTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCAACCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACAGTATGATCGCACTGGACATTGACCCTTTGGAAAACACCGATTTTCGCGTCCTTGAGCTGTACTCCCAGAAAGAACTGCGCTCTAGCAACGTCTTTGATCTTGAGGAGATCATGAGGGAGTTTAACTCTTATAAACAGAGGGTGAAGTATGTGGAGGATAAAGTGGTCAAGATCGAGGAGATCCTGTCCAAGCAGTACCACATCGAGAACGAGATCGCCCGCATCAAGAAGCTGATCGGCCMV gB2556: V23..V702 gBVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH263CMV (ACM48044) of SEQ IDGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNO: 1 (Towne) includingNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRtrimerization domain (GCN4RSYAGHRTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVICC tri2)AGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVTVKDQW[YIH to GHR (155-157) D217CHSRGSTALYRETSNLNCMVTITTARSKYPYHFFATSTGDVVDISW240A C246S N524C Y589CPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLI675S M684C/GCN4 CC tri2]VAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKICPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEICREFNSYKQRVKYVEDKVVKIEEILSKQYHIENEIARIKKLIGGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC264ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCGGTCATCGTACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACAGCACTGTATAGAGAGACCTCTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGATGACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGGTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCTGTCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACAGTATGATCGCACTGGACATTGACCCTTTGGAAAACACCGATTTTCGCGTCCTTGAGCTGTACTCCCAGAAAGAACTGCGCTCTAGCAACGTCTTTGATCTTGAGGAGATCTGTAGGGAGTTTAACTCTTATAAACAGAGGGTGAAGTATGTGGAGGATAAAGTGGTCAAGATCGAGGAGATCCTGTCCAAGCAGTACCACATCGAGAACGAGATCGCCCGCATCAAGAAGCTGATCGGCCMV gB2796 (Prefusion,VSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH265ectodomain) V23 to D646 ofGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERSEQ ID NO: 1 (Towne)NIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFR[YIH to GHR (155-157) D217CRSYAGHRTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIW240A C246S M371C W506CAGTVFVAYHRDSYENKTMQLMPDCYSNTHSTRYVTVKDQWY589C]HSRGSTALYRETSNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKCTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEACCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSCVQYGQLGEDNElLLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDGTATCCTCCAGCTCTACTAGAGGAACGAGCGCGACCCATTC266ACACCACAGCTCCCACACTACGAGCGCTGCCCATTCTAGGAGTGGCTCCGTGTCACAACGCGTTACTAGTAGCCAGACCGTGTCTCACGGCGTCAACGAGACAATTTACAACACGACGCTGAAATACGGCGATGTCGTGGGTGTGAATACTACCAAGTACCCCTATAGAGTCTGTAGCATGGCCCAGGGCACCGACCTGATCAGGTTCGAACGGAACATCGTTTGCACATCAATGAAGCCTATCAACGAAGACCTTGACGAGGGGATTATGGTGGTATACAAACGGAATATCGTGGCTCACACCTTCAAAGTGCGAGTGTATCAGAAGGTTCTGACATTCCGAAGAAGTTACGCCGGTCATCGTACCACCTATCTGCTGGGCTCCAACACTGAGTACGTGGCGCCCCCAATGTGGGAAATCCACCACATCAACAGCCATTCACAGTGCTACTCTTCCTACAGCAGGGTGATTGCGGGCACAGTGTTTGTGGCCTACCACAGGGACAGCTATGAGAACAAGACGATGCAGTTGATGCCAGATTGTTACAGTAACACTCACAGTACACGGTATGTTACAGTTAAGGATCAGTGGCATTCACGCGGAAGCACAGCACTGTATAGAGAGACCTCTAATTTGAATTGTATGGTAACTATCACTACTGCACGGAGCAAGTACCCTTATCATTTCTTTGCTACAAGCACGGGCGATGTGGTAGACATCAGCCCCTTCTATAATGGCACAAATCGGAACGCAAGCTATTTCGGGGAGAACGCCGACAAGTTTTTCATTTTTCCTAATTATACTATTGTTTCTGACTTCGGGAGACCCAACTCCGCCCTGGAAACTCACAGACTGGTTGCGTTCCTCGAAAGAGCAGATTCTGTGATATCCTGGGACATTCAGGATGAAAAGAACGTCACGTGTCAGCTGACCTTCTGGGAGGCCTCAGAGCGGACGATCCGGTCTGAGGCCGAGGACTCTTACCACTTTAGCAGCGCCAAGTGCACCGCAACCTTCCTGTCTAAAAAACAGGAAGTGAACATGTCCGATTCTGCTTTGGACTGCGTTCGCGATGAGGCCATCAACAAGCTTCAACAAATTTTCAATACCTCCTACAATCAGACATATGAAAAATACGGAAACGTGAGTGTCTTTGAAACCACCGGGGGCCTGGTCGTGTTCTGGCAGGGTATCAAACAGAAGAGCCTGGTGGAACTGGAACGCCTGGCCAACAGAAGCAGTTTGAACCTCACGCACAACCGGACAAAGAGGAGCACCGACGGAAACAATGCTACACACCTTTCCAACATGGAGTCTGTCCACAATCTGGTTTATGCACAGCTTCAGTTCACTTATGACACACTGCGGGGCTACATAAACAGGGCTCTGGCACAGATAGCCGAGGCTTGCTGTGTGGACCAGCGGAGAACCCTGGAGGTATTTAAAGAACTGTCTAAGATCAACCCCTCTGCGATTCTGAGCGCTATTTACAACAAACCCATTGCCGCACGCTTCATGGGGGACGTCCTCGGTCTTGCCTCCTGTGTGACAATTAACCAGACGAGCGTGAAGGTGCTGCGAGATATGAACGTGAAGGAATCCCCTGGGCGGTGTTACAGTAGGCCTGTGGTGATTTTCAACTTCGCCAACTCTTCCTGTGTCCAATACGGTCAACTCGGTGAAGATAACGAGATTCTGCTGGGCAATCATCGGACAGAAGAATGCCAGTTGCCAAGCCTTAAAATCTTTATTGCAGGAAATTCAGCCTACGAGTACGTCGACTATCTGTTTAAAAGAATGATTGATCTGAGCAGCATTTCCACTGTGGACCMV gB ectodomain, V23 toVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSH267P707 of SEQ ID NO: 1GVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFER(Towne strain)NIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPP*signal sequence M1..A22 (SEQ ID NO: 268) removed Signal Sequence of wt HCMV gBMESRIWCLVVCVNLCIVCLGAA268(Towne)GCN4 CC tri2 trimerizationKIEEILSKQYHIENEIARIKKLIG269domain (Table 9)AAGATCGAGGAGATCCTGTCCAAGCAGTACCACATCGA270GAACGAGATCGCCCGCATCAAGAAGCTGATCGGCT4 fibritin foldon domainGYIPEAPRDGQAYVRKDGEWVLLSTFL271GCN4 (Table 9)RIKQIEDKIEEILSKQYHIENEIARIKKLIG272GCN4 (Table 9)KIYHIENEIARIKKLIG273C-terminal fusion sequenceLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL274(Table 9) Embodiments of the present invention are set out in the following numbered clauses:C1. A polypeptide comprising at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB), wherein the polypeptide comprises a conformation that is not an HCMV gB postfusion conformation.C2. A polypeptide that binds to an HCMV gB prefusion-specific antibody.C3. A polypeptide comprising at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB), wherein the polypeptide is capable of binding to an HCMV gB prefusion-specific antibody.C4. A polypeptide comprising at least one introduced amino acid mutation relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB), wherein the polypeptide is capable of binding to a bis(aryl)thiourea compound.C5. The polypeptide according to clause C3, wherein the compound is N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide.C6. The polypeptide according to clause C1, wherein said polypeptide is characterized by structure coordinates comprising a root mean square deviation (RMSD) of conserved residue backbone atoms when superimposed on backbone atoms described by structural coordinates of Table 1A.C7. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a cysteine substitution.C8. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a mutation that allows a disulfide bond to form.C9. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises an electrostatic mutation.C10. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a phenylalanine substitution.C11. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the amino acid mutation comprises a leucine substitution.C12. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB polypeptide, wherein the mutation stabilizes prefusion conformation of the polypeptide, and wherein the polypeptide specifically binds to an HCMV gB prefusion-specific antibody.C13. A polypeptide comprising a cysteine at any one of the amino acid positions listed in column (ii) of Table 2, as compared to SEQ ID NO: 1.C14. A polypeptide comprising an amino acid substitution at any one of the amino acid positions listed in column (ii) of Table 2, as compared to SEQ ID NO: 1.C15. A polypeptide comprising the mutations Q98C and I653C according to the numbering of SEQ ID NO: 1.C16. A polypeptide comprising the mutations T100C and S269C according to the numbering of SEQ ID NO: 1.C17. A polypeptide comprising the mutations D217C and F584C according to the numbering of SEQ ID NO: 1.C18. A polypeptide comprising the mutations Y242C and K710C according to the numbering of SEQ ID NO: 1.C19. A polypeptide comprising the mutations Y242C and D714C according to the numbering of SEQ ID NO: 1.C20. A polypeptide comprising the mutations S367C and L499C according to the numbering of SEQ ID NO: 1.C21. A polypeptide comprising the mutations T372C and W506C according to the numbering of SEQ ID NO: 1.C22. A polypeptide comprising the mutations S550C and D652C according to the numbering of SEQ ID NO: 1.C23. A polypeptide comprising the mutations T608C and D679C according to the numbering of SEQ ID NO: 1.C24. A polypeptide comprising the mutations K695C and K724C according to the numbering of SEQ ID NO: 1.C25. A polypeptide comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-43, wherein the polypeptide comprises a mutation as compared to SEQ ID NO: 1.C26. The polypeptide according to any one of clauses C1-C25, wherein the polypeptide does not comprise a mutation at any one of the following positions: R562, P577, S587, Y588, G592, G595, L601/H605, C610, L612, P613, Y625, Y627, F632, and K633.C27. The polypeptide according to any one of clauses C1-C26, wherein the polypeptide does not comprise the cytoplasmic tail of HCMV gB.C28. The polypeptide according to any one of clauses C1-C26, wherein the polypeptide does not comprise the transmembrane region.C29. The polypeptide according to any one of clauses C1-C26, wherein the polypeptide comprises the cytoplasmic tail of HCMV gB and does not comprise the transmembrane region.C30. The polypeptide according to any one of clauses C1-C29, wherein the polypeptide does not contain an insect cell pattern of glycosylation.C31. The polypeptide according to any one of clauses C1-C30, wherein the polypeptide exhibits improved solubility or stability, as compared to a native gB in a postfusion conformation.C32. The polypeptide according to any one of clauses C1-C31, wherein the polypeptide is immunogenic.C33. A nucleic acid encoding the polypeptide according to any one of clauses C1-C32.C34. The nucleic acid according to clause C33, wherein the nucleic acid comprises a self-replicating RNA molecule.C35. The nucleic acid according to clause C33, wherein the nucleic acid comprises a modified RNA molecule.C36. A composition comprising a nucleic acid according to any one of clauses C33-C35.C37. A composition comprising the polypeptide according to any one of clauses C1-C32, and further comprising a CMV antigen.C38. The composition according to any one of clauses C36-C37, further comprising any one of the following polypeptides: gO, gH, gL, pUL128, pUL130, pUL131, and any combination thereof.C39. A composition comprising the polypeptide according to any one of clauses C1-C32, and a diluent.C40. A composition comprising the polypeptide according to any one of clauses C1-C32, and an adjuvant.C41. The composition according to any one of clauses C36-C40, wherein the composition is immunogenic.C42. The composition according to any one of clauses C36-C41, for use in eliciting an immune response against cytomegalovirus.C43. A method of eliciting an immune response in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide according to any one of clauses C1-C32.C44. A method for reducing cytomegalovirus viral shedding in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide according to any one of clauses C1-C32.C45. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-43.C46. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-106.C47. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 47-106.C48. A polypeptide comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 57.C49. A composition comprising at least one polynucleotide encoding an HCMV polypeptide selected from any one of gH, gL, UL128, UL130, and UL131; a polynucleotide encoding HCMV gB or a fragment thereof; a polynucleotide encoding pp65 or a fragment thereof; and a pharmaceutically acceptable carrier or dilent.C50. A composition comprising at least one polynucleotide comprising a sequence having at least 95% identity to a sequence selected from any one of SEQ ID NOS: 141-210; a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C51. A composition comprising at least one polynucleotide comprising a sequence having at least 95% identity to a sequence selected from any one of SEQ ID NOS: 224-254; a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C52. A composition comprising at least one polypeptide comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from any one of SEQ ID NOS: 211-223; a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C53. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-43, wherein the polypeptide comprises a mutation as compared to SEQ ID NO: 1.C54. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-106, wherein the polypeptide comprises a mutation as compared to SEQ ID NO: 1.C55. A polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type HCMV gB, wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 47-106, wherein the polypeptide comprises a mutation as compared to SEQ ID NO: 1.C56. A polypeptide comprising the sequence set forth in SEQ ID NO: 57.C57. A composition comprising at least one polynucleotide encoding an HCMV polypeptide selected from any one of gH, gL, UL128, UL130, and UL131; a polynucleotide encoding HCMV gB or a fragment thereof; a polynucleotide encoding pp65 or a fragment thereof; and a pharmaceutically acceptable carrier or dilent.C58. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NOS: 141-210; a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C59. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NOS: 224-254; a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C60. A composition comprising at least one polypeptide comprising a sequence selected from any one of SEQ ID NOS: 211-223; a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C61. The composition according to any one of clause C49-051 and C57-059, wherein the polynucleotide is DNA.C62. The composition according to any one of clause C49-051 and C57-059, wherein the polynucleotide is RNA.C63. The composition according to any one of clause C49-051 and C57-059, wherein at least one polynucleotide comprises at least one chemical modification.C64. The composition according to clause C61, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-I-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.C65. The composition according to any one of clause C49-051 and C57-059, wherein the composition is formulated within a cationic lipid nanoparticle.C66. A composition comprising at least one polynucleotide comprising a sequence selected from any one of SEQ ID NO: 153, SEQ ID NO: 156, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 210, SEQ ID NO: 152, and SEQ ID NO: 158; a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C67. A composition comprising at least one polypeptide comprising a sequence selected from any one of SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, and SEQ ID NO: 217; a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-106; and a pharmaceutically acceptable carrier or dilent.C68. A polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-106.C69. The polypeptide according to clause C68, wherein the sequence comprises SEQ ID NO: 56.C70. The polypeptide according to clause C68, wherein the sequence comprises SEQ ID NO: 57.C71. The polypeptide according to clause C68, wherein the sequence comprises SEQ ID NO: 58.C72. The polypeptide according to clause C68, wherein the sequence comprises SEQ ID NO: 75.C73. A polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-106.C74. A composition comprising a polypeptide comprising the sequence set forth in any one of SEQ ID NOs:1-106; and a diluent.C75. The composition according to clause C74, wherein the sequence does not comprise any one of SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 71, SEQ ID NO: 52, SEQ ID NO: 96, and SEQ ID NO: 50.C76. The composition according to clause C74, further comprising a polypeptide comprising any one sequence selected from SEQ ID NOS: 211-224.C77. A composition comprising a polynucleotide encoding a polypeptide comprising the sequence selected from any one of SEQ ID NOs:1-106; and a diluent.C78. The composition according to clause C77, further comprising a polynucleotide comprising a sequence selected from any one of SEQ ID NOS: 141-210.C79. The composition according to clause C77, further comprising a polynucleotide comprising a sequence selected from any one of SEQ ID NOS: 224-254.C80. A method of eliciting an immune response in a mammal, comprising administering an effective amount of a composition comprising a polypeptide comprising the sequence set forth in any one of SEQ ID NOs:1-106; and a diluent.C81. A method of eliciting an immune response in a mammal, comprising administering an effective amount of a composition comprising a polynucleotide encoding a polypeptide comprising the sequence set forth in any one of SEQ ID NOs:1-106; and a diluent. | 262,623 |
11857623 | DETAILED DESCRIPTION OF THE INVENTION The description and specific examples below, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested. Embodiments of this technology include, but are not limited to the following. One embodiment of the invention is directed to a protein construct comprising an IgE motif segment and a scaffold, preferably an exogenous non-human protein scaffold, into which the IgE motif segment is embedded. The scaffold holds the IgE segment in a conformation suitable for inducing an immune response against IgE. This protein construct may be used as an antigen, immunogen or vaccine to induce immune responses against the IgE motif segment. The induced anti-IgE immune responses reduce the severity of IgE mediated allergy and other immunological phenomena and thus may be used to treat a subject having a disease, disorder or condition associated with or mediated by IgE. Another embodiment of the invention is directed to a protein construct comprising a scaffold segment and a motif segment. The scaffold segment is selected to present the motif segment which comprises T cell or B cell epitopes or determinants of IgE, especially of human IgE. Typically, the scaffold segment is a protein other than IgE or is exogenous to human IgE, or exogenous to humans. A protein construct may comprise the entire scaffold protein substituted with the IgE motif or active or epitopic fragments thereof, for example, a protein construct where immunologically nonessential amino acid sequences are removed. A protein construct may also comprise a complex or conjugate containing the protein construct or an active fragment thereof, such as a larger chimeric or fusion protein, a bead or other substrate to which the protein construct is noncovalently or covalently bound, or a composition, such as an emulsion or liposome containing the protein construct or an immunogenically active fragment thereof capable of inducing antibodies that recognize the IgE motif. Typically the scaffold segment is modified, by replacement of a scaffold subsegment with the amino acid residues comprising the IgE motif segment. In other words, the scaffold protein is modified by insertion or replacement of the amino acid residues of the IgE motif. In one embodiment, the motif comprises an IgE FG loop (SEQ ID NO: 2) or an IgE R loop (SEQ ID NO: 3) or epitopic portions thereof. However, other epitopes of IgE may be used instead of, or in addition to, the FG or R loop, such as the BC or DE loops, or epitopic portions thereof described byFIG.2. In one embodiment the protein construct as disclosed herein comprises a scaffold segment corresponding to an extracellular adherence protein (“EAP”) domain of a member of the genusStaphylococcus, especiallyStaphylococcus aureus, that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the EAP of SEQ ID NO: 5 or which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or >20 deletions, insertions, or substitutions of amino acid residues to the sequence of SEQ ID NO:5. In some preferred embodiments, the protein construct may comprise the extracellular adherence protein 1YN3 chain A (SEQ ID NO: 5) or a protein that is at least 90, 95 or 99% identical thereto. Other scaffold proteins may be used in some embodiments such as scaffold proteins comprising 3LDZ chain A comprising SEQ ID NO: 17 or a protein that is at least 90-95% identical thereto; scaffolds comprising 3EGR chain A comprising SEQ ID NO: 19 or a protein that is at least 90-95% identical thereto; or scaffolds comprising 3Q4H chain B comprising SEQ ID NO: 21 or a protein that is at least 90-95% identical thereto. In the 3Q4H structure, residues QGDTGMTY (SEQ ID NO: 23) at positions 44-51 were replaced by the R motif; in the 3LDZ structure residues ATSEMNTAED (SEQ ID NO: 24) at positions 16-25 were replaced by the FG motif; and in the 3EGR structure residues VRSKQGLEHK (SEQ ID NO: 25) at positions 13-22 were replaced by the FG motif. In some embodiments, the protein construct comprises an IgE motif segment comprising the FG loop described by SEQ ID NO: 2. For example, the IgE motif may comprise any one of the protein sequences described by SEQ ID NOS: 7 to 16 which each contain said FG loop. In another embodiment, the IgE motif comprises an IgE R loop (SEQ ID NO: 3), BC loop, or DE loop segment in combination with a scaffold segment of the extracellular adherence protein 1YN3 chain A (SEQ ID NO: 5), 3LDZ (SEQ ID NO: 17) or 3EGR (SEQ ID NO: 19) or a protein that is at least 90, 95 or 100% identical thereto. In some embodiments, the protein construct disclosed herein will comprise a variant scaffold protein or variant IgE motif, that can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, insertions or substitutions of an amino acid residue of the scaffold or motif or have at least 70, 80, 90, 95, 99 or up to 100% sequence identity with a disclosed amino acid sequence, such as the IgE motif and scaffold proteins identified by sequence identifiers herein. BLASTP may be used to identify an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence similarity to a reference amino acid sequence, such as those described herein using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. Default settings for BLASTP are described by and incorporated by reference to http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last accessed Jan. 9, 2020). This disclosure also encompasses degenerate polynucleotide sequences encoding the proteins disclosed herein which are deduced from the corresponding amino acid sequences using the genetic code. Any scaffold protein that effectively presents the IgE epitopes in the IgE motif segment to a subject's immune system may be used. However, the inventors have discovered that only some protein constructs are capable of efficient presentation of IgE epitopes. Based on structural analysis, preferred scaffolds include the extracellular adherence protein 1YN3 chain A (SEQ ID NO: 5) or a protein that is at least 90, 95, 96, 97, 98, 99 or 100% identical thereto or aStaphylococcusextracellular adherence protein that comprises ITVNGTSQNI (SEQ ID NO: 6) into which epitopes of the FG loop or other IgE loops described herein may be substituted or inserted. Compositions. Another aspect of the invention is directed to a composition, including, but not limited to, an antigenic, immunogenic, or vaccinogenic composition that comprises a protein construct as disclosed herein comprising a scaffold segment and an IgE motif segment. Typically, such a composition will include a pharmaceutically acceptable excipient or carrier and may further contain an adjuvant or other active agents. The term carrier encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, for example, for intravenous administration a carrier may be sodium chloride 0.9% or mixtures of normal saline with glucose or mannose. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g.,Remington's Pharmaceutical Sciences,21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which is incorporated herein by reference in its entirety. An adjuvant is a pharmacological or agent that modifies the effect of other agents. Adjuvants may be added to a protein construct as disclosed herein to boost the humoral or cellular immune responses and produce more anti-IgE antibodies and longer-lasting immunity, thus minimizing the dose of protein construct needed. Adjuvants that may be compounded with, or otherwise used along with the protein construct disclosed herein include, but are not limited to, inorganic compounds including alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oil or paraffin oil; bacterial products or their immunologically active fractions, such as those derived killedBordatella pertussis, Mycobacterium bovis, or bacterial toxoids; organics such as squalene; detergents such as Quil A, saponins such asQuillaja, soybean orpolygala senega; cytokines such as IL-1, IL-2 or IL-12; Freund's complete adjuvant or Freund's incomplete adjuvant; and food based oils like Adjuvant 65, which is a product based on peanut oil. Those skilled in the medical or immunological arts may select an appropriate adjuvant based on the type of patient and mode of administration of the protein construct of the invention. For therapeutic purposes, formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. The term parenteral, as used herein, includes intravenous, intravesical, intraperitoneal, subcutaneous, intramuscular, intralesional, intracranial, intrapulmonal, intracardial, intrasternal, and sublingual injections, or infusion techniques. These solutions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration, preferably in a digestion-resistant form such as an enteric coating. The active ingredient can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting ingredients and suspending ingredients. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting ingredients such as those discussed above are also useful. Administration to the respiratory system may be accomplished using a drug delivery device such as a nebulize to administer the protein construct, DNA encoding it, or antibodies induced to the protein construction in an inhalable form. Nebulizers for treatment of cystic fibrosis, asthma, COPD and other respiratory diseases are known and incorporated by reference to hypertext transfer protocol secure://en.wikipedia.org/wiki/Nebulizer. These include soft mist inhalers, jet nebulizers, ultrasonic wave nebulizers, and nebulizers using vibrating mesh technology. A metered-dosage inhaler is another drug delivery device that delivers a selected or metered amount of a medication, such as the protein construct disclosed herein, DNA encoding it, or an antibody induced to it. Typically, this device produces and releases an aerosol of micrometer-sized particles that are inhaled. In some cases, the particles may be a dry powder in others as a mist or in a semiliquid form. Metered-dose inhalers and their various components, propellants, excipients and other elements are described by and incorporated by reference to hypertext transfer protocol secure://en.wikipedia.org/wiki/Metered-dose_inhaler. An inhalable composition may be formulated in the form of a hydrofluoroalkane inhaler or HFA (metered dose inhaler or MDI), dry powder inhaler (DPI), or as a nebulizer solution. Methods of Treatment. Another aspect of the invention is directed to method for preventing, treating or reducing the severity of a disease, disorder or condition associated with IgE comprising, consisting essentially of, or consisting administering the protein construct as disclosed herein to subject in need thereof; an antibody induced against the protein construct, or DNA encoding the protein construct. Active Vaccination. An active vaccine containing an IgE motif containing protein construct disclosed herein may be administered to prevent, treat or reduce the severity of IgE-mediated symptoms. Such symptoms include those commonly associated with allergy or anaphylaxis including, but not limited to “hives” (red blotches or welts that itch), mild to severe swelling on the skin; tearing, redness, or itching of the eyes; a clear discharge, itch, congestion of the nose; an itch, lip swelling, or tongue swelling of the mouth; a tightness, trouble speaking, or trouble inhaling associated with the throat; shortness of breath, rapid breathing, cough, or wheeze associated with the lungs; repeated vomiting, nausea, abdominal pain, or diarrhea associated with the stomach; a weak pulse, loss of consciousness associated with the circulatory system; or anxiety, agitation, loss of consciousness associated with brain or nervous system functions. The immunogen or vaccine comprising the protein construction may be used to treat IgE-mediated food allergies. Such allergies cause a child's or adult's system to react abnormally when exposed to one or more specific foods such as milk, egg, wheat or nuts. IgE mediated food allergies typically occur quickly within a few minutes to a few hours and are caused by pre-existing allergen-specific immunoglobulin E (IgE) antibody found in a subject's blood stream. The most common food allergens include: milk, egg, soy, wheat, peanut, tree nuts, fish, and shellfish. All of these foods can trigger anaphylaxis, a severe, whole-body allergic reaction) in patients who are allergic. The protein construct as disclosed herein may be used in the treatment or prevention of IgE associated or mediated disorders such as those described above. In some embodiments, the subject will be a male or female no more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 21 years of age. In other embodiments, the subject will be a male or female more than 18, 21, 25, 30, 40, 50, 60, 70, 80 or 90 years of age. In some embodiments the protein construct is administered parenterally to the subject in need thereof to induce antibodies to IgE. For example, the protein construct or a composition containing it may be administered by subcutaneous, intramuscular, or intravenous routes. In other embodiments it may be administered to the respiratory system of the subject in need thereof. For example, it may be administered as an aerosol or powder that is sized or formulated to reach and deposit in various areas of the upper respiratory tract such as the nasal cavity, sinuses, pharynx or larynx; or sized and formulated reach and deposit in various areas of the lower respiratory tract such as the trachea, bronchi, diaphragm or lungs. In other embodiments, the protein construct is administered onto a non-respiratory system mucous membrane, such as those of the eyes, gastrointestinal tract, urinary tract or sexual organs or to the skin of a subject in need thereof. Passive Vaccination. In an alternative embodiment, the protein construct is used to induce antibodies against IgE which are passively administered to a subject in need of treatment for an IgE mediated condition. For example, the protein construct is used to produce polyclonal or monoclonal antibodies that recognize IgE. Such antibodies may be of any isotype, such as IgM, IgG, IgA or IgE. Such passive vaccines may be administered parenterally, into the respiratory system, or onto a mucous membrane or skin. DNA vaccination is a technique for protecting against a disorder or disease by injection with engineered DNA so cells directly produce antigenic IgE determinants or epitopes, producing a protective immunological response or a response the reduces the severity of allergy or other IgE mediated phenomena. DNA vaccines have potential advantages over conventional vaccines, including the ability to induce a wider range of immune response types. In some embodiments of the invention, the protein construct as disclosed herein may be encoded and expressed by a DNA vaccine. The DNA encoding the protein construct is injected into the body and taken up by cells, whose normal metabolic processes synthesize proteins based on the genetic code in the plasmid or other construct that they have taken up. A DNA vaccine may be administered by intramuscular or intradermal delivery, by gene gun, by jet injection or by administration of DNA in liposomal form. In some embodiments, a DNA vaccine, such as a liposome containing DNA encoding the protein construct may be administer to the nasal mucosa or other surfaces of the respiratory system. In some embodiments, DNA encoding the protein construct as disclosed herein may be replicated or synthesized by known methods. The DNA is then formulated for administration to a subject, for example, by pulmonary, intravenous, subcutaneous, intramuscular, intrapulmonary, or intralymphatic administration. DNA-based vaccines and methods of their use are known and are incorporated by reference to Tregoning, J S, et al.,Using Plasmids as DNA Vaccines for Infectious Diseases. Microbiol Spectr. 2014 Dec.; 2(6). doi: 10.1128/microbiolspec.PLAS-0028-2014; Ramirez, L A, et al.,Therapeutic and prophylactic DNA vaccines for HIV-1. Expert Opin Biol Ther. 2013 April; 13(4):563-73. doi: 10.1517/14712598.2013.758709; Williams, J A,Improving DNA vaccine performance through vector design. Curr Gene Ther. 2014; 14(3):170-89. Method for Recombinant Production of a Protein Construct. Another aspect of the invention is directed to a method for making the protein construct disclosed herein. This method typically involves construction of a chimeric DNA molecule that encodes the scaffold and IgE motifs of the protein construct and optionally a protein tag. The chimeric DNA molecule may be constructed by methods known in the art. Once constructed it is transformed or transfected into a host cell usually in the form of an expression vector which then expresses the protein construct. Recombinant or chimeric DNA encoding a protein construct as disclosed herein, including both scaffold segments and IgE motif segments, can be designed by careful selection and evaluation of putative scaffold and IgE motif sequences as shown by the Example. Typically, this will involve screening a protein data base for a suitable scaffold protein, evaluating the conformation of particular scaffolds and IgE motifs by modelling then on a computer, for example, the conformation of combinations of an IgE motif comprising an FG loop of SEQ ID NO: 2 or a R loop of SEQ ID NO: 3, and scaffolds such as that of 1YN3 chain, and engineering the sequence of the protein construct so that it folds in a way to efficiently present the IgE FG loop or R loop to the immune system. A search on the entire PDB database for possible scaffolds for the IgE motif was performed using Rosetta which can also be performed using the MotifGraftMover from PyRosetta. Once several scaffolds were found each scaffold was sequence designed using the RosettaDesign fixed-backbone protocol to find a sequence that will fold to the desired backbone, this procedure was followed by forward folding using the AbinitioRelax protocol in Rosetta to simulate the folding of the newly designed structure. Several rounds of design and forward folding were performed on each structure (sometimes using manual residue mutations) until a successful forward fold was achieved. Since the backbone of the Y1N3 structure was ideal all the sequence designs were successful at the forward fold simulations. A flowchart showing the preferred steps of this process is shown inFIG.8. Additionally, the 1YN3, 3LDZ, and 3EGR scaffolds were sequence designed and their sequences were changed from the original published sequences. The computational algorithm used was standard and simple. In addition to a high level (e.g, at least 95%) of sequence identity, the backbone similarity can be described through a “root means square deviation” metric value. Advantageously a cutoff point of a RMSD<1, 2, 3, 4, 5, 6, 7, 8, 9 to 10 angstroms can be used because the closer the RMSD value is to 0 the more identical the backbone is to a designed scaffold; see e.g. Kufareva, Irina, and Ruben Abagyan.Methods of protein structure comparison. Methods in molecular biology (Clifton, N.J.) vol. 857 (2012): 231-57. doi:10.1007/978-1-61779-588-6_10 which is incorporated by reference. The root-mean-square deviation of atomic positions or simply root-mean-square deviation, RMSD, is a measure of the average distance between the atoms, usually backbone atoms, of superimposed proteins. Root mean square deviation (RMSD) between two structures can be determined as is well-known to one of skill in the art, for example, using MOE v2016.0802 (Chemical Computing Group). Similarity is typically measured in three-dimensional structure by the RMSD of the Ca atomic coordinates after optimal rigid body superposition. For a variant scaffold protein or variant IgE motif as disclosed herein the atomic positions of backbone atoms may vary by up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 angstroms from a reference protein such as a protein described by SEQ ID NOS: 5, 17, 19 or 21 or to a segment of reference protein having at least 10, 15, 20, 25 or 30 contiguous residues. Preferably, the RMSD is calculated from the full length of the reference protein structure and not from a shorter segment. Recombinant Expression. The protein constructs as disclosed herein may be made by any technique known to those of skill in the art, including by expression of the constructs through standard molecular biological techniques including by recombinant protein expression or by chemical synthesis. Typically, for recombinant expression of a protein construct, DNA encoding the construct is synthesized or spliced together from a source of the scaffold and motif DNA segments, and is cloned downstream of a promoter in an expression vector. This vector is then introduced into a host cell, and the cell's protein synthesis machinery produces the protein construct. Thus, another embodiment of the invention is a method for expressing DNA encoding a protein construct as disclosed herein, thereby producing the protein construct. The term “protein construct” as used herein refers to a protein comprising a scaffold segment as described herein and an IgE motif segment, preferably where the IgE motif segment is substituted for a stretch of amino acids in the scaffold protein segment. A “recombinant host cell,” as used herein, is a cell comprising one or more recombinant nucleic acid sequences or transgenes not naturally present in the cell which encode the protein construct of the invention. These transgenes are expressed in the host cell to produce recombinant protein constructs comprising the IgE motif described herein that are encoded by these nucleic acid sequences when these cells are cultured under conditions conducive to expression of nucleic acid sequences. In some instances, the host cell may be cell within a subject which received a DNA-based vaccine. The host cell, as used herein, can be present in the form of a culture from a clone that is derived from a single host cell wherein the recombinant DNA or transgenes have been introduced. It is well known to those skilled in the art that sequences capable of driving such expression can be functionally linked to the nucleic acid sequences encoding the recombinant proteins, such as the protein construct disclosed herein. “Functionally linked” is meant to describe that the nucleic acid sequences encoding the protein construct or fragments or precursors thereof which are linked to the sequences capable of driving expression such that these sequences can drive expression of the protein construct or precursors thereof. Useful expression systems are available in the art, for example, the mammalian protein expression, insect protein expression, yeast protein expression, bacterial protein expression, or algal protein expression systems of Invitrogen. Where the sequence encoding the polypeptide of interest, namely the protein construct of the invention, is properly inserted with reference to sequences governing the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful to produce the polypeptide of interest, referred to as expression. Sequences driving expression may include promoters, enhancers and the like, and combinations thereof. These should be capable of functioning in the host cell, thereby driving expression of the nucleic acid sequences that are functionally linked to them. Promoters can be constitutive or regulated and can be obtained from various sources, including viruses, prokaryotic or eukaryotic sources, or artificially designed. Expression of nucleic acids of interest may be from the natural promoter or derivative thereof or from an entirely heterologous promoter. Any promoter or enhancer/promoter capable of driving expression of the sequence of interest in the host cell is suitable in the invention. The skilled artisan will be aware that the expression sequences used in the invention may suitably be combined with elements that can stabilize or enhance expression. These may enhance the stability and/or levels of expression. Protein production in recombinant host cells has been extensively described, e.g., in Current Protocols in Protein Science,Production of Recombinant Proteins, Jan. 1, 2018 (updated Oct. 7, 2019), Online ISSN:1934-3663, the entirety of which is incorporated herein by reference. Culturing a cell is done to enable it to metabolize, grow, divide, and/or produce recombinant proteins of interest. This can be accomplished by methods well known to persons skilled in the art and includes, but is not limited to, providing nutrients for the cell. Such methods comprise growth adhering to surfaces, growth in suspension, or combinations thereof. Several culturing conditions can be optimized by methods well known in the art to optimize protein production yields. Culturing can be done, for instance, in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, hollow fiber, and the like. “Host cells,” may be any host cell capable of expressing recombinant DNA molecules encoding the protein construct presenting IgE epitopes, including bacteria such asEscherichia(e.g.,E. coli),Enterobocter, Salmonella, Bacillus, Pseudomonas, Streptomyces, yeasts such asS. cerevisiae, K lactis, P. pastoris, Candida, oryarrowia, filamentous fungi such asNeurospora, Aspergillus oryzae, Aspergillus nidulansandAspergillus niger, insect cells such asSpodoptera frugiperdaSF-9 or SF-21 cells, mammalian cells such as Chinese hamster ovary (CHO) cells, BHK cells, mouse cells including SP2/0 cells and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3A cells, and the like. The protein constructs are expressed in the host cells and may be recovered from the cells or, preferably, from the cell culture medium, by methods generally known to persons skilled in the art. Such methods may include precipitation, centrifugation, filtration, size-exclusion chromatography, affinity chromatography, cation- and/or anion-exchange chromatography, hydrophobic interaction chromatography, and the like. Codon optimization. A protein construct as disclosed herein may be encoded by a polynucleotide or vector comprising a polynucleotide that has been codon optimized for expression in a particular host cell. Thus, different functionally equivalent codons that encode the same amino acid may be substituted for one another, such as the six codons for arginine, leucine, or serine, the four codons for alanine, glycine, proline, threonine, or valine etc. Codon optimization methods or programs which optimize codon usage for various host cells including those disclosed herein, are incorporated by reference to hypertext transfer protocol secure://www.novoprolabs.com/tools/codon-optimization; hypertext transfer protocol secure://academic.oup.com/bioinformatics/article/30/15/2210/2391162; and hypertext transfer protocol://bioinfo.bti.a-star.edu.sg/COOL/. In some embodiments, a polynucleotide sequence encoding a protein construct as disclosed herein may be optimized using one of the above mentioned methods or programs or a publically or commercially available program for expression by human or mammalian cells, such as by Chinese hamster ovary cells, myeloma lymphoblastoid cells such as NS0 cells, or by fully human host cells such as human embryonic kidney cells like HEK-293, human embryonic retinal cells like Crucell's Per.C6, human amniocyte cells like Glycotope and CEVEC; by baculovirus-infected cells such as Sf9, Sf21, High Five strains; filamentous fungi such as by yeasts such asS. cerevisiaeorPichia pastoris, or by prokaryotic expression systems such as those usingEscherichia coli, corynebacterium, Bacillus subtilis, orPseudomonas fluorescens. Purification of the protein construct. The recombinantly expressed protein construct can be purified by methods known in the art including by solvent or surfactant extraction, removal of insoluble components by filtration or centrifugation and affinity purification. In some embodiments, the protein construct is purified by affinity purification using antibodies that bind to the scaffold, IgE motif or to an expression or purification tag attached to the protein construct. Protein tags may be produced by grafting DNA encoding them onto a polynucleotide construct encoding the protein construct and then expressing the grafted protein construct. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes including to facilitate their purification by antibodies or other ligands that bind to the tag. Such tags include but are not limited to affinity tags which are appended to a protein so that it can be purified from a crude biological source using an affinity technique. These include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag, which binds to metal matrices. Other protein tags include those described by and incorporated by reference to hypertext transfer protocol secure://en.wikipedia.org/wiki/Protein tag (as last accessed Jan. 8, 2020). Those skill in the art may select an appropriate tag and/or other mode of purification to isolate the protein construct as disclosed herein. The proteins may be expressed in systems, such as those using prokaryotic vectors and cells, that do not glycosylate proteins or in systems using eukaryotic vectors and cells, such as Chinese Hamster Ovary cells, which glycosylate protein. Preferably in terms of function, these proteins are soluble in a culture medium, in the cytoplasm of host cells, and in physiological acceptable solvents. The degree of solubility depends also on selection of the expression vector and host cells as well as choice of solvent and other chemical and physical factors such as pH, salt content, and temperature. Examples The following steps were used to generate a database of scaffold structures as well as isolate the IgE motif, graft it onto a scaffold, then design the scaffold to fold onto the designed structure. Motif Determination and Excision. The FG motif is part of the Ig epsilon C region, chain B (2Y7Q_B; SEQ ID NO: 1). This motif comprises residues 420-429 of this sequence (residues 200-209 of SEQ ID NO: 1): VTHPHLPRAL (SEQ ID NO: 2) and chosen due to its very close proximity to the receptor binding site (“R”) which comprises residues 331-338 of 2Y7Q chain B: SNPRGVSA, residues 111-118 of SEQ ID NO: 3). The numbering is PBD centric and takes into account that chain B of the 1YN3 structure starts at position 229 of the PBD structure (and not the FASTA sequence from NLM). The FG motif is a ridged structure and the motif resembles a heart shape with an anchoring 425 lysine pointing into the core fixing its shape. The FG motif was selected for further work because the receptor binding site (“R”) was not anchored and had a higher degree of movement its surrounding area and the was not as well modelled in the crystal structure. The FG motif was isolated along with the IgE receptor, 2Y7Q chain A; SEQ ID NO: 4, as separate files in preparation for grafting. Scaffold Database Generation. The scaffold database was generated by downloading the entire PDB database, then isolating only protein structures and separating each chain into separate .pdb files. Each structure was cleaned (removed of any non-peptide atoms) then passed through (scored) by the Rosetta modelling software to make sure each structure will not crash the software. Structures that were not satisfactory were discarded. Such procedures are described by and incorporated by reference to Leaver-Fay, et al. 2011. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol 487: 545-74. doi: 10.1016/b978-0-12-381270-4.00019-6. For an alternative embodiment a script was developed that produces a better, smaller, and more targeted database, but was not used here. Motif Grafting. The desired motif between positions 420 and 429 in the 2Y7Q chain B protein was isolated along with the receptor in chain A then a grafting search was performed that matched the backbone of the motif to backbones within the database, if there was a match within an RMSD value of 1.0 A or less the motif was grafted onto the scaffold structure (replacing the original backbone) and measured for its clash with the receptor (to make sure the backbone was not grafted inward or was buried within the structure). This protocol was developed by and is incorporated by reference to Azoitei, M. L., et al., 2011. Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334: 373-376. doi: 10.1126/science.1209368; and Azoitei, M. L., et al., 2012. Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope. J Mol Biol 415: 175-92. doi: 10.1016/j.jmb.2011.10.003. Selective Fixed-Backbone Design. The final structure was tested for folding and failed. Accordingly, some human guided mutations were employed to push the structure to fold onto its designed structure. After many failed attempts the fixed-backbone design protocol was employed, where the side chain sequence of the structure was stochastically mutated and packed using a rotated library to find the lowest energy structure that would fold into the designed structure. Such procedures are described by and incorporated by reference to Kuhlman, B., et al., 2003. Design of a novel globular protein fold with atomic-level accuracy. Science 302: 1364-1368. doi: 10.1126/science.1089427; Dantas, G., et al., 2003. A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. J Mol Biol 332: 449-60; Leaver-Fay, A., et al., 2005. An adaptive dynamic programming algorithm for the side chain placement problem. Pac Symp Biocomput 16-27; Hu, X., et al., 2007. High-resolution design of a protein loop. Proc Natl Acad Sci USA 104: 17668-73. doi: 10.1073/pnas.0707977104; and Andrew Leaver-Fay, et al. 2005. Rotamer-pair energy calculations using a trie data structure. Mallorca, Spain: Springer-Verlag. Using this protocol, the inventors changed the Rosetta Energy Function 2015 (REF15). energy function weights to include aa_rep 1.0, aspartimid_penalty 1.0, buried_unsatisfied_penalty 1.0, and approximate_buried_unsat_penalt 5.0 which assisted in designing an adequate sequence that both fits the backbone structure and increases the energy gap between the desired structure and any other possible undesired fold. Folding Simulation. To get inside into whether the design process was successful, the structures were simulated for their folding using the Abinitio protocol, where the sequence is folded using first principals and some statistical weights through the REF15 scoring function, and to reduce the folding space and speed up the search for the global minima fragment were developed from the FASTA sequence, were backbone torsion angles are statistically analysed and inserted to help the algorithm fold the structure. Such procedures are described by an incorporated by reference to Raman, S., et al., 2009. Structure prediction for GASPS with all atom refinement using Rosetta. Proteins 77 Suppl 9: 89-99. doi: 10.1002/prot.22540; Bradley, P., et al., 2005. Toward high-resolution de novo structure prediction for small proteins. Science 309: 1868-71. doi: 10.1126/science.1113801; Bonneau, R., et al. 2002. De novo prediction of three-dimensional structures for major protein families. J Mol Biol 322: 65-78; Bonneau, R., et al., 2001. Rosetta in CASP4: progress in ab initio protein structure prediction. Proteins Suppl 5: 119-26; Simons, K. T., et al. 1999. Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins. Proteins 34: 82-95; and Simons, K. T., et al., 1997. Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. J Mal Biol 268: 209-25. doi: 10.1006/jmbi.1997.0959. Results. Analysis of the motif position revealed that the R loop and the FG loop from the human IgE (1Y7Q) were the best candidates for an IgE-targeted vaccine due to their proximity to the binding site on the a chain of the FcεRI receptor (FIG.2). After several attempts at grafting and designing the R loop, the FG loop was selected for further work because it has an inward pointing leucine resulting in a ridged loop structure. In contrast, the R loop was found to have a high degree of angle freedom which resulted in a wide range of different structures when grafted, seeFIG.3. The scaffold search algorithm resulted in the FG loop motif being grafted onto the 1YN3 structure as well as several other structures; seeFIG.4. The structure and other description of 1YN3 and EAPs in general is incorporated by reference to Geisbrecht, B. V., et al. 2005. The crystal structures of extracellular adherence protein(“EAP”)domains from Staphylococcus aureus reveal an unexpected homology to bacterial superantigens. J Biol Chem 280: 17243-50. doi: 10.1074/jbc.M412311200. The 1YN3 structure (SEQ ID NO: 5) was chosen since it had a backbone that was easily simulated by forward folding using the AbinitioRelax protocol; seeFIG.5and because it would be easy to crystallize for structural evaluation. The 1YN3 protein is an EAP domain fromStaphylococcus aureuswhich has been previously expressed inEscherichia colihost cells prior to its crystallization. The FG motif was grafted between positions 164 and 173 in the 1YN3 structure and replaced the sequence ITVNGTSQNI (SEQ ID NO: 6) with VTHPHLPRAL (SEQ ID NO: 2); seeFIG.6. However, the inventors determined using AbinitioRelax that the freshly grafted structure failed a forward fold, and attributed this failure to the addition of the motif backbone and side chains which severely disrupted the stability of the entire structure. To overcome this problem, with the exception of the FG motif, the entire secondary structure was redesigned by changing the side chains while fixing the backbone to stabilize the structure and accommodate the redesigned motif backbone and side chains. To compensate for a failure rate between a successful forward fold and a successful crystal structure, the inventors repeated the sequence design step ten times producing ten structures (SEQ ID NOS: 7-16) all of which passed a successful forward fold;FIGS.7A-7AD. This process increases the probability of synthesizing a correctly folded vaccine structure as determined by a crystallography evaluation. The ten structures of SEQ ID NOS: 7-16 each contain the IgE motif sequence, but flanking sequences have been designed using a Rosetta Design fixed-backbone computational protocol. This protocol uses a Monte Carlo based method to design protein sequences that fit the backbone of 1YN3. The RosettaDesign protocol also uses the REF2015 energy function to determine the best sequence for each run. All structures were predicted to fold within a sub angstrom level of the designed structure, giving high confidence that a biologically synthesized protein construct would retain the same structure. Preferably, prior to structural evaluation in vivo, each structure is crystallized to definitively confirm the resulting protein construct is correctly folded. As shown herein, the inventors have developed a protocol for computationally designing proteins that correctly display the three dimensional structure of a strategic motif of the IgE molecule, where the motif is grafted onto scaffold proteins, opening the possibility of using such protein structures as a vaccine against self-IgE and permanently shutting down the allergy pathway regardless of the offending allergen (a pan-anti-allergy vaccine). The resulting structures showed agreement in their final folds when simulated with different computational folding algorithms and can be verified by solving their structures through X-Ray crystallography. Furthermore, the efficacy of the proteins in pushing the immune system into developing high-affinity antibodies against self-IgE at a higher binding affinity than IgE/FceRI receptor's binding affinity could not be computationally simulated, and thus must be tested on animals to reach a definitive answer. The script that was used to design these proteins is available at this GitHub repository (hypertext transfer protocol secure://github.com/sarisabban/VaxDesign; last accessed Apr. 22, 2020) which includes an extensive README file that explains how to use it. Terminology. Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any or all combinations of one or more of the associated items listed and may be abbreviated as “/”. As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology. Polynucleotide sequences encoding the protein sequences disclosed herein are described by reference to the corresponding protein sequence. Such polynucleotide sequences are deduced using the genetic code and encompass any codons which encode the corresponding protein. Due to the degeneracy of the genetic code multiple polynucleotide codons may be used to encode the same amino acid residue. In some embodiments, polynucleotide sequences may be codon optimized for expression in a particular host cell. However, the polynucleotide sequences described herein also encompass those native polynucleotide sequences already known in the art, such as those associated with the various accession numbers of the proteins disclosed herein. All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears. Specifically, unless otherwise specified the accession numbers such as those from the PDB refer to the last versions available on the filing date of this application. All such accession numbers, and the ancillary information they contain, are incorporated by reference. | 46,679 |
11857624 | EXAMPLE 1: OPCML TUMOR SUPPRESSOR FUNCTIONS AS A REPRESSOR-ADAPTOR, NEGATIVELY REGULATING RECEPTOR TYROSINE KINASES IN BOTH NORMAL OVARIAN SURFACE EPITHELIUM AND OVARIAN CANCER Summary Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy. Its molecular basis is poorly understood but involves dysfunction of p53 (Hall et al (2004) “Critical evaluation of p53 as a prognostic marker in ovarian cancer”. Expert Reviews in Molecular Medicine 6: 1-20), BRCA1 and −2 (Radice (2002) “Mutations of BRCA genes in hereditary breast and ovarian cancer”J Exp Clin Cancer Res.21(3 Suppl): 9-12), PI3K (Meng et al (2002) “Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway”Cellular Signaling18(12): 2262-2271), and growth factor and angiogenic signaling pathways (Maihle et al (2002) “EGF/ErbB receptor family in ovarian cancer” Cancer Treat Res. 107: 247-58; Le Page et al (2006) “Gene expression profiling of primary cultures of ovarian epithelial cells identifies novel molecular classifiers of ovarian cancer”British Journal of Cancer94: 436-445; Birrer et al (2007) “Whole genome oligonucleotide-based array comparative genomic hybridization analysis identified Fibroblast Growth Factor 1 as a prognostic marker for advanced-stage serous ovarian adenocarcinomas”Journal of Clinical Oncology25(16): 2281-2287; Trinh et al (2009) “The VEGF pathway and the AKT/mTOR/p70S6K1 signaling pathway in human epithelial ovarian cancer”British Journal of Cancer100: 971-978; and Lafky et al (2008) “Clinical implications of the ErbB/epidermal growth factor (EGF) receptor family and its ligands in ovarian cancer”Biochim Biophys Acta.1785(2): 232-65). We previously identified opioid binding protein cell adhesion molecule (OPCML) as epigenetically inactivated in 83% of ovarian cancers and demonstrated that it was a functional tumor suppressor in vitro and in vivo (Sellar et al (2003) “OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer”Nat. Genet.34(3): 337-43). Here, we show that OPCML interacts with and downregulates HER2 and FGFR1 proteins, leading to inhibition of those signaling pathways, with consequent inhibition of in-vitro growth in SK-OV-3 ovarian cancer cells. siRNA knockdown of physiologically expressed OPCML in OSE-C2 normal ovarian surface epithelial cells strongly upregulated HER2 and FGFR1. OPCML sensitized HER2 positive ovarian cancer cells to lapatinib and trastuzumab in vitro and was a good prognostic indicator in patients with HER2 positive ovarian cancer. The finding that OPCML actively mediates negative regulation of multiple RTK pathways opens novel research avenues in normal cell and cancer biology. Experimental Procedures Antibodies The polyclonal goat and monoclonal mouse anti-OPCML antibodies were purchased from R&D. Anti-HER2 antibodies were purchased from Calbiochem (anti-ErbB2 (Ab-4) and (3B5) mouse MAbs). Anti-EGFR antibody was from R&D Systems. Anti-EGFR goat pAb-cat no AF-231. Phospho-specific EGFR and HER2 antibodies were purchased from AbCam. Anti-HA antibody was from Santa Cruz Biotechnology (Santa Cruz CA) HRP-conjugated secondary antibodies were from Dako. Alexa-Fluor 488 goat anti-rabbit IgG, Alexa-Fluor 555 goat anti-mouse were from Molecular Probes (Eugene, OR). Cell Culture The SK-OV-3 derived OPCML expressing lines (SKOBS-3.5, BKS2.1 and empty vector SKOBS-V1.2) were described previously (Sellar et al, 2003). Stimulation time courses were undertaken with 50 ng/ml human recombinant epidermal growth factor (hrEGF-Promega) following serum-starvation overnight. Plasmid Constructs The OPCML cDNA expression plasmids in pcDNA3.1zeo previously described (Sellar et al, 2003) were used for transient transfections. The cDNAs encoding all three Ig domains and domains 1 and 2 were generated by PCR and introduced into the bacterial GST-fusion expression vector pGEX-6P-1 (GE-Healthcare) and sequenced to confirm their fidelity. Vector pIRES-AcGFP1 (Clontech) was employed in transient transfections of OPCML complete cDNA. The HA-tagged Ubiquitin pRK5-HA-Ubiquitin-WT was obtained from Dr. Luke Gaughan, Newcastle University, and the EGFR and HER2 cDNA in pcDNA-3.1zeo was provided by Prof. Bill Gullick, University of Kent. FGFR1 cDNA clones was provided by Prof. Graeme Guy, FGFR1 extracellular domain clones provided by Prof. Kyung Hyun Kim. Expression of Recombinant OPCML and FGFR Ectodomain Recombinant proteins were produced in the BL21 bacterial cell line (Promega) as described. Solubilisation and Refolding of Inclusion Bodies Inclusion bodies were solubilised in denaturation buffer (8 M Urea, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 10 mM DTT) to a final concentration of 5 mg/ml. The suspension was centrifuged and filtered through 0.45 μm membrane filter. Refolding of proteins was undertaken by extensive dialysis against cold PBS in 10 kDa MWCO dialysis tubing. The suspension was then centrifuged and filtered to remove insoluble protein precipitates and soluble aggregates. Protein concentrations were monitored throughout the experiment with protein assay reagent (Bio-Rad Laboratories, California) using bovine serum albumen as a standard RNA Extraction and cDNA Synthesis Total RNA was extracted from cell pellets using TriReagent® (Sigma-Aldrich, Dorset, UK) following their protocol. Synthesis of cDNA was from 1 μg of RNA template with OligodT15primers (Promega, UK), by Moloney-Murine Leukaemia Virus Reverse Transcriptase (MMLV-RT) (Promega, UK) and cDNA was stored at −20° C. qRT-PCR Primers were designed using PerlPrimer v.1.14 open source software. Custom oligonucleotide synthesis was carried out by Invitrogen, UK. Quantitative reverse-transcription PCR (qRT-PCR) analysis of gene expression was carried out on an Applied Biosystems 7900HT thermal cycler using SYBR green I technology. Premixed qPCR reagent, Platinum® Quantitative PCR SuperMix-UDG with ROX (Invitrogen, UK), was used for amplification. The expression of specific genes was normalized to the expression of the endogenous control gene HPRT1. Co-Immunoprecipitation and Pull-Down Assays Cell layers were washed in PBS and incubated for 30 minutes in lysis buffer (1% TritonX-100, 10 mM Tris pH8.0, 150 mM NaCl, 2.5 mM MgCl2, 5 mM EGTA, 1 mM Na3VO4, 50 mM NaF and protein inhibitor cocktail (Roche). Cell Lysates were then cleared by centrifugation at 13,000 rpm for 20 minutes at 4° C. and aliquots containing equal amounts of protein were incubated with the appropriate antibody before addition of secondary antibody conjugated to sepharose resin. Beads were then washed 3× with lysis buffer and eluted by heating for 5 minutes in 50 μl of SDS sample buffer. Pull-down assays were performed using recombinant GST-OPCML fusion proteins bound to magnetic glutathione beads (Promega). Cell lysates prepared as for immunoprecipitation, proteins produced using TNT in vitro Rabbit reticulocyte lysate expression system (Promega) or expressed in bacteria were used analysed for interactions. Immunofluorescent Microscopy Cells grown on glass slides were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Cells were then permeabilized for 20 minutes with PBS containing 0.2% Saponin prior to blocking in PBS containing 10% goat serum, 2% albumen 2% fetal calf serum for 1 h. Slides were incubated with appropriate combinations of mAb OPCML, mAb HER2 and pAb EGFR primary antibodies for 1 h at room temperature, followed by incubation for 1 h with animal anti-mouse Alexa-555 (OPCML), animal anti-rabbit Alexa 488 (HER2) before mounting and imaging on a Zeiss LSM 510 confocal microscope. siRNA Knockdown Endogenous OPCML was knocked down in OSE-C2 cells by transient transfection of a specific pool of 3 siRNAs (Stealth knockdown-Invitrogen) using lipofectamine RNAiMAX reagent. MTT Proliferation Assay Cell proliferation assays were carried out in quadruplicate using the thiazolyl blue tetrazolium bromide (MTT) assay. Cells were plated out in 96-well plates at a density of 2,000 cells/well and cultured in low serum medium (0.25% FCS) or low serum medium supplemented with 50 ng/ml EGF. At appropriate time points, the medium was removed from cells and replaced with 100 μl PBS and 11 μl of 5 mg/ml MTT (w/v). Cells were incubated in this solution for 2 hours at 37° C. and the purple fomazan product was solubilised in 100 μl DMSO, resuspended and read on plate reader at 540 nm. Statistical Analyses Data are expressed as mean±SEM. Differences were analysed by Fishers exact or Student's t test. P<% 0.05 was considered significant. Progression-free survival curves were estimated using the Kaplan-Meier method and analysed by the log-rank test. Correlation between the mRNA expression indices of genes was analysed using Pearson's correlation analysis. Statistical Analysis and Mining of Tothill Data Gene expression data on the 251 epithelial ovarian cancers within 285 ovarian tumors (published by Tothill et al (2008)Clinical Cancer Research14: 5198) were obtained from the Gene Expression Omnibus (GEO). OPCML, EGFR and ERBB2 gene expression Pearson correlation coefficients were computed for all probe-sets. For survival analyses included all patients followed up to 5-years, and excluded patients with borderline/low malignant potential histology in view of their distinct natural history compared to invasive tumors. The effect of gene expression (probe: OPCML 206215_at, ERBB2 210930_s_at) on survival was assessed as a continuous variable using Cox-regression, and after transformation to categorical variables by median dichotomization or quartiles using Kaplan-Meier curves and the log-rank test. Results OPCML is Rapidly Induced by EGF and FGF 1/2 Serum starved SK-OV-3 cells (low OPCML expression) {Sellar, 2003 #2} were stimulated with 50 ng/ml EGF or 10 ng/ml FGF. EGF induced OPCML rapidly, achieving maximal mRNA expression at 30 min, with return to basal levels of expression by 60 min (FIG.1A(i)), with maximal OPCML protein at 60 min (FIG.1A(ii)). Similarly, FGF1/2 also induced OPCML mRNA, by 15 minutes (FIG.1B(i)) with protein peaking at 90 minutes (FIG.1B(ii)). These data were replicated for several other cell lines (data not shown). Specifically, induction of OPCML expression in a panel of ovarian cancer cell lines upon EGF stimulation (50 ng/ml) demonstrated consistent induction of OPCML mRNA by 5 to 10-fold, with varied timescale of peak induction. OPCML Interacts with HER2 and FGFR1 Via Different Binding Sites To determine if OPCML interacted with RTKs, co-immunoprecipitation (co-IP) using an OPCML polyclonal antibody was performed in a SK-OV-3 cell lines stably transfected with OPCML (BKS2.1) and vector-only controls (SKOBS-V1.2). Other OPCML stable transfected clones have been reported previously and behave identically as BKS2.1 (Sellar et al, 2003). Immunoblotting with anti-HER2 and anti-EGFR demonstrated that both interacted with OPCML, however reciprocal Co-IP using anti-HER2 and anti-EGFR antibodies confirmed the Co-IP only for OPCML with HER2 and not with EGFR (FIG.2A(i & ii)). We further used GST/OPCML domain fusion proteins in pull-down experiments with either SK-OV-3 cell lysates (expressing HER2 and EGFR) or with purified TnT HER2 ECD fragments (structures shown inFIG.2B). HER2 interacted with a full length OPCML extracellular domain (ECD) fused to GST (GST-OPCML D1+2+3) but not the truncated OPCML ECD lacking Ig domain 3 (GST-OPCML D1+2) from SK-OV-3 lysates (FIG.2C(i)), in addition to in vitro translated HER2 ECD (FIG.2C(ii)), suggesting that the third (juxtamembrane) Ig domain (Ig-Ill) of OPCML is crucial for interaction with HER2. We then explored whether OPCML interacted with the fibroblast growth factor receptors 1 and 2 (FGFR1 & 2). Co-IP of SKOBS-V1.2 and BKS2.1 with OPCML antibody revealed that OPCML bound to FGFR1, confirmed by reciprocal co-IP (FIG.2A(ii)). We used the GST/OPCML fusion proteins in pull-downs using cell lysates transiently transfected with full length FGFR1 (FIG.2C(i)) and separately in in vitro studies with His-tagged FGFR1 (FIG.2C(ii)). These experiments showed that both GST-OPCML D1+2+3 and GST-OPCML D1+2 interacted with FGFR1, therefore domain 3 was not essential for FGFR1 binding, implying that FGFR1 and HER2 bound to different sites on OPCML. Further experiments showed that GST-OPCML D2+3 interacted with FGFR1 but not GST-OPCML D3, showing that domain 2 is essential for FGFR1 binding (data not shown). OPCML Downregulates HER2 and FGFR1, and Abrogates Phosphorylation of HER and EGFR, Together with Downstream Signaling of the MEK-ERK Cascade We then explored the functional consequences of these OPCML-RTK interactions. OPCML expressing BKS2.1 demonstrated strong downregulation of HER2 but not EGFR protein as compared with SKOBS-V1.2 (FIG.3A(i)), implying that OPCML specifically regulates HER2 protein. We extended our investigations to the FGF receptor family and demonstrated downregulation of FGFR1 but not FGFR2 in BKS2.1 (FIG.3A(ii)). Immunofluorescence microscopy (IFM) confirmed that OPCML expression in BKS2.1 dramatically reduced the levels of HER2 and FGFR1 but not EGFR or FGFR2 (FIGS.3B(i & ii) and3E). We explored the impact of OPCML on cellular RTK phospho-activation and signaling in ovarian cancer cells. Phosphorylation of 2 analogous autophosphorylation sites, HER2-Y1248 and EGFR-Y1173 was abrogated in BKS2.1 (FIG.3C(i)) (an independent OPCML stable transfectant, previously described (Sellar et al (2003)) (data not shown), and OSE-C2 expressing physiological levels of OPCML (data not shown). Similarly, FGF mediated phosphorylation of FGFR1-Y766 (known to transactivate phospho lipase Cγ) was abolished in BKS2.1 cell lines (FIG.3C(i)). In both EGFR and FGFR signaling systems, we noted inhibition of phospho-PLC. and phospho-ERK 1& 2 (T202 & Y204—FIG.3C(ii)) but not phospho-Akt S473 or T308 (FIG.3C(iii)) suggesting that OPCML principally affected the MEK-ERK cascade. These signaling findings were phenotypically confirmed in growth assays; BKS2.1 and SKOBS-3.5 lines and OSE-C2 were profoundly growth-inhibited compared with vector control SKOBS-V1.2 (p<0.0001, student's t-test) (FIG.3C(iv)). To explore the physiological role of OPCML, normal epithelial cell line OSE-C2 (OPCML expressing) was transfected with OPCML siRNA, which abolished OPCML protein. This resulted in a strong induction of HER2 and FGFR1 (but not EGFR or FGFR2) and phospho activation of HER2-Y1248 and EGFR-Y1173 levels (FIG.3D(ii)). Since OPCML expression and physiological function seems to be regulated by growth factor signaling and it is downregulating at least two members of two different families of RTKs, we decided to extend our analysis to other RTKs. SiRNA against OPCML was used to verify whether RTKs appearing downregulated in the OPCML-expressing lines would show reciprocal upregulation if OPCML is knocked down in OSE-C2 cells. From this analysis, in addition to HER2, HER4 also appears downregulated in both SKOBS-3.5 and BKS2.1 cells, whereas FGFR1 and FGFR3 appear downregulated in predominantly the BKS2.1 line expressing five times more OPCML than SKOBS-3.5 (FIG.3D(i)). The reciprocal analysis of looking at RTK expression after OPCML knockdown revealed HER2, HER4, FGFR1, all showing substantive upregulation in siRNA lane. FGFR3 exhibits a slight increase in expression level with knockdown (FIG.3D(ii)). In contrast OPCML does not affect EGFR, HER3, FGFR2, FGFR4, EPHA50, VEGFR1 and VEGFR3. OPCML Prevents HER2/EGFR Hetero Dimer Formation and Reduces EGF Receptor Availability. SKOBS-V1.2 and BKS2.1 cell extracts were subjected to Co-IP and immunoblotted with antibodies as shown inFIG.4A, demonstrating loss of hetero-dimerisation in the presence of OPCML. Further, OPCML reduced EGF receptor availability (FIG.4B). OPCML is Localized in the Detergent—Resistant (Raft) Membrane Fraction and Co-Localizes with EGFR and HER2 in Ovarian Cancer Cells. To define the mechanism of OPCML-based RTK degradation, we used HER2 as a paradigm for further study. Initially, we investigated the influence of OPCML expression upon the mode of HER2 degradation linked to immunofluorescent confocal microscopy (IFM) analysis to examine the trafficking of OPCML and HER2 in cells. It has been previously reported that GPI-anchored proteins are sequestered in the detergent insoluble ‘lipid-raft’ membrane microdomain of cells (Sangiorgio et al (2004)Ital J Biochem53(2): 98-111). To examine the localisation of OPCML (a GPI anchored protein) within lipid rafts, purified membrane of OPCML negative (SKOBS-V1.2) and positive (BKS-2.1) were subjected to solubilisation in 1% Triton X100 (for detailed method see Materials and Methods) and samples subjected to ultracentrifugation to separate detergent solubilised and insoluble proteins (FIG.5A(i)). This experiment revealed that the majority of OPCML was localized within the detergent insoluble fraction, along with Caveolin-1 (a marker of caveolae—a distinct form of lipid raft domain). Interestingly, HER2, in the OPCML-expressing line, was reduced as previously shown inFIGS.3A and3Bbut also sequestered in the detergent insoluble fraction when compared to the OPCML negative line, where HER2 was equally distributed. The distribution of EGFR was only marginally effected by the expression of OPCML. IFM was employed to examine the trafficking of OPCML in cells; EEA-1 (a marker of the early endosome) and caveolin-1 (a marker of the raft-caveolar pathway) were used to distinguish between Clathrin-coated pit and caveolar endocytic vesicles. These studies revealed that the majority of the internalized protein co-localized with Caveolin-1 compared to EEA-1 (OPCML+cell line: cav-1 co-localisation=23%, EEA-1=7.5% of total HER2; OPCML-cell line: cav-1 co-localisation=4.5%, EEA-1=32% of total HER2). Furthermore, vesicular staining was seen to be markedly different within the cell, consistent with these representing distinct compartments (FIG.5A(ii)). IFM also confirmed that OPCML co-localized with EGFR and HER2 in ovarian cancer cells (FIG.5A(iii)). We next transfected both OPCML-expressing and non-expressing cell lines with a HA-tagged ubiquitin construct to analyze the levels of receptor ubiquitination +/−OPCML. Twenty four hours post transfection, cells were serum starved and subjected to acute stimulation with EGF (50 ng/ml) for 60 minutes. Consistent with the significant reduction in receptor levels, OPCML expression was associated with enhanced ubiquitination of HER2, which was strongly increased upon EGF stimulation (FIG.4B(i&ii)). IFM and quantification of co-localisation demonstrated that OPCML expression induced a shift in proportion of the HER2 into caveolin-1 positive vesicles compared to a predominant co-localisation with EEA-1 in the OPCML negative cell line (+OPCML: HER2/CAV-1, 22.863%±1.859; HER2/EEA-1 8.767±1.852.−OPCML: HER2/CAV-1, 4.767%±1.559; HER2/EEA-1 30.667±3.756) (FIG.5C(i&ii)). Transmembrane proteins in the EEA-1 compartment can enter either the late-endosome-lysosome for degradation, or the Rab11-positive recycling endosome. Whilst Caveolin-1 positive vesicles have been reported to be non-recycling and result in proteasomal degradation of their cargo (Di Guglielmo et al (2003)Nat Cell Biol5: 410-421). Consistent with degradation by the proteasome, chloroquine (CQ), a weak base that alkalinises the lysosome, was ineffective, but MG-132, a potent antagonist of the proteasomal 26S proteinase, inhibited HER2 degradation in the OPCML expressing cell line with no effect on EGFR expression found (FIG.5D(i&ii)). Furthermore, disruption of cholesterol using methyl-R-cyclodextrin (MP-CD) also inhibited the degradation of HER2 and increased the phosphorylation at Y1248 (FIG.5D(iii)) suggesting an important role for the lipid-raft in the OPCML-specific regulation and degradation of HER2. In conclusion, OPCML binds specifically to HER2, sequesters the receptor in lipid-rafts, enhancing caveolar-based endocytosis, ubiquitination and subsequent proteasomal degradation of the oncogenic receptor. OPCML Regulates/Predicts Response to Lapatinib in Ovarian and Breast Cancer. The finding that OPCML could regulate activity of HER2 and EGFR led us to explore whether OPCML might influence the efficacy of anti-EGFR/HER2 therapeutics. OPCML transfected and control cells were pre-incubated with lapatinib, trastuzumab, cituximab, erlotinib and gefitinib. We then used EGF induced phospho-ERK activation as an assay to define the effectiveness of therapeutic inhibition. The dual inhibitor of EGFR and HER2 tyrosine kinases, lapatinib, exhibited strong OPCML mediated sensitization, reducing the effective concentration of lapatinib required to abolish the phospho-ERK signal by 10-fold for BKS2.1 compared with SKOBS-V1.2 (FIGS.6A(i) and6C). We noted enhanced down-regulation of phospho-AKT in OPCML expressing cells. Lesser sensitization than for lapatinib was observed with trastuzumab (FIG.6A(ii)). Notably, cetuximab, erlotinib and gefitinib, inhibitors of EGFR showed no sensitization in OPCML transfected cells (data not shown) consistent with the hypothesis that OPCML interacts with HER2 and not EGFR. We then investigated whether siRNA knockdown of physiological OPCML expression in normal OSE-C2 cells could affect sensitivity to lapatinib. We observed that the lapatinib-mediated reduction in phospho-ERK signal strength was significantly reversed by OPCML siRNA knockdown in these normal ovarian surface epithelial cells (FIG.6B). These data demonstrate that OPCML modulates sensitivity to lapatinib through regulation of the level of HER2, however the mechanism of this finding remains to be clarified. We next tested whether OPCML could be used to predict response to lapatinib in ovarian and breast cancer. Histology was obtained by new biopsy of recurrent disease and TTP (time in months to progression from start of therapy until progression) assessed. Docetaxel and anthracyclines were administered for a maximum of 6 cycles and capecitabine was administered until disease progression or unacceptable toxicity. HER2 immunohistochemistry (IHC) was performed using the Dako Herceptest kit: 3+ in all cases. The results are shown in Table 1 below. TABLE 1Previous treatment chronologyResponse toOPCMLCase numberHistology(TTP in months)*lapatinibScore1.G2 IDC, ERAdjuvant: antracyclines and taxanesSD−6/8, PR 0/8,(23) then adjuvant trastuzumab (12)HER2 3+Metastatic: Hormonal therapy (12)Capecitabine (1)2.G3 IDC,Adjuvant: anthracyclines (48)SD−ER/PR (0/8),Metastatic: Trastuzumab (8)HER2 3+,Capecitabine (2)Vinorelbine (8)Taxanes (5)3.G3 IDC,Metastatic: Anthracyclines (12)PD+ER++, PR++,Hormonal therapy (12)HER2 3+Trastuzumab (5)(metastaticTaxanes (5)presentation)Capecitabine (1)4.G3 IDC, ERMetastatic: Capecitabine (14)PD+(8/8), PRTrastuzumab (5)(8/8), HER2 3+Vinorelbine (4)(metastaticTaxanes (4)presentation)Hormonal therapy (4)5.G3 ILC,Metastatic: Anthracyclines (3)PR++ER/PR (0/8),Taxanes (4)HER2 3+Trastuzumab (4)(metastaticGemcitabine and vinorelbine (7)presentation)Capecitabine (1)6.G2 IDC,Adjuvant: anthracyclines (7)PR++ER/PR (0/8),Metastatic: Taxanes (10)HER2 3+Trastuzumab (12)Vinorelbine (9)7.G2 IDC,Adjuvant: anthracyclines (36)PR++ER/PR (0/8),Metastatic: Trastuzumab (5)HER2 3+Taxanes (4)Capecitabine (2)Vinorelbine (4)8.G3 IDC,Adjuvant: Anthracyclines andPR+++ER/PR (0/8),taxanes (14), trastuzumab (8)HER2 3+,inflammatoryEstrogen receptor (ER) and progesterone receptor (PR) are either scored using H scores (out of 8) or IHC.IDC = invasive ductal carcinoma, ILC = invasive lobular carcinoma. SD = stable disease, PR = partial response, PD = progressive disease, by RECIST criteria. Examples of OPCML IHC - to +++ are shown. OPCML is a Prognostic Factor in Strongly HER2 Expressing Ovarian Cancer In view of the strong tumor suppressor role of OPCML and these findings, we explored whether its expression was related to ovarian cancer prognosis. We used a recently published expression microarray dataset of 251 ovarian cancers (Tothill et al, 2008) with full clinical annotation and follow-up of patients for progression free survival (PFS). The relationship between OPCML mRNA expression and PFS was examined for all 251 ovarian cancer patients with epithelial ovarian cancers in the dataset. Overall high OPCML expression demonstrated a significant association with better survival, as shown by the Kaplan-Meier curve inFIG.7A(i) (Log-rank p=0.061 Breslow test p=0.034), although the difference was of modest magnitude. However, because the findings described herein suggested that OPCML's tumor suppressor role was related to repression of HER2 and FGFR1 function we specifically analysed the patient cohort according to their HER2 RNA expression and explored the impact of OPCML expression on this group of patients. We found that OPCML mRNA level was strongly prognostic only for patients expressing top quartile HER2 mRNA levels; patients with above median expression of OPCML had 27 months median PFS (FIG.7A(ii)) and Table 1 & 2) compared with top quartile HER2 and below median OPCML expression with 13 month median PFS (Log-Rank p=0.004). In contrast, bottom quartile HER2 expressing patients showed similar survival regardless of OPCML expression. This validated our hypothesis that OPCML impacts on an intact HER2 pathway. No significant association was observed for EGFR or FGFR1 and OPCML with PFS in this dataset (data not shown). A possible explanation for this clinical data is that strong OPCML expression (in the context of strong HER2 expression) regulates HER2 protein level/activity and abrogates HER2 pro-oncogenic signaling with consequent better patient prognosis, whereas tumors with weak OPCML expression and strong HER2 expression have unrestrained HER2 pro-oncogenic signaling and consequently poor prognosis. DISCUSSION OPCML therefore has repressor-adaptor function, interacting with HER2 and FGFR1, targeting them for degradation and thereby physiologically negatively regulating both the EGF and FGF signaling pathways in normal ovarian surface epithelium, and conversely, by CpG island somatic methylation, activating both these pathways concurrently in epithelial ovarian cancer. In summary the OPCML tumor suppressor functions by concurrently negatively regulating HER2 and FGFR1 in normal ovarian surface epithelial cells and in ovarian cancer. This finding has general implications for understanding the relationship of IgLONs to the RTK pathways, and their role in both biology of ovarian surface epithelial cells and also in the understanding of how somatic methylation of this tumor suppressor uncovers such strong pro-oncogenic phenotype driven by several RTK pathways. Table 2a and b: show case processing summary, means and median survival and data comparisons for the at a depicted in Kaplan Meier curves (FIG.7(i)and (ii)). P TABLE 2ACase Processing summaryB OPCMLCensored215Total NoNo of EventsNoPercentLow OPCML122932923.80%High OPCML129913829.50%Overall2511846726.70%Means and Medians for Survival TimeMean (a)Median95% confidence interval95% confidence intervalB OPCML 215EstimateStd. ErrorLower BoundUpper BoundEstimateStd. ErrorLower BoundUpper BoundLow OPCML20.7881.65217.5524.027140.62512.77515.225High OPCML24.7711.66921.528.043191.10116.34321.157Overall22.8661.18720.53925.193151.13812.7717.23(a) Estimation is limited to the largest survival time if it is censoredOverall ComparisonsChi-SquaredfSig.Log Rank (Mantel-Cox)3.52210.061Breslow (Generalised Wilcoxon)4.50210.034Tarone-Ware4.45110.035Test of Equality of survival distribution for the different levels of B_OPCML_215 TABLE 2BCase Processing summaryCensoredQ-HER2-930Total No.No. of EventsNo.percentLow HER2/Low OPCML413337.30%Low HER2/High OPCML2117419.00%High HER2/Low OPCML1713423.50%High HER2/High OPCML42261638.10%Overall121942722.30%Means and Medians for Survival TimeMean (a)Median95% confidence interval95% confidence intervalQ-HER2-930-LowerUpperStd.LowerUpperOPCML-215-10-11-40-41EstimateStd. ErrorBoundBoundEstimateErrorBoundBoundLow HER2/Low OPCML16.0161.7312.62419.407131.23210.58615.414Low HER2/High OPCML17.0262.39312.33721.716122.5117.07916.921High HER2/Low OPCML16.2432.90910.54121.946131.15910.72815.272High HER2/High OPCML30.0973.29923.6327275.75315.72538.275Overall21.4791.61418.31515151.4712.11917.881(a) Estimation is limited to the largest survival time if it is censoredOverall ComparisonsChi-SquaredfSig.Log Rank (Mantel-Cox)13.5353.004Breslow (Generalized9.2003.027Wilcoxon)Test of equality of survival distributions for the different levels of Q_erb_930_Opcm1_215_10_11_40_41. EXAMPLE 2: EXOGENOUS OPCML INHIBITS RECEPTOR TYROSINE KINASE SIGNALING AND OVARIAN AND BREAST CANCER CELL GROWTH IN VITRO, WHILE SPARING NORMAL OVARIAN SURFACE EPITHELIAL CELLS To complement the findings described in Example 1, we expressed and purified recombinant human OPCML and assessed its affect on in vitro tyrosine kinase signaling and cell growth. The results are in agreement with those in Example 1. FIG.8shows purification of recombinant human OPCML expressed inE. coli. Expressed OPCML was present in inclusion bodies and was successfully refolded by dialysis into PBS. Recombinant OPCML is a 272 amino acid polypeptide whereas physiologically synthesized OPCML is a 345 amino acid polypeptide. Post-translationally modified OPCML (including N-linked glycosylation) is 55 kDa, whereas without glycosylation, the signal peptide region and the GPI anchor region, OPCML is 31 kDa. The protein and polynucleotide sequences of recombinant human OPCML correspond to SEQ ID Nos: 5 and 6 respectively. FIG.9shows cellular uptake of recombinant OPCML Cells which took up exogenous OPCML demonstrate downregulation of HER2, as confirmed by IFM. FIG.10shows that administration of exogenous OPCML inhibits receptor tyrosine kinase signaling in vitro. Specifically, administration of exogenous OPCML downregulates HER2 and ERK protein levels. FIGS.11A and11Bshow that administration of exogenous OPCML inhibits SKOV-3 cell growth in vitro, as assessed by a MTT cell growth assay. FIG.11Cshows that OPCML inhibits growth of a range of ovarian and breast cancer cell lines, while sparing normal ovarian surface epithelial cells. Interestingly, growth of HER2+ and HER2− breast cancer cell line was profoundly inhibited, suggesting that the mechanism is not solely restricted to HER2+ cells but may be mediated through FGFR pathways or other EGFR components that interact with HER2 and are phospho-inactivated as part of OPCML therapy. Only one cell line showed resistance to OPCML (PEA2). EXAMPLE 3: OPCML IS A PROGNOSTIC FACTOR IN BREAST, LUNG AND GLIOMA CANCERS Having established that OPCML is a prognostic factor in strongly HER2 expressing ovarian cancer (see Example 1), we assessed whether its expression was related to the prognosis of other cancers. This was done by a Kaplan Meier analysis of overall survival according to OPCML dichotomized survival. FIG.12A(first graph—A1) shows that OPCML expression is a good prognostic factor in EGFR/HER2 positive node negative breast cancers in patients receiving no adjuvant systemic therapy. OPCML expressing tumors show better survival, with a 10 year relapse free survival of 72% vs 52%. (‘cum survival’=cumulative survival). Analysis was based on a published dataset of 149 node negative breast cancer patients (Wang et al 2005, Lancet365(9460): 671). The second two graphs (A2 and A3) show that OPCML expression is a particularly good prognostic factor in ER-breast cancer. FIG.12Bshows that OPCML is a prognostic factor in lung cancer. OPCML expressing tumors show better overall survival (OS). Analysis was based on a published dataset of 115 lung cancer patients (Takeuchi et al 2006, J Clin Oncol24(11): 1679-1688). FIGS.12C and12Dshow that OPCML is a prognostic factor in brain high grade gliomas. OPCML expressing tumors show better overall survival (OS). Analysis was based on a published dataset of high grade glioma patients (Phillips et al 2006Cancer Cell9(3): 157-73). EXAMPLE 4: FUNCTIONAL EFFECTS OF OPCML EXPRESSION IN SKOV3 IN VITRO AND IN VIVO We next investigated the effects of OPCML expression in SKOV3 cells in vivo, using methods described in Sellar et al (2003). FIG.13Ashows the effect of OPCML expression on in vitro growth of SKOV3 cells, with OPCML expression inhibiting growth relative to normal SKOV3 cells or to SKOV3 cells in which OPCML expression was knocked-down, in agreement with the data described above. FIGS.13B and13Cshow the effect of OPCML expression on in vivo growth of SKOV3 cells. OPCML expression reduced mean tumor volume relative to normal SKOV3 cells or to SKOV3 cells in which OPCML expression was knocked-down. EXAMPLE 5: FURTHER STUDIES ON THE ROLE OF OPCML Summary OPCML, a GPI anchored tumor suppressor gene is inactivated by somatic methylation in multiple cancers. We previously identified this gene by LOH mapping and demonstrated that it was inactivated by somatic methylation in 80% of ovarian cancers. Restoring OPCML expression by stable transfection suppressed in-vitro growth and in-vivo tumorigenicity. We investigated the role of OPCML in growth signaling pathways. In SKOV-3 and PEO1, ovarian cancer cell lines with no expression of OPCML, we demonstrated that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1, FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA knockdown in normal ovarian surface epithelial cells up-regulates these same RTKs. OPCML has no effect on the RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 and VEGFR3. Example immunoprecipitation experiments revealed that OPCML binds to EphA2, FGFR1 and HER2 extracellular domains with no such interaction to EGFR, thus OPCML binds directly to RTKS that it negatively regulates. We demonstrate that OPCML is located exclusively in the raft membrane fraction and sequesters RTKs that it binds to the raft fraction, leading to polyubiquitination and proteosomal degradation via a cav-1 endosomal mechanism resulting in systems depletion of this specific RTK repertoire, that does not occur with RTKs that OPCML does not bind. We demonstrate that OPCML abrogates EGF mediated phosphorylation of FGFR1, HER2 and EGFR and the downstream phosphosignaling of pErk and pAKT. A recombinant modified OPCML-like protein without a GPI anchor, signal peptide or glycosylation was constructed and expressed inE. coli. This rOPCML tumor suppressor protein therapeutic caused growth inhibition by apoptosis in 6/7 ovarian cancer cell lines tested, with no effect on OPCML expressing normal ovarian surface epithelium, by an identical mechanism to the transfected normal protein. rOPCML was then injected intraperitoneally twice weekly in two murine intraperitoneal models of ovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profound inhibition of tumour weight, ascites volume and peritoneal dissemination compared with BSA control. Mechanism of OPCML TSG Function OPCML is a non-transmembrane, external lipid leaflet GPI-anchored protein, and is frequently lost from cells by somatic inactivation of the gene. We hypothesised that it may mediate its tumour suppressor properties via interactions with transmembrane signalling proteins, and so we analysed the effect of receptor tyrosine kinase (RTK) growth factor stimulation on OPCML gene expression. Treatment of 4/4 ovarian cancer cell lines with EGF or FGF 1/2 resulted in rapid OPCML RNA and concomitant protein expression (data not shown) suggesting that OPCML may be a putative suppressor-type immediate-early negative feedback regulator. Stable transfection of OPCML in the basal unstimulated or ligand-stimulated SKOV-3 ovarian cancer cells, resulted in the profound protein down-regulation of a specific repertoire of RTKs: EPHA2; FGFR1; FGFR3; HER2 and HER4 (FIG.16A) and this RTK down-regulation spectrum is reproducible by transient transfection of a different ovarian cancer cell line, PEO1 (FIG.16B). These same RTKs were also reciprocally up-regulated when physiological OPCML was knocked down by siRNA in OSE-C2, a normal ovarian surface epithelial cell line (Davies et al, (2003)Experimental Cell Research288: 390-402) (FIG.16C). This specific inactivation by OPCML was not seen for other RTKs we have investigated so far including: EPHA10; FGFR2; FGFR4; EGFR; HER3; VEGFR1 and VEGFR3 (FIG.16). The phenotypic consequences of these signalling effects were confirmed in growth assays in ligand-supplemented media where OPCML-transfectants were significantly growth-inhibited compared with vector control (data not shown). Negative Regulation of Specific RTKs by OPCML is Related to Direct Protein Interaction We further explored as examples EPHA2, FGFR1 and HER2, RTKs that are strongly inactivated at the protein level upon OPCML expression. We also analysed EGFR as an example of a protein that is unaffected by OPCML. Immunoprecipitation (IP) experiments demonstrated protein/protein interactions with EPHA2, FGFR1 and HER2, but no such binding to EGFR (FIG.17A). These findings were further confirmed using a recombinant OPCML (GST-OPCML D1-3) pull-down assay (FIG.17B) which was then used to determine that the extracellular domains (ECDs) of the RTKs FGFR1 and HER2 (as examples) were capable of interacting specifically with OPCML (FIGS.17C&D), showing that the site of interaction lay within the ECD of the RTKs and domain 1-3 of OPCML, defining the site of OPCML action as extracellular. Downstream Signalling Upon acute ligand stimulation, OPCML expression led to profound abrogation of phospho-FGFR1-Y766, phospho-HER2-Y1248 and, also, phospho-EGFR-Y1173. Whilst EGFR total protein down-regulation is NOT observed, presumably due to the absence of an RTK ECD physical interaction with OPCML, the consequence of OPCML mediated loss of the activating dimerisation partners of EGFR, (HER2 and HER4), coupled with the continuing availability of the HER3 family member (that results in an inhibitory dimerisation with EGFR), explain the down-regulation of EGFR signalling even though total EGFR levels are unaffected (FIG.18A). Analysis of FGFR1 signalling showed a similar pattern of phospho inhibition relating to protein down-regulation (FIG.18B). Analysis of downstream signalling demonstrated abrogation of phospho-ERK 1 & 2 (T202 & T204) and phospho-AKT-S473 (FIG.18C), suggesting that both pro-growth and pro-survival pathways are inhibited by OPCML re-expression, via a systems level abrogation of this specific RTK spectrum. OPCML-Mediated RTK Degradation Mechanism Using HER2 as a paradigm molecule of OPCML-RTK regulation, we found that the available HER2 in OPCML expressing cells was sequestered in the detergent resistant membrane (DRM) fraction. In the OPCML non-expressing line, HER2 was found equally distributed between the DRM and the detergent soluble (non-raft) fractions. The total level of EGFR was not affected by the expression of OPCML and its distribution showed a much less pronounced but discernible shift to the DRM fraction (FIG.19A). These data indicate that OPCML expression leads to redistribution of HER2 to the DRM fraction in the plasma membrane (that broadly correlates with membrane “rafts”). IFM was employed to examine the trafficking of OPCML in cells; EEA-1 (a marker of the early endosome) and Caveolin-1 (a marker of the raft-caveolar pathway) were used to investigate this apparent redistribution. A decrease in HER2 co-localisation with EEA1 shows that the sequestration of HER2 to the DRM fraction decreases its endocytosis via clathrin-mediated pathways. While an increase in co-localisation with caveolin-1 was observed, the immunofluorescence pattern suggests this is a function of the redistribution of HER2 into the DRM fraction (housing lipid-raft domains) where caveolin is also localised, as HER2 did not appear to be exclusively localised to caveolae in the presence of OPCML expression. Furthermore, in the presence of OPCML the staining was organised into specific sub-cellular particles, suggestive of distinct vesicular compartments (FIGS.19B&C). This analysis demonstrated that OPCML expression was associated with increased ubiquitination of HER2 (that binds OPCML), which was strongly increased upon EGF stimulation (FIGS.19D&E). Exposure to MG-132, a potent inhibitor of the proteasomal 26S proteinase, attenuated HER2 degradation with no such effect on EGFR expression (that does not bind OPCML). In contrast, chloroquine, a weak base that alkalinises the lysosome, showed no inhibition of HER2 degradation (FIGS.19H&I). This suggested that the proteasomal pathway was preferentially utilised for OPCML-mediated HER2 degradation. Furthermore, disruption of cholesterol (a component of DRM fraction/lipid rafts) using methyl-β-cyclodextrin (Mβ-CD) also inhibited the degradation of HER2 and increased HER2 phosphorylation (FIG.19J) suggesting that cholesterol-rich lipid-raft structures are important for OPCML-specific internalisation and degradation of HER2. These findings suggest that OPCML-mediated negative regulation of this specific repertoire of RTKs is the result of direct binding of OPCML to the ECD of that RTK. These multiple but specific binding events result in ‘lipid-raft’ sequestration, enhanced ubiquitination, and a switch away from clathrin-mediated endocytosis to proteasomal degradation of those specific RTKs negatively regulating their signaling through reducing their protein level. Our data, in the context of very recent publications (Howes et al (2010)J. Cell Biol.190(4): 675-91; Howes et al (2010)Curr. Opin. Cell Bio.22(4): 519-527)), would suggest that CLIC/GEEC bulk internalization route is a strong candidate pathway for OPCML-mediated degradation of HER2 and that this is linked to RTK inactivation and the observable strong tumour suppressor phenotype of OPCML. Recombinant OPCML (r-OPCML) inhibits tumour growth in vitro and in vivo Purified recombinant human OPCML domain 1-3 protein (r-OPCML) (FIG.8) was produced from the bacterial expression vector (pHis-Trx) subcloned with domains 1-3 of OPCML, excluding the signal peptide and GPI anchor sequences (FIG.8A). Addition of r-OPCML protein to growth media demonstrated a specific, dose-dependent inhibition of cell growth in OPCML non-expressing SKOV-3 ovarian cancer cells, without affecting normal ovarian surface epithelial cells, OSE-C2 (FIG.20A). We have confirmed that r-OPCML profoundly inhibited cell growth in 6 of 7 additional OPCML non-expressing epithelial ovarian cancer cell lines; 2 of 2 breast HER2-positive and negative cells; and 5 of 5 lung cancer cell lines (FIG.20B). To determine the mechanism of this pharmacological growth inhibition we performed Annexin V FACS apoptosis assay in SKOV-3 and A2780 demonstrating evidence of early apoptosis induced by r-OPCML at 2-6 hours post exposure depending on cell line (FIG.20C). We then performed caspase-glo apoptosis assays across a concentration range in SKOV-3 and A2780 ovarian cancer cells and demonstrated that r-OPCML induces apoptosis in both these cell lines in a dose dependent fashion, demonstrating the underlying mechanism of the observed growth inhibition (FIGS.20D&E). Immunoblotting confirmed that addition of r-OPCML protein to media potently downregulated the same spectrum of RTKs as seen by transfecting OPCML into cancer cells, as well as abrogating pERK and pAKT in both SKOV-3 and A2780 (FIG.21). This suggests that pharmacological use of extracellular unanchored r-OPCML utilises the same mechanism of action as transfection induced intracellular re-expression of the normal GPI-anchored, glycosylated, OPCML protein. These data were confirmed by IFM for HER2 in SKOV-3, closely mirroring stable transfection of the normal protein in the same cell line (see,FIG.9). In view of these in-vitro findings, we proceeded to determine whether r-OPCML protein had potential and relevance as an in-vivo tumour suppressor therapy. Mice with either SKOV-3 or A2780 cancer cells injected intraperitoneally (IP), after tumour establishment, received twice-weekly IP injections of either 1 ml (10 μM) bovine serum albumin (BSA) or 1 ml (10 μM) r-OPCML. The experiment was terminated after 3 weeks due to obvious extensive IP tumour growth and deteriorating condition of BSA-treated control animals whereas r-OPCML treated mice remained well (FIG.22A). r-OPCML significantly and profoundly suppressed both IP tumour growth and ascites formation in-vivo in both IP models (FIG.22B-D), and in A2780 tumour bearing mice, profoundly inhibited the number of IP peritoneal deposits compared with BSA control (FIG.22E). Western blotting of SKOV3 IP tumour recovered from BSA treated and r-OPCML treated animals clearly demonstrated the same spectrum of RTKs inhibited as predicted from the in-vitro analysis (FIG.22F). CONCLUSION OPCML mediates its tumour suppressor function by systems level negative regulation of at least 5 RTKs and a recombinant modified OPCML derivative is a potent tumor suppressor protein therapeutic in-vitro and in-vivo. | 45,294 |
11857625 | DETAILED DESCRIPTION OF THE INVENTION The invention is based on the combination of polysorbate 80 and a buffer solution comprising an acetate buffer (as a buffering agent) and glycine (as a zwitterion) at pH of from about 4.6 to about 5.5 for preparing a suitable pharmaceutical composition for human use of an antibody, or an antigen binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2, at concentration of from 80 mg/ml to 200 mg/ml, without affecting the processability of the pharmaceutical composition and the long-term stability of the antibody. It is a finding from the inventors that the pharmaceutical compositions according to the invention are stable over time, in particular when stored at 2-25° C., as shown for example at 2-8° C. and 25° C. The term “stable formulation” refers to a formulation in which the protein of interest (here an antibody or an antigen binding fragment thereof) essentially retains its physical, chemical and/or biological properties upon storage. In order to measure the protein stability in a formulation, various analytical methods are well within the knowledge of the skilled person (see some examples in the example section). Stability is typically assessed at a selected temperature (for instance −70° C., 2-8° C., 25° C., 35° C. or more) for a selected time period (e.g. 3 months, 6 months, 12 months or more). As an antibody; once formulated, is typically stored in the fridge (typically 2-8° C.) or at room temperature (typically 15-25° C.) before being administered to a patient, it is important that said formulated antibody is stable over time at least at 2-25° C., as shown for example at 2-8° C. and 25° C. Various values can be used to conclude about stability over a given time period (in comparison of the initial data), such as (and not limited to): 1) no more than 10% of alteration of the monomeric form of the antibody, 2) no more than 5% of increase in High Molecular Weight Species (HMW or HMWS; also herein referred to as aggregates), 3) no more than 10% of increase in Low Molecular Weight species (LMW or LMWS), or 4) no more than +1-0.3 unit variation of the pH. In all the embodiments of the invention, “pharmaceutical composition” can also be referred to as “stable pharmaceutical composition” without any differentiation. In one embodiment of the invention, the pharmaceutical composition according to the invention preferably comprises from or from about 80 mg/ml to or to about 200 mg/mL, preferably from or from about 120 mg/ml to or to about 185 mg/ml or from or from about 120 mg/ml to or to about 180 mg/mL of the antibody; or antigen-binding fragment thereof, such as about 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 mg/ml, even preferably about 160 mg/mL of the antibody, or antigen-binding fragment thereof. In another embodiment, the pharmaceutical composition according to the invention composition comprises from or from about 0.01% to or to about 0.07% (w/v) polysorbate 80, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.07% (w/v polysorbate 80). In another embodiment, the pharmaceutical composition according to the invention comprises from or from about 140 mM to or to about 350 mM of glycine. Preferably, the pharmaceutical composition according to the invention comprises from or from about 160 mM to or to about 300 mM of glycine, such as about 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mM of glycine. Glycine, in the context of the present invention as a whole, is not a buffering agent. Indeed, at pHs of from about 4.6 to about 5.5, Glycine is a zwitterion. As a zwitterion, it has the ability to interact with hydrophobic and hydrophilic parts of the antibody or antigen-binding fragment thereof therefore possibly reducing self-interaction between the antibody or antigen-binding fragment thereof, providing a stabilizing effect. In yet another embodiment, the pharmaceutical composition according to the invention as a whole comprises from about 20 mM to about 100 mM acetate, and preferably from about 40 mM to about 90 mM acetate, such as about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 mM acetate. Alternatively, the pharmaceutical composition according to the invention as a whole comprises from about 50 mM to about 90 mM acetate, such as about 50, 55, 60, 65, 70, 75, 80, 85 or 90 mM acetate. In one embodiment, the pharmaceutical composition comprises about 55 mM acetate. Acetate, in the context of the present invention as a whole, is the buffering agent. Any kind of acetates can be used such as calcium acetate, magnesium acetate, sodium acetate or zinc acetate. Preferably, sodium acetate is used. Preferably, the pharmaceutical composition according to the invention does not comprise any sugar (such as monosaccharides, disaccharides or polysaccharides) nor polyol. The antibody or antigen-binding fragment thereof comprised in the pharmaceutical composition according to the invention specifically binds to human IL-17A and IL-17F. The term “specifically binds to human IL-17A and IL-17F”, “specifically binding to human IL-17A and IL-17F”, and equivalents as used herein means the antibody will bind to human IL-17A and IL-17F with sufficient affinity and specificity to achieve a biologically meaningful effect. The antibody selected will normally have a binding affinity for human IL-17A and IL-17F, for example, the antibody may bind human IL-17A and IL-17F with a Kd value of between 100 nM and 1 pM. Antibody affinities may be determined for example by a surface plasmon resonance bases assay, such as the BIAcore assay; enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's). Within the meaning of the present invention an antibody or antigen-binding fragment thereof specifically binding to human IL-17A and IL-17F may also bind to another molecule, e.g. cyno IL-17A and IL-17F or such as by way of a non-limiting example, in the case where the antibody or antigen-binding fragment thereof is incorporated into a bi- or multi-specific antibody. In particular, the present antibody or antigen-binding fragment thereof does not bind any other human IL-17 isoform other than IL-17A, IL-17F and the IL-17A and IL-17F heterodimer. IL-17A (originally named CTLA-8) is a pro-inflammatory cytokine and the first IL-17 of the IL-17 family to have been discovered. Subsequently, 5 additional members of the family have been identified (IL-17B to F). IL-17A and F have approximately 55% amino acid sequence homology, they express as homodimers and as heterodimers, signal through the receptors IL-17R, IL-17RC or IL-17RA/RC and have been associated with a number of autoimmune diseases. The antibody or antigen-binding fragment thereof binding specifically to human IL-17A and IL-17F, preferably also neutralizes human IL-17A and IL-17F. The term “neutralizes” as used herein refers to an antibody that inhibits or substantially reduces the biological effect of the molecule to which it specifically binds. Therefore, the expression “the antibody neutralizes human IL-17A and IL-17F” refers to an antibody that specifically binds to human IL-17A and IL-17F and inhibits or substantially reduces the biological effect thereof such as by blocking IL-17A and IL-17F binding to their receptor. The term “antibody” or “antibodies” as used herein refers to monoclonal or polyclonal antibodies and is not limited to recombinant antibodies that are generated by recombinant technologies as known in the art. The antibody, or antigen-binding fragment thereof, having a variable heavy chain comprising SEQ ID NO:1 and a variable light chain comprising SEQ ID NO:2, as shown in Table 1, is described in more detail in WO2012095662, which content is incorporated herein by reference. The term “antibody” or “antibodies” also refers to humanized antibodies. Humanized antibodies are antibodies that contain a sequence derived from non-human antibodies. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region or complementarity determining region (CDR) of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken or non-human primate, having the desired specificity, affinity, and activity. In most instances residues of the human (recipient) antibody outside of the CDR; i.e. in the framework region (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human diseases. Humanized antibodies and several different technologies to generate them are well known in the art. The term “antibody” or “antibodies” also refers to human antibodies, which can be generated as an alternative to humanization. For example, it is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous murine antibodies. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies with specificity against a particular antigen upon immunization of the transgenic animal carrying the human germline immunoglobulin genes with said antigen. Technologies for producing such transgenic animals and technologies for isolating and producing the human antibodies from such transgenic animals are known in the art. Alternatively, in the transgenic animal; e.g. mouse, only the immunoglobulin genes coding for the variable regions of the mouse antibody are replaced with corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes coding for the antibody constant regions remain unchanged. In this way, the antibody effector functions in the immune system of the transgenic mouse and consequently the B cell development are essentially unchanged, which may lead to an improved antibody response upon antigenic challenge in vivo. Once the genes coding for a particular antibody of interest have been isolated from such transgenic animals the genes coding for the constant regions can be replaced with human constant region genes in order to obtain a fully human antibody. The term “antibody” or “antibodies” as used herein, also refers to an aglycosylated antibody. The term “antigen-binding fragment thereof” or its grammatical variations as used herein refers to an antibody fragment. Examples of antibody fragments according to the invention include Fab, Fab′, F(ab′)2, and Fv, scFv fragments, single-chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. Antibody fragments as defined above are known in the art. Preferably, the pharmaceutical composition according to the invention comprises (Table 1):1) an antibody which comprises a heavy chain having the sequence as defined in SEQ ID NO: 1 and a light chain having the sequence as defined in SEQ ID NO:22) an antibody which comprises a heavy chain having the sequence as defined in SEQ ID NO: 3 and a light chain having the sequence as defined in SEQ ID NO: 4; or3) an antibody which comprises a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the variable region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the variable region of the sequence as defined in SEQ ID NO: 4. TABLE 1Region and SEQID identifierAmino acid sequenceHeavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYNMAWVRQAPGKGLvariable regionEWVATITYEGRNTYYRDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSEQ ID NO: 1AVYYCASPPQYYEGSIYRLWFAHWGQGTLVTVSSLight chainAIQLTQSPSSLSASVGDRVTITCRADESVRTLMHWYQQKPGKAPKLvariable regionLIYLVSNSEIGVPDRFSGSGSGTDFRLTISSLQPEDFATYYCQQTWSSEQ ID NO: 2DPWTFGQGTKVEIKHeavy ChainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYNMAWVRQAPGKGLSEQ ID NO: 3EWVATITYEGRNTYYRDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPPQYYEGSIYRLWFAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)**The final K may be absentLight chainAIQLTQSPSSLSASVGDRVTITCRADESVRTLMHWYQQKPGKAPKLSEQ ID NO: 4LIYLVSNSEIGVPDRFSGSGSGTDFRLTISSLQPEDFATYYCQQTWSDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody molecules may be typically produced by culturing a host cell containing a vector encoding the antibody sequence under conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule. For production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides. An antibody or an antigen-binding fragment thereof that can be manufactured according to industrial scales can be produced by culturing eukaryotic host cells transfected with one or more expression vectors encoding the recombinant antibody fragment. The eukaryotic host cells are preferably mammalian cells, more preferably Chinese Hamster Ovary (CHO) cells. Mammalian cells may be cultured in any medium that will support their growth and expression of the recombinant protein, preferably the medium is a chemically defined medium that is free of animal-derived products such as animal serum and peptone. There are different cell culture mediums available to the person skilled in the art comprising different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or an equivalent energy source, present at appropriate concentrations to enable cell growth and protein production. Additional cell culture media components may be included in the cell culture medium at appropriate concentrations at different times during a cell culture cycle that would be known to those skilled in the art. Mammalian cell culture can take place in any suitable container such as a shake flask or a bioreactor, which may or may not be operated in a fed-batch mode depending on the scale of production required. These bioreactors may be either stirred-tank or air-lift reactors. Various large-scale bioreactors are available with a capacity of more than 1,000 L to 50,000 L, preferably between 5,000 L and 20,000 L, or to 10,000 L. Alternatively, bioreactors of a smaller scale such as between 2 L and 100 L may also be used to manufacture an antibody or antibody fragment. An antibody or antigen-binding fragment thereof is typically found in the supernatant of a mammalian host cell culture, typically a CHO cell culture. For CHO culture processes wherein the protein of interest such as an antibody or antigen-binding fragment thereof is secreted in the supernatant, said supernatant is collected by methods known in the art, typically by centrifugation. Therefore, the antibody or antigen-binding fragment thereof production method comprises a step of centrifugation and supernatant recovery after cell culture and prior to protein purification. In a further particular embodiment said centrifugation is continuous centrifugation. For avoidance of doubt, supernatant denotes the liquid lying above the sedimented cells resulting from the centrifugation of the cell culture. Alternatively, host cells are prokaryotic cells, preferably gram-negative bacteria. More preferably, the host cells areE. colicells. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. Appl Microbial Biotechnol 72, 211-222 (2006)). The host cells are recombinant cells which have been genetically engineered to produce the protein of interest such as an antigen-binding fragment of an antibody. The recombinantE. colihost cells may be derived from any suitableE. colistrain including from MC4100, TG1, TG2, DHB4, DH5α, DH1, BL21, K12, XL1 Blue and JM109. One example isE. colistrain W3110 (ATCC 27,325) a commonly used host strain for recombinant protein fermentations. Antibody fragments can also be produced by culturing modifiedE. colistrains, for example metabolic mutants or protease deficientE. colistrains. E. colihost cell cultures (fermentations) may be cultured in any medium that will support the growth ofE. coliand expression of the recombinant protein. The medium may be any chemically defined medium such as e.g. described in Durany O, et al. (2004). Studies on the expression of recombinant fuculose-1-phosphate aldolase inEscherichia coli. Process Biochem 39, 1677-1684. Culturing of theE. colihost cells can take place in any suitable container such as a shake flask or a fermenter depending on the scale of production required. Various large-scale fermenters are available with a capacity of more than 1,000 liters up to about 100,000 liters, Preferably, fermenters of 1,000 to 50,000 liters are used, more preferably 1,000 to 25,000, 20,000, 15,000, 12,000 or 10,000 liters. Smaller scale fermenters may also be used with a capacity of between 0.5 and 1,000 liters. Other methods for obtaining antigen-binding fragment of a human antibody in vitro are based on display technologies such as phage display or ribosome display technology, wherein recombinant DNA libraries are used that are either generated at least in part artificially or from immunoglobulin variable (V) domain gene repertoires of donors. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies may also be generated from isolated human B cells that are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas which can then be screened for the optimal human antibody. It will be understood by one skilled in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R J. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the antibody heavy chain may be absent. The pharmaceutical composition according to the invention as a whole has a pH of from about 4.6 to about 5.5, such as 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5. Alternatively, it has a pH of from about 4.6 to about 5.3, such as 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 or 5.3. In all the embodiments of the present invention, unless otherwise indicated, the pH value was measured at 23-25° C. and it is within ±0.1 or ±0.2 of a pH unit. The present invention provides for a method for preparing a pharmaceutical composition comprising an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2. The method comprises the steps of preparing a) a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of the antibody, or antigen-binding fragment thereof, with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5; and then b) preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof in the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml; and finally c) adding polysorbate 80 to the high concentration formulation obtained in b). Optionally, before step c) the concentration of the antibody or antigen-binding fragment thereof may be adjusted with the buffer solution comprising glycine and acetate. Additional excipients for use within the pharmaceutical compositions according to the invention include, but are not limited to, viscosity enhancing agents, bulking agents, solubilising agents or combinations thereof. The present invention also provides for a container comprising the pharmaceutical composition according to the invention. In particular, the container may be, without any limitations, a vial, an ampoule, a tube, a bottle or a syringe (such as a pre-filled syringe) comprising the pharmaceutical composition. The container may be part of a kit-of-parts comprising one or more containers comprising the pharmaceutical compositions according to the invention and delivery devices such as a syringe, pre-filled syringe, an autoinjector, a needleless device, an implant or a patch, or other devices for parental administration and instructions of use. In one embodiment of the present invention, a container comprises the pharmaceutical composition comprising:a. from about 80 mg/ml to about 200 mg/mL, alternatively from about 120 mg/ml to about 185 mg/ml of antibody, or antigen-binding fragment thereof having:i. a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2; orii. a heavy chain comprising SEQ ID NO:3 and a light chain comprising SEQ ID NO:4; oriii. a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4.b. acetate;c. glycine;d. polysorbate 80, wherein the composition has a pH of from about 4.6 to about 5.5. In one preferred embodiment of the present invention, a container comprises the pharmaceutical composition comprising:a. about 160 mg/mL of antibody, or antigen-binding fragment thereof having:i. a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2; orii. a heavy chain comprising SEQ ID NO:3 and a light chain comprising SEQ ID NO:4; oriii. a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4.b. sodium acetate;c. glycine;d. polysorbate 80, wherein the composition has a pH of about 4.6 to about 5.5. Also preferably, the present invention provides for a container comprising a pharmaceutical composition obtained by the method according to the present invention, which method comprises the steps of:a. preparing a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2 with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5;b. preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof of the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml;c. adding polysorbate 80 to the high concentration formulation obtained in b), preferably at from about 0.01 to 0.07% (w/v);d. optionally, before step c) adjusting the concentration of the antibody or antigen-binding fragment thereof with the buffer solution comprising glycine and acetate. The pharmaceutical composition obtained by the method of the present invention and comprised in the container has a pH of from about 4.6 to 5.5. Preferably, the buffer solution comprises from about 20 mM to about 100 mM acetate, preferably from about 40 mM to about 90 mM acetate and from about 140 mM to about 350 mM glycine. The pharmaceutical compositions or the liquid pharmaceutical formulations according to the invention are for use in therapy. In one embodiment, the pharmaceutical composition for use in therapy comprises from 80 mg/mL to 200 mg/mL, preferably from about 120 to about 185 mg/mL of an antibody or antigen-binding fragment thereof, acetate, glycine, polysorbate 80, at a pH of from about 4.6 to about 5.5; wherein the antibody or antigen-binding fragment thereof (as it may apply) comprises:1) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2; or2) a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO:4; or3) a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4; preferably from about 20 mM to about 100 mM acetate, from about 140 mM to about 350 mM glycine and from about 0.01% to about 0.07% (w/v) polysorbate 80 at a pH of from about 4.6 to about 5.5. In another embodiment, the pharmaceutical composition for use in therapy is obtained by the method according to the present invention, which method comprises the steps of:a. preparing a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2 with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5;b. preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof of the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml;c. adding polysorbate 80 to the high concentration formulation obtained in b), preferably at from about 0.01 to 0.07 (w/v) %;d. optionally, before step c) adjusting the concentration of the antibody or antigen-binding fragment thereof with the buffer solution comprising glycine and acetate. The pharmaceutical composition according to the invention is also for use in the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F. In one embodiment, the pharmaceutical composition for use in the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F comprises from 80 mg/mL to 200 mg/mL, preferably from about 120 to about 185 mg/mL of an antibody or antigen-binding fragment thereof, acetate, glycine, polysorbate 80, at a pH of from about 4.6 to about 5.5; wherein the antibody or antigen-binding fragment thereof (as it may apply) comprises:1) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2; or2) a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO:4; or3) a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4; preferably from about 20 mM to about 100 mM acetate, from about 140 mM to about 350 mM glycine and from about 0.01% to about 0.07% (w/v) polysorbate 80 at a pH of from about 4.6 to about 5.5. In one preferred embodiment, the pharmaceutical composition for use in the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F is obtained by the method according to the present invention, which method comprises the steps of:a. preparing a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2 with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5;b. preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof of the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml;c. adding polysorbate 80 to the high concentration formulation obtained in b), preferably at from about 0.01 to about 0.07 (w/v) %;d. optionally, before step c) adjusting the concentration of the antibody or antigen-binding fragment thereof with the buffer solution comprising glycine and acetate. The present invention also provides for the use of the pharmaceutical composition in the manufacture of a medicament for the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F, wherein the pharmaceutical composition comprises from about 80 mg/mL to about 200 mg/mL, preferably from about 120 to about 180 mg/mL of an antibody or antigen-binding fragment thereof, acetate, glycine, polysorbate 80, at a pH of from about 4.6 to about 5.5; wherein the antibody or antigen-binding fragment thereof (as it may apply) comprises:1) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2; or2) a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO:4; or3) a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4; preferably from about 20 mM to about 100 mM sodium acetate, from about 140 mM to about 350 mM glycine and from about 0.01 to about 0.07 (w/v) % polysorbate 80 at a pH of from about 4.6 to about 5.5. In one preferred embodiment, the use of the pharmaceutical composition in the manufacture of a medicament for the treatment or prophylaxis of a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F, wherein the pharmaceutical composition is obtained by the method according to the present invention, which method comprises the steps of:a. preparing a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2 with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5;b. preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof of the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml;c. adding polysorbate 80 to the high concentration formulation obtained in b), preferably at from about 0.01 to about 0.07 (w/v) %;d. optionally, before step c) adjusting the concentration of the antibody or antigen-binding fragment thereof with the buffer solution comprising glycine and acetate. Also contemplated by the present invention is a method of treating or preventing a pathological disorder mediated by IL-17A and/or IL-17F, or that is associated with increased levels of IL-17A and/or IL-17F in a mammalian subject comprising administering pharmaceutical composition comprising from about 80 mg/mL to about 200 mg/mL, preferably from about 120 to about 185 mg/mL of an antibody or antigen-binding fragment thereof, acetate, glycine, polysorbate 80, at a pH of from about 4.6 to about 5.5; wherein the antibody or antigen-binding fragment thereof (as it may apply) comprises:1) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2; or2) a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO:4; or3) a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4; preferably from about 20 mM to about 100 mM acetate, from about 150 mM to about 250 mM glycine and from about 0.01 to about 0.07 (w/v) % polysorbate 80 at a pH of from about 4.6 to about 5.5. Preferably; the pathological disorder is selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), asthma, chronic obstructive airways disease (COAD), chronic obstructive pulmonary disease (COPD), acute lung injury; pelvic inflammatory disease, Alzheimer's Disease, Crohn's disease, inflammatory bowel disease, irritable bowel syndrome, Ulcerative colitis, Castleman's disease, ankylosing spondylitis, axial spondyloarthritis and other spondyloarthropathies, dermatomyositis, myocarditis, uveitis, exophthalmos, autoimmune thyroiditis; Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, atopic dermatitis, vasculitis, surgical adhesions, stroke, autoimmune diabetes, Type I Diabetes, lyme arthritis, meningoencephalitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis and Guillain-Barr syndrome, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, fibrosing disorders including pulmonary fibrosis, liver fibrosis, renal fibrosis, scleroderma or systemic sclerosis, cancer (both solid tumours such as melanomas, hepatoblastomas, sarcomas, squamous cell carcinomas, transitional cell cancers, ovarian cancers and hematologic malignancies and in particular acute myelogenous leukaemia, chronic myelogenous leukemia, chronic lymphatic leukemia, gastric cancer and colon cancer), heart disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, periodontitis and hypochlorhydria. More preferably, the pathological disorder is selected from the group consisting of arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), asthma, chronic obstructive airway disease, chronic obstructive pulmonary disease, atopic dermatitis, scleroderma, systemic sclerosis, lung fibrosis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, axial spondyloarthritis and other spondyloarthropathies; and even more preferably the pathological disorder is selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis and axial spondyloarthritis. Even more preferably, the pathological disorder is selected is selected from the group consisting of rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and axial spondyloarthritis. In one preferred embodiment of the present invention, the pharmaceutical composition is for use in the treatment or prophylaxis of rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis and axial spondyloarthritis and comprises about 160 mg/mL of an antibody or antigen-binding fragment thereof, acetate, glycine, polysorbate 80, at a pH of from about 4.6 to about 5.5; wherein the antibody or antigen-binding fragment thereof (as it may apply) comprises:1) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2; or2) a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO:4; or3) a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 3 and a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the constant region of the sequence as defined in SEQ ID NO: 4. In another preferred embodiment, the pharmaceutical composition is for use in the treatment or prophylaxis of rheumatoid arthritis, Crohn's disease, ulcerative colitis psoriasis, psoriatic arthritis, ankylosing spondylitis and axial spondyloarthritis, wherein the pharmaceutical composition is obtained by the method according to the present invention, which method comprises the steps of:a. preparing a low concentration formulation by combining from about 40 mg/ml to about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2 with a buffer solution comprising glycine and acetate at pH of from about 4.6 to about 5.5;b. preparing a high concentration formulation by concentrating the antibody or antigen-binding fragment thereof of the low concentration formulation obtained in a) to a concentration of about 160 mg/ml to 180 mg/ml;c. adding polysorbate 80 to the high concentration formulation obtained in b), preferably at from about 0.01 to about 0.07 (w/v)°/o;d. optionally, before step c) adjusting the concentration of the antibody or antigen-binding fragment thereof with the buffer solution comprising glycine and acetate. The pharmaceutical composition according to the invention may be administered in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent (i.e. an antibody) needed to treat, ameliorate or prevent a targeted disease, disorder or condition, or to exhibit a detectable therapeutic, pharmacological or preventative effect. For any antibody or antigen-binding fragments thereof, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, a therapeutically effective amount of antibody will be from 0.01 mg/kg to 500 mg/kg, for example 0.1 mg/kg to 200 mg/kg or 1 to 100 mg/kg. For the treatment of the above diseases and/or disorders, the appropriate dosage will vary depending upon, for example, the particular antibody to be employed, the subject treated, the mode of administration and the nature and severity of the condition being treated. In a particular embodiment, the pharmaceutical composition according to the invention is administered by intravenous or subcutaneous route. When administered via intravenous injection, it may be administered as a bolus injection or as a continuous infusion. The pharmaceutical composition according to any of the embodiments of the invention may also be administered by intramuscular injection. The pharmaceutical composition may be injected using a syringe, an injection device such as an autoinjector, a needleless device, an implant and a patch. The liquid pharmaceutical formulation of the invention is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards; it may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the conditions as described herein before. The antibody or antigen-binding fragment thereof may be the sole active ingredient in the liquid pharmaceutical formulation. Alternatively, the antibody or antigen-binding fragment thereof may be administered in combination, e.g. simultaneously, sequentially or separately, with one or more other therapeutically active ingredients. Active ingredient as employed herein refers to an ingredient with a pharmacological effect, such as a therapeutic effect, at a relevant dose. In some embodiments the antibody or antigen-binding fragment thereof in the pharmaceutical composition may be accompanied by other active ingredients including other antibodies or non-antibody ingredients, administered by the same or by a different route of administration, to treat other inflammatory or autoimmune diseases. In one embodiment, the subject is administered, simultaneously or in sequence (before and/or after) other antibody ingredients, such as anti-TNF antibodies or non-antibody ingredients such as small molecule drug molecules. The invention will now be further described by way of examples with references to embodiments illustrated in the accompanying drawings EXAMPLES Abbreviations Anti-IL-17A/F Ab: anti IL-17A and IL-17F antibody having sequences as defined in SEQ ID NOs: 3 and 4 of Table 1; AFT: Accelerated Freeze/thaw; BPP: Biologics Pilot Plant; CCPS: Cell Culture Process Sciences; CEX-HPLC: Cation Exchange Chromatography—High Pressure Liquid Chromatography; cIEF: capillary Isoelectric Focusing; DSL: dynamic light scattering; DFS: differential scanning fluorimetry; DPS: Downstream Process Sciences; DTT: Dithiothreitol; FZT: Freeze/thaw; HMW: Heavy Molecular Weight; H PLC: high pressure liquid chromatography; HSC: High throughput Self-interaction Chromatography; IAA: Iodoacetamide ICE: Imaged Capillary Electrophoresis; LMW: Low Molecular Weight; MW: Molecular Weight; NP: Not available; PCR: polymerase chain reaction; PEG: Poly Ethylene Glycol; PES: Poly Ether Sulfone; P520: Polysorbate 20; P580: Polysorbate 80; PTFE: Polytetrafluoroethylene; PVDF: Polyvinylidene fluoride; RH: relative humidity; % RSD: % Relative Standard Deviation; SDS-PAGE: Sodium Dodecyl Sulphate—Polyacrylamide Gel Electrophoresis; s: Seconds; SEC: Size Exclusion Chromatography; SEC—HPLC: Size Exclusion Chromatography—High Pressure Liquid Chromatography; SIC: Self-Interaction Chromatography; Trp: Tryptophan; WFI: Water for Injection; w/v: weight/volume. Example 1: Additives Screening Initial screening studies were performed by interrogating the effect of different additives on the anti-IL-17A and IL-17F antibody at 1 mg/mL by determining the B22 (second viral coefficient) value by SIC and selecting 5 top formulations with the greatest likelihood of success (HSC™ Technology). Positive values of B22 suggest which additive may help mitigating more effectively protein-protein interactions that can lead to aggregation and other types of degradation in a formulation comprising the Anti-IL-17A/F Ab. Different additives were screened. The top additives returning the highest B22 values while not harming the conformational stability of the antibody were identified. From an incomplete factorial design of the possible combinations of the best 9 additives and 3 buffer systems a total of 36 formulations were generated and the resulting B22 values were measured through the HSC™ Technology (Soluble Therapeutics™). From the analysis of these 36 formulations, 5 formulations (Table 2) were predicted and validated though SIC (data not shown). In Table 2, M means measured B22 value and C means calculated B22 value TABLE 2MC10.04M Citrate3 mM1.5% (w/v)3.83.6pH 5.5KH2PO4PEG335020.04M Citrate50 mM35 uM75 mM3.53.4pH 5.5K2HPO4PS20Sucrose30.04M Acetate15 mM0.1% (w/v)100 mM2.92.7pH 5.7NaOAcBenzyl AlcoholSucrose40.04M Citrate25 mM17.5 uM100 mM2.82.5pH 5.5K2HPO4PS20Sucrose50.04M Acetate25 mM125 mM2.82.4pH 5.7K2HPO4Sucrose Example 2: Effect of High Concentration of Antibody on Best Performing Formulations The results of the study performed in Example 1, performed with the Anti-IL-17A/F Ab at 1 mg/ml, were subsequently verified at concentration of about 160 mg/ml of Anti-IL-17A/F Ab and were found not to be representative. The formulations which were included in the screen were (Table 3): TABLE 3140 mM Sodium Citrate3 mM1.5% (w/v)pH 5.6KH2PO4PEG 3350240 mM Sodium Citrate50 mM75 mM0.00044%pH 5.6K2HPO4Sucrose(w/v) PS20340 mM Sodium Acetate15 mM100 mM0.1% (w/v)pH 5.7NaOAcSucroseBenzyl Alcohol440 mM Sodium Citrate25 mM100 mM0.00022%pH 5.5K2HPO4Sucrose(w/v) PS20540 mM Sodium Acetate25 mM125 mMpH 5.7K2HPO4Sucrose640 mM Sodium Citrate50 mM6 mMpH 5.5K2HPO4Histidine The Anti-IL-17A/F Ab at 88.7 mg/mL in 20 mM Histidine, 250 mM Sorbitol pH6.0 was concentrated using vivaflow 50 cassettes with a MWCO of 30 kDa PES membrane. Antibody loss was observed and to account for this predicted loss, the antibody was concentrated up to 175 mg/mL before exchanging the buffer into the relevant formulation buffers using PD10 columns containing Sephadex G-25 medium. After buffer exchange the final formulations were adjusted to 160 mg/mL using the relevant formulation buffers (Table 3—formulations 1 to 6); the amounts are negligible and do not affect the concentration of buffer components). Following this adjustment, PS20 was spiked into formulations 2 and 4 to reach a final content of 0,00044 and 0.00022% (w/v) respectively and benzyl alcohol was spiked into formulation 3 to reach a final content of 0.1% (w/v). Under a laminar flow hood, formulations 1 to 6 were transferred into 2 mL 96 deep well plate then sub-aliquoted into twenty sterile 96-well half area plates (80 μL per well). One mL of each formulation was also transferred into sterile 2 mL Schott Type I Glass vials sealed with Flurotec coated Westar rubber stoppers and Tru-Edge Flip off seals for initial testing. The plates were stored as the following (Table 4; a: 2 mL Schott vials—remaining in 96-well plate; b: only for formulation 1; c: for formulations 2-6; d: for formulation 2-6 this measurement was taken at 5 weeks): TABLE 4Time points in weeks2612ConditionsInitialweeks4 weeksweeks8 weeksweeksSpares5° C.✓a✓✓✓c✓✓✓25° C./✓✓✓c✓✓✓60% RH35° C./✓✓✓c✓✓✓75% RHFZT✓b✓cAFT✓b✓c, d The Freeze/thaw stress was performed by freezing/thawing the formulation five times using a Cryomed Controlled rate freezer with freezing and thawing rates set at 0.5° C./min for the low rate freeze/thaw experiment (FZT) and 2° C./min for the accelerated rate freeze/thaw experiment (AFT). In each case, the probe of the freezer was inserted in the well of an additional plate containing a water/glycerol solution of similar viscosity to the samples. The FZT analysis was performed after 4 weeks at 5° C. for formulation 1 whilst it was performed after 8 weeks at 5° C. for formulations 2 to 6. The AFT analysis was performed after 1 week at 5° C. for formulation 1 and after 5 weeks at 5° C. formulations 2 to 6. Visual Assessment The plates were scanned using an Epson Scanner Expression v750 Pro model J221A in colour (1200 dpi, 24 bits, no image treatment) and greyscale (1200 dpi, 16 bits, no image treatment). Automated visual inspection scan was performed using the Molecular Devices M5 plate reader performing a 1 point well scan measuring absorbance at 600 nm. By visual inspection, for all time points and conditions formulation 1 always looked more turbid than all remaining formulations. By A600, at all conditions formulations 1 and 2 seem to show an increase in absorbance at 600 nm (Table 8) however the increase was minor. After FZT and AFT stress, formulation 1 shows an increase in absorbance at 600 nm (Table 5) with the increase being less pronounced after AFT stress. TABLE 5Timepoint inFormulationsConditionsweeks1234565° C.Tinitial0.100.090.080.120.100.13T040.120.200.090.120.080.10T080.160.200.130.110.080.10T12NPa0.210.0|90.080.080.0925° C./Tinitial0.100.090.080.120.100.1360% RHT040.270.150.090.090.100.07T080.100.240.180.150.150.09T12NPa0.240.120.110.150.0935° C./Tinitial0.100.090.080.120.100.1375% RHT040.190.160.140.100.100.08T080.160.190.110.250.150.11T12NPa0.220.150.170.140.11FZTTinitial0.100.090.080.120.100.13T040.25—————T08—0.210.120.130.090.11AFTTinitial0.100.090.030.120.100.13T040.150.200.110.140.100.10 Protein Concentration Determination by UV at 280 nm Samples were diluted to a nominal concentration of 20 mg/mL then to a nominal concentration of 0.5 mg/mL with filtered de-ionized water. The concentration was determined using absorbance at 280 nm combined with a standard curve in a flat bottom UV transparent 96-well plate with an extinction coefficient of 1.56 mL/(mg*cm) using a Molecular devices M5 plate reader (sample volume: 100 μL). No obvious decreasing or increasing trends can be observed in this data over the course of the study (Table 6A). TABLE 6ATimepoint inFormulationsConditionsweeks1234565° C.Tinitial162.8171.1170.5168.7174.8169.2T04159.0196.2208.4193.6195.8192.2T08150.9177.7191.8169.9179.0178.1T12—141.9158.9136.8144.5150.925° C./Tinitial162.8171.1170.5168.7174.8169.260% RHT04162.9196.1206.0189.6210.9185.3T08165.6189.1203.1177.1196.4180.8T12—195.1162.9152.6197.8165.635° C./Tinitial162.8171.1170.5168.7174.8169.275% RHT04169.1195.0217.5192.9205.0181.4T08168.8208.4191.4182.0200.0211.6T12—184.3163.2160.0186.2168.8FZTTinitial162.8171.1170.5168.7174.8169.2T04160.1—————T08—176.5188.9175.8187.0182.7AFTTinitial162.8171.1170.5168.7174.8169.2T04169.2177.2183.0177.3176.2178.8 pH Measurement The pH was determined on a Mettler Toledo S47 pH meter at 23-25° C. No dilution was performed prior to the measurement. For samples stored at 5° C., during the course of the study the pH value for all formulations was within 0.2 pH unit of the initial value (Table 6B). TABLE 6BBuffers1A234565.575.555.755.495.705.53Timepoint inFormulationsConditionsweeks1234565° C.Tinitial5.565.555.895.515.765.56T045.605.545.925.545.76NPaT085.745.565.905.585.775.57T12NPb5.555.915.695.765.5525° C./Tinitial5.565.555.895.515.765.5660% RHT048.395.555.885.895.765.49T088.175.635.966.075.795.68T12NPbNPa5.966.56NPaNPa35° C./Tinitial5.565.555.895.515.765.5675% RHT047.485.555.906.505.765.58T088.665.716.00NPa5.875.90T12NPbNPa6.448.25NPa5.62FZTTinitial5.565.555.895.515.765.56T045.56—————T08—5.555.905.605.785.57AFTTinitial5.565.555.895.515.765.56T045.585.565.895.595.775.57aNo sample remaining in the plate;bformulation 1 had to be re-prepared impairing time collection at T12. For formulation 3, the initial value was 0.14 higher than the buffer alone value which suggests that in this buffer the antibody drives the pH value up. Formulations 1 and 4, even though the pH is within 0.2 pH unit of initial, there seems to be a trend showing a gradual increase over 2 and 3 months respectively which is not observed in the other formulations. For samples stored at 25° C., during the course of the study, the pH value for formulations 2, 3, 5 and 6 was within 0.2 pH unit with 2, 5 and 6 not being able to be determined due to no remaining sample at T12. For samples stored at 25° C., for formulations 1 and 4 a significant increase in pH can be observed from T04 onwards at 25° C. and 35° C. suggesting a degradation of the sample or contamination. For samples stored at 35° C., formulations 2, 5 and 6 display pH values within 0.2 pH unit of initial with an out of trend value observed at T08 for formulation 6. As no sample was remaining for measurement at T12 for formulations 2 and 5 no conclusions can be drawn regarding possibility of an increase in pH over time as values are still within 0.2 pH unit of initial. Formulation 3 at T12 shows an abnormal increase in pH. Size Exclusion Chromatography Analyses were performed on sample aliquots diluted to 5 mg/mL in filtered mobile phase (0.2 M Na Phosphate pH7.0) using Agilent 1200 series HPLC with 96-well plate auto-sampler. Analyses were performed as follows:Sample load: 50 μL (250 μg) at 5 mg/mLColumn: Tosoh BioScience TSK Gel G3000 SWXL, 250 Å, 5 μm, 7.8×300 mm (part number: 8541)Eluent A: 0.2 M Sodium Phosphate, pH7.0Flow rate: 1 mL/minDetection: UV (Wavelength: 280 nm, Resolution: 8 nm, reference: off)Column Temperature: 25° C.Sample Temperature: 4° C.Gradient: IsocraticMax Pressure: 70 barRun time: 15 minPost time: 5 min Data analysis was performed using Empower 2 software. The % increase of HMW species by SEC for formulation 1 to 6 in comparison to reference formulations DS (anti-IL-17A/F Ab at 80 mg/ml in 20 mM histidine, 250 mM Sorbitol, 0.02% Polysorbate 80, pH 6.0) and DP (same as DS but packaged in a glass vial) is shown in Table 7. For samples stored at 5° C., after 12 weeks, formulations 2, 4, 5 and 6 show a similar degree of aggregation than the DS and DP formulations, with formulation 3 showing a higher increase in aggregation than the DS and DP formulations. Generation of fragments is minimal for formulations 2, 3, 5 and 6 (data not shown). For samples stored at 25° C., formulation 3 was the best performer however all formulations perform worse than the DS and DP formulations. All formulations showed an increase in fragmentation levels with formulations (data not shown). Formulation 2 shows a decrease in % HMW species over the 12 weeks and a significant increase in fragmentation. For samples stored at 35° C., formulation 6 was the best performer, however all formulations perform worse than the DS and DP formulations. All formulations show an increase in fragmentation levels (data not shown). TABLE 1Time points in weeksConditionsFormulationsT04T08T125° C.10.210.36NP20.220.180.4430.620.591.0340.140.060.3150.380.380.6260.120.160.38DS0.200.500.70DP0.000.200.4025° C./60% RH13.883.04NP21.000.76−0.3631.872.232.4741.582.564.6451.712.093.0961.131.391.93DS0.801.301.70DP0.400.901.3035° C./75% RH13.444.66NP22.212.493.2933.493.704.5944.756.417.8253.154.195.1162.083.063.1940° C./75% RHDP1.602.603.80 iCE Imaged Capillary Electrophoresis was performed using a Protein Simple iCE3 system. Analyses were Performed as Follows: Formulations 1 to 6 were diluted to a nominal concentration of 20 mg/mL then to a concentration of 2 mg/mL (using the concentration determined by A280) with filtered de-ionized water. Analyses were performed on samples at 0.2 mg/mL (1/10 dilution in master mix of the samples at 2 mg/mL). A master mix with the following components was prepared (Table 8): TABLE 8PharmalytesDI water1% MC3-10pI marker 4.65pI marker 9.50100 μL70 μL8 μL1 μL1 μL The focus parameters were as follows: 1 min at 1500 Volts followed by 6 min at 3000 Volts. The results are reported in Table 9A (% acidic species) and Table 9B (% basic species). At 5° C., no significant changes in % acidic species can be observed across the conditions and formulations with values all within 2-3%. For samples stored at 25° C., an increase in % acidic species can be observed for all formulations over 12 weeks with formulations 1 and 4 showing a significant increase. This observation is likely linked to the increase in pH observed in those 2 formulations after initial mixing. For samples stored at 35° C., a significant increase in % acidic species is observed after initial mixing and subsequent time points for all formulations with formulation 4 showing the most increase over 12 weeks. Freeze/thaw stress does not affect the % of acidic species in any formulations. With respect to the % basic species, at 5° C., there were no significant changes observed across the formulations and time points. For samples stored at 25° C., formulations 3, 5 and 6 show a slight increase in % basic species over time with formulations 1 and 4 showing a more significant increase in basic species (ca. 2.5%). For samples stored at 35° C., all formulations show an increase in % basic species with formulation 3 showing the lowest increase. Freeze/thaw stress did not affect the % of basic species in any formulations. TABLE 9ATimepoint inFormulationsConditionsweeks1234565° C.Tinitial59.8057.3957.1157.7257.5656.63T0456.6958.4259.0657.8260.4559.30T0860.0957.2657.3558.1459.8156.38T12NPa59.7059.3759.7659.7159.0225° C./Tinitial59.8057.3957.1157.7257.5656.6360% RHT0465.1261.2659.9965.1160.1260.46T0868.0763.3859.9969.5260.0562.35T12NPa55.1662.3775.95NPb63.5535° C./Tinitial59.8057.3957.1157.7257.5656.6375% RHT0468.2067.6564.4869.1564.3966.34T0867.7971.7467.7679.2968.7771.24T12NPa75.1171.4387.65NPb83.81FZTTinitial59.8057.3957.1157.7257.5656.63T0457.21—————T08—57.8358.7158.0258.0057.93AFTTinitial59.8057.3957.1157.7257.5656.63T0458.3257.4157.6357.8757.5558.14anot availablebno sample remaining to be tested TABLE 9BTimepoint inFormulationsConditionsweeks1234565° C.Tinitial2.672.232.342.152.162.28T042.422.532.622.762.832.83T082.972.252.192.702.102.53T12NPa2.772.192.452.412.4425° C./Tinitial2.672.232.342.152.162.2860% RHT043.203.283.043.593.273.49T085.243.922.904.343.102.42T12NPa18.612.894.80NPb2.9035° C./Tinitial2.672.232.342.152.162.2875% RHT043.644.293.563.644.004.10T085.214.753.535.484.204.14T12NPa6.093.405.02NPb5.13Slow FZTTinitial2.672.232.342.152.162.28T042.48—————T08—2.512.393.162.262.69Fast FZTTinitial2.672.232.342.152.162.28T042.372.632.492.282.362.24anot availablebno sample remaining to be tested Intrinsic Fluorescence Analysis was performed on 100 μL of sample from each formulation at 0.5 mg/mL of antibody. This method is based upon the intrinsic fluorescent properties of Trp. Trp is known to fluoresce strongly at 340 nm when excited at 280 nm and shielded from water; Trp exposed to water fluoresces weakly. This property can be used to assess the stability of proteins, as the protein begins to unfold the shielded Trp's are exposed to water resulting in a reduction in fluorescence, as proteins aggregate more Trp's are shielded the fluorescence should increase. The method was carried out in a flat bottom 96-well opaque black fluorescence plate using the molecular devices M5 plate reader (read from top, no shake) with an excitation wavelength of 280 nm and an emission wavelength of from 310 nm to 370 nm with 6 flashes per read. The blank plate was water and the reference standard was 5× Reference standard at 0.5 mg/mL. The results were normalized for concentration against the reference standard and reported as a response factor (Table 11) using the following calculation: ((FLU/Concentration)/FLU Reference Standard)*100 At 5° C., over 12 weeks, formulations 2 and 3 show less of an increase of the response factor than formulations 4, 5 and 6 suggesting that the molecule is less susceptible to aggregation for formulations 2 and 3 (Table 10). For all formulations 2 to 6 there is a significant increase at T04 which is also observed at the other conditions. A possible explanation for this could be increased susceptibility to aggregation in all formulations 2 to 6 after 1 month which would be reversible (non-covalent aggregation) as the effect is much less pronounced for subsequent time points. However, as the effect is similar across the conditions and we would expect to see less of an effect at lower temperatures, this observation seems to be an outlier as all measurements at T04 are higher. This could also be linked to an error in the preparation or measurement. At 25° C., over 12 weeks formulation 2 shows the least increase in response factor compared to formulations 3, 4, 5 and 6 suggesting that the molecule is less susceptible to aggregation for this formulation. At 35° C., over 12 weeks, formulations 2 and 4 show the least increase in response factor compared to formulations 3, 5 and 6 suggesting that the molecule is less susceptible to aggregation for formulations 2 and 4. After slow and fast freeze/thaw stress, formulations 2, 4 and 6 show less of an increase of the response factor than formulations 3 and 5 suggesting that the molecule is less susceptible to aggregation for formulations 2, 4 and 6. In the case of formulation 1, the response factor decreases after slow and fast freeze/thaw stress as well as after 12 weeks at all conditions, which could be linked to unfolding of the protein in this formulation. To be noted that it is not known at which point the magnitude of change of the response factor becomes significant in order to differentiate between formulations. Consequently, it is difficult to judge performance of the formulations based on intrinsic fluorescence only as it does not correlate with the SEC data. TABLE 10Timepoint inFormulationsConditionsweeks1234565° C.T04−13.8987.4689.3697.5699.41102.29T08−8.1717.7434.6528.2240.1720.01T12NPa35.3840.1248.3953.8645.3025° C./T04−35.6658.6088.0259.8888.2065.4060% RHT08−26.3510.1027.2816.1733.5418.20T12NPa14.5541.6131.2430.2232.7635° C./T04−27.2593.2878.4289.2793.71104.9475% RHT08−39.643.6333.485.0523.621.26T12NPa18.5842.6517.4834.1729.55FZTT04−17.49—————T08—17.5135.8021.7639.9422.19AFTT04−16.5923.3437.2325.3746.3926.76 Dynamic Light Scattering Analyses were performed on sample aliquots diluted to ca. 5 mg/mL in the relevant filtered buffers without polysorbate 80 or sucrose (where those excipients are part of the formulation) using a Malvern APS Zetasizer with 96-well plate auto-sampler. Analyses were performed as indicated on Table 11 with a scattering angle 90° and an upper size range limit of 0.5 μm, TABLE 11Size StandardParameter(60 μm)Protein SampleMaterialLatexProteinSolventWaterWaterTemperature25° C.25° C.Equilibration time120 sec120 secSampling SpeedDefaultDefaultCleaningVigorous washaVigorous washaMeasurement DurationAutomaticAutomaticMeasurement Number35Extended duration—Yesfor large particlesRelaxation time multiplier—1000000Automatic attenuationYesYesselectionData processingGeneral PurposeProtein AnalysisaRinse Solvent: filtered deionised water; Wash Solvent: 1M NaOH The parameters considered from the DLS measurements were the % monomer and % Pd (polydispersity) by intensity distribution. The % monomer is not strictly speaking monomer as DLS cannot differentiate between molecules unless they have a size that is at least 6 times bigger than the monomer. The % Pd gives an indication whether the distribution is monodisperse; however, the technique might not be able to detect dimers species. According to the manufacturer literature a distribution with a % Pd less than 23% is monodisperse, less than 28% is nearly monodisperse and more than 28% is polydisperse. At initial temperature the % monomer by intensity distribution is low (below ca. 80%) for all formulations (data not shown) but formulation 1 (above 90%). Throughout the whole study at 5° C. the values are between ca. 82 and 98% for formulations 2 to 6 except for formulation 3 for which the values observed are ca 66-68. At 25° C., formulations 1, 3 and 4 are the worst performing formulations with % monomer by intensity changing to values below 80% after Tinitial for formulations 1 and 3 and T04 for formulation 4. At 35° C., formulations 1, 3 and 4 are the worst performing formulations with % monomer by intensity changing to values ≤80% after the initial time point. For formulation 5, this change occurs after T04. With regard to the % Pd, at 5° C. and 25° C. no real trends can be observed across the conditions and formulations with all values being below 23% meaning monodisperse distributions. At 35° C., even though the % Pd is below 23% an increasing trend can be observed across all formulations, Differential Scanning Fluorimetry The method was carried out by Thermofluor using an Applied BioSystem 7500 Fast Real Time PCR Oven. The basis of this method is that when proteins are subjected to increases in temperature they begin to unfold. A dye (the dye is quenched in an aqueous environment but not in a non-polar environment) is added to the protein and as the unfolding takes place the dye binds to exposed hydrophobic regions and emits a fluorescence response which is detected by the detector of the PCR oven. Different regions of the protein have different thermal stabilities hence will unfold at different temperatures. The temperature at which the unfolding occurs is known as the mid-point of thermal denaturation. The higher the temperature, the higher the thermal stability of the protein in a specific environment. All samples were diluted to 0.12 mg/mL using the relevant formulation buffer. Quadruple replicate preparations were made. As the effect of polysorbate 20, PEG3350 and Benzyl alcohol was not known the following measurements were performed: Formulation 1 was measured diluted in formulation buffer with and without PEG3350. Formulations 2 and 4 were measured diluted in formulation buffer with and without PS20. Formulation 3 was measured diluted in formulation buffer with and without Benzyl alcohol. Formulations 5 and 6 were measured in their respective formulation buffer. The dye solution was prepared by mixing 2 μL of 1000× Protein Thermal Shift dye to 250 ul of de-ionised water to obtain an 8× Protein Thermal Shift dye solution. Sample preparation for the assay is as indicated in Table 12A. TABLE 12AComponentVolume (μL)Protein Thermal Shift Buffer5Sample at 0.12 mg/mL12.58X Protein Thermal Shift Dye2.5 Triplicate samples were prepared in an Applied Biosystems MicroAmp Fast Optical 96-well plate and sealed with an Applied Biosystems MicroAmp Optical adhesive film, Protein Thermal Shift Software was used for data analysis. Only Tm2 could be determined automatically. Tm1 was manually estimated using the first derivative of the thermogram. In order to be able to differentiate between formulations by DSF the difference in Tm would need to be greater than 2° C. (Table 12B). The results indicated that, at least by DSF, all formulations are similar. TABLE 12BFormulationsTm1aTm2b170-7274.5270-7275.0370-7275.2470-7274.7570-7275.2670-7275.2aFirst derivative shoulder;bFirst derivative main peak Osmolality Analyses were performed on an Advanced Micro-Osmometer Model 3320 by freezing point depression using the manufacturer's protocol. Samples were measured in triplicates. Formulations 1 and 6 are below 240 mOsm/kg which is not suitable for sub-cutaneous injection (Table 13). TABLE 13FormulationOsmolality in mOsm/kg116822553251440853916173 Viscosity Analyses were performed on 76 μL sample aliquots using a TA Instruments DHR-1 Rheometer using a steady state sensing flow sweep method. The geometry used was a 20 mm, 1.99° cone with a solvent trap containing di-ionized water to reduce the evaporation of material during the measurement. For the steady state sensing flow sweep method, the viscosity was averaged for all points at which steady state was reached (acceptance criteria: less or equal to 5% RSD between the points). Steady State Sensing Flow SweepTemperature: 25° C.Soak Time: 10 secSweep: logarithmicShear rate: 2.9 to 287.9 s−1Points per decade: 5Steady state sensing: yesMaximum equilibration time: 180 sSample period: 25 s% tolerance: 5Consecutive within: 3Controlled rate: motor mode autoData acquisition: ‘save point display’Step termination: none All formulations apart from formulation 6 were considered suitable (Table 14). TABLE 14Viscosity at 25° C. in cPFormulations(steady state flow sweep)113.5213.6312.8414.0512.86aa: failed. Conclusions SEC and DLS have been identified as the differentiating assays. Considering SEC at 5° C., formulation 3 shows an increased rate of aggregation over 12 weeks yet the B22 value is similar to formulations 4 and 5. Also, formulation 2 having a higher B22 value (3.5) than formulations 4, 5 and 6 (2.8-2.9) does not perform better than formulations 4, 5 and 6 while formulation 6 having a negative B22 which would be synonymous with protein-protein net attraction leading to higher aggregation propensity performs similar to 2, 4 and 5. Considering DLS, when stored at 5° C., formulation 6 seemed to behave the best with formulation 3 performing the worst. It is known that protein self-association is mainly related to colloidal stability, while formation of partially unfolded intermediates is mainly related to conformational stability. However, those 2 aggregation pathways are sometimes difficult to distinguish. Often relative B22 values do not indicate aggregation tendency as similar B22 values could be obtained in different solution conditions irrespective of the different aggregation tendencies or conditions where the measured B22 were more negative that showed less aggregation propensity (Bajaj, H., Sharma, V. K and Kalonia, D. S., 2004, Biophys. J. 87(6), 4048-4054). In the case of the anti-IL-17A/F Ab exemplified herein, the screening approach using B22 values does not correlate with the aggregation behaviour of this molecule at 160 mg/mL. This could be due to the fact that the mechanisms of aggregation are different at 1 mg/mL and 160 mg/mL or that the tendency of protein self-association is not what primarily governs the degradation/aggregation of this molecule. In addition, formulations 1 and 6 are below the 240 mOsm/kg threshold, formulations with osmolality below such value are not suitable for sub-cutaneous injection; hence they were not included in further long-term stability evaluation. Formulation 3 was also excluded because of the aggregation rate over 12 weeks. Example 3: Aggregation Studies To assess the kinetics of HMW species formation of anti-IL-17A/F antibody according to the invention was studied in 2 formulation buffers:A: 20 mM Histidine, 250 mM Sorbitol pH6.0 andB: 55 mM Sodium Acetate, 220 mM Glycine, pH5.0 at 4 different concentrations (80, 120, 160 and 200 mg/mL) of the antibody and of polysorbate 80 (0.02, 0.03, 0.04 and 0.05%, depending on the concentration of the antibody) by SEC over 3 months with numerous time points at 3 storage conditions (5° C., 25° C./60% RH and 35° C./75% RH) of HMW species formation. The anti-IL-17A/F antibody according to the invention was in an original buffer of 20 mM Histidine; 250 mM Sorbitol pH6.0 at ca 88 mg/mL, so buffer exchange was only performed for buffer B without polysorbate 80 using Vivaflow 50 cassettes with a PES membrane and a WACO of 30 kDa. Three cycles of 2 volumes of formulation buffer B were performed. The antibody in formulation buffers A and B was concentrated to a nominal target of 120 mg/ml, 160 mg/ml and 200 mg/mL. The concentration values that were not within 5% of the target value were adjusted with the relevant buffer. Concentrations were measured using the SoloVPE (Variable Path Extension system from C. Technologies connected to a Cary50 spectrophotometer) with an extinction coefficient of 1.56 at 280 nm. All prepared formulations were sterile filtered using Steriflip tubes with a 0.22 μm PVDF membrane except formulation B at 200 mg/mL where PES membrane was used after PVDF membrane was blocked. Sample in formulation A at 200 mg/mL was more easily filtered using PVDF membrane filters while sample in formulation B at 200 mg/mL were more easily filtered using PES membrane filters. All formulations were spiked with the relevant amount of polysorbate 80 so as to obtain the values listed in Table 15. This was performed in a laminar flow hood. TABLE 15PS80 concentrationFormulations(% w/v)Formulations A and B at0.0280 mg/mLFormulations A and B at0.03120 mg/mLFormulations A and B at0.04160 mg/mLFormulations A and B at0.05200 mg/mL Three 2 mL vials with 1 mL fill volume were prepared for each formulation at each concentration. At each time point the vials were transferred into a laminar flow hood and 2×10 μl aliquots per sample were taken for analysis by SEC followed by sealing of the vial and placement in the relevant storage condition. The reduction in head space by the last time point would not impact the results of the study as the total volume taken from 1 vial was only 260 μL. Storage was performed at 5° C., 25° C./60% RH and 35° C./75% RH at initial time, 1, 2, 3, 4, 5, 7, 10, 14, 18, 28, 42, 56 and 84 days. A further measurement at 168 days was performed for formulation B at 160 mg/ml only. Size Exclusion Chromatography Analyses were performed on sample aliquots diluted to 5 mg/mL in filtered mobile phase (0.2 M Na Phosphate pH7.0) using Agilent 1200 series HPLC with 96-well plate auto-sampler. Analyses were performed as follows:Sample load: 50 μL (250 μg) at 5 mg/mLColumn: Tosoh BioScience TSK Gel G3000 SWXL, 250 Å, 5 μm, 7.8×300 mmEluent A: 0.2 M Sodium Phosphate, pH7.0Flow rate: 1 mL/minDetection: UV (Wavelength: 280 nm, Resolution: 8 nm, reference: off)Column Temperature: 25° C.Sample Temperature: 4° C.; Gradient: IsocraticMax Pressure: 70 bar Run time: 15 min Post time: 5 min Data analysis was performed using Empower 2 software. Aggregation rates reported in the Tables 16 and 17 refer to the average monthly rate increase for each formulation, based on the aggregation measured after 3 months or after 6 months, in comparison to the one at TO. At all concentrations, formulations B were the best performing formulations with a lower aggregation rate over time at 5° C. (Table 16). In addition, after 6 months, the formulation with 55 mM Na acetate, 220 mM Glycine, 0.04% (w/v) PS80 pH 5.0 at a concentration of anti-IL-17A/F Ab of 160 mg/mL showed a similar aggregation rate to the DP formulation or to the rate after 3 months of the formulation at a concentration of anti-IL-17A/F Ab of 80 mg/mL in 20 mM Histidine, 250 mM Sorbitol, 0.02% (w/v) PS80 pH 6.0 at 5° C. (Table 16). There is no significant % LMW species increase over time at 5° C. (data not shown). TABLE 16AntibodyconcentrationRate afterRate afterFormulation(mg/ml)3 months6 monthsA800.10NPB0.08NPA1200.18NPB0.13NPA1600.25NPB0.170.10A2000.33NPB0.21NPDP800.130.10DS800.230.17 At 25° C., for each concentration, formulations A and B exhibit a comparable aggregation rate over time (Table 17). However, formulation B shows a slightly higher propensity to fragment overtime at 25° C., which results in formulation A being the better performing formulation at 25° C. for all concentrations (data not shown). In particular, at 25° C., the anti-IL17A/F antibody exemplified herein in 55 mM Na acetate, 220 mM Glycine, 0.04% (w/v) PS80 pH5.0 at 160 mg/mL exhibits a slightly higher aggregation rate than when formulated in 20 mM Histidine, 250 mM Sorbitol, 0.02% (w/v) PS80 pH6.0 at 80 mg/mL (Table 17). When compared to the DP and DS (prepared as per example 2), both formulations shown higher aggregation rate at 160 mg/mL and 200 mg/mL whilst at 80 mg/ml and 120 mg/mL formulation B shows a similar aggregation rate than the DP and DS material. TABLE 17AntibodyconcentrationRate afterRate afterFormulation(mg/ml)3 months6 monthsA800.34NPB0.37NPA1200.56NPB0.59NPA1600.77NPB0.790.62A2000.97NPB0.97NPDP800.430.30DS800.570.37 At 35° C., at all concentrations, formulations A performed better overtime with a lower aggregation and fragmentation rate (Table 18 showing the % HMW species), In particular, formulation A at 160 mg/mL exhibits a similar aggregation rate as DP at 80 mg/mL at 40° C. (data not shown). DS and DP were prepared as in Example 2. TABLE 18ConcentrationdaysFormulation(mg/ml)01234710141828425684168A801.371.441.471.551.581.681.781.972.082.162.682.883.43NPB1.171.271.321.401 411.531.691.882.112.213.133.604.75NPA1201.561.781.872.042.042.32.432.692.903.053.764.054.80NPB1.361.571.641.771.792.042.232.532.842.954.244.846.36NPA1601.732.102.272.452.522.843.043.373.613.844.665.015.92NPB1.491.771.892.032.132.382.642.993.363.495.075.767.5312.14A2001.972.492.682.953.043.433.654.074.404.695.676.127.24NPB1.702.102.252.432.532.883.23.664.124.256.477.379.59NP Example 4: Aggregation Study 2 Given the aggregation rate shown by formulation B with 160 mg/ml of anti-IL-17A/F antibody, at both 5° C. and 25° C./60% RH over the DS and DP material (prepared as in example 2), a second aggregation study was performed to validate the results of the first study (example 3) by using non-aged anti-IL-17A/F antibody. Only formulation 55 mM Sodium Acetate, 220 mM Glycine, pH5.0 with 0.02%, 0.03%, 0.04% or 0.05% (w/v) PS80 (PS80 concentration depending on concentration of antibody) at 4 different antibody concentrations (80 mg/ml, 120 mg/ml, 160 mg/ml and 200 mg/mL) at 3 storage conditions (5° C., 25° C./60% RH, 35° C./75% RH) was investigated. As with aggregation study 1, SEC was used to investigate the sample over 3 months. After 3 months, the formulation at 160 mg/mL was still a good performer that could be considered in a long-term stability evaluation hence this formulation was also tested at 6 months. Buffers' and vials' preparation, storage and SEC methodology was as described for example 3. Aggregation rates reported in the Tables 19B, 20B and 21B refer to the average monthly rate increase for each formulation, based on the aggregation measured after 3 months or after 6 months, in comparison to the one at TO. The results of aggregation study 2 confirm the results of aggregation study 1 at 5° C. (Table 19A % HMW species and % LMW species—Table 19B, aggregation rate comparison of studies 1 and 2), at 25° C./60% RH (Table 20A, % HMW species and % LMW species—Table 20B, aggregation rate comparison of studies 1 and 2) and 35° C./75% RH (Table 21A, % HMW species and % LMW species—Table 21B, aggregation rate comparison of studies 1 and 2). TABLE 19AConcentrationdays(mg/ml)071421285684168% HMW species800.760.770.790.800.850.870.941.021200.90NP0.960.961.021.101.181.311601.001.031.081.121.211.281.411.582001.091.141.221.251.321.481.601.78% LMW species800.650.680.690.750.660.690.720.781200.59NP0.640.660.640.690.730.791600.620.690.730.700.630.700.740.772000.580.700.690.720.680.670.740.82 TABLE 19BAntibodyAggregationconcentrationRate afterRate afterstudy(mg/ml)3 months6 months1800.08NP20.060.0411200.13NP20.090.0711600.170.1020.140.1012000.21NP20.170.12DP800.130.10DS800.230.17 TABLE 20AConcentrationdays(mg/ml)071421285684168% HMW species800.760.880.991.051.161.411.692.301200.901.121.291.381.511.922.283.101601.001.341.611.771.962.512.964.052001.091.541.852.002.202.823.324.53% LMW species800.650.780.900.991.041.391.882.541200.590.770.840.980.961.321.822.461600.620.770.810.940.981.371.792.542000.580.780.890.950.991.361.802.31 TABLE 20BAntibodyAggregationconcentrationRate afterRate afterstudy(mg/ml)3 months6 months1800.37NP20.310.2611200.59NP20.460.3711600.790.6220.650.5112000.97NP20.740.57DP800.430.30DS800.570.37 The results shown in Tables 19B, 20B and 21B show that in aggregation study 2 (performed with fresh antibody material) the aggregation rate and fragmentation was slightly lower than in aggregation study 1. TABLE 21AConcentrationdays(mg/ml)071421285684168% HMW species800.761.141.451.661.912.853.856.601200.901.451.932.212.543.804.958.151601.001.852.452.833.234.716.119.912001.092.102.773.173.645.246.7410.81% LMW species800.650.981.341.621.822.994.086.661200.590.981.351.581.782.944.056.411600.620.941.321.601.742.853.876.212000.580.961.301.861.742.803.785.95 TABLE 21BAntibodyAggregationconcentrationRate afterRate afterstudy(mg/ml)3 months6 months1801.19NP21.030.9711201.67NP21.351.2111602.011.7821.701.4912002.63NP21.881.62DP801.27a1.02aDS801.19NPaat 40° C./75% RH Example 5: Long Term Stability Study Considering the results of both aggregation studies (examples 3 and 4) and the additive screening study at 160 mg/mL (example 2) the following formulations were selected for long term stability evaluation (Table 22). All formulations comprised 160 mg/ml of anti-IL-17A/F Ab exemplified herein and were also subjected to 5 cycles of freeze/thaw. TABLE 22A40 mM Sodium Citrate, 50 mM K2HPO4,Formulation 275 mM Sucrose, 0.00044% (w/v) PS20, pH 5.6examples 1 and 2B40 mM Sodium Citrate, 25 mM K2HPO4,Formulation 4100 mM Sucrose, 0.00022% (w/v) PS20, pH 5.5examples 1 and 2C40 mM Sodium Acetate, 25 mM K2HPO4,Formulation 5125 mM Sucrose pH 5.7examples 1 and 2D55 mM Sodium Acetate, 220 mM Glycine,Formulation B0.04% (w/v) PS80, pH 5.0examples 3 and 4E20 mM Histidine, 250 mM Sorbitol,Formulation A0.04% PS80, pH 6.0examples 3 and 4 During the formulation preparation buffer exchange cycle for formulations A, B and C took longer than formulation E (˜90 minutes for formulations A-C and 50 minutes for formulation E). Formulations A and B behaved similarly and during both the buffer exchange and concentration steps both formulations were cloudy. In addition, the flush for each of these formulations was cloudy and milky in colour. Formulation C was also cloudy during the buffer exchange and concentration steps. There were no noticeable differences for formulations D and E, although E seemed to concentrate and filter the best compared to the other formulations. Osmolality (measured with Precision System Multi-Osmetter 2430), viscosity (measured with Anton Paar Automated Multi Viscometer), pH (Mettler Toledo SevenMulti), visual appearance, absorbance at 280 nm (Agilent 8453 spectrophotometer), SDS-PAGE analysis, cIEF (measured with Protein Simple iCE280 system, see example 2), binding activity (measured with a GE Healthcare Biacore T100 system, subvisible particle analysis by light obscuration (HIAC 9703 system from Hach Lange), CEX-HPLC and SEC-HPLC measurements were performed over a period of 6 months (viscosity and osmolality were only measured at time point 0) at time points 0, 1 month, 2 months, 3 months, 4 months, 6 months. Unless specified, the methods were as described in example 2. After the six-month time point, only formulation D samples were evaluated. For all formulations, a 1.0 mL sample fill volume was used. For the freeze/thaw study, all of the formulations were stored at −70° C. for 12 hours then stored at room temperature until completely thawed (≥2 hours). This was repeated for a total of 5 cycles of freeze/thaw. Viscosity Viscosity was measured using an Anton Paar Automated Multi Viscometer. The viscosity values of formulation A to E were tested at time zero. All formulations displayed viscosity values ranging from 2.3-17.3 cP. Samples were evaluated at room temperature (ca.25.00±0.01° C.). Osmolality Osmolality has been performed using Precision Systems Multi-Osmetter 2430. No dilution was performed. The osmolality was tested at time zero for all formulations. Osmolality values ranged from 316 mOsm/kg to 450 mOsm/kg. pH pH has been measured at 25° C. using a Mettler Toledo sevenMulti pH meter. No dilution was performed. The pH was tested for each formulation at each data point. During the first 6 months of stability, the pH of formulations A and B at each time point was 5.5±0.1, the pH of formulation C was 5.7±0.1, and the pH of formulation E was 6.2±0.2. Throughout the 12-month stability, the pH of formulation D was 5.1. No change in pH was observed for any of the five formulations evaluated during the freeze thaw study as compared to time zero. Appearance For each time point during the 12 months of stability, including freeze thaw, the appearance for each formulation was evaluated. For all formulations for the first 6 months of stability, the appearance was “yellow brown, clear solution, free of visible particulates”. After the 4-month time point, the appearance was determined to be “clear, yellow liquid, free of visible particulates”. The change observed in appearance of all formulations can be attributed to analyst variability. As such there were no differences between formulations during the 12 months of stability. Absorbance at 280 nm The protein concentrations were determined using an Agilent 8453 spectrophotometer. Samples were diluted gravimetrically to 0.5 mg/mL in their respective buffers. Prior to analysis of each formulation the system was blanked using formulation buffer. No clear trends were observed for the freeze thaw study or over the course of the 12-month stability study suggesting that any changes observed in the concentration were within the variability of the assay. No consistent decrease in concentration was observed for any of the 5 formulations. SDS-PAGE SDS-PAGE analysis was performed using a 4-20% Tris-Glycine gel with a 3 μg (non-reducing conditions with IAA) or 4 μg (reducing conditions with DTT) per lane loading. Denaturation was performed by incubating samples at 70° C. for 5 min. Staining used was Colloidal Blue. Analyses of the stability samples by reduced SDS-PAGE showed no trend (increasing or decreasing) in the % heavy chains (HC)+light chain (LC) for any of the formulations except for the measurements carried out for the accelerated and stressed conditions. A decrease in % HC+LC was observed in all formulations for the 25° C./60% RH condition with a greater decrease observed in the formulations at 40° C./75% RH. For each of the formulations at those conditions, the formation of a new species was observed. No change was observed for any of the formulations evaluated during the freeze thaw study. Analyses of the stability samples by non-reduced SDS-PAGE showed a general decrease in % IgG observed across all formulations and conditions. The greatest decrease in % IgG was observed for formulations stored at 25° C./60% RH and 40° C./75% RH. The formation of aggregate species was particularly evident in formulation D at 40° C./75% RH. No change was observed for any of the formulations evaluated during the freeze thaw study. cIEF The % main peak for formulations stored at −70° C. and 2-8° C. showed little to no change during the 12 months of stability. A decrease was observed in the % main peak area for all formulations stored at 25° C./60% RH with a greater decrease observed when stored at 40° C./75% RH formulations. This decrease was slightly higher in formulation D and slightly lower in formulation E. For all formulations, there was no change observed in the % acidic species at −70° C. and 2-8° C. An increase in % acidic species for the formulations stored at 25° C./60% RH and 40″C/75% RH formulation was observed. For formulation D, there was a slightly higher increase in % acidic species observed at 25° C./60% RH. At 40° C./75% RH, formulations B and D showed a slightly higher increase in % acidic species while formulation E showed a slightly lower increase. No significant change in the % basic species was observed for formulations stored at −70° C. and 2-8° C. A slight increase was observed in formulations stored at 25° C./60% RH with an even larger increase in % basic species observed in formulation A with storage at 40° C./75% RH. At the 2-month time point, there was a loss of resolution due to capillary issues causing a decrease in % basic species for all formulations and conditions. For all time points, there was greater variability in % basic species. No changes in acidic, main or basic species were observed in freeze thaw formulations A to E. Biacore Binding of IL-17A and IL-17F was measured using a Biacore T100 (GE Healthcare). All experiments were performed at 25° C. Affinipure F(ab′)2 fragment goat anti-human IgG, Fc fragment specific (Jackson ImmunoResearch, category #109-006-098, lot #83295) was immobilized on a CM5 Sensor Chip (Biacore AB, category #BR1000-14, different chips used from lot #10030608) via amine coupling chemistry to a capture level of ca. 7000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, GE Healthcare) was used as the running buffer with a flow rate of 10 μL/min. A 10 μL injection of each sample of antibody at 0.5 μg/mL was used for capture. Recombinant Human IL-17A (R&D Systems, catalog number 317-ILB) and IL-17F (R&D Systems, catalog number 1335-IL) were titrated over the captured anti-IL17AF antibody at doubling dilutions from 10 nM to 2.5 nM and from 10 nM to 1.25 nM respectively at a flow rate of 30 μL/min. The surface was regenerated at a flow rate of 10 uL/min by a 10 μL injection of 40 mM HCl, followed by a 5 μL injection of 5 mM NaOH. Double referenced background subtracted binding curves were analyzed using the BIAevaluation software (version 3.2) following standard procedures. Kinetic parameters were determined from the fitting algorithm (Biacore 1:1 Langmuir binding fit). No trends were observed in the binding activity over the first 12 months of stability suggesting that changes in KDwere within the variation of the assay. An evaluation of freeze thaw study samples was performed in parallel with the 1-month stability samples. There were no changes observed for these samples and slight changes in KDare within the variation of the assay. Size Exclusion Chromatography SEC was performed on sample aliquots diluted to 1 mg/mL in filtered eluent A using an Agilent 1200 series system with the following parameters:Sample load: 20 μL (20 μg) at 1 mg/mLColumn: Tosoh BioScience TSK Gel G3000 SWXL, 250 Å, 5 μm, 7.8×300 mm (part number: 8541)Eluent A: 0.05 M Na2HPO4, 0.25 M NaCl pH7.2Flow rate: 05 mL/minDetection: UV (Wavelength: 280 nm, Resolution: 8 nm, reference: off)Column Temperature: 20±5° C.Sample Temperature: 6±2° C.Gradient: IsocraticMax Pressure: 70 barRun time: 35 min For all formulations, there was no change observed in the % main peak at −70° C. and a slight change observed at 2-8° C. A decrease in the % main peak was observed for all formulations stored at 25° C./60% RH and an even greater decrease for formulations stored at 40° C./75% RH. Overall, formulation D showed the greatest decrease in % main peak over the 6 months of stability at 25° C./60% RH and 3 months stability at 40° C./75% RH. No significant change was observed in the % HMW and % LMW species in all formulations stored at −70° C. There was a slight increase in % HMW but no noticeable change in % LMW for formulations stored at 2-8° C. Over the six months of stability, the rate of aggregation was lower for formulations A, B and D compared to formulation C and E. For the 25° C./60% RH and 40° C./75% RH conditions an increase was observed for both the HMW and LMW species. Formulation D showed the greatest increase for both the HMW and LMW species over the 6 months stability at 25° C./60% RH and over the 3 months stability at 40° C./75% RH. No changes were observed for any of the formulations evaluated during freeze thaw study. Cation Exchange Chromatography CEX was performed on sample aliquots diluted to 1 mg/mL in eluent A using an Agilent 1100 system with the following parameters:Sample load: 20 μL (20 μg)Column: BioMAb, NPS, PK, 4.6*250 mm #Agilent 5190-2407Eluent A: 10 mM Sodium phosphate pH6.0Eluent B: 10 mM Sodium phosphate, 1 M NaCl, pH6.0Flow rate: 1 mL/minWavelength: 220 nm bandwidth 8 nm/220 nm bandwidth 8 nm, reference 360 nm bandwidth 100, slit 4 nmPeak width: 0.1 min (2 s)Column temperature: 25° C.Sample temperature: 4° C. Gradient: Time (min)% B02401540.021004510045.022602 For all formulations and conditions during the 12 months stability, there was no change in % main peak area for the −70° C. or 2-8° C. conditions. A decrease was observed at 25° C./60% RH and even greater decrease at the stressed condition 40° C./75% RH. At 25° C./60% RH, all formulations behaved similarly within the variability of the assay. At 40° C./75% RH, formulations A, B, C and D behaved similarly with formulation E showing less of a decrease. The % area for the acidic and basic species did not change over the 12-month study for formulations stored at −70° C. and 2-8° C. However, the decrease in % main peak for formulations stored at 25° C./60% RH and 40° C./75% RH corresponds mainly to an increase in the % area of the acidic species with a smaller increase observed in the % area of the basic species. All formulations behaved similarly within the variability of the assay. The CEX method has a high variability—5% for acidic species and 9% for the basic species. No changes were observed for the freeze thaw stress samples in the % main, % acidic or % basic peak areas. HIAC Sub-visible particle analysis by light obscuration was performed using a HACH Lange HIAC9703 system by diluting 200 μL of sample into 1000 μL of WFI. Two 500 μL draws were analysed for particles ≥2, 5, 10 and 25 μm particles and the data from the second draw was corrected for dilution (results multiplied by 5) and reported as particles/mL. The results were fairly consistent over the course of 12 months with differences occurring due to the variability within the assay. No changes were observed for the freeze thaw stress samples. Conclusions When samples are stored at 2-8′C condition, only the SEC results showed any differentiation between the formulations. All formulations show similar low levels of fragmentation, however, formulations A, B and D show the lowest aggregation over the course of the study with D exhibiting a lower initial level of HMW species. The SEC results combined with the processing observations leads to the conclusion that, given that the formulation's shelf-life is intended to be at 5° C. and not under conditions similar to those used in the accelerated or stress studies, formulation D was the best performer at 2-8° C. followed by formulation E, also in light of the fact that formulation D at 160 mg/ml of anti-IL-17A/F Ab had a comparable profile to the DP at 80 mg/ml and reduced processing issues. Example 6: 3-Months Formulation Robustness Screen Study of Selected Formulations A DoE using a fractional factorial design with 3 centre points was generated with the JMP version 11 statistical software from SAS. It was intended for a primary screening of the following formulation variables by testing the main effects and interactions over 12 weeks at 2 conditions 5±3° C. and 25±2° C./60±5% RH:acetate concentration (55 mM±20%)glycine concentration (220 mM±20%)Polysorbate 80 concentration (0.04%±0.02%)pH (4.9±0.3)Protein concentration (160±15%) The formulations comprising the anti-IL17A/F antibody exemplified herein were made as detailed in Table 23. Formulation 20 was added to evaluate the impact on stability of low protein and excipients concentrations. The formulations were prepared by buffer exchanging the anti-IL17A/F antibody exemplified herein at 50 mg/mL in the relevant formulation detailed in Table 23 without PS80. Seven cycles were performed before the protein concentration was adjusted to the target concentration listed in Table 23. Formulation 20 was prepared by dilution of formulation 9. A 10% stock solution of PS80 was used to spike each formulation to reach the target PS80 concentration detailed in Table 23. TABLE 23target proteinmeasuredAcetateGlycinePolysorbateDF bufferMeasured pHpHFormulationsconcconc(mM)(mM)80 (w/v)osmolalitypHat 24.0° C.shiftF1160160.0552200.043504.905.020.12F2136138.2662640.024055.215.280.07F3136135.8661760.022914.604.750.15F4136134.8441760.062624.604.800.21F5184186.4441760.063105.205.350.15F6184188.1662640.024324.604.810.21F7136135.1442640.063635.215.310.10F8136134.8662640.063774.614.760.15F9136134.4441760.022775.205.310.10F10184190.3442640.063984.604.900.30F11136137.0442640.023544.614.840.23F12184184.5441760.022934.604.880.28F13184185.8442640.024025.205.350.15F14184187.1662640.064425.215.300.08F15160161.9552200.043494.905.050.15F16160160.3552200.043444.905.040.14F17136137.2661760.063115.185.250.07F18184184.5661760.063264.604.810.21F19184184.3661760.023555.205.310.11F206869.9441760.022625.205.260.06 After sterile filtration, 1 mL of each formulation was transferred into 2 mL Schott Type 1 Glass vials sealed with Flurotec coated Westar stoppers and Tru-Edge Flip off seals for initial testing and storage at 5±3° C. and 25±2° C./60±5% RH. pH and osmolality detailed in Table 23 were measured at Tinitial while visual assessment as well as SEC and iCE were performed at 4, 8 and 12 weeks (T12w). SEC was performed on sample aliquots diluted to 1 mg/mL in filtered eluent A (0.05 M Na2HPO4, 0.25 M NaCl pH7.2) using an Agilent 1200 series system with the following parameters:Sample load: 20 μL (20 μg) at 1 mg/mLColumn: Tosoh BioScience TSK Gel G3000 SWXL 250 Å, 5 μm, 7.8×300 mm (part number: 8541)Flow rate: 0.5 mL/minDetection: UV (Wavelength: 280 nm, Resolution: 8 nm, reference: off)Column Temperature: 20±5° C.Sample Temperature: 6±2° C.Gradient: IsocraticMax Pressure: 70 barRun time: 35 min Data analysis was performed using Empower 3 software. For all formulations, no significant changes were observed in the % LMW species in all formulations stored at 5° C., but with an increase observed for all formulations when stored at 25° C. As expected, some increase in % HMW species over 12 weeks across all the formulations was observed, with formulations stored at 25° C. showing a more pronounced increase than those stored at 5° C. (Table 24). TABLE 24T12wDeltaT12wDeltaT12wDeltaSECoverSECoverSECoverFormulations% HMW3M% Mono3M% LMW3M5° C.F12.390.6596.53−0.761.080.11F22.380.6596.57−0.751.050.10F32.060.5596.86−0.661.080.12F42.020.5096.90−0.611.090.11F52.880.8196.06−0.911.060.09F62.410.7196.50−0.831.090.12F72.380.6596.54−0.791.080.14F81.990.4996.93−0.581.080.09F92.420.6696.52−0.761.060.10F102.500.7396.43−0.821.060.09F111.990.5096.92−0.611.080.10F122.470.7296.46−0.821.070.09F132.780.8696.18−0.931.050.08F142.870.8696.07−0.941.060.08F152.300.6096.64−0.691.060.09F162.370.6596.57−0.741.050.08F172.410.6596.53−0.741.060.09F182.440.6996.47−0.791.090.10F192.930.9096.01−0.991.060.09F201.850.3997.07−0.511.080.1225° C.F14.212.4793.56−3.732.231.26F24.432.7093.60−3.721.971.02F33.451.9493.89−3.632.661.70F43.381.8694.13−3.382.491.51F55.113.0493.00−3.971.880.91F64.562.8692.81−4.522.631.66F74.302.5793.73−3.601.971.03F83.542.0493.80−3.712.661.67F94.152.3993.90−3.381.950.99F104.682.9192.91−4.342.411.44F113.502.0194.01−3.522.481.50F124.442.6993.21−4.072.351.37F135.433.5192.67−4.441.910.94F145.433.4292.65−4.361.920.94F154.232.5393.59−3.742.181.21F164.392.6793.44−3.872.171.20F174.122.3693.90−3.371.991.02F184.432.6893.02−4.242.551.56F195.303.2792.78−4.221.920.95F204.212.4793.56−3.732.231.26 Imaged Capillary Electrophoresis was performed using a Protein Simple iCE3 system. Analyses were Performed as Follows: Samples were diluted to a nominal concentration of 20 mg/mL then to a concentration of 2 mg/mL with filtered de-ionized water. Analyses were performed on samples at 0.2 mg/mL (1/10 dilution in master mix of the samples at 2 mg/mL). A master mix with the following components was prepared (Table 25). TABLE 25PharmalytespI markerpI marker1% MC3-104.659.504M Urea70 μL8 μL1 μL1 μL100 μL The focus parameters were as follows: 1 min at 1500 Volts followed by 6 min at 3000 Volts. As shown in Table 26, for all formulations, no significant changes were observed in % acidic and % basic species for formulations stored at 5° C. As for the % HMW species, formulations stored at 25° C. showed a more pronounced increase in % acidic and basic species than those stored at 5° C. (Table 26). TABLE 26DeltaDeltaDeltaiCE %overiCE %overiCE %overFormulationsacidic3MMain3Mbasic3M5° C.F150.30.845.4−1.04.20.2F250.40.845.6−0.34.0−0.5F349.80.446.1−0.24.1−0.2F449.9−0.245.80.74.2−0.5F550.50.245.5−0.64.10.4F650.20.345.7−0.24.1−0.1F750.30.145.70.14.1−0.3F849.6−0.445.90.54.4−0.1F950.20.946.0−0.53.9−0.4F1050.50.445.1−0.74.40.3F1150.40.545.5−0.64.20.1F1250.41.445.3−1.44.30.0F1350.21.345.6−1.14.2−0.2F1450.50.645.3−0.54.2−0.1F1550.00.345.6−0.54.40.2F1650.31.245.7−1.04.1−0.2F1750.01.246.1−0.94.0−0.3F1849.8−0.346.00.24.20.2F1950.50.645.4−0.34.2−0.3F2049.9−0.845.90.84.20.025° C.F155.345.7939.4−7.05.31.2F255.996.3939.3−6.64.70.2F354.134.7539.9−6.35.91.6F454.994.8839.3−5.85.71.0F555.755.5039.7−6.44.60.9F654.905.0139.2−6.85.91.8F756.506.3938.6−6.94.90.5F855.215.1338.9−6.55.91.4F955.536.2339.6−6.94.90.6F1055.345.2439.1−6.75.61.5F1155.335.5238.9−7.25.81.7F1254.665.7339.3−7.46.01.7F1356.057.1439.5−7.34.50.1F1456.086.1739.2−6.64.70.4F1555.115.3739.6−6.55.31.1F1654.665.5440.0−6.75.31.1F1755.306.5039.8−7.25.00.7F1854.204.1039.6−6.26.22.1F1955.095.2039.6−6.15.30.9F2054.894.2740.2−4.94.90.6 | 101,543 |
11857626 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified formulations or methods as such may, of course, vary. Thus, although a number of formulations and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred formulations and methods are described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains. Further, all publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Finally, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Definitions The terms “glycerin” and “glycerin-based water” are used interchangeably herein, and mean and include a solution comprising water (i.e., H2O) and glycerol. The term “structured water,” as used herein, means and includes H2O comprising a hydrogen bond angle greater than 110°, more preferably, a hydrogen bond angle in the range of approximately 113° to 115°. According to the invention, the term “structured water” also means and includes H2O that is processed according to at least one of the methods disclosed in U.S. application Ser. No. 16/559,986, which is incorporated by reference herein. The term “vibrational energy platform,” as used herein, means and includes biologically targeted complex, stable, and efficient energetic blanks and glycerol water-soluble molecules, which, when programmed with a laser charged imprint of herbs, minerals, vitamins, amino acids, or pharmaceutical properties (creating energy-signature templates), help stimulate/enable/enhance vital cellular biochemical processes necessary to maintain homeostasis. The term “biochemical agent” as used herein, means and includes any element, agent, drug, compound, composition of matter or mixture thereof comprising an energy signature component. The terms “energy signature” and “energy signature component,” are used interchangeably herein, mean and include the specific energetic or electromagnetic identity of a selective herb or biochemical agent and, hence, molecular structure(s) thereof when the herb or biochemical agent is exposed to radiation energy, such as radiation energy generated via harmonic oscillation. The terms “energy signature” and “energy signature component,” as used interchangeably herein, also mean and include the properties and functions of an herb or biochemical agent associated with the energetic identity of the herb or biochemical agent. The term “Krebs cycle modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces and/or modulates a Krebs cycle metabolic reaction, process and/or pathway, including, without limitation, Krebs cycle product inhibition and/or substrate availability. According to the invention, suitable Krebs cycle modulators can comprise, without limitation, eleuthero root (or extract), maca, an amino acid, e.g., L-arginine and L-citrulline, and vitamins B1, B2, B3, B5, and B9. The term “neurotransmitter modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces the generation or proliferation of at least one neurotransmitter and/or modulates the transmission thereof by and between neurons and, hence, cells. According to the invention, suitable neurotransmitter modulators comprise, without limitation, epimedium, stinging nettle leaf (also referred to herein as “stinging nettle”), maca root, eleuthero root, ginger root, Yohimbe, vitamin B1, vitamin B6, lion's mane mushroom (hericium erinaceus), waterhyssop (bacopa monnieri), gotu kola (centella asiatica), huperzine A, vitamin E and phosphatidylserine. The term “glutathione modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces the generation or proliferation of glutathione and/or the glutathione family, including, without limitation, glutathione peroxidase. The term “glutathione modulator” also means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces catalase synthesis. According to the invention, suitable glutathione modulators comprise, without limitation, herbs, including, without limitation,schisandra chinensisberry, damiana, epimedium, maca, and stinging nettle leaf; metal ions including iron (Fe) and copper (Cu); and B-vitamins selected from the group comprising vitamins B2, B5, B6, and B7. The term “DNA modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, that induces and/or modulates mitochondrial DNA, including protecting and/or facilitating the repair of mitochondrial DNA. According to the invention, a suitable DNA modulator comprises, without limitation, vitamin B12. The term “endocannabinoid system modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces and/or modulates cell receptor activity; particularly, cannabinoid receptor activity, i.e., the activity of CB1 or CB2. According to the invention, a suitable endocannabinoid system modulator comprises, without limitation, cannabidiol (CBD). The term “nuclear hormone receptor modulator,” as used herein, means and includes an element, agent, drug, compound, composition of matter or mixture thereof, including its formulation, which induces and/or modulates cell receptor activity; particularly, nuclear hormone receptor activity, e.g., the activity of estrogen receptor-α (ERα), estrogen receptor-β (ERβ), androgen receptor (AR), and mineralocorticoid receptor (MR). According to the invention, a suitable nuclear hormone receptor modulator comprises, without limitation, red Koreanginseng. The terms “cellular dysfunction” and “cell dysfunction” are used interchangeably herein and mean and include a reduction or impairment in physical structure or function of a cell. The term “organ dysfunction”, as used herein, means and includes a reduction or impairment in physical structure or function of a mammalian organ, including, without limitation, the cardiovascular vascular system (heart and lungs), digestive system (salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, and anus), endocrine system (hypothalamus, pituitary gland, pineal body, thyroid, parathyroids, and adrenals), excretory system (kidneys, ureters, bladder, and urethra), immune system (lymphatic system, tonsils, adenoids, thymus, and spleen), integumentary system (skin, hair and nails), muscular system, nervous system (brain and spinal cord), reproductive system (ovaries, fallopian tubes, uterus, vagina, mammary glands, prostate, and penis), respiratory system (pharynx, larynx, trachea, bronchi, and diaphragm) and the skeletal system (bones, cartilage, ligaments, and tendons). The terms “prevent” and “preventing” are used interchangeably herein, and mean and include reducing the frequency or severity of a disease, condition, dysfunction or disorder. The term does not require an absolute preclusion of the disease, condition, dysfunction, or disorder. Rather, this term includes decreasing the chance for disease occurrence. The terms “treat” and “treatment” are used interchangeably herein, and mean and include medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, dysfunction or disorder. The terms include “active treatment”, i.e., treatment directed specifically toward the improvement of a disease, pathological condition, dysfunction, or disorder, and “causal treatment”, i.e., treatment directed toward removal of the cause of the associated disease, pathological condition, dysfunction, or disorder. The terms “treat” and “treatment” further include “palliative treatment”, i.e., treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, dysfunction, or disorder, “preventative treatment”, i.e., treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, dysfunction, or disorder, and “supportive treatment”, i.e., treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, dysfunction, or disorder. The terms “pharmacological agent,” “active agent” and “drug” are used interchangeably herein, and mean and include an agent, drug, compound, composition of matter or mixture thereof, including its formulation, which provides some therapeutic, often beneficial, effect. This includes any physiologically or pharmacologically active substance that produces a localized or systemic effect or effects in animals, including warm blooded mammals, humans, and primates; avians; domestic household or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like. The terms “pharmacological agent,” “active agent” and “drug” thus mean and include, without limitation, antibiotics, anti-viral agents, analgesics, steroidal anti-inflammatories, non-steroidal anti-inflammatories, anti-neoplastics, anti-spasmodics, modulators of cell-extracellular matrix interactions, proteins, hormones, enzymes and enzyme inhibitors, anticoagulants and/or antithrombotic agents, DNA, RNA, modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or protein synthesis, polypeptides, oligonucleotides, polynucleotides, nucleoproteins, compounds modulating cell migration, and vasodilating agents. The term “therapeutically effective”, as used herein, means that the amount of a Krebs cycle modulator, glutathione modulator, neurotransmitter modulator, endocannabinoid system modulator, nuclear hormone receptor modulator or DNA modulator and/or biochemical scaffold formed therefrom, or pharmacological or bioactive agent administered to a subject is of sufficient quantity to ameliorate one or more causes, symptoms, or sequelae of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination, of the cause, symptom, or sequelae of a disease or disorder. The terms “delivery” and “administration” are used interchangeably herein, and mean and include providing a Krebs cycle modulator, glutathione modulator, neurotransmitter modulator, endocannabinoid system modulator, nuclear hormone receptor modulator or DNA modulator and/or biochemical scaffold formed therefrom to a subject through any method appropriate to deliver formulations and/or scaffolds to a subject. Non-limiting examples of delivery methods include oral, sublingual, nasal, direct injection, topical application, etc. The terms “patient” and “subject” are used interchangeably herein, and mean and include warm blooded mammals, humans and primates; avians; domestic household or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like. The term “comprise” and variations of the term, such as “comprising” and “comprises,” means “including, but not limited to” and is not intended to exclude, for example, other additives, components, integers or steps. The following disclosure is provided to further explain in an enabling fashion the best modes of performing one or more embodiments of the present invention. The disclosure is further offered to enhance an understanding and appreciation for the inventive principles and advantages thereof, rather than to limit in any manner the invention. The invention is defined solely by the appended claims including any amendments made during the pendency of this application and all equivalents of those claims as issued. As indicated above, the present invention is directed to biochemical scaffolds and associated methods that induce and/or modulate at least one, more preferably, a plurality of molecular and cellular activities, including, without limitation, (i) at least one Krebs cycle metabolic reaction, process and/or pathway, (ii) generation or proliferation of glutathione and/or a member of the glutathione family and, thereby, induced catalase synthesis, (iii) generation or proliferation of at least one neurotransmitter, and/or modulating the transmission of a neurotransmitter by and between neurons, (iv) mitochondrial DNA activity, and (v) cell receptor activity. In a preferred embodiment of the invention, the biochemical scaffolds comprise two platforms (and components associated therewith): a vibrational energy platform and a bioenergetic platform. In some embodiments of the invention, the bioenergetic platforms further comprise a liquid medium. According to the invention, the biochemical scaffold can comprise any suitable medium, such as glycerol water solution (also referred to herein as “glycerin-based water”) and distilled water. In some embodiments, the liquid medium comprises oxygen enriched glycerin infused water molecules. In some embodiments of the invention, the liquid medium comprises structured water. As indicated above, structured water comprises H2O comprising a hydrogen bond angle greater than 110°, more preferably, a hydrogen bond angle in the range of approximately 113° to 115°. In some embodiments, the liquid medium comprises glycerin-based water and structured water. According to the invention, structured water enhances the molecular activities induced by the biochemical scaffolds of the invention; particularly, molecular activities that modulate Krebs cycle metabolic reactions, processes and/or pathways. As set forth in priority U.S. application Ser. No. 14/223,392, in some embodiments, the biochemical scaffold comprises a glycerol water solution comprising at least 1200 mg/oz. of glycerin. In some embodiments of the invention, the bioenergetic platforms comprise at least one of the following modulators: a Krebs cycle modulator, glutathione modulator, neurotransmitter modulator, DNA modulator, endocannabinoid modulator or nuclear hormone receptor modulator. Thus, in some embodiments, the bioenergetic platforms comprise a Krebs cycle modulator and/or glutathione modulator and/or neurotransmitter modulator and/or DNA modulator and/or endocannabinoid modulator and/or nuclear hormone receptor modulator. In some embodiments, the bioenergetic platforms comprise a plurality of Krebs cycle modulators, and/or glutathione modulators, and/or neurotransmitter modulators, and/or DNA modulators, and/or endocannabinoid modulators and/or nuclear hormone receptor modulators. As discussed in detail herein, according to the invention, the Krebs cycle modulators induce and/or modulate a Krebs cycle metabolic reaction, process and/or pathway, including, without limitation, Krebs cycle product inhibition and/or substrate availability. As set forth inFIG.4Aand discussed in detail below, in some embodiments, the Krebs cycle modulators also induce multiple Krebs cycle reactions and/or pathways, resulting in the production of CO2, and/or acetyl-CoA, and/or FADH2, and enhanced adenosine-5′-triphosphate (ATP) energy potential. As set forth in Applicant's priority U.S. application Ser. Nos. 14/223,392, 16/116,539, 17/732,639, and 17/961,836, ATP is a multifunctional nucleoside triphosphate that is used as a coenzyme in cells. ATP is one of the end products of photophosphorylation and cellular respiration, and is used by structural proteins in many cellular processes, including biosynthetic reactions, motility, and cell division. Mammalian mitochondria are organelles that produce more than 90% of cellular ATP. In addition to supplying ATP, i.e., cellular energy, mitochondria are also involved in other cellular mechanisms, including cellular differentiation, apoptosis, as well as cell cycle modulation and cell growth. Mitochondria provide intracellular ATP via a process called glycolysis, which breaks down monosaccharides into ATP through a series of biochemical processes. Mitochondria contain, among other things, the Krebs cycle enzymes that are involved in heme biosynthesis and the electron transport chain, i.e., the Oxidative Phosphorylation pathway (OxPHOS) system. Due to the large flux of redox reactions necessary to maintain oxidative phosphorylation, mitochondria are the primary site of production of reactive oxygen species (ROS). It has, however, been found that increased production of ROS and interference with the OxPhos system can cause cell cycle dysfunction and arrest. The OxPHOS system is composed of five large multi-protein enzyme complexes, which collectively transform the reducing energy of NADH and FADH2to ATP. NADH ubiquinone oxidoreductase (Complex I) contains 45 different subunits, and succinate ubiquinone reductase (Complex II), ubiquinone-cytochrome c oxidoreductase (Complex III), cytochrome c oxidase (Complex IV), and the ATP synthase (Complex V) contain 4, 11, 13 and 16 subunits, respectively. Four of the OxPHOS enzyme complexes (Complexes I, III, IV and V) have a dual genetic origin, i.e., they are composed of both nuclear DNA-encoded proteins and mitochondrial DNA-encoded proteins. Transient ischemia (anoxia) results in the local production of extremely high levels of reactive oxygen species (ROS), which can cause long term damage to mitochondria. In the initial phase of transient ischemia, oxygen is scarce, but tissue demands for ATP remain high, resulting in continued functioning of the OxPhos system except for the terminal reduction of oxygen to water by Complex IV. Therefore, reduced electron acceptors “upstream” of Complex IV accumulate to abnormally high levels. Upon resupply of oxygen, these excess reduced carriers react directly with oxygen to generate highly toxic partially reduced ROS, which are capable of protein, lipid, and DNA modifying reactions. The resulting oxidative damage is deemed to occur mainly inside the mitochondrion, because such ROS are so reactive that they are short lived and cannot diffuse far before finding a target for reaction. Accordingly, OxPHOS proteins and DNA are deemed the cellular molecules most affected by such oxidative stress. The resulting defects in DNA and OxPHOS proteins can, and in most instances will, result in continued increased production of ROS. However, it has been found that modulating the OxPhos system and, thereby, ROS production, which can be achieved by the Krebs cycle modulators of the invention, oxidative stress of cells can be substantially reduced or eliminated. In a preferred embodiment of the invention, the Krebs cycle modulators of the invention comprise, without limitation,schisandra chinensisberry, epimedium, stinging nettle, yohimbe, red Koreanginseng, eleuthero root (or extract), damiana, ashwagandha, maca, L-arginine and L-citrulline, and vitamins B1, B2, B3, B5, B7, B9, and B12. As discussed in detail herein, in a preferred embodiment of the invention, the glutathione modulators of the invention induce the generation or proliferation of glutathione and/or a member of the glutathione family, including, without limitation, glutathione peroxidase, and/or catalase synthesis. In a preferred embodiment, the glutathione modulators of the invention comprise, without limitation,schisandra chinensisberry, epimedium, stinging nettle, yohimbe, red Koreanginseng, eleuthero root (or extract), damiana, ashwagandha, maca, iron (Fe), copper (Cu), and vitamins B1, B2, B3, B5, B7, B9, and B12. As discussed in detail herein, in a preferred embodiment of the invention, the neurotransmitter modulators of the invention induce and/or modulate the generation of neurotransmitters and modulate the transmission thereof by and between neurons and, hence, cells. In a preferred embodiment, the neurotransmitter modulators of the invention comprise, without limitation, epimedium, stinging nettle, maca, eleuthero root, yohimbe, lion's mane mushroom (hericium erinaceus), waterhyssop (bacopa monnieri), gotu kola (centella asiatica), huperzine A, vitamin B1, vitamin B6, vitamin E and phosphatidylserine. As also discussed in detail herein, in a preferred embodiment, the DNA modulators support and/or enhance mitochondrial DNA activity by protecting and/or facilitating the repair of mitochondrial DNA. In a preferred embodiment of the invention, the DNA modulators comprise, without limitation, vitamin B12. In a preferred embodiment of the invention, the endocannabinoid system modulators induce cell receptor and endocannabinoid system activity. In a preferred embodiment, the endocannabinoid system modulators comprise, without limitation, cannabidiol (CBD) or a component thereof. In some embodiments of the invention, the bioenergetic platforms further comprise a nuclear hormone receptor modulator. In a preferred embodiment of the invention, the nuclear hormone receptor modulators induce cell receptor activity; preferably, nuclear hormone receptor modulator activity, e.g., the activity of nuclear hormone receptor modulators estrogen receptor-α (ERα), estrogen receptor-β (ERβ), androgen receptor (AR), and mineralocorticoid receptor (MR). In a preferred embodiment of the invention, the nuclear hormone receptor modulator comprises red Koreanginseng. Vibrational Energy Platform As discussed in detail below, in a preferred embodiment of the invention, the vibrational energy platforms of the invention comprise at least one energy signature component derived from at least one biochemical scaffold formulation component, i.e., an herb or biochemical agent. It has been found and Applicant has confirmed that specific, critical frequencies of radiation energy create an interaction by and between a selective herb or biochemical agent and a suitable medium; more particularly, by and between the herb or biochemical agent and electric dipole structures of water molecules in a glycerin-based water, i.e., a glycerol water solution, whereby permanent polarization of the glycerol water molecules, i.e., coherent glycerol water molecules, is generated. It has also been found and Applicant has also confirmed that water molecules behave as an “active” medium that can capture, replicate, and retain energy signatures of an herb or biochemical agent through defined harmonic oscillation frequencies. Indeed, Applicant has confirmed that highly specific short-range hydrogen bond and electric dipole-to-dipole static interactions between water molecules can be modulated by defined harmonic oscillation frequencies to generate quantum coherent water molecules (also referred to as “energetic blanks”), which form self-assembled coherence domains (CDs) that capture, replicate, and retain energy signatures of herbs and biochemical agents in energy blank regions of the coherence domains. Referring now toFIG.5, there is shown a coherence domain100in a glycerol water solution104that comprises a series of quantum coherent water molecules102, which are bound via short-range hydrogen bonds106. As illustrated inFIG.5, the coherence domain100comprises an energy signature108of a selective herb or biochemical agent that is retained within the energy blank region110. The coherence domain100preferably oscillates in unison with the energy signature108retained within the energy blank region110. According to the invention, when at least one herb and/or biochemical agent of the invention and a glycerol water solution104are subjected to harmonic oscillation at a defined frequency range or sequential harmonic oscillation at defined frequency ranges for a defined, predetermined period of time, the water molecules101in the glycerol water solution104exhibit quantum coherence, whereby a plurality of distinct quantum coherent water molecules102and, hence, coherent domains100, are generated in the glycerol water solution104, and, thereby, a unique glycerol water solution (i.e., vibrational energy platform) comprising the following two separate and distinct forms of water molecules is formed: (i) complex, stable quantum coherent water molecules102, i.e., “energetic blanks,” and (ii) water molecules101. The quantum coherent water molecules102of the glycerol water solution104, i.e., distinct energetic blanks, form coherent domains100that capture, replicate, and retain defined energy signatures108of the selective herb and/or biochemical agent of the invention and, hence, chemical components thereof in the energy blank region110of the coherence domains100, i.e., the energy signature108of the selective herb and/or biochemical agent is imparted to, captured, replicated, and retained by the coherent domains100formed by the quantum coherent water molecules102of the glycerol water solution104. Applicant has found that when a glycerol water solution comprising coherent domains with a retained energy signature of a selective herb or biochemical agent, i.e., a biochemical scaffold, is delivered to and, hence, is in communication with biological tissue, the biochemical scaffold induces specific biochemical activities through the resonant transfer of the retained energy signatures to the biological tissue and, hence, endogenous cells thereof, whereby a mechanism for the precise regulation of biochemical activities in vivo (based on the properties and function of the transferred energy signature) is provided. Applicant has thus specifically found that (i) when the biochemical scaffolds of the invention comprise a glycerol-based water solution and defined quantities of epimedium, stinging nettle, eleuthero root, damiana,schisandra chinensisberry, maca root, red Koreanginseng, ashwagandha, yohimbe, lion's mane mushroom (hericium erinaceus), vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin E and cannabidiol (CBD) or waterhyssop (bacopa monnieri), ginger root, gotu kola (centella asiatica), huperzine A, lion's mane mushroom (hericium erinaceus), vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin E, phosphatidylserine and CBD, (ii) are subjected to harmonic oscillation for a predetermined period of time and thereafter delivered to a subject, and (iii) delivered to a subject, the biochemical scaffolds induce enhanced seminal molecular and cell activity and, thereby, enhanced mental function and acuity of the subject, and thus also abate cognitive degradation associated with neurodegenerative diseases and disorders, if presented by the subject. According to the invention, the diseases and disorders associated with cognitive degradation can comprise any disease or disorder, such as Alzheimer's Disease, vascular dementia, Lewy body dementia, Parkinson's disease, frontotemporal dementia, Creutzfeldt-Jakob disease, Wernicke-Korsakoff syndrome, mixed dementia, normal pressure hydrocephalus and Huntington's disease. According to the invention, the biochemical scaffolds, i.e., liquid compositions thereof, can be subjected to various harmonic oscillations to achieve the above referenced enhanced seminal molecular and cell activity and, thereby, mental function and acuity. Thus, in some embodiments, the harmonic oscillation comprises a frequency in the range of approximately 0.02 kHz to 10.5 kHz for a period of time in the range of at least 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, and a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes, a third frequency in the range of 9.5 kHz to 11.0 kHz for a third period of time in the range of 3.0 minutes to 60.0 minutes, a fourth frequency in the range of 0.01 kHz to 0.03 kHz for a fourth period of time in the range of 3.0 minutes to 60.0 minutes, and a fifth frequency in the range of 0.004 kHz to 0.010 kHz for a fifth period of time in the range of 3.0 minutes to 60.0 minutes. Applicant has further found that when a glycerol water solution, such as glycerol water solution104illustrated inFIG.5, further comprises structured water, i.e., a glycerol structured water solution, and when at least one herb of the invention and the glycerol structured water solution are subjected to sequential harmonic oscillation at frequencies in the range of approximately 0.9-1.5 kHz, 9.5-10.5 kHz, 9.5-11.0 kHz, 0.01-0.03 kHz, and 0.004-0.010 kHz for a time period in the range of 3-60 minutes per frequency range, the water molecules in the glycerol structured water solution exhibit enhanced quantum coherence and, thus, form an enhanced plurality of energetic blanks comprising retained energy signatures of the herb. Applicant has additionally found that when the glycerol structured water solution referenced above is delivered to and, hence, in communication with biological tissue the glycerol structured water solution, i.e., biochemical scaffold, induces enhanced biochemical activity via the resonant transfer of the retained energy signatures to the biological tissue and, hence, endogenous cells thereof. Bioenergetic Platform In some embodiments of the invention, the bioenergetic platforms of the invention comprise at least one Krebs cycle modulator, at least one glutathione modulator, at least one neurotransmitter modulator, at least one DNA modulator, at least one endocannabinoid system modulator, or at least one nuclear hormone receptor modulator. In some embodiments of the invention, the bioenergetic platforms comprise at least one Krebs cycle modulator, at least one glutathione modulator, at least one neurotransmitter modulator, at least one DNA modulator, at least one endocannabinoid system modulator, and at least one nuclear hormone receptor modulator. As indicated above, in one embodiment of the invention, the bioenergetic platforms comprise a plurality of Krebs cycle modulators, a plurality of neurotransmitter modulators, a glutathione modulator, a DNA modulator and an endocannabinoid system modulator. As discussed in detail below, according to the invention, the Krebs cycle modulators, glutathione modulators, neurotransmitter modulators, DNA modulators, endocannabinoid system modulators, and nuclear hormone receptor modulators of the invention, alone and, particularly, in combination, when delivered to a subject, induce seminal molecular and cell activity, which (i) enhance mental function and acuity and (ii) abate cognitive degradation associated with neurodegenerative diseases and disorders, if presented. Each of the noted modulators are discussed in detail below. Krebs Cycle Modulators As indicated above, the Krebs cycle modulators of the invention preferably compriseschisandra chinensisberry, epimedium, stinging nettle, yohimbe, red Koreanginseng, eleuthero root (or extract), damiana, ashwagandha, maca, L-arginine, L-citrulline, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B7, vitamin B9, and vitamin B12. As also indicated above, according to the invention, the Krebs cycle modulators of the invention induce and/or modulate at least one Krebs cycle metabolic reaction, process and/or pathway, including, without limitation, Krebs cycle product inhibition and/or substrate availability. As discussed in detail below, by virtue of the induced Krebs cycle activity, the Krebs cycle modulators of the invention enhance mental function and acuity, and abate cognitive degradation, if presented. As set forth in priority U.S. application Ser. Nos. 14/223,392, 16/116,539, 17/732,639, and 17/961,836, a seminal process associated with the Krebs cycle is the catabolism of carbohydrates, fats, and proteins, which results in the production of a two carbon organic product, i.e., acetate in the form of acetyl-CoA. Acetyl-CoA and two equivalents of water (H2O) are consumed during the Krebs cycle, producing two equivalents of carbon dioxide (CO2) and one equivalent of HS-CoA. In addition, one complete cycle of the Krebs cycle converts three equivalents of nicotinamide adenine dinucleotide (NAD+) into three equivalents of reduced NAD+(NADH), one equivalent of ubiquinone (Q) into one equivalent of reduced ubiquinone (QH2), and one equivalent each of guanosine diphosphate (GDP) and inorganic phosphate (Pi) into one equivalent of guanosine triphosphate (GTP). The NADH and QH2generated during the Krebs cycle are in turn used by the oxidative phosphorylation pathway to generate energy-rich adenosine triphosphate (ATP). A primary source of acetyl-CoA is carbohydrates, which are broken down by glycolysis to produce pyruvate. Pyruvate is decarboxylated by the enzyme pyruvate dehydrogenase to generate acetyl-CoA. Regulation of the Krebs cycle is largely dependent upon product inhibition and substrate availability. For example, NADH, a product of all dehydrogenases in the cycle (with the exception of succinate dehydrogenase) inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and citrate synthase. Acetyl-CoA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. In a preferred embodiment, the Krebs cycle modulators of the invention also upregulate seminal Krebs cycle components and, thereby, induce enhanced seminal molecular and cell activity, support immune function and, hence, (i) enhance mental function and acuity and (ii) abate cognitive degradation associated with neurodegenerative diseases and disorders, if presented. As discussed in detail herein, Krebs cycle converts three equivalents of nicotinamide adenine dinucleotide (NAD+) into three equivalents of reduced NAD+(NADH), which is an essential metabolite that is known to induce anti-inflammatory and antioxidant activity in brain tissue that is often associated with cognitive degradation due to inflammation-mediated neurodegenerative diseases and disorders, and support genomic stability, mitochondrial homeostasis, adaptive stress responses, and cell survival. NAD+ is also a substrate for different NAD+ consuming proteins, which catabolize NAD+ to NAM. A seminal class of NAD+ consuming protein includes class III histone deacetylases sirtuins (SIRTs), which are upregulated in the presence of higher concentrations of NAD+ and regulate seminal cellular pathways, including pathways associated with neuronal survival and, hence, enhance mental function and acuity and abate neurodegenerative diseases and disorders presenting with cognitive degradation. SIRTs are NAD+ dependent enzymes that regulate a wide spectrum of cellular pathways involved in neurodegenerative diseases and disorders presenting with cognitive degradation. By way of example, a seminal SIRT, i.e., SIRT1, promotes neurite outgrowth and associated axon development, in addition to regulating dendritic arborization, i.e., fine branching at distal ends of a nerve fibers, long-term potentiation and, thereby, enhances mental function and acuity by supporting learning and memory capabilities. SIRT1 is also neuroprotective and, thus, protects neurons and, hence, brain tissue from neurodegenerative diseases and disorders presenting with cognitive degradation by interfering with and/or inhibiting cell signaling pathways responsible for the generation of amyloid precursor protein (APP). By virtue of interfering with and/or inhibiting cell signaling pathways responsible for the generation of amyloid precursor protein (APP), SIRT1 significantly reduces generation of amyloid beta peptide (Aβ) and, thereby, formation of amyloid plaques i.e., neurofibrillary tangles of Aβ peptide fibrils in the brain, which are often observed in neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. Further, SIRT1 directly deacetylates histone residue H3K9 of the p53 promoter, which results in reduced expression of the p53 gene and, thereby, promotes cell survival signaling pathways, hence, and protects healthy neurons from p53-mediated programmed cell death associated with neurotoxic cascades that cause deleterious cytoskeletal changes and neuronal dysfunction in a plurality of neurodegenerative diseases and disorders. SIRT1 also retains the integrity of brain tissue; more particularly, white matter of the brain, which is often compromised by lesions in individuals afflicted with neurodegenerative diseases and/or disorders that present with a degradation in mental function and acuity. SIRT1 retains, and often restores, the integrity of white matter by inducing increased regenerative glial progenitor cell activity, e.g., differentiation, and, thereby, promoting white matter regeneration, which ameliorates the neurodegenerative diseases and/or disorders that present (or are associated) with degradation of mental function and acuity. As indicated above, the Krebs cycle modulators of the invention are also capable of inducing and/or modulating product and/or substrate availability. By way of example, Applicant has found that eleuthero root facilitates the formation of glucose-6-phosphate, which, as indicated above, converts to pyruvate, which enters the Krebs cycle as Acetyl-CoA and, thereby, facilitates conversion of NAD+ into reduced NAD+(NADH). The conversion of NAD+ into reduced NAD+(NADH) and, hence, the provision of an optimal NAD+/NADH ratio induces anti-inflammatory and antioxidant activity in brain tissue that is often associated with cognitive degradation due to inflammation-mediated neurodegenerative diseases and disorders. The noted conversion also supports genomic stability, mitochondrial homeostasis, adaptive stress responses, and cell survival. As set forth in priority U.S. application Ser. Nos. 14/223,392 and 16/116,539, Applicant has also found that maca root works synergistically with eleuthero root by inducing co-factor proliferation, including the co-factor NAD+, which supports activation of the Krebs cycle and, thereby, numerous cell signaling pathways that are associated with enhancement of mental function and acuity. Maca root also facilitates the production of super oxide dismutase, i.e., an important antioxidant. Intracellular super oxide dismutase converts a highly undesirable reactive oxygen species (ROS) known as superoxide to hydrogen peroxide and oxygen and, thereby, abates oxidative stress generated by superoxide in the brain that is associated with the injury and death of neurons during the progression of neurodegenerative diseases that present with cognitive degeneration, e.g., Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS). As is well established, vitamin B1, i.e., thiamine, is involved in RNA and DNA production, as well as nerve and mental function. Vitamin B1's active form is a coenzyme called thiamine pyrophosphate (TPP), which converts pyruvate to acetyl Coenzyme A (CoA). Vitamin B1is also a neuroprotective, site-directed antioxidant that neutralizes ROS proximate to neurons and, hence, protects the neurons from oxidative damage that is often observed in numerous neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. Vitamin B2, i.e., riboflavin, is involved in energy production for the electron transport chain and catabolism of fatty acids, i.e., beta oxidation. Vitamin B2also ameliorates oxidative stress, mitochondrial dysfunction, neuroinflammation, and glutamate excitotoxicity often observed in numerous neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. Vitamin B3, i.e., niacin, is composed of two co-enzyme forms of niacin: nicotinamide adenine dinucleotide (NAD), i.e., an NAD+ precursor, and nicotinamide adenine dinucleotide phosphate (NADP). As indicated above, the co-factor NAD+ induces anti-inflammatory and antioxidant activity in brain tissue that is often associated with cognitive degradation due to inflammation-mediated neurodegenerative diseases and disorders. Vitamin B3also increases the ratio of NAD+/NADH in brain tissue and, thereby, also reduces DNA damage, neuroinflammation, and apoptosis of hippocampal neurons associated with a reduced NAD+/NADH ratio, which is often observed in neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. The increased ratio of NAD+/NADH in brain tissue also upregulates activity of neuroprotective SIRTs, e.g., SIRT3. Vitamin B3thus enhances mental function and acuity by ameliorating defects in brain-energy metabolism and oxidative stress associated therewith, which are also often observed in neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. Vitamins B5, B7and B9also enhance mental function and abate cognitive degradation by inducing and/or supporting seminal neurometabolic activities. Vitamin B5, i.e., pantothenic acid, is involved in the oxidation of fatty acids and carbohydrates. Coenzyme A, which can be synthesized from Vitamin B5, is involved in the synthesis of several biological elements, including amino acids, phospholipids and most importantly, neurotransmitters, such as acetylcholine (ACh), which, as discussed in detail below, enhances mental function and acuity, and abates cognitive degradation. Vitamin B7, i.e., biotin, also plays a key role in the metabolism of lipids, proteins, and carbohydrates. Vitamin B7is a critical co-enzyme of four carboxylases: (i) acetyl CoA carboxylase, which is involved in the synthesis of fatty acids from acetate; (ii) propionyl CoA carboxylase, which is involved in gluconeogenesis; (iii) β-methylcrotonyl CoA carboxylase, which is involved in the metabolism of leucin; and (iv) pyruvate CoA carboxylase, which is involved in the metabolism of energy, amino acids, and cholesterol. Vitamin B7also regulates and maintains optimal concentrations of glutamate, glutamine and dopamine, and optimal protein kinase A (PKA) activity in the hippocampus of the brain and, thereby, enhances mental function and acuity. Vitamin B9, i.e., folate or folic acid, acts as a co-enzyme in the form of tetrahydrofolate (THF), which is involved in the transfer of single-carbon units in the metabolism of nucleic acids and amino acids. THF is involved in pyrimidine nucleotide synthesis, which is required for normal cell division. Folate also aids in erythropoiesis, i.e., the production of red blood cells. Further, Vitamin B9is a critical co-enzyme required for nucleotide synthesis in the hippocampus of the brain, which is one of the unique regions in the brain where cell renewal and DNA replication occurs. Folate thus enhances mental function and acuity by facilitating cell renewal and DNA replication in the hippocampus of the brain. Vitamin B12, i.e., cobalamin, is a coordination complex of cobalt, which occupies the center of a corrin ligand and is further bound to a benzimidazole ligand and adenosyl group. Vitamin B12induces and/or supports Krebs cycle activity by binding to methylmalonyl—coenzyme A (CoA) mutase and, thereby, mediates the isomerization of methylmalonyl CoA to succinyl CoA. Vitamin B12also supports nervous system function and, thereby, enhances mental function and acuity by facilitating myelinogenesis, i.e., the formation and development of myelin sheath structures, which are critical structures of neurons that insulate the axon of neurons and enable consistent transmission of neurological signals through the axon. Glutathione Modulators According to the invention, the glutathione modulators of the invention enhance mental function and acuity, and abate cognitive degradation, if presented, by inducing several seminal neurometabolic and antioxidant activities. As indicated above, the glutathione modulators of the invention also comprise one of the aforementioned herbs, i.e.,schisandra chinensisberry, epimedium, stinging nettle, yohimbe, red Koreanginseng, eleuthero root (or extract), damiana, ashwagandha, maca root, iron (Fe), copper (Cu), and vitamins B1, B2, B3, B5, B7, B9and B12. As also indicated above, the glutathione modulators of the invention induce (i) the generation or proliferation of glutathione and/or the glutathione family, including, without limitation, glutathione peroxidase, and/or (ii) catalase synthesis. As also set forth in priority U.S. application Ser. Nos. 14/223,392 and 16/116,539, glutathione; specifically, glutathione peroxidase, is a key intracellular antioxidant that induces conversion of reactive oxygen species (ROS), such as hydrogen peroxide, to H2O and O2. As is well established, oxidative stress generated by ROS in the brain is associated with the injury and death of neurons during the progression of neurodegenerative diseases, e.g., Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Glutathione reacts directly with ROS in nonenzymatic reactions to lower concentrations of ROS in the brain and, thereby, enhance mental function and acuity and abate cognitive degradation associated with neurodegenerative diseases and disorders, if presented. Glutathione also lowers concentrations of ROS in the brain by serving as an electron donor for the reduction of peroxides in the glutathione peroxidase reaction. In the glutathione peroxidase reaction process, glutathione is converted to its oxidized form, i.e., glutathione disulfide (GSSG), which is also referred to as L-(−)-glutathione. After glutathione is converted to GSSG, the GSSG is reduced back to glutathione by glutathione reductase using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor and, hence, regenerates glutathione by transferring electrons from NADPH to GSSG, thus, forming a positive feedback loop that is adapted to continuously remove clinically deleterious ROS from the brain and, thereby, enhance mental function and acuity and abate cognitive degradation associated with neurodegenerative diseases and disorders, if presented. As additionally set forth in priority U.S. application Ser. Nos. 14/223,392 and 16/116,539, vitamin B6, i.e., pyridoxine, is stored in the body as pyridoxal 5′-phosphate (PLP), which is the co-enzyme form of vitamin B6. Pyridoxine is also involved in the metabolism of amino acids and lipids; in the synthesis of key neurotransmitters, including gamma-aminobutyric acid (GABA) and serotonin, and hemoglobin, as well as in the production of nicotinic acid (vitamin B3). Pyridoxine also plays an important role in gluconeogenesis. Further, vitamin B6is also required for the synthesis of sphingolipids, which are critical components required to conduct myelinogenesis, i.e., the formation and development of myelin sheath structures, which, as discussed herein, are critical structures of neurons that insulate the axon of neurons and enable consistent transmission of neurological signals through the axon, and thereby, enhance mental function and acuity. Neurotransmitter Modulators It is well established that the human brain contains large numbers of highly specialized cells called neurons. As illustrated inFIG.3, the neurons10connect to and communicate with other neurons and, hence, cells via neurotransmitters12, i.e., endogenous electrochemical signals, over synapses14. As further illustrated inFIG.3and discussed in detail below, when a sender neuron10generates and transmits neurotransmitters12, the neurotransmitters12activate target receptors16on the receiver neuron10and, hence, initiate at least one seminal biological activity conducted by receiver neuron10. As indicated above, the neurotransmitter modulators of the invention preferably comprise epimedium, stinging nettle, maca root, eleuthero root, ginger root, Yohimbe, vitamin B1, vitamin B6, lion's mane mushroom (hericium erinaceus), waterhyssop (bacopa monnieri), gotu kola (centella asiatica), huperzine A, vitamin E and phosphatidylserine. As also indicated above, according to the invention, the neurotransmitter modulators of the invention induce (and/or modulate) the generation or proliferation of at least one neurotransmitter, including acetylcholine (ACh), dopamine and norepinephrine, and/or the transmission thereof by and between neurons. According to the invention, the neurotransmitter modulators of the invention similarly enhance mental function and acuity, and abate cognitive degradation, if presented, by inducing several seminal neurometabolic activities. As set forth in priority U.S. application Ser. Nos. 14/223,392 and 16/116,539, ACh is a key neurotransmitter that stimulates the central nervous system to enhance mental acuity, i.e., learning ability, short term memory and mental focus. Acetylcholine (ACh), dopamine and norepinephrine are also key neurotransmitters. ACh is functions as an inhibitory and excitatory neurotransmitter that modulates neuronal excitability, influences synaptic transmission, induces synaptic plasticity and coordinates the firing of groups of neurons. ACh also improves memory, motivation, and attention. Dopamine similarly functions as an inhibitory and excitatory neurotransmitter. As inhibitory neurotransmitter, it causes balance and general sense of well-being. As excitatory neurotransmitter, it improves cognition, concentration and focus. Norepinephrine also functions as an inhibitory and excitatory neurotransmitter that similarly improves cognition, as well as mood and mental concentration. Phosphatidylserine, a seminal neurotransmitter modulator of the invention, induces the generation (or release) of ACh, dopamine and norepinephrine, and/or the transmission thereof by and between neurons. Epimedium, yet another seminal neurotransmitter modulator of the invention, comprises the active element icariin, which lowers the amyloid precursor protein (APP) level and, hence, reduces amyloid beta peptide (Aβ) and, thereby, formation of amyloid plaques i.e., neurofibrillary tangles of AP peptide fibrils in the brain, which, as indicated above, are often observed in neurodegenerative diseases and disorders that present with cognitive degradation and, hence, impaired mental function and acuity. As is well known, Tau proteins are used in the brain as axonal microtubule stabilizers. However, when the Tau proteins are hyperphosphorylated via glycogen synthase kinase-3 (GSK-3), amyloid beta proteins are generated, which can, and often will result in formation of amyloid plaques. The hyperphosphorylation of Tau proteins is initiated in the locus coeruleus. Icariin abates hyperphosphorylation and, thus, reduces AP generation. Icariin also facilitates the generation (or release) of ACh and, thereby, enhanced mental acuity by inhibiting acetylcholinesterase enzyme formation. Applicant has also found that eleuthero root and lion's mane mushroom (hericium erinaceus) also stimulate the generation (or release) of ACh, which, as indicated above, enhances mental function and acuity. Applicant has further found that (i) waterhyssop (bacopa monnieri) facilitates removal of formed amyloid beta plaques; (ii) that stinging nettle, ginger root, lion's mane mushroom (hericium erinaceus), and vitamin E reduce inflammation of brain tissue and, hence, brain cells, which is associated with impaired mental function; and (iii) that ginger root, waterhyssop (bacopa monnieri), gotu kola (centella asiatica), huperzine A and vitamin E also inhibit acetylcholinesterase enzyme formation and, hence, preserve ACh. As set forth in priority U.S. application Ser. Nos. 14/223,392 and 16/116,539, Applicant has further found (i) stinging nettle increases the level of neurotransmitters available to act on the neuron receptors; particularly, the neurotransmitters dopamine and acetylcholine, thus improving several mental processes, e.g., learning and recollection abilities, (ii) in addition to inducing the Krebs cycle functions discussed above, eleuthero root also enhances neuron activities, e.g., short term memory, and (iii) Yohimbe also induces elevation of norepinephrine from the locus coeruleus, resulting in enhanced memory. DNA Modulators According to the invention, the DNA modulator(s) of the invention can comprise various biological or pharmacological agents. As indicated above, the DNA modulators of the invention support and/or enhance mitochondrial DNA activity by, among other activities, protecting and/or facilitating the repair of mitochondrial DNA. According to the invention, the DNA modulators of the invention enhance mental function and acuity, and abate cognitive degradation, if presented, by abating DNA damage and degradation and protecting and/or facilitating the repair of mitochondrial DNA to achieve optimal cell function and, thereby, physiological functioning. As is well established and indicated above, mammalian mitochondria are organelles that produce more than 90% of cellular ATP. In addition to supplying ATP, i.e., cellular energy, mitochondria are also involved in other cellular mechanisms, including cellular differentiation, apoptosis, as well as cell cycle modulation and cell growth. When a cell has temporarily or reversibly stopped dividing or regenerating it is often deemed to have entered a quiescent or senescent state referred to as the Go phase of the cell cycle. Non-proliferative cells generally enter the senescent Go phase or state from the Gi phase and may remain senescent for long periods of time, possibly indefinitely (as is often the case for neurons). Senescence is very common for “adult” cells that are fully differentiated. The maximum number of cell divisions that a cell can undergo, varies from cell type to cell type and organism. In fibroblasts, this number is about 50 divisions, after which cell division ceases. However, some cells become senescent after fewer replication cycles as a result of DNA damage or degradation, e.g., DNA mutations, DNA oxidation, and chromosome losses, which would make a cell's progeny nonviable. If the DNA damage cannot be easily repaired, the cells either prematurely age or self-destruct (i.e., apoptosis or programmed cell death). By way of example, individuals afflicted with a neurodegenerative disease or disorder, such as Alzheimer's disease, often have clinically significant populations of senescent neurons, which exhibit increased expression of cyclin-dependent kinase inhibitor 2D (CDKN2D/p19), in their brains caused in-part by DNA damage or degradation. The process of cellular senescence can also be triggered by several additional mechanisms, including telomere shortening (i.e., a form of DNA damage or degradation). Due to DNA replication mechanisms and oxidative stress, telomeres become progressively shorter with each round of replication. As increasing numbers of cell division occur, the telomeres reach a critically short length, which present as double-stranded DNA breaks, resulting in telomere-initiated senescence. Protecting and/or facilitating the repair of mitochondrial DNA, which can be achieved by virtue of the DNA modulators of the invention, is thus essential to achieve optimal cell function and, thereby, physiological functioning. Healthy mitochondrial DNA also provides healthy enzymatic processes, which are required for oxidative phosphorylation and, hence, continued energy production. As indicated above, a preferred DNA modulator comprises vitamin B12. According to the invention, B12supports DNA activity; specifically, DNA synthesis and, in most instances, enhances mental function and acuity and abates cognitive degradation associated with neurodegenerative diseases and disorders. Vitamin B12also supports nervous system function and, thereby, enhances mental function and acuity by facilitating myelinogenesis, i.e., the formation and development of myelin sheath structures, which are critical structures of neurons that insulate the axon of neurons and enable consistent transmission of neurological signals through the axon. Vitamin B12is also involved in the cellular metabolism of carbohydrates, proteins, and lipids. It functions as a co-enzyme in intermediary metabolism for the methionine synthase reaction with methylcobalamin, and the methylmalonyl CoA mutase reaction with adenosylcobalamin. Endocannabinoid System Modulators According to the invention, the endocannabinoid system modulators of the invention enhance mental function and acuity, and abate cognitive degradation, if presented, by modulating inflammation, i.e., reducing the neurochemical effects of beta-amyloid proteins and, thereby, reactive oxidative stress and reactive oxygen, and reducing adverse neuroinflammation. As indicated above, the endocannabinoid system modulators of the invention induce cell receptor activity; preferably, cannabinoid receptor activity, i.e., receptors CB1 or CB2. As also indicated above, a preferred endocannabinoid system modulator comprises cannabidiol (CBD). CBD is one of many cannabinoid molecules produced by plants from the genuscannabis, second only to THC in abundance. CBD activates the two seminal cannabinoid receptors (CB1 and CB2) and, hence, as discussed below, induces several significant physiological activities. One significant physiological activity induced by activating the CB1 and CB2 receptors is modulation of inflammatory activity of tissue and, hence cells, including brain tissue. The inflammation modulation, i.e., reduction thereof, is achieved by (among other factors) reducing the neurochemical effects of beta-amyloid proteins and, thereby, reactive oxidative stress and reactive oxygen. As discussed below, in addition to activating the CB1 and CB2 receptors, CBD can, and in many instances will, enhance the levels of naturally-produced endocannabinoids, e.g., anandamide and 2-arachidonoyl glycerol (2-AG), by inhibiting the enzymes that break them down. CBD also activates multiple serotonin receptors in the brain; particularly, serotonin 1A receptors. As a result, CBD can, and often will, abate cognitive degradation and enhance mental acuity. Applicant has also found that CBD is also an effective neurotransmitter modulator. As indicated above, CBD activates the two seminal cannabinoid receptors CB1 and CB2. By activating the CB1 receptors, anandamide is increased and the associated elevation of corticosterone (stress hormone) and 2-arachidonoyl glycerol (2-AG) are reduced, which have a direct effect (and in many instances a calming effect) on the amygdala, i.e., the seminal emotional center of the brain. Although CBD is a cannabinoid, CBD does not directly interact with and, hence, activate the CB1 and CB2 receptors. Instead, CBD indirectly activates the CB1 and CB2 receptors by modulating signaling through the CB1 and CB2 receptors by inhibiting the enzyme fatty acid amide hydrolase (FAAH). FAAH inactivates anandamide and also converts 2-AG to mono acylglycerol. By inhibiting FAAH more of anandamide and 2-AG available, which further enhances the calming effect on the amygdala. In a preferred embodiment, the endocannabinoid system modulators(s) of the invention also upregulate seminal cannabinoid receptor activity and, thereby, induce enhanced seminal molecular and cell activity, and further modulate inflammation by reducing adverse neuroinflammation and, hence, enhances mental function and acuity. CBD reduces adverse neuroinflammation by inducing (i) upregulation of the neuroprotective cytokines “interleukin-33” (IL-33) and “triggering receptor expressed on myeloid cells 2” (TREM2) expression in glial cells and (ii) downregulating pro-inflammatory “interleukin-6” (IL-6) expression in peripheral blood leukocytes proximate to the glial cells. The upregulation of TREM2 expression induced in glial cells is also associated with reduced concentrations of amyloid precursor protein (APP) and, hence, amyloid beta peptide (Aβ) in the brain. The upregulation of IL-33 expression signals the recruitment of endogenous immune cells, such as microglia, to a biological insult site in the brain, thus, rapidly facilitating remodeling and regeneration of brain tissue and also abating glial scarring in the brain associated with cognitive degradation. Nuclear Hormone Receptor Modulators According to the invention, the nuclear hormone receptor modulators of the invention also enhance mental function and acuity, and abate cognitive degradation, if presented. As indicated above, the nuclear hormone receptor modulators of the invention induce cell receptor activity; preferably, nuclear hormone receptor modulator activity, e.g., the activity of nuclear hormone receptor modulators estrogen receptor-α (ERα), estrogen receptor-(ERβ), androgen receptor (AR), and mineralocorticoid receptor (MR). As also indicated above, a preferred nuclear hormone receptor modulator comprises red Koreanginseng. As is well established, red Koreanginsengcomprises a plurality of ginsenosides, including, without limitation, ginsenoside Rb-1, ginsenoside Rg-1, ginsenoside Re, ginsenoside Rg3, ginsenoside Rg5, ginsenoside Rh2, ginsenoside Rh1, ginsenoside Rh3, ginsenoside Rh4, ginsenoside Rs3, ginsenoside Rb-2, ginsenoside Rd, ginsenoside Rp-1, and ginsenoside F4. As is also well established, ginsenosides (also referred to as “panaxosides”) are classified as both steroid glycosides and triterpene saponins. Ginsenosides are derived exclusively from plants belonging to the genuspanax(i.e.,ginseng), and exhibit a multitude of biological effects that mimic seminal biological activities of anti-inflammatory steroidal drugs that bind to and activate nuclear hormone receptors. Ginsenosides are thus lipophilic in nature, and by virtue of their steroidal backbone, they can traverse cell membranes of mammalian cells by simple diffusion and regulate cellular functions by binding to specific intracellular target proteins in the cytoplasm and nucleus of the mammalian cells. It has also been found and Applicant has confirmed that ginsenosides activate seminal nuclear hormone receptors (e.g., ERα, ERβ, AR, and MR) and, hence, as discussed below, induce several significant physiological activities. One significant physiological activity induced by activating the nuclear hormone receptors is the modulation of inflammatory activity and diseases associated therewith, including neuroinflammatory activity associated with impaired mental acuity and memory. The modulation of inflammatory activity, i.e., reduction thereof, is achieved by (among other factors) suppressing the production of seminal proinflammatory cytokines and, thereby modulating the activities of inflammatory signaling pathways, such as nuclear factor-κB (NF-κB) and activator protein-1 signaling pathways. Ginsenoside Rb1 inhibits TNF-α production in macrophages and suppresses the activation of NF-κB, which is a key regulator of inflammatory activity, as well as a modulator of TNF-α production in macrophages. Ginsenoside Rb1 also significantly reduces activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK-1), which is an inhibitor of κB (IκB) kinase (IKK)-α, NF-κB, and mitogen-activated protein kinases (MAPKs). Ginsenosides also exhibit seminal neuroprotective effects by modulating, i.e., reducing neuroinflammatory activity in brain tissue and in the central nervous system (CNS), by, among other activities, regulating free radical scavenging pathways and reducing inflammatory responses in late-stage ischemia by the inhibiting the expression of inducible nitric oxide synthase (iNOS) and prostaglandin-endoperoxide synthase 2 (COX-2). As is well established, lipopolysaccharide (LPS)-induced neuroinflammatory activity is associated with various neurodegenerative diseases, including Parkinson disease, Alzheimer's disease (AD), and multiple sclerosis. LPS compounds activate microglial cells in the brain and CNS that promote and induce neuroinflammatory activities associated with the noted neurodegenerative diseases. Ginsenoside Re, in particular, reduces inflammatory activity in the brain and CNS by inhibiting proinflammatory mediators (i.e., iNOS and COX2) that are generated in response to mammalian cell exposure to LPS and inhibiting activation of the p38-MAPK signaling pathway in microglial cells. Ginsenoside Rg1 similarly modulates microglial cell activation by reducing the production of tumor necrosis factor-α (TNF-α) and NO as well as the expression of iNOS and ionized calcium-binding adapter molecule 1 (Iba-1) by inhibiting the activation of NF-κB and MAPKs pathways. It has also been found that ginsenoside Rg1 also facilitates protection of other biological tissues, e.g., hepatic tissue, from ischemia/reperfusion (IR) injury by reducing inflammatory activity and associated apoptosis events resulting therefrom by modulating NF-κB and ROS—NO—hypoxia-inducible factor signaling pathways. IR injury; more particularly, IR injury of the brain and CNS is also ameliorated via Ginsenoside Rg1 mediated activation of peroxisome proliferator-activated receptor-γ/heme oxygenase-1 (HO-1), suppression of protease-activated receptor-1 expression, and inhibition of mitogen-activated protein kinase 14 (p38a MAPK), and, hence, inhibited activation of the p38-MAPK cell signaling pathway. Thus, in some embodiments of the invention, the biochemical scaffolds of the invention comprise a liquid composition comprising a liquid medium and at least one of the aforementioned Krebs cycle modulators, neurotransmitter modulators, glutathione modulators, DNA modulators, endocannabinoid system modulators or nuclear hormone receptor modulators. In some embodiments of the invention, the biochemical scaffolds of the invention comprise a liquid composition comprising a liquid medium and a Krebs cycle modulator, neurotransmitter modulator, glutathione modulator, DNA modulator, endocannabinoid system modulator and nuclear hormone receptor modulator. As indicated above, in one preferred embodiment, the biochemical scaffolds of the invention comprise a liquid composition comprising a liquid medium, a plurality of Krebs cycle modulators, a plurality of neurotransmitter modulators, a glutathione modulator, a DNA modulator and an endocannabinoid system modulator. In a preferred embodiment, the liquid medium comprises glycerin-based water. In a preferred embodiment, the Krebs cycle modulators comprise vitamin B1, vitamin B2, vitamin B3, vitamin B5and vitamin B9. In a preferred embodiment, the neurotransmitter modulators comprise ginger root, lion's mane mushroom (hericium erinaceus), waterhyssop (bacopa monnieri), gotu kola (centella asiatica), huperzine A, vitamin E, phosphatidylserine and vitamin B6. In a preferred embodiment, the glutathione modulator comprises vitamin B7. In a preferred embodiment, the DNA modulator comprises vitamin B12. In a preferred embodiment, the endocannabinoid system modulator comprises cannabidiol (CBD). In a preferred embodiment, the liquid medium, i.e., glycerin-based water, comprises in the range of approximately 235 ml to 245 ml. The preferred quantities of the biochemical scaffold components referenced above are set forth in Table I below. TABLE IBiochemicalScaffold ComponentQuantitywaterhyssop (bacopa monnieri)approx. 330.0 mg to approx. 1000.0 mgginger rootapprox. 500.0 mg to approx. 3000.0 mggotu kola (centella asiatica)approx. 50.0 mg to approx. 1000.0 mglion's mane mushroomapprox. 250.0 mg to approx. 1000.0 mg(hericium erinaceus)huperzine Aapprox. 0.2 mg to approx. 0.5 mgvitamin B1approx. 5.0 mg to approx. 7.0 mgvitamin B2approx. 0.60 mg to approx. 0.80 mgvitamin B3approx. 8.0 mg to approx. 12.0 mgvitamin B5approx. 8.0 mg to approx. 12.0 mgvitamin B6approx. 1.0 mg to approx. 3.0 mgvitamin B7approx. 0.055 mg to approx. 0.065 mgvitamin B9approx. 0.155 mg to approx. 0.165 mgvitamin B12approx. 0.995 mg to approx. 1.005 mgvitamin Eapprox. 170.0 IU to approx. 190 IUphosphatidylserineapprox. 250.0 mg to approx. 500.0 mgcannabidiol (CBD)approx. 12.0 mg to approx. 13.0 mg In another preferred embodiment of the invention, the Krebs cycle modulators compriseschisandra chinensisberry, stinging nettle, yohimbe, red Koreanginseng, eleuthero root (or extract), damiana, ashwagandha, maca root, vitamin B1, vitamin B2, vitamin B3, vitamin B5and vitamin B9. In the noted preferred embodiment, the neurotransmitter modulators comprise epimedium, lion's mane mushroom (hericium erinaceus), vitamin E and vitamin B6, the glutathione modulator similarly comprises vitamin B7, the DNA modulator similarly comprises vitamin B12and the endocannabinoid system modulator similarly comprises cannabidiol (CBD). The preferred quantities of the biochemical scaffold components referenced above are set forth in Table II below. TABLE IIBiochemicalScaffold ComponentQuantityepimediumapprox. 1175.0 mg to approx. 2350.0 mgstinging nettleapprox. 1175.0 mg to approx. 2350.0 mgyohimbeapprox. 1175.0 mg to approx. 2350.0 mgred Korean ginsengapprox. 1175.0 mg to approx. 2350.0 mgeleuthero rootapprox. 1175.0 mg to approx. 2350.0 mgdamianaapprox. 1175.0 mg to approx. 2350.0 mgschisandra chinensisberryapprox. 1175.0 mg to approx. 2350.0 mgashwagandhaapprox. 1175.0 mg to approx. 2350.0 mgmaca rootapprox. 1175.0 mg to approx. 2350.0 mglion's mane mushroomapprox. 250.0 mg to approx. 1000.0 mg(hericium erinaceus)vitamin B1approx. 5.0 mg to approx. 7.0 mgvitamin B2approx. 0.60 mg to approx. 0.80 mgvitamin B3approx. 8.0 mg to approx. 12.0 mgvitamin B5approx. 8.0 mg to approx. 12.0 mgvitamin B6approx. 1.0 mg to approx. 3.0 mgvitamin B7approx. 0.055 mg to approx. 0.065 mgvitamin B9approx. 0.155 mg to approx. 0.165 mgvitamin B12approx. 0.995 mg to approx. 1.005 mgvitamin Eapprox. 170.0 IU to approx. 190 IUcannabidiol (CBD)approx. 12.0 mg to approx. 13.0 mg As indicated above, in a preferred embodiment of the invention, the biochemical scaffolds of the invention are subjected to harmonic oscillation at a defined frequency or frequencies and a defined period of time or times. In some embodiments, the harmonic oscillation comprises a frequency in the range of approximately 0.02 kHz to 10.5 kHz for a period of time in the range of at least 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, and a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes, a third frequency in the range of 9.5 kHz to 11.0 kHz for a third period of time in the range of 3.0 minutes to 60.0 minutes, a fourth frequency in the range of 0.01 kHz to 0.03 kHz for a fourth period of time in the range of 3.0 minutes to 60.0 minutes, and a fifth frequency in the range of 0.004 kHz to 0.010 kHz for a fifth period of time in the range of 3.0 minutes to 60.0 minutes. According to the invention, there are thus also provided methods for enhancing mental function and acuity of a subject. In one embodiment, the method comprises:(i) providing a biochemical scaffold comprising a liquid composition comprising glycerin-based water, waterhyssop (bacopa monnieri), lion's mane mushroom (hericium erinaceus), vitamin E and cannabidiol (CBD);(ii) subjecting the biochemical scaffold to harmonic oscillation; and(iii) delivering a therapeutically effective amount of the biochemical scaffold to the subject. In some embodiments, the biochemical scaffold further comprises ginger root. In some embodiments, the biochemical scaffold further comprises huperzine A. In some embodiments, the biochemical scaffold further comprises phosphatidylserine. In some embodiments, the biochemical scaffold further comprises vitamin B6. The preferred quantities of the aforementioned biochemical scaffold components set forth in Table II. In some embodiments, the harmonic oscillation comprises a frequency in the range of approximately 0.02 kHz to 10.5 kHz for a period of time in the range of at least 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, and a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes. In some embodiments, the harmonic oscillation comprises sequential harmonic oscillation comprising a first frequency in the range of 0.9 kHz to 1.5 kHz for a first period of time in the range of 3.0 minutes to 60.0 minutes, a second frequency in the range of 9.5 kHz to 10.5 kHz for a second period of time in the range of 3.0 minutes to 60.0 minutes, a third frequency in the range of 9.5 kHz to 11.0 kHz for a third period of time in the range of 3.0 minutes to 60.0 minutes, a fourth frequency in the range of 0.01 kHz to 0.03 kHz for a fourth period of time in the range of 3.0 minutes to 60.0 minutes, and a fifth frequency in the range of 0.004 kHz to 0.010 kHz for a fifth period of time in the range of 3.0 minutes to 60.0 minutes. As set forth in priority U.S. application Ser. No. 14/223,392, the biochemical scaffolds of the invention can be delivered to host tissue by various conventional means, including, without limitation, oral, sublingual, nasal, direct injection, topical application, etc. As will readily be appreciated by one having ordinary skill in the art, the present invention provides numerous advantages compared to prior art formulations and methods for enhancing cell function and, thereby mental function and acuity. Among the advantages are the following:The provision of biochemical scaffolds and methods associated therewith that enhance physical and mental function by inducing and/or modulating a plurality of seminal molecular and cell activities.The provision of biochemical scaffolds and methods associated therewith that enhance mental function and acuity by inducing at least one Krebs cycle metabolic reaction, process and/or pathway.The provision of biochemical scaffolds biochemical scaffolds and methods associated therewith that enhance mental function and acuity by inducing the enhanced generation of neurotransmitters and/or modulating the transmission thereof by and between neurons.The provision of biochemical scaffolds biochemical scaffolds and methods associated therewith that enhance mental function and acuity by inducing cell receptor activity.The provision of biochemical scaffolds biochemical scaffolds and methods associated therewith that enhance mental function and acuity by modulating the endocannabinoid system.The provision of biochemical scaffolds biochemical scaffolds and methods associated therewith that enhance mental function and acuity by inducing and/or modulating mitochondria DNA activity. Without departing from the spirit and scope of this invention, one of ordinary skill can make various changes and modifications to the invention to adapt it to various usages and conditions. As such, these changes and modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims. | 77,117 |
11857627 | DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS This invention is described in preferred embodiments in the following description with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment.” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment. The described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. Dodecafluoropentane emulsion (DDFPe) was previously tested as a sensitizer for radiotherapy with a single fraction of radiotherapy. Tumors (xenografts) were irradiated, the tumors removed from the animals, the cells disaggregated and tested for viability. Neither tumor growth nor survival was assessed and tumor pO2was not directly assessed. Nor were the effects of administration of DDFPe with chemotherapy assessed. Multi-dose administration of DDFPe with fractionated radiotherapy was not assessed. In certain embodiments, the animal to be treated is a mammal. In certain embodiments, the animal to be treated is a human. In certain embodiments, doses range from about 0.01 cc/kg to about 1.0 cc/kg (2% w/vol DDFPe). In certain embodiments, the doses range from about 0.05 cc/kg to 0.3 cc/kg administered by infusion for up to 30 minutes or a single bolus. As one skilled in the art would recognize, if the concentration of DDFP in the emulsion is increased, e.g. to 5% or 10% by weight, the volume administered will generally be decreased accordingly. Preferably the subject is breathing oxygen or a mixture of oxygen and CO2, e.g. carbogen, between 95% oxygen with 5% CO2to 98% oxygen with 2% CO2. Applicant has discovered that use of carbogen and oxygen are comparable, but carbogen is problematic. It has to be ordered specially whereas oxygen is available everywhere. DDFPe is administered IV prior to each fraction of radiation therapy. In certain embodiments, the DDFPe is administered as a product currently in clinical development under the name NVX-108. In the prior art, relatively high molecular weight fluorocarbons have been studies as radiosensitizers. Materials that have been studied as radiosensitizers include F-1,3-dimethyladamantane, F-trimethylbicyclo[3.3.1]nonane, F-tributylamine (FC-43,” 3M Company), perfluorodecalin and perfluorooctylbromide. The inventor has discovered that lower molecular weight fluorocarbons (FC), most particularly with boiling points from about −4 degrees centigrade to about 100 degrees centigrade are far more effective than the higher molecular weight, higher boiling point FCs. More preferably the boiling point of the FC is from about 20 to about 80 degrees C. and still more preferably from about 28 degrees C. to about 60 degrees C. FCs useful in this invention include perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane and perfluorooctane. Most preferred are perfluoropentane and perfluorohexane with the most preferred being perfluoropentane. Most preferably the FC is prepared as an emulsion by high pressure, temperature controlled homogenization. A variety of surfactants may be used to prepare the emulsions. The preferred surfactants are phospholipids and a preferred composition of phospholipids includes dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine-PEG-5,000 (DOPE-PEG 5 k). Another preferred mixture of phospholipids include dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidylethanolamine-PEG-5,000 (DPPE-PEG 5 k). A preferred ratio of lipids is 92 mole percent DPPC with 8 mole percent DPPE-PEG. The same ratio of lipids is also preferred for the unsaturated phosphatidyl moieties. Other lipids such as cholesterol, phosphatidic acid, stearic acid, palmitic acid, oleic acid and phosphatidylethanolamine may be mixed with the above mentioned lipids. Other useful surfactants include fluorosurfactants such as PEG Telomer B and CAPSTONE (DuPont). Mixtures of phospholipids and the fluorosurfactants may be used. Other surfactants include polyoxyethylene-polyoxypropylene copolymer surfactant e.g. “Pluronic” F-68. Preferably a viscogen is included in the formulation to increase the viscosity in the product to decrease settling of the nano-emulsion. Viscogens include sucrose, carboxymethylcellulose, trehalose, starch, Hextend®, xanthan gum, propylene glycol, glycerol and polyethylene glycol ranging in molecular weight from about 400 to 8,000 MW. Preferably the formulation also includes a buffer such as sodium phosphate to stabilize the pH near neutrality, i.e. pH=7.0. Because of the greater efficacy of the present invention using the lower boiling point FCs much lower doses can be administered to effectively reverse radiation resistance. For example, NVX-108 only has 2% w/vol DDFP. Prior materials had >10% w/vol FC. For this invention the preferred weight range is from about 1% to 5% w/vol FC with 2% w/vol FC most preferred, e.g. 2% w/vol DDFP or perfluorohexane. Experiments in animals bearing tumor xenografts showed that the effects of NVX-108 on tumor pO2were comparable on animals breathing carbogen and oxygen. But the effect on tumor pO2was less in animals breathing room air. Therefore for the purpose of this invention the subject may breathe either carbogen or supplemental oxygen during and/or after administration of the emulsion and during radiotherapy or administration of chemotherapy. The material may be administered concomitantly with radiotherapy or prior to radiotherapy, e.g. up to about 120 minutes prior to radiotherapy. Optionally, in addition to radiotherapy, or alone, chemotherapy is administered concomitantly with DDFPe. A variety of anti-neoplastic agents may be employed in the invention including but not limited to alkylating agents, antimetabolites, anthracyclines, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, miscellaneous chemotherapy drugs, targeted therapies, hormone therapy and immunotherapy. NVX-108 comprises a formulation having the components recited in Table 1. TABLE 1CONCINGREDIENTSPECIFICATIONPURPOSE(MG/ML)DODECAFLUORO-MEDICALACTIVE20PENTANEGRADESUCROSEMEDICALEXCIPIENT300GRADEPEG TELOMER BPURIFIEDEXCIPIENT3CHEMICALGRADEWATER FORUSPSOLVENTQ.S. TOINJECTION1 MLNITROGENMEDICALHEAD SPACEQ.S.GRADEAIR FLUSHSODIUMUSPBUFFER0.01MPHOSPHATEHYDROCHLORICUSPEXCIPIENTQ.S.ACID As a general matter, Applicant's fluorocarbon emulsion does not comprise any amidoamine oxide compounds. More specifically. Applicant's fluorocarbon emulsion does not comprise any fluorinated amidoamine oxide compounds. Alkylating agents include but are not limited to nitrogen mustards: such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; nitrosoureas: which include streptozocin, carmustine (BCNU), and lomustine; alkyl sulfonates: busulfan; triazines: dacarbazine (DTIC) and temozolomide (TEMODAR); ethylenimines: thiotepa and altretamine (hexamethylmelamine). The platinum drugs (cisplatin, carboplatin, and oxalaplatin) are sometimes grouped with alkylating agents because they kill cells in a similar way. Examples of antimetabolites include: 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (XELODA), cladribine, clofarabine, cytarabine (ARA-C), floxuridine, fludarabine, gemcitabine (GEMZAR), hydroxyurea, methotrexate, pemetrexed (ALIMTA), pentostatin and thioguanine. Anthracyclines include: daunorubicin, doxorubicin (ADRIAMYCIN) and epirubicin. Idarubicin anti-tumor antibiotics that are not anthracyclines include: actinomycin-bleomycin, and mitomycin-C. Mitoxantrone is an anti-tumor antibiotic that is similar to doxorubicin in many ways. Examples of topoisomerase I inhibitors include topotecan and irinotecan (CPT-11). Examples of topoisomerase II inhibitors include etoposide (VP-16) and teniposide. Mitoxantrone also inhibits topoisomerase II. Examples of mitotic inhibitors include: taxanes: paclitaxel (TAXOI) and docetaxel (TAXOTERE). Epothilones include ixabepilone (IXEMPRA). Vinca alkaloids include vinblastine (VELBAN), vincristine (ONCOVIN), and vinorelbine (NAVELBINE) and estramustine (EMCYT). Examples of corticosteroids include prednisone, methylprednisolone (SOLUMEDROL), and dexamethasone (DECADRON). Examples of targeted therapies include imatinib (GLEEVEC), gefitinib (IRESSA), sunitinib (SUTENT) and bortezomib (VELCADE). Examples of differentiating agents include the retinoids, tretinoin (ATRA or ATRALIN) and bexarotene (TARGRETIN), as well as arsenic trioxide (ARSENOX). Examples of hormone therapy agents include the anti-estrogens: fulvestrant (FASLODEX), tamoxifen, and toremifene (FARESTON). Aromatase inhibitors include: anastrozole (ARIMIDEX), exemestane (AROMASIN), and letrozole (FEMARA). Progestins include megestrol acetate (MEGACE) and estrogens. Anti-androgens include bicalutamide (CASODEX), flutamide (EULEXIN), and nilutamide (NILANDRON). Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH) agonists or analogs include leuprolide (LUPRON) and goserelin (ZOLADEX). Types of immunotherapies and some examples include: monoclonal antibody therapy (passive immunotherapies), such as rituximab (RITUXAN) and alemtuzumab (CAMPATH). Non-specific immunotherapies and adjuvants (other substances or cells that boost the immune response) include BCG, interleukin-2 (IL-2), and interferon-alfa. Immunomodulating drugs, for instance, thalidomide and lenalidomide (REVLIMID). Cancer vaccines (active specific immunotherapies such as PROVENGE vaccine for advanced prostate cancer may be used with DDFPe and other vaccines currently under development. Administration of the FC increases the activity of the immunotherapy, for example Yervoy® (Ipilimumab) as the immune cells are more active in an oxidative environment achieved through tumor re-oxygenation. Other immune modulating drugs that can be used with the FC emulsions include Inhibitors of PD-L1 expression. Monoclonal antibodies are particularly useful with the invention. The antibodies can be used as vaccines to trigger an immune response to reject the cancer. Non-specific stimulators of the immune system can be used in the invention. Examples include cytokines such as interleukins and interferons such as Interferon-alpha and Interleukin-2: Antibodies useful in this invention include Alemtuzumab, Bevacizumab, Brentuximab vedotin, Cetuximab, Gemtuzumab ozogamicin, Ibritumomab tiuxetan, Ipilimumab, Nivolumab, Ofatumumab, Panitumumab, Rituximab, Tositumomab and Trastuzumab. The invention can be used with Adoptive T-cell therapy Anti-CD47 antibodies, Anti-GD2 antibodies, Immune checkpoint blockade and EGF receptor antibodies. Administration of the emulsion can be used to increase the oxygen in the tumor tissue so that immune mechanisms accelerated of oxidative stress through increasing oxygen and making the immune system more efficient but also by changing gene expression. By decreasing expression of hypoxia related genes the oxygen therapeutic converts the aggression hypoxia mediated phenotype to a less aggressive phenotype that is more easily defeated by the immune system. In certain embodiments, Applicant's invention includes a method to alter gene expression by administering to a patient in need thereof, a therapeutically effective dosage of an oxygen therapeutic, such as and without limitation, NVX-108. Administration of DDFPe, preferably while breathing supplemental oxygen, or carbogen, increases tumor oxygenation, enabling the chemotherapeutic drugs to work more effectively. Synergy is furthermore attained by simultaneous radiotherapy. The following examples are presented to further illustrate to persons skilled in the art how to make and use the invention. These examples are not intended as a limitation, however, upon the scope of the invention. Example 1 Preparation of Dodecafluoropentane Emulsion A 30% sucrose solution was prepared by dissolving appropriate amount of USP grade sucrose in water for injection at room temperature followed by sodium dihydrogen phosphate to buffer the system at a pH of 7.0. In a second vessel a suspension of DDFP (dodecafluoropentane) in Peg Telomer B in the ratio of DDFP:PEG Telomer B:7:1 (w:w), was prepared as follows. PEG Telomer B was dispersed in water for injection by stirring in a jacketed vessel cooled to 4° C.: Pre-cooled (4° C.) DDFP was added to the stirred PEG Telomer B and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C.). The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water; the resulting solution was stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations were carried out at <8° C. in cold jacketed vessels due to the volatility of the active ingredient (DDFP). Compensation for losses during processing were accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. The resulting product comprised 2% w/vol DDPE. Particle sizing by Nycomps showed mean particle size of about 250 nm. Example 2 An experiment was performed in Hs-766t pancreatic cancer xenografts implanted in mice. The Hs-766T (pancreatic; ATCC Cat #HTB-134) cell line utilized for this study was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and was handled, stored and managed by the University of Arizona Experimental Mouse Shared Services (EMSS, University of Arizona). Cells were grown in DMEM (Mediatech) with high glucose, L-glutamic and 10% fetal bovine serum and maintained at 37° C. with 5% CO2. Tumor cells were authenticated through ATCC Cell Authentication testing services by way of PCR:short tandem repeat (STR) profiling. Cells were routinely tested for mycoplasma using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K), and found to be free of contamination. Twenty-nine female, SCID mice between the ages of 5-8 weeks of age were used in these studies. All mouse feeding, husbandry and veterinary was managed by the EMSS under IACUC approved guidelines and protocols. Mice were caged in groups of four or less, and fed and watered ad libitum. Prior to injection, mice were shaved to ascertain a suitable site for the tumor xenografts. Tumor cells (10×106cells in Matrigel™; BD Bioscience) were injected subcutaneously on the left rear flank of each mouse. Tumor burden evaluations were made twice weekly using electronic calipers to determine tumor size ((a2×b2)/2). When tumors reached a mean volume of 500-700 mm3the mice were randomized to one of 6 groups: 3 groups of mice for tumor O2measurements and 3 groups of mice for tumor growth measurements. Tumor pO2measurements were performed in 9 of the mice. NVX-108 doses of 0.3 (2 mice), 0.45 (3 mice) and 0.6 (4 mice) mL/kg (2% w/vol emulsion) were administered. Tumor growth rate was studied in the other 20 mice. These mice were designated to the following 3 treatment groups: Group 1: No treatment (4 mice), Group 2: breathing carbogen while being treated with a 12 Gy radiation dose (8 mice), Group 3: Treated with NVX-108 (0.6 cc per kg, 2% w/vol dodecafluoropentane (DDFP) administered IV over 30 minutes with radiation at end of infusion) and breathing carbogen while being treated with a 12 Gy radiation dose (8 mice). Each mouse received ketamine (20 mg/kg IP) with xylazine (5 mg/kg IP) for immobilization and was fitted with a tail-vein catheter for dosing by tail vein injection (TVI). The tail-vein catheterization was achieved by using a fabricated 27-gauge needle catheter retrofitted to PE 50 tubing. The catheter was firmly affixed to each animal's tail using 3-0 suture thread and specialized adhesive tape on both sides of the tail. Following catheterization, the mice were placed within a custom-built gas chamber introducing carbogen (95% O2, 5% CO2) which was ventilated at the 10-minute mark. They were held in custom restraints in order to be positioned under a lead shield isolating the flank tumor xenograft for radiation fractionation. All mice underwent a single fractionation of 12 Gy to the tumor within a custom-built gas chamber introducing carbogen (95% O2, 5% CO2) which was ventilated at the 10-minute mark. The calculation for the XRT dose duration was based on “prescribed dose/dose rate”=(1200 cGyV (87.9 cGy/min) amounts to a 13.65 minute (13m39s) single fraction delivered to the tumor. For group #2, 200 μL sterile saline was introduced via tail vein injection (TVI) (Time 0:00) and served as the sham injection commencing 10 minutes prior to carbogen breathing and 16.35 minutes (16 m21 s) before irradiation. For group #3, 200 μL of NVX-108 was injected by way of TVI (Time 0:00) 10 minutes before carbogen breathing and 16.35 minutes before irradiation. Tail vein injections were performed with a multi-syringe pump for simultaneous administration to each group. Simultaneous injections of NVX-108 and saline initiated the study at time 0:00 minutes. This was followed by carbogen breathing at 10 minutes and then radiation at 16.35 minutes. Once radiation was completed, the mice were allowed to recover and have food and water ad libitum. Tumor size bi-dimensional measurements were performed twice per week. When tumor size was greater than 2000 mm3, mice were sacrificed. Tumor oxygenation and blood flow levels were monitored using OxyLab (Oxford Optronics, Oxford, UK) triple parameter E-series fiber-optic probes stereotactically inserted into all tumors. Oxygen partial pressure signals from these monitors were recorded in real-time using a multi-channel data acquisition system (PowerLab KSP, ADInstruments, Australia) running under Chart™ for Windows™ (Ver.5.02. ADInstruments, Australia). Anesthetized mice (Isoflurane®), 100% O2) were restrained on a custom immobilization platform to prevent movement and retrofitted with a heating pad to sustain body core temperature. Precaution was taken to prevent any movement of the hypoxia probes and eliminate interference from external light sources to prevent probe artifact. Tumors were penetrated using a 19 gauge needle to a depth of 2-4 mm and microprobes (OD ˜450 μm) were fed through the needle into the tumor xenografts and fixed in position using stereotactic methods. Microprobes were carefully marked with gradations in order to reach the same depth in all tumors. Once the probes were stabilized and immobilized the output signals were monitored (5-10 min) until a stable baseline was observed. Real-time measurements were taken for 10 minutes at baseline on carbogen and following a 200 μL IV injection via tail vein of doses of 0.3, 0.45 or 0.6 cc/kg NVX-108 (NuvOx Pharma Tucson, Arizona) while animals continued to breathe carbogen. Example 3 Treatment of Glioblastoma Multiforme (GBM) A patient with GBM undergoes surgery. The post-surgery gadolinium enhanced MRI scan shows residual enhancing tumor. The patient is treated with 30 fractions of radiotherapy of 2 Gray (Gy) each over 6 weeks for a total of 60 Gy with oral administration of temozolamide day, at a dose of 75 mg per square meter per day given 7 days per week from the first day of radiotherapy until the last day of radiotherapy. The patient receives an intravenous PICC line. DDFPc is administered at a dose of 0.05 cc/kg (2% w/vol) as IV infusion over 30 minutes with infusion commencing about 30 minutes prior to initiation of each radiotherapy session. Magnetic resonance TOLD scan is performed to show reversal of tumor hypoxia. Follow-up MRI scans performed with intravenous gadolinium contrast show decrease in tumor compared to patients treated without DDFPe. Baseline post-operative MRI scan is shown hereinbelow to left. White arrow designates residual enhancing tumor seen in medial left temporal lobe. Scan to right is 4 weeks after completion of chemo-irradiation and treatment with DDFPe. White arrow designates residual enhancing tumor. Enhancing tumor has decreased by about 80%. Patient is now alive and doing well more than 6-months after completion of therapy. Example 4 Treatment of Glioblastoma Multiforme (GBM) Another patient with glioblastoma undergoes surgery and has residual tumor visualized on contrast enhanced MRI. The patient is treated as in Example 1 (above) except using a dose of 0.1 cc/kg of DDFPe. The patient tolerates the treatment well. The next patient, presently being consented will be treated with a dose of 0.17 cc/kg of DDFPe during each fraction of chemo-irradiation. Prophetic Example 1 A patient with non-small cell lung cancer is treated with thoracic radiotherapy and concomitant chemotherapy as described by the protocol by Belani, et al. “Sequential chemotherapy consisted of two 3-week cycles of paclitaxel 200 mg/m2administered over 3 hours, immediately followed by carboplatin at an area under the plasma concentration time curve (AUC)=6 mg/mL min as an intravenous infusion over 30 minute. Thoracic radiotherapy is initiated on day 42 and consists of 1.8 Gy daily, five times per week (45.0 Gy target dose in 5 weeks to the initial field), followed by a total of 18.0 Gy fractions delivered at 2.0 Gy fractions daily to the initial tumor volume with reduced fields (total dose, 63.0 Gy in 34 fractions over 7 weeks), but including enlarged lymph nodes ≥2.0 cm.3” DDFPe is administered IV as an infusion over 15 minutes, commencing 30 minutes before the initiation or radiotherapy (RT), for each fraction of RT. Patients treated with DDFPe show improved response to the regimen. Prophetic Example 2 A patient with Stage I non-small cell lung cancer is treated with a hypo-fractionated radiotherapy schedule with three fractions of 15 Gy to a total of 45 Gy during 1 week. This represents a biological equivalent dose (BED) of 112.5 Gy. Between 30 to 60 minutes prior to each radiation dose, the patient is administered a bolus IV dose of 0.17 cc/kg NVX-108 (2% w/vol DDFPe). Follow-up shows greater eradication of the treated tumor than would be observed without DDFPe. Prophetic Example 3 A female patient with cervical carcinoma is treated with combined radiation therapy and chemotherapy+NVX-108. Radiation dosage is 45 Gray (Gy) in 20 fractions followed by low dose-rate intracavitary application of 30 Gy to the cervical region. Chemotherapy consists of intravenous cisplatin 40 mg/m2 every week for up to 6 weekly cycles. The patient is administered a bolus IV dose of 0.2 cc/kg NVX-108 (2% w/vol DDFPe) 60 minutes prior to each dose of radiation. Follow-up shows complete response to treatment. Prophetic Example 4 A patient with squamous cell carcinoma of the head and neck is treated with 0.50 units/kg (20 units/m2) of bleomycin intravenously twice weekly. During each administration of bleomycin the patient is administered 0.2 cc/kg of 2% w/vol perfluorohexane emulsion while breathing carbogen (98% O2/2% CO2. The increased oxygen levels attained in the tumor tissue increase the activity of the bleomycin and an improved response is attained. Prophetic Example 5 An adult patient with germ cell ovarian cancer is treated with dactinomycin 500 mcg/day for 5 day: every 4 weeks. Each vial of dactinomycin contains 0.5 mg (500 meg) of dactinomycin and 20 mg of mannitol and is administered IV to the patient. DDFPe (0.2 cc/kg, 2% w/vol DDFP) is infused as an IV bolus concomitantly with each administration of dactinomycin. The patient breathes carbogen for 30 minutes during and after the infusion. Increased levels of oxygen in the tumor tissue are attained, enhancing the activity of the drug. Prophetic Example 6 An adult patient with rhabomyosarcoma is treated with IV Vincristine at a dose of 1.4 mg/m2. Concomitantly the patient is administered 0.1 cc kg of DDFPc while breathing room air. Despite breathing room air, increased oxygen levels am still attained in the tumor tissue resulting in increased activity of the drug. Prophetic Example 7 A patient with multiple myeloma is treated with BiCNU® (carmustine for injection), a nitrosourea (1,3-bis(2-chloroethyl)-1-nitrosourea) in combination with prednisone. The dose of BiCNU administered to this previously untreated patient is 200 mg/m2 intravenously every 6 weeks. This is divided into daily injections of 100 mg/m2 on 2 successive days. DDFPe is administered as an IV bolus (dose=0.2 cc/kg, 2% w/vol DDFP) during each dose of BiCNU while the patient breathes supplemental oxygen for 60 minutes. A repeat course of BiCNU is again administered once the circulating blood elements have returned to acceptable levels (platelets above 100,000/mm3, leukocytes above 4,000/mm3), in 6 weeks, and again DDFPe is administered concomitantly with BiCNU. Prophetic Example 8 A patient with prostate cancer is treated with combination external beam radiotherapy and high temporary seed implant high dose rate brachytherapy. About 3 weeks after receiving 45Gy of external beam radiotherapy for prostate cancer temporary seed implant with high dose rate brachytherapy is performed. The patient is brought to the operating room and anesthesia is induced. A transrectal ultrasound probe is introduced into the rectum and the probe is then secured into a floor mounted stepping device. A needle guide/perineal template is attached to the stepping unit and pushed up against the perineal skin. Twenty metal needles are placed through the template, pushed through the perineum, and advanced to the mid-prostate gland. The needles are replaced with plastic catheters. Upon recovery, the patient is brought to the Radiation Oncology department. CT scanning is performed to confirm the accuracy of the catheter placements. This computer-controlled high dose radiotherapy unit contains a source drive mechanism that moves the radioactive Iridium wire through the interstitial catheters sequentially in accordance with the loading pattern determined by the dosimetry plan. It takes about 10-15 minutes for the high dose rate brachytherapy procedure to be performed wherein the iridium wire is advanced into each of the interstitial catheters. This is repeated one more time and the patient is transferred to the hospital room for 6-hours, and the process is repeated again, for two more high dose rate brachytherapy administrations, e.g. the iridium wire is advanced into each catheter a total of four times, twice in the morning and twice in the afternoon, each session taking a total of about 30-minutes. During each treatment session the patient is administered a bolus dose of 0.2 cc/kg DDFPc over 30 minutes while breathing carbogen. http://prostate-cancer.org/temporary-seed-implant-with-high-dose-rate-brachytherapy/. Prophetic Example 9 A pediatric patient with Stage IV Wilms tumor is treated with dactinomycin, doxorubicin, cyclophosphamide and vincristine for 65 weeks. Doses of the drugs are as follows: dactinomycin (15 mcg/kg/d [IV]), vincristine (1.5 mg/m 2 wk [IV)), Adriamycin (doxorubicin 20 mg/m2/d [IV]), and cyclophosphamide (10 mg/kg/d [IV]). Dactinomycin courses are given postoperatively and at 13, 26, 39, 52, and 65 weeks. Vincristine is given on days 1 and 8 of each Adriamycin course. Adriamycin is given for three daily doses at 6, 19, 32, 45, and 58 weeks. Cyclophosphamide is given for three daily doses during each Adriamycin and each dactinomycin course except the postoperative dactinomycin course. During each administration of dactinomycin and vincristine a dose of 0.2 cc/kg of DDFPe is administered while the patient breathes supplemental oxygen. *D'angio, Giulio J., et al. “Treatment of Wilms' tumor. Results of the third national Wilms' tumor study.” Cancer 64.2 (1989): 349-360. Prophetic Example 10 A patient with unresectable hepatocellular carcinoma is under treatment with sorafenib. The patient is receiving 400 mg per day of oral sorafenib (2×200 mg). In a single setting the patient is also treated with TheraSphere which consists of insoluble glass microspheres where yttrium-90 is bound within the spheres. The hepatic artery is catheterized and the tumor vascular bed is embolized with TheraSpehere delivering a target dose of TheraSphere of 100 Gy by injection through the hepatic artery. A dose of 0.1 cc per kg of DDFPe is mixed with oxygen and is also infused into the hepatic artery during the embolization procedure. Prophetic Example 11 Xenograft tumors were generated in mice with cell lines of UTSCC33 (oral carcinoma), FADUDD (a subline of FaDu, an undifferentiated hypopharyngeal carcinoma) and SiHa uterine cervix carcinoma. HPV-positive, (obtained from the American Type Culture Collection) as previously described. See, Toustrup, Kasper, et al. “Development of a hypoxia gene expression classifier with predictive impact for hypoxic modification of radiotherapy in head and neck cancer.” Cancer research 71.17 (2011): 5923-5931 (hereinafter “Toustrup”). Mice bearing each kind of tumor were randomly assigned to two groups, DDFPe treatment and control (injected with same volume of saline). DDFPe treatment comprised administration of 0.3 cc/kg of DDFPe IV as bolus each day for 14 days. After the 14th day the mice were sacrificed and the tumors assayed for expression of hypoxia related genes. See, Toustrup RNA from fresh-frozen tissue was extracted by using RNeasy-kit (Qiagen) according to the manufacturer's instructions. cDNA was generated by using the High Capacity cDNA Archive kit (Applied Biosystems; ABI) and gene expression was quantified by using qPCR. cDNA based on FFPE samples was preamplified according to the manufacturer's details (TaqMan PreAmp, ABI) before real time qPCR. To detect transcripts of interest, TaqMan Gene Expression assay (ABI) was used for all potential classifier and reference genes. Genes of interest (known to be upregulated in hypoxic tumors and associated with tumors most likely to progress) included the following: ADM (stress response), ALDOA (glucose metabolism), ANKRD37 (protein-protein interactions), BNIP3 (apoptosis), BNIP3L (apoptosis), C3orf28 (unknown), EGNL3 (regulation of HIF-1 activity), KCTD11 (apoptosis), LOX (extracellular matrix metabolism), NDRG1 (stress response), P4HA1 (extracellular-matrix metabolism). P4HA2 (extracellular matrix metabolism), PDK1 (energy metabolism), PFKFB3 (glucose metabolism) and SLC2A1 (glucose metabolism). Assay of gene expression in the tumor xenografts from the animals treated with DDFPe showed significantly lower expression of the hypoxia related genes than in tumor tissue specimen derived from the animals treated with saline control injections. Prophetic Example 12 The following prophetic example is meant to show how administration of DDFPe can downregulate expression of genes that are over expressed in hypoxic tumor tissue and upregulate expression of genes that are expressed in normoxic tissue (i.e. normalize gene expression). Fischer 344 rats (F344/Ncr; National Cancer Institute, Frederick, MD) were used to generate 9 L glioma tumor models. Pieces of 9 L glioma were tied into the epigastric artery/epigastric vein pair as previously described. The animals received daily IV injections of either 0.45 cc/kg DDFPe or saline until the tumors weighed approximately 1.5-g at which time the animals were euthanized, the tumors removed and flash frozen. Gene expression in the tumors was assayed similarly to that described above. Up-regulated genes seen in the control group included BCL2/adenovirus E1B 19 kDa-interacting protein 3, hemc oxygenase (decycling) 1, activating transcription factor 3, heat shock protein (HSP27), N-myc downstream regulated gene 1, carbonic anhydrase 9 and others. Genes that were downregulated in the control group included Ly6-C antigen, solute carrier family 44 (member2), sterile alpha motif domain containing 9-like, DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 and CD3 molecule delta polypeptide and others. Comparison of gene expression from 9-L glioma tissues from the animals treated with DDFPe showed significant decrease in expression of the genes that were upregulated in the control animals and significant increase in the genes that were downregulated in the control animals; i.e. there was normalization of gene expression in the tumors from animals treated with DDFPe. See, Marotta, Diane, et al. “In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection.” Cancer research 71.3 (2011): 779-789. Prophetic Example 13 A 30% sucrose solution was prepared as described in example 1. In a second vessel a suspension of a mixture of phospholipids with the following composition. DPPC and DPPE-PEG 5 k in a mole ratio of 92% DPPC and 8 mole percent DPPE-PEG was prepared by warming them in water to above the phase transition temperature of the all the lipids. Once the lipids were dispersed the suspension was cooled to 4 C and stirred in a jacketed vessel. Pre-cooled (4° C.) DDFP was added to the stirred phospholipid suspension at weight ratio of 7 to 1, and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C. The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water; the resulting solution is stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations were carried out at <8° C. in cold jacketed vessels due to the volatility of the active ingredient (DDFP). Compensation for losses during processing are accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. Prophetic Example 14 A 30% sucrose solution was prepared as described in example 1. In a second vessel a suspension of a mixture of phospholipids with the following composition, DPPC and DPPE-PEG 5 k was prepared by warming them in water to above the phase transition temperature of the all the lipids. Once the lipids are dispersed the suspension was cooled to 4° C. and stirred in a jacketed vessel. Pre-cooled (4° C.) perfluorohexane was added to the stirred phospholipid suspension at weight ratio of 7 to 1, and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C.). The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water, the resulting solution was stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations were carried out at <80° C. in cold jacketed vessels due to the volatility of the active ingredient (perfluorohexane). Compensation for losses during processing were accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. Prophetic Example 15 A 30% sucrose solution was prepared as described in example 1. In a second vessel a suspension of a mixture of phospholipids with the following composition, DPPC, cholesterol and DPPE-PEG 5 k was prepared by warming them in water to above the phase transition temperature of the all the lipids. Once the lipids were dispersed the suspension was cooled to 4° C. and stirred in a jacketed vessel. Pre-cooled (4° C.) perfluoroheptane was added to the stirred phospholipid suspension at weight ratio of 7 to 1, and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C. The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water; the resulting solution was stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations were carried out at <8° C. in cold jacketed vessels due to the volatility of the active ingredient (perfluoroheptane). Compensation for losses during processing were accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. Prophetic Example 16 A 30% sucrose solution was prepared as described in example 1. In a second vessel a suspension of a mixture of phospholipids with the following composition, DPPC, phosphatidic acid (DPPA) and DPPE-PEG 5 k was prepared by warming them in water to above the phase transition temperature of the all the lipids. Once the lipids were dispersed the suspension was cooled to 4° C. and stirred in a jacketed vessel. Pre-cooled (4° C.) perfluorooctane was added to the stirred phospholipid suspension at weight ratio of 7 to 1, and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C. The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water; the resulting solution is stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations are carried out at <8° C. in cold jacketed vessels due to the volatility of the active ingredient (perfluorooctane). Compensation for losses during processing were accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. Prophetic Example 17 A suspension of a mixture of phospholipids with the following composition, dioleoylphosphatidylcholine (DOPC), cholesterol and dioleoylphosphatidylethanolamine-PEG-5,000 was prepared by warming them in water to above the phase transition temperature of the all the lipids. The resulting suspension of lipids was suspended in a mixture of propylene glycol/glycerol to achieve 80:10:10 weight percent phosphate buffered saline:propylene glycol:glycerol. Once the lipids were dispersed the suspension was cooled to 4 C and stirred in a jacketed vessel. Pre-cooled (4° C.) perfluorohexane was added to the stirred phospholipid suspension at weight ratio of 7 to 1, and allowed to stir until a uniformly milky suspension was achieved. This suspension was homogenized under high pressure in an Avestin model C50 homogenizer for up to 18 minutes keeping the temperature below 7° C. The emulsion was transferred via the homogenizer under low pressure to a vessel containing 30% sucrose solution in water; the resulting solution is stirred for up to 20 minutes, and then transferred through the homogenizer under low pressure to a second vessel. This solution was then transferred through a 0.2 micron filter into a third vessel. The product was dispensed to vials, which were capped and crimped. These operations are carried out at <8° C. in cold jacketed vessels due to the volatility of the active ingredient (perfluorooctane). Compensation for losses during processing were accounted for by the use of an overage of the active component. Product fill volume was also tightly controlled to produce vials to meet release and shelf-life specifications. While the preferred embodiments of the present invention have been illustrated in detail, it should be apparent that modifications and adaptations to those embodiments may occur to one skilled in the art without departing from the scope of the present invention as set forth herein. | 41,705 |
11857628 | DETAILED DESCRIPTION The present invention is described more fully hereinafter using illustrative, non-limiting embodiments, and references to the accompanying figures. This invention may, however, be embodied in many different forms and should not be construed as to be limited to the embodiments set forth below. Rather, these embodiments are provided so that this disclosure is thorough and conveys the scope of the invention to those skilled in the art. In order that the present invention may be more readily understood, certain terms are defined below. Additional definitions may be found within the detailed description of the invention. As used in the specification and the appended claims, the terms “a,” “an” and “the” include both singular and the plural referents unless the context clearly dictates otherwise. As used in the specification and the appended claims, the term “and/or” when referring to two species, A and B, means at least one of A and B. As used in the specification and the appended claims, the term “and/or” when referring to greater than two species, such as A, B, and C, means at least one of A, B, or C, or at least one of any combination of A, B, or C (with each species in singular or multiple possibility). Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components). Throughout this specification, the term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably. The term “amino acid residue” or “amino acid” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide. The term “polypeptide” includes any polymer of amino acids or amino acid residues. The term “polypeptide sequence” refers to a series of amino acids or amino acid residues from which a polypeptide is physically composed. A “protein” is a macromolecule comprising one or more polypeptides or polypeptide “chains.” A “peptide” is a small polypeptide of sizes less than about 15 to 20 amino acid residues. The term “amino acid sequence” refers to a series of amino acids or amino acid residues which physically comprise a peptide or polypeptide depending on the length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right representing their order from an amino terminus to a carboxy terminus. For purposes of the claimed invention and with regard to a Shiga toxin protein sequence or Shiga toxin derived polypeptide, the term “wild-type” generally refers to a naturally occurring, Shiga toxin protein sequence(s) found in a living species, such as, e.g., a pathogenic bacterium, wherein that Shiga toxin protein sequence(s) is one of the most frequently occurring variants. This is in contrast to infrequently occurring Shiga toxin protein sequences that, while still naturally occurring, are found in less than one percent of individual organisms of a given species out of individual organisms of that same species when sampling a statistically powerful number of naturally occurring individual organisms of that species which comprise at least one Shiga toxin protein variant. A clonal expansion of a natural isolate outside its natural environment (regardless of whether the isolate is an organism or molecule comprising biological sequence information) does not alter the naturally occurring requirement as long as the clonal expansion does not introduce new sequence variety not present in naturally occurring populations of that species and/or does not change the relative proportions of sequence variants to each other. The terms “amino acid,” “amino acid residue,” “amino acid sequence,” or polypeptide sequence include naturally occurring amino acids (including L and D isosteriomers) and, unless otherwise limited, also include known analogs of the twenty common natural amino acids that can function in a similar manner, such as, e.g., selenocysteine, pyrrolysine, N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine (see e.g. Nagata K et al.,Bioinformatics30: 1681-9 (2014)). The amino acids referred to herein are described by shorthand designations as follows in Table A: TABLE AAmino Acid NomenclatureName3-letter1-letterAlanineAlaAArginineArgRAsparagineAsnNAspartic Acid or AspartateAspDCysteineCysCGlutamic Acid or GlutamateGluEGlutamineGlnQGlycineGlyGHistidineHisHIsoleucineIleILeucineLeuLLysineLysKMethionineMetMPhenylalaninePheFProlineProPPyrroline-carboxy-lysinePclSelenocysteineSecSerineSerSThreonineThrTTryptophanTrpWTyrosineTyrYValineValV The phrase “conservative substitution” with regard to a protein, polypeptide, or polypeptide region refers to a change in the amino acid composition of the polypeptide that does not substantially alter the function and structure of the overall protein, polypeptide, or polypeptide region (see Creighton,Proteins: Structures and Molecular Properties(W. H. Freeman and Company, New York (2nd ed., 1992)). As used herein, the terms “expressed,” “expressing,” or “expresses,” and grammatical variants thereof, refer to translation of a polynucleotide or nucleic acid into a protein. The expressed protein may remain intracellular, become a component of the cell surface membrane or be secreted into an extracellular space. As used herein, the phrase “target-expressing cell” encompasses any cell that expresses, at a cellular surface, a target biomolecule bound by a binding region of the cell-targeting molecule of the present invention. As used herein, cells which express a significant amount of an extracellular target biomolecule at least one cellular surface are “target positive cells” or “target+ cells” and are cells physically coupled to the specified, extracellular target biomolecule. As used herein, cells which express a significant amount of target biomolecule at least one cellular surface are “target-positive cells” or “target+ cells” and are cells physically coupled to the extracellular target biomolecule. A significant amount of target biomolecule is defined below. As used herein, the symbol “α” is shorthand for an immunoglobulin-type binding region capable of binding to the biomolecule following the symbol. The symbol “α” is used to refer to the functional characteristic of an immunoglobulin-type binding region based on its ability to bind to the biomolecule following the symbol with a binding affinity described by a dissociation constant (KD) of 10−5or less. The terms “associated,” “associating,” “linked,” or “linking” with regard to the claimed invention refers to the state of two or more components of a molecule being joined, attached, connected, or otherwise coupled to form a single molecule or the act of making two molecules associated with each other to form a single molecule by creating an association, linkage, attachment, and/or any other connection between the two molecules. For example, the term “linked” may refer to two or more components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions may be covalent and/or non-covalent. Non-limiting examples of covalent associations between two components include peptide bonds and cysteine-cysteine disulfide bonds. Non-limiting examples of non-covalent associations between two molecular components include ionic bonds. For purposes of the present invention, the term “linked” refer to two or more molecular components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions includes at least one covalent bond. For purposes of the present invention, the term “linking” refers to the act of creating a linked molecule as described above. For purposes of the present invention, the term “fused” refers to two or more proteinaceous components associated by at least one covalent bond which is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxyl acid group or involves another carbon atom, such as, e.g., the α-carbon, β-carbon, γ-carbon, σ-carbon, etc. Non-limiting examples of two proteinaceous components fused together include, e.g., an amino acid, peptide, or polypeptide fused to a polypeptide via a peptide bond such that the resulting molecule is a single, continuous polypeptide. For purposes of the present invention, the term “fusing” refers to the act of creating a fused molecule as described above, such as, e.g., a fusion protein generated from the recombinant fusion of genetic regions which when translated produces a single proteinaceous molecule. The symbol “::” means the proteinaceous molecules before and after the symbol are physically linked together to form a continuous polypeptide. The symbol “_” with regard to a cell-targeting molecule means the molecules before and after the symbol are covalently linked together, either directly or indirectly. For purposes of the present invention, the term “effector” means providing a biological activity, such as cytotoxicity, biological signaling, enzymatic catalysis, subcellular routing, and/or intermolecular binding resulting in the recruitment of one or more factors and/or allosteric effect(s). For example, a Shiga toxin effector polypeptide provides one or more biological activities present in a Shiga toxin, Shiga toxin component, and/or fragment thereof. As used herein, the phrase “multivalent target-binding molecule” refers to a target-binding molecule or plurality of target-binding molecules comprising two or more high-affinity binding regions, such as, e.g. a protein comprising two or more binding regions wherein each individual binding region has a dissociation constant of 10−5to 10−12moles per liter toward an extracellular part of a target biomolecule. For purposes of the present invention, the phrase “derived from” when referring to a polypeptide or polypeptide region means that the polypeptide or polypeptide region comprises amino acid sequences originally found in a “parental” protein and which may now comprise certain amino acid residue additions, deletions, truncations, rearrangements, or other alterations relative to the original polypeptide or polypeptide region as long as a certain function(s) and a structure(s) of the “parental” molecule are substantially conserved. The skilled worker will be able to identify a parental molecule from which a polypeptide or polypeptide region was derived using techniques known in the art, e.g., protein sequence alignment software. For purposes of the present invention, a Shiga toxin effector function is a biological activity conferred by a polypeptide region derived from a Shiga toxin A Subunit. Non-limiting examples of Shiga toxin effector functions include cellular internalization, subcellular routing, catalytic activity, and cytotoxicity. Shiga toxin catalytic activities include, for example, ribosome inactivation, protein synthesis inhibition, N-glycosidase activity, polynucleotide:adenosine glycosidase activity, RNAase activity, and DNAase activity. Shiga toxins are ribosome inactivating proteins (RIPs). RIPs can depurinate nucleic acids, polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (and polyA), and viral nucleic acids (see e.g. Brigotti M et al.,Toxicon39: 341-8 (2001); Brigotti M et al.,FASEB J16: 365-72 (2002)). Some RIPs show antiviral activity and superoxide dismutase activity. Shiga toxin catalytic activities have been observed both in vitro and in vivo. Non-limiting examples of assays for Shiga toxin effector activity measure protein synthesis inhibitory activity, depurination activity, inhibition of cell growth, cytotoxicity, supercoiled DNA relaxation activity, and nuclease activity. As used herein, the retention of Shiga toxin effector function refers to being capable of exhibiting a level of Shiga toxin functional activity, as measured by an appropriate quantitative assay with reproducibility, comparable to a wild-type, Shiga toxin effector polypeptide control (e.g. a Shiga toxin A1 fragment) or cell-targeting molecule comprising a wild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1 fragment) under the same conditions. For the Shiga toxin effector function of ribosome inactivation or ribosome inhibition, retained Shiga toxin effector function is exhibiting an IC50of 10,000 picomolar (pM) or less in an in vitro setting, such as, e.g., by using an assay known to the skilled worker and/or described herein. For the Shiga toxin effector function of cytotoxicity in a target positive cell-kill assay, retained Shiga toxin effector function is exhibiting a CD50of 1,000 nanomolar (nM) or less, depending on the cell type and its expression of the appropriate extracellular target biomolecule, as shown, e.g., by using an assay known to the skilled worker and/or described herein. As used herein, the retention of “significant” Shiga toxin effector function refers to a level of Shiga toxin functional activity, as measured by an appropriate quantitative assay with reproducibility comparable to a wild-type Shiga toxin effector polypeptide control. For in vitro ribosome inhibition, significant Shiga toxin effector function is exhibiting an IC50of 300 pM or less depending on the source of the ribosomes (e.g. bacteria, archaea, or eukaryote (algae, fungi, plants, or animals)). This is significantly greater inhibition as compared to the approximate IC50of 100,000 pM for the catalytically inactive SLT-1A 1-251 double mutant (Y77S/E167D). For cytotoxicity in a target-positive cell kill assay in laboratory cell culture, significant Shiga toxin effector function is exhibiting a CD50of 100, 50, or 30 nM or less, depending on the cell line and its expression of the appropriate extracellular target biomolecule. This is significantly greater cytotoxicity to the appropriate target cell line as compared to an SLT-1A subunit alone, without a cell targeting binding region, which has a CD50of 100-10,000 nM, depending on the cell line. For some samples, accurate values for either IC50or CD50might be unobtainable due to the inability to collect the required data points for an accurate curve fit. For example, theoretically, neither an IC50nor CD50can be determined if greater than 50% ribosome inhibition or cell death, respectively, does not occur in a concentration series for a given sample. Data insufficient to accurately fit a curve as described in the analysis of the data from exemplary Shiga toxin effector function assays, such as, e.g., assays described in the Examples, should not be considered as representative of actual Shiga toxin effector function. A failure to detect activity in Shiga toxin effector function may be due to improper expression, polypeptide folding, and/or polypeptide stability rather than a lack of cell entry, subcellular routing, and/or enzymatic activity. Assays for Shiga toxin effector functions may not require much cell-targeting molecule of the invention to measure significant amounts of Shiga toxin effector function activity. To the extent that an underlying cause of low or no effector function is determined empirically to relate to protein expression or stability, one of skill in the art may be able to compensate for such factors using protein chemistry and molecular engineering techniques known in the art, such that a Shiga toxin functional effector activity may be restored and measured. As examples, improper cell-based expression may be compensated for by using different expression control sequences; improper polypeptide folding and/or stability may benefit from stabilizing terminal sequences, or compensatory mutations in non-effector regions which stabilize the three-dimensional structure of the protein, etc. When new assays for individual Shiga toxin functions become available, Shiga toxin effector regions or polypeptides may be analyzed for any level of those Shiga toxin effector functions, such as for being within a certain-fold activity of a wild-type Shiga toxin effector polypeptide. Examples of meaningful activity differences are, e.g., Shiga toxin effector regions that have 1000-fold or 100-fold or less the activity of a wild-type Shiga toxin effector polypeptide; or that have 3-fold to 30-fold or more activity compared to a functional knock-down or knockout Shiga toxin effector polypeptide. Certain Shiga toxin effector functions are not easily measurable, e.g. subcellular routing functions. Currently there is no routine, quantitative assay to distinguish whether the failure of a Shiga toxin effector polypeptide to be cytotoxic is due to improper subcellular routing, but at a time when tests are available, Shiga toxin effector polypeptides may be analyzed for any significant level of subcellular routing as compared to the appropriate wild-type Shiga toxin effector region. It should be noted that even if the cytotoxicity of a Shiga toxin effector polypeptide is reduced relative to wild-type, in practice, applications using attenuated, Shiga toxin effector polypeptides may be equally or more effective than those using wild-type, Shiga toxin effector polypeptides because the highest potency variants might exhibit undesirable effects which are minimized or reduced in reduced-potency variants. Wild-type Shiga toxin effector polypeptides are very potent, being able to kill with only one molecule reaching the cytosol or perhaps 40 molecules being internalized (Tam P, Lingwood C,Microbiology153: 2700-10 (2007)). Shiga toxin effector polypeptides with even considerably reduced Shiga toxin effector functions, such as, e.g., subcellular routing or cytotoxicity, as compared to wild-type Shiga toxin effector polypeptides may still be potent enough for practical applications involving targeted cell killing and/or detection of certain subcellular compartments of specific cell types. And such effector polypeptides may also be useful for delivering cargos (e.g. additional exogenous material) to certain intracellular locations or subcellular compartments. The term “selective cytotoxicity” with regard to the cytotoxic activity of a cytotoxic, cell-targeting molecule refers to the relative levels of cytotoxicity between a targeted cell population and a non-targeted bystander cell population, which can be expressed as a ratio of the half-maximal cytotoxic concentration (CD50) for a targeted cell type over the CD50for an untargeted cell type to show the preferentiality of cell killing of the targeted cell type as a metric for selectivity. As used in the specification and the claims herein, the phrase “physiological temperature appropriate for the cell” refers to temperatures known in the art and/or identifiable by the skilled worker which fall within a range suitable for healthy growth, propagation, and/or function of that particular cell or cell type; corresponding to the core temperature of the species from which the cell is derived; and/or corresponding to a healthy, living organism comprising the cell. For example, temperatures around 37° C. are appropriate for many mammalian cells depending on the species. For purposes of the present invention, the phrase “internalization of a molecular complex comprising the cell-targeting molecule bound to target biomolecule” means the cellular internalization of the cell-targeting molecule is target-mediated in that the internalization begins with the cell-targeting molecule and cell-surface target biomolecule forming a complex at an extracellular position and ends with both the cell-targeting molecule and target biomolecule(s) entering the cell prior to dissociation of the cell-targeting molecule from the target biomolecule(s) to which the cell-targeting molecule has bound. For purposes of the present invention, the phrase “target biomolecule natively present on the surface of a cell” means a cell expresses the target biomolecule using its own internal machinery and localizes the target biomolecule to a cellular surface using its own internal machinery such that the target biomolecule is physically coupled to said cell and at least a part of the target biomolecule is accessible from an extracellular space, i.e. on the surface of a cell. For purposes of the present invention, the phrase “cell-targeting molecule altering agent” refers to any of a number of different types of atoms or molecules known to the skilled worker and/or described herein which may be conjugated to a molecule of the invention in order to alter one or more properties of the molecule of the invention. For the purposes of certain embodiments of the present invention, cellular internalization is considered rapid if the time for internalization to occur due to the binding of the cell-targeting molecule of the present invention is reduced as compared to the time for internalization of a prior art reference molecule at the same percent target biomolecule occupancy as determined by the same assay using the same cell type at the same temperature. As used in the specification and the claims herein, the phrase “rapid cellular internalization” refers to the ability of a cell-targeting molecule of the present invention to decrease the time on average for cellular internalization of an extracellular target antigen or cell surface localized target biomolecule as compared to the time on average required for cellular internalization of an extracellular target antigen or cell surface localized target biomolecule, as measured by any one of a number of cell internalization assays known in the art or described herein. As used in the specification and the claims herein, the phrase “rapid internalization” includes internalization which may be assayed as compared to a basal target biomolecule internalization rate and/or molecular binding induced internalization rate for target biomolecule after administration of an immunoglobulin-type binding molecule (e.g. a monoclonal antibody) known in the art to bind an extracellular part of target biomolecule. The scope of the phrase “rapid cellular internalization” is intended to encompass internalization rates, on average, faster than those observed when testing a target-specific antibody or immunoglobulin-derived protein molecule with an Fc region. In general, an internalization rate constant may be defined as the time after administration of a target-specific binding molecule of interest to target-positive cells at which 50% of cell surface target antigens, target biomolecules, and/or the target-specific binding molecule is internalized at a given administered concentration, mass, molarity, or target biomolecule occupancy-adjusted concentration, to a particular cell type, and at a particular temperature. Cell-surface target biomolecule internalization, whether basally or in response to administration of a target-binding molecule, may be assayed by various methods known to the skilled worker. For the purposes of certain embodiments of the present invention, cellular internalization is considered rapid if the time for internalization to occur due to the binding of the cell-targeting molecule of the present invention is reduced as compared to the time for internalization of the target biomolecule with the binding of a well-characterized antibody recognizing an extracellular target biomolecule antigen. The term “rapid” as used throughout the present description is intended to indicate that a cell-targeting molecule of the present invention enters one or more target-expressing and/or target-positive cells in less than six hours. In certain embodiments, rapid can be as quickly as less than about thirty minutes, but can also encompass a range of from about 1 hour to about 2 hours, to about 3 hours, to about 4 hours, to about 5 hours; a range of about 2 hours to about 3 hours, to about 4 hours, to about 5 hours; a range of about 3 hours to about 4 hours, to about 5 hours; and a range of about 4 hours to about 5 hours. For purposes of the present invention, the phrase “one or more non-covalent linkages,” with regard to a molecule comprising two or more components linked together, includes the types of linkages connecting the components that in certain molecules may be observed as being eliminated (i.e., no longer connecting two or more components) when changing the molecule from native protein-folding conditions to protein-denaturing conditions. For example, when using techniques known in the art and/or described herein, such as, e.g., electrophoretic and/or chromatographic assays, for assaying the sizes of proteinaceous molecules, a multi-component molecule that appears as a single-sized species under native protein-folding conditions (e.g. pH-buffered environments intended to be similar to the lumen of the endoplasmic reticulum of a eukaryotic cell or to an extracellular environment within an organism), can also be observed as being composed of two or more smaller-sized, proteinaceous molecules under denaturing conditions and/or after being subjected to a denaturing condition. “Protein-denaturing” conditions are known to the skilled worker and include conditions markedly different from native protein-folding conditions, such as, e.g., environments with a high temperature (e.g., greater than 50 degrees Celsius) and/or those characterized by the presence of chemical denaturants and/or detergents, such as, e.g., 1-10% sodium dodecyl sulfate, polysorbates, Triton® X-100, sarkosyl, and other detergents whether ionic, non-ionic, zwitterionic, and/or chaotropic. As used herein, the term “monomeric” with regard to describing a protein and/or proteinaceous molecule refers to a molecule comprising only one polypeptide component consisting of a single, continuous polypeptide, regardless of its secondary or tertiary structure, which may be synthesized by a ribosome from a single polynucleotide template, including a continuous linear polypeptide which later forms a cyclic structure. In contrast, a multimeric molecule comprises two or more polypeptides (e.g. subunits) which together do not form a single, continuous polypeptide that may be synthesized by a ribosome from a single polynucleotide template. As used herein, the term “multimeric” with regard to describing a protein and/or proteinaceous molecule refers to a molecule that comprises two or more, individual, polypeptide components associated together and/or linked together, such as, e.g., a molecule consisting of two components each of which is its own continuous polypeptide. For example, the association or linkage between components of a molecule may include 1) one or more non-covalent interactions; 2) one or more post-translational, covalent interactions; 3) one or more, covalent chemical conjugations; and/or 4) one or more covalent interactions resulting in a single molecule comprising a non-linear polypeptide, such as, e.g., a branched or cyclic polypeptide structure, resulting from the arrangement of the two or more polypeptide components. A molecule comprising two, discontinuous polypeptides as a result of the proteolytic cleavage of one or more peptide bonds in a single, continuous polypeptide synthesized by a ribosome from a single polynucleotide templates is “multimeric” and not “monomeric.” As used herein, the terms “disrupted,” “disruption,” or “disrupting,” and grammatical variants thereof, with regard to a polypeptide region or feature within a polypeptide refers to an alteration of at least one amino acid within the region or composing the disrupted feature. Amino acid alterations include various mutations, such as, e.g., a deletion, inversion, insertion, or substitution which alter the amino acid sequence of the polypeptide. Amino acid alterations also include chemical changes, such as, e.g., the alteration one or more atoms in an amino acid functional group or the addition of one or more atoms to an amino acid functional group. As used herein, “de-immunized” means reduced antigenic and/or immunogenic potential after administration to a chordate as compared to a reference molecule, such as, e.g., a wild-type peptide region, polypeptide region, or polypeptide. This includes a reduction in overall antigenic and/or immunogenic potential despite the introduction of one or more, de novo, antigenic and/or immunogenic epitopes as compared to a reference molecule. For certain embodiments, “de-immunized” means a molecule exhibited reduced antigenicity and/or immunogenicity after administration to a mammal as compared to a “parental” molecule from which it was derived, such as, e.g., a wild-type Shiga toxin A1 fragment. In certain embodiments, the de-immunized, Shiga toxin effector polypeptide of the present invention is capable of exhibiting a relative antigenicity compared to a reference molecule which is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than the antigenicity of the reference molecule under the same conditions measured by the same assay, such as, e.g., an assay known to the skilled worker and/or described herein like a quantitative ELISA or Western blot analysis. In certain embodiments, the de-immunized, Shiga toxin effector polypeptide of the present invention is capable of exhibiting a relative immunogenicity compared to a reference molecule which is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater than the immunogenicity of the reference molecule under the same conditions measured by the same assay, such as, e.g., an assay known to the skilled worker and/or described herein like a quantitative measurement of anti-molecule antibodies produced in a mammal(s) after receiving parenteral administration of the molecule at a given time-point. The relative immunogenicities of exemplary cell-targeting molecules may be determined using an assay for in vivo antibody responses to the cell-targeting molecules after repeat, parenteral administrations over periods of many. For purposes of the present invention, the terms “terminus,” “amino-terminus,” or “carboxy-terminus” with regard to a cell-targeting molecule refers generally to the last amino acid residue of a polypeptide chain of the cell-targeting molecule (e.g., a single, continuous polypeptide chain). A cell-targeting molecule may comprise more than one polypeptides or proteins, and, thus, a cell-targeting molecule of the present invention may comprise multiple amino-terminals and carboxy-terminals. For example, the “amino-terminus” of a cell-targeting molecule may be defined by the first amino acid residue of a polypeptide chain representing the amino-terminal end of the polypeptide, which is generally characterized by a starting, amino acid residue which does not have a peptide bond with any amino acid residue involving the primary amino group of the starting amino acid residue or involving the equivalent nitrogen for starting amino acid residues which are members of the class of N-alkylated alpha amino acid residues. Similarly, the “carboxy-terminus” of a cell-targeting molecule may be defined by the last amino acid residue of a polypeptide chain representing the carboxyl-terminal end of the polypeptide, which is generally characterized by a final, amino acid residue which does not have any amino acid residue linked by a peptide bond to the alpha-carbon of its primary carboxyl group. For purposes of the present invention, the terms “terminus,” “amino-terminus,” or “carboxy-terminus” with regard to a polypeptide region refers to the regional boundaries of that region, regardless of whether additional amino acid residues are linked by peptide bonds outside of that region. In other words, the terminals of the polypeptide region regardless of whether that region is fused to other peptides or polypeptides. For example, a fusion protein comprising two proteinaceous regions, e.g., a binding region comprising a peptide or polypeptide and a Shiga toxin effector polypeptide, may have a Shiga toxin effector polypeptide region with a carboxy-terminus ending at amino acid residue 251 of the Shiga toxin effector polypeptide region despite a peptide bond involving residue 251 to an amino acid residue at position 252 representing the beginning of another proteinaceous region, e.g., the binding region. In this example, the carboxy-terminus of the Shiga toxin effector polypeptide region refers to residue 251, which is not a terminus of the fusion protein but rather represents an internal, regional boundary. Thus, for polypeptide regions, the terms “terminus,” “amino-terminus,” and “carboxy-terminus” are used to refer to the boundaries of polypeptide regions, whether the boundary is a physically terminus or an internal, position embedded within a larger polypeptide chain. For purposes of the present invention, the phrase “furin-cleavage resistant” means a molecule or specific polypeptide region thereof exhibits reproducibly less furin cleavage than (i) the carboxy-terminus of a Shiga toxin A1 fragment in a wild-type Shiga toxin A Subunit or (ii) the carboxy-terminus of the Shiga toxin A1 fragment derived region of construct wherein the naturally occurring furin-cleavage site natively positioned at the junction between the A1 and A2 fragments is not disrupted; as assayed by any available means to the skilled worker, including by using a method described herein. For purposes of the present invention, the phrase “disrupted furin-cleavage motif” refers to (i) a specific furin-cleavage motif as described herein in Section I-B and (ii) which comprises a mutation and/or truncation that can confer a molecule with a reduction in furin-cleavage as compared to a reference molecule, such as, e.g., a reduction in furin-cleavage reproducibly observed to be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for no cleavage) than the furin-cleavage of a reference molecule observed in the same assay under the same conditions. The percentage of furin-cleavage as compared to a reference molecule can be expressed as a ratio of cleaved:uncleaved material of the molecule of interest divided by the cleaved:uncleaved material of the reference molecule (see e.g. WO 2015/191764). Non-limiting examples of suitable reference molecules include certain molecules comprising a wild-type Shiga toxin furin-cleavage motif and/or furin-cleavage site are described in WO 2015/191764 and WO 2016/196344. Introduction The present invention provides Shiga toxin effector polypeptides and cell-targeting molecules comprising specific attachment sites for conjugating other molecules. A unique amino acid residue with a unique functional group that is solvent accessible and/or one or more position-ectopic residues in a Shiga toxin effector polypeptide or cell-targeting molecule scaffold provides an attachment point for the site-specific linking of various atoms and molecules. The atoms or molecules may function as (1) cargos designed for intracellular delivery, including for controlled liberation once inside a target cell, and/or (2) agents having extracellular function(s), such as, e.g., biologically inert moieties which prolong half-life in a vertebrate, mask immunogenic portions of the scaffold, and/or block proteolytic cleavage. The Shiga toxin effector polypeptides and cell-targeting molecules of the present invention may be conjugated to a variety of atoms and molecules in a controlled and convenient manner using routine methods in order to obtain homogenous products. The present invention also provides Shiga toxin effector polypeptides and cell-targeting molecules conjugated to other molecules, such as, e.g., a molecular cargo for intracellular delivery or a cell-targeting molecule altering agent. Certain cell-targeting molecules of the present invention, and compositions thereof, may be used to selectively deliver conjugated cargo(s) to a target-expressing cell type(s) in the presence of one or more other cell types based on its cell-targeting and cellular internalization activity(ies), such as, e.g., a cargo having a desired, intracellular function. In addition, certain cell-targeting molecules of the present invention, and compositions thereof, may be used to selectively kill a target-expressing cell in the presence of one or more other cell types based on its cell-targeting activity and cellular internalization activity(ies), such as, e.g., by delivering into the interior of the targeted, target-expressing cell a component of the cell-targeting molecule which is cytotoxic at an intracellular location. The conjugated atoms, molecules, cargos, and/or cell-targeting molecule altering agents contemplated as suitable for use in the present invention include linkers, cell-targeting moieties, antibiotics, peptides, nucleic acids, proteins, protein-nucleic acid complexes, cytotoxic agents, solubility-altering agents, pharmacokinetic-altering agents, immunogenicity-altering agents, and pharmacodynamics-altering agents. The molecules of the present invention, and compositions thereof, have uses, e.g., for the selective delivery of cargos to target-expressing cells and as therapeutics for the treatment of a variety of diseases, disorders, and conditions. I. General Structures of the Shiga Toxin Effector Polypeptides and Cell-Targeting Molecules of the Present Invention Certain embodiments of the present invention are Shiga toxin A Subunit effector polypeptides, such as, e.g., (1) a Shiga toxin effector polypeptide conjugated to a heterologous molecule; and (2) a Shiga toxin effector polypeptide comprising one or more ectopic amino acid residues relative to wild-type, Shiga toxin polypeptides. The present invention provides cell-targeting molecules comprising (1) a cell-targeting moiety (e.g. a cell-targeting agent and/or binding region) and (2) a toxin effector polypeptide region. Certain further embodiments are cell-targeting molecules comprising a Shiga toxin effector polypeptide of the present invention. In addition, the present invention provides cell-targeting molecules lacking any Shiga toxin effector polypeptide but comprising a linker or binding region (e.g. an immunoglobulin-type polypeptide) comprising functional group(s) for site specific attachment of other molecules. All of the molecules of the present invention may be optionally conjugated to another molecule, such as, e.g., a cargo for cell-targeted delivery, a cell-targeting molecule altering agent, and/or an additional exogenous material. A. Shiga Toxin Effector a Subunit Polypeptides of the Present Invention A Shiga toxin effector polypeptide of the present invention is a polypeptide derived from a Shiga toxin A Subunit of at least one member of the Shiga toxin family wherein the Shiga toxin effector region is capable of exhibiting at least one Shiga toxin function. Shiga toxin functions include, e.g., promoting cell entry, deforming lipid membranes, stimulating clathrin-mediated endocytosis, directing retrograde transport, directing subcellular routing, avoiding intracellular degradation, catalytically inactivating ribosomes, effectuating cytotoxicity, and effectuating cytostatic effects. There are numerous Shiga toxin effector polypeptides known to the skilled worker (see e.g., Cheung M et al.,Mol Cancer9: 28 (2010); WO 2014/164693; WO 2015/113005; WO 2015/113007; US20150259428; WO 2015/191764; US20160177284; WO 2016/126950) that are suitable for use in the present invention or to use as parental polypeptides to be modified into a Shiga toxin effector polypeptide of the present invention using techniques known the art. Shiga toxin effector polypeptides of the present invention comprise or consist essentially of a polypeptide derived from a Shiga toxin A Subunit dissociated from any form of its native Shiga toxin B Subunit. In addition, the cell-targeting molecules of the present invention need not comprise any polypeptide comprising or consisting essentially of a functional binding domain of a native Shiga toxin B subunit. Rather, the Shiga toxin effector polypeptides of the present invention may be functionally associated with heterologous binding regions to effectuate cell targeting. In certain embodiments, a Shiga toxin effector polypeptide of the present invention may comprise or consist essentially of a full-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), or SLT-2A (SEQ ID NO:3)), noting that naturally occurring Shiga toxin A Subunits may comprise precursor forms containing signal sequences of about 22 amino acids at their amino-terminals which are removed to produce mature Shiga toxin A Subunits and are recognizable to the skilled worker. In other embodiments, the Shiga toxin effector polypeptide of the invention comprises or consists essentially of a truncated Shiga toxin A Subunit which is shorter than a full-length Shiga toxin A Subunit, such as, e.g., a truncation known in the art (see e.g., WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764; US20160177284; WO 2016/126950). Although derived from a wild-type Shiga toxin A Subunit polypeptide, for certain embodiments of the molecules of the present invention, the Shiga toxin effector polypeptide differs from a naturally occurring Shiga toxin A Subunit by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by no more than that which retains at least 85%, 90%, 95%, 99%, or more amino acid sequence identity). In certain embodiments, the Shiga toxin effector polypeptide of the present invention comprises only one lysine residue. The cytotoxic activity of the SLT-1 holotoxin was not affected by the removal of all lysine residues from the A Subunit (McCluskey, A J, “Shiga-like Toxin 1: Molecular Mechanism of Toxicity and Discovery of Inhibitors”, thesis, University of Toronto, (2012), Appendix B). Moreover, the removal of both amino-terminal lysines of the A Subunit of SLT-1 did not affect its cytotoxicity (McCluskey, AJ, “Shiga-like Toxin 1: Molecular Mechanism of Toxicity and Discovery of Inhibitors”, thesis, University of Toronto, (2012), Appendix B). Thus, all but one of the naturally occurring lysine residues in SLT-1A, StxA, and/or SLT-2A may be removed by amino acid residue substitution thereby leaving only one natively occurring lysine residue in a Shiga toxin effector polypeptide. Alternatively, all of the naturally occurring lysine residues in SLT-1A, StxA, and/or SLT-2A may be removed from a Shiga toxin effector polypeptide by amino acid residue substitution and an ectopic lysine residue may be engineered into a suitable position for site-specific conjugation and retention of one or more Shiga toxin functions. B. Cell-Targeting Molecules of the Present Invention The cell-targeting molecules of the present invention all comprise a cell-targeting agent or moiety, such as, e.g., a binding region described herein. Cell-targeting agents or moieties of the cell-targeting molecules of the present invention comprise molecular structures, that when linked to a polypeptide of the present invention, are each capable of bringing the cell-targeting molecule within close proximity to specific cells based on molecular interactions on the surfaces of those specific cells. Cell-targeting moieties include ligand and polypeptides which bind to cell-surface targets. One type of cell-targeting moiety is a proteinaceous binding region. Binding regions of the cell-targeted molecules of the present invention comprise one or more polypeptides capable of selectively and specifically binding an extracellular target biomolecule. Binding regions may comprise one or more various polypeptide moieties, such as ligands whether synthetic or naturally occurring ligands and derivatives thereof, immunoglobulin derived domains, synthetically engineered scaffolds as alternatives to immunoglobulin domains, and the like. The use of proteinaceous binding regions in cell-targeting molecules of the invention allows for the creation of cell-targeting molecules which are single-chain, cell-targeting proteins. Certain embodiments of the cell-targeting molecules of the present invention comprise a cell-targeting binding region capable of specifically binding to an extracellular part of a target biomolecule physically coupled to a cell. The binding region of a cell-targeting molecule of the present invention comprises a peptide or polypeptide region capable of binding specifically to a target biomolecule. In certain embodiments, the binding region of a cell-targeting molecule of the invention comprises one or more polypeptides capable of selectively and specifically binding an extracellular target biomolecule. Binding region may comprise one or more various peptidic or polypeptide moieties, such as randomly generated peptide sequences, naturally occurring ligands or derivatives thereof, immunoglobulin derived domains, synthetically engineered scaffolds as alternatives to immunoglobulin domains, and the like. There are numerous binding regions known in the art that are useful for targeting polypeptides to specific cell-types via their binding characteristics, such as ligands, monoclonal antibodies, engineered antibody derivatives, and engineered alternatives to antibodies (see e.g., Cheung M et al.,Mol Cancer9: 28 (2010); WO 2014/164680; WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138435; WO 2015/138452; US20150259428; WO 2015/191764; US20160177284; WO 2016/126950). According to one specific, but non-limiting aspect, the binding region of the molecule of the invention comprises a naturally occurring ligand or derivative thereof that retains binding functionality to an extracellular target biomolecule, commonly a cell surface receptor. For example, various cytokines, growth factors, and hormones known in the art may be used to target the cell-targeting molecule to the cell-surface of specific cell types expressing a cognate cytokine receptor, growth factor receptor, or hormone receptor. According to certain other embodiments, the binding region comprises a synthetic ligand capable of binding an extracellular target biomolecule. According to one specific, but non-limiting aspect, the binding region may comprise an immunoglobulin-type binding region. The term “immunoglobulin-type binding region” as used herein refers to a polypeptide region capable of binding one or more target biomolecules, such as an antigen or epitope. Binding regions may be functionally defined by their ability to bind to target molecules. Immunoglobulin-type binding regions are commonly derived from antibody or antibody-like structures; however, alternative scaffolds from other sources are contemplated within the scope of the term. Immunoglobulin (Ig) proteins have a structural domain known as an Ig domain. Ig domains range in length from about 70-110 amino acid residues and possess a characteristic Ig-fold, in which typically 7 to 9 antiparallel beta strands arrange into two beta sheets which form a sandwich-like structure. The Ig fold is stabilized by hydrophobic amino acid interactions on inner surfaces of the sandwich and highly conserved disulfide bonds between cysteine residues in the strands. Ig domains may be variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgI or I-set). Some Ig domains may be associated with a complementarity determining region or complementary determining region (CDR) which is important for the specificity of antibodies binding to their epitopes. Ig-like domains are also found in non-immunoglobulin proteins and are classified on that basis as members of the Ig superfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC) provides a list of members of the Ig-like domain containing family. As used herein, the term “heavy chain variable (VH) domain” or “light chain variable (VL) domain” respectively refer to any antibody VHor VLdomain (e.g. a human VHor VLdomain) as well as any derivative thereof retaining at least qualitative antigen binding ability of the corresponding native antibody (e.g. a humanized VHor VLdomain derived from a native murine VHor VLdomain). A VHor VLdomain consists of a “framework” region interrupted by the three CDRs or ABRs. The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. From amino-terminus to carboxyl-terminus, both VHand VLdomains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. For camelid VHH fragments, IgNARs of cartilaginous fish, VNARfragments, and derivatives thereof, there is a single heavy chain variable domain comprising the same basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An immunoglobulin-type binding region may be a polypeptide sequence of an antibody or antigen-binding fragment thereof wherein the amino acid sequence has been varied from that of a native antibody or an Ig-like domain of a non-immunoglobulin protein, for example by molecular engineering or selection by library screening. Because of the relevance of recombinant DNA techniques and in vitro library screening in the generation of immunoglobulin-type binding regions, antibodies can be redesigned to obtain desired characteristics, such as smaller size, cell entry, or other therapeutic improvements. The possible variations are many and may range from the changing of just one amino acid to the complete redesign of, for example, a variable region. Typically, changes in the variable region will be made in order to improve the antigen-binding characteristics, improve variable region stability, or reduce the potential for immunogenic responses. There are numerous immunoglobulin-type binding regions contemplated as components of the molecules of the present invention, such as, e.g., the cell-targeting molecules of the present invention. An immunoglobulin binding region generally comprises one or more CDRs. In certain embodiments, the immunoglobulin-type binding region is derived from an immunoglobulin binding region, such as an antibody paratope capable of binding an extracellular target biomolecule. In certain other embodiments, the immunoglobulin-type binding region comprises an engineered polypeptide not derived from any immunoglobulin domain but which functions like an immunoglobulin binding region by providing high-affinity binding to an extracellular target biomolecule. This engineered polypeptide may optionally include polypeptide scaffolds comprising or consisting essentially of complementary determining regions from immunoglobulins as described herein. There are also numerous binding regions in the prior art that are useful for targeting polypeptides to specific cell-types via their high-affinity binding characteristics. In certain embodiments, the binding region of the present proteins is selected from the group which includes single-domain antibody domains (sdAbs), nanobodies, heavy-chain antibody domains derived from camelids (VHH fragments), bivalent nanobodies, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), VNARfragments, single-chain variable (scFv) fragments, autonomous VHdomains, single-domain antibody domains (sdAbs), heavy-chain antibody domains derived from camelids (VHH fragments or VHdomain fragments), heavy-chain antibody domains derived from camelid VHH fragments or VHdomain fragments, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), VNARfragments, single-chain variable (scFv) fragments, nanobodies, Fd fragments consisting of the heavy chain and CHI domains, single chain Fv-CH3 minibodies, dimeric CH2 domain fragments (CH2D), Fc antigen binding domains (Fcabs), isolated complementary determining region 3 (CDR3) fragments, constrained framework region 3, CDR3, framework region 4 (FR3-CDR3-FR4) polypeptides, small modular immunopharmaceutical (SMIP) domains, multimerizing VHH fragments, scFv-Fc fusions, multimerizing scFv fragments (diabodies, triabodies, tetrabodies), disulfide stabilized antibody variable (Fv) fragments, disulfide stabilized antigen-binding (Fab) fragments consisting of the VL, VH, CLand CH1 domains, bivalent nanobodies, bivalent minibodies, bivalent F(ab′)2fragments (Fab dimers), bispecific tandem VHH fragments, bispecific tandem scFv fragments, bispecific nanobodies, bispecific minibodies, and any genetically manipulated counterparts of the foregoing that retain its paratope and binding function (see e.g. Ward E et al.,Nature341: 544-6 (1989); Davies J, Riechmann L,Biotechnology(NY) 13: 475-9 (1995); Reiter Y et al.,Mol Biol290: 685-98 (1999); Riechmann L, Muyldermans S,J Immunol Methods231: 25-38 (1999); Tanha J et al.,J Immunol Methods263: 97-109 (2002); Vranken W et al.,Biochemistry41: 8570-9 (2002); Dottorini T et al.,Biochemistry43: 622-8 (2004); Jespers L et al.,J Mol Biol337: 893-903 (2004); Jespers L et al.,Nat Biotechnol22: 1161-5 (2004); To R et al.,J Biol Chem280: 41395-403 (2005); Spinelli S et al.,FEBS Lett564: 35-40 (2004); Saerens D et al.,Curr Opin Pharmacol8: 600-8 (2008); Dimitrov D,MAbs1: 26-8 (2009); Baral T et al.,PLoS One7: e30149 (2012); Ahmad Z et al.,Clin Dev Immunol2012: 980250 (2012); Weiner L,Cell148: 1081-4 (2012); Richard G et al.,PLoS One8: e69495 (2013)). There are a variety of binding regions comprising polypeptides derived from the constant regions of immunoglobulins, such as, e.g., engineered dimeric Fc domains, monomeric Fcs (mFcs), VHH-Fc fusions, scFv-Fc fusions, CH2 domains, monomeric CH3s domains (mCH3s), synthetically reprogrammed immunoglobulin domains, and/or hybrid fusions of immunoglobulin domains with ligands (Hofer T et al.,Proc Natl Acad Sci USA105: 12451-6 (2008); Xiao J et al.,J Am Chem Soc131: 13616-13618 (2009); Xiao X et al.,Biochem Biophys Res Commun387: 387-92 (2009); Wozniak-Knopp G et al.,Protein Eng Des Sel23 289-97 (2010); Gong R et al.,PLoS ONE7: e42288 (2012); Wozniak-Knopp G et al.,PLoS ONE7: e30083 (2012); Ying T et al.,J Biol Chem287: 19399-408 (2012); Ying T et al.,J Biol Chem288: 25154-64 (2013); Chiang M et al.,J Am Chem Soc136: 3370-3 (2014); Rader C,Trends Biotechnol32: 186-97 (2014); Ying T et al.,Biochimica Biophys Acta1844: 1977-82 (2014). In accordance with certain other embodiments, the binding region includes engineered, alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high-affinity and specific binding of target biomolecules, and enables the engineering of improved characteristics, such as greater stability or reduced immunogenicity. For certain embodiments of the cell-targeting molecules of the present the invention, the binding region is selected from the group which includes engineered Armadillo repeat polypeptides (ArmRPs), engineered, fibronectin-derived, 10thfibronectin type III (10Fn3) domain (monobodies, AdNectins™, or AdNexins™); engineered, tenascin-derived, tenascin type III domain (Centryns™); engineered, ankyrin repeat motif containing polypeptide (DARPins™); engineered, low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (Avimers™); lipocalin (anticalins); engineered, protease inhibitor-derived, Kunitz domain; engineered, Protein-A-derived, Z domain (Affibodies™); engineered, gamma-B crystallin-derived scaffold or engineered, ubiquitin-derived scaffold (Affilins); Sac7d-derived polypeptides (Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domain (Fynomers®); and engineered antibody mimic and any genetically manipulated counterparts of the foregoing that retains its binding functionality (Worn A, Plückthun A,J Mol Biol305: 989-1010 (2001); Xu L et al.,Chem Biol9: 933-42 (2002); Wikman M et al.,Protein Eng Des Sel17: 455-62 (2004); Binz H et al.,Nat Biotechnol23: 1257-68 (2005); Holliger P, Hudson P,Nat Biotechnol23: 1126-36 (2005); Gill D, Damle N,Curr Opin Biotech17: 653-8 (2006); Koide A, Koide S,Methods Mol Biol352: 95-109 (2007); Byla P et al.,J Biol Chem285: 12096 (2010); Zoller F et al.,Molecules16: 2467-85 (2011); Alfarano P et al.,Protein Sci21: 1298-314 (2012); Madhurantakam C et al.,Protein Sci21: 1015-28 (2012); Varadamsetty G et al.,J Mol Biol424: 68-87 (2012)). Among certain embodiments of the present invention, the immunoglobulin-type binding region is derived from a nanobody or single domain immunoglobulin-derived region VHH. Generally, nanobodies are constructed from fragments of naturally occurring single, monomeric variable domain antibodies (sdAbs) of the sort found in camelids and cartilaginous fishes (Chondrichthyes). Nanobodies are engineered from these naturally occurring antibodies by truncating the single, monomeric variable domain to create smaller and more stable molecules, such as, e.g., IgNAR, VHH, and VNARconstructs. Due to their small size, nanobodies are able to bind to antigens that are not accessible to whole antibodies. In certain embodiments of the cell-targeting molecule of the present invention, the binding region comprises a polypeptide(s) selected from the group consisting of: a) a heavy chain variable (VH) domain comprising (i) a HABR1 or HCDR1, (ii) a HABR2 or HCDR2, and (iii) a HABR3 or HCDR3; and/or b) a light chain variable (VL) domain comprising (i) a LABR1 or LCDR1, (ii) a LABR2 or LCDR2, and (iii) a LABR3 or LCDR3; wherein each of the aforementioned ABRs and/or CDRs is selected from the polypeptide comprising or consisting essentially of one of the amino acid sequence as shown in SEQ ID NOs: 844-1100. In certain further embodiments, the binding region comprises or consists essentially of 269-499 of any one of SEQ ID NOs: 807-808 and 812-813, comprises or consists essentially of amino acids of 269-519 of any one of SEQ ID NOs: 814-815 and 818-829, or comprises or consists essentially of amino acids 268-386 of any one of SEQ ID NOs: 816-817. In certain embodiments of the present invention, the binding region polypeptide comprises a free cysteine residue suitable for conjugation to another molecule, wherein the cysteine is at or proximal to a carboxy-terminus of the binding region polypeptide. In certain further embodiments, the binding region of a cell-targeting molecule of the present invention comprises a series of amino acid residues represented by (X)n-C—X or (X)n-C, where X refers to any amino acid, (X)n refers to a polypeptide comprising a binding domain, and C refers to a cysteine residue. In certain embodiments of the present invention, the binding region polypeptide comprises a free cysteine residue suitable for conjugation to another molecule, wherein the cysteine is at or proximal to an amino-terminus of the binding region polypeptide. In certain further embodiments, the binding region of a cell-targeting molecule of the present invention comprises a series of amino acid residues represented by C—(X)n or M-C—(X)n, where X refers to any amino acid, (X)n refers to a polypeptide comprising a binding domain, C refers to a cysteine residue, and M refers to a starting methionine. In certain embodiments of the present invention, the molecule of the present invention comprises an immunoglobulin binding region which lacks cysteine residues. Such immunoglobulin binding region structures are known to the skilled worker and/or can be created using routine methods (see e.g. Proba K,J Mol Biol275: 245-53 (1998)). Any of the above binding regions may be used as a component of the cell-targeting molecules of the present invention as long as the binding region component has a dissociation constant of 10−5to 10−12moles per liter, preferably less than 200 nanomolar (nM), towards an extracellular target biomolecule. Cell-specific targeting can be accomplished by attaching molecules of the present invention to cell targeting carriers, such as, e.g., liposomes, polymers, nanocarriers, microspheres, nanospheres, dendrimers, polymeric micelles, silicon or carbon materials, such as e.g., nanotubes, nanorods and nanohorns, magnetic nanoparticles, microemulsions, and other nanostructures (Sinha R et al.,Molecular Cancer Therapeutics5: 1909-17 (2006); L Brinton et al.,Journal of the National Cancer Institute100: 1643-8 (2008); Tanaka T et al.,Biomed Micro Devices11: 49-63 (2009)). Attachment may be accomplished using one or more covalent bonds and/or encapsulation. Extracellular Target Biomolecules The binding region of the molecule of the invention comprises a polypeptide region capable of binding specifically to an extracellular target biomolecule, preferably which is physically-coupled to the surface of a cell type of interest, such as a cancer cell, tumor cell, plasma cell, infected cell, or host cell harboring an intracellular pathogen. The term “target biomolecule” refers to a biological molecule, commonly a protein or a protein modified by post-translational modifications, such as glycosylation, which is capable of being bound by a binding region to target a protein to a specific cell-type or location within an organism. Extracellular target biomolecules may include various epitopes, including unmodified polypeptides, polypeptides modified by the addition of biochemical functional groups, and glycolipids. There are numerous extracellular target biomolecules known to the skilled worker that may be targeted by the binding region of a cell-targeting molecule of the present invention and polypeptide binding domains known to bind such target biomolecules (see e.g. WO 2014/164680; WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138435; WO 2015/138452; US20150259428; WO 2015/191764; US20160177284; WO 2016/126950). For purposes of the present invention, the term “extracellular” with regard to modifying a target biomolecule refers to a biomolecule that has at least a portion of its structure exposed to the extracellular environment. Extracellular target biomolecules include cell membrane components, transmembrane spanning proteins, cell membrane-anchored biomolecules, cell-surface-bound biomolecules, and secreted biomolecules. With regard to the present invention, the phrase “physically coupled” when used to describe a target biomolecule means both covalent and/or non-covalent intermolecular interactions that couple the target biomolecule, or a portion thereof, to the outside of a cell, such as a plurality of non-covalent interactions between the target biomolecule and the cell where the energy of each single interaction is on the order of about 1-5 kiloCalories (e.g. electrostatic bonds, hydrogen bonds, Van der Walls interactions, hydrophobic forces, etc.). All integral membrane proteins can be found physically coupled to a cell membrane, as well as peripheral membrane proteins. For example, an extracellular target biomolecule might comprise a transmembrane spanning region, a lipid anchor, a glycolipid anchor, and/or be non-covalently associated (e.g. via non-specific hydrophobic interactions and/or lipid binding interactions) with a factor comprising any one of the foregoing. The binding regions of the cell-targeting molecules of the present invention may be designed or selected based on numerous criteria, such as the cell-type specific expression of their target biomolecules and/or the physical localization of their target biomolecules with regard to specific cell types. For example, certain cell-targeting molecules of the present invention comprise binding domains capable of binding cell-surface targets which are expressed exclusively by only one cell-type to the cell surface. Among certain embodiments of the present invention, the cell-targeting molecule comprises a binding region derived from an immunoglobulin-type polypeptide selected for specific and high-affinity binding to a surface antigen on the cell surface of a cancer cell, where the antigen is restricted in expression to cancer cells (see Glokler J et al.,Molecules15: 2478-90 (2010); Liu Y et al.,Lab Chip9: 1033-6 (2009)). In accordance with other embodiments, the binding region is selected for specific and high-affinity binding to a surface antigen on the cell surface of a cancer cell, where the antigen is over-expressed or preferentially expressed by cancer cells as compared to non-cancer cells. Some representative target biomolecules include, but are not limited to, the following enumerated targets associated with cancers and/or specific immune cell types. Many immunoglobulin-type binding regions that recognize epitopes associated with cancer cells exist in the prior art, such as binding regions that target annexin A1, B3 melanoma antigen, B4 melanoma antigen, B7-H3 (CD276, B7RP-2), B-cell maturation antigen (BCMA, BCM, TNRSF17, CD269), CD2, CD3, CD4, CD19, CD20 (B-lymphocyte antigen protein CD20), CD22, CD25 (interleukin-2 receptor IL2R), CD30 (TNFRSF8), CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 (hyaluronan receptor), protein tyrosine phosphatase receptor type C (CD45, PTPRC, LCA), ITGAV (CD51), CD56, CD66, CD70, CD71 (transferrin receptor), CD73, CD74 (HLA-DR antigens-associated invariant chain), CD79 (e.g. CD79a or CD79b), CD98, endoglin (END, CD105), CD106 (VCAM-1), CD138, chemokine receptor type 4 (CDCR-4, fusin, CD184), CD200, insulin-like growth factor 1 receptor (CD221), mucin1 (MUC1, CD227, CA6, CanAg), basal cell adhesion molecule (B-CAM, CD239), CD248 (endosialin, TEM1), tumor necrosis factor receptor 10b (TNFRSF10B, CD262), tumor necrosis factor receptor 13B (TNFRSF13B, TACI), vascular endothelial growth factor receptor 2 (KDR, CD309), epithelial cell adhesion molecule (EpCAM, CD326), human epidermal growth factor receptor 2 (HER2, Neu, ErbB2, CD340), cancer antigen 15-3 (CA15-3), cancer antigen 19-9 (CA 19-9), cancer antigen 125 (CA125, MUC16), CA242, carcinoembryonic antigen-related cell adhesion molecules (e.g. CEACAM3 (CD66d) and CEACAM5), carcinoembryonic antigen protein (CEA), choline transporter-like protein 4 (SLC44A4), chondroitin sulfate proteoglycan 4 (CSP4, MCSP, NG2), CTLA4, delta-like proteins (e.g. DLL3, DLL4), ectonucleotide pyrophosphatase/phosphodiesterase proteins (e.g. ENPP3), endothelin receptors (ETBRs), epidermal growth factor receptor (EGFR, ErbB1), Epstein-Barr virus latent membrane protein 1 (LMP1), folate receptors (FOLRs, e.g. FRα), G-28, ganglioside GD2, ganglioside GD3, HLA-Dr10, HLA-DRB, human epidermal growth factor receptor 1 (HER1), HER3/ErbB-3, Ephrin type-B receptor 2 (EphB2), epithelial cell adhesion molecule (EpCAM), fibroblast activation protein (FAP/seprase), guanylyl cyclase c (GCC), insulin-like growth factor 1 receptor (IGF1R), interleukin 2 receptor (IL-2R), interleukin 6 receptor (IL-6R), integrins alpha-V beta-3 (αvβ3), integrins alpha-V beta-5 (αvβ5), integrins alpha-5 beta-1 (α5β1), L6, zinc transporter (LIV-1), MPG, melanoma-associated antigen 1 protein (MAGE-1), melanoma-associated antigen 3 (MAGE-3), mesothelin (MSLN), metalloreductase STEAPI, MPG, MS4A, NaPi2b, nectins (e.g. nectin-4), p21, p97, polio virus receptor-like 4 (PVRL4), protease-activated-receptors (such as PAR1), prostate-specific membrane antigen proteins (PSMAs), SAIL (C16orf54), SLIT and NTRK-like proteins (e.g. SLITRK6), Thomas-Friedenreich antigen, transmembrane glycoprotein (GPNMB), trophoblast glycoproteins (TPGB, 5T4, WAIF1), and tumor-associated calcium signal transducers (TACSTDs, e.g. Trop-2, EGP-1, etc) (see e.g. Lui B et al.,Cancer Res64: 704-10 (2004); Novellino L et al.,Cancer Immunol Immunother54: 187-207 (2005); Bagley R et al.,Int J Oncol34: 619-27 (2009); Gerber H et al., mAbs 1: 247-53 (2009); Beck A et al.,Nat Rev Immunol10: 345-52 (2010); Andersen J et al.,J Biol Chem287: 22927-37 (2012); Nolan-Stevaux O et al.,PLoS One7: e50920 (2012); Rust S et al.,Mol Cancer12: 11 (2013); Kim S et al.,Blood Cancer Journal5: e316 (2015)). This list of target biomolecules is intended to be non-limiting. It will be appreciated by the skilled worker that any desired target biomolecule associated with a cancer cell or other desired cell type may be used to design or select a binding region to be coupled with a toxin effector polypeptide to produce a cell-targeting molecule of the present invention. Examples of other target biomolecules which are strongly associated with cancer cells and immunoglobulin-type binding regions known to bind them include BAGE proteins (B melanoma antigens), basal cell adhesion molecules (BCAMs or Lutheran blood group glycoproteins), bladder tumor antigen (BTA), cancer-testis antigen NY-ESO-1, cancer-testis antigen LAGE proteins, CD19 (B-lymphocyte antigen protein CD19), CD21 (complement receptor-2 or complement 3d receptor), CD26 (dipeptidyl peptidase-4, DPP4, or adenosine deaminase complexing protein 2), CD33 (sialic acid-binding immunoglobulin-type lectin-3), CD52 (CAMPATH-1 antigen), CD56, CS1 (SLAM family number 7 or SLAMF7), cell surface A33 antigen protein (gpA33), Epstein-Barr virus antigen proteins, GAGE/PAGE proteins (melanoma associated cancer/testis antigens), hepatocyte growth factor receptor (HGFR or c-Met), MAGE proteins, melanoma antigen recognized by T-cells 1 protein (MART-1/MelanA, MARTI), mucins, Preferentially Expressed Antigen of Melanoma (PRAME) proteins, prostate specific antigen protein (PSA), prostate stem cell antigen protein (PSCA), Receptor for Advanced Glycation Endroducts (RAGE), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptors (VEGFRs), and Wilms' tumor antigen. Examples of other target biomolecules which are strongly associated with cancer cells are carbonic anhydrase IX (CA9/CAIX), claudin proteins (CLDN3, CLDN4), ephrin type-A receptor 3 (EphA3), folate binding proteins (FBP), ganglioside GM2, insulin-like growth factor receptors, integrins (such as CD11a-c), receptor activator of nuclear factor kappa B (RANK), receptor tyrosine-protein kinase erB-3, SAIL (C16orf54), tumor necrosis factor receptor 10A (TRAIL-R1/DR4), tumor necrosis factor receptor 10B (TRAIL-R2), tenascin C, and CD64 (FcTRI) (see Hough C et al.,Cancer Res60: 6281-7 (2000); Thepen T et al.,Nat Biotechnol18: 48-51 (2000); Pastan I et al.,Nat Rev Cancer6: 559-65 (2006); Pastan,Annu Rev Med58: 221-37 (2007); Fitzgerald D et al.,Cancer Res71: 6300-9 (2011); Scott A et al.,Cancer Immun12: 14-22 (2012); Kim S et al.,Blood Cancer Journal5: e316 (2015)). This list of target biomolecules is intended to be non-limiting. In addition, there are numerous other examples of contemplated, target biomolecules such as ADAM metalloproteinases (e.g. ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17), ADP-ribosyltransferases (ART1, ART4), antigen F4/80, bone marrow stroma antigens (BST1, BST2), break point cluster region-c-abl oncogene (BCR-ABL) proteins, C3aR (complement component 3a receptors), CD7, CD13, CD14, CD15 (Lewis X or stage-specific embryonic antigen 1), CD23 (FC epsilon RII), CD49d, CD53, CD54 (intercellular adhesion molecule 1), CD63 (tetraspanin), CD69, CD80, CD86, CD88 (complement component 5a receptor 1), CD115 (colony stimulating factor 1 receptor), IL-1R (interleukin-1 receptor), CD123 (interleukin-3 receptor), CD129 (interleukin 9 receptor), CD183 (chemokine receptor CXCR3), CD191 (CCR1), CD193 (CCR3), CD195 (chemokine receptor CCR5), CD203c, CD225 (interferon-induced transmembrane protein 1), CD244 (Natural Killer Cell Receptor 2B4), CD282 (Toll-like receptor 2), CD284 (Toll-like receptor 4), CD294 (GPR44), CD305 (leukocyte-associated immunoglobulin-like receptor 1), ephrin type-A receptor 2 (EphA2), FceRIa, galectin-9, alpha-fetoprotein antigen 17-A1 protein, human aspartyl (asparaginyl) beta-hydroxylase (HAAH), immunoglobulin-like transcript ILT-3, lysophosphatidlglycerol acyltransferase 1 (LPGAT1/IAA0205), lysosome-associated membrane proteins (LAMPs, such as CD107), melanocyte protein PMEL (gp100), myeloid-related protein-14 (mrp-14), NKG2D ligands (e.g., MICA, MICB, ULBP1, ULBP2, UL-16-binding proteins, H-60s, Rae-1s, and homologs thereof), receptor tyrosine-protein kinase erbB-3, SART proteins, scavenger receptors (such as CD64 and CD68), Siglecs (sialic acid-binding immunoglobulin-type lectins), syndecans (such as SDC1 or CD138), tyrosinase, tyrosinease-related protein 1 (TRP-1), tyrosinease-related protein 2 (TRP-2), tyrosinase associated antigen (TAA), APO-3, BCMA, CD2, CD3, CD4, CD8, CD18, CD27, CD28, CD29, CD41, CD49, CD90, CD95 (Fas), CD103, CD104, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), chemokine receptors, complement proteins, cytokine receptors, histocompatibility proteins, ICOS, interferon-alpha, interferon-beta, c-myc, osteoprotegerin, PD-1, RANK, TACI, TNF receptor superfamily member (TNF-R1, TNFR-2), Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 (see Scott A et al.,Cancer Immunity12: 14 (2012); Cheever M et al.,Clin Cancer Res15: 5323-37 (2009)), for target biomolecules and note the target molecules described therein are non-limiting examples). It will be appreciated by the skilled worker that any desired target biomolecule may be used to design or select a binding region to be coupled with a toxin effector polypeptide to produce a cell-targeting molecule of the present invention. In certain embodiments, the binding region comprises or consists essentially of an immunoglobulin-type polypeptide selected for specific and high-affinity binding to the cellular surface of a cell type of the immune system. For example, immunoglobulin-type binding domains are known that bind to programmed death ligand 1 (PD-L1), CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD41, CD56, CD61, CD62, CD66, CD95, CD117, CD123, CD235, CD146, CD326, interleukin-2 receptor (IL-2R), receptor activator of nuclear factor kappa B (RANKL), SLAM-associated protein (SAP), and TNFSF18 (tumor necrosis factor ligand 18 or GITRL). Extracellular target biomolecules of the binding region of the cell-targeting molecules of the present invention may include biomarkers over-proportionately or exclusively present on cancer cells, immune cells, and cells infected with intracellular pathogens, such as viruses, bacteria, fungi, prions, or protozoans. This general structure is modular in that any number of diverse cell-targeting binding regions may be linked to one or more toxin effector polypeptides to produce a cell-targeting molecule of the present invention, and compositions thereof. C. Toxin Effector Polypeptides Derived from Toxins In certain embodiments, the cell-targeting molecule of the present invention comprises a toxin effector polypeptide region derived from a proteinaceous toxin other than a Shiga toxin(s). In certain embodiments, the cell-targeting molecule of the present invention does not comprise a Shiga toxin effector polypeptide. In certain embodiments, the cell-targeting molecule of the present invention comprises a toxin effector region derived from a toxin other than a member of the Shiga toxin family, such as, e.g., from an ABx toxin other than a Shiga toxin, a ribosome inactivating protein toxin other than Shiga toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin,Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, and subtilase cytotoxin (see e.g., WO 2015/113005; WO 2015/120058). In certain embodiments, the cell-targeting molecule of the present invention does not comprise either a toxin effector region or any polypeptide derived from a toxin. In certain embodiments, the cell-targeting molecule of the present invention comprises a toxin effector polypeptide which is not a Shiga toxin effector polypeptide. The present invention contemplates the use of various polypeptides derived from toxins as toxin effector polypeptides. For example, many toxins represent optimal sources of cytotoxic polypeptides and/or proteasome delivering effector polypeptides because of the wealth of knowledge about their intracellular routing behaviors (see e.g. WO 2015/113005). Any protein toxin with the intrinsic ability to intracellularly route from an early endosomal compartment to either the cytosol or the ER represents a source for a proteasome delivery effector polypeptide which may be exploited for the purposes of the present invention, such as a starting component for modification or as a source for mapping a smaller proteasome delivery effector region therein. In certain embodiments, the cell-targeting molecule of the present invention comprises a toxin effector lacking any lysine residues or comprising exactly one lysine residue for site-specific conjugation. The catalytic domains of many toxins, especially toxins which use retrograde routing pathways via the endoplasmic reticulum, are devoid of lysine residues (DeLange R et al.,Proc Natl Acad Sci USA73: 69-72 (1976); London E, Luongo C,Biochem Biophys Res Commun160: 333-9 (1989); Hazes B, Read R,Biochemistry36: 11051-4 (1997); Deeks E et al.,Biochemistry41: 3405-13 (2002); Worthington Z, Carbonetti N,Infect Immun75: 2946-53 (2007)). In certain embodiments, the cell-targeting molecule of the present invention comprises the toxin effector polypeptide which is a Shiga toxin effector polypeptide. In certain further embodiments, the Shiga toxin effector polypeptide is a SLT-1A-Cys1-variant (e.g. SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, and 75). In certain further embodiments, the Shiga toxin effector polypeptide is carboxy-terminal to another proteinaceous component of the cell-targeting molecule (e.g. a cell-targeting binding region or starting methionine) such that the cysteine residue at position 1 of the Shiga toxin effector polypeptide (C1) forms a peptide bond via its amino group. The general structure of the cell-targeting molecules of the present invention is modular, in that various, diverse binding regions may be used with the same toxin effector polypeptide (e.g. a Shiga toxin effector polypeptide of the present invention) to provide for diverse targeting of various extracellular target biomolecules and thus targeting of cytotoxicity, cytostasis, and/or exogenous material delivery to various diverse cell types. In the cell-targeting molecules of the invention, the binding region(s) and Shiga toxin effector polypeptide(s) may be directly linked to each other and/or suitably linked to each other via one or more linkers well known in the art. For the purposes of the cell-targeting molecules of the present invention, the specific order or orientation is not fixed for the toxin effector polypeptide and the cell-targeting, binding region in relation to each other (see e.g.FIG.1-B). Toxin effector polypeptides which are not cytotoxic may still be useful for delivering exogenous materials into cells, certain subcellular compartments, and/or providing efficient subcellular routing to the cytosol. Optionally, a cell-targeting molecule of the present invention may further comprise a carboxy-terminal endoplasmic retention/retrieval signal motif, such as, e.g., the amino acids KDEL (SEQ ID NO:1142) at the carboxy-terminus of a proteinaceous component of the cell-targeting molecule (see e.g. WO 2015/138435). D. Amino Acid Residues and Structures Amenable for Site-Specific Conjugation Any amino acid residue having a bio-orthogonal reactive moiety (e.g. a side chain or functional group) may be suitable as conjugation site in the molecules of the present invention. The skilled worker can select an amenable and suitable amino acid from the prior art or using routine techniques can identify through experimentation a novel amino acid which is amenable and suitable for use in a molecule of the present invention. In certain embodiments, a molecule of the present invention comprises a Shiga toxin effector polypeptide having a unique amino acid residue, which may be either ectoptically positioned or naturally occurring at that position. In certain other embodiments, the invention may involve a unique amino acid or a non-unique amino acid residue which is uniquely accessible, i.e. not buried in the interior of a proteinaceous structure as are all other) residues of the same amino acid type. In certain embodiments of the present invention, a single lysine, histidine, or cysteine is engineered into a cell-targeting molecule to provide a specific residue site for conjugation of another molecule. In general, the strongly nucleophilic functional groups of the amino acids lysine, histidine, and cysteine make these three amino acid residues the most amenable attachment points for chemically conjugating of an atom or molecule to a protein. However, the number of lysines, histidines, and/or cysteines vary from protein to protein, which affects the suitability of site-specific attachment and/or control of conjugate stoichiometry. In the examples below, all the cysteine residues were removed from a parental, Shiga toxin A Subunit polypeptide. Then, an ectopic cysteine residue was engineered into the polypeptide to provide a unique attachment position for controlled conjugation. This was repeated at different positions to provide a group of cysteine-engineered Shiga toxin effector polypeptides, and these polypeptides are tested for retention of Shiga toxin effector functions and ability to deliver a conjugated molecule to the inside of a cell. In certain embodiments of the present invention, a single, unnatural amino acid residue is engineered into a cell-targeting molecule to provide a specific residue site for conjugation of another molecule (see e.g. Liu W et al.,Nat Methods4: 239-44 (2007); Liu C, Schultz P,Annu Rev Biochem79: 413-44 (2010); Young T et al.,J Mol Biol395: 361-74 (2010); Young T, Schultz P,J Biol Chem285: 11039-44 (2010); Young D et al.,Biochemistry50: 1894-900 (2011); Hoesl M, Budisa N,Curr Opin Biotechnol23: 751-7 (2012); Ozawa K et al.,Biochem Biophys Res Commun418: 652-6 (2012); Chin J,Annu Rev Biochem83: 379-408 (2014); Ozawa K, Loh C,Methods Mol Biol1118: 189-203 (2014)). For example, unnatural amino acid residues such as selenocysteine and para-acetylphenylalanine, are known in the art to provide useful conjugation sites. Similarly, amino acid residues having an azide group can be used for site specific conjugation. In certain embodiments of the present invention, the site-specific attachment site is the primary amine or carboxy-terminal of a polypeptide component of the cell-targeting molecule of the present invention. In certain embodiments of the present invention, a short polypeptide motif is engineered into a cell-targeting molecule to provide a specific residue site for conjugation of another molecule. For example, cysteine residues in certain motifs like CxPxR can be modified by formylglycine generating enzymes into formylglycine, and then the resulting aldehyde functional group may be conjugated to another molecule using hydrozino-Pictet-Spengler chemistry (see e.g. Carrico I et al.,Nat Chem Biol3: 321-2 (2007); Rabuka D, et al.,Nat Protoc7: 1052-67 (2012)). It is important to note that the solvent accessibility of the engineered residue may affect its ability to provide a suitable conjugation site; however, suitability may vary with the conjugated molecule and specific application of the resulting conjugate. For example, the size and steric hindrances of the conjugated molecule may affect the stability of the final conjugated product such that certain engineered sites and/or ectopic residues are suitable for certain conjugated molecules but not others. While highly solvent accessible amino acid residues may be predicted to provide better sites for conjugation, it is important to note that the Shiga toxin effector polypeptide or cell-targeting molecule with the greatest therapeutic utility may require a conjugation site which is less solvent accessible than other possible conjugation sites (see e.g. Shen B et al.,Nat Biotechnol30: 184-9 (2012)). There are several common conjugation strategies for linking a proteinaceous molecule to another molecule, such as, e.g., via a lysine in a protein to an amine-reactive linker or molecule, via a cysteine in a protein to a sulfhydryl reactive linker or molecule (e.g. involving an activated maleimide group), via enzyme-mediated conjugation, and/or via the incorporation of an unnatural amino acid such as para-acetylphenylalanine (see e.g. Kline T et al.,Pharm Res32: 3480-93 (2015)). For example, sulfhydryl-reactive chemical groups are highly amenable to conjugation chemistry, such as via alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond). Non-limiting examples of sulfhyryl-reactive groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. For example, a cysteine residue can be conjugated to another molecule, e.g., using maleimide or bromoacetamide groups on linkers or cargos and/or using click chemistry. Unnatural amino acid residues can be conjugated using oxime groups on linkers or cargos and/or via click chemistry. For example, para-acetylphenylalanine can be conjugated to another molecule comprising an alkoxy-amine via oxime ligation. For example, selenocysteine can be conjugated to another molecule using maleimide groups and/or click chemistry (see e.g. Hofer T et al.,Biochemistry48: 12047-57 (2009); Young T et al.,J Mol Biol395: 361-74 (2010); Kiick K et al.,Proc Natl Acad Sci USA99: 19-24 (2002)). For certain embodiments, the molecule of the present invention is made using a haloalkyl derivative, such as, e.g., an iodoacetamide or maleimide, present in the cargo or linker to link the cargo to a cysteine, methionine, and/or histidine residue(s) present in a polypeptide component of a molecule of the present invention. In certain embodiments, a maleimide agent is specifically used to avoid conjugation to a tyrosine, histidine, and/or methionine residue(s). A cysteine residue already present in a polypeptide component of a molecule of the present invention may be used, such as, e.g., after a reduction reaction to make free its thiol group and make it available for conjugation. The skilled worker can reduce a thiol linkage using methods known in the art, such as, e.g., using TCEP (tris(2-carboxyethyl)phosphine hydrochloride, dithiothreitol (DTT), and/or beta-mercaptoethanol (BME). For certain embodiments, the molecule of the present invention is made using a nitrosylated thiol derivative, such as, e.g., a thiosulfate, present in the cargo or linker to link the cargo to a cysteine, methionine, and/or histidine residue(s) present in a polypeptide component of a molecule of the present invention. For certain embodiments, a homobifunctional maleimide, homobifunctional sulfhydryl-reactive maleimide, heterobifunctional maleimide, and/or heterobifunctional amine-to-sulfhydryl maleimide crosslinker(s). For certain embodiments, a haloacetyl reagent is used, such as, e.g., a haloacetyl crosslinker contain an iodoacetyl or a bromoacetyl group and/or using a NHS ester amine-to sulfhydryl crosslinker. For certain embodiments, an iodoacetyl reactive group is used for chemical conjugation to a sulfhydryl group, such as, e.g., a homobifunctional or heterobifunctional iodoacetyl crosslinker. For certain embodiments, a bromoacetyl reactive group is used for chemical conjugation to a sulfhydryl group, such as, e.g., a homobifunctional or heterobifunctional bromoacetyl crosslinker. For certain embodiments, a sulfhydryl group is added using a chemical reaction, such as, e.g., using 2-iminothiolane, SATA, SATP, SAT(PEG)4, or a pyridyl disulfide. In certain embodiments, the cell-targeting molecule is treated with Traut's Reagent (2-iminothiolane, 2-IT) or SATA to add sulfhydryl groups onto primary amine sites. In certain embodiments, the linking to the Shiga toxin effector polypeptide involves a chemical reaction involving a sulfhydryl group, such as, e.g., a sulfhydryl group of a cysteine, methionine, N-formylmethionine, homocysteine, or taurine residue of the Shiga toxin effector polypeptide, the linker connecting the Shiga toxin effector polypeptide to a cell-targeting binding region, or the cargo being conjugated. Chemical reactions suitable for use in conjugating an atom or molecule to a polypeptide or polypeptide component of the present invention include: carbodiimide-mediated reaction, EDC-mediated amide bond formation, hydrazide activation reaction, pyridyl disulfide reaction, iodoacetyl reaction, and/or the Mannich reaction. For example, a carboxyl group of a polypeptide or polypeptide component of the present invention may be linked to a cargo via a carbodiimide-mediated reaction, such as, e.g., using a water-soluble carbodiimide crosslinker, that results in amide bond formation with an amino, amine, and/or hydrazine group of the cargo. The carboxyl group of the polypeptide may be part of a surface accessible amino acid residue, such as, e.g., the carboxy-terminal residue, an aspartic acid, and/or glutamic acid. For examples, EDC-mediated amide bond formation may be used for the linking of a carboxylate group of a cargo with the primary amine group of the amino-terminal amine group of a polypeptide or polypeptide component of the present invention. Alternatively, EDC-mediated amide bond formation may be used for the linking of an amine group of a cargo with the carboxy-terminal carboxylate group of a polypeptide or polypeptide component of the present invention. The skilled worker may then use a purification step(s) to further isolate and purify the desired conjugate molecule(s). For example, the Mannich reaction may be used for the condensation of an aldehyde group of a cargo with the active hydrogen of an amine group of an amino acid residue, such as, e.g., an amine group of an arginine, histidine, lysine, asparagine, glutamine, proline, or tryptophan residue, and/or the amino-terminal amine group of a polypeptide or polypeptide component of the present invention. The skilled worker may then use a purification step(s) to further isolate and purify the desired conjugate molecule(s). For example, a pyridyl disulfide of a cargo or linker may be used to react with a sulfhydryl group of a polypeptide or polypeptide component of the present invention. The pyridyl disulfide may be pre-activated via other chemical reactions before the coupling reaction between the cargo and the protein or polypeptide component of the present invention. For example, an iodoacetyl of a cargo may be used to react with a sulfhydryl group of a polypeptide or polypeptide component of the present invention. The pyridyl disulfide may be pre-activated via other chemical reactions before the coupling reaction between the cargo and the protein or polypeptide component of the present invention. In certain embodiments, the cargo and Shiga toxin effector polypeptide of the present invention are linked via a disuccinimidyl suberate (DSS) linker. E. Linkers for Conjugating Polypeptide Components of the Present Invention to a Heterologous Molecule, Cargo, and/or Additional Exogenous Material In certain embodiments, the molecule of the present invention comprises a conjugated molecule, such as, e.g., a heterologous molecule, a cargo, an additional exogenous material, and/or a cell-targeting molecule altering agent. In certain further embodiments, the molecule of the present invention is indirectly conjugated via one or more linkers, referred to herein as a “conjugation linker.” The skilled worker can select conjugation linker from the prior art or using routine techniques can identify through experimentation a novel conjugation linker suitable for use in a molecule of the present invention. In certain embodiments, the conjugation linkage involves an amine reactive compound and/or a sulfhydryl reactive compound, such as, e.g., a thiol. Conjugation linkers are generally those which allow stable linkage of the conjugate molecule to a Shiga toxin effector polypeptide and/or proteinaceous component of the cell-targeting molecule of the present invention. Stability in this sense refers to both during storage and in the circulatory system of a vertebrate; however, in certain embodiments the conjugation linker allows for the selective dissociation of the conjugate molecule from the linker and/or the remainder of the cell-targeting molecule, and/or the degradation of the linker thereby breaking the linkage between the conjugate molecule and the remainder of the cell-targeting molecule. For example, suitable conjugation linkers known to the skilled worker include or involve hydrazone linkages, thio-ether linkages, disulfide linkages, proteolytically-cleaved linkers, valine-citrulline linkers, β-glucuronide linkers, self-immolative linkages, reversible amino-thiol linkages, and SpaceLink linkers (see e.g. Højfeldt J et al.,J Org Chem71: 9556-9 (2006); Ducry L, Stump B,Bioconjugate Chem21: 5-13 (2010)). In certain embodiments, a molecule of the present invention comprises the conjugation linker which is homobifunctional, whereas in other embodiments, the conjugation linker is heterobifunctional. Examples of homobifunctional linkers suitable for use in certain embodiments of the present invention include NHS esters, haloacetly, aryl azide, diazirine, imidoesters, carbodiimide, maleimide, hydrazide, pyridyldithiol, isocyanate, psoralen. In certain embodiments, the conjugation linker comprises a polyethylene glycol (PEG) spacer. Examples of homobifunctional and/or heterobifunctional linkers suitable for use in certain embodiments of the present invention include bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydrosuccinimide, and succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester. In certain embodiments, a molecule of the present invention comprises a maleimide type conjugation linker. In certain further embodiments, a molecule of the present invention comprises a dibenzocyclooctyne (DBCO) maleimide linker linking a cargo molecule to a proteinaceous component of the cell-targeting molecule, such as, e.g., via a cysteine residue in the proteinaceous component. In certain embodiments, the conjugation linker is conjugated to another molecule via a disulfide bond(s) having a sterically-hindered carbon(s) near the sulfur atom(s) (see e.g. Erickson H et al.,Cancer Res66: 4426-33 (2006)). In certain embodiments, the conjugation linker is a disulfide type linker (see e.g. Hamilton G,Biologicals43: 318-32 (2015)). In certain embodiments of the present invention, the components of a Shiga toxin effector polypeptide (e.g. a cargo or to a Shiga toxin effector polypeptide) or cell-targeting molecule (e.g. a binding region to a toxin effector polypeptide) may be suitably linked to each other via one or more linkers well known in the art and/or described herein, such as, e.g., proteinaceous linkers capable of being genetically fused between other proteinaceous components of the cell-targeting molecules of the present invention. Suitable linkers are generally those which allow each polypeptide component of the present invention to fold with a three-dimensional structure very similar to the polypeptide components produced individually without any linker or other component. Suitable linkers include single amino acids, peptides, polypeptides, and linkers lacking any of the aforementioned such as various non-proteinaceous carbon chains, whether branched or cyclic (see e.g. Alley S et al.,Bioconjug Chem19: 759-65 (2008) Ducry L, Stump B,Bioconjug Chem21: 5-13 (2010); Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). Suitable linkers may be proteinaceous and comprise one or more amino acids, peptides, and/or polypeptides. Proteinaceous linkers are suitable for both recombinant fusion proteins and chemically linked conjugates. A proteinaceous linker typically has from about 2 to about 50 amino acid residues, such as, e.g., from about 5 to about 30 or from about 6 to about 25 amino acid residues. The length of the linker selected will depend upon a variety of factors, such as, e.g., the desired property or properties for which the linker is being selected (see e.g. Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). Suitable linkers may be non-proteinaceous, such as, e.g. chemical linkers (see e.g. Dosio F et al.,Toxins3: 848-83 (2011); Feld J et al.,Oncotarget4: 397-412 (2013)). Various non-proteinaceous linkers known in the art may be used to link cell-targeting moieties to toxin effector polypeptide and other components to form a cell-targeting molecule of the present invention, such as linkers commonly used to conjugate immunoglobulin-derived polypeptides to heterologous polypeptides. For example, polypeptide regions of the cell-targeting molecules of the present invention may be linked using the functional side chains of their amino acid residues and carbohydrate moieties such as, e.g., a carboxy, amine, sulfhydryl, carboxylic acid, carbonyl, hydroxyl, and/or cyclic ring groups. For example, disulfide bonds and thioether bonds may be used to link two or more polypeptides (see e.g. Fitzgerald D et al.,Bioconjugate Chem1: 264-8 (1990); Pasqualucci L et al.,Haematologica80: 546-56 (1995)). In addition, non-natural amino acid residues may be used with other functional side chains, such as ketone groups (see e.g. Axup J et al.,Proc Natl Acad Sci U.S.A.109: 16101-6 (2012); Sun S et al.,ChembiochemJul. 18 (2014); Tian F et al.,Proc Natl Acad Sci USA111: 1766-71 (2014)). Examples of non-proteinaceous chemical linkers include but are not limited to N-succinimidyl (4-iodoacetyl)-aminobenzoate, S—(N-succinimidyl) thioacetate (SATA), N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene (SMPT), N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate, 4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio) toluene, sulfosuccinimidyl-6-(α-methyl-α-(pyridyldithiol)-toluamido) hexanoate, N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP), succinimidyl 6(3(-(-2-pyridyldithio)-proprionamido) hexanoate, sulfosuccinimidyl 6(3(-(-2-pyridyldithio)-propionamido) hexanoate, maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), alpha-alkyl derivatives, sulfoNHS-ATMBA (sulfosuccinimidyl N-[3-(acetylthio)-3-methylbutyryl-beta-alanine]), sulfodicholorphenol, 2-iminothiolane, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine (see e.g. Thorpe P et al.,Eur J Biochem147: 197-206 (1985); Thorpe P et al.,Cancer Res47: 5924-31 (1987); Thorpe P et al.,Cancer Res48: 6396-403 (1988); Grossbard M et al.,Blood79: 576-85 (1992); Lui C et al.,Proc Natl Acad Sci USA93: 8618-23 (1996); Doronina S et al.,Nat Biotechnol21: 778-84 (2003); Feld J et al.,Oncotarget4: 397-412 (2013)). Suitable linkers, whether proteinaceous or non-proteinaceous, may include, e.g., protease sensitive, environmental redox potential sensitive, pH sensitive, acid cleavable, photocleavable, and/or heat sensitive linkers (see e.g. Dosio F et al.,Toxins3: 848-83 (2011); Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013); Feld J et al.,Oncotarget4: 397-412 (2013)). Proteinaceous linkers may be chosen for incorporation into recombinant, fusion protein, cell-targeting molecules of the present invention. For recombinant fusion cell-targeting proteins of the invention, linkers typically comprise about 2 to 50 amino acid residues, preferably about 5 to 30 amino acid residues (Argos P, JMolBiol 211: 943-58 (1990);Williamson M, Biochem J297: 240-60 (1994);George R, Heringa J, Protein Eng15: 871-9 (2002); Kreitman R,AAPS J8: E532-51 (2006)). Commonly, proteinaceous linkers comprise a majority of amino acid residues with polar, uncharged, and/or charged residues, such as, e.g., threonine, proline, glutamine, glycine, and alanine (see e.g. Huston J et al.Proc Natl Acad Sci U.S.A.85: 5879-83 (1988); Pastan I et al.,Annu Rev Med58: 221-37 (2007); Li J et al.,Cell Immunol118: 85-99 (1989); Cumber A et al.Bioconj Chem3: 397-401 (1992); Friedman P et al.,Cancer Res53: 334-9 (1993); Whitlow M et al.,Protein Engineering6: 989-95 (1993); Siegall C et al.,J Immunol152: 2377-84 (1994); Newton et al.Biochemistry35: 545-53 (1996); Ladurner et al. J Mol Biol 273: 330-7 (1997); Kreitman R et al.,Leuk Lymphoma52: 82-6 (2011); U.S. Pat. No. 4,894,443). Non-limiting examples of proteinaceous linkers include alanine-serine-glycine-glycine-proline-glutamate (ASGGPE) (SEQ ID NO:1190), valine-methionine (VM), alanine-methionine (AM), AM(G2 to 4S)xAM (SEQ ID NO:1191) where G is glycine, S is serine, and x is an integer from 1 to 10. Proteinaceous linkers may be selected based upon the properties desired. Proteinaceous linkers may be chosen by the skilled worker with specific features in mind, such as to optimize one or more of the fusion protein's folding, stability, expression, solubility, pharmacokinetic properties, pharmacodynamic properties, and/or the activity of the fused domains in the context of a fusion construct as compared to the activity of the same domain by itself. For example, proteinaceous linkers may be selected based on flexibility, rigidity, and/or cleavability (see e.g. Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). The skilled worker may use databases and linker design software tools when choosing linkers. Certain linkers may be chosen to optimize expression (see e.g. Turner D et al.,J Immunol Methods205: 43-54 (1997)). Certain linkers may be chosen to promote intermolecular interactions between identical polypeptides or proteins to form homomultimers or different polypeptides or proteins to form heteromultimers. For example, proteinaceous linkers may be selected which allow for desired non-covalent interactions between polypeptide components of the cell-targeting proteins of the invention, such as, e.g., interactions related to the formation dimers and other higher order multimers (see e.g. U.S. Pat. No. 4,946,778). Flexible proteinaceous linkers are often greater than 12 amino acid residues long and rich in small, non-polar amino acid residues, polar amino acid residues, and/or hydrophilic amino acid residues, such as, e.g., glycines, serines, and threonines (see e.g. Bird R et al.,Science242: 423-6 (1988); Friedman P et al.,Cancer Res53: 334-9 (1993); Siegall C et al.,J Immunol152: 2377-84 (1994)). Flexible proteinaceous linkers may be chosen to increase the spatial separation between components and/or to allow for intramolecular interactions between components. For example, various “GS” linkers are known to the skilled worker and are composed of multiple glycines and/or one or more serines, sometimes in repeating units, such as, e.g., (GxS)n(SEQ ID NO: 1192), (SxG)n(SEQ ID NO: 1193), (GGGGS)n(SEQ ID NO: 1194), and (G)n(SEQ ID NO: 1195). in which x is 1 to 6 and n is 1 to 30 (see e.g. WO 96/06641). Non-limiting examples of flexible proteinaceous linkers include GKSSGSGSESKS (SEQ ID NO: 1196), GSTSGSGKSSEGKG (SEQ ID NO: 1197), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 1198), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1199), EGKSSGSGSESKEF (SEQ ID NO: 1200), SRSSG (SEQ ID NO: 1201), and SGSSC (SEQ ID NO: 1202). Rigid proteinaceous linkers are often stiff alpha-helical structures and rich in proline residues and/or one or more strategically placed prolines (see Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). Rigid linkers may be chosen to prevent intramolecular interactions between linked components. Suitable linkers may be chosen to allow for in vivo separation of components, such as, e.g., due to cleavage and/or environment-specific instability (see Dosio F et al.,Toxins3: 848-83 (2011); Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). The skilled worker knows how to make and use linkers designed to be stable, particularly in extracellular environments like in the blood plasma of a chordate's circulatory system, but to be cleaved in certain intracellular environments having unique characteristics, such as, e.g., in certain cell-types and/or intracellular compartments due to the presence of certain proteolytic activities, redox environments, and/or pH environments, thereby separating linked components of the molecule of the present invention. In vivo cleavable proteinaceous linkers are capable of unlinking by proteolytic processing and/or reducing environments often at a specific site within an organism or inside a certain cell type (see e.g. Doronina S et al.,Bioconjug Chem17: 144-24 (2006); Erickson H et al.,Cancer Res66: 4426-33 (2006)). In vivo cleavable proteinaceous linkers often comprise protease sensitive motifs and/or disulfide bonds formed by one or more cysteine pairs (see e.g. Pietersz G et al.,Cancer Res48: 4469-76 (1998); The J et al.,J Immunol Methods110: 101-9 (1998); see Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). In vivo cleavable proteinaceous linkers may be designed to be sensitive to proteases that exist only at certain locations in an organism, compartments within a cell, and/or become active only under certain physiological or pathological conditions (such as, e.g., proteases with abnormally high levels, proteases overexpressed at certain disease sites, and proteases specifically expressed by a pathogenic microorganism). For example, there are proteinaceous linkers known in the art which are cleaved by proteases present only intracellularly, proteases present only within specific cell types, and proteases present only under pathological conditions like cancer or inflammation, such as, e.g., R-x-x-R motif and AMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:1203). In certain embodiments of the cell-targeting molecules of the present invention, a linker may be used which comprises one or more protease sensitive sites to provide for cleavage by a protease present within a target cell. In certain embodiments of the cell-targeting molecules of the invention, a linker may be used which is not cleavable to reduce unwanted toxicity after administration to a vertebrate organism (see e.g. Polson et al.,Cancer Res69: 2358-(2009)). Suitable linkers may include, e.g., protease sensitive, environmental redox potential sensitive, pH sensitive, acid cleavable, photocleavable, and/or heat sensitive linkers, whether proteinaceous or non-proteinaceous (see Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013)). Suitable cleavable linkers may include linkers comprising cleavable groups which are known in the art such as, e.g., linkers noted by Zarling D et al.,J Immunol124: 913-20 (1980); Jung S, Moroi M,Biochem Biophys Acta761: 152-62 (1983); Bouizar Z et al.,Eur J Biochem155: 141-7 (1986); Park L et al.,J Biol Chem261: 205-10 (1986); Browning J, Ribolini A,J Immunol143: 1859-67 (1989); Joshi S, Burrows R,J Biol Chem265: 14518-25 (1990); Choi W et al.,J Bioact Compat Polym14: 447-56 (1999); Jensen K et al.,Bioconjug Chem13: 975-84 (2002); Christie R, Grainger, D,Adv Drug Deliv Rev55: 421-37 (2003)). Suitable linkers may include pH sensitive linkers. For example, certain suitable linkers may be chosen for their instability in lower pH environments to provide for dissociation inside a subcellular compartment of a target cell. For example, linkers that comprise one or more trityl groups, derivatized trityl groups, bismaleimideothoxy propane groups, adipic acid dihydrazide groups, and/or acid labile transferrin groups, may provide for release of components of the cell-targeting molecules of the invention, e.g. a polypeptide component, in environments with specific pH ranges (see e.g. Welhoner H et al.,J Biol Chem266: 4309-14 (1991); Fattom A et al.,Infect Immun60: 584-9 (1992)). Certain linkers may be chosen which are cleaved in pH ranges corresponding to physiological pH differences between tissues, such as, e.g., the pH of tumor tissue is lower than in healthy tissues (see e.g. U.S. Pat. No. 5,612,474). Photocleavable linkers are linkers that are cleaved upon exposure to electromagnetic radiation of certain wavelength ranges, such as light in the visible range (see e.g. Goldmacher V et al.,Bioconj Chem3: 104-7 (1992)). Photocleavable linkers may be used to release a component of a cell-targeting molecule of the invention, e.g. a polypeptide component, upon exposure to light of certain wavelengths. Non-limiting examples of photocleavable linkers include a nitrobenzyl group as a photocleavable protective group for cysteine, nitrobenzyloxycarbonyl chloride cross-linkers, hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer, and methylrhodamine copolymer (Hazum E et al.,Pept Proc Eur Pept Symp,16th,Brunfeldt K, ed.,105-110 (1981); Senter et al.,Photochem Photobiol42: 231-7 (1985); Yen et al.,Makromol Chem190: 69-82 (1989); Goldmacher V et al.,Bioconj Chem3: 104-7 (1992)). Photocleavable linkers may have particular uses in linking components to form cell-targeting molecules of the invention designed for treating diseases, disorders, and conditions that can be exposed to light using fiber optics. In certain embodiments of the cell-targeting molecules of the present invention, a cell-targeting binding region is linked to a toxin effector polypeptide using any number of means known to the skilled worker, including both covalent and noncovalent linkages (see e.g. Chen X et al.,Adv Drug Deliv Rev65: 1357-69 (2013); Behrens C, Liu B,MAbs6: 46-53 (2014)). In certain embodiments of the cell-targeting molecules of the present invention, the protein comprises a binding region which is a scFv with a linker connecting a heavy chain variable (VH) domain and a light chain variable (VL) domain. There are numerous linkers known in the art suitable for this purpose, such as, e.g., the 15-residue (Gly4Ser)3peptide (SEQ ID NO:1204). Suitable scFv linkers which may be used in forming non-covalent multivalent structures include GGS, GGGS (Gly3Ser or G3S) (SEQ ID NO:1205), GGGGS (Gly4Ser or G4S) (SEQ ID NO:1206), GGGGSGGG (SEQ ID NO:1207), GGSGGGG (SEQ ID NO:1208), GSTSGGGSGGGSGGGGSS (SEQ ID NO:1209), and GSTSGSGKPGSSEGSTKG (SEQ ID NO:1210) (Plückthun A, Pack P,Immunotechnology3: 83-105 (1997); Atwell J et al.,Protein Eng12: 597-604 (1999); Wu A et al.,Protein Eng14: 1025-33 (2001); Yazaki P et al., J Immunol Methods 253: 195-208 (2001); Carmichael J et al.,J Mol Biol326: 341-51 (2003); Arndt M et al.,FEBS Lett578: 257-61 (2004); Bie C et al.,World J Hepatol2: 185-91 (2010)). In certain embodiments of the cell-targeting molecule of the present invention, the molecule comprises a linker between the Shiga toxin effector polypeptide and the binding region wherein the linker has one or more cysteine residues. In certain further embodiments, there is a molecule covalently linked to the cysteine residue via its sulfhydryl functional group. In certain embodiments of the cell-targeting molecules of the present invention, the cell-targeting molecule comprises the linker comprising or consisting essentially of any one of SEQ ID NOs: 757-761 and 768-772. In certain embodiments of the cell-targeting molecule of the present invention, the molecule comprises the linker comprising the polypeptide GGGC (SEQ ID NO:1211), such as, e.g., the linker comprising or consisting essentially of GGGGCGG (SEQ ID NO:1212), GGGGSGGGGCGG (SEQ ID NO:1213), GGGGCGGGGSGG (SEQ ID NO:1214), GGGGSGGGGSGGGGC (SEQ ID NO:1215), GGGGSGGGGCGGGGS (SEQ ID NO:1216), GGGGCGGGGCSGGGS (SEQ ID NO:1217), GGGGSGGGGCGGGGSSGGGGSSGGGGS (SEQ ID NO:1218), GGGGCGGGGSGGGGSSGGGGSSGGGGS (SEQ ID NO:1219), GGGGSGGGGSGGGGCSGGGGSSGGGGS (SEQ ID NO:1220), GGGGSGGGGSGGGGSCGGGGSSGGGGS (SEQ ID NO:1221), GGGGSGGGGSGGGGSSGGGGCSGGGGS (SEQ ID NO:1222), GGGGSGGGGSGGGGSSGGGGSCGGGGS (SEQ ID NO:1223), or GGGGSGGGGSGGGGSGGGGSSGGGGC (SEQ ID NO:1224). In certain embodiments of the cell-targeting molecule of the present invention, the molecule comprises the linker comprising the polypeptide GGGK (SEQ ID NO:1225), such as, e.g., the linker comprising or consisting essentially of GGGGKGG (SEQ ID NO:1226), GGGGSGGGGKGG (SEQ ID NO:1227), GGGGKGGGGSGG (SEQ ID NO:1228), GGGGSGGGGSGGGGK (SEQ ID NO:1229), GGGGSGGGGKGGGGS (SEQ ID NO:1230), GGGGKGGGGCSGGGS (SEQ ID NO:1231), GGGGCGGGGKSGGGS (SEQ ID NO:1232), GGGGSGGGGKGGGGSSGGGGSSGGGGS (SEQ ID NO:1233), GGGGKGGGGSGGGGSSGGGGSSGGGGS (SEQ ID NO:1234), GGGGSGGGGSGGGGKSGGGGSSGGGGS (SEQ ID NO:1235), GGGGSGGGGSGGGGSKGGGGSSGGGGS (SEQ ID NO:1236), GGGGSGGGGSGGGGSSGGGGKSGGGGS (SEQ ID NO:1237), GGGGSGGGGSGGGGSSGGGGSKGGGGS (SEQ ID NO:1238), or GGGGSGGGGSGGGGSGGGGSSGGGGK (SEQ ID NO:1239). Suitable methods for linkage of the components of the cell-targeting molecules of the present invention may be by any method presently known in the art for accomplishing such, as long as the attachment does not substantially impede the binding capability of the cell-targeting agent or binding region, the cellular internalization of the cell-targeting molecule, the intracellular delivery of a cargo to a subcellular compartment or specific location, and/or the subcellular routing of the toxin effector polypeptide, each of which can be determined by an appropriate assay, including by assays described herein. F. Cargos, Heterologous Matter, Conjugated Moieties, Cell-Targeting Molecule Altering Agents, and Additional Exogenous Materials In certain embodiments, a molecule of the present invention comprises matter heterologous to Shiga toxins, such as, e.g., a cargo, conjugated molecule, additional exogenous material, and/or cell-targeting molecule altering agent. A molecule conjugated to a cell-targeting molecule represents a conjugated moiety. Such a heterologous matter (e.g. an atom or molecule) linked to a Shiga toxin effector polypeptide may be a matter which is foreign to the target cell and/or is not present in intended target cells in desirable amounts. In certain embodiments, the conjugated matter is an atomic or molecular cargo, heterologous molecule, additional exogenous material, and/or cell-targeting molecule altering agent, such as, e.g., a CD8+ T-cell epitope and/or antigen, radionucleide, peptide, detection-promoting agent, protein, small molecule chemotherapeutic agent, and/or polynucleotide. In certain embodiments, the conjugated matter is or comprises an atom, such as a radionucleide. In certain embodiments, the radionucleide is211At,131I,125I,90Y,111In,186Re,188Re,153Sm,212Bi,32P,60C, and/or a radioactive isotope of lutetium. In certain embodiments, the conjugated matter is a lipid, serum albumin binding molecule, antibiotic, cytotoxic agent, detection-promoting agent, peptide, protein, enzyme, nucleic acid, and/or protein-nucleic acid complex (see e.g. Liu B,Brief Funct Genomic Proteomic6: 112-9 (2007); Endoh T, Ohtsuki T,Adv Drug Deliv Rev61: 704-9 (2009)). In certain embodiments, the cell-targeting molecules of the present invention comprise a conjugated matter which is a molecule altering agent meant to function prior to or during target cell internalization of the cell-targeting molecule. In certain other embodiments, the cell-targeting molecules of the present invention comprise a conjugated matter which is a cargo (e.g. an additional exogenous material) meant to function after target cell internalization of the cell-targeting molecule. In certain embodiments, the cell-targeting molecules of the present invention comprise a conjugated matter which is an additional exogenous material or cargo, such as, e.g., a polypeptide comprising a pro-apoptotic effector like, e.g., fragments of caspase-3, caspase-6, granzyme B, tBid, and apoptosis inducing factor (AIF) (see e.g., Jia L et al.,Cancer Res63: 3257-62 (2003); Xu Y et al.J Immunol173: 61-7 (2004); Wang T et al.,Cancer Res67: 11830-9 (2007); Shan L et al.,Cancer Biol Ther11: 1717-22 (2008); Qiu X et al.,Mol Cancer Ther7: 1890-9 (2008)). In certain embodiments, the conjugated matter is a peptide comprising or consisting essentially of a CD8+ T-cell epitope and/or antigen. Non-limiting examples of cell-targeting molecule altering agents include various polyethylene glycol molecules, lipids, and liposomes because these agents can alter, e.g., the immunogenicity and/or pharmacokinetics of the cell-targeting molecule. In certain embodiments, the conjugated matter is a non-proteinaceous polymer, e.g., a polyethylene glycol, polypropylene glycol, or polyoxyalkylene (see e.g., U.S. Pat. Nos. 4,179,337; 4,301,144; 4,640,835; 4,670,417; 4,791,192; and 4,496,689). Any one of these can function as a cell-targeting molecule altering agent. In certain embodiments, the cell-targeting molecule of the present invention comprises a cargo molecule covalently conjugated to an amino acid residue in a proteinaceous component of the cell-targeting molecule. In certain further embodiments, the cell-targeting molecule of the present invention comprises a cargo molecule covalently conjugated to a cysteine, lysine, or histidine residue in the cell-targeting molecule. In certain further embodiments, the cell-targeting molecule of the present invention comprises a cargo molecule covalently conjugated to a Shiga toxin effector polypeptide component of the cell-targeting molecule via a cysteine, lysine, or histidine residue in the Shiga toxin effector polypeptide. In certain further embodiments, the cargo is covalently linked to the cysteine residue via its sulfhydryl functional group. In certain further embodiments, the cargo molecule is released from the cell-targeting molecule upon reaching the endoplasmic reticulum or another compartment having a reducing environment due to the reduction of a disulfide bond between the cargo and the Shiga toxin effector polypeptide (see e.g. El Alaoui A et al.,Angew Chem Int Ed Engl46: 6469-72 (2007)). In certain embodiments, the cell-targeting molecule of the present invention comprises a nucleic acid cargo conjugated to a proteinaceous component of the cell-targeting molecule via a disulfide bond (see e.g. Muratovska A, Eccles M,FEBS Lett558: 63-8 (2004); Ishihara T et al.,Drug Deliv16: 153-9 (2009)). In certain embodiments, the cargo is a protein or polypeptide comprising an enzyme. In certain other embodiments, the cargo is a nucleic acid, such as, e.g. a ribonucleic acid that functions as a small inhibiting RNA (siRNA) or microRNA (miRNA). In certain embodiments, the cargo is an antigen, such as antigens derived from pathogens, bacterial proteins, viral proteins, proteins mutated in cancer, proteins aberrantly expressed in cancer, or T-cell complementary determining regions. For example, cargos may include antigens, such as those antigens presented by antigen-presenting cells infected by bacteria, and T-cell complementary determining regions capable of functioning as exogenous antigens. Cargo molecules comprising polypeptides or proteins may optionally comprise one or more antigens whether known or unknown to the skilled worker. In certain embodiments of the cell-targeting molecules of the present invention, all heterologous antigens and/or epitopes associated with the Shiga toxin effector polypeptide are arranged in the cell-targeting molecule amino-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region of the Shiga toxin effector polypeptide. In certain further embodiments, all heterologous antigens and/or epitopes associated with the Shiga toxin effector polypeptide are associated, either directly or indirectly, with the Shiga toxin effector polypeptide at a position amino-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region of the Shiga toxin effector polypeptide. In certain further embodiments, all additional exogenous material(s) which is an antigen is arranged amino-terminal to the Shiga toxin effector polypeptide, such as, e.g., fused directly or indirectly to the amino terminus of the Shiga toxin effector polypeptide. In certain embodiments, the cell-targeting molecule of the present invention comprises a protein-nucleic acid complex cargo conjugated to a proteinaceous component of the cell-targeting molecule via a disulfide bond. In certain embodiments, the cargo and/or detection-promoting agent is a fluorophore, such as, e.g., a maleimide derivative of an Alexa Fluor® fluorophore for conjugating to a thiol functional group of an amino acid residue of a molecule of the present invention. In certain embodiments, the cell-targeting molecule of the present invention comprises a zymoxin, which is an inactive viral enzyme that may be activated by proteolytic cleavage (see e.g. Shapira A et al.,PLoS One6: e15916 (2011)). In certain embodiments, the cargo is a proapoptotic peptide, polypeptide, or protein, such as, e.g., BCL-2, caspases (e.g. fragments of caspase-3 or caspase-6), cytochromes, granzyme B, apoptosis-inducing factor (AIF), BAX, tBid (truncated Bid), and proapoptotic fragments or derivatives thereof (see e.g., Ellerby H et al.,Nat Med5: 1032-8 (1999); Mai J et al.,Cancer Res61: 7709-12 (2001); Jia L et al.,Cancer Res63: 3257-62 (2003); Liu Y et al.,Mol Cancer Ther2: 1341-50 (2003); Perea S et al.,Cancer Res64: 7127-9 (2004); Xu Y et al.,J Immunol173: 61 7 (2004); Dalken B et al.,Cell Death Differ13: 576-85 (2006); Wang T et al.,Cancer Res67: 11830-9 (2007); Kwon M et al.,Mol Cancer Ther7: 1514-22 (2008); Qiu X et al.,Mol Cancer Ther7: 1890-9 (2008); Shan L et al.,Cancer Biol Ther11: 1717-22 (2008); Wang F et al.,Clin Cancer Res16: 2284-94 (2010); Kim J et al.,J Virol85: 1507-16 (2011)). In certain embodiments, the cargo is a cytotoxic agent, such as, e.g., a small molecule chemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor. Non-limiting examples of cytotoxic agents suitable for use with the present invention include aziridines, cisplatins, tetrazines, procarbazine, hexamethylmelamine, vinca alkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, amanitin, amatoxins, bleomycin, centanamycin (indolecarboxamide), plicamycin, mitomycin, daunorubicin, epirubicin, idarubicins, dolastatins, maytansines, maytansionoids, duromycin, docetaxel, duocarmycins, adriamycin, calicheamicin, auristatins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers (PBDs), carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, gemcitabine, aceglatone, acetogenins (e.g. bullatacin and bullatacinone), aclacinomysins, AG1478, AG1571, aldophosphamide glycoside, alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan), alkylating agents (e.g. thiotepa and cyclosphosphamide), aminolevulinic acid, aminopterin, amsacrine, ancitabine, anthramycin, arabinoside, azacitidine, azaserine, aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa), azauridine, bestrabucil, bisantrene, bisphosphonates (e.g. clodronate), bleomycins, bortezomib, bryostatin, cactinomycin, callystatin, carabicin, carminomycin, carmofur, carmustine, carzinophilin, CC-1065, chlorambucil, chloranbucil, chlornaphazine, chlorozotocin, chromomycinis, chromoprotein enediyne antibiotic chromophores, CPT-11, cryptophycins (e.g. cryptophycin 1 and cryptophycin 8), cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunomycin, defofamine, demecolcine, detorubicin, diaziquone, 6-diazo-5-oxo-L-norleucine, dideoxyuridine, difluoromethylornithine (DMFO), doxifluridine, doxorubicins (e.g., morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), dynemicins, edatraxate, edatrexate, eleutherobins, elformithine, elliptinium acetate, enediyne antibiotics (e.g. calicheamicins), eniluracil, enocitabine, epirubicins, epothilone, esorubicins, esperamicins, estramustine, ethylenimines, 2-ethylhydrazide, etoglucid, fludarabine, folic acid analogues (e.g., denopterin, methotrexate, pteropterin, and trimetrexate), folic acid replenishers (e.g. frolinic acid), fotemustine, fulvestrant, gacytosine, gallium nitrate, gefitinib, gemcitabine, hydroxyurea, ibandronate, ifosfamide, imatinib mesylate, erlotinib, fulvestrant, letrozole, PTK787/ZK 222584 (Novartis, Basel, CH), oxaliplatin, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, methylamelamines (e.g., altretamine, triethy lenemelamine, triethy lenephosphoramide, triethylenethiophosphoramide and trimethylomelamine), pancratistatins, sarcodictyins, spongistatins, nitrogen mustards (e.g., chlorambucil, chlornaphazine, cyclophosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard), nitrosureas (e.g., carmustine, fotemustine, lomustine, nimustine, and ranimnustine), dynemicins, neocarzinostatin chromophores, anthramycin, detorubicin, epirubicins, marcellomycins, mitomycins (e.g. mitomycin C), mycophenolic acid, nogalamycins, olivomycins, peplomycins, potfiromycins, puromycins, quelamycins, rodorubicins, ubenimex, zinostatins, zorubicins, purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine), aceglatone, lentinan, lonidainine, maytansinoids (e.g. maytansins and ansamitocins), mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″trichlorotriethylamine, trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine), urethan, vindesine, mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside, cyclophosphamide, toxoids (e.g. paclitaxel and doxetaxel), 6-thioguanine, mercaptopurine, platinum, platinum analogs (e.g. cisplatin and carboplatin), etoposide (VP-16), mitoxantrone, vinorelbine, novantrone, daunomycin, xeloda, topoisomerase inhibitor RFS 2000, retinoids (e.g. retinoic acid), capecitabine, lomustine, losoxantrone, mercaptopurines, nimustine, nitraerine, rapamycin, razoxane, roridin A, spongistatins, streptonigrins, streptozocins, sutent, T-2 toxin, thiamiprine, thiotepa, toxoids (e.g. paclitaxel and doxetaxel), tubercidins, verracurin A, vinblastine, vincristine, and structural analogs of any of the aforementioned (e.g. synthetic analogs), and/or derivatives of any of the aforementioned (see e.g., Lindell T et al.,Science170: 447-9 (1970); Remillard S et al.,Science189: 1002-5 (1975); Ravry M et al.,Am J Clin Oncol8: 148-50 (1985); Ravry M et al.,Cancer Treat Rep69: 1457-8 (1985); Sternberg C et al.,Cancer64: 2448-58 (1989); Bai R et al.,Biochem Pharmacol39: 1941-9 (1990); Boger D, Johnson D,Proc Natl Acad Sci USA92: 3642-9 (1995); Beck J et al.,Leuk Lymphoma41: 117-24 (2001); Cassady J et al.,Chem Pharm Bull(Tokyo) 52: 1-26 (2004); Sapra P et al.,Clin Cancer Res11: 5257-64 (2005); Okeley N et al.,Clinc Cancer Res16: 888-97 (2010); Oroudjev E et al.,Mol Cancer Ther9: 2700-13 (2010); Ellestad G,Chirality23: 660-71 (2011); Kantarjian H et al.,Lancet Oncol13: 403-11 (2012); Moldenhauer G et al.,J Natl Cancer Inst104: 622-34 (2012); Gromek S, Balunas M,Curr Top Med Chem14: 2822-34 (2015); Meulendijks D et al.,Invest New Drugs34: 119-28 (2016)). In certain embodiments of the cell-targeting molecules of the present invention, the additional exogenous material is a polypeptide characterized as highly efficient in delivering various molecules into cells, such polypeptides also known as “cell penetrating peptides” (CPPs) or “protein transduction domains” (PTDs) (see Futaki S et al.,Biochemistry41: 7925-30 (2002); Wender P et al.,J Am Chem Soc124: 13382-3 (2002); Dietz G, Bahr M,Mol Cell Neurosci27: 85-131 (2004); Vives E,J Control Release109: 77-85 (2005); Vives E et al.,Biochim Biophys Acta17866: 126-38 (2008); van den Berg A, Dowdy S,Curr Opin Biotechnol22: 888-93 (2011); Copolovici D et al.,ACS Nano8: 1972-94 (2014); Kauffman W et al.,Trends Biochem Sci40: 749-64 (2015); WO 2003106491; U.S. Pat. Nos. 7,579,318; 7,943,581; 8,242,081). In certain embodiments, the additional exogenous material comprises both a CPP and/or PTD and a nucleic acid and optionally a cationic peptide (see e.g. Lehto T et al.,Expert Opin Drug Deliv9: 823-36 (2012); Shukla R et al.,Mol Pharm11: 3395-408 (2014); Beloor J et al.,Ther Deliv6: 491-507 (2015); Chuah J et al.,Biomacromolecules10.1021/acs.biomac.6b01056 (2016); Cerrato C et al.,Expert Opin Drug Deliv1-11 (2016); Tai W, Gao X,Adv Drug Deliv Revpii: 50169-409X: 30236-8 (2016); Wada S et al.,Bioorg Med Chem15: 4478-85 (2016)). In certain embodiments, the additional exogenous material comprises a CPP and/or PTD for intracellular targeting of another additional exogenous material (see e.g. Sakhrani N, Padh H,Drug Des Devel Ther7: 585-99 (2013); Li H et al.,Int J Mol Sci16: 19518-36 (2015); Cerrato C et al.,Expert Opin Drug Deliv1-11 (2016)). II. Examples of Structural Variants of the Cell-Targeting Molecules of the Present Invention In certain embodiments of the cell-targeting molecules of the present invention, many of the molecule's components have already been described, such as the binding region, linker, and/or toxin effector polypeptide (see e.g. WO 2005/092917, WO 2007/033497, US2009/0156417, JP4339511, EP1727827, DE602004027168, EP1945660, JP4934761, EP2228383, US2013/0196928, WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, US20150259428, WO 2015/191764, WO 2016/126950). The skilled worker will recognize that variations may be made to the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention, and polynucleotides encoding any of the former, without diminishing their biological activities, e.g., by maintaining the overall structure and function of the Shiga toxin effector polypeptide, such as in conjunction with one or more 1) endogenous epitope disruptions which reduce antigenic and/or immunogenic potential, 2) furin-cleavage motif disruptions which reduce proteolytic cleavage, and/or 3) embedded or inserted epitopes which reduce antigenic and/or immunogenic potential and/or are capable of being delivered to a MHC I molecule for presentation on a cell surface. For example, some modifications may facilitate expression, facilitate purification, improve pharmacokinetic properties, and/or improve immunogenicity. Such modifications are well known to the skilled worker and include, for example, a methionine added at the amino-terminus to provide an initiation site, additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons, and biochemical affinity tags fused to either terminus to provide for convenient detection and/or purification. A common modification to improve the immunogenicity of a polypeptide produced using a non-chordate system (e.g. a prokaryotic cell) is to remove, after the production of the polypeptide, the starting methionine residue, which may be formylated during production, such as, e.g., in a bacterial host system, because, e.g., the presence of N-formylmethionine (fMet) might induce undesirable immune responses in chordates. Also contemplated herein is the inclusion of additional amino acid residues at the amino and/or carboxy termini of a Shiga toxin effector polypeptide of the present invention, a cell-targeting molecule of the present invention, or a proteinaceous component of a cell-targeting molecules of the present invention, such as sequences for epitope tags or other moieties. The additional amino acid residues may be used for various purposes including, e.g., facilitating cloning, facilitating expression, post-translational modification, facilitating synthesis, purification, facilitating detection, and administration. Non-limiting examples of epitope tags and moieties are chitin binding protein domains, enteropeptidase cleavage sites, Factor Xa cleavage sites, FIAsH tags, FLAG tags, green fluorescent proteins (GFP), glutathione-S-transferase moieties, HA tags, maltose binding protein domains, myc tags, polyhistidine tags, ReAsH tags, strep-tags, strep-tag II, TEV protease sites, thioredoxin domains, thrombin cleavage site, and V5 epitope tags. In certain of the above embodiments, the polypeptide sequence of the Shiga toxin effector polypeptides and/or cell-targeting molecules of the present invention are varied by one or more conservative amino acid substitutions introduced into the polypeptide region(s) as long as all required structural features are still present and the Shiga toxin effector polypeptide is capable of exhibiting any required function(s), either alone or as a component of a cell-targeting molecule. As used herein, the term “conservative substitution” denotes that one or more amino acids are replaced by another, biologically similar amino acid residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids (see, for example, Table B). An example of a conservative substitution with a residue normally not found in endogenous, mammalian peptides and proteins is the conservative substitution of an arginine or lysine residue with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. For further information concerning phenotypically silent substitutions in peptides and proteins see, e.g., Bowie J et al.,Science247: 1306-10 (1990). TABLE BExamples of Conservative Amino AcidSubstitutionsIIIIIIIVVVIVIIVIIIIXXXIXIIXIIIXIVADHCFNACFACAADGEKIWQGMHCDCCEPQRLYSIPWFEDDGSNMTLYGHGEKTVVHKNGPINPHQLQSKRMRTNSRSVQTTTRVSWPYT In the conservative substitution scheme in Table B, exemplary conservative substitutions of amino acids are grouped by physicochemical properties—I: neutral, hydrophilic; II: acids and amides; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilic uncharged, VII aliphatic uncharged, VIII non-polar uncharged, IX cycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIII turn-permitting, and XIV flexible. For example, conservative amino acid substitutions include the following: 1) S may be substituted for C; 2) M or L may be substituted for F; 3) Y may be substituted for M; 4) Q or E may be substituted for K; 5) N or Q may be substituted for H; and 6) H may be substituted for N. Additional conservative amino acid substitutions include the following: 1) S may be substituted for C; 2) M or L may be substituted for F; 3) Y may be substituted for M; 4) Q or E may be substituted for K; 5) N or Q may be substituted for H; and 6) H may be substituted for N. In certain embodiments, the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention may comprise functional fragments or variants of a polypeptide region of the present invention described herein that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a polypeptide sequence recited herein, as long as it (1) comprises at least one embedded or inserted, heterologous T-cell epitope and at least one amino acid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12), wherein the disrupted amino acid does not overlap with the embedded or inserted epitope; (2) comprises at least one embedded or inserted, heterologous T-cell epitope and a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region; or (3) comprises a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region and comprises at least one amino acid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12), wherein the disrupted amino acid does not overlap with the disrupted furin-cleavage motif. Variants of the Shiga toxin effector polypeptides and cell-targeting molecules of the invention are within the scope of the present invention as a result of changing a polypeptide described herein by altering one or more amino acid residues or deleting or inserting one or more amino acid residues, such as within the binding region or Shiga toxin effector polypeptide region, in order to achieve desired properties, such as changed cytotoxicity, changed cytostatic effects, changed immunogenicity, and/or changed serum half-life. The Shiga toxin effector polypeptides and cell-targeting molecules of the present invention may further be with or without a signal sequence. Accordingly, in certain embodiments, the Shiga toxin effector polypeptides of the present invention comprise or consists essentially of amino acid sequences having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, overall sequence identity to a naturally occurring Shiga toxin A Subunit or fragment thereof, such as, e.g., Shiga toxin A Subunit, such as SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3), wherein the Shiga toxin effector polypeptide (1) comprises at least one embedded or inserted, heterologous T-cell epitope and at least one amino acid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12), and wherein the disrupted amino acid does not overlap with the embedded or inserted epitope; (2) comprises at least one embedded or inserted, heterologous T-cell epitope and a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region; or (3) comprises a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region and comprises at least one amino acid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12), and wherein the disrupted amino acid does not overlap with the disrupted furin-cleavage motif. In certain embodiments of the Shiga toxin effector polypeptides of the present invention, one or more amino acid residues may be mutated, inserted, or deleted in order to increase the enzymatic activity of the Shiga toxin effector polypeptide. In certain embodiments of the Shiga toxin effector polypeptides of the present invention, one or more amino acid residues may be mutated or deleted in order to reduce or eliminate catalytic and/or cytotoxic activity of the Shiga toxin effector polypeptide. For example, the catalytic and/or cytotoxic activity of the A Subunits of members of the Shiga toxin family may be diminished or eliminated by mutation or truncation. The cytotoxicity of the A Subunits of members of the Shiga toxin family may be altered, reduced, or eliminated by mutation and/or truncation. The positions labeled tyrosine-77, glutamate-167, arginine-170, tyrosine-114, and tryptophan-203 have been shown to be important for the catalytic activity of Stx, Stx1, and Stx2 (Hovde C et al.,Proc Natl Acad Sci USA85: 2568-72 (1988); Deresiewicz R et al.,Biochemistry31: 3272-80 (1992); Deresiewicz R et al.,Mol Gen Genet241: 467-73 (1993); Ohmura M et al.,Microb Pathog15: 169-76 (1993); Cao C et al.,Microbiol Immunol38: 441-7 (1994); Suhan M, Hovde C,Infect Immun66: 5252-9 (1998)). Mutating both glutamate-167 and arginine-170 eliminated the enzymatic activity of Slt-I A1 in a cell-free ribosome inactivation assay (LaPointe P et al.,J Biol Chem280: 23310-18 (2005)). In another approach using de novo expression of Slt-I A1 in the endoplasmic reticulum, mutating both glutamate-167 and arginine-170 eliminated Slt-I A1 fragment cytotoxicity at that expression level (LaPointe P et al.,J Biol Chem280: 23310-18 (2005)). A truncation analysis demonstrated that a fragment of StxA from residues 75 to 268 still retains significant enzymatic activity in vitro (Haddad J et al.,J Bacteriol175: 4970-8 (1993)). A truncated fragment of Slt-I A1 containing residues 1-239 displayed significant enzymatic activity in vitro and cytotoxicity by de novo expression in the cytosol (LaPointe P et al.,J Biol Chem280: 23310-18 (2005)). Expression of a Slt-I A1 fragment truncated to residues 1-239 in the endoplasmic reticulum was not cytotoxic because it could not retrotranslocate to the cytosol (LaPointe P et al.,J Biol Chem280: 23310-18 (2005)). The most critical residues for enzymatic activity and/or cytotoxicity in the Shiga toxin A Subunits were mapped to the following residue-positions: asparagine-75, tyrosine-77, tyrosine-114, glutamate-167, arginine-170, arginine-176, and tryptophan-203 among others (Di R et al.,Toxicon57: 525-39 (2011)). In particular, a double-mutant construct of Stx2A containing glutamate-E167-to-lysine and arginine-176-to-lysine mutations was completely inactivated; whereas, many single mutations in Stx1 and Stx2 showed a 10-fold reduction in cytotoxicity. Further, truncation of Stx1A to 1-239 or 1-240 reduced its cytotoxicity, and similarly, truncation of Stx2A to a conserved hydrophobic residue reduced its cytotoxicity. The most critical residues for binding eukaryotic ribosomes and/or eukaryotic ribosome inhibition in the Shiga toxin A Subunit have been mapped to the following residue-positions arginine-172, arginine-176, arginine-179, arginine-188, tyrosine-189, valine-191, and leucine-233 among others (McCluskey A et al.,PLoS One7: e31191 (2012). However, certain modification may increase a Shiga toxin functional activity exhibited by a Shiga toxin effector polypeptide of the present invention. For example, mutating residue-position alanine-231 in Stx1A to glutamate increased Stx1A's enzymatic activity in vitro (Suhan M, Hovde C,Infect Immun66: 5252-9 (1998)). In certain embodiments of Shiga toxin effector polypeptides of the present invention derived from SLT-1A (SEQ ID NO:1) or StxA (SEQ ID NO:2), the one or more amino acid residues mutated include substitution of the asparagine at position 75, tyrosine at position 77, tyrosine at position 114, glutamate at position 167, arginine at position 170, arginine at position 176, and/or substitution of the tryptophan at position 203. Examples of such substitutions will be known to the skilled worker based on the prior art, such as asparagine at position 75 to alanine; tyrosine at position 77 to serine; substitution of the tyrosine at position 114 to serine; substitution of the glutamate position 167 to glutamate, glutamine, or lysine; substitution of the arginine at position 170 to alanine, glycine, or lysine; substitution of the arginine at position 176 to lysine; substitution of the tryptophan at position 203 to alanine; and/or substitution of the alanine at 231 with glutamate. Other mutations which either enhance or reduce Shiga toxin enzymatic activity and/or cytotoxicity are within the scope of the invention and may be determined using well-known techniques and assays disclosed herein. In certain embodiments of the Shiga toxin effector polypeptide scaffolds of the present invention, the Shiga toxin effector polypeptide component of the scaffold is selected from any one of SEQ ID NOs: 233-756. In certain further embodiments of the Shiga toxin effector polypeptide scaffolds of the present invention, the Shiga toxin effector polypeptide component of the scaffold is selected from any one of SEQ ID NOs: 233-756 and the Shiga toxin effector polypeptide further comprises a linker. In certain further embodiments of the Shiga toxin effector polypeptide scaffolds of the present invention, the Shiga toxin effector polypeptide component of the scaffold is selected from any one of SEQ ID NOs: 233-756 and the Shiga toxin effector polypeptide scaffold further comprises a linker selected from any one of SEQ ID NOs: 757-761. In certain further embodiments of the Shiga toxin effector polypeptide scaffolds of the present invention, the Shiga toxin effector polypeptide component of the scaffold is selected from any one of SEQ ID NOs: 233-756 and the Shiga toxin effector polypeptide scaffold further comprises a linker selected from any one of SEQ ID NOs: 757-761 as long as there is a single, unique cysteine or lysine residue present in the scaffold outside of the Shiga toxin effector polypeptide. In certain further embodiments of the Shiga toxin effector polypeptide scaffolds of the present invention, the Shiga toxin effector polypeptide scaffold comprises or consists essentially of any one of SEQ ID NOs: 762-767. In certain embodiments of the cell-targeting molecules of the present invention, the cell-targeting molecule comprises a Shiga toxin effector polypeptide component selected from any one of SEQ ID NOs: 5-756. In certain embodiments of the cell-targeting molecules of the present invention, the cell-targeting molecule comprises a Shiga toxin effector polypeptide scaffold of the present invention. In certain further embodiments, the Shiga toxin effector polypeptide scaffold comprises only one cysteine and/or lysine residue. In certain further embodiments, the Shiga toxin effector polypeptide scaffold is selected from any one of SEQ ID NOs: 762-767. In certain embodiments of the cell-targeting molecules of the present invention, the cell-targeting molecule comprises one or more of SEQ ID NOs: 757-761 and 768-772. Certain embodiments of the cell-targeting molecules of the present invention comprise or consist essentially of any one of SEQ ID NOs: 773-829. In certain embodiments of the cell-targeting molecules of the present invention, the cell-targeting molecule comprises a binding region comprising an immunoglobulin domain. In certain further embodiments of the cell-targeting molecule of the present invention, the binding region comprises a polypeptide(s) selected from the group consisting of: (a) a heavy chain variable (VH) domain comprising a HCDR1 comprising or consisting essentially of the amino acid sequences as shown in any one of SEQ ID NOs: 844, 850, 857, 863, 869, 875, 881, 885, 891, 897, 903, 909, 915, 921, 927, 933, 939, 948, 954, 960, 966, 972, 978, 984, 990, 996, 1002, 1008, 1014, 1020, 1026, 1032, 1035, 1041, 1044, 1050, 1056, 1062, 1065, 1071, 1077, 1083, 1089, and 1095, a HCDR2 comprising or consisting essentially of the amino acid sequences as shown in SEQ ID NOs: 845, 851, 856, 858, 864, 876, 886, 892, 898, 904, 910, 916, 922, 928, 934, 940, 949, 955, 961, 967, 973, 979, 985, 991, 997, 1003, 1009, 1015, 1021, 1027, 1036, 1042, 1045, 1051, 1057, 1063, 1066, 1072, 1078, 1084, 1090, and 1096, and a HCDR3 comprising or consisting essentially of the amino acid sequences as shown in any one of SEQ ID NOs: 846, 852, 859, 865, 870, 872, 877, 882, 887, 893, 899, 905, 911, 917, 923, 929, 935, 941, 950, 956, 962, 968, 974, 980, 982, 986, 992, 998, 1004, 1010, 1016, 1022, 1028, 1037, 1043, 1046, 1064, 1052, 1058, 1067, 1073, 1079, 1085, 1091, and 1097; and (b) a light chain variable (VL) domain comprising a LCDR1 comprising or consisting essentially of the amino acid sequences as shown in any one of SEQ ID NOs: 847, 853, 860, 866, 871, 888, 894, 900, 906, 912, 918, 924, 930, 936, 942, 947, 953, 959, 965, 971, 977, 983, 989, 995, 1001, 1007, 1013, 1019, 1025, 1032, 1038, 1047, 1053, 1059, 1068, 1074, 1080, 1086, 1092, and 1098, a LCDR2 comprising or consisting essentially of the amino acid sequences as shown in any one of SEQ ID NOs: 848, 854, 861, 867, 883, 889, 895, 901, 907, 913, 919, 925, 931, 937, 948, 954, 960, 966, 972, 978, 984, 990, 996, 1002, 1008, 1014, 1020, 1026, 1033, 1039, 1048, 1054, 1060, 1069, 1075, 1081, 1087, 1093, and 1099, and a LCDR3 comprising or consisting essentially of the amino acid sequences as shown in any one of SEQ ID NOs: 849, 855, 862, 868, 884, 890, 896, 902, 908, 914, 920, 926, 932, 938, 949, 955, 961, 967, 973, 979, 985, 991, 997, 1003, 1009, 1015, 1021, 1027, 1034, 1040, 1049, 1055, 1061, 1070, 1076, 1082, 1088, 1094, and 1100. The Shiga toxin effector polypeptides, the Shiga toxin effector polypeptide scaffolds, and cell-targeting molecules of the present invention may optionally be conjugated to one or more additional agents, which may include therapeutic agents, diagnostic agents, and/or other additional exogenous materials known in the art, including such agents as described herein. In certain embodiments, the Shiga toxin effector polypeptide, the Shiga toxin effector polypeptide scaffold or cell-targeting molecule of the present invention is PEGylated or albuminated, such as, e.g., to provide de-immunization, disrupt furin-cleavage by masking the extended loop and/or the furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region, improve pharmacokinetic properties, and/or improve immunogenicity (see e.g., Wang Q et al.,Cancer Res53: 4588-94 (1993); Tsutsumi Y et al.,Proc Natl Acad Sci USA97: 8548-53 (2000); Buse J, El-Aneed A,Nanomed5: 1237-60 (2010); Lim S et al.,J Control Release207-93 (2015)). III. General Functions of the Cell-Targeting Molecules of the Present Invention The molecules of the present invention are useful in a variety of applications involving cell-targeting molecules (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/113005, WO 2015/113007, WO 2015/120058, WO 2015/138435, WO 2015/138452, US 2015/0259428, WO 2015/191764, US2016/177284, WO 2016/126950, WO 2016/196344, and WO 2017/019623). The Shiga toxin effector polypeptides, binding region polypeptides, and linkers of the present invention may be used as components to create cell-targeting molecules with improved properties (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/113005, WO 2015/113007, WO 2015/120058, WO 2015/138435, WO 2015/138452, US 2015/0259428, WO 2015/191764, US2016/177284, WO 2016/126950, WO 2016/196344, and WO 2017/019623). The Shiga toxin effector polypeptides, binding region polypeptides, and linkers of the present invention are useful as components of various therapeutic and/or diagnostic molecules, such as, e.g. ligand-toxin fusions, immunotoxins, and/or immuno-conjugates (see e.g. WO 2015/113005, WO 2015/113007, WO 2015/138435, US 2015/0259428, WO 2015/191764, US2016/177284, WO 2016/126950, WO 2016/196344, and WO 2017/019623). The functional association of Shiga toxin effector polypeptides with cell-targeting binding regions enables the creation of cell-targeting molecules which selectively kill, inhibit the growth of, deliver exogenous material to, and/or detect specific cell types. For example, certain cell-targeting molecules of the present invention may be potently cytotoxic to target-expressing cells via their abilities to efficiently deliver into the interior of a target-expressing cell a catalytically active, Shiga toxin effector polypeptide(s) that is able to effectively route to the cytosol. The Shiga toxin effector polypeptides and cell-targeting molecules of the present invention are useful in diverse applications involving, e.g., cell-killing; cell growth inhibition; intracellular, cargo delivery; biological information gathering; immune response stimulation, and/or remediation of a health condition. The cell-targeting molecules of the present invention are useful as therapeutic and/or diagnostic molecules, such as, e.g., as cell-targeting, cytotoxic, therapeutic molecules; cell-targeting, nontoxic, delivery vehicles; and/or cell-targeting, diagnostic molecules; for examples in applications involving the in vivo targeting of specific cell types for the diagnosis or treatment of a variety of diseases, including cancers, immune disorders, and microbial infections. The Shiga toxin effector polypeptides of the present invention allow for controlled and site-specific conjugation of molecular cargos to form cell-targeting molecules capable of cell-targeted delivery of their cargo(s). For example, conjugation can be chemically limited to occur at a single amino acid residue or linker attached thereto in order to produce a homogeneous product with a defined conjugate stoichiometry using strategies such as engineered cysteine residues, unnatural amino acid residues, and/or enzymatic conjugation. Similarly, the cell-targeting molecules of the present invention allow for controlled and site-specific conjugation of heterologous molecules to form useful cell-targeting molecules conjugates, such as, e.g., conjugates involving therapeutic molecules, immunogenicity-reducing agents, half-life extending agents, and various other cargos. The cell-targeting molecules of the present invention, and compositions thereof, have uses, e.g., for the selective delivery of cargos to target-expressing cells and as therapeutics for the treatment of a variety of diseases, disorders, and conditions, which include genetic disorders, genetic predispositions, infections, cancers, tumors, growth abnormalities, and/or immune disorders. Certain cell-targeting molecules of the present invention, and compositions thereof, may be used to selectively deliver conjugated cargo(s) to a target-expressing cell type(s) in the presence of one or more other cell types based on its cell-targeting and cellular internalization activity(ies), such as, e.g., a cargo having a desired, intracellular function. In addition, certain cell-targeting molecules of the present invention, and compositions thereof, may be used to selectively kill a target-expressing cell in the presence of one or more other cell types based on its cell-targeting activity and cellular internalization activity(ies), such as, e.g., by delivering into the interior of the targeted, target-expressing cell a component of the cell-targeting molecule which is cytotoxic at an intracellular location. Depending on the embodiment, a Shiga toxin effector polypeptide or cell-targeting molecule of the present invention may have or provide one or more of the following characteristics or functionalities: (1) de-immunization (see e.g. WO 2015/113005; WO 2015/113007), (2) protease-cleavage resistance (see e.g. WO 2015/191764), (3) potent cytotoxicity at certain concentrations, (4) intracellular delivery of a cargo consisting of an additional material (e.g. a heterologous, T-cell epitope) (see e.g. WO 2015/113005), (4) selective cytotoxicity, (6) low off-target toxicity in multicellular organisms at certain doses or dosages (see e.g. WO 2015/191764), (7) delivery of a heterologous, T-cell epitope to the MHC class I presentation pathway of a target cell (see e.g. WO 2015/113005), and/or (8) stimulation of CD8+ T-cell immune response(s). Certain embodiments of the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention are multi-functional because the molecules have two or more of the characteristics or functionalities described herein. In certain embodiments, the cell-targeting molecules of the present invention are capable of binding extracellular target biomolecules associated with the cell surface of particular cell types and entering those cells. Once internalized within a targeted cell type, certain embodiments of the cell-targeting molecules of the invention are capable of routing an enzymatically active, cytotoxic, Shiga toxin effector polypeptide fragment into the cytosol of the target cell and eventually killing the cell. Alternatively, nontoxic or reduced-toxicity variants of the cell-targeting molecules of the present invention may be used to deliver additional exogenous materials into target cells, such as B-cell or T-cell epitopes, peptides, proteins, polynucleotides, and detection-promoting agents. This system is modular, in that any number of diverse binding regions can be used to target a Shiga toxin effector polypeptide of the present invention to various, diverse cell types. A. Cell-Kill Via Shiga Toxin a Subunit Cytotoxicity Certain embodiments of the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention are cytotoxic. Certain further embodiments of the cell-targeting molecules of the present invention are cytotoxic only due to the presence of one or more Shiga toxin effector polypeptide components. The A Subunits of members of the Shiga toxin family each comprise an enzymatically active polypeptide region capable of killing a eukaryotic cell once in the cell's cytosol. Because members of the Shiga toxin family are adapted to killing eukaryotic cells, molecules derived from Shiga toxins, such as, e.g., molecules comprising certain embodiments of the Shiga toxin effector polypeptides of the present invention can exhibit potent cell-kill activities. For certain embodiments of the cell-targeting molecules of the present invention, upon contacting a cell physically coupled with an extracellular target biomolecule of the binding region of the cell-targeting molecule (e.g. a target positive cell), the cell-targeting molecule is capable of causing death of the cell. For certain further embodiments, the CD50value of the cell-targeting molecule is less than 5, 2.5, 1, 0.5, or 0.25 nM, which is vastly more potent than an untargeted, wild-type, Shiga toxin effector polypeptide (e.g. SEQ ID NO:830). Cell-kill may be accomplished using a molecule of the present invention under varied conditions of target cells, such as, e.g., an ex vivo manipulated target cell, a target cell cultured in vitro, a target cell within a tissue sample cultured in vitro, or a target cell in an in vivo setting like within a multicellular organism. In certain embodiments, the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention comprise (1) a de-immunized, Shiga toxin effector sub-region, (2) a protease-cleavage resistant region near the carboxy-terminus of a Shiga toxin A1 fragment derived region, (3) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif, and/or (4) a heterologous, T-cell epitope embedded or inserted region; however, for certain further embodiments, these structural modifications do not significantly alter the potency of Shiga toxin cytotoxicity as compared to a reference molecules comprising a wild-type Shiga toxin A Subunit polypeptide, such as, e.g., a wild-type Shiga toxin A1 fragment. Thus, Shiga toxin effector polypeptides and cell-targeting molecules of the present invention which are de-immunized, protease cleavage resistant, and/or carrying embedded or inserted, heterologous, epitopes can maintain potent cytotoxicity while providing one or more various other functionalities or properties. Already cytotoxic cell-targeting molecules comprising Shiga toxin effector polypeptides may be engineered by the skilled worker using the information and methods provided herein to be more cytotoxic and/or to have redundant, backup cytotoxicities operating via completely different mechanisms. These multiple cytotoxic mechanisms may complement each other by their diversity of functions (such as by providing potent killing via two mechanisms of cell-killing, direct and indirect, as well as mechanisms of immuno-stimulation to the local area), redundantly backup each other (such as by providing one cell-killing mechanism in the absence of the other mechanisms-like if a target cell is resistant to or acquires some immunity to a subset of previously active mechanisms), and/or protect against developed resistance (by limiting resistance to the less probable situation of the malignant or infected cell blocking multiple, different cell-killing mechanisms simultaneously). B. Delivery of a T-Cell Epitope for MHC Class I Presentation on a Cell Surface In certain embodiments, the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention comprise a T-cell epitope, which enables the engineering of “T-cell epitope delivering” molecules with virtually unlimited choices of epitope-peptide cargos for delivery and cell-surface presentation by a nucleated, chordate cell. For certain embodiments, the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention are each capable of delivering one or more T-cell epitopes, associated with the Shiga toxin effector polypeptides and/or cell-targeting molecules, to the proteasome of a cell. The delivered T-cell epitope are then proteolytic processed and presented by the MHC class I pathway on the surface of the cell. By conjugating MHC class I epitopes to cell-targeting molecules, the targeted delivery and presentation of immuno-stimulatory antigens may be accomplished in order to harness and direct a beneficial function(s) of a chordate immune system. For certain embodiments, the Shiga toxin effector polypeptide or cell-targeting molecule of the present invention is capable of delivering a T-cell epitope to a MHC class I molecule of a cell for cell-surface presentation. In certain embodiments, the Shiga toxin effector polypeptide or cell-targeting molecule of the present invention comprises a heterologous, T-cell epitope, whether as an additional exogenous material or embedded or inserted within a Shiga toxin effector polypeptide. For certain further embodiments, the Shiga toxin effector polypeptide or cell-targeting molecule of the present invention is capable of delivering an embedded or inserted T-cell epitope to a MHC class I molecule for cell-surface presentation. For certain embodiments, the Shiga toxin effector polypeptide of the present invention is capable of delivering a T-cell epitope, which is conjugated to the Shiga toxin effector polypeptide, to a MHC class I molecule of a cell in which the Shiga toxin effector polypeptide is present for presentation of the T-cell epitope by the MHC class I molecule on a surface of the cell. For certain further embodiments, the T-cell epitope is a heterologous, T-cell epitope. For certain further embodiments, the T-cell epitope functions as CD8+ T-cell epitope, whether already known or identified in the future using methods which are currently routine to the skilled worker. For certain embodiments, the cell-targeting molecule of the present invention is capable of delivering a T-cell epitope, which is associated with the cell-targeting molecule, to a MHC class I molecule of a cell for presentation of the T-cell epitope by the MHC class I molecule on a surface of the cell. For certain further embodiments, the T-cell epitope is a heterologous, T-cell epitope which is conjugated to the Shiga toxin effector polypeptide. For certain further embodiments, the T-cell epitope functions as CD8+ T-cell epitope, whether already known or identified in the future using methods which are currently routine to the skilled worker. For certain embodiments, upon contacting a cell with the cell-targeting molecule of the present invention, the cell-targeting molecule is capable of delivering a T-cell epitope-peptide, which is associated with the cell-targeting molecule, to a MHC class I molecule of the cell for presentation of the T-cell epitope-peptide by the MHC class I molecule on a surface of the cell. For certain further embodiments, the T-cell epitope-peptide is a heterologous epitope which is conjugated to a Shiga toxin effector polypeptide. For certain further embodiments, the T-cell epitope-peptide functions as CD8+ T-cell epitope, whether already known or identified in the future using methods which are currently routine to the skilled worker. The addition of a heterologous epitope into or presence of a heterologous epitope in a cell-targeting molecule of the present invention, whether as an additional exogenous material or embedded or inserted within a Shiga toxin effector polypeptide, enables methods of using such cell-targeting molecules for the cell-targeted delivery of a chosen epitope for cell-surface presentation by a nucleated, target cell within a chordate. One function of certain, CD8+ T-cell hyper-immunized, Shiga toxin effector polypeptides and cell-targeting molecules of the present invention is the delivery of one or more T-cell epitope-peptides to a MHC class I molecule for MHC class I presentation by a cell. Delivery of exogenous, T-cell epitope-peptides to the MHC class I system of a target cell can be used to induce the target cell to present the T-cell epitope-peptide in association with MHC class I molecules on the cell surface, which subsequently leads to the activation of CD8+ effector T-cells to attack the target cell. The skilled worker, using techniques known in the art, can associate, couple, and/or link certain, Shiga toxin effector polypeptides of the present invention to various other cell-targeting binding regions to create cell-targeting molecules of the present invention which target specific, extracellular, target biomolecules physically coupled to cells and promote target-cell internalization of these cell-targeting molecules. All nucleated vertebrate cells are believed to be capable of presenting intracellular epitopes using the MHC class I system. Thus, extracellular target biomolecules of the cell-targeting molecules of the invention may in principle target any nucleated vertebrate cell for T-cell epitope delivery to a MHC class I presentation pathway of such a cell. The epitope-delivering functions of the Shiga toxin effector polypeptides and cell-targeting molecules of the present invention can be detected and monitored by a variety of standard methods known in the art to the skilled worker and/or described herein (see e.g. WO 2015/113005). Certain assays to monitor this function of the polypeptides and molecules of the present invention involve the direct detection of a specific MHC class I/peptide antigen complex in vitro or ex vivo. Common methods for direct visualization and quantitation of peptide-MHC class I complexes involve various immuno-detection reagents known to the skilled worker. For example, specific monoclonal antibodies can be developed to recognize a particular MHC/class I/peptide antigen complex. Similarly, soluble, multimeric T cell receptors, such as the TCR-STAR reagents (Altor Bioscience Corp., Mirmar, FL, U.S.) can be used to directly visualize or quantitate specific MHC I/antigen complexes (Zhu X et al.,J Immunol176: 3223-32 (2006)). These specific mAbs or soluble, multimeric T-cell receptors may be used with various detection methods, including, e.g. immunohistochemistry, flow cytometry, and enzyme-linked immuno assay (ELISA). An alternative method for direct identification and quantification of MHC I/peptide complexes involves mass spectrometry analyses, such as, e.g., the ProPresent Antigen Presentation Assay (ProImmune, Inc., Sarasota, FL, U.S.) in which peptide-MCH class I complexes are extracted from the surfaces of cells, then the peptides are purified and identified by sequencing mass spectrometry (Falk K et al.,Nature351: 290-6 (1991)). In certain assays to monitor the T-cell epitope delivery and MHC class I presentation function of the polypeptides and molecules of the present invention involve computational and/or experimental methods to monitor MHC class I and peptide binding and stability. Several software programs are available for use by the skilled worker for predicting the binding responses of peptides to MHC class I alleles, such as, e.g., The Immune Epitope Database and Analysis Resource (IEDB) Analysis Resource MHC-I binding prediction Consensus tool (Kim Y et al.,Nucleic Acid Res40: W525-30 (2012). Several experimental assays have been routinely applied, such as, e.g., cell surface binding assays and/or surface plasmon resonance assays to quantify and/or compare binding kinetics (Miles K et al.,Mol Immunol48: 728-32 (2011)). Additionally, other MHC-peptide binding assays based on a measure of the ability of a peptide to stabilize the ternary MHC-peptide complex for a given MHC class I allele, as a comparison to known controls, have been developed (e.g., MHC-peptide binding assay from ProImmmune, Inc.). Alternatively, measurements of the consequence of MHC class I/peptide antigen complex presentation on the cell surface can be performed by monitoring the cytotoxic T-cell (CTL) response to the specific complex. These measurements by include direct labeling of the CTLs with MHC class I tetramer or pentamer reagents. Tetramers or pentamers bind directly to T cell receptors of a particular specificity, determined by the Major Histocompatibility Complex (MHC) allele and peptide complex. Additionally, the quantification of released cytokines, such as interferon gamma or interleukins by ELISA or enzyme-linked immunospot (ELIspot) is commonly assayed to identify specific CTL responses. The cytotoxic capacity of CTL can be measured using a number of assays, including the classical 51 Chromium (Cr) release assay or alternative non-radioactive cytotoxicity assays (e.g., CytoTox96® non-radioactive kits and CellTox™ CellTiter-GLO® kits available from Promega Corp., Madison, WI, U.S.), Granzyme B ELISpot, Caspase Activity Assays or LAMP-1 translocation flow cytometric assays. To specifically monitor the killing of target cells, carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vitro or in vivo investigation to monitor killing of epitope specific CSFE labeled target cells (Durward M et al.,J Vis Exp45 pii 2250 (2010)). In vivo responses to MHC class I presentation can be followed by administering a MHC class I/antigen promoting agent (e.g., an immunogenic peptide, protein or inactivated/attenuated virus vaccine) followed by challenge with an active agent (e.g. a virus) and monitoring responses to that agent, typically in comparison with unvaccinated controls. Ex vivo samples can be monitored for CTL activity with methods similar to those described previously (e.g. CTL cytotoxicity assays and quantification of cytokine release). HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicated cells after lysis using immune affinity (e.g., an anti-MHC antibody “pulldown” purification) and the associated peptides (i.e., the peptides presented by the isolated MHC molecules) are recovered from the purified complexes. The recovered peptides are analyzed by sequencing mass spectrometry. The mass spectrometry data is compared against a protein database library consisting of the sequence of the exogenous (non-self) peptide (T-cell epitope X) and the international protein index for humans (representing “self” or non-immunogenic peptides). The peptides are ranked by significance according to a probability database. All detected antigenic (non-self) peptide sequences are listed. The data is verified by searching against a scrambled decoy database to reduce false hits (see e.g. Ma B, Johnson R,Mol Cell Proteomics11: O111.014902 (2012)). The results will demonstrate that peptides from the T-cell epitope X are presented in MHC complexes on the surface of intoxicated target cells. The set of presented peptide-antigen-MHC complexes can vary between cells due to the antigen-specific HLA molecules expressed. T-cells can then recognize specific peptide-antigen-MHC complexes displayed on a cell surface using different TCR molecules with different antigen-specificities. Because multiple T-cell epitopes may be delivered by a cell-targeting molecule of the invention, such as, e.g., by embedding two or more different T-cell epitopes in a single proteasome delivering effector polypeptide, a single cell-targeting molecule of the invention may be effective chordates of the same species with different MHC class variants, such as, e.g., in humans with different HLA alleles. This may allow for the combining within a single molecule of different T-cell epitopes with different effectiveness in different sub-populations of subjects based on MHC complex protein diversity and polymorphisms. For example, human MHC complex proteins, HLA proteins, vary among humans based on genetic ancestry, e.g. African (sub-Saharan), Amerindian, Caucasiod, Mongoloid, New Guinean and Australian, or Pacific islander. The applications involving the T-cell epitope delivering polypeptides and molecules of the present invention are vast. Every nucleated cell in a mammalian organism may be capable of MHC class I pathway presentation of immunogenic, T-cell epitope-peptides on their cell outer surfaces complexed to MHC class I molecules. In addition, the sensitivity of T-cell epitope recognition is so exquisite that only a few MIC-I peptide complexes are required to be presented to result in an immune response, e.g., even presentation of a single complex can be sufficient for recognition by an effector T-cell (Sykulev Y et al.,Immunity4: 565-71 (1996)). The activation of T-cell responses are desired characteristics of certain anti-cancer, anti-neoplastic, anti-tumor, and/or anti-microbial biologic drugs to stimulate the patient's own immune system toward targeted cells. Activation of a robust and strong T-cell response is also a desired characteristic of many vaccines. The presentation of a T-cell epitope by a target cell within an organism can lead to the activation of robust immune responses to a target cell and/or its general locale within an organism. Thus, the targeted delivery of a T-cell epitope for presentation may be utilized for as a mechanism for activating T-cell responses during a therapeutic regime. The presentation of a T-cell immunogenic epitope-peptide by the MHC class I system targets the presenting cell for killing by CTL-mediated lysis and also triggers immune stimulation in the local microenvironment. By engineering immunogenic epitope sequences within Shiga toxin effector polypeptide components of target-cell-internalizing therapeutic molecules, the targeted delivery and presentation of immuno-stimulatory antigens may be accomplished. The presentation of immuno-stimulatory non-self antigens, such as e.g. known viral antigens with high immunogenicity, by target cells signals to other immune cells to destroy the target cells as well as to recruit more immune cells to the area. The presentation of an immunogenic, T-cell epitope-peptide by the MHC class I complex targets the presenting cell for killing by CTL-mediated cytolysis. The presentation by targeted cells of immuno-stimulatory non-self antigens, such as, e.g., known viral epitope-peptides with high immunogenicity, can signal to other immune cells to destroy the target cells and recruit more immune cells to the target cell site within a chordate. Thus, already cytotoxic molecules, such as e.g. therapeutic or potentially therapeutic molecules comprising Shiga toxin effector polypeptides, may be engineered using methods of the present invention into more cytotoxic molecules and/or to have an additional cytotoxic mechanism operating via delivery of a T-cell epitope, presentation, and stimulation of effector T-cells. These multiple cytotoxic mechanisms may complement each other (such as by providing both direct target-cell-killing and indirect (CTL-mediated) cell-killing, redundantly backup each other (such as by providing one mechanism of cell-killing in the absence of the other), and/or protect against the development of therapeutic resistance (by limiting resistance to the less probable situation of the malignant or infected cell evolving to block two different cell-killing mechanisms simultaneously). In addition, a cytotoxic molecule comprising a Shiga toxin effector polypeptide component that exhibits catalytic-based cytotoxicity may be engineered by the skilled worker using routine methods into enzymatically inactive variants. For example, the cytotoxic Shiga toxin effector polypeptide component of a cytotoxic molecule may be conferred with reduced activity and/or rendered inactive by the introduction of one or mutations and/or truncations such that the resulting molecule can still be cytotoxic via its ability to deliver a T-cell epitope to the MHC class I system of a target cell and subsequent presentation to the surface of the target cell. In another example, a T-cell epitope may be inserted or embedded into a Shiga toxin effector polypeptide such that the Shiga toxin effector polypeptide is inactivated by the added epitope (see e.g. WO 2015/113005). This approach removes one cytotoxic mechanism while retaining or adding another and may also provide a molecule capable of exhibiting immuno-stimulation to the local area of a target cell(s) within an organism via delivered T-cell epitope presentation or “antigen seeding.” Furthermore, non-cytotoxic variants of the cell-targeting molecules of the present invention which comprise embedded or inserted, heterologous, T-cell epitopes may be useful in applications involving immune-stimulation within a chordate and/or labeling of target cells within a chordate with MHC class I molecule displayed epitopes. The ability to deliver a T-cell epitope of certain Shiga toxin effector polypeptides and cell-targeting molecules of the present invention may be accomplished under varied conditions and in the presence of non-targeted bystander cells, such as, e.g., an ex vivo manipulated target cell, a target cell cultured in vitro, a target cell within a tissue sample cultured in vitro, or a target cell in an in vivo setting like within a multicellular organism. C. Cell-Kill Via Targeted Cytotoxicity and/or Engagement of Cytotoxic T-Cells For certain embodiments, the cell-targeting molecule of the present invention can provide 1) delivery of a T-cell epitope for MHC class I presentation by a target cell and/or 2) potent cytotoxicity. For certain embodiments of the cell-targeting molecules of the present invention, upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting binding region, the cell-targeting molecule of the invention is capable of causing death of the cell. The mechanism of cell-kill may be direct, e.g. via the enzymatic activity of a toxin effector polypeptide region, or indirect via CTL-mediated cytolysis. 1. Indirect Cell-Kill Via T-Cell Epitope Delivery and MHC Class I Presentation Certain embodiments of the cell-targeting molecules of the present invention are cytotoxic because they comprise a CD8+ T-cell epitope capable of being delivered to the MHC class I presentation pathway of a target cell and presented on a cellular surface of the target cell. For example, T-cell epitope delivering, Shiga toxin effector polypeptides of the present invention, with or without endogenous epitope de-immunization, may be used as components of cell-targeting molecules for applications involving indirect cell-killing (see e.g. WO 2015/113005). In certain embodiments of the cell-targeting molecules of the present invention, upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting binding region, the cell-targeting molecule of the invention is capable of indirectly causing the death of the cell, such as, e.g., via the presentation of one or more T-cell epitopes by the target cell and the subsequent recruitment of CTLs which kill the target cell. The presentation of an antigenic peptide complexed with a MHC class I molecule by a cell sensitizes the presenting cell to targeted killing by cytotoxic T-cells (CTLs) via the induction of apoptosis, lysis, and/or necrosis. In addition, the CTLs which recognize the target cell may release immuno-stimulatory cytokines, such as, e.g., interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF), macrophage inflammatory protein-1 beta (MIP-1beta), and interleukins such as IL-17, IL-4, and IL-22. Furthermore, CTLs activated by recognition of a presented epitope may indiscriminately kill other cells proximal to the presenting cell regardless of the peptide-MHC class I complex repertoire presented by those proximal cells (Wiedemann A et al.,Proc Natl Acad Sci USA103: 10985-90 (2006)). Because of MHC allele diversity within different species, a cell-targeting molecule of the present invention comprising only a single epitope may exhibit varied effectiveness to different patients or subjects of the same species. However, certain embodiments of the cell-targeting molecules of the present invention may each comprise multiple, T-cell epitopes that are capable of being delivered to the MHC class I system of a target cell simultaneously. Thus, for certain embodiments of the cell-targeting molecules of the present invention, a cell-targeting molecule is used to treat different subjects with considerable differences in their MHC molecules' epitope-peptide binding affinities (i.e. considerable differences in their MHC alleles and/or MHC genotypes). In addition, certain embodiments of the cell-targeting molecules of the present invention reduce or prevent target cell adaptations to escape killing (e.g. a target cancer cell mutating to escape therapeutic effectiveness or “mutant escape”) by using multiple cell-killing mechanisms simultaneously (e.g. direct killing and indirect killing via multiple different T-cell epitopes simultaneously). 2. Direct Cell-Kill Via Cell-Targeted, Shiga Toxin Cytotoxicity Certain embodiments of the cell-targeting molecules of the present invention are cytotoxic because they comprise a catalytically active, Shiga toxin effector polypeptide and regardless of the presence of any cytotoxic agent or immunogenic, CD8+ T-cell epitope in the molecule. For example, Shiga toxin effector polypeptides of the present invention, with or without endogenous epitope de-immunization, may be used as components of cell-targeting molecules for applications involving direct cell-killing, such as, e.g., via the ribotoxic, enzymatic activity of a Shiga toxin effector polypeptide or ribosome binding and interference with ribosome function due to a non-catalytic mechanism(s). For certain embodiments of the CD8+ T-cell hyper-immunized, cell-targeting molecules of the present invention, upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting binding region, the cell-targeting molecule of the invention is capable of directly causing the death of the cell, such as, e.g., without the involvement of an untargeted, cytotoxic T-cell (see Section III-D, supra). D. Selective Cytotoxicity Among Cell Types Certain cell-targeting molecules of the present invention have uses in the selective killing of specific target cells in the presence of untargeted, bystander cells. By targeting the delivery of Shiga toxin effector polypeptides of the present invention to specific cells via a cell-targeting binding region(s), the cell-targeting molecules of the present invention can exhibit cell-type specific, restricted cell-kill activities resulting in the exclusive or preferential killing selected cell types in the presence of untargeted cells. Similarly, by targeting the delivery of immunogenic T-cell epitopes to the MHC class I pathway of target cells, the subsequent presentation of T-cell epitopes and CTL-mediated cytolysis of target cells induced by the cell-targeting molecules of the invention can be restricted to exclusively or preferentially killing selected cell types in the presence of untargeted cells. In addition, both the cell-targeted delivery of a cytotoxic, Shiga toxin effector polypeptide region and an immunogenic, T-cell epitope can be accomplished by a single cell-targeting molecule of the present invention such that deliver of both potentially cytotoxic components is restricted exclusively or preferentially to target cells in the presence of untargeted cells. For certain embodiments, the cell-targeting molecule of the present invention is cytotoxic at certain concentrations. In certain embodiments, upon administration of the cell-targeting molecule of the present invention to a mixture of cell types, the cytotoxic cell-targeting molecule is capable of selectively killing those cells which are physically coupled with an extracellular target biomolecule compared to cell types not physically coupled with an extracellular target biomolecule. For certain embodiments, the cytotoxic cell-targeting molecule of the present invention is capable of selectively or preferentially causing the death of a specific cell type within a mixture of two or more different cell types. This enables targeting cytotoxic activity to specific cell types with a high preferentiality, such as a 3-fold cytotoxic effect, over “bystander” cell types that do not express the target biomolecule. Alternatively, the expression of the target biomolecule of the binding region may be non-exclusive to one cell type if the target biomolecule is expressed in low enough amounts and/or physically coupled in low amounts with cell types that are not to be targeted. This enables the targeted cell-killing of specific cell types with a high preferentiality, such as a 3-fold cytotoxic effect, over “bystander” cell types that do not express significant amounts of the target biomolecule or are not physically coupled to significant amounts of the target biomolecule. For certain further embodiments, upon administration of the cytotoxic cell-targeting molecule to two different populations of cell types, the cytotoxic cell-targeting molecule is capable of causing cell death as defined by the half-maximal cytotoxic concentration (CD50) on a population of target cells, whose members express an extracellular target biomolecule of the binding region of the cytotoxic cell-targeting molecule, at a dose at least three-times lower than the CD50dose of the same cytotoxic cell-targeting molecule to a population of cells whose members do not express an extracellular target biomolecule of the binding region of the cytotoxic cell-targeting molecule. For certain embodiments, the cytotoxic activity of a cell-targeting molecule of the present invention toward populations of cell types physically coupled with an extracellular target biomolecule is at least 3-fold higher than the cytotoxic activity toward populations of cell types not physically coupled with any extracellular target biomolecule of the binding region. According to the present invention, selective cytotoxicity may be quantified in terms of the ratio (a/b) of (a) cytotoxicity towards a population of cells of a specific cell type physically coupled with a target biomolecule of the binding region to (b) cytotoxicity towards a population of cells of a cell type not physically coupled with a target biomolecule of the binding region. In certain embodiments, the cytotoxicity ratio is indicative of selective cytotoxicity which is at least 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 250-fold, 500-fold, 750-fold, or 1000-fold higher for populations of cells or cell types physically coupled with a target biomolecule of the binding region compared to populations of cells or cell types not physically coupled with a target biomolecule of the binding region. For certain embodiments, the preferential cell-killing function or selective cytotoxicity of a cell-targeting molecule of the present invention is due to an additional exogenous material (e.g. a cytotoxic material) and/or heterologous, T-cell epitope present in a Shiga toxin effector polypeptide of the present invention and not necessarily a result of the catalytic activity of a Shiga toxin effector polypeptide. This preferential cell-killing function allows a targeted cell to be killed by certain cytotoxic, cell-targeting molecules of the present invention under varied conditions and in the presence of non-targeted bystander cells, such as ex vivo manipulated mixtures of cell types, in vitro cultured tissues with mixtures of cell types, or in vivo in the presence of multiple cell types (e.g. in situ or in a native location within a multicellular organism). E. Delivery of Molecular Cargos into Interior Compartments of Target Cells In addition to cytotoxic, cytostatic, and immune stimulation applications, cell-targeting molecules of the present invention optionally may be used for targeted intracellular delivery functions, such as, e.g., in applications involving information gathering and diagnostic functions. Because the cell-targeting molecules of the invention, including reduced cytotoxicity and/or nontoxic forms thereof, are capable of entering cells physically coupled with an extracellular target biomolecule recognized by the cell-targeting molecule's binding region, certain embodiments of the cell-targeting molecules of the invention may be used to deliver additional exogenous materials or “cargos” (e.g. conjugated molecules) into the interior of targeted cell types. For example, non-toxic variants of the cytotoxic, cell-targeting molecules of the invention, or optionally cytotoxic variants, may be used to deliver additional exogenous materials to and/or label the interiors of cells physically coupled with an extracellular target biomolecule of the binding region of the cell-targeting molecule. Various types of cells and/or cell populations which express target biomolecules to at least one cellular surface may be targeted by the cell-targeting molecules of the invention for receiving exogenous materials. The functional components of the present invention are modular, in that various Shiga toxin effector polypeptides, additional exogenous materials, and binding regions may be associated with each other to provide cell-targeting molecules suitable for diverse applications involving cargo delivery, such as, e.g., non-invasive, in vivo imaging of tumor cells. This delivery of exogenous material function of certain cell-targeting molecules of the present invention may be accomplished under varied conditions and in the presence of non-targeted bystander cells, such as, e.g., an ex vivo manipulated target cell, a target cell cultured in vitro, a target cell within a tissue sample cultured in vitro, or a target cell in an in vivo setting like within a multicellular organism. Furthermore, the selective delivery of exogenous material to certain cells by certain cell-targeting molecules of the present invention may be accomplished under varied conditions and in the presence of non-targeted bystander cells, such as ex vivo manipulated mixtures of cell types, in vitro cultured tissues with mixtures of cell types, or in vivo in the presence of multiple cell types (e.g. in situ or in a native location within a multicellular organism). Shiga toxin effector polypeptides and cell-targeting molecules which are not capable, such as a certain concentration ranges, of killing a target cell and/or delivering an embedded or inserted epitope for cell-surface presentation by a MHC molecule of a target cell may still be useful for delivering exogenous materials into cells, such as, e.g., detection-promoting agents. For certain embodiments, the Shiga toxin effector polypeptides of the present invention exhibits low to zero cytotoxicity and thus are referred to herein as “noncytotoxic and/or reduced cytotoxic.” For certain embodiments, the cell-targeting molecule of the present invention exhibits low to zero cytotoxicity and may be referred to as “noncytotoxic” and/or “reduced cytotoxic variants.” For example, certain embodiments of the molecules of the present invention do not exhibit a significant level of Shiga toxin based cytotoxicity wherein at doses of less than 1,000 nM, 500 nM, 100 nM, 75 nM, 50 nM, there is no significant amount of cell death as compared to the appropriate reference molecule, such as, e.g., as measured by an assay known to the skilled worker and/or described herein. For certain further embodiments, the molecules of the present invention do not exhibit any toxicity at dosages of 1-100 microgram (μg) per kilogram (kg) of a mammalian recipient. Reduced-cytotoxic variants may still be cytotoxic at certain concentrations or dosages but exhibit reduced cytotoxicity, such as, e.g., are not capable of exhibiting a significant level of Shiga toxin cytotoxicity in certain situations. Shiga toxin effector polypeptides of the present invention, and certain cell-targeting molecules comprising the same, can be rendered non-cytotoxic, such as, e.g., via the addition of one or more amino acid substitutions known to the skilled worker to inactivate a Shiga toxin A Subunit and/or Shiga toxin effector polypeptide, including exemplary substitutions described herein. The non-cytotoxic and reduced cytotoxic variants of the cell-targeting molecules of the present invention may be in certain situations more suitable for delivery of additional exogenous materials than more cytotoxic variants. F. Information Gathering for Diagnostic Functions Certain cell-targeting molecules of the present invention have uses in the in vitro and/or in vivo detection of specific cells, cell types, and/or cell populations, as well as specific subcellular compartments of any of the aforementioned. Reduced-cytotoxicity and/or nontoxic forms of the cytotoxic, cell-targeting molecules of the invention that are conjugated to detection-promoting agents optionally may be used for diagnostic functions, such as for companion diagnostics used in conjunction with a therapeutic regimen comprising the same or a related binding region, such as, e.g., a binding region with high-affinity binding to the same target biomolecule, an overlapping epitope, and/or the same epitope. In certain embodiments, the cell-targeting molecules described herein are used for both diagnosis and treatment, or for diagnosis alone. When the same cytotoxic cell-targeting molecule is used for both diagnosis and treatment, for certain embodiments of the present invention the cell-targeting molecule variant which incorporates a detection-promoting agent for diagnosis may have its cytotoxicity reduced or may be rendered nontoxic by catalytic inactivation of its Shiga toxin effector polypeptide region(s) via one or more amino acid substitutions, including exemplary substitutions described herein. For example, certain nontoxic variants of the cell-targeting molecules of the present invention exhibit less than 5%, 4%, 3%, 2%, or 1% death of target cells after administration of a dose less than 1 mg/kg. Reduced-cytotoxicity variants may still be cytotoxic at certain concentrations or dosages but exhibit reduced cytotoxicity, such as, e.g., are not capable of exhibiting a significant level of Shiga toxin cytotoxicity as described herein. The ability to conjugate detection-promoting agents known in the art to various cell-targeting molecules of the present invention provides useful compositions for the detection of certain cells, such as, e.g., cancer, tumor, immune, and/or infected cells. These diagnostic embodiments of the cell-targeting molecules of the invention may be used for information gathering via various imaging techniques and assays known in the art. For example, diagnostic embodiments of the cell-targeting molecules of the invention may be used for information gathering via imaging of intracellular organelles (e.g. endocytotic, Golgi, endoplasmic reticulum, and cytosolic compartments) of individual cancer cells, immune cells, and/or infected cells in a patient or biopsy sample. Various types of information may be gathered using the diagnostic embodiments of the cell-targeting molecules of the invention whether for diagnostic uses or other uses. This information may be useful, for example, in diagnosing neoplastic cell types, determining therapeutic susceptibilities of a patient's disease, assaying the progression of anti-neoplastic therapies over time, assaying the progression of immunomodulatory therapies over time, assaying the progression of antimicrobial therapies over time, evaluating the presence of infected cells in transplantation materials, evaluating the presence of unwanted cell types in transplantation materials, and/or evaluating the presence of residual tumor cells after surgical excision of a tumor mass. For example, subpopulations of patients might be ascertained using information gathered using the diagnostic variants of the cell-targeting molecules of the invention, and then individual patients could be further categorized into subpopulations based on their unique characteristic(s) revealed using those diagnostic embodiments. For example, the effectiveness of specific pharmaceuticals or therapies might be a criterion used to define a patient subpopulation. For example, a nontoxic diagnostic variant of a particular cytotoxic, cell-targeting molecule of the invention may be used to differentiate which patients are in a class or subpopulation of patients predicted to respond positively to a cytotoxic variant of that cell-targeting molecule of the invention. Accordingly, associated methods for patient identification, patient stratification, and diagnosis using cell-targeting molecules of the present invention, including non-toxic variants of cytotoxic, cell-targeting molecules of the present invention, are considered to be within the scope of the present invention. The expression of the target biomolecule by a cell need not be native in order for cell-targeting by a cell-targeting molecule of the present invention, such as, e.g., for direct cell-kill, indirect cell-kill, delivery of exogenous materials like T-cell epitopes, and/or information gathering. Cell surface expression of the target biomolecule could be the result of an infection, the presence of a pathogen, and/or the presence of an intracellular microbial pathogen. Expression of a target biomolecule could be artificial such as, for example, by forced or induced expression after infection with a viral expression vector, see e.g. adenoviral, adeno-associated viral, and retroviral systems. An example of inducing expression of a target biomolecule is the upregulation of CD38 expression of cells exposed to retinoids, like all-trans retinoic acid and various synthetic retinoids, or any retinoic acid receptor (RAR) agonist (Drach J et al.,Cancer Res54: 1746-52 (1994); Uruno A et al.,J Leukoc Biol90: 235-47 (2011)). Expression of CD30 can be induced in both B-cells and T-cells by exposure to by mitogens, phytohemagglutinin (PHA), staphylococcal protein A, EBV virus, human T-cell leukemia virus 1 or 2 (HTLV-1 or HTLV-2) (see e.g. Stein H et al.,Blood66: 848-58 (1985)). In another example, CD20, HER2, and EGFR expression may be induced by exposing a cell to ionizing radiation (Wattenberg M et al.,Br J Cancer110: 1472-80 (2014)). Further, PSMA expression is upregulated in response to androgen deprivation (see e.g. Chang S et al.,Cancer88: 407-15 (2000); Meller B et al.,EJNMMI Res5: 66 (2015)). In certain embodiments, the molecule of the present invention is useful for tracking the behavior of an inert Shiga toxin effector polypeptide and/or cell-targeting molecule, such as, e.g., in vivo, tissue culture, and/or by a laboratory sensor. For example, a dye-conjugated Shiga toxin effector polypeptide of the present invention may be tracked using a light sensor during an experiment, e.g., both before and after an anti-Shiga toxin antibody incubation step. IV. Pharmaceutical and Diagnostic Compositions Comprising Cell-Targeting Molecules of the Present Invention The present invention provides Shiga toxin effector polypeptides and cell-targeting molecules for use, alone or in combination with one or more additional therapeutic agents, in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases, disorders, or symptoms described in further detail below (e.g. cancers, malignant tumors, non-malignant tumors, growth abnormalities, immune disorders, and microbial infections). The present invention further provides pharmaceutical compositions comprising a Shiga toxin polypeptide or cell-targeting molecule of the present invention, or a pharmaceutically acceptable salt or solvate thereof, according to the invention, together with at least one pharmaceutically acceptable carrier, excipient, or vehicle. In certain embodiments, the pharmaceutical composition of the present invention may comprise homo-multimeric and/or hetero-multimeric forms of a Shiga toxin effector polypeptides or cell-targeting molecule of the present invention. The pharmaceutical compositions of the invention are useful in methods of treating, ameliorating, or preventing a disease, condition, disorder, or symptom described in further detail below. Each such disease, condition, disorder, or symptom is envisioned to be a separate embodiment with respect to uses of a pharmaceutical composition according to the invention. The invention further provides pharmaceutical compositions for use in at least one method of treatment according to the invention, as described in more detail below. As used herein, the terms “patient” and “subject” are used interchangeably to refer to any organism, commonly vertebrates such as humans and animals, which presents symptoms, signs, and/or indications of at least one disease, disorder, or condition. These terms include mammals such as the non-limiting examples of primates, livestock animals (e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g. cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats, etc.). As used herein, “treat,” “treating,” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (e.g. not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treat,” “treating,” or “treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g. a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The terms “treat,” “treating,” or “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder, or condition. With regard to tumors and/or cancers, treatment includes reduction in overall tumor burden and/or individual tumor size. As used herein, the terms “prevent,” “preventing,” “prevention” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease, or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g. a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition. As used herein, an “effective amount” or “therapeutically effective amount” is an amount or dose of a composition (e.g. a therapeutic composition, compound, or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition. The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type, disease stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a composition and adjusting the dosage accordingly (see e.g.Remington: The Science and Practice of Pharmacy(Gennaro A, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)). Diagnostic compositions of the present invention comprise a cell-targeting molecule of the present invention and one or more detection-promoting agents. When producing or manufacturing a diagnostic composition of the present invention, a cell-targeting molecule of the present invention may be directly or indirectly linked to one or more detection-promoting agents. There are numerous standard techniques known to the skilled worker for incorporating, affixing, and/or conjugating various detection-promoting agents to proteins or proteinaceous components of molecules, especially to immunoglobulins and immunoglobulin-derived domains. There are numerous detection-promoting agents known to the skilled worker, such as isotopes, dyes, colorimetric agents, contrast enhancing agents, fluorescent agents, bioluminescent agents, and magnetic agents, which can be operably linked to the polypeptides or cell-targeting molecules of the invention for information gathering methods, such as for diagnostic and/or prognostic applications to diseases, disorders, or conditions of an organism (see e.g. Cai W et al.,J Nucl Med48: 304-10 (2007); Nayak T, Brechbiel M,Bioconjug Chem20: 825-41 (2009); Paudyal P et al.,Oncol Rep22: 115-9 (2009); Qiao J et al.,PLoS ONE6: e18103 (2011); Sano K et al.,Breast Cancer Res14: R61 (2012)). These agents may be associated with, linked to, and/or incorporated within the polypeptide or cell-targeting molecule of the invention at any suitable position. For example, the linkage or incorporation of the detection-promoting agent may be via an amino acid residue(s) of a molecule of the present invention or via some type of linkage known in the art, including via linkers and/or chelators. The incorporation of the agent is in such a way to enable the detection of the presence of the diagnostic composition in a screen, assay, diagnostic procedure, and/or imaging technique. Similarly, there are numerous imaging approaches known to the skilled worker, such as non-invasive in vivo imaging techniques commonly used in the medical arena, for example: computed tomography imaging (CT scanning), optical imaging (including direct, fluorescent, and bioluminescent imaging), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, and x-ray computed tomography imaging. V. Molecules of the Present Invention Immobilized on Solid Substrates Certain embodiments of the present invention include a molecule of the present invention (e.g. a Shiga toxin effector polypeptide, a Shiga toxin effector polypeptide scaffold, a cell-targeting molecule, fusion protein, or polynucleotide of the present invention), or any effector fragment thereof, immobilized on a solid substrate. Solid substrates contemplated herein include, but are not limited to, microbeads, nanoparticles, polymers, matrix materials, microarrays, microtiter plates, or any solid surface known in the art (see e.g. U.S. Pat. No. 7,771,955). In accordance with these embodiments, a molecule of the present invention may be covalently or non-covalently linked to a solid substrate, such as, e.g., a bead, particle, or plate, using techniques known to the skilled worker (see e.g. Jung Y et al.,Analyst133: 697-701 (2008)). Immobilized molecules of the invention may be used for screening applications using techniques known in the art (see e.g. Bradbury A et al.,Nat Biotechnol29: 245-54 (2011); Sutton C,Br J Pharmacol166: 457-75 (2012); Diamante L et al.,Protein Eng Des Sel26: 713-24 (2013); Houlihan G et al.,J Immunol Methods405: 47-56 (2014)). Non-limiting examples of solid substrates to which a molecule of the invention may be immobilized on include: microbeads, nanoparticles, polymers, nanopolymers, nanotubes, magnetic beads, paramagnetic beads, superparamagnetic beads, streptavidin coated beads, reverse-phase magnetic beads, carboxy terminated beads, hydrazine terminated beads, silica (sodium silica) beads and iminodiacetic acid (IDA)-modified beads, aldehyde-modified beads, epoxy-activated beads, diaminodipropylamine (DADPA)-modified beads (beads with primary amine surface group), biodegradable polymeric beads, polystyrene substrates, amino-polystyrene particles, carboxyl-polystyrene particles, epoxy-polystyrene particles, dimethylamino-polystyrene particles, hydroxy-polystyrene particles, colored particles, flow cytometry particles, sulfonate-polystyrene particles, nitrocellulose surfaces, reinforced nitrocellulose membranes, nylon membranes, glass surfaces, activated glass surfaces, activated quartz surfaces, polyvinylidene difluoride (PVDF) membranes, polyacrylamide-based substrates, poly-vinyl chloride substrates, poly-methyl methacrylate substrates, poly(dimethyl siloxane) substrates, and photopolymers which contain photoreactive species (such as nitrenes, carbenes, and ketyl radicals) capable of forming covalent linkages. Other examples of solid substrates to which a molecule of the invention may be immobilized on are commonly used in molecular display systems, such as, e.g., cellular surfaces, phages, and virus particles. VI. Production or Manufacture of Pharmaceutical and/or Diagnostic Compositions Comprising Cell-Targeting Molecules of the Present Invention Pharmaceutically acceptable salts or solvates of any of the Shiga toxin effector polypeptides, Shiga toxin effector polypeptide scaffolds, and cell-targeting molecules of the present invention are within the scope of the present invention. In addition, pharmaceutical compositions comprising a salt or solvate of a cell-targeting molecule(s) of the present invention are within the scope of the present invention. The term “solvate” in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a proteinaceous compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate. Polypeptides and proteins of the present invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a molecule of the present invention, or a salt thereof, in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic molecule use are well known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences(Mack Publishing Co. (A. Gennaro, ed., 1985). As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically acceptable, i.e. compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic, and absorption delaying agents, and the like. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. Exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). Depending on selected route of administration, the protein or other pharmaceutical component may be coated in a material intended to protect the compound from the action of low pH and other natural inactivating conditions to which the active protein may encounter when administered to a patient by a particular route of administration. Therapeutic compositions of the present invention are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, solvate, salt, powder, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixture. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, the pharmaceutical composition of the present invention may comprise one or more isotonic agents, such as, e.g., a sugar, polyalcohol, and/or ions like mannitol, sorbitol, and sodium chloride A pharmaceutical composition of the present invention optionally includes a pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include arginine, arginine sulfate, glycerol, mannitol, methionine, polysorbate, sodium chloride, sorbitol, sucrose, and/or trehalose. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and at least one pharmaceutically acceptable excipient. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising at least one pharmaceutically acceptable excipient. In certain embodiments of the pharmaceutical composition of the present invention, the excipient functions to reduce and/or limit the immunogenicity and/or immunogenic potential of the cell-targeting molecule, such as, e.g. after administration and/or repeated administration to a mammal. The pharmaceutical compositions of the present invention may comprise one or more adjuvants such as a buffer, tonicity-adjusting agent (isotonic agent), antioxidant, surfactant, stabilizer, preservative, emulsifying agent, cryoprotective agent, wetting agent, and/or dispersing agent or other additives well known to those of skill in the art, such as, e.g. a binding agent. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and a pharmaceutically acceptable adjuvant or other additive. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable adjuvant or other additive. A non-limiting example of a pharmaceutically suitable stabilizer is human albumin. The pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable buffers. Non-limiting examples of suitable buffers include acetate, citrate, histidine, phosphate, and succinate buffers. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier comprising a pharmaceutically acceptable buffer. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable buffer. The pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable isotonic agents or tonicity-adjusting agents. Non-limiting examples of suitable isotonic agents include sugars (e.g. dextrose), sugar alcohols, sodium chloride, and the like. Further examples of suitable sugars include disaccharides like sucrose and trehalose. Exemplary, pharmaceutically acceptable sugar alcohols include glycerol, mannitol, and sorbitol. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and a pharmaceutically acceptable isotonic agent. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable isotonic agent. The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable antioxidants. Exemplary pharmaceutically acceptable antioxidants include water soluble antioxidants, such as, e.g., ascorbic acid, cysteine hydrochloride, methionine, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as, e.g., ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal-chelating agents, such as, e.g., citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and a pharmaceutically acceptable antioxidant. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable antioxidant. A pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable surfactants and/or emulsifying agents (emulsifiers). Non-limiting examples of suitable surfactants and/or emulsifiers include polysorbates such as, e.g., polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20) sorbitan monopalmitate (polysorbate 40), polyoxyethylene (20) sorbitan monostearate (polysorbate 60), and (polyoxyethylene (20) sorbitan monooleate (polysorbate 80). In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and a pharmaceutically acceptable surfactant and/or emulsifier. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable surfactant and/or emulsifier. One or more surfactants and/or emulsifying agents may also be desirable in a pharmaceutical composition of the present invention to help prevent aggregation of the cell-targeting molecule of the present invention. The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable preservative agents. For example, preventing the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, such as, e.g., paraben, chlorobutanol, phenol, sorbic acid, and the like in the compositions of the present invention. A pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable cryoprotective agents (cryoprotectants). Non-limiting examples of suitable cryoprotectants include ethylene glycol, glycerol, sucrose, and trehalose. In certain embodiments, the pharmaceutical composition of the present invention comprises an aqueous carrier and a pharmaceutically acceptable cryoprotectant. In certain other embodiments, the pharmaceutical composition of the present invention comprises a salt and/or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and/or cryodessicated composition comprising a pharmaceutically acceptable cryoprotectant. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, e.g, a monostearate salt, aluminum monostearate, and/or gelatin. In another aspect, the present invention provides pharmaceutical compositions comprising one or a combination of different polypeptides and/or cell-targeting molecules of the invention, or an ester, salt or amide of any of the foregoing, and at least one pharmaceutically acceptable carrier. The pH of the pharmaceutical composition of the present invention can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with acetate, citrate, histidine, succinate, phosphate, and the like. Non-limiting examples of pharmaceutically acceptable solvents or carriers for use in a pharmaceutical composition of the present invention include aqueous solutions comprising a cell-targeting molecule of the present invention and a buffer such as, e.g., citrate, histidine, phosphate, or succinate adjusted to pH 5.0, 6.0, 7.0, or 4.0, respectively. Certain embodiments of the present invention include compositions comprising one of the aforementioned solvents and/or carriers of the present invention. Pharmaceutical compositions of the present invention that are solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid, cysteine hydrochloride, methionine, sodium bisulfate, sodium metabisulfite, and sodium sulfite; chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid; surfactants such as a polysorbate; buffers such as acetate, citrate, histidine, and phosphate buffers; and tonicity adjusting agents such as, e.g., dextrose, glycerol, mannitol, sodium chloride, sorbitol, sucrose, and trehalose. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of a glass or plastic. Sterile injectable solutions may be prepared by incorporating a protein or cell-targeting molecule of the present invention in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and other ingredients, such as those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof. In certain embodiments, the pharmaceutical composition of the present invention comprises a powder comprising sorbitol, trehalose, sodium citrate, and polysorbate-20, and optionally, further comprises glycerol and/or methionine. In certain embodiments, the pharmaceutical composition of the present invention comprises sodium citrate, trehalose, and polysorbate-20, and optionally, further comprises glycerol and/or methionine. When a therapeutically effective amount of a polypeptide and/or cell-targeting molecule of the invention is designed to be administered by, e.g. intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. In certain embodiments, the pharmaceutical composition of the present invention comprises sorbitol, sodium citrate, and polysorbate-20, and optionally, further comprises albumin, glycerol, and/or methionine. In certain embodiments, the pharmaceutical composition of the present invention comprises sorbitol, histidine, and polysorbate-20, and optionally, further comprises albumin, glycerol, and/or methionine. The formulations of the pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of an autoinjector or pen. Compositions of the present invention may be formulated for any suitable route and means of administration. Subcutaneous or transdermal modes of administration may be particularly suitable for therapeutic molecules described herein. As described elsewhere herein, a polypeptide and/or cell-targeting molecule of the present invention may be prepared with carriers that will protect the active therapeutic agent against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art (see e.g.Sustained and Controlled Release Drug Delivery Systems(Robinson J, ed., Marcel Dekker, Inc., NY, U.S., 1978)). In certain embodiments, the composition of the present invention (e.g. a pharmaceutical and/or diagnostic composition) may be formulated to ensure a desired in vivo distribution of a cell-targeting molecule of the present invention. For example, the blood-brain barrier excludes many large and/or hydrophilic compounds. To target a therapeutic molecule or composition of the present invention to a particular in vivo location, they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery. Exemplary targeting moieties include folate or biotin, mannosides, antibodies, surfactant protein A receptor, p120 catenin, and the like. Pharmaceutical compositions include parenteral formulations designed to be used as implants or particulate systems. Examples of implants are depot formulations composed of polymeric or hydrophobic components such as emulsions, ion exchange resins, and soluble salt solutions. Examples of particulate systems are microspheres, microparticles, nanocapsules, nanospheres, and nanoparticles (see e.g. Honda M et al.,Int J Nanomedicine8: 495-503 (2013); Sharma A et al.,Biomed Res Int2013: 960821 (2013); Ramishetti S, Huang L,Ther Deliv3: 1429-45 (2012)). Controlled release formulations may be prepared using polymers sensitive to ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite. VII. Polynucleotides, Expression Vectors, and Host Cells of the Present Invention Beyond the polypeptides and cell-targeting molecules of the present invention, the polynucleotides that encode the polypeptides, proteins, and cell-targeting molecules of the invention, or functional portions thereof, are also encompassed within the scope of the present invention. The term “polynucleotide” is equivalent to the term “nucleic acid,” each of which includes one or more of: polymers of deoxyribonucleic acids (DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs or RNAs generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The polynucleotide of the present invention may be single-, double-, or triple-stranded. Such polynucleotides are specifically disclosed to include all polynucleotides capable of encoding an exemplary protein, for example, taking into account the wobble known to be tolerated in the third position of RNA codons, yet encoding for the same amino acid as a different RNA codon (see Stothard P,Biotechniques28: 1102-4 (2000)). In one aspect, the present invention provides polynucleotides which encode a Shiga toxin effector polypeptide and/or cell-targeting molecule of the present invention, or a fragment or derivative thereof. The polynucleotides may include, e.g., a nucleic acid sequence encoding a polypeptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, identical to a polypeptide comprising one of the amino acid sequences of a polypeptide or cell-targeting molecule of the present invention. The invention also includes polynucleotides comprising nucleotide sequences that hybridize under stringent conditions to a polynucleotide which encodes Shiga toxin effector polypeptide and/or cell-targeting molecule of the invention, or a fragment or derivative thereof, or the antisense or complement of any such sequence. Derivatives or analogs of the molecules of the present invention (e.g., Shiga toxin effector polypeptides of the present invention and cell-targeting molecules comprising the same) include, inter alia, polynucleotide (or polypeptide) molecules having regions that are substantially homologous to the polynucleotides (or Shiga toxin effector polypeptides and cell-targeting molecules of the present invention), e.g. by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a polynucleotide (or polypeptide) sequence of the same size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art. An exemplary program is the GAP program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI, U.S.) using the default settings, which uses the algorithm of Smith T, Waterman M,Adv Appl Math2: 482-9 (1981). Also included are polynucleotides capable of hybridizing to the complement of a sequence encoding the cell-targeting proteins of the invention under stringent conditions (see e.g. Ausubel F et al.,Current Protocols in Molecular Biology(John Wiley & Sons, New York, NY, U.S., 1993)), and below. Stringent conditions are known to those skilled in the art and may be found, e.g., inCurrent Protocols in Molecular Biology(John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6 (1989)). The present invention further provides expression vectors that comprise the polynucleotides within the scope of the present invention. The polynucleotides capable of encoding the Shiga toxin effector polypeptides and/or cell-targeting molecules of the invention may be inserted into known vectors, including bacterial plasmids, viral vectors and phage vectors, using material and methods well known in the art to produce expression vectors. Such expression vectors will include the polynucleotides necessary to support production of contemplated Shiga toxin effector polypeptides and/or cell-targeting molecules of the invention within any host cell of choice or cell-free expression systems (e.g. pTxb1 and pIVEX2.3). The specific polynucleotides comprising expression vectors for use with specific types of host cells or cell-free expression systems are well known to one of ordinary skill in the art, can be determined using routine experimentation, and/or may be purchased. The term “expression vector,” as used herein, refers to a polynucleotide, linear or circular, comprising one or more expression units. The term “expression unit” denotes a polynucleotide segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration. An expression vector contains one or more expression units. Thus, in the context of the present invention, an expression vector encoding a Shiga toxin effector polypeptide and/or cell-targeting molecule of the invention comprising a single polypeptide chain includes at least an expression unit for the single polypeptide chain, whereas a protein comprising, e.g. two or more polypeptide chains (e.g. one chain comprising a VLdomain and a second chain comprising a VHdomain linked to a toxin effector polypeptide) includes at least two expression units, one for each of the two polypeptide chains of the protein. For expression of multi-chain cell-targeting proteins of the invention, an expression unit for each polypeptide chain may also be separately contained on different expression vectors (e.g. expression may be achieved with a single host cell into which expression vectors for each polypeptide chain has been introduced). Expression vectors capable of directing transient or stable expression of polypeptides and proteins are well known in the art. The expression vectors generally include, but are not limited to, one or more of the following: a heterologous signal sequence or peptide, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is well known in the art. Optional regulatory control sequences, integration sequences, and useful markers that can be employed are known in the art. The term “host cell” refers to a cell which can support the replication or expression of the expression vector. Host cells may be prokaryotic cells, such asE. colior eukaryotic cells (e.g. yeast, insect, amphibian, bird, or mammalian cells). Creation and isolation of host cell lines comprising a polynucleotide of the invention or capable of producing a polypeptide and/or cell-targeting molecule of the present invention can be accomplished using standard techniques known in the art. Shiga toxin effector polypeptides and/or proteins within the scope of the present invention may be variants or derivatives of the polypeptides and molecules described herein that are produced by modifying the polynucleotide encoding a polypeptide and/or proteinaceous component of a cell-targeting molecule by altering one or more amino acids or deleting or inserting one or more amino acids that may render it more suitable to achieve desired properties, such as more optimal expression by a host cell. VIII. Delivery Devices and Kits In certain embodiments, the invention relates to a device comprising one or more compositions of matter of the present invention, such as a pharmaceutical composition or diagnostic composition, for delivery to a subject in need thereof. Thus, a delivery device comprising one or more compositions of the present invention can be used to administer to a patient a composition of matter of the present invention by various delivery methods, including: intravenous, subcutaneous, intramuscular or intraperitoneal injection; oral administration; transdermal administration; pulmonary or transmucosal administration; administration by implant, osmotic pump, cartridge or micro pump; or by other means recognized by a person of skill in the art. Also within the scope of the present invention are kits comprising at least one composition of matter of the invention, and optionally, packaging and instructions for use. Kits may be useful for drug administration and/or diagnostic information gathering. A kit of the invention may optionally comprise at least one additional reagent (e.g., standards, markers and the like). Kits typically include a label indicating the intended use of the contents of the kit. The kit may further comprise reagents and other tools for detecting a cell type (e.g. a tumor cell) in a sample or in a subject, or for diagnosing whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition, or related method of the present invention, e.g., such as a method described herein. IX. Methods for Using Cell-Targeting Molecules of the Present Invention and/or Pharmaceutical and/or Diagnostic Compositions Thereof Generally, it is an object of the present invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the prevention and/or treatment of diseases, disorders, and conditions, such as certain cancers, tumors, growth abnormalities, immune disorders, or further pathological conditions mentioned herein. Accordingly, the present invention provides methods of using the polypeptides, cell-targeting molecules, and pharmaceutical compositions of the invention for the targeted killing of cells, for delivering additional exogenous materials into targeted cells, for labeling of the interiors of targeted cells, for collecting diagnostic information, for the delivering of T-cell epitopes to the MHC class I presentation pathway of target cells, and for treating diseases, disorders, and conditions as described herein. For example, the methods of the present invention may be used to prevent or treat cancers, cancer initiation, tumor initiation, metastasis, and/or disease reoccurrence. In particular, it is an object of the invention to provide such pharmacologically active agents, compositions, and/or methods that have certain advantages compared to the agents, compositions, and/or methods that are currently known in the art. Accordingly, the present invention provides methods of using Shiga toxin effector polypeptides and cell-targeting molecules with specified protein sequences and pharmaceutical compositions thereof. For example, any of the amino acid sequences in SEQ ID NOs: 4-1140 may be specifically utilized as a component of the cell-targeting molecule used in the following methods or any method for using a cell-targeting molecule known to the skilled worker, such as, e.g., various methods described in WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005; WO 2015/113007, WO 2015/138435, WO 2015/138452, US20150259428, WO 2015/191764, US20160177284, and WO 2016/126950. The present invention provides methods of killing a cell comprising the step of contacting the cell, either in vitro or in vivo, with a Shiga toxin effector polypeptide, cell-targeting molecule, or pharmaceutical composition of the present invention. The Shiga toxin effector polypeptides, cell-targeting molecules, and pharmaceutical compositions of the present invention can be used to kill a specific cell type upon contacting a cell or cells with one of the claimed compositions of matter. In certain embodiments, a cell-targeting molecule or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as mixtures comprising cancer cells, infected cells, and/or hematological cells. In certain embodiments, a cell-targeting molecule, or pharmaceutical composition of the present invention can be used to kill cancer cells in a mixture of different cell types. In certain embodiments, a cytotoxic Shiga cell-targeting molecule, or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as pre-transplantation tissues. In certain embodiments, a Shiga toxin effector polypeptide, cell-targeting molecule, or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of cell types, such as pre-administration tissue material for therapeutic purposes. In certain embodiments, a cell-targeting molecule or pharmaceutical composition of the present invention can be used to selectively kill cells infected by viruses or microorganisms, or otherwise selectively kill cells expressing a particular extracellular target biomolecule, such as a cell surface biomolecule. The Shiga toxin effector polypeptides, cell-targeting molecules, and pharmaceutical compositions of the present invention have varied applications, including, e.g., uses in depleting unwanted cell types from tissues either in vitro or in vivo, uses in modulating immune responses to treat graft versus host, uses as antiviral agents, uses as anti-parasitic agents, and uses in purging transplantation tissues of unwanted cell types. In certain embodiments, certain Shiga toxin effector polypeptides, cell-targeting molecules, and pharmaceutical compositions of the present invention, alone or in combination with other compounds or pharmaceutical compositions, can show potent cell-kill activity when administered to a population of cells, in vitro or in vivo in a subject such as in a patient in need of treatment. By targeting the delivery of enzymatically active Shiga toxin A Subunit effector polypeptides and/or T-cell epitopes using high-affinity binding regions to specific cell types, cell-kill activities can be restricted to specifically and selectively killing certain cell types within an organism, such as certain cancer cells, neoplastic cells, malignant cells, non-malignant tumor cells, and/or infected cells. The present invention provides a method of killing a cell in a patient in need thereof, the method comprising the step of administering to the patient at least one cell-targeting molecule of the present invention or a pharmaceutical composition thereof. In certain embodiments, the cell-targeting molecule of the present invention or pharmaceutical compositions thereof can be used to kill a cancer cell in a patient by targeting an extracellular biomolecule found physically coupled with a cancer or tumor cell. The terms “cancer cell” or “cancerous cell” refers to various neoplastic cells which grow and divide in an abnormally accelerated and/or unregulated fashion and will be clear to the skilled person. The term “tumor cell” includes both malignant and non-malignant cells. Generally, cancers and/or tumors can be defined as diseases, disorders, or conditions that are amenable to treatment and/or prevention. The cancers and tumors (either malignant or non-malignant) which are comprised of cancer cells and/or tumor cells which may benefit from methods and compositions of the invention will be clear to the skilled person. Neoplastic cells are often associated with one or more of the following: unregulated growth, lack of differentiation, local tissue invasion, angiogenesis, and metastasis. The diseases, disorders, and conditions resulting from cancers and/or tumors (either malignant or non-malignant) which may benefit from the methods and compositions of the present invention targeting certain cancer cells and/or tumor cells will be clear to the skilled person. Certain embodiments of the cell-targeting molecules and compositions of the present invention may be used to kill cancer stem cells, tumor stem cells, pre-malignant cancer-initiating cells, and tumor-initiating cells, which commonly are slow dividing and resistant to cancer therapies like chemotherapy and radiation. For example, acute myeloid leukemias (AMLs) may be treated with the present invention by killing AML stem cells and/or dormant AML progenitor cells (see e.g. Shlush L et al.,Blood120: 603-12 (2012)). Cancer stem cells often overexpress cell surface targets, such as, e.g., CD44, CD200, and others listed herein, which can be targets of certain binding regions of certain embodiments of the cell-targeting molecules of the present invention (see e.g. Kawasaki B et al.,Biochem Biophys Res Commun364:778-82 (2007); Reim F et al.,Cancer Res69: 8058-66 (2009)). Because of the Shiga toxin A Subunit based mechanism of action, compositions of matter of the present invention may be more effectively used in methods involving their combination with, or in complementary fashion with other therapies, such as, e.g., chemotherapies, immunotherapies, radiation, stem cell transplantation, and immune checkpoint inhibitors, and/or effective against chemoresistant/radiation-resistant and/or resting tumor cells/tumor initiating cells/stem cells. Similarly, compositions of matter of the present invention may be more effectively used in methods involving in combination with other cell-targeted therapies targeting other than the same epitope on, non-overlapping, or different targets for the same disease disorder or condition. Certain embodiments of the cell-targeting molecules of the present invention, or pharmaceutical compositions thereof, can be used to kill an immune cell (whether healthy or malignant) in a patient by targeting an extracellular biomolecule found physically coupled with an immune cell. It is within the scope of the present invention to utilize a cell-targeting molecule of the present invention, or pharmaceutical composition thereof, for the purposes of purging patient cell populations (e.g. bone marrow) of malignant, neoplastic, or otherwise unwanted T-cells and/or B-cells and then reinfusing the T-cell and/or B-cells depleted material into the patient (see e.g. van Heeckeren W et al.,Br J Haematol132: 42-55 (2006); (see e.g. Alpdogan O, van den Brink M,Semin Oncol39: 629-42 (2012)). It is within the scope of the present invention to utilize the cell-targeting molecule of the present invention, or pharmaceutical composition thereof, for the purposes of ex vivo depletion of T cells and/or B-cells from isolated cell populations removed from a patient. In one non-limiting example, the cell-targeting molecule of the invention can be used in a method for prophylaxis of organ and/or tissue transplant rejection wherein the donor organ or tissue is perfused prior to transplant with a cytotoxic, cell-targeting molecule of the invention or a pharmaceutical composition thereof in order to purge the organ of donor T-cells and/or B-cells (see e.g. Alpdogan O, van den Brink M,Semin Oncol39: 629-42 (2012)). It is also within the scope of the present invention to utilize the cell-targeting molecule of the invention, or pharmaceutical composition thereof, for the purposes of depleting T-cells and/or B-cells from a donor cell population as a prophylaxis against graft-versus-host disease, and induction of tolerance, in a patient to undergo a bone marrow and or stem cell transplant (see e.g. van Heeckeren W et al.,Br J Haematol132: 42-55 (2006); (see e.g. Alpdogan O, van den Brink M,Semin Oncol39: 629-42 (2012)). In certain embodiments of the Shiga toxin effector polypeptide or cell-targeting molecule of the present invention, or pharmaceutical compositions thereof, can be used to kill an infected cell in a patient by targeting an extracellular biomolecule found physically coupled with an infected cell. In certain embodiments of the cell-targeting molecules of the present invention, or pharmaceutical compositions thereof, can be used to “seed” a locus within a chordate with non-self, T-cell epitope-peptide presenting cells in order to activate the immune system to enhance policing of the locus. In certain further embodiments of this “seeding” method of the present invention, the locus is a tumor mass or infected tissue site. In preferred embodiments of this “seeding” method of the present invention, the non-self, T-cell epitope-peptide is selected from the group consisting of: peptides not already presented by the target cells of the cell-targeting molecule, peptides not present within any protein expressed by the target cell, peptides not present within the proteome or transcriptome of the target cell, peptides not present in the extracellular microenvironment of the site to be seeded, and peptides not present in the tumor mass or infect tissue site to be targeting. This “seeding” method functions to label one or more target cells within a chordate with one or more MHC class I presented T-cell epitopes for recognition by effector T-cells and activation of downstream immune responses. By exploiting the cell internalizing, intracellularly routing, and T-cell epitope delivering functions of the cell-targeting molecules of the present invention, the target cells which display the delivered T-cell epitope are harnessed to induce recognition of the presenting target cell by host T-cells and induction of further immune responses including target-cell-killing by CTLs. This “seeding” method of using a cell-targeting molecule of the present invention can provide a temporary vaccination-effect by inducing adaptive immune responses to attack the cells within the seeded microenvironment, such as, e.g. a tumor mass or infected tissue site, whether presenting a cell-targeting molecule-delivered T-cell epitope(s) or not. This “seeding” method may also induce the breaking of immuno-tolerance to a target cell population, a tumor mass, and/or infected tissue site within a chordate. Certain methods of the present invention involving the seeding of a locus within a chordate with one or more antigenic and/or immunogenic epitopes may be combined with the administration of immunologic adjuvants, whether administered locally or systemically, to stimulate the immune response to certain antigens, such as, e.g., the co-administration of a composition of the present invention with one or more immunologic adjuvants like a cytokine, bacterial product, or plant saponin. Other examples of immunologic adjuvants which may be suitable for use in the methods of the present invention include aluminum salts and oils, such as, e.g., alums, aluminum hydroxide, mineral oils, squalene, paraffin oils, peanut oils, and thimerosal. Additionally, the present invention provides a method of treating a disease, disorder, or condition in a patient comprising the step of administering to a patient in need thereof a therapeutically effective amount of at least one of the cell-targeting molecules of the present invention, or a pharmaceutical composition thereof. Contemplated diseases, disorders, and conditions that can be treated using this method include cancers, malignant tumors, non-malignant tumors, growth abnormalities, immune disorders, and microbial infections. Administration of a “therapeutically effective dosage” of a composition of the present invention can result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The therapeutically effective amount of a composition of the present invention will depend on the route of administration, the type of organism being treated, and the physical characteristics of the specific patient under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person. An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g. topical administration of a cream, gel or ointment, or by means of a transdermal patch). “Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration. For administration of a pharmaceutical composition of the present invention, the dosage range will generally be from about 0.001 to 10 milligrams per kilogram (mg/kg), and more, usually 0.001 to 0.5 mg/kg, of the subject's body weight. Exemplary dosages may be 0.01 mg/kg body weight, 0.03 mg/kg body weight, 0.07 mg/kg body weight, 0.9 mg/kg body weight or 0.1 mg/kg body weight or within the range of 0.01 to 0.1 mg/kg. An exemplary treatment regime is a once or twice daily administration, or a once or twice weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months or once every three to 6 months. Dosages may be selected and readjusted by the skilled health care professional as required to maximize therapeutic benefit for a particular patient. Pharmaceutical compositions of the present invention will typically be administered to the same patient on multiple occasions. Intervals between single dosages can be, for example, two to five days, weekly, monthly, every two or three months, every six months, or yearly. Intervals between administrations can also be irregular, based on regulating blood levels or other markers in the subject or patient. Dosage regimens for a composition of the present invention include intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight with the composition administered every two to four weeks for six dosages, then every three months at 3 mg/kg body weight or 1 mg/kg body weight. A pharmaceutical composition of the present invention may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for cell-targeting molecules and pharmaceutical compositions of the present invention include, e.g. intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. For other embodiments, a cell-targeting molecule or pharmaceutical composition of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Therapeutic cell-targeting molecules or pharmaceutical compositions of the present invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art. The cell-targeting molecule or pharmaceutical composition of the present invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include a cell-targeting molecule of the present invention, or pharmaceutical composition thereof, combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated. Examples of other such agents include, inter alia, a cytotoxic, anti-cancer or chemotherapeutic agent, an anti-inflammatory or anti-proliferative agent, an antimicrobial or antiviral agent, growth factors, cytokines, an analgesic, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates one or more signaling pathways, and similar modulating therapeutic molecules which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen. Treatment of a patient with cell-targeting molecule or pharmaceutical composition of the present invention preferably leads to cell death of targeted cells and/or the inhibition of growth of targeted cells. As such, cytotoxic, cell-targeting molecules of the present invention, and pharmaceutical compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which killing or depleting target cells may be beneficial, such as, inter alia, cancer, tumors, other growth abnormalities, immune disorders, and infected cells. The present invention provides methods for suppressing cell proliferation, and treating cell disorders, including neoplasia, overactive B-cells, and overactive T-cells. In certain embodiments, the cell-targeting molecules and pharmaceutical compositions of the present invention can be used to treat or prevent cancers, tumors (malignant and non-malignant), growth abnormalities, immune disorders, and microbial infections. In a further aspect, the above ex vivo method can be combined with the above in vivo method to provide methods of treating or preventing rejection in bone marrow transplant recipients, and for achieving immunological tolerance. In certain embodiments, the present invention provides methods for treating malignancies or neoplasms and other blood cell associated cancers in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a cytotoxic cell-targeting molecule or pharmaceutical composition of the present invention. The cell-targeting molecules and pharmaceutical compositions of the present invention have varied applications, including, e.g., uses in removing unwanted T-cells, uses in modulating immune responses to treat graft versus host, uses as antiviral agents, uses as antimicrobial agents, and uses in purging transplantation tissues of unwanted cell types. The cell-targeting molecules and pharmaceutical compositions of the present invention are commonly anti-neoplastic agents—meaning they are capable of treating and/or preventing the development, maturation, or spread of neoplastic or malignant cells by inhibiting the growth and/or causing the death of cancer or tumor cells. In certain embodiments, the cell-targeting molecule or pharmaceutical composition of the present invention is used to treat a B-cell-, plasma cell- or antibody-mediated disease or disorder, such as for example leukemia, lymphoma, myeloma, Human Immunodeficiency Virus-related diseases, amyloidosis, hemolytic uremic syndrome, polyarteritis, septic shock, Crohn's Disease, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, psoriasis, asthma, Sjögren's syndrome, graft-versus-host disease, graft rejection, diabetes, vasculitis, scleroderma, and systemic lupus erythematosus. In another aspect, certain embodiments of the cell-targeting molecules and pharmaceutical compositions of the present invention are antimicrobial agents—meaning they are capable of treating and/or preventing the acquisition, development, or consequences of microbiological pathogenic infections, such as caused by viruses, bacteria, fungi, prions, or protozoans. It is within the scope of the present invention to provide a prophylaxis or treatment for diseases or conditions mediated by T-cells or B-cells by administering the cell-targeting molecule the present invention, or a pharmaceutical composition thereof, to a patient for the purpose of killing T-cells or B-cells in the patient. This usage is compatible with preparing or conditioning a patient for bone marrow transplantation, stem cell transplantation, tissue transplantation, or organ transplantation, regardless of the source of the transplanted material, e.g. human or non-human sources. It is within the scope of the present invention to provide a bone marrow recipient for prophylaxis or treatment of host-versus-graft disease via the targeted cell-killing of host T-cells using a cytotoxic cell-targeting molecule or pharmaceutical composition of the present invention. Certain embodiments of the cell-targeting molecules and pharmaceutical compositions of the present invention can be utilized in a method of treating cancer comprising administering to a patient, in need thereof, a therapeutically effective amount of a cell-targeting molecule and/or pharmaceutical composition of the present invention. In certain embodiments of the methods of the present invention, the cancer being treated is selected from the group consisting of: bone cancer (such as multiple myeloma or Ewing's sarcoma), breast cancer, central/peripheral nervous system cancer (such as brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancer and bladder cancer), liver cancer, lung/pleura cancer (such as mesothelioma, small cell lung carcinoma, or non-small cell lung carcinoma), prostate cancer, sarcoma (such as angiosarcoma, fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (such as basal cell carcinoma, squamous cell carcinoma, or melanoma), and uterine cancer. Certain embodiments of the cell-targeting molecules and pharmaceutical compositions of the present invention can be utilized in a method of treating an immune disorder comprising administering to a patient, in need thereof, a therapeutically effective amount of the cell-targeting molecules and/or pharmaceutical composition of the present invention. In certain embodiments of the methods of the present invention, the immune disorder is related to an inflammation associated with a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-vs.-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjögren's syndrome, ulcerative colitis, and vasculitis. Among certain embodiments of the present invention is using the Shiga toxin effector polypeptide or cell-targeting molecule of the present invention as a component of a pharmaceutical composition or medicament for the treatment or prevention of a cancer, tumor, other growth abnormality, immune disorder, and/or microbial infection. For example, immune disorders presenting on the skin of a patient may be treated with such a medicament in efforts to reduce inflammation. In another example, skin tumors may be treated with such a medicament in efforts to reduce tumor size or eliminate the tumor completely. Certain cytotoxic cell-targeting molecules of the present invention, and compositions thereof, may be used in molecular neurosurgery applications such as immunolesioning and neuronal tracing. For example, the targeting domain may be selected or derived from various ligands, such as neurotransmitters and neuropeptides, which target specific neuronal cell types by binding neuronal surface receptors, such as a neuronal circuit specific G-protein coupled receptor. Similarly, the targeting domain may be selected from or derived from antibodies that bind neuronal surface receptors. Because certain Shiga toxin effector polypeptides robustly direct their own retrograde axonal transport, certain cell-targeting molecules of the present invention may be used to kill a neuron(s) which expresses the extracellular target at a site of cytotoxic protein injection distant from the cell body, These targeted cytotoxic molecules of the invention that specifically target neuronal cell types have uses in neuroscience research, such as for elucidating mechanisms of sensations, and creating model systems of neurodegenerative diseases, such as Parkinson's and Alzheimer's. Among certain embodiment of the present invention is a method of using a Shiga toxin effector polypeptide, cell-targeting molecule, pharmaceutical composition, and/or diagnostic composition of the present invention to label or detect the interiors of neoplastic cells and/or immune cell types. This method may be based on the ability of certain cell-targeting molecules of the present invention to enter specific cell types and route within cells via retrograde intracellular transport, to the interior compartments of specific cell types are labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g. biopsy material. Among certain embodiment of the present invention is a method of using a Shiga toxin effector polypeptide, cell-targeting molecule, pharmaceutical composition, and/or diagnostic composition of the present invention to detect the presence of a cell type for the purpose of information gathering regarding diseases, conditions and/or disorders. The method comprises contacting a cell with a diagnostically sufficient amount of a cell-targeting molecule of the present invention in order to detect the molecule by an assay or diagnostic technique. The phrase “diagnostically sufficient amount” refers to an amount that provides adequate detection and accurate measurement for information gathering purposes by the particular assay or diagnostic technique utilized. Generally, the diagnostically sufficient amount for whole organism in vivo diagnostic use will be a non-cumulative dose of between 0.001 to 10 milligrams of the detection-promoting agent linked cell-targeting molecule of the invention per kg of subject per subject. Typically, the amount of Shiga toxin effector polypeptide or cell-targeting molecule of the invention used in these information gathering methods will be as low as possible provided that it is still a diagnostically sufficient amount. For example, for in vivo detection in an organism, the amount of Shiga toxin effector polypeptide, cell-targeting molecule, or pharmaceutical composition of the invention administered to a subject will be as low as feasibly possible. The cell-type specific targeting of cell-targeting molecules of the present invention combined with detection-promoting agents provides a way to detect and image cells physically coupled with an extracellular target biomolecule of a binding region of the molecule of the invention. Imaging of cells using the cell-targeting molecules of the present invention may be performed in vitro or in vivo by any suitable technique known in the art. Diagnostic information may be collected using various methods known in the art, including whole body imaging of an organism or using ex vivo samples taken from an organism. The term “sample” used herein refers to any number of things, but not limited to, fluids such as blood, urine, serum, lymph, saliva, anal secretions, vaginal secretions, and semen, and tissues obtained by biopsy procedures. For example, various detection-promoting agents may be utilized for non-invasive in vivo tumor imaging by techniques such as magnetic resonance imaging (MRI), optical methods (such as direct, fluorescent, and bioluminescent imaging), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, x-ray computed tomography, and combinations of the aforementioned (see, Kaur S et al.,Cancer Lett315: 97-111 (2012), for review). Among certain embodiment of the present invention is a method of using a Shiga toxin effector polypeptide, cell-targeting molecule, or pharmaceutical composition of the present invention in a diagnostic composition to label or detect the interiors of a hematologic cell, cancer cell, tumor cell, infected cell, and/or immune cell (see e.g., Koyama Y et al.,Clin Cancer Res13: 2936-45 (2007); Ogawa M et al.,Cancer Res69: 1268-72 (2009); Yang L et al.,Small5: 235-43 (2009)). Based on the ability of certain cell-targeting molecules of the invention to enter specific cell types and route within cells via retrograde intracellular transport, the interior compartments of specific cell types are labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g. biopsy material. Diagnostic compositions of the present invention may be used to characterize a disease, disorder, or condition as potentially treatable by a related pharmaceutical composition of the present invention. In certain compositions of matter of the present invention may be used to determine whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition or related method of the present invention as described herein or is well suited for using a delivery device of the invention. Diagnostic compositions of the present invention may be used after a disease, e.g. a cancer, is detected in order to better characterize it, such as to monitor distant metastases, heterogeneity, and stage of cancer progression. The phenotypic assessment of disease disorder or infection can help prognostic and prediction during therapeutic decision making. In disease reoccurrence, certain methods of the invention may be used to determine if local or systemic problem. Diagnostic compositions of the present invention may be used to assess responses to therapies regardless of the type of the type of therapy, e.g. small molecule drug, biological drug, or cell-based therapy. For example, certain embodiments of the diagnostics of the invention may be used to measure changes in tumor size, changes in antigen positive cell populations including number and distribution, or monitoring a different marker than the antigen targeted by a therapy already being administered to a patient (see Smith-Jones P et al.,Nat. Biotechnol22: 701-6 (2004); Evans M et al.,Proc. Natl. Acad. Sci. USA108: 9578-82 (2011)). In certain embodiments of the method used to detect the presence of a cell type may be used to gather information regarding diseases, disorders, and conditions, such as, for example bone cancer (such as multiple myeloma or Ewing's sarcoma), breast cancer, central/peripheral nervous system cancer (such as brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancer and bladder cancer), liver cancer, lung/pleura cancer (such as mesothelioma, small cell lung carcinoma, or non-small cell lung carcinoma), prostate cancer, sarcoma (such as angiosarcoma, fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (such as basal cell carcinoma, squamous cell carcinoma, or melanoma), uterine cancer, AIDS, amyloidosis, ankylosing spondylitis, asthma, autism, cardiogenesis, Crohn's disease, diabetes, erythematosus, gastritis, graft rejection, graft-versus-host disease, Grave's disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, lymphoproliferative disorders(including post-transplant lymphoproliferative disorders), multiple sclerosis, myasthenia gravis, neuroinflammation, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjögren's syndrome, systemic lupus erythematosus, ulcerative colitis, vasculitis, cell proliferation, inflammation, leukocyte activation, leukocyte adhesion, leukocyte chemotaxis, leukocyte maturation, leukocyte migration, neuronal differentiation, acute lymphoblastic leukemia (ALL), T acute lymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloid leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B-cell prolymphocytic lymphoma, Burkitt's lymphoma (BL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), intravascular large B-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM), natural killer cell leukemia, nodal marginal B-cell lymphoma, Non-Hodgkin's lymphoma (NHL), plasma cell leukemia, plasmacytoma, primary effusion lymphoma, pro-lymphocytic leukemia, promyelocytic leukemia, small lymphocytic lymphoma, splenic marginal zone lymphoma, T-cell lymphoma (TCL), heavy chain disease, monoclonal gammopathy, monoclonal immunoglobulin deposition disease, myelodusplastic syndromes (MDS), smoldering multiple myeloma, and Waldenstrom macroglobulinemia. The present invention is further illustrated by the following non-limiting examples of 1) Shiga toxin effector polypeptides of the present invention, 2) cell-targeting molecules of the present invention, and 3) cytotoxic, cell-targeting molecules of the present invention comprising the aforementioned polypeptides and capable of specifically targeting certain cell types. EXAMPLES The following examples demonstrate certain embodiments of the present invention. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this invention. The experiments in the following examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Site-specific conjugation strategies based on unique amino acid residues can help control homogeneity of the product(s) of a conjugation reaction, such as, e.g., strategies using engineered cysteine residues, unnatural amino acid residues, and/or enzymatic conjugation. By using unique conjugation sites, heterogeneity may be minimized and the resulting compositions become more consistent from batch to batch and have predictable properties. These examples describe the creation and testing of various molecules, each comprising an amino acid residue for site-specific conjugation to various molecules, such as, e.g., a unique, free cysteine or lysine residue in a Shiga toxin effector polypeptide or Shiga toxin effector polypeptide scaffold, whether the unique, free cysteine or lysine residue is positioned ectopically or at a naturally occurring position). In certain examples, the Shiga toxin effector polypeptides are de-immunized as described in WO 2015/113007. In certain examples, the Shiga toxin effector polypeptides comprise an embedded, heterologous, CD8+ T-cell epitope as described in WO 2015/113005. In certain examples, the Shiga toxin effector polypeptides comprise a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment derived region that confers reduced protease cleavage sensitivity to the Shiga toxin effector polypeptide as a component of certain molecules as described in WO 2015/191764. In certain examples, the Shiga toxin effector polypeptides are de-immunized, comprise an embedded, heterologous, CD8+ T-cell epitope, and comprise a disrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxin A1 fragment as described in WO 2016/196344. Exemplary, Shiga toxin effector polypeptides were tested for the retention of Shiga toxin effector function(s) as components of exemplary, cell-targeting molecules of the present invention. The retention of potent cytotoxicity by a Shiga toxin effector polypeptide component of a cell-targeting molecules implicates the retention by that Shiga toxin effector polypeptide of a certain, minimum level of retrograde intracellular routing to the cytosol. The presence of free cysteine residues in exemplary cell-targeting molecules was demonstrated by the formation of intermolecular disulfide bonds linking cell-targeting protein monomers. The presence of one or more ectopic, free cysteine residues and/or a unique lysine residue in a Shiga toxin A Subunit effector scaffold provides an amenable attachment point for linking various molecules to the Shiga toxin effector polypeptide scaffold at specific residues in a controlled fashion. Such linked molecules may be (1) cell-targeting agents; (2) cell-targeting, proteinaceous molecules; (3) cargos designed for intracellular delivery, including for controlled liberation; and/or (4) agents having extracellular function(s), such as, e.g., biologically inert moieties which prolong half-life in a vertebrate and/or mask immunogenic portions of the scaffold. The conjugation of cell-targeting molecules comprising Shiga toxin A Subunit effectors to a cargo (such as a cytotoxic “payload”) allows for the targeted delivery of the cargo or “payload” to an internal location of target cells. Examples of cargos include DNA, RNA, nucleic acid complexes, enzymes, proteins, peptides, fluorescent proteins or peptides, and cytotoxic small molecules. Certain examples show exemplary, cell-targeting molecules comprising a Shiga toxin effector polypeptide having an ectopic, cysteine residue conjugated using standard techniques to a cargo, such as, e.g., a peptide, nucleic acid, protein, protein-nucleic acid complex, cytotoxic agent, antibiotic, and/or detection-promoting agent. Example 1. Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention Comprising a Cysteine Residue(s) for Site-Specific Conjugation In this Example, exemplary Shiga toxin A Subunit effector polypeptides of the present invention (SLT-1A-Cys(p)-variant), where Cys(p) represents a cysteine residue engineered at a unique position), each comprising one ectopic cysteine residue, were created and tested as components of exemplary cell-targeting molecules of the present invention. A. Constructing Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention by Adding Free Cysteine Residues Via Amino Acid Residue Substitution This section describes the creation of various scaffolds comprising Shiga toxin effector polypeptides, each comprising one ectopic, cysteine residue. The ectopic, cysteine residues were engineered into Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. SLT-1A-Cys(p)-variant::scFv(n)) were created using these Shiga toxin effector polypeptides. In this Example, cell-targeting molecules, each comprising a Shiga toxin A Subunit effector polypeptide having one ectopic, cysteine residue, were created and tested. These cell-targeting molecules each comprised a cell-targeting, immunoglobulin-type, binding region comprising a polypeptide capable of binding to an extracellular target biomolecule with high-affinity. The parental, Shiga toxin effector polypeptide of this Example is the A1 fragment of Shiga-like toxin (SLT-1A1) comprising the substitution C242S to remove the only endogenous, cysteine residue (SEQ ID NO:4). The mutation of the cysteine at position 242 to a serine (C242S) had no apparent effect on catalytic activity of the A1 fragment. Using standard techniques, the parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides, each having one ectopic cysteine residue and no other cysteine residues (see e.g. Table 1). Cell-targeting molecules (see e.g. SEQ ID NOs: 773-783) comprising Shiga toxin effector polypeptides (see e.g. SEQ ID NOs: 5-84) having exactly one of the ectopic cysteine residues described in Table 1 were made using standard techniques and tested in one or more of the experiments described below. TABLE 1Ectopic, Cysteine Residues Engineered into Shiga Toxin Effector PolypeptidesExemplary, Shiga toxinCysteineSequence Variantseffector polypeptidesSubstitution(SLT-1A-Cys(p)-variant))SLT-1A-Cys1K1CSEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105,and 115SLT-1A-Cys2S8CSEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106,and 116SLT-1A-Cys3S16CSEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107,and 117SLT-1A-Cys4S22CSEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108,and 118SLT-1A-Cys5S33CSEQ ID NOs: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109,119, and 1101SLT-1A-Cys6S43CSEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,and 120SLT-1A-Cys7S45CSEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111,121, and 1102SLT-1A-Cys8S146CSEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112,122, and 1103SLT-1A-Cys9S186CSEQ ID NOs: 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 123,and 1104SLT-1A-Cys10V54CSEQ ID NOs: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114,and 124 B. Testing Shiga Toxin a Subunit Effector Polypeptides for Retention of Shiga Toxin Functions Shiga toxin effector polypeptides and molecules comprising the same can be tested for Shiga toxin functions using techniques known to the skilled worker (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/113005; WO 2015/113007; WO 2015/138435, WO 2015/138452, WO 2015/191764, US20160177284; WO 2016/126950). Exemplary, cell-targeting molecules were tested for Shiga toxin A Subunit functions. The Shiga toxin A Subunit functions analyzed were: catalytic activity, inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. 1. Testing Shiga Toxin Catalytic Activity and Ability to Inhibit Ribosome Function The catalytic activities of Shiga toxin effector polypeptide components of exemplary, cell-targeting molecules were tested using a ribosome inhibition assay. Catalytic activities were tested for exemplary, cell-targeting molecules comprising exemplary, Shiga toxin effector polypeptides of the present invention (SEQ ID NOs: 9, 11, and 14). The ribosome inactivation capabilities of exemplary, cell-targeting molecules of this Example were determined using a cell-free, in vitro protein translation assay based on the TNT® Quick Coupled Transcription/Translation Kit (L1170, Promega Corp., Madison, WI, U.S.). The ribosome activity reaction was prepared according to manufacturer's instructions. A series of 10-fold dilutions of the cell-targeting molecule to be tested was prepared in an appropriate buffer and a series of identical, TNT reaction mixtures were created for each dilution. Each sample in the dilution series was combined with each of the TNT reaction mixtures along with Luciferase T7 Control DNA (L4821, Promega Corp., Madison, WI, U.S.). The test samples were incubated for 1.5 hours at 30 degrees Celsius (° C.). After the incubation, Luciferase Assay Reagent (E1483, Promega Corp., Madison, WI, U.S.) was added to all test samples and the amount of luciferase protein present was measured by luminescence according to manufacturer's instructions. A positive control was tested: the wild-type, Shiga-like toxin A1 fragment (SLT-1A1-WT) (SEQ ID NO:830). This cell-free, in vitro, protein translation assay was used to determine the ribosome inactivation capabilities of SLT-1A-Cys5::scFv1 (SEQ ID NO:781), SLT-1A-Cys7::scFv1 (SEQ ID NO:782), and SLT-1A-Cys10::scFv1 (SEQ ID NO:783). The level of translational inhibition was determined by non-linear regression analysis of log-transformed concentrations of total protein versus relative luminescence units (RLU). Using Prism software (GraphPad Prism, San Diego, CA, U.S.), the half maximal inhibitory concentration (IC50) value was calculated for each sample using the Prism software function of log(inhibitor) vs. response (three parameters) [Y=Bottom+((Top−Bottom)/(1+10{circumflex over ( )}(X−Log IC50)))] under the heading dose-response-inhibition. The IC50values for each cell-targeting molecule from one or more experiments was calculated and is shown in Table 2 in picomolar (pM). In this assay, measurements of the inhibition of protein synthesis represent the ribosome inactivation activity of the sample molecule, which is one metric of the catalytic activity of a Shiga toxin effector polypeptide. Any molecule which exhibits an IC50within 10-fold of a reference molecule is considered herein to exhibit ribosome inhibition activity comparable to the reference molecule. As reported in the Examples, a molecule exhibiting an IC50less than or within 10 percent of an IC50exhibited by a reference molecule is considered to exhibit ribosome inhibition activity equivalent to that reference molecule. TABLE 2Ribosome Inhibition Activities ofExemplary, Cell-Targeting MoleculesCysteineRibosome InhibitionMolecule TestedpositionIC50(pM)SLT-1A-Cys5::scFv1C33190(SEQ ID NO: 781)SLT-1A-Cys7::scFv1C45109(SEQ ID NO: 782)SLT-1A-Cys10::scFv1C5478(SEQ ID NO: 783)SLT-1A1-WT (wild-type)none185(SEQ ID NO: 830) The ribosome inactivation activities of all the Shiga toxin effector polypeptides SLT-1A-Cys(p) tested in the context of a cell-targeting molecule were equivalent to the catalytic activity of a wild-type, Shiga toxin A1 fragment (Table 2). As shown in Table 2, exemplary cell-targeting molecules comprising or consisting of SEQ ID NO: 781, 782, or 783 exhibited potent ribosome inhibition equivalent to the positive control of an isolated Shiga toxin A Subunit effector polypeptide consisting of a wild-type, Shiga toxin A1 fragment (SEQ ID NO:830). The results of this assay showed that multiple, exemplary, Shiga toxin effector polypeptides of the present invention (SEQ ID NOs: 9, 11, and 14) exhibited catalytic activity in the context of a cell-targeting molecule equivalent to the activity of a wild-type, Shiga toxin effector polypeptide that comprised a single, endogenous cysteine residue at position 242 (Table 2). In other experiments, it was demonstrated that cell-targeting molecules comprising mutated Shiga toxin effector polypeptides having no cysteine residues or only a single free cysteine residue, the endogenous cysteine residue at position 242 (e.g. a wild-type SLT-1A1 (amino acids 1-251 of SEQ ID NO: 1)), exhibited catalytic activity comparable to the activity of each other and to the activity of a cell-targeting molecule comprising a wild-type, Shiga toxin effector polypeptide (amino acids 1-251 of SEQ ID NO:1). 2. Testing the Cytotoxic Activities of Exemplary, Cell-Targeting Molecules of the Invention The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules were tested to assess the functions of their Shiga toxin effector polypeptide components. The cytotoxic activities of exemplary, cell-targeting molecules, which each comprised a Shiga toxin effector polypeptide SLT-1A-Cys(p), were determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker (“cell-kill assay”). The cytotoxicities of exemplary, cell-targeting molecules were determined using cells expressing, at a cellular surface, significant amounts of the appropriate, extracellular target biomolecule, such as, a target of the binding region scFv1, scFv2, scFv3, and/or scFv4. The cells used in this Example were immortalized, human tumor cells available from the ATCC (Manassas VA, U.S.), National Cancer Institute of the U.S. (Frederick, MD, U.S.), and/or DSZM (Braunschweig, DE). The cells referred to below were HCC-1954, MDA-MB-231, H929, ST486, L1236, HCC827, Daudi, HDLM-2, and U-266, or more simply cell-lines A, B, C, D, E, F, G, H and I, respectively. Certain cell-targeting molecules were tested using cell-kill assays involving both target biomolecule positive and target biomolecule negative cells with respect to the target biomolecule of each cell-targeting molecule's binding region. The cytotoxicity assays were performed as follows. Certain, human tumor cell-line cells were plated (typically at 2×103cells per well for adherent cells, plated the day prior to cell-targeting molecule addition, or 7.5×103cells per well for suspension cells, plated the same day as cell-targeting molecule addition) in 20 μL cell culture medium in 384-well plates. A series of 10-fold dilutions of the molecules to be tested was prepared in an appropriate buffer, and 5 μL of the dilutions or buffer control were added to the plated cells. Control wells containing only cell culture medium were used for baseline correction. The cell samples were incubated with the cell-targeting molecule or just buffer for 3 or 5 days at 37° C. and in an atmosphere of 5% carbon dioxide (CO2). The total cell survival or percent viability was determined using a luminescent readout using the CellTiter-Glo® Luminescent Cell Viability Assay or RealTime-Glo® MT Cell Viability Assay (Promega Corp., Madison, WI, U.S.) according to the manufacturer's instructions. The Percent Viability of experimental wells was calculated using the following equation: (Test RLU−Average Media RLU)÷(Average Cells RLU−Average Media RLU)×100. The logarithm of the cell-targeting molecule protein concentration versus Percent Viability was plotted in Prism (GraphPad Prism, San Diego, CA, U.S.) and log (inhibitor) versus response (3 parameter) analysis or and log (inhibitor) versus normalized response analysis were used to determine the half-maximal cytotoxic concentration (CD50) value for the tested molecule. The CD50value(s) for each molecule tested were calculated, when possible, and shown in Table 3. When CD50values could not be calculated based on the shape of the curve over the concentrations tested, then a maximum CD50value was noted as being beyond the maximum tested value, e.g., greater than 100 nanomolar (“>100 nM”) or 200 nanomolar (“>200 nM”), for samples which did not kill 50% of the cells at the highest, tested, sample concentration, e.g., 100 or 200 nanomolar (nM). In some experiments, biomolecule target negative cells treated with the maximum concentration of the cell-targeting molecule did not show any change in viability as compared to a buffer only control. As reported in the Examples herein, a molecule exhibiting a CD50within 10-fold of a CD50exhibited by a reference molecule is considered to exhibit cytotoxic activity comparable to that reference molecule. Cell-targeting molecules that exhibited a CD50to a biomolecule target positive cell population within 100-fold to 10-fold of a reference molecule comprising the same binding region and a related Shiga toxin effector polypeptide component lacking any cysteine residue is referred to herein as active but “attenuated.” The CD50values for exemplary, cell-targeting molecules are shown in Table 3 and associated cell-kill assay data is shown inFIGS.2-3. The molecules tested for cytotoxic activity in this Example included cell-targeting molecules comprising or consisting of SLT-1A-Cys5::scFv1 (SEQ ID NO:781), SLT-1A-Cys7::scFv1 (SEQ ID NO:782), SLT-1A-Cys10::scFv1 (SEQ ID NO:783), SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773), SLT-1A-Cys2-D1::scFv3 (SEQ ID NO:780), SLT-1A-Cys3-D1::scFv3 (SEQ ID NO:779), SLT-1A-Cys5-D1::scFv3 (SEQ ID NO:778), SLT-1A-Cys6-D1::scFv2 (SEQ ID NO:774), SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:776), SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:777), SLT-1A1-WT (SEQ ID NO:830), SLT-1A-D1::scFv2 (SEQ ID NO:838), SLT-1A-D1 (SEQ ID NO:831), SLT-1A-D1-C242::scFv3 (SEQ ID NO:837), and SLT-1A-D1::scFv3 (SEQ ID NO:839). TABLE 3Cytotoxic Activities of Exemplary, Cell-Targeting MoleculesSLT-1ACell-type testedCell-type testedCysteine(target positiveCytotoxicity(target positiveCytotoxicityMolecule testedpositionor negative)CD50(nM)or negative)CD50(nM)Experiment 1SLT-1A-Cell Line A0.67Cell Line B>200 nMCys5::scFv1(positive)(negative)SLT-1A-C45Cell Line A0.44Cell Line B>200 nMCys7::scFv1(positive)(negative)SLT-1A-C54Cell Line A0.42Cell Line B>200 nMCys10::scFv1(positive)(negative)SLT-1A1-WTC242Cell Line A373.00Cell Line B>200 nM(n/a)(n/a)Experiment 2SLT-1A-Cys2-C8Cell Line C0.0023Cell Line D0.0074D1::scFv2(positive)(positive)SLT-1A-Cys6-C43Cell Line C0.0041Cell Line D0.0588D1::scFv2(positive)(positive)SLT-1A-Cys7-C45Cell Line C0.0012Cell Line D0.0063D1::scFv2(positive)(positive)SLT-1A-Cys8-C146Cell Line C0.0010Cell Line D0.0038D1::scFv2(positive)(positive)SLT-1A-Cys9-C186Cell Line C0.0046Cell Line D0.0176D1::scFv2(positive)(positive)SLT-1A-D1::noneCell Line C0.0026Cell Line D0.0073scFv2(positive)(positive)SLT-1A-D1noneCell Line C>100 nMCell Line D>100 nM(n/a)(n/a)Experiment 3SLT-1A-Cys2-C8Cell Line E54.8D1::scFv3(positive)SLT-1A-Cys3-C16Cell Line E34.8D1::scFv3(positive)SLT-1A-Cys5-C33Cell Line E17.4D1::scFv3(positive)SLT-1A-D1-C242Cell Line E40.3C242::scFv3(positive)SLT-1A-noneCell Line E55.7D1 ::scFv3(positive)* “n/a” denotes that the presence or absence of a particular target biomolecule is irrelevant because the molecule tested was untargeted The exemplary cell-targeting molecules of the present invention represented by SLT-1A-Cys5::scFv1 (SEQ ID NO:781), SLT-1A-Cys7::scFv1 (SEQ ID NO:782), SLT-1A-Cys10::scFv1 (SEQ ID NO:783), SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773), SLT-1A-Cys2-D1::scFv3 (SEQ ID NO:780), SLT-1A-Cys3-D1::scFv3 (SEQ ID NO:779), SLT-1A-Cys5-D1::scFv3 (SEQ ID NO:778), SLT-1A-Cys6-D1::scFv2 (SEQ ID NO:774), SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:776), and SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:777) were each cytotoxic to target positive cells, commonly exhibiting CD50values less than 100 nM. The results reported in Table 3 show that cell-targeting molecules comprising any of the Shiga toxin effector polypeptides SLT-1A-Cys5 (SEQ ID NO:9), SLT-1A-Cys7 (SEQ ID NO:11), SLT-1A-Cys10 (SEQ ID NO:14) were cytotoxic to target positive cells at potencies associated with CD50values generally less than 100 nM to 5 micromolar (μM) depending on the cell-line tested (see Table 3;FIGS.2-3). The cytotoxicities of many of the Shiga toxin effector polypeptides tested as a component of a cell-targeting molecule were comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide (either lacking any cysteine residues (SEQ ID NO:831) or having the endogenous cysteine at position 242 (SEQ ID NO:832), as a component of a related, cell-targeting molecule (SEQ ID NO:837) (Table 3;FIGS.2-3). FIG.2shows the potent cytotoxic activities exhibited by three different exemplary cell-targeting molecules of the present invention SLT-1A-Cys5::scFv1 (SEQ ID NO:781), SLT-1A-Cys7::scFv1 (SEQ ID NO:782), and SLT-1A-Cys10::scFv1 (SEQ ID NO:783) as compared to the untargeted SLT-1A1-WT alone (SEQ ID NO:830) toward target positive cells (top panel), and as compared to their activities to target negative cells (bottom panel). FIG.3shows the cytotoxic activities exhibited by three different exemplary cell-targeting molecules of the present invention: SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773), SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), and SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:776); and the cytotoxic activities shown inFIG.3were comparable to the activities displayed by a related cell-targeting molecule (SLT-1A-D1::scFv2 (SEQ ID NO:838), which comprised a Shiga toxin effector polypeptide lacking any cysteine residue (SEQ ID NO:831). The specificity of the cytotoxic activity of a given cell-targeting molecule can be determined by comparing cell-kill activities toward cells expressing a significant amount of a target biomolecule of the binding region of the cell-targeted molecule (target positive cells) with cell-kill activities toward cells which do not have any significant amount of any target biomolecule of the binding region of the cell-targeted molecule physically coupled to any cellular surface (target negative cells). This was accomplished by comparing the CD50value(s) of a given cell-targeting molecule toward a cell population(s) which were positive for cell surface expression of the target biomolecule of the cell-targeting molecule being analyzed to the CD50value(s) of the same molecule toward a cell population(s) which were negative for cell surface expression of any target biomolecule of the molecule being analyzed. The results of the cell-kill assays described above and shown in Table 3 andFIGS.2-3indicated that the introduction of various, ectopic, cysteine residues in various, Shiga toxin effector polypeptide scaffolds was possible without significantly impairing the Shiga toxin A Subunit functions of catalytic activity, catalytic inactivation of eukaryotic ribosomes, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. Example 2. Exemplary, Cell-Targeting Molecules of the Present Invention That Comprise a Free Cysteine Residue Outside the Toxin Effector Region In this Example, exemplary cell-targeting molecules of the present invention, each comprising one free cysteine residue outside any toxin effector region, were created and tested. A. Constructing Exemplary, Cell-Targeting Molecules of the Present Invention that Comprise a Free Cysteine Residue Outside all Shiga Toxin Effector Polypeptide Components This section describes the creation of various cell-targeting molecules comprising a free cysteine residue. Using standard techniques, cysteine residues were engineered into cell-targeting proteins as genetically encoded substitutions which did not change the overall number of amino acid residues in the cell-targeting protein. In this Example, each cell-targeting molecule that was tested comprised a cell-targeting, immunoglobulin-type, binding region comprising a polypeptide capable of binding to an extracellular target biomolecule with high-affinity (see Table 4). Protein expression and purification of these cell-targeting molecules was performed using standard techniques known to the skilled worker and/or as previously described (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/191764, and WO 2016/126950), such as using Capto™-L (GE Healthcare, Marlborough, MA, U.S.). TABLE 4Free Cysteine Residues Engineered into Cell-Targeting ProteinsCell-TargetingExemplary, Cell-MoleculeTargeting MoleculesComponentFree Cysteine locationwith Componentlinker-Cys1between Binding RegionSEQ ID NOs: 803-806(SEQ ID NO: 757)and Toxin Effector RegionIinker-Cys2between Binding Region(SEQ ID NO: 758)and Toxin Effector RegionIinker-Cys3between Binding Region(SEQ ID NO: 759)and Toxin Effector RegionIinker-Cys4between Binding Region(SEQ ID NO: 760)and Toxin Effector RegionscFv-linker-Cys1between the scFv's heavySEQ ID NOs: 807-808(SEQ ID NO: 769)and light variable regionsscFv-linker-Cys2between the scFv's heavySEQ ID NOs: 809-811(SEQ ID NO: 770)and light variable regionsscFv-linker-Cys3between the scFv's heavy(SEQ ID NO:771)and light variable regionsscFv-linker-Cys4between the scFv's heavy(SEQ ID NO: 772)and light variable regionsscFv(n)-Cys-C2penultimate C-terminalSEQ ID NOs: 812-815residue of Binding RegionVHH(n)-Cys-C2penultimate C-terminalSEQ ID NOs: 816-817residue of Binding Region B. Testing the Cytotoxic Activities of Exemplary. Cell-Targeting Molecules of the Invention The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of the present invention were tested to assess the functionalities of their Shiga toxin effector polypeptide components. The cytotoxic activities of exemplary, cell-targeting molecules, which each comprised one of the following components: linker-Cys(p), scFv(n)-linker-Cys(p), or scFv(n)-Cys(p) were determined using the cell-kill assay described in Example 1 (above). The cytotoxicities of exemplary, cell-targeting molecules were determined using cells expressing, at a cellular surface, significant amounts of the appropriate, extracellular target biomolecule of scFv2. Using the cytotoxicity assay, the CD50value(s) for each molecule tested were calculated (Table 5), and associated cell-kill assay data is shown inFIG.4. When CD50values could not be calculated based on the shape of the curve over the concentrations tested, then a maximum CD50value was noted as being beyond the maximum tested value, e.g., greater than 100 nM (“>100 nM”), for samples which did not kill 50% of the cells at the highest, tested, sample concentration, e.g., 100 nM. Cytotoxic activities were tested for exemplary, cell-targeting molecules of the present invention such as, e.g., cell-targeting molecules comprising or consisting of SEQ ID NO: 803, 807, and 812. TABLE 5Cytotoxic Activities of Exemplary, Cell-Targeting MoleculesFreeCell-type testedCytotoxicityCell-type testedCytotoxicityCysteine(target positiveCD50(target positiveCD50Molecule Testedlocationor negative)(nM)or negative)(nM)SLT-1A-D1::betweenCell Line C0.0006Cell Line D0.0089linker-Cys1::scFv2Toxin(positive)(positive)(SEQ ID NO: 803)Effector andBindingRegionSLT-1A-D1::betweenCell Line C0.0038Cell Line D0.0273scFv2-linker-Cys1variable(positive)(positive)(SEQ ID NO: 807)domains ofscFv2SLT-1A-D1::in variableCell Line C0.0002Cell Line D0.002scFv2-Cys-C2domain of(positive)(positive)(SEQ ID NO: 812)scFv2SLT-1A-D1::scFv2noneCell Line C0.0026Cell Line D0.0073(SEQ ID NO: 838)(positive)(positive)SLT-1A-D1noneCell Line C>100 nMCell Line D>100 nM(SEQ ID NO: 831)(n/a)(n/a) Table 5 andFIG.4show experimental results from examples of cell targeting molecules comprising a free, cysteine residue outside any Shiga toxin effector polypeptide component(s) (e.g., SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), and SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO:812). The cytotoxic activities of SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), and SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO: 812) were comparable to the activity of the parental molecule SLT-1A-D1::scFv2 (SEQ ID NO:838), which lacks any cysteine residue in its Shiga toxin effector polypeptide component. The results reported in Table 5 show that the cell-targeting molecules comprising or consisting of SLT-1A-D1::linker-Cys1::scFv2, SLT-1A-D1::scFv2-linker-Cys1, and SLT-1A-D1::scFv2-Cys-C2 were all cytotoxic to target positive cells, with CD50values of less than 30 pM. The cytotoxicities of the Shiga toxin effector polypeptide component (SLT-1A-D1 (SEQ ID NO:831)) of these cell-targeting molecules was comparable to the cytotoxicity of the identical Shiga toxin effector polypeptide (SLT-1A-D1 (SEQ ID NO:831)) as a component of the positive control, reference molecule SLT-1A-D1::scFv2 (SEQ ID NO:837) (see Table 5;FIG.4). Example 3. Testing Purified, Cell-Targeting Molecules for Intermolecular Disulfide Bonding Certain, exemplary, cell-targeting molecules of this Example where produced and purified using routine methods known to the skilled worker and then analyzed for intermolecular associations. The cell-targeting molecules tested in this Example included those which comprise a single free cysteine residue such as #1) SLT-1A-D1-C242::scFv3 (SEQ ID NO:838), #2) SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773), #3) SLT-1A-Cys2-D1::scFv3 (SEQ ID NO:780), #4) SLT-1A-Cys3-D1::scFv3 (SEQ ID NO:779), #5) SLT-1A-Cys5-D1::scFv3 (SEQ ID NO:778), #6) SLT-1A-Cys6-D1::scFv2 (SEQ ID NO:774), #7) SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), #8) SLT-1A-Cys8-D1::scFv2 (SEQ ID NO: 776), #9) SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:777), #10) SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), #11) SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), and #12) SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO:812) as well as other cell-targeting molecules used as various controls such as SLT-1A-D1::scFv2 (SEQ ID NO:838) and SLT-1A-D1::scFv3 (SEQ ID NO:839). Exemplary, cell-targeting molecule preparations were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE). Exemplary, cell-targeting molecules and the reference molecules SLT-1A-D1::scFv(n) were loaded in equal concentrations to replicate, 4-20%, SDS-PAGE gels (Lonza, Basel, CH) and electrophoresed under denaturing conditions or denaturing and reducing conditions. The resulting gels were analyzed by COOMASSIE staining for intermolecular associations indicative of disulfide bond formation (shown inFIGS.5and6). InFIGS.5-6, the left side shows COOMASSIE-stained SDS-PAGE gels run under reducing and denaturing conditions, and the right side shows replicate, COOMASSIE-stained SDS-PAGE gels run under non-reducing, denaturing conditions. The Figure legends list the samples loaded and run in each lane of the replicate gels. The first lane marked “MW Marker” shows the migration pattern of a protein molecular weight ladder, and the approximate size of each ladder protein band is labeled in kiloDaltons (kDa). InFIG.5, the samples loaded and run in lanes numbered 2-6 are indicated in the figure legend: #2) SLT-1A-D1::scFv3 (SEQ ID NO:839), #3) SLT-1A-D1-C242::scFv3 (SEQ ID NO:837), #4) SLT-1A-Cys5-D1::scFv3 (SEQ ID NO:778), #5) SLT-1A-Cys3-D1::scFv3 (SEQ ID NO:779), and #6) SLT-1A-Cys2-D1::scFv3 (SEQ ID NO:780). InFIG.6, the samples loaded and run in lanes numbered 2-10 are indicated in the figure legend: #2) SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773), #3) SLT-1A-Cys6-D1::scFv2 (SEQ ID NO:774), #4) SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:776), #5) SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:777), #6) SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), #7) SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), #8) SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO: 812), #9) SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), and #10) SLT-1A-D1::scFv2 (SEQ ID NO:838). The results of this analysis and as shown inFIGS.5-6was that intermolecular disulfide bond based multimerization had occurred in the samples with SLT-1A-Cys5-D1::scFv3 (SEQ ID NO:778), SLT-1A-Cys6-D1::scFv2 (SEQ ID NO:774), SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775), SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:776), and SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:777). In addition, truncations of certain, cell-targeting molecules were observed for some of the samples analyzed, and the size of the truncation matches the expected size of a purification intermediate. To further analyze the nature of intermolecular associations formed among purified, exemplary, cell-targeting molecules, two samples were further purified and analyzed. Samples of the exemplary, cell-targeting molecules SLT-1A-Cys2-D1::scFv2 and SLT-1A-Cys7-D1::scFv2 used in the experiments above were run over a size exclusion chromatography (SEC) column (Superdex 200 Increase, GE Healthcare, Marlborough, MA, U.S.) and compared to commercial, molecular-size migration reference standards to estimate sizes (see e.g.FIGS.7-8). Through the use of molecular-size migration standards and knowledge of possible molecular species present in a sample, the size of molecular species in a peak may be estimated and the identity of the molecular species in a peak may be inferred. Alternatively or in addition, complimentary methods (e.g. SDS-PAGE or mass spectroscopy) known to the skilled worker and/or described herein may be used to more definitively determine the molecule(s) composed in certain peaks. Molecules of known sizes and migration characteristics (standards) were analyzed in order to calibrate which retention times corresponded to which protein sizes and/or protein sizes were predicted from the amino acid composition and/or SDS-PAGE analyses of the purified protein present in the sample to be analyzed. Chromatographic data collected from commercial size standards were used to create calibration curves to help estimate sizes of molecular species in samples and focus analyses on specific retention time ranges. The results of the experiments shown inFIGS.6and7indicate that the dimeric protein in the SLT-1A-Cys2-D1::scFv2 (SEQ ID NO:773) sample was not predominantly comprised by covalent dimers but instead was comprised predominantly by non-covalent dimeric complexes. This may indicate that the cysteine residue at position 8 in SEQ ID NO:26 was merely unavailable to intermolecularly pair with other cysteine residues at position 8 of SEQ ID NO:26 or that more generally the cysteine residue at position 8 of SEQ ID NO:26 is not available for pairing to molecules of a certain size (e.g. polypeptides larger than 25 amino acid residues) or to any molecule at all (e.g., buried in the hydrophobic core of the protein and/or tightly packed within a secondary structure). The results of the experiments shown inFIGS.6and8indicate that the protein in the SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:775) sample was comprised by redox-sensitive, covalent dimeric complexes as well as by non-covalent dimeric complexes, indicating that the cysteine residue at position 45 in SEQ ID NO:31 is available for pairing to proteinaceous molecules, including relatively large polypeptides and proteins (e.g. having a mass of about 50-60 kDa). The ectopic cysteine at position 45 of the Shiga toxin effector polypeptide mediated a covalent interaction between two molecules of SLT-1A-Cys7-D1:scFv2. Thus, it can be inferred that the cysteine residue at position 45 of the Shiga toxin effector polypeptide is surface-accessible and more generally unavailable for pairing with cysteine residues in other molecules. Example 4. Constructing Cargo-Linked, Cell-Targeting Molecules Comprising Exemplary, Shiga Toxin A Subunit Effector Polypeptides of the Present Invention Exemplary cell-targeting molecules of the present invention (SEQ ID NOs: 789, 791-793, 804, 808, 813, and 1141) were produced, purified and conjugated to a maleimide-activated fluorescent dye using routine methods known to the skilled worker. The maleimide-activated fluorescent dyes used were Alexa Fluor® 488 C5 Maleimide and Alexa Fluor® 555 C2 Maleimide (Thermo Fisher Scientific, Waltham, MA, U.S.). For each sample, 200 microgram (μg) of an exemplary cell-targeting molecule was incubated with 2 mM TCEP at pH 7 and room temperature for twenty minutes. A 5-fold molar excess of either the 488 or 555 maleimide-activated dye was added to each sample, and chemical reactions were allowed to proceed at room temperature for one hour. Then for each sample, protein was separated from unbound dye using a purification resin according to the manufacturer's instructions (Antibody Conjugate Purification Kit, Catalog #A33088, Thermo Fisher Scientific, Waltham, MA, U.S.), and the protein concentration was estimated by measuring each sample's absorbance of light of a wavelength of 280 nM. The method of conjugation used (i.e. the chemical reaction during the one-hour incubation period) was intended to link the maleimide functional group of the dye to a free cysteine residue of a polypeptide component of the cell-targeting molecule. As a control, a related cell-targeting molecule lacking any free cysteine was subjected to the same chemical reaction and method steps described above. In certain embodiments, exemplary cell-targeting molecules of the present invention comprise a mutation known in the art to cause the inactivation of the toxin effector component(s) of the cell-targeting molecule. For example, Shiga toxin effector polypeptides comprising the mutation E167D, wherein 167 refers to the native position of a Shiga toxin A Subunit, is referred to in the Examples herein as IA-SLT-1A, with the IA indicating catalytically inactive or catalytically impaired for noncytotoxic or reduced-cytotoxicity variants as a result of severely reduced catalytic activity consistent with descriptions in the scientific literature and as would be recognized by the skilled worker (see e.g. Hovde C et al.,Proc Natl Acad Sci USA85: 2568-72 (1988); Jackson M et al.,J Bacteriol172: 3346-50 (1990); Gordon V et al.,Infect Immun60: 485-90 (1992); Ohmura M et al.,Microb Pathog15: 169-76 (1993)). Exemplary, cell-targeting molecules were created and tested in Example 5, including cell-targeting molecules comprising a Shiga toxin effector polypeptide(s) comprising the E167D mutation: IA-SLT-1A-D1 (SEQ ID NO:834), IA-SLT-1A-Cys5-D1 variant 2 (SEQ ID NO:1101), IA-SLT-1A-Cys7-D1 (SEQ ID NO: 1102), IA-SLT-1A-Cys8-D1 (SEQ ID NO:1103), or IA-SLT-1A-Cys9-D1 (SEQ ID NO:1104)). Example 5. Testing Cargo-Linked Cell-Targeting Molecules of the Present Invention that Comprise a Cargo Linked to a Site-Specific Free Cysteine Residue Cargo-linked cell-targeting molecules were tested for cell-targeting molecule functions (e.g. target binding, cell binding, cellular internalization, intracellular routing efficiency, and cell-killing) using standard techniques known to the skilled worker (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/113005, WO 2015/113007, WO 2015/138435, WO 2015/138452, US 2015/0259428, and WO 2015/191764). Exemplary, cell-targeting molecules were tested for Shiga toxin A Subunit functions of cellular internalization and cytotoxicity, and by inference catalytic activity, inhibition of eukaryotic ribosome function, and self-directing subcellular routing to the cytosol. The potency and specificity of cytotoxic activities of exemplary, cargo-linked cell-targeting molecules of the present invention were tested based on the exemplary cell-targeting molecule comprising or consisting of SEQ ID NO:789. In addition, cellular internalization of cargo by cargo-linked cell-targeting molecules was tested to demonstrate cargo delivery functionality of cell-targeting molecules which each comprised a cargo linked to a specific cysteine residue. A. Testing Cargo-Linked Cell-Targeting Molecules for Retention of Cell-Targeting Exemplary, cargo-linked cell-targeting molecules were tested for cell binding, and by inference target biomolecule binding function, using routine techniques known to the skilled worker. The cell-binding ability of each dye-linked cell-targeting molecule was measured by standard flow cytometry assays known to the skilled worker (e.g. fluorescence-activated cell sorting (FACS)) using immortalized human cell lines that either expressed at a cellular surface the target of the cell-targeting molecule (cell lines C and G) or, as a negative control, cells that did not express the target of the cell-targeting molecule being tested on the cell surface (Cell line H). For each sample and cell-type tested, 300,000 cells were incubated on ice for one hour in media containing 200 nM of each dye-linked cell-targeting molecule sample. Then the cells were washed, re-suspended in PBS, and analyzed using a BD Accuri™ C6 flow cytometer according to the manufacturer's instructions (BD Biosciences, San Jose, CA, U.S.). As assay controls for each specific target, each cell line was tested in the same assay using a monoclonal antibody specific to the same target conjugated to fluorescein isothiocyanate (FITC) as a positive control and an isotype negative monoclonal antibody specific to the same target conjugated to FITC as a negative control. Thus, 300,000 cells from each cell line tested were incubated on ice for one hour in media containing one μg of either a positive control antibody or a negative control antibody and analyzed with the same method and parameters. The FACS analysis of each sample involved the sorting of living cells by gating based on forward versus side scatter. The FACS gate for positive was based on the isotype negative control samples (encompassing the negative population) and confirmed with a positive control monoclonal antibody (e.g. anti-target2 mAb-FITC). The exemplary cell-targeting molecules tested in this assay included: IA-SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:1141), SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:789), IA-SLT-1A-Cys7-D1::scFv2 (SEQ ID NO:793), IA-SLT-1A-Cys8-D1::scFv2 (SEQ ID NO:791), IA-SLT-1A-Cys9-D1::scFv2 (SEQ ID NO:792), IA-SLT-1A-DI::linker-Cys1::scFv2 (SEQ ID NO:804), IA-SLT-1A-DI::scFv2-linker-Cys1 (SEQ ID NO:808), and IA-SLT-1A-DI::scFv2-Cys1 (SEQ ID NO:813). The results of the FACS cell-binding assay measurements for cells incubated with 200 nM of cell-targeting molecule or control antibody are listed in Table 6 and some representative FACS histogram overlay data is shown inFIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C. InFIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C, the number of cells counted (cell count) is plotted over the fluorescence intensity in the FL1-A channel. InFIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C, the gray line denotes the negative population as measured using the isotype negative control sample and the black line denotes the dye-linked cell-targeting molecule or antibody positive control (anti-target2 mAb-FITC). In Table 6, the percentage of cells counted in the positive gate is indicated as “percent positive” for each cell line tested. Table 6 also lists the iMFI (indexed mean fluorescent intensity), the product of the MFI and the percentage positive, for each target positive cell line tested. In Table 6, “N/A” refers to ‘not applicable’ as the monoclonal antibody listed here was used as a positive control for cell surface binding to the target biomolecule of the experimental cell-targeting molecules. TABLE 6Exemplary, Cargo-Linked Cell-Targeting Molecules Exhibited Binding to TargetPositive Cells at Cell-Targeting Molecule Concentrations of 200 nMtargettarget positivetarget positivenegativeCell Line CCell Line GCell Line HMolecule Tested bytargettargettargetlinking to dye andCysteinepositivepositivepositiveassaying cell bindingpositionpercentageiMFIpercentageiMFIpercentageIA-SLT-1A-D1::scFv2none5%1,1306%3617%(SEQ ID NO: 843)IA-SLT-1A-Cys5-3399%52,462100%83,4439%D1::scFv2(SEQ ID NO: 1141)SLT-1A-Cys5-3399%52,466100%92,1737%D1::scFv2(SEQ ID NO: 789)IA-SLT-1A-Cys7-4598%39,793100%81,6587%D1::scFv2(SEQ ID NO: 793)IA-SLT-1A-Cys8-14689%26,388100%103,0757%D1::scFv2(SEQ ID NO: 791)IA-SLT-1A-Cys9-18697%33,677100%60,4596%D1::scFv2(SEQ ID NO: 792)IA-SLT-1A-DI::linker-linker97%37,627100%101,1056%Cys1::scFv2(SEQ ID NO: 804)IA-SLT-1A-DI::scFv2-linker99%64,287100%131,9206%linker-Cys1(SEQ ID NO: 808)IA-SLT-1A-DI::scFv2-scFv-Cys190%18,634100%35,5285%Cys1(SEQ ID NO: 813)anti-target 2 mAbN/A98%48,505100%156,7832% The results in Table 6 demonstrated that exemplary, cargo-linked cell-targeting molecules bound to target positive cells and labeled them with their dye-cargos. However, the control protein IA-SLT-1A-D1::scFv2 (SEQ ID NO:843), which lacks any free cysteine residue, was not capable of labeling cells after being subjected to the same dye-conjugation procedure (Table 6). The exemplary, cargo-linked cell-targeting molecules did not label appreciable numbers of target negative cells (Table 6). In addition, the cell-binding results summarized in Table 6 indicate that some cargo and cell-targeting molecule pairs were not as successful as others. For example, the samples which resulted in lower MFI values and iMFI for target positive cell lines suggest those samples had less efficient maleimide-cysteine conjugation reactions and/or lower effective cell binding properties than other samples tested. The results of the FACS cell-binding assay measurements for cells incubated with 200 nM of cell-targeting molecule or antibody are shown in histogram overlays inFIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C.FIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C show cell binding assay results for two, different, exemplary, cargo-linked cell-targeting molecules (SEQ ID NOs: 789 and 1141) which each comprise the same cysteine residue (Cys 5) linked to the same dye cargo. These two molecules differ only in that one has been rendered catalytically impaired by the amino acid residue substitution E167D in the Shiga toxin effector polypeptide component. The results shown inFIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C demonstrate the cell binding abilities of two, cargo-linked cell-targeted molecules of the present invention to different cell-types. These FACS results show that exemplary, cell-targeting molecules SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:789) and IA-SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:1141) bind target positive cells after conjugation to an Alexa Fluor® dye cargo (Table 6;FIGS.9-A,9-B,9-C,10-A,10-B,10-C,11-A,11-B, and11-C). Similar results is observed for other exemplary, cargo-linked cell-targeting molecules of the present invention after conjugation to an Alexa Fluor® dye cargo, such as, e.g., cell-targeting molecules comprising or consisting of any one of SEQ ID NOs: 791, 792, 804, 808, 813, 793, and 1141 conjugated to ALEXA-488 or ALEXA-555. B. Testing Cargo-Linked Cell-Targeting Molecules for Cell Internalization of their Cargos Exemplary, cargo-linked cell-targeting molecules were tested for cellular internalization using routine microscopy-based fluorescent techniques known to the skilled worker (see e.g. WO 2014/164680; WO 2015/138452; US20150259428). The behavior and localization of a fluorescent molecule administered to a cell(s) may be observed using fluorescent microscopy. To examine cellular internalization of the exemplary, cell-targeting molecules of the present invention, a fluorescence microscopy-based assay was used to observe the localization of cargo-linked cell-targeting molecules after administration to cells. The molecules tested in this section were an active or inactive version of a scFv2 targeted molecule (SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:789) and IA-SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:1141)), which comprised the Shiga toxin effector polypeptide of either SLT-1A-Cys5-D1 (SEQ ID NO:29) or IA-SLT-1A-Cys5-D1 variant 2 (SEQ ID NO:1101) conjugated to an Alexa Fluor® dye, such as, ALEXA-488 or ALEXA-555. In this cell-internalization assay, approximately 500,000 cells per sample were incubated at 37° C. for 1 hour in media containing 200 nM of an exemplary, dye-linked, cell-targeting molecule, a cocktail of protease inhibitors, and human BD Fc Block™. The cell samples were then washed and fixed with BD Cytofix/Cytoperm™ (BD Biosciences, San Jose, CA, U.S.). Then the cell samples were re-suspended in PBS and spread on poly-L-lysine coated glass slides. For each slide, a coverslip was mounted using a 4′,6-diamidino-2-phenylindole (DAPI)-containing VECTASHIELD® solution (catalog #NC9524612, Fisher Scientific, Waltham, MA, U.S.), and the slides were viewed using fluorescent microscopy. FIGS.12-A,12-B,13-A,13-B,14-A, and14-B show some of the cell internalization results from this assay, specifically involving SLT-1A-Cys5-D1::scFv2-ALEXA-555 and IA-SLT-1A-Cys5-D1::scFv2-ALEXA-555 observed using target positive cell lines (C and G) and a target negative cell line (H).FIGS.12-A,12-B,13-A,13-B,14-A, and14-B show merged images of two different fluorophore measurements, one of DAPI shown in blue and one for ALEXA-555 shown in red. Both ALEXA-488 and ALEXA-555 conjugated cell-targeting molecules produced similar qualitative results, though the ALEXA-555 signal appeared more intense than the ALEXA-488 signal. The results from this cell internalization assay, some of which are shown inFIGS.12-A,12-B,13-A,13-B,14-A, and14-B, were that SLT-1A-Cys5-D1::scFv2_ALEXA-555, IA-SLT-1A-Cys5-D1::scFv2_ALEXA-555, SLT-1A-Cys5-D1::scFv2_ALEXA-488, and IA-SLT-1A-Cys5-D1::scFv2_ALEXA-488 each bound to and entered target positive cells within one hour or less (FIGS.12-A,12-B,13-A,13-B,14-A, and14-B). C. Testing Cell-Targeting Molecules for Conjugation Using a Size Shift Cargo-linked cell-targeting molecule samples were analyzed by SDS-PAGE in attempt to observe conjugates having a greater mass than unconjugated molecules. FIG.15shows the results of the SDS-PAGE analysis of samples of SLT-1A-Cys5-D1::scFv2 (SEQ ID NO:789) before and after being linked to the cargo ALEXA-488 or ALEXA-555. The results inFIG.15showed a change in the mass of the target band, as expected after the conjugation to the 1,000 Dalton dye molecule. The SDS-PAGE analysis shows relatively pure samples of exemplary, cargo-linked, cell-targeting molecules of the present invention wherein the majority of protein species present are conjugated to a dye-cargo (FIG.15). D. Testing Cargo-Linked Cell-Targeting Molecules for Retention of Cytotoxicity Exemplary, cargo-linked cell-targeting molecules were tested for the Shiga toxin A Subunit function of cell-targeted cytotoxicity, and by inference catalytic activity, inhibition of eukaryotic ribosome function, and self-directing subcellular routing to the cytosol. The cytotoxicity assay was performed as described in Example 1 and Example 2. Table 7 andFIGS.16-A,16-B, and16-C show the result of cell-kill assays for exemplary, cargo-linked cell-targeting molecules of the present invention. Unlike in Examples 1-2, the cytotoxicity of samples of cargo-linked cell targeting molecules was compared to the cytotoxicity of samples of the same cell-targeting molecules lacking any conjugated cargo. TABLE 7Exemplary, Cargo-Linked Cell-Targeting Molecules ExhibitedPotent and Specific Cytotoxicity to Target Positive CellsSLT-1Atarget positive cellstarget negativeCysteineCell Line CCell Line GCell Line HMolecule TestedpositionCargoIC50(ng/mL)IC50(ng/mL)IC50(ng/mL)SLT-1A-D1::nonenone1.1122>10,000scFv2(SEQ ID NO: 838)SLT-1A-Cys5-D1::33none0.6712>10,000scFv2(SEQ ID NO: 789)SLT-1A-Cys5-D1::33ALEXA-4881.3248>10,000scFv2_ALEXA-488SLT-1A-Cys5-D1::33ALEXA-5551.9371>10,000scFv2_ALEXA-555 The results of the cytotoxicity assay shown inFIGS.16-A,16-B, and16-C and Table 7 demonstrate that cargo-linked cell-targeting molecules of this Example retained potent and specific cytotoxicity to target positive cells. The dye-linked cell-targeting molecules exhibited a cytotoxicity within 6-fold of their corresponding, unconjugated cell-targeting molecule and similar to the cytotoxicity of a related cell-targeting molecule that contained no free cysteine residue (Table 7). The results of the experiments in Examples 4-5 demonstrated that a cargo molecule can be conjugated to a free cysteine residue of a cell-targeting molecule of the present invention such that a useful level of (1) the cell-targeting, target binding function of the binding region component is present, (2) cargo molecule delivery to the interior of a target positive cell, and/or (3) the cytotoxic function of the Shiga toxin A Subunit effector polypeptide component is present. Example 6. Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention Comprising a Unique Lysine Residue for Site-Specific Conjugation This example describes the creation of various scaffolds comprising Shiga toxin effector polypeptides, each comprising one, unique, lysine residue (SLT-1A-Lys(p)-variant), where Lys(p) represents a lysine residue at a unique position) were created and tested as components of exemplary cell-targeting molecules of the present invention. All other lysine residues were removed from the Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. SLT-1A-Lys(p)::scFv(n)) were created using these Shiga toxin effector polypeptides. The parental, Shiga toxin effector polypeptide of this Example is the A Subunit of Shiga-like toxin 1 (SEQ ID NO:1). Using standard techniques, the parental, Shiga toxin effector polypeptide are used to make various Shiga toxin effector polypeptides, each having one unique lysine residue (see e.g. Table 8). Cell-targeting molecules (see e.g. SEQ ID NOs: 818-823) comprising Shiga toxin effector polypeptides (see e.g. SEQ ID NOs: 197, 198, 200-202, and 205) having exactly one of the unique lysine residues described in Table 8 were made using standard techniques and tested for retention of Shiga toxin A Subunit functions as described in Example 1. TABLE 8Shiga Toxin Effector Polypeptides Engineered to have Unique Lysine ResiduesUniqueExemplary, Shiga ToxinLysineSequence VariantsEffector PolypeptideResidue(Shiga Toxin A Subunit-Lys(p)-variant)SLT-1A-Lys1K1SEQ ID NOs: 125-128, 134-137, 143-146, 152-155,161-164, 170-173, 179-182, 188-191, 197-200,206-209, 215-218, and 224-227SLT-1A-Lys2K11SEQ ID NOs: 129-133, 138-142, 147-151, 156-160,165-169, 174-178, 183-187, 192-196, 201-205,210-214, 219-223, and 228-232 Exemplary, cell-targeting molecules of the present invention were tested for retention of Shiga toxin A Subunit functions as described in Example 1 after removal of an endogenous lysine residue from their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed were: inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide SLT-1A-Lys(p), were determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker, mainly to assess the functions of their Shiga toxin effector polypeptide components. The cytotoxicities of exemplary, cell-targeting molecules were determined using cells expressing, at a cellular surface, significant amounts of the appropriate, extracellular target biomolecule, such as, a target of the binding region scFv4. The cells used in this Example were immortalized, human tumor cells available from the ATCC (Manassas VA, U.S.), National Cancer Institute of the U.S. (Frederick, MD, U.S.), and/or DSZM (Braunschweig, DE), such as HCC-1954, MDA-MB-231, Daudi, and U-266 cells, or more simply cell-lines A, B, G, and I, respectively. Certain cell-targeting molecules were tested using cell-kill assays involving both target biomolecule positive and target biomolecule negative cells with respect to the target biomolecule of each cell-targeting molecule's binding region. The cytotoxicity assays were performed as follows. Certain, human tumor cell-line cells were plated (typically at 2×103cells per well for adherent cells, plated the day prior to cell-targeting molecule addition, or 7.5×103cells per well for suspension cells, plated the same day as cell-targeting molecule addition) in 20 μL cell culture medium in 384-well plates. A series of 10-fold dilutions of the molecules to be tested was prepared in an appropriate buffer, and 5 μL of the dilutions or buffer control were added to the plated cells. Control wells containing only cell culture medium were used for baseline correction. The cell samples were incubated with the cell-targeting molecule or just buffer for 3 or 5 days at 37° C. and in an atmosphere of 5% carbon dioxide (CO2). The total cell survival or percent viability was determined using a luminescent readout using the CellTiter-Glo® Luminescent Cell Viability Assay or RealTime-Glo® MT Cell Viability Assay (Promega Corp., Madison, WI, U.S.) according to the manufacturer's instructions. The Percent Viability of experimental wells was calculated using the following equation: (Test RLU−Average Media RLU)÷(Average Cells RLU−Average Media RLU)×100. The logarithm of the cell-targeting molecule protein concentration versus Percent Viability was plotted in Prism (GraphPad Prism, San Diego, CA, U.S.) and log (inhibitor) versus response (3 parameter) analysis or and log (inhibitor) versus normalized response analysis were used to determine the half-maximal cytotoxic concentration (CD50) value for the tested molecule. The CD50value(s) for each molecule tested were calculated, when possible, and shown in Table 9. When CD50values could not be calculated based on the shape of the curve over the concentrations tested, then a maximum CD50value was noted as being beyond the maximum tested value, e.g., greater than 20,000 nanogram/milliliter (“>20,000 ng/mL”), for samples which did not kill 50% of the cells at the highest, tested, sample concentration, e.g., 100 or 200 nanomolar (nM). In some experiments, biomolecule target negative cells treated with the maximum concentration of the cell-targeting molecule did not show any change in viability as compared to a buffer only control. The CD50values for exemplary, cell-targeting molecules are shown in Table 9 and associated cell-kill assay data is shown inFIGS.17-18. TABLE 9Cytotoxic Activities of Exemplary, Cell-Targeting MoleculesSLT-1ACell-type testedCytotoxicityCell-type testedCytotoxicitylysine(target positiveCD50(target positiveCD50Molecule testedpositionor negative)(ng/mL)or negative)(ng/mL)SLT-1A-Lys1-D3-K1Cell Line A1.61Cell Line G>20,000variant-1::scFv4(positive)(negative)SEQ ID NO: 818SLT-1A-Lys1-D3-K1Cell Line A1.89Cell Line G>20,000variant-2::scFv4(positive)(negative)SEQ ID NO: 819SLT-1A-Lys1-D3-K1Cell Line A2.99Cell Line G>20,000variant-4::scFv4(positive)(negative)SEQ ID NO: 820SLT-1A-Lys2-D3-K11Cell Line A0.58Cell Line G>20,000variant-1::scFv4(positive)(negative)SEQ ID NO: 821SLT-1A-Lys2-D3-K11Cell Line A2.40Cell Line G>20,000variant-2::scFv4(positive)(negative)SEQ ID NO: 822SLT-1A-Lys2-D3-K11Cell Line A2.99Cell Line G>20,000variant-5::scFv4(positive)(negative)SEQ ID NO: 823SLT-1A-K1/K11Cell Line A1.22Cell Line G>20,000D3::scFv4 control(positive)(negative)SEQ ID NO: 828 The molecules tested for cytotoxic activity in this Example included cell-targeting molecules comprising or consisting of SLT-1A-Lys1-D3-variant−1::scFv4 (SEQ ID NO:818), SLT-1A-Lys1-D3-variant-2::scFv4 (SEQ ID NO:819), SLT-1A-Lys1-D3-variant4::scFv4 (SEQ ID NO: 820), SLT-1A-Lys2-D3-variant-1:scFv4 (SEQ ID NO:821), SLT-1A-Lys2-D3-variant2::scFv4 (SEQ ID NO:822), and SLT-1A-Lys2-D3-variant-5::scFv4 (SEQ ID NO:823). The exemplary cell-targeting molecules of the present invention represented by SEQ ID NOs: 818-823 were each cytotoxic to target positive cells, commonly exhibiting CD50values less than 3 ng/mL, depending on the cell-line tested. The cytotoxicities of the Shiga toxin effector polypeptides (SEQ TD NOs: 197, 198, 200-202, and 205) tested as a component of a cell-targeting molecule were comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide having two or more endogenous lysine residues (see e.g. cytotoxicities measured for SLT-1A-D3::scFv4 (SEQ TD NO: 828) in Table 9 andFIGS.17-18). Similar to the use of cysteine residues in Examples 1-5, a unique lysine residue may be used for site-specific attachment of other molecules using conjugation chemical reactions known to the skilled worker. Commonly, two molecules are linked via an acylation of the functional group of the lysine. Example 7. Constructing Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention by Removing Lysine Residues Via Amino Acid Residue Substitution This example describes the creation of various scaffolds comprising Shiga toxin effector polypeptides, each comprising one, unique, lysine residue. All other lysine residues are removed from the Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. scFv(n)::SLT-1A-Lys(p) and SLT-1A-Lys(p)::scFv(n), where ‘p’ numbers the unique lysine residue) are created using these Shiga toxin effector polypeptides. The parental, Shiga toxin effector polypeptide of this Example is the A Subunit of Shiga toxin (SEQ ID NO:2), Shiga-like toxin 1 (SEQ ID NO:1), and Shiga-like toxin 2 (SLT-2A) (SEQ ID NO:3). Using standard techniques, the parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides, each having one unique lysine residue (see e.g. Table 10). Cell-targeting molecules (see e.g. SEQ ID NOs: 773-783) comprising Shiga toxin effector polypeptides (see e.g. SEQ ID NOs: 125-232, 1109-1140) having exactly one of the unique lysine residues described in Table 10 are made using standard techniques and tested for retention of Shiga toxin A Subunit function as in Example 1. TABLE 10Shiga Toxin Effector Polypeptides Engineeredto have Unique Lysine ResiduesExemplary, ShigaUniqueToxin EffectorLysineSequence VariantsPolypeptideResidue(Shiga Toxin A Subunit-Lys(p)-variant)StxA-Lys1K1SEQ ID NOs: 1109 and 1112StxA-Lys2K11SEQ ID NOs: 1110 and 1113-1114StxA-Lys3K274SEQ ID NOs: 1111 and 1115-1116SLT-1A-Lys1K1SEQ ID NOs: 1117 and 1120SLT-1A-Lys2K11SEQ ID NOs: 1118 and 1121-1122SLT-1A-Lys3K274SEQ ID NOs: 1119 and 1123-1124SLT-2A-Lys1K11SEQ ID NOs: 1125 and 1130SLT-2A-Lys2K255SEQ ID NOs: 1126, 1131, 1138 and 1135SLT-2A-Lys3K257SEQ ID NOs: 1127, 1132, 1136, 1139SLT-2A-Lys4K270SEQ ID NOs: 1128, 1133, 1137 and 1140SLT-2A-Lys5K288SEQ ID NOs: 1129 and 1134 Exemplary, cell-targeting molecules of the present invention are tested for retention of Shiga toxin A Subunit functions after removal of endogenous lysine residues from their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed are: catalytic activity, inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The catalytic activities of Shiga toxin effector polypeptide components of exemplary, cell-targeting molecules of this Example are tested using a ribosome inhibition assay. The ribosome inactivation activities of all the Shiga toxin effector polypeptides SLT-1A-Lys(p) tested in the context of a cell-targeting molecule are equivalent to the catalytic activity of similarly de-immunized Shiga toxin effector polypeptides comprising all the natively occurring lysine residues and/or wild-type, Shiga toxin A1 fragments (amino acids 1-251 of SEQ ID NO:1 or SEQ ID NO:2; or amino acids 1-250 of SEQ ID NO:3). The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide SLT-1A-Lys(p), are determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker. The exemplary cell-targeting molecules of the present invention are each cytotoxic to target positive cells, commonly exhibiting CD50values less than 100 nM, depending on the cell-line tested. The cytotoxicities of many of the Shiga toxin effector polypeptides tested as a component of a cell-targeting molecule are comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide having two or more endogenous lysine residues. Exemplary cell-targeting molecules of this Example are produced, purified and conjugated to another molecule via a lysine residue of the Shiga toxin effector polypeptide component of the cell-targeting molecule using routine methods known to the skilled worker, such as, e.g., wherein the other molecule comprises a succinimidyl ester-fluorescent dye or iso/isothio-cyanate-fluorescent dye. These cell-targeting molecules are tested for cell-targeting, internalization, multimerization, and/or cytotoxicity as described in Example 5. The results of cytotoxicity assays show that cargo-linked cell-targeting molecules of this Example retain potent and specific cytotoxicity to target positive cells that is comparable in potency and activity to unconjugated variants of these cell-targeting molecules. The results of the experiments in this Example demonstrate that a cargo molecule can be conjugated to a unique lysine residue of a Shiga toxin effector polypeptide component of a cell-targeting molecule of the present invention, and, furthermore, such conjugated cell-targeting molecules exhibit a useful level of cargo molecule delivery to the interior of a target positive cell and/or cytotoxicity to targeted cells. Example 8. Constructing Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention by Removing Lysine Residues Via Amino Acid Residue Substitution This example describes the creation of various scaffolds comprising Shiga toxin effector polypeptides, each comprising zero lysine residues. All lysine residues respectively are removed from the Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. SLT-1A-Lys(null)::scFv(n) where ‘null’ refers to the lack of any lysine residue in the Shiga toxin A Subunit component) are created using these Shiga toxin effector polypeptides. The parental, Shiga toxin effector polypeptide of this Example is the A Subunit of Shiga toxin (SEQ ID NO:2), Shiga-like toxin 1 (SEQ ID NO:1), and Shiga-like toxin 2 (SLT-2A) (SEQ ID NO:3). Using standard techniques, the parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides, each lacking any lysine residues (see e.g. Table 11). Cell-targeting molecules (see e.g. SEQ ID NOs: 773-783 and 824-827) comprising Shiga toxin effector polypeptides (see e.g. SEQ ID NOs: 233-720) are made using standard techniques and tested for retention of Shiga toxin A Subunit function as in Examples 1 and 6. TABLE 11Shiga Toxin Effector Polypeptides Engineered to have Unique Lysine ResiduesExemplary, Shiga ToxinSequence VariantsEffector PolypeptideLysine Null(Shiga Toxin A Subunit-Lys(null)-variant)SLT-1A-Lys(null)no lysinesSEQ ID NOs: 233-252, 314-333, 477-496, and558-577SLT-1A-Lys(null)-FRno lysinesSEQ ID NOs: 253-272, 334-353, 497-516, and578-597SLT-1A-Lys(null)-D1no lysinesSEQ ID NOs: 273-292, 354-373, 517-536, and598-617SLT-1A-Lys(null)-D2no lysinesSEQ ID NOs: 293-313, 374-394, 537-557, and618-638SLT-1A-Lys(null)-D3no lysinesSEQ ID NOs: 395-414, 436-455, 639-658, and680-699SLT-1A-Lys(null)-D4no lysinesSEQ ID NOs: 415-435, 456-476, 659-679, and700-720 Exemplary, cell-targeting molecules of the present invention were tested for retention of Shiga toxin A Subunit functions after removal of all lysine residues from their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed are: catalytic activity, inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The catalytic activities of Shiga toxin effector polypeptide components of exemplary, cell-targeting molecules of this Example are tested using a ribosome inhibition assay. The ribosome inactivation activities of Shiga toxin effector polypeptides StxA-Lys(null) variants, SLT-1A-Lys(null) variants, and SLT-2A-Lys(null) variants are tested in the context of a cell-targeting molecule and found to be equivalent to the catalytic activity of similarly de-immunized Shiga toxin effector polypeptides comprising all the endogenous, natively occurring lysine residues and/or wild-type, Shiga toxin A1 fragments (such as, e.g., amino acids 1-251 of SEQ ID NO:1 or SEQ ID NO:2; or amino acids 1-250 of SEQ ID NO:3). The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide StxA-Lys(null), SLT-1A-Lys(null), or SLT-2A-Lys(null) are determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker. Exemplary, cell-targeting molecules of the present invention were tested for retention of Shiga toxin A Subunit functions as described in Example 6 after removal of all endogenous lysine residues from their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed were: inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide SLT-CA-Lys(null) variant as described in Table 11, were determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker, mainly to assess the functions of their Shiga toxin effector polypeptide components. The CD5values for exemplary, cell-targeting molecules are shown in Table 12 and associated cell-kill assay data is shown inFIGS.17-19. TABLE 12Cytotoxic Activities of Exemplary, Cell-Targeting MoleculesSLT-1ACell-type testedCytotoxicityCell-type testedCytotoxicityLysine(target positiveCD50(target positiveCD50Molecule testedpositionor negative)(ng/mL)or negative)(ng/mL)SLT-1A-noneCell Line A1.62Cell Line G>20,000Lys(null)-D3-(positive)(negative)variant-21::scFv4SEQ ID NO: 824SLT-1A-noneCell Line A2.4Cell Line G>20,000Lys(null)-varaint-(positive)(negative)27::scFv4SEQ ID NO: 825SLT-1A-noneCell Line A8.81Cell Line G>20,000Lys(null)-D3-(positive)(negative)variant-40::scFv4SEQ ID NO: 826SLT-1A-noneCell Line A1.04Cell Line I>20,000Lys(null)-D4-(positive)(negative)variant-42::scFv4SEQ ID NO: 827SLT-1A-K1/K11Cell Line A1.01Cell Line G>20,000D3::scFv4 control(positive)(negative)SEQ ID NO: 828SLT-1A-K1/K11Cell Line A1.36D5::scFv4 control(positive)SEQ ID NO: 829 The exemplary cell-targeting molecules of the present invention (SEQ ID NOs: 824-827) are each cytotoxic to target positive cells, commonly exhibiting CD50values less than 10 ng/mL, depending on the cell-line tested (see Table 12 andFIGS.17-19). The cytotoxicities of many of the Shiga toxin effector polypeptides (SEQ ID NOs: 639, 645, 658, and 679) tested as a component of a cell-targeting molecule were comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide having two or more endogenous lysine residues (see e.g. the cytotoxicities measured for SLT-1A-D3::scFv4 (SEQ ID NO:828) and SLT-1A-D5::scFv4 (SEQ ID NO:829) in Table 12 andFIG.19). Exemplary cell-targeting molecules of this Example are produced, purified and conjugated to another molecule via a lysine residue of the cell-targeting molecule using routine methods known to the skilled worker, such as, e.g., wherein the other molecule comprises a succinimidyl ester-fluorescent dye or iso/isothio-cyanate-fluorescent dye. These cell-targeting molecules are tested for cell-targeting, internalization, multimerization, and/or cytotoxicity as described in Example 5. The results of cytotoxicity assays show that cargo-linked cell-targeting molecules of this Example retain potent and specific cytotoxicity to target positive cells that is comparable in potency and activity to unconjugated variants of these cell-targeting molecules. The results of the experiments in this Example demonstrate that a cargo molecule can be conjugated to a free lysine residue of a component of a cell-targeting molecule of the present invention comprising only those Shiga toxin effector polypeptide(s) lacking lysine residues, and, furthermore, such conjugated cell-targeting molecules exhibit a useful level of cargo molecule delivery to the interior of a target positive cell and/or cytotoxicity to targeted cells. Example 9. Exemplary, Cell-Targeting Molecules of the Present Invention Comprising a Lysine or Free Cysteine for Site-Specific Conjugation Outside the Toxin Effector Region This example describes the creation of various scaffolds comprising Shiga toxin effector polypeptides, each lacking either any cysteine residue or any lysine residue. Many of these scaffolds involved a linker or extension consisting of a peptide or polypeptide fused directly to a Shiga toxin A Subunit effector polypeptide lacking any cysteine or lysine residue. As used in this example, a “linker” represents a structure in a cell-targeting molecule which links a Shiga toxin effector polypeptide to a cell-targeting binding region capable of binding an extracellular target biomolecule with high affinity wherein the linker is neither part of the Shiga toxin effector polypeptide region structure nor part of the cell-targeting binding region structure. In this Example, Shiga toxin A Subunit effector polypeptides lacking any cysteine and/or lysine residue (such as, e.g., SLT-1A-Lys(null) variants described in Example 8) were created and fused to cell-targeting moieties. This allows for the creation of cell-targeting molecules comprising such a Shiga toxin A Subunit effector polypeptide wherein there is a unique lysine or free cysteine present outside of the toxin effector region. The removal of all cysteine or lysine residues from the toxin effector region prevents other conjugation sites from being available when certain conjugation reactions are performed on such cell-targeting molecules. A free cysteine or unique lysine residue outside the Shiga toxin component of a cell-targeting molecule of the present invention may be used for site-specific attachment of other molecules using conjugation chemical reactions known to the skilled worker. Using routine techniques known to the skilled worker, all cysteine and/or lysine residues are removed from Shiga toxin effector polypeptides by introducing one or more amino acid residue substitutions as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. The parental, Shiga toxin effector polypeptide of this Example is the A Subunit of Shiga toxin (SEQ ID NO:2), Shiga-like toxin 1 (SEQ ID NO:1), and Shiga-like toxin 2 (SLT-2A) (SEQ ID NO:3). The parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides (see e.g. Table 11). Cell-targeting molecules (see e.g. SEQ ID NOs: 773-783 and 824-827) comprising such ‘null’ Shiga toxin A Subunit effector polypeptides (see e.g. SEQ ID NOs: 233-756) are made using standard techniques and tested for retention of Shiga toxin A Subunit function as in Examples 1 and 6. Using standard techniques, Shiga toxin effector polypeptides of this Example were fused to linkers comprising exactly one cysteine or lysine residue to create exemplary Shiga toxin A Subunit effector polypeptide scaffolds of the present invention (also referred to herein as “Shiga toxin effector scaffolds”). Whichever amino acid residue was absent from the Shiga toxin effector polypeptide (e.g. cysteine or lysine) was included at a single unique position in the Shiga toxin effector scaffold. Exemplary Shiga toxin A Subunit effector polypeptide scaffolds of the present invention, each comprising one or more lysine and/or free cysteine residues outside any toxin effector region, were created and tested in the context of a cell-targeting molecule for retention of Shiga toxin A Subunit function as in Examples 1 and 6. In this Example, each exemplary Shiga toxin effector scaffold of the present invention that was tested comprised additional proteinaceous structure outside of the Shiga toxin effector polypeptide region (see Tables 13). TABLE 13Exemplary, Shiga Toxin Effector Polypeptide Scaffolds of the PresentInvention Comprising Only One Lysine Residue for ConjugationToxin EffectorResidue Available forShiga Toxin A SubunitScaffoldNull forConjugationScaffold SequenceSLT-1A-Lys(null)cysteinecysteine at position 257SEQ ID NO: 762scaffold variant 1SLT-1A-Lys(null)cysteinecysteine at position 257SEQ ID NO: 763scaffold variant 2SLT-1A-Lys(null)lysine andlysine at position 255SEQ ID NO: 764scaffold variant 1cysteineSLT-1A-Lys(null)lysine andlysine at position 255SEQ ID NO: 765scaffold variant 2cysteineSLT-1A-Lys(null)lysine andlysine at position 255SEQ ID NO: 766scaffold variant 3cysteineSLT-1A-Lys(null)lysine andlysine at position 255SEQ ID NO: 767scaffold variant 4cysteine Using standard techniques, exemplary, cell-targeting molecules of the present invention (named e.g. SLT-1A-Lys(null)::scFv(n) or SLT-1A-Cys(null)::scFv(n)), each comprising one or more lysine and/or free cysteine residues outside any toxin effector region, were created using the ‘null’ Shiga toxin effector polypeptides (e.g. SLT-1A-Lys(null) or SLT-1A-Cys(null), where ‘null’ represents the lack of any lysine or cysteine residue, respectively). Some of the exemplary cell-targeting molecules of this Example comprises an exemplary, Shiga toxin effector polypeptide scaffolds of this Example (selected from SEQ ID NOs: 762-767, see also Table 13). Cell-targeting molecules (see e.g. SEQ ID NOs: 773-783 and 824-827) based on such ‘null’ Shiga toxin A Subunit effector polypeptides (SEQ ID NOs: 233-756) were made using standard techniques and tested for retention of Shiga toxin A Subunit function as in Examples 1 and 6. In this Example, each cell-targeting molecule that was tested comprised a cell-targeting, immunoglobulin-type, binding region comprising a polypeptide capable of binding to an extracellular target biomolecule with high-affinity (see Tables 13-15). Protein expression and purification of these cell-targeting molecules was performed using standard techniques known to the skilled worker and/or as previously described (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/191764, and WO 2016/126950), such as using Capto™-L (GE Healthcare, Marlborough, MA, U.S.). TABLE 14Exemplary Cell-Targeting Molecule Components of the Present InventionComprising an Engineered, Free Cysteine Available for ConjugationCell-Targeting MoleculeExemplary, Cell-TargetingComponentConjugation site locationMolecules with Componentlinker-Cys1between Binding Region and ToxinSEQ ID NOs: 803-806(SEQ ID NO: 757)Effector RegionIinker-Cys2between Binding Region and Toxin(SEQ ID NO: 758)Effector RegionIinker-Cys3between Binding Region and Toxin(SEQ ID NO: 759)Effector RegionIinker-Cys4between Binding Region and Toxin(SEQ ID NO: 760)Effector RegionscFv-linker-Cys1between the scFv's heavy and lightSEQ ID NOs: 807-808(SEQ ID NO: 769)variable regionsscFv-linker-Cys2between the scFv's heavy and lightSEQ ID NOs: 809-811(SEQ ID NO: 770)variable regionsscFv-linker-Cys3between the scFv's heavy and light(SEQ ID NO: 771)variable regionsscFv-linker-Cys4between the scFv's heavy and light(SEQ ID NO: 772)variable regionsscFv(n)-Cys-C2penultimate C-terminal residue ofSEQ ID NOs: 812-815Binding RegionVHH(n)-Cys-C2penultimate C-terminal residue ofSEQ ID NOs: 816-817Binding Region The cytotoxicities of exemplary, cell-targeting molecules were determined using cells expressing, at a cellular surface, significant amounts of the appropriate, extracellular target biomolecule. Using the cytotoxicity assay, the CD50value(s) for each molecule tested were calculated (and reported in Table 15), and associated cell-kill assay data is shown inFIGS.4, and17-18. When CD50values could not be calculated based on the shape of the curve over the concentrations tested, then a maximum CD50value was noted as being beyond the maximum tested value, e.g., greater than 100 nM (“>100 nM”), for samples which did not kill 50% of the cells at the highest, tested, sample concentration, e.g., 100 nM. Cytotoxic activities were measured for exemplary, cell-targeting molecules of the present invention which each comprised one of the following components: a SLT-1A-Lys(null) scaffold variant, linker-Cys(p), scFv(n)-linker-Cys(p), or scFv(n)-Cys(p). These experiments included measuring the cytotoxic activities of the exemplary, cell-targeting molecules of the present invention comprising or consisting of SEQ TD NOs: 803, 807, 812, and 824-826. The exemplary, cell-targeting molecules SLT-1A-Lys(null)-D3-variant-21::scFv4 (SEQ ID NO:824), SLT-1A-Lys(null)-D3-variant-27::scFv4 (SEQ ID NO:825), and SLT-1A-Lys(null)-D3-variant-40::scFv4 (SEQ ID NO:826) comprise the Shiga toxin effector scaffolds SEQ ID NOs: 764, 765, and 766, respectively (see e.g. Table 13). TABLE 15Cytotoxic Activities of Exemplary, Cell-Targeting MoleculesFreeCell-type testedCytotoxicityCell-type testedCytotoxicityCysteine(target positiveCD50(target positiveCD50Molecule Testedlocationor negative)(nM)or negative)(nM)SLT-1A-D1::between ToxinCell Line C0.0006Cell Line D0.0089linker-Cys1::scFv2Effector and(positive)(positive)(SEQ ID NO: 803)Binding RegionSLT-1A-D1::betweenCell Line C0.0038Cell Line D0.0273scFv2-linker-Cys1variable(positive)(positive)(SEQ ID NO: 807)domains ofscFv2SLT-1A-D1::in variableCell Line C0.0002Cell Line D0.002scFv2-Cys-C2domain of(positive)(positive)(SEQ ID NO: 812)scFv2SLT-1A-D1::scFv2noneCell Line C0.0026Cell Line D0.0073(SEQ ID NO: 838)(positive)(positive)Cell-type testedCytotoxicityCell-type testedCytotoxicityLysine(target positiveCD50(target positiveCD50Molecule Testedlocationor negative)(ng/mL)or negative)(ng/mL)SLT-1A-Lys(null)-K256 betweenCell Line A1.62Cell Line G>20,000D3-variant-Toxin Effector(positive)(negative)21::scFv4and Binding(SEQ ID NO: 824)RegionSLT-1A-Lys(null)-K256 betweenCell Line A2.4Cell Line G>20,000D3-variant-Toxin Effector(positive)(negative)27::scFv4and Binding(SEQ ID NO: 825)RegionSLT-1A-Lys(null)-K256 betweenCell Line A8.81Cell Line G>20,000D3-variant-Toxin Effector(positive)(negative)40::scFv4and Binding(SEQ ID NO: 826)RegionSLT-1A-D3::scFv4K1, K11, andCell Line A1.22Cell Line G>20,000(SEQ ID NO: 828)K256 between(positive)(negative)Toxin Effectorand BindingRegionSLT-1A-D3::scFv4K1, K11, andCell Line A1.01Cell Line G>20.000(SEQ ID NO: 828)K256 between(positive)(negative)Toxin Effectorand BindingRegionSLT-1A-D3::scFv4K1, K11Cell Line A1.36variant 2(positive)(SEQ ID NO: 829) Table 15 andFIG.4shows experimental results from examples of cell targeting molecules comprising a free, cysteine residue outside any Shiga toxin effector polypeptide component(s) (e.g., SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), and SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO:812). The cytotoxic activities of SLT-1A-D1::linker-Cys1::scFv2 (SEQ ID NO:803), SLT-1A-D1::scFv2-linker-Cys1 (SEQ ID NO:807), and SLT-1A-D1::scFv2-Cys-C2 (SEQ ID NO: 812) were comparable to the activity of the parental molecule SLT-1A-D1::scFv2 (SEQ ID NO:838), which lacks any cysteine residue in its Shiga toxin effector polypeptide component. The results reported in Table 15 show that the cell-targeting molecules comprising or consisting of SLT-1A-D1::linker-Cys1::scFv2, SLT-1A-D1::scFv2-linker-Cys1, and SLT-1A-D1::scFv2-Cys-C2 were all cytotoxic to target positive cells, with CD50values of less than 30 pM. The cytotoxicities of the Shiga toxin effector polypeptide component (SLT-1A-D1 (SEQ ID NO:831)) of these cell-targeting molecules was comparable to the cytotoxicity of the identical Shiga toxin effector polypeptide (SLT-1A-D1 (SEQ ID NO:831)) as a component of the positive control, reference molecule SLT-1A-D1::scFv2 (SEQ ID NO:837) (Table 15;FIG.4). Table 15 andFIGS.17-18show experimental results from the testing of exemplary, cell targeting molecules (SEQ ID NOs: 824-826), which each comprise a Shiga toxin effector scaffold of the present invention (SEQ ID NO: 764, 765, or 766) based on a lysine-null Shiga toxin effector polypeptide of the present invention (e.g. SLT-1A-Lys(null)-variant). Each one of these molecules SLT-1A-Lys(null)-D3-variant-21::scFv4 (SEQ ID NO:824), SLT-1A-Lys(null)-D3-variant-27::scFv4 (SEQ ID NO:825), and SLT-1A-Lys(null)-D3-variant-40::scFv4 (SEQ ID NO:826) comprises a lysine at position 256, which is outside any Shiga toxin effector polypeptide region. The cytotoxic activities of SLT-1A-Lys(null)-D3-variant-21::scFv4 (SEQ ID NO:824), SLT-1A-Lys(null)-D3-variant-27::scFv4 (SEQ ID NO:825), and SLT-1A-Lys(null)-D3-variant-40::scFv4 (SEQ ID NO:826) were comparable to the activity of the reference molecules SLT-1A-D3::scFv4 (SEQ ID NO:828) and SLT-1A-D3::scFv4 variant 2 (SEQ ID NO:829), which both comprise all the endogenous, natively occurring lysine residues present in the wild-type SLT-1A1 fragment. The results reported in Table 15 show that the cell-targeting molecules comprising or consisting of SEQ ID NO: 824, 825, or 826 were each cytotoxic to target positive cells, with CD50values of less than 10 ng/mL (see Table 15;FIGS.17-18). Thus, cell-targeting molecules comprising SEQ ID NO: 764, 765, or 766 are capable, in the context of a cell-targeting molecule, of exhibiting cytotoxicity to target positive cells, with CD50values of less than 10 ng/mL. Example 10. Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention Comprising a Unique Selenocysteine Residue for Site-Specific Conjugation In this Example, exemplary Shiga toxin A Subunit effector polypeptides of the present invention (SLT-1A-Sec(p)-variant), where Sec(p) represents a selenocysteine residue engineered at a unique position), each comprising one ectopic selenocysteine residue, are created and tested as components of exemplary cell-targeting molecules of the present invention. The ectopic, selenocysteine residues are engineered into Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. SLT-1A-Sec(p)-variant::scFv(n), scFv(n)::SLT-1A-Sec(p)-variant), StxA-Sec(p)-variant::scFv(n), and scFv(n)::StxA-Sec(p)-variant) are created using these Shiga toxin effector polypeptides. In this Example, cell-targeting molecules, each comprising a Shiga toxin A Subunit effector polypeptide having one ectopic, selenocysteine residue, are created and tested. These cell-targeting molecules each comprised a cell-targeting, immunoglobulin-type, binding region comprising a polypeptide capable of binding to an extracellular target biomolecule with high-affinity. The parental, Shiga toxin effector polypeptide of this Example is the A1 fragment of the A Subunit of Shiga toxin (SEQ ID NO:2), Shiga-like toxin 1 (SEQ ID NO:1), and Shiga-like toxin 2 (SLT-2A) (SEQ ID NO:3), optionally comprising the substitution C241S or C242S to remove the only endogenous, cysteine residue in the parental molecule. Using standard techniques, the parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides, each having one unique and ectopic selenocysteine residue and optionally no cysteine residues (see e.g. Table 16). Cell-targeting molecules comprising Shiga toxin effector polypeptides having exactly one of the ectopic selenocysteine residues described in Table 16 are made using standard techniques and tested as described in the Examples above. In Table 16, the code “Se—C” refers to selenocysteine. TABLE 16Ectopic, Selenocysteine Residues Engineeredinto Shiga Toxin Effector PolypeptidesExemplary, Shiga toxin effector polypeptidesSelenocysteineStxA-Sec(p) and SLT-1A-Sec(p)SubstitutionStxA-Sec1 and SLT-1A-Sec1K1Se-CStxA-Sec2 and SLT-1A-Sec2S8Se-CStxA-Sec3 and SLT-1A-Sec3S16Se-CStxA-Sec4 and SLT-1A-Sec4S22Se-CStxA-Sec5 and SLT-1A-Sec5S33Se-CStxA-Sec6 and SLT-1A-Sec6S43Se-CStxA-Sec7 or SLT-1A-Sec7 (identical)S45Se-CStxA-Sec8 and SLT-1A-Sec8V54Se-CStxA-Sec9 and SLT-1A-Sec9S146Se-CStxA-Sec10 and SLT-1A-Sec10S186Se-C Exemplary, cell-targeting molecules of the present invention are tested for retention of Shiga toxin A Subunit functions after introduction of selenocysteine residues into their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed are: catalytic activity, inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The catalytic activities of Shiga toxin effector polypeptide components of exemplary, cell-targeting molecules of this Example are tested using a ribosome inhibition assay. The ribosome inactivation activities of all the Shiga toxin effector polypeptides SLT-1A-Sec(p) tested in the context of a cell-targeting molecule are equivalent to the catalytic activity of similarly de-immunized Shiga toxin effector polypeptides comprising all the natively occurring cysteine residues and/or wild-type, Shiga toxin A1 fragments (amino acids 1-251 of SEQ ID NO:1 or SEQ ID NO:2; or amino acids 1-250 of SEQ ID NO:3). The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide SLT-1A-Sec(p), are determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker. The exemplary cell-targeting molecules of the present invention are each cytotoxic to target positive cells, commonly exhibiting CD50values less than 100 nM, depending on the cell-line tested. The cytotoxicities of many of the Shiga toxin effector polypeptides tested as a component of a cell-targeting molecule are comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide having all its endogenous cysteine residues. Exemplary cell-targeting molecules of this Example are produced, purified and conjugated to another molecule via a selenocysteine of the cell-targeting molecule using routine methods known to the skilled worker, such as, e.g., wherein the other molecule comprises a succinimidyl ester-fluorescent dye or iso/isothio-cyanate-fluorescent dye. These cell-targeting molecules are tested for cell-targeting, internalization, multimerization, and/or cytotoxicity as described in Example 5. The results of cytotoxicity assays show that cargo-linked cell-targeting molecules of this Example retain potent and specific cytotoxicity to target positive cells that is comparable in potency and activity to unconjugated variants of these cell-targeting molecules. The results of the experiments in this Example demonstrate that a cargo molecule can be conjugated to a selenocysteine residue of a Shiga toxin effector polypeptide component of a cell-targeting molecule of the present invention, and, furthermore, such conjugated cell-targeting molecules exhibit a useful level of cargo molecule delivery to the interior of a target positive cell and/or cytotoxicity to targeted cells. Example 11. Exemplary, Shiga Toxin Effector Polypeptides of the Present Invention Comprising a Unique Pyrroline-Carboxy-Lysine Residue for Site-Specific Conjugation In this Example, exemplary Shiga toxin A Subunit effector polypeptides of the present invention (SLT-1A-Pcl(p)-variant), where Pcl(p) represents a pyrroline-carboxy-lysine residue engineered at a unique position), each comprising one ectopic pyrroline-carboxy-lysine residue, are created and tested as components of exemplary cell-targeting molecules of the present invention. The ectopic, pyrroline-carboxy-lysine residues are engineered into Shiga toxin effector polypeptides as genetically encoded substitutions which did not change the overall number of amino acid residues in the polypeptide. Exemplary, cell-targeting molecules of the present invention (e.g. SLT-1A-Pcl(p)-variant::scFv(n), scFv(n)::SLT-1A-Pcl(p)-variant), StxA-Pcl(p)-variant::scFv(n), and scFv(n)::StxA-Pcl(p)-variant) are created using these Shiga toxin effector polypeptides. In this Example, cell-targeting molecules, each comprising a Shiga toxin A Subunit effector polypeptide having one ectopic, pyrroline-carboxy-lysine residue, are created and tested. These cell-targeting molecules each comprised a cell-targeting, immunoglobulin-type, binding region comprising a polypeptide capable of binding to an extracellular target biomolecule with high-affinity. The parental, Shiga toxin effector polypeptide of this Example is the A1 fragment of the A Subunit of Shiga toxin (SEQ ID NO:2), Shiga-like toxin 1 (SEQ ID NO:1), and Shiga-like toxin 2 (SLT-2A) (SEQ ID NO:3), optionally comprising the substitution C241S or C242S to remove the only endogenous, cysteine residue of the parental molecule. Using standard techniques, the parental, Shiga toxin effector polypeptide is used to make various Shiga toxin effector polypeptides, each having one unique and ectopic pyrroline-carboxy-lysine residue and optionally no lysine residues (see e.g. Table 17). Cell-targeting molecules comprising Shiga toxin effector polypeptides having exactly one of the ectopic pyrroline-carboxy-lysine residues described in Table 17 are made using standard techniques and tested as described in the Examples above. In Table 17, the code “Pcl” refers to pyrroline-carboxy-lysine. TABLE 17Ectopic, Pyrroline-Carboxy-Lysine Residues Engineeredinto Shiga Toxin Effector PolypeptidesShiga Toxin Effector PolypeptideStxA-Pcl(p), SLT-1A-Pcl(p), orUnique Pcl ResidueSLT-2A-Pcl(p)Substitution PositionStxA-Pcl1K1PclStxA-Pcl2K11PclStxA-Pcl3K27PclSLT-1A-Pcl1K1PclSLT-1A-Pcl2K11PclSLT-1A-Pcl3K274PclSLT-2A-Pcl1K11PclSLT-2A-Pcl2K255PclSLT-2A-Pcl3K257PclSLT-2A-Pcl4K270PclSLT-2A-Pcl5K288Pcl Exemplary, cell-targeting molecules of the present invention are tested for retention of Shiga toxin A Subunit functions after introduction of pyrroline-carboxy-lysine residues into their Shiga toxin effector polypeptide components. The Shiga toxin A Subunit functions analyzed are: catalytic activity, inhibition of eukaryotic ribosome function, cytotoxicity, and by inference self-directing subcellular routing to the cytosol. The catalytic activities of Shiga toxin effector polypeptide components of exemplary, cell-targeting molecules of this Example are tested using a ribosome inhibition assay. The ribosome inactivation activities of all the Shiga toxin effector polypeptides SLT-1A-Pcl(p) tested in the context of a cell-targeting molecule are equivalent to the catalytic activity of similarly de-immunized Shiga toxin effector polypeptides comprising all the natively occurring lysine residues and/or wild-type, Shiga toxin A1 fragments (amino acids 1-251 of SEQ ID NO:1 or SEQ ID NO:2; or amino acids 1-250 of SEQ ID NO:3). The potency and specificity of cytotoxic activities of exemplary, cell-targeting molecules of this Example, which each comprised a Shiga toxin effector polypeptide SLT-1A-Pcl(p), are determined using a tissue culture cell-based cytotoxicity assay known to the skilled worker. The exemplary cell-targeting molecules of the present invention are each cytotoxic to target positive cells, commonly exhibiting CD50values less than 100 nM, depending on the cell-line tested. The cytotoxicities of many of the Shiga toxin effector polypeptides tested as a component of a cell-targeting molecule are comparable to the cytotoxicities of a related, Shiga toxin effector polypeptide comprising all the natively occurring lysine residues and no pyrroline-carboxy-lysine residues or alternatively a wild-type, Shiga toxin A1 fragment. Exemplary cell-targeting molecules of this Example are produced, purified and conjugated to another molecule via a pyrroline-carboxy-lysine residue of the cell-targeting molecule using routine methods known to the skilled worker, such as, e.g., wherein the other molecule comprises a succinimidyl ester-fluorescent dye or iso/isothio-cyanate-fluorescent dye. These cell-targeting molecules are tested for cell-targeting, internalization, multimerization, and/or cytotoxicity as described in Example 5. The results of cytotoxicity assays show that cargo-linked cell-targeting molecules of this Example retain potent and specific cytotoxicity to target positive cells that is comparable in potency and activity to unconjugated variants of these cell-targeting molecules. The results of the experiments in this Example demonstrate that a cargo molecule can be conjugated to a pyrroline-carboxy-lysine residue of a Shiga toxin effector polypeptide component of a cell-targeting molecule of the present invention, and, furthermore, such conjugated cell-targeting molecules exhibit a useful level of cargo molecule delivery to the interior of a target positive cell and/or cytotoxicity to targeted cells. Example 12. Cell-Targeting Molecules Targeting Various Cell Types, Each Comprising a Shiga Toxin A Subunit Effector Polypeptide of the Present Invention In this example, cell-targeting molecules of the present invention are created using one or more Shiga toxin effector polypeptides described herein to provide a unique amino acid residue for site-specific attachment of a cargo and/or cell-targeting molecule altering agent wherein the Shiga toxin effector polypeptide(s) also provide two or more of the following: 1) de-immunization, 2) protease-cleavage resistance, and/or 3) an embedded or inserted, heterologous, T-cell epitope. An immunoglobulin-type binding region is derived from the molecules chosen from column 1 of Table 18 and which binds the extracellular target biomolecule indicated in column 2 of Table 18. The selected immunoglobulin-type binding region and Shiga toxin effector polypeptide are associated with each other. The exemplary proteins of this example are optionally created with a carboxy-terminal KDEL-type signal motif (“KDEL” disclosed as SEQ ID NO:1142) using techniques known in the art and optionally linked to an additional exogenous material, such as, e.g., a useful agent: detection-promoting agent, solubility-altering agent, pharmacokinetic-altering agent, immunogenicity-altering agent, and/or pharmacodynamic-altering agent like a polyethylene glycol or serum albumin, and/or an additional exogenous material: a peptide, protein, nucleic acid, protein-nucleic acid complex, cytotoxic agent, or antibiotic like an antigen, enzyme, or messenger RNA. The resulting molecules are tested for any function required to be categorized as a cell-targeting molecule of the present invention as described in the previous examples using cells expressing the appropriate extracellular target biomolecules. TABLE 18Various Binding Regions for Cell-Targeting Molecules of the Present InventionSource ofbinding regionExtracellular targetApplication(s)alemtuzumabCD52B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersbasiliximabCD25T-cell disorders, such as prevention of organ transplantrejections, and some B-cell lineage cancersbrentuximabCD30hematological cancers, B-cell related immune disorders,and T-cell related immune disorderscatumaxomabEpCAMvarious cancers, such as ovarian cancer, malignant ascites,gastric cancercetuximabEGFRvarious cancers, such as colorectal cancer and head andneck cancerdaclizumabCD25B-cell lineage cancers and T-cell disorders, such asrejection of organ transplantsdaratumumabCD38hematological cancers, B-cell related immune disorders,and T-cell related immune disordersdinutuximabganglioside GD2Various cancers, such as breast cancer, myeloid cancers,and neuroblastomaefalizumabLFA-1 (CD11a)autoimmune disorders, such as psoriasisenoblituzumabCD276 (B7-H3)various cancers and immune disordersertumaxomabHER2/neuvarious cancers and tumors, such as breast cancer andcolorectal cancergemtuzumabCD33myeloid cancer or immune disorderibritumomabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersipilimumabCD152T-cell related disorders and various cancers, such asleukemia, melanomamuromonabCD3prevention of organ transplant rejectionsnatalizumabintegrin α4autoimmune disorders, such as multiple sclerosis andCrohn's diseaseobinutuzumabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersocaratuzumabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersocrelizumabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersofatumumabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisorderspalivizumabF protein of respiratorytreat respiratory syncytial virussyncytial viruspanitumumabEGFRvarious cancers, such as colorectal cancer and head andneck cancerpertuzumabHER2/neuvarious cancers and tumors, such as breast cancer andcolorectal cancerpro 140CCR5HIV infection and T-cell disordersramucirumabVEGFR2various cancers and cancer related disorders, such as solidtumorsrituximabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisorderstocilizumab orIL-6 receptorautoimmune disorders, such as rheumatoid arthritisatlizumabtositumomabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisorderstrastuzumabHER2/neuvarious cancers and tumors, such as breast cancer andcolorectal cancerublituximabCD20B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmunedisordersvedolizumabintegrin α4β7autoimmune disorders, such as Crohn's disease andulcerative colitisCD20 bindingCD20B-cell cancers, such as lymphoma and leukemia, and B-antibodies,cell related immune disorders, such as autoimmunescFv(s), anddisorders (see e.g. Gcng S et al.,Cell Mol Immunol3: 439-fibronectin43 (2006); Olafesn T et al.,Protein Eng Des Sel23: 243-9domain(s)(2010); Natarajan A et al.,Clin Cancer Res19: 6820-9FN3CD20(2013))CD22 bindingCD22B-cell cancers or B-cell related immune disorders (see e.g.scFv(s)Kawas S et al.,MAbs3: 479-86 (2011))CD24 bindingCD24various cancers, such as bladder cancer (see e.g.monoclonalKristiansen G et al.,Lab Invest90: 1102-16 (2010))antibody(ies)CD25 bindingCD25various cancers of the B-cell lineage and immunescFv(s)disorders related to T-cells (see e.g. Muramatsu H et al.,Cancer Lett225: 225-36 (2005))CD30 bindingCD30B-cell cancers or B-cell/T-cell related immune disordersmonoclonal(see e.g. Klimka A et al.,Br J Cancer83: 252-60 (2000))antibody(ies)CD33 bindingCD33myeloid cancer or immune disorder (see e.g. Benedict C etmonoclonalal.,J Immunol Methods201: 223-31 (1997))antibody(ies)CD38 bindingCD38hematological cancers, B-cell related immune disorders,immunoglobulinand T-cell related immune disorders (see e.g. U.S. Pat. No.domains8,153,765)CD40 bindingCD40various cancers and immune disorders (see e.g. Ellmark PscFv(s)et al.,Immunology106: 456-63 (2002))CD45 bindingCD45Hematological cancers and myelodysplastic syndromesmonoclonal(see e.g. Matthews D et al.,Blood94: 1237-47 (1999); Linantibody(ies)Y et al.,Cancer Res66: 3884-92 (2006); Pagel J et al.,and scFv(s)Blood107: 2184-91 (2006))CD52 bindingCD52B-cell cancers, such as lymphoma and leukemia, and B-monoclonalcell related immune disorders, such as autoimmuneantibody(ies)disorders (see e.g. U.S. Pat. No. 7,910,104)CD56 bindingCD56immune disorders and various cancers, such as lungmonoclonalcancer, Merkel cell carcinoma, myeloma (see e.g. Shin J etantibody(ies)al.,Hybridoma18: 521-7 (1999))CD79 bindingCD79B-cell cancers or B-cell related immune disorders (see e.g.monoclonalZhang L et al.,Ther Immunol2: 191-202 (1995))antibody(ies)CD133 bindingCD133various cancers, hematologic malignancies, and immunemonoclonaldisorders (see e.g. Bidlingmaier S et al.,J Mol Med86:antibodies and1025-32 (2008); Pavlon L et al.,J Microsc231: 374-83scFv(s)(2008); Rappa G et al.,Stem Cells26: 3008-17 (2008);Swaminathan S et al.,J Immunol Methods361: 110-5(2010); Wang J et al.,Hybridoma29: 241-9 (2010); ZhuX et al.,Mol Cancer Ther9: 2131-41 (2010); Xia J et al.,Sci Rep3: 3320 (2013))CD248 bindingCD248various cancers, such as inhibiting angiogenesis (see e.g.scFv(s)Zhao A et al.,J Immunol Methods363: 221-32 (2011))CEA-bindingCEAvarious cancers, such as gastrointestinal cancer, pancreaticantibody(ies),cancer, lung cancer, and breast cancer (see e.g. NeumaierscFv(s), andM et al.,Cancer Res50: 2128-34 (1990); Pavoni E et al.,engineered,BMC Cancer6: 4 (2006); Yazaki P et al.,Nucl Med Biolfibronectin35: 151-8 (2008); Zhao J et al.,Oncol Res17: 217-22domain(s)(2008); Pirie C et al.,J Biol Chem286: 4165-72 (2011))FN3CEAEGFR-bindingEGFRvarious cancers (see e.g. GenBank Accession: 3QWQ_B;Adnectin(s) andGenBank Accession: 2KZI_A; U.S. Pat. No. 8,598,113)Affibody(ies)EpCAM bindingEpCAMvarious cancers, such as ovarian cancer, malignant ascites,monoclonalgastric cancer (see e.g. Schanzer J et al.,J Immunother29:antibody(ies)477-88 (2006))PSMA bindingPSMAprostate cancer (see e.g. Frigerio B et al.,Eur J Cancer49:monoclonal2223-32 (2013))antibody(ies)Eph-B2 bindingEph-B2various cancers such as colorectal cancer and prostatemonoclonalcancer (see e.g. Abéngozar M et al.,Blood119: 4565-76antibody(ies)(2012))EndoglinEndoglinvarious cancers, such as breast cancer and colorectalbindingcancers (see e.g. Völkel T et al.,Biochim Biophys ResmonoclonalActa1663: 158-66 (2004))antibody(ies)FAP bindingFAPvarious cancers, such as sarcomas and bone cancers (seemonoclonale.g. Zhang J et al.,FASEB J27: 581-9 (2013))antibody(ies)HER2-bindingHER2/neuvarious cancers, such as breast cancer and colorectalmonoclonalcancer (see e.g. Zahnd C et al.,J Mol Biol369: 1015-28antibodies,(2007); WO 1993/21319; WO 1994/00136; WOscFvs, VHHs,1997/00271; WO 1998/77797; U.S. Pat. No. 5,772,997; U.S. Pat. No.and DARPins5,783,186; U.S. Pat. No. 5,821,337; U.S. Pat. No. 5,840,525; U.S. Pat. No. 6,949,245;U.S. Pat. No. 7,625,859; US2011/0059090; Goldstein R et al.,Eur JNucl Med Mol Imaging42: 288-301 (2015))LewisY antigenLewisY antigensvarious cancers, such as cervical and uterine cancer (seebindinge.g. Power B et al.,Protein Sci12: 734-47 (2003);monoclonalFeridani A et al.,Cytometry71: 361-70 (2007))antibody(ies)and scFv(s)Neurotensinneurotensin receptorsvarious cancers (see e.g. GenBank Accession: 2P2C_R);receptor bindingOvigne J et al.,Neuropeptides32: 247-56 (1998); Haase Cantibodies andet al.,Anticancer Res3527-33 (2006))DARPin(s)adalimumabTNF-αvarious cancers and immune disorders, such as rheumatoidarthritis, Crohn's Disease, plaque psoriasis, psoriaticarthritis, ankylosing spondylitis, juvenile idiopathicarthritis, hemolytic disease of the newbornafelimomabTNF-αvarious cancers and immune disordersald518IL-6various cancers and immune disorders, such as rheumatoidarthritisanrukinzumab orIL-13various cancers and immune disordersima-638briakinumabIL-12, IL-23various cancers and immune disorders, such as psoriasis,rheumatoid arthritis, inflammatory bowel diseases,multiple sclerosisbrodalumabIL-17various cancers and immune disorders, such asinflammatory diseasescanakinumabIL-1various cancers and immune disorders, such as rheumatoidarthritiscertolizumabTNF-αvarious cancers and immune disorders, such as Crohn'sdiseasefezakinumabIL-22various cancers and immune disorders, such as rheumatoidarthritis, psoriasisganitumabIGF-Ivarious cancersgolimumabTNF-αvarious cancers and immune disorders, such as rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitisinfliximabTNF-αvarious cancers and immune disorders, such as rheumatoidarthritis, ankylosing spondylitis, psoriatic arthritis,psoriasis. Crohn's disease, ulcerative colitisixekizumabIL-17Avarious cancers and immune disorders, such asautoimmune diseasesmepolizumabIL-5various immune disorders and cancers, such as B-cellcancersnerelimomabTNF-αvarious cancers and immune disordersolokizumabIL6various cancers and immune disordersozoralizumabTNF-αinflammationperakizumabIL-17Avarious cancers and immune disorders, such as arthritisplaculumabhuman TNFvarious immune disorders and cancerssarilumabIL6various cancers and immune disorders, such as rheumatoidarthritis, ankylosing spondylitissiltuximabIL-6various cancers and immune disorderssirukumabIL-6various cancers and immune disorders, such as rheumatoidarthritistabalumabBAFFB-cell cancersticilimumab orCTLA-4various cancerstremelimumabtildrakizumabIL23immunologically mediated inflammatory disorderstnx-650IL-13various cancers and immune disorders, such as B-cellcancerstocilizumab orIL-6 receptorvarious cancers and immune disorders, such as rheumatoidatlizumabarthritisustekinumabIL-12, IL-23various cancers and immune disorders, such as multiplesclerosis, psoriasis, psoriatic arthritisVarious growthVEGFR, EGFR, FGFRvarious cancer, such as breast cancer and colon cancer,factors: VEGF,and to inhibit vascularizationEGF1, EGF2,FGFVariousIL-2R, IL-6R, IL-23R,various immune disorders and cancerscytokines: IL-2,CD80/CD86,IL-6, IL-23,TNFRSF13/TNFRSF17,CCL2, BAFFs,TNFRTNFs, RANKLVariousEpstein-Barr virus latentvarious cancers, viral infections, and immune disordersantibodiesmembrane protein(see e.g. Chan B et al.,In J Cancer102: 492-8 (2002);(LMP1)Fang C et al.,J Immunol Methods287: 21-30 (2004));Nichols J et al.,J Virol Methods116: 79-88 (2004); Sim Aet al.,Sci Rep3: 3232 (2013))BroadlyInfluenza surfaceviral infections (see e.g. Prabakaran P et al.,Frontneutralizingantigens (e.g.Microbiol3: 277 (2012))antibodieshemaglutinins andidentified frommatrix protein 2)patient samplesBroadlyCoronavirus surfaceviral infections (see e.g. Prabakaran P et al.,FrontneutralizingantigensMicrobiol3: 277 (2012))antibodiesidentified frompatient samplesVariousFilovirus surfaceviral infections (see e.g. Olinger G et al.,Proc Natl Acadantibodiesantigens (e.g. VP35,Sci USA109: 18030-5 (2012); Pettitt J et al.,Sci TranslVP40, and glycoprotein)Med5: 199ra113 (2013); Stahelin R,Expert Opin TherTargets18: 115-20 (2014); Becquart P et al.,PLoS One9:e96360 (2014); Stahelin R,Fron Microbiol5: 300 (2014);Tran E et al.,J Virol88: 10958-62 (2014); Murin C et al.,Proc Natl Acad Sci USA111: 17182-7 (2014))BroadlyHenipavirus surfaceviral infections (see e.g. Prabakaran P et al.,FrontneutralizingantigensMicrobiol3: 277 (2012))antibodiesidentified frompatient samplesVariousHIV surface antigensviral infections (see e.g. Kitidee K et al.,BMC Biotechnolantibodies(e.g. matrix protein10: 80 (2010); Yu L, Guan Y,Front Immunol5: 250includingMap17)(2014))broadlyneutralizingantibodies andscFvs While some embodiments of the invention have been described by way of illustration, it will be apparent that the invention may be put into practice with many modifications, variations and adaptations, and with the use of numerous equivalents or alternative solutions that are within the scope of persons skilled in the art, without departing from the spirit of the invention or exceeding the scope of the claims. All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The patent application publications WO 2005/092917, WO 2007/033497, US 2013/196928, WO 2014/164680, WO 2014/164693, WO 2015/113005, WO 2015/113007, WO 2015/120058, WO 2015/138435, WO 2015/138452, US 2015/0259428, WO 2015/191764, US2016/177284, WO 2016/126950, WO 2016/196344, and WO 2017/019623 are each incorporated herein by reference in its entirety. The disclosure of U.S. patent application 62/431,036 is incorporated herein by reference in its entirety. The complete disclosures of all electronically available biological sequence information from GenBank (National Center for Biotechnology Information, U.S.) for amino acid and nucleotide sequences cited herein are each incorporated herein by reference in their entirety. Sequence ListingID NumberText DescriptionBiological SequenceSEQ ID NO: 1Shiga-like toxin 1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSubunit A (SLT-SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN1A)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ ID NO: 2Shiga toxinKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSubunit A (StxA)TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ ID NO: 3Shiga-like toxin 2DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHSubunit A (SLT-VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTE2A)TNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKQKTECQIVGDRAAIKVNNVLWEANTIAALLNRKPQDLTEPNQSEQ ID NO: 4parental, ShigaKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGtoxin effectorSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNpolypeptideNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 5SLT-1A-Cys1CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 6SLT-1A-Cys2KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 7SLT-1A-Cys3KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 8SLT-1A-Cys4KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 9SLT-1A-Cys5KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 10SLT-1A-Cys6KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 11SLT-1A-Cys7KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 12SLT-1A-Cys8KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 13SLT-1A-Cys9KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 14SLT-1A-Cys10KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 15SLT-1A-Cys1-FRCEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 16SLT-1A-Cys2-FRKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 17SLT-1A-Cys3-FRKEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 18SLT-1A-Cys4-FRKEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 19SLT-1A-Cys5-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 20SLT-1A-Cys6-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 21SLT-1A-Cys7-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 22SLT-1A-Cys8-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 23SLT-1A-Cys9-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 24SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGFRSGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 25SLT-1A-Cys1-D1CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 26SLT-1A-Cys2-D1KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 27SLT-1A-Cys3-D1KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 28SLT-1A-Cys4-D1KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 29SLT-1A-Cys5-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 30SLT-1A-Cys6-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 31SLT-1A-Cys7-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 32SLT-1A-Cys8-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 33SLT-1A-Cys9-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 34SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD1IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 35SLT-1A-Cys1-D2CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 36SLT-1A-Cys2-D2KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 37SLT-1A-Cys3-D2KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 38SLT-1A-Cys4-D2KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 39SLT-1A-Cys5-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 40SLT-1A-Cys6-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 41SLT-1A-Cys7-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 42SLT-1A-Cys8-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 43SLT-1A-Cys9-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 44SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD2IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 45SLT-1A-Cys1CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 46SLT-1A-Cys2KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 47SLT-1A-Cys3KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 48SLT-1A-Cys4KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 49SLT-1A-Cys5KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 50SLT-1A-Cys6KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 51SLT-1A-Cys7KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 52SLT-1A-Cys8KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 53SLT-1A-Cys9KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 54SLT-1A-Cys10KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 55SLT-1A-Cys1-FRCEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 56SLT-1A-Cys2-FRKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 57SLT-1A-Cys3-FRKEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 58SLT-1A-Cys4-FRKEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 59SLT-1A-Cys5-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 60SLT-1A-Cys6-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 61SLT-1A-Cys7-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 62SLT-1A-Cys8-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 63SLT-1A-Cys9-FRKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 64SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGFR (inactivated)SGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 65SLT-1A-Cys1-D1CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 66SLT-1A-Cys2-D1KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 67SLT-1A-Cys3-D1KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 68SLT-1A-Cys4-D1KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 69SLT-1A-Cys5-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 70SLT-1A-Cys6-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 71SLT-1A-Cys7-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 72SLT-1A-Cys8-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 73SLT-1A-Cys9-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 74SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD1 (inactivated)IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 75SLT-1A-Cys1-D2CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 76SLT-1A-Cys2-D2KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 77SLT-1A-Cys3-D2KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 78SLT-1A-Cys4-D2KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 79SLT-1A-Cys5-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 80SLT-1A-Cys6-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 81SLT-1A-Cys7-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 82SLT-1A-Cys8-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 83SLT-1A-Cys9-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 84SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD2 (inactivated)IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 85SLT-1A-Cys1-D3CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 86SLT-1A-Cys2-D3KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 87SLT-1A-Cys3-D3KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 88SLT-1A-Cys4-D3KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 89SLT-1A-Cys5-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 90SLT-1A-Cys6-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 91SLT-1A-Cys7-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 92SLT-1A-Cys8-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 93SLT-1A-Cys9-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 94SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD3IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 95SLT-1A-Cys1-D4CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 96SLT-1A-Cys2-D4KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 97SLT-1A-Cys3-D4KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 98SLT-1A-Cys4-D4KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 99SLT-1A-Cys5-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 100SLT-1A-Cys6-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 101SLT-1A-Cys7-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 102SLT-1A-Cys8-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 103SLT-1A-Cys9-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 104SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD4IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 105SLT-1A-Cys1-D3CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 106SLT-1A-Cys2-D3KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 107SLT-1A-Cys3-D3KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 108SLT-1A-Cys4-D3KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 109SLT-1A-Cys5-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 110SLT-1A-Cys6-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 111SLT-1A-Cys7-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 112SLT-1A-Cys8-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 113SLT-1A-Cys9-D3KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 114SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD3 (inactivated)IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 115SLT-1A-Cys1-D4CEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 116SLT-1A-Cys2-D4KEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 117SLT-1A-Cys3-D4KEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 118SLT-1A-Cys4-D4KEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 119SLT-1A-Cys5-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 120SLT-1A-Cys6-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 121SLT-1A-Cys7-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)CGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 122SLT-1A-Cys8-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 123SLT-1A-Cys9-D4KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 124SLT-1A-Cys10-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGD4 (inactivated)IGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 125SLT-1A-Lys1KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 126SLT-1A-Lys1KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 127SLT-1A-Lys1KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 128SLT-1A-Lys1KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 129SLT-1A-Lys2REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 130SLT-1A-Lys2HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 131SLT-1A-Lys2DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 132SLT-1A-Lys2QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 133SLT-1A-Lys2SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 134SLT-1A-Lys1-FRKEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 135SLT-1A-Lys1-FRKEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 136SLT-1A-Lys1-FRKEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 137SLT-1A-Lys1-FRKEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 138SLT-1A-Lys2-FRREFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 139SLT-1A-Lys2-FRHEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 140SLT-1A-Lys2-FRDEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 141SLT-1A-Lys2-FRQEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 142SLT-1A-Lys2-FRSEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 143SLT-1A-Lys1-D1KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 144SLT-1A-Lys1-D1KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 145SLT-1A-Lys1-D1KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 146SLT-1A-Lys1-D1KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 147SLT-1A-Lys2-D1REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 148SLT-1A-Lys2-D1HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 149SLT-1A-Lys2-D1DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 150SLT-1A-Lys2-D1QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 151SLT-1A-Lys2-D1SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 152SLT-1A-Lys1-D2KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 153SLT-1A-Lys1-D2KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 154SLT-1A-Lys1-D2KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 155SLT-1A-Lys1-D2KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 156SLT-1A-Lys2-D2REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 157SLT-1A-Lys2-D2HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 158SLT-1A-Lys2-D2DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 159SLT-1A-Lys2-D2QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 160SLT-1A-Lys2-D2SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 161SLT-1A-Lys1KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 162SLT-1A-Lys1KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 163SLT-1A-Lys1KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 164SLT-1A-Lys1KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 165SLT-1A-Lys2REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 166SLT-1A-Lys2HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 167SLT-1A-Lys2DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 168SLT-1A-Lys2QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 169SLT-1A-Lys2SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 170SLT-1A-Lys1-FRKEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 171SLT-1A-Lys1-FRKEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 172SLT-1A-Lys1-FRKEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 173SLT-1A-Lys1-FRKEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 174SLT-1A-Lys2-FRREFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 175SLT-1A-Lys2-FRHEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 176SLT-1A-Lys2-FRDEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 177SLT-1A-Lys2-FRQEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 178SLT-1A-Lys2-FRSEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 179SLT-1A-Lys1-D1KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 180SLT-1A-Lys1-D1KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 181SLT-1A-Lys1-D1KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 182SLT-1A-Lys1-D1KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 183SLT-1A-Lys2-D1REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 184SLT-1A-Lys2-D1HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 185SLT-1A-Lys2-D1DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 186SLT-1A-Lys2-D1QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 187SLT-1A-Lys2-D1SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 188SLT-1A-Lys1-D2KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 189SLT-1A-Lys1-D2KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 190SLT-1A-Lys1-D2KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 191SLT-1A-Lys1-D2KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 192SLT-1A-Lys2-D2REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 193SLT-1A-Lys2-D2HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 194SLT-1A-Lys2-D2DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 195SLT-1A-Lys2-D2QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 196SLT-1A-Lys2-D2SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 197SLT-1A-Lys1-D3KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 198SLT-1A-Lys1-D3KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 199SLT-1A-Lys1-D3KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 200SLT-1A-Lys1-D3KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 201SLT-1A-Lys2-D3REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 202SLT-1A-Lys2-D3HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 203SLT-1A-Lys2-D3DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 204SLT-1A-Lys2-D3QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 205SLT-1A-Lys2-D3SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 206SLT-1A-Lys1-D4KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 207SLT-1A-Lys1-D4KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 208SLT-1A-Lys1-D4KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 209SLT-1A-Lys1-D4KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 210SLT-1A-Lys2-D4REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 211SLT-1A-Lys2-D4HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 212SLT-1A-Lys2-D4DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 213SLT-1A-Lys2-D4QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 214SLT-1A-Lys2-D4SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 215SLT-1A-Lys1-D3KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 216SLT-1A-Lys1-D3KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 217SLT-1A-Lys1-D3KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 218SLT-1A-Lys1-D3KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 219SLT-1A-Lys2-D3REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 220SLT-1A-Lys2-D3HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 221SLT-1A-Lys2-D3DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 222SLT-1A-Lys2-D3QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 223SLT-1A-Lys2-D3SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 224SLT-1A-Lys1-D4KEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 225SLT-1A-Lys1-D4KEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 226SLT-1A-Lys1-D4KEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 227SLT-1A-Lys1-D4KEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 228SLT-1A-Lys2-D4REFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 229SLT-1A-Lys2-D4HEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 230SLT-1A-Lys2-D4DEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 231SLT-1A-Lys2-D4QEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 232SLT-1A-Lys2-D4SEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGvariant 5IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 233SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 234SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 235SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 236SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN4NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 237SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN5NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 238SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN6NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 239SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN7NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 240SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN8NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 241SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN9NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 242SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN10NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 243SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN11NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 244SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN12NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 245SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN13NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 246SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN14NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 247SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN15NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 248SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN16NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 249SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN17NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 250SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN18NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 251SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN19NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 252SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 253SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 254SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 255SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 256SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 257SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 258SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 259SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 260SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 261SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 262SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 263SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 264SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 265SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 266SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 267SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 268SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 269SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 270SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 271SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 272SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 273SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 274SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 275SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 276SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 277SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 278SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 279SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 280SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 281SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 282SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 283SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 284SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 285SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 286SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 287SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 288SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 289SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 290SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 291SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 292SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D1GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 20VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 293SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 294SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 295SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 296SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 297SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 298SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 299SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 300SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 301SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 302SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 303SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 304SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 305SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 306SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 307SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 308SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 309SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 310SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 311SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D2GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 19VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 312SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 313SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 314SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN1 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 315SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN2 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 316SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN3 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 317SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN4 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 318SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN5 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 319SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN6 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 320SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN7 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 321SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN8 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 322SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN9 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 323SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN10 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 324SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN11 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 325SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN12 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 326SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN13 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 327SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN14 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 328SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN15 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 329SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN16 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 330SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN17 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 331SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN18 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 332SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN19 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 333SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN20 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 334SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 335SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 336SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 337SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 338SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 339SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 340SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 341SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 342SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 343SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 344SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 345SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 346SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 347SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 348SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 349SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 350SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 351SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 352SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 353SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 354SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 355SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 356SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 357SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 358SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 359SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 360SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 361SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 362SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 363SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 364SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 365SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 366SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 367SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 368SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 369SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 370SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 371SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 372SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 373SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D1GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 20VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 374SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 375SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 376SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 377SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 378SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 379SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 380SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 381SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 382SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 383SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 384SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 385SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 386SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 387SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 388SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 389SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 390SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 391SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 392SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D2GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 19VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 393SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 394SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 395SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 396SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 397SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 398SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 399SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 400SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 401SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 402SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 403SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 404SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 405SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 406SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 407SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 408SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 409SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 410SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 411SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 412SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 413SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 414SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 20VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 415SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 416SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 417SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 418SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 419SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 420SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 421SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 422SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 423SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 424SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 425SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 426SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 427SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 428SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 429SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 430SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 431SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 432SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 433SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D4GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 19VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 434SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 435SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 436SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 437SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 438SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 439SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 440SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 441SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 442SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 443SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 444SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 445SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 446SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 447SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 448SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 449SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 450SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 451SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 452SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 453SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 454SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 19NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 455SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 20VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 456SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 457SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 458SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 459SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 460SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 461SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 6NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 462SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 7NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 463SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 8NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 464SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 9NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 465SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 10NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 466SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 11NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 467SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 12NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 468SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 13NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 469SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 14NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 470SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 15NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 471SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 16NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 472SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 17NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 473SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 18NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 474SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D4GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 19VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 475SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 20NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 476SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 477SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN21NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 478SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN22NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 479SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN23NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 480SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN24NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 481SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN25NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 482SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN26NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 483SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN27NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 484SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN28NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 485SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN29NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 486SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN30NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 487SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN31NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 488SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN32NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 489SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN33NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 490SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN34NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 491SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN35NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 492SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN36NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 493SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN37NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 494SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN38NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 495SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN39NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 496SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN40NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 497SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 498SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 499SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 500SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 501SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 502SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 503SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 504SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 505SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 506SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 507SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 508SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 509SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 510SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 511SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 512SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 513SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 514SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 515SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 516SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 40NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 517SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 518SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 519SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 520SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 521SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 522SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 523SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 524SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 525SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 526SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 527SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 528SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 529SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 530SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 531SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 532SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 533SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 534SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 535SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 536SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D1GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 537SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 538SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 539SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 540SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 541SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 542SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 543SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 544SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 545SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 546SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 547SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 548SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 549SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 550SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 551SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 552SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 553SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 554SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 555SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D2GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 556SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 41NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 557SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 42NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 558SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN21 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 559SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN22 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 560SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN23 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 561SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN24 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 562SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN25 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 563SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN26 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 564SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN27 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 565SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN28 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 566SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN29 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 567SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN30 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 568SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN31 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 569SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN32 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 570SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN33 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 571SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN34 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 572SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN35 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 573SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN36 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 574SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN37 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 575SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN38 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 576SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN39 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 577SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN40 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 578SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 579SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 580SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 581SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 582SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 583SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 584SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 585SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 586SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 587SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 588SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 589SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 590SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 591SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 592SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 593SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 594SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 595SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 596SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 597SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 40NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 598SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 599SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 600SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 601SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 602SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 603SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 604SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 605SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 606SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 607SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 608SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 609SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 610SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 611SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 612SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 613SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 614SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 615SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 616SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 617SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D1GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 618SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 619SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 620SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 621SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 622SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 623SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 624SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 625SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 626SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 627SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 628SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 629SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 630SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 631SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 632SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 633SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 634SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 635SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 636SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D2GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 637SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 41NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 638SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 42NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 639SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 640SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 641SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 642SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 643SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 644SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 645SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 646SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 647SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 648SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 649SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 650SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 651SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 652SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 653SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 654SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 655SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 656SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 657SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 658SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 659SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 660SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 661SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 662SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 663SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 664SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 665SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 666SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 667SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 668SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 669SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 670SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 671SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 672SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 673SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 674SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 675SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 676SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 677SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D4GDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 678SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 41NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 679SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 42NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 680SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 21NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 681SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 682SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 683SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 684SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 685SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 686SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 687SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 688SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 689SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 690SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 691SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 692SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 693SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 694SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 695SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 696SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 697SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 698SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 699SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 700SLT-1A-HEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 22NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 701SLT-1A-DEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 23NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 702SLT-1A-QEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 24NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 703SLT-1A-SEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 25NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 704SLT-1A-REFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 26NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 705SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 27NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 706SLT-1A-DEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 707SLT-1A-QEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 708SLT-1A-SEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 709SLT-1A-REFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 31NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 710SLT-1A-HEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 32NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 711SLT-1A-DEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 712SLT-1A-QEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 713SLT-1A-SEFTLDFSTAQTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 35NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 714SLT-1A-REFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 36NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 715SLT-1A-HEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 37NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 716SLT-1A-DEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 38NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 717SLT-1A-QEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 39NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 718SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-D4GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNvariant 40VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQ(inactivated)INRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 719SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 41NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 720SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 42NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 721SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 722SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 723SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 724SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 725SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN1 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 726SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 727SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 728SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 729SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 730SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 731SLT-1A-kEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGICys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNN(inactivated)VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQvariant 1INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 732SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 1NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 733SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 734SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 735SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 736SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 737SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTN2 (inactivated)NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 738SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 739SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 740SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASRVARSEQ ID NO: 741SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 742SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 743SLT-1A-kEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGICys(null)-D3GDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNN(inactivated)VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQvariant 2INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 744SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNAHHHASAVAASEQ ID NO: 745SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTN3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 746SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 747SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 748SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 749SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null) variantIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN3 (inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 750SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-FRSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 751SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D1IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 752SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D2IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASRVARSEQ ID NO: 753SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 754SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 755SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D3IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTN(inactivated)NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMvariant 3QINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 756SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-D4IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNvariant 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGM(inactivated)QINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNVHHHASAVAASEQ ID NO: 757linker-Cys1EFPKPCTPPGSSGGAPSEQ ID NO: 758linker-Cys2EFPKPSTPPGSCGGAPSEQ ID NO: 759linker-Cys3AHHCEDPSSKAPKAPSEQ ID NO: 760linker-Cys4AHHSEDPSCKAPKAPSEQ ID NO: 761linker-Lys1EFPKPSTPPGSSGGAPSEQ ID NO: 762SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-scaffoldIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1 C257NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPSEQ ID NO: 763SLT-1A-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGCys(null)-scaffoldIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2 C257NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPSEQ ID NO: 764SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-scaffoldIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 1 K255NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPSEQ ID NO: 765SLT-1A-HEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-scaffoldIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2 K255NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPSEQ ID NO: 766SLT-1A-SEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILys(null)-scaffoldGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNvariant 3 K255VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPSEQ ID NO: 767SLT-1A-REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGLys(null)-scaffoldIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 4 K255NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPSEQ ID NO: 768linker-IgA hingeSPSTPPTPSPSTPPASSEQ ID NO: 769scFv-linker-Cys1GGGGCSEQ ID NO: 770scFv-linker-Cys2GGGGSGGGGSGGGGCGGGGSGGGGSSEQ ID NO: 771scFv-linker-Cys3GSTSGCGKPGSGEGSSEQ ID NO: 772scFv-linker-Cys4GSTSGCGKPGSGEGSTKGSEQ ID NO: 773cell-targetingMKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 1SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 774cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 2CGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 775cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 3SGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 776cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 4SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 777cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 5SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 778cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDmolecule 6SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMEIWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 779cell-targetingMKEFTLDFSTAKTYVDCLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 7SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMEIWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 780cell-targetingMKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 8SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMEIWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 781cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDmolecule 9SGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITVRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYAQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSVAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYASRWGGDGFYAMDVWGQGTLVTVSSASEQ ID NO: 782cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 10SGCGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITVRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYAQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSVAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYASRWGGDGFYAMDVWGQGTLVTVSSASEQ ID NO: 783cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 11SGSGDNLFAVDCRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITVRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYAQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSVAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYASRWGGDGFYAMDVWGQGTLVTVSSASEQ ID NO: 784cell-targetingMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGmolecule 12QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 785cell-targetingMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGmolecule 13QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDCGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 786cell-targetingMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGmolecule 14QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 787cell-targetingMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGmolecule 15QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 788cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 16SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSASEQ ID NO: 789cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDmolecule 17SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 790cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 18CGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 791cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 19SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 792cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 20SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 793cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 21SGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 794cell-targetingMKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 22SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 795cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 23SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 796cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 24SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 797cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 25SGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 798cell-targetingMKEFTLDFCTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 26SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 799cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 27SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 800cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 28SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 801cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 29SGCGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 802cell-targetingMKEFTLDFSTAKTYVDSLNVIRCAIGTPLQTISSGGTSLLMIDmolecule 30SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 803cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 31SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 804cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 32SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 805cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 33SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 806cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 34SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPCTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 807cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 35SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGCQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 808cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 36SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGCQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 809cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 37SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGCGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 810cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 38SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGCGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 811cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 39SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGCGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 812cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 40SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVCSSEQ ID NO: 813cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 41SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVCSSEQ ID NO: 814cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 42SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVCSSEQ ID NO: 815cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 43SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVCSSEQ ID NO: 816cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 44SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSCASEQ ID NO: 817cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 45SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSCASEQ ID NO: 818cell-targetingMKEFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 46SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 819cell-targetingMKEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 47SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 820cell-targetingMKEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSmolecule 48GIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 821cell-targetingMREFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 49SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 822cell-targetingMHEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 50SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 823cell-targetingMSEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSmolecule 51GIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 824cell-targetingMREFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSmolecule 52GIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 825cell-targetingMHEFTLDFSTAHTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 53SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 826cell-targetingMSEFTLDFSTASTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSmolecule 54GIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 827cell-targetingMREFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSmolecule 55GIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASPSTPPTPSPSTPPASQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 828cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 56SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 829cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDmolecule 57SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASPSTPPTPSPSTPPASQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO: 830SLT-1A1-WTKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 831SLT-1A-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 832SLT-1A-D1-KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGC242IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAASEQ ID NO: 833SLT-1A-D2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 834IA- SLT-1A-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ ID NO: 835cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDprotein 1SGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 836cell-targetingMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGprotein 2QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ ID NO: 837cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDprotein 3SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 838reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDtargetingSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRmolecule 1TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 839reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDtargetingSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRmolecule 2TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMEIWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 840reference cell-MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGtargetingQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVmolecule 3YYCQQRSDWPLTFGGGTKVEIKGSTSGSGKPGSGEGSAVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVTVSSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVARSEQ ID NO: 841reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDtargetingSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRmolecule 4TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSASEQ ID NO: 842reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDtargetingSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRmolecule 5TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 843reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDtargetingSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRmolecule 1TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 844heavy chain ABR1YTFTSYVMHSEQ ID NO: 845heavy chain ABR2WIGYINPYNDGTKYSEQ ID NO: 846heavy chain ABR3RGTYYYGSRVFDYSEQ ID NO: 847light chain ABR1KSLLNSNGNTYLYSEQ ID NO: 848light chain ABR2LLIYRMSNLASSEQ ID NO: 849light chain ABR3MQHLEYPFSEQ ID NO: 850heavy chain ABR1YAFSSYWMNSEQ ID NO: 851heavy chain ABR2WIGQIWPGDGDTNYSEQ ID NO: 852heavy chain ABR3RRETTTVGRYYYAMDYSEQ ID NO: 853light chain ABR1QSVDYDGDSYLNSEQ ID NO: 854light chain ABR2LLIYDASNLVSSEQ ID NO: 855light chain ABR3QQSTEDPWSEQ ID NO: 856heavy chain ABR2WIGQIWPGDGDTNYNGSEQ ID NO: 857heavy chain ABR1GSISTSGMGVGSEQ ID NO: 858heavy chain ABR2WIGHIWWDDDKRYSEQ ID NO: 859heavy chain ABR3RMELWSYYFDYSEQ ID NO: 860light chain ABR1SSVSYMHSEQ ID NO: 861light chain ABR2LLIYDTSKLASSEQ ID NO: 862light chain ABR3FQGSVYPFSEQ ID NO: 863heavy chain CDR1GYTFTSYNMHSEQ ID NO: 864heavy chain CDR2AIYPGNGDTSYNQKFKGSEQ ID NO: 865heavy chain CDR3AQLRPNYWYFDVSEQ ID NO: 866light chain CDR1RASSSVSYMHSEQ ID NO: 867light chain CDR2ATSNLASSEQ ID NO: 868light chain CDR3QQWISNPPTSEQ ID NO: 869heavy chain CDR1GYTFTSYNVHSEQ ID NO: 870heavy chain CDR3SNYYGSSYVWFFDVSEQ ID NO: 871light chain CDR1RASSSVSYMDSEQ ID NO: 872heavy chain CDR3STYYGGDWYFNVSEQ ID NO: 873light chain CDR1RASSSVSYIHSEQ ID NO: 874light chain CDR3QQWTSNPPTSEQ ID NO: 875heavy chain CDR1GFTFNDYAMHSEQ ID NO: 876heavy chain CDR2TISWNSGSIGYADSVKGSEQ ID NO: 877heavy chain CDR3DIQYGNYYYGMDVSEQ ID NO: 878light chain CDR1RASQSVSSYLASEQ ID NO: 879light chain CDR2DASNRATSEQ ID NO: 880light chain CDR3QQRSNWPITSEQ ID NO: 881heavy chain CDR1GYTFTSYNMHSEQ ID NO: 882heavy chain CDR3VVYYSNSYWYFDVSEQ ID NO: 883light chain CDR2APSNLASSEQ ID NO: 884light chain CDR3QQWSFNPPTSEQ ID NO: 885heavy chain CDR1GYAFSYSWINSEQ ID NO: 886heavy chain CDR2RIFPGDGDTDYNGKFKGSEQ ID NO: 887heavy chain CDR3NVFDGYWLVYSEQ ID NO: 888light chain CDR1RSSKSLLHSNGITYLYSEQ ID NO: 889light chain CDR2QMSNLVSSEQ ID NO: 890light chain CDR3AQNLELPYTSEQ ID NO: 891heavy chain ABR1YRFTNYWIHSEQ ID NO: 892heavy chain ABR2WIGGINPGNNYATYRRSEQ ID NO: 893heavy chain ABR3TREGYGNYGAWFAYSEQ ID NO: 894light chain ABR1QSLANSYGNTFLSSEQ ID NO: 895light chain ABR2LLIYGISNRFSSEQ ID NO: 896light chain ABR3LQGTHQPYSEQ ID NO: 897heavy chain ABR1FAFSIYDMSSEQ ID NO: 898heavy chain ABR2WVAYISSGGGTTYYSEQ ID NO: 899heavy chain ABR3RHSGYGTHWGVLFAYSEQ ID NO: 900light chain ABR1QDISNYLASEQ ID NO: 901light chain ABR2LLIYYTSILHSSEQ ID NO: 902light chain ABR3QQGNTLPWSEQ ID NO: 903heavy chain ABR1YTFTSYWLHSEQ ID NO: 904heavy chain ABR2WIGYINPRNDYTEYSEQ ID NO: 905heavy chain ABR3RRDITTFYSEQ ID NO: 906light chain ABR1QSVLYSANHKNYLASEQ ID NO: 907light chain ABR2LLIYWASTRESSEQ ID NO: 908light chain ABR3HQYLSSWSEQ ID NO: 909heavy chain ABR1YEFSRSWMNSEQ ID NO: 910heavy chain ABR2WVGRIYPGDGDTNYSGKFSEQ ID NO: 911heavy chain ABR3RDGSSWDWYFDVSEQ ID NO: 912light chain ABR1QSIVHSVGNTFLESEQ ID NO: 913light chain ABR2LLIYKVSNRFSSEQ ID NO: 914light chain ABR3FQGSQFPYSEQ ID NO: 915heavy chain CDR1GYRFTNYWIHSEQ ID NO: 916heavy chain CDR2GINPGNNYATYRRKFQGSEQ ID NO: 917heavy chain CDR3EGYGNYGAWFAYSEQ ID NO: 918light chain CDR1RSSQSLANSYGNTFLSSEQ ID NO: 919light chain CDR2GISNRFSSEQ ID NO: 920light chain CDR3LQGTHQPYTSEQ ID NO: 921heavy chain CDR1GFAFSIYDMSSEQ ID NO: 922heavy chain CDR2YISSGGGTTYYPDTVKGSEQ ID NO: 923heavy chain CDR3HSGYGTHWGVLFAYSEQ ID NO: 924light chain CDR1RASQDISNYLASEQ ID NO: 925light chain CDR2YTSILHSSEQ ID NO: 926light chain CDR3QQGNTLPWTSEQ ID NO: 927heavy chain CDR1GYTFTDYYITSEQ ID NO: 928heavy chain CDR2WIYPGSGNTKYNEKFSEQ ID NO: 929heavy chain CDR3YGNYWFAYSEQ ID NO: 930light chain CDR1KASQSVDFDGDSYMNSEQ ID NO: 931light chain CDR2AASNLESSEQ ID NO: 932light chain CDR3QQSNEDPWTSEQ ID NO: 933heavy chain CDR1YTFTTYWMHSEQ ID NO: 934heavy chain CDR2WIGYINPSTGYTDYSEQ ID NO: 935heavy chain CDR3TRRGPSYGNHGAWFPYSEQ ID NO: 936light chain CDR1ENVDTYVSSEQ ID NO: 937light chain CDR2LLIYGASNRYTSEQ ID NO: 938light chain CDR3GQSYRYPPSEQ ID NO: 939heavy chain CDR1GYTFTGYYMHSEQ ID NO: 940heavy chain CDR2WIDPNSGATTYAQKFSEQ ID NO: 941heavy chain CDR3KTTQTTWGFPFSEQ ID NO: 942light chain CDR1RASQGVYQWLASEQ ID NO: 943light chain CDR2KASHLYNSEQ ID NO: 944light chain CDR3QQLNSYPLTSEQ ID NO: 945heavy chain CDR1GYTFTDYWMHSEQ ID NO: 946heavy chain CDR2WIGYINPNTAYTDYSEQ ID NO: 947light chain CDR1KASENVDSFVSSEQ ID NO: 948light chain CDR2GASNRYTSEQ ID NO: 949light chain CDR3GQNYRYPLTSEQ ID NO: 950heavy chain ABR1FSLISYGVHSEQ ID NO: 951heavy chain ABR2WLGVIWRGGSTDYSEQ ID NO: 952heavy chain ABR3KTLITTGYAMDYSEQ ID NO: 953light chain ABR1EDIYNRLASEQ ID NO: 954light chain ABR2LLISGATSLETGSEQ ID NO: 955light chain ABR3QQYWSTPSEQ ID NO: 956heavy chain ABR1FTFNSFAMSSEQ ID NO: 957heavy chain ABR2WVSAISGSGGGTYYSEQ ID NO: 958heavy chain ABR3KDKILWFGEPVFDYSEQ ID NO: 959light chain ABR1QSVSSYLASEQ ID NO: 960light chain ABR2LLIYDASNRATSEQ ID NO: 961light chain ABR3QQRSNWPPSEQ ID NO: 962heavy chain ABR1FSLTSYGVHSEQ ID NO: 963heavy chain ABR2WIGVMWRGGSTDYSEQ ID NO: 964heavy chain ABR3KSMITTGFVMDSSEQ ID NO: 965light chain ABR1EDIYNRLTSEQ ID NO: 966light chain ABR2LLISGATSLETSEQ ID NO: 967light chain ABR3QQYWSNPYSEQ ID NO: 968heavy chain ABR1FDFSRSWMNSEQ ID NO: 969heavy chain ABR2WIGEINPDSSTINYSEQ ID NO: 970heavy chain ABR3RYGNWFPYSEQ ID NO: 971light chain ABR1QNVDTNVASEQ ID NO: 972light chain ABR2ALIYSASYRYSSEQ ID NO: 973light chain ABR3QQYDSYPLSEQ ID NO: 974heavy chain ABR1GTFSSYAFSSEQ ID NO: 975heavy chain ABR2WMGRVIPFLGIANSSEQ ID NO: 976heavy chain ABR3RDDIAALGPFDYSEQ ID NO: 977light chain ABR1QGISSWLASEQ ID NO: 978light chain ABR2SLIYAASSLQSSEQ ID NO: 979light chain ABR3QQYNSYPRSEQ ID NO: 980heavy chain ABR1YTFTDYWMQSEQ ID NO: 981heavy chain ABR2WIGTIYPGDGDTGYSEQ ID NO: 982heavy chain ABR3RGDYYGSNSLDYSEQ ID NO: 983light chain ABR1QDVSTVVASEQ ID NO: 984light chain ABR2RLIYSASYRYISEQ ID NO: 985light chain ABR3QQHYSPPYSEQ ID NO: 986heavy chain CDR1GFSLTSYGVHSEQ ID NO: 987heavy chain CDR2VMWRGGSTDYNAAFMSSEQ ID NO: 988heavy chain CDR3SMITTGFVMDSSEQ ID NO: 989light chain CDR1KASEDIYNRLTSEQ ID NO: 990light chain CDR2GATSLETSEQ ID NO: 991light chain CDR3QQYWSNPYTSEQ ID NO: 992heavy chain CDR1GFSLISYGVHSEQ ID NO: 993heavy chain CDR2VIWRGGSTDYNAAFMSSEQ ID NO: 994heavy chain CDR3TLITTGYAMDYSEQ ID NO: 995light chain CDR1KASEDIYNRLASEQ ID NO: 996light chain CDR2GATSLETSEQ ID NO: 997light chain CDR3QQYWSTPTSEQ ID NO: 998heavy chain CDR1GFDFSRSWMNSEQ ID NO: 999heavy chain CDR2EINPDSSTINYTTSLKDSEQ ID NO: 1000heavy chain CDR3YGNWFPYSEQ ID NO: 1001light chain CDR1KASQNVDTNVASEQ ID NO: 1002light chain CDR2SASYRYSSEQ ID NO: 1003light chain CDR3QQYDSYPLTSEQ ID NO: 1004heavy chain ABR1FDFSRYWMSSEQ ID NO: 1005heavy chain ABR2WIGEINPTSSTINFSEQ ID NO: 1006heavy chain ABR3RGNYYRYGDAMDYSEQ ID NO: 1007light chain ABR1KSVSTSGYSYLHSEQ ID NO: 1008light chain ABR2LLIYLASNLESSEQ ID NO: 1009light chain ABR3QHSRELPFSEQ ID NO: 1010heavy chain ABR1STFTTYWIHSEQ ID NO: 1011heavy chain ABR2WIGYINPNTGYTEYSEQ ID NO: 1012heavy chain ABR3VRFITVVGGSEQ ID NO: 1013light chain ABR1SSVSSSHLHSEQ ID NO: 1014light chain ABR2LWIYSTSNLASSEQ ID NO: 1015light chain ABR3HQYHRSPLSEQ ID NO: 1016heavy chain ABR1FSLTTYGIGVGSEQ ID NO: 1017heavy chain ABR2WLTHIWWNDNKYYSEQ ID NO: 1018heavy chain ABR3YGYTYSEQ ID NO: 1019light chain ABR1QSLLYSNGNTYLHSEQ ID NO: 1020light chain ABR2LLIYKLSNRFSSEQ ID NO: 1021light chain ABR3SQSTHVPWSEQ ID NO: 1022heavy chain ABR1FNIKDTYIHSEQ ID NO: 1023heavy chain ABR2WVARIYPTNGYTRYSEQ ID NO: 1024heavy chain ABR3RWGGDGFYAMDYSEQ ID NO: 1025light chain ABR1QDVNTAVASEQ ID NO: 1026light chain ABR2LLIYSASFLYSSEQ ID NO: 1027light chain ABR3QQHYTTPPSEQ ID NO: 1028heavy chain ABR3RWGGDGFYAMDVSEQ ID NO: 1029heavy chain ABR1YSFTSYWIASEQ ID NO: 1030heavy chain ABR2YMGLIYPGDSDTKYSEQ ID NO: 1031heavy chain ABR3RHDVGYCSSSNCAKWPEYFQHSEQ ID NO: 1032light chain ABR1SSNIGNNYVSSEQ ID NO: 1033light chain ABR2LLIYGHTNRPASEQ ID NO: 1034light chain ABR3AAWDDSLSGWSEQ ID NO: 1035heavy chain ABR1YPFTNYGMNSEQ ID NO: 1036heavy chain ABR2WMGWINTSTGESTFSEQ ID NO: 1037heavy chain ABR3RWEVYHGYVPYSEQ ID NO: 1038light chain ABR1QDVYNAVASEQ ID NO: 1039light chain ABR2LLIYSASSRYTSEQ ID NO: 1040light chain ABR3QQHFRTPFSEQ ID NO: 1041heavy chain ABR1ITFSINTMGSEQ ID NO: 1042heavy chain ABR2LVALISSIGDTYYASEQ ID NO: 1043heavy chain ABR3KRFRTAAQGTDYSEQ ID NO: 1044heavy chain CDR1GFNIKDTYIHSEQ ID NO: 1045heavy chain CDR2RIYPTNGYTRYADSVKGSEQ ID NO: 1046heavy chain CDR3WGGDGFYAMDYSEQ ID NO: 1047light chain CDR1RASQDVNTAVASEQ ID NO: 1048light chain CDR2SASFLYSSEQ ID NO: 1049light chain CDR3QQHYTTPPTSEQ ID NO: 1050heavy chain CDR1GFNIKDTYIHSEQ ID NO: 1051heavy chain CDR2RIYPTNGYTRYADSVKGSEQ ID NO: 1052heavy chain CDR3WGGDGFYAMDVSEQ ID NO: 1053light chain CDR1RASQDVNTAVASEQ ID NO: 1054light chain CDR2SASFLYSSEQ ID NO: 1055light chain CDR3QQHYTTPPTSEQ ID NO: 1056heavy chain CDR1GYSFTSYWIASEQ ID NO: 1057heavy chain CDR2LIYPGDSDTKYSPSFQGSEQ ID NO: 1058heavy chain CDR3HDVGYCSSSNCAKWPEYFQHSEQ ID NO: 1059light chain CDR1SGSSSNIGNNYVSSEQ ID NO: 1060light chain CDR2GHTNRPASEQ ID NO: 1061light chain CDR3AAWDDSLSGWVSEQ ID NO: 1062heavy chain CDR1GITFSINTMGSEQ ID NO: 1063heavy chain CDR2LISSIGDTYYADSVKGSEQ ID NO: 1064heavy chain CDR3FRTAAQGTDYSEQ ID NO: 1065heavy chain ABR1FTFSDSWIHSEQ ID NO: 1066heavy chain ABR2WVAWISPYGGSTYYSEQ ID NO: 1067heavy chain ABR3RRHWPGGFDYSEQ ID NO: 1068light chain ABR1QDVSTAVASEQ ID NO: 1069light chain ABR2LLIYSASFLYSSEQ ID NO: 1070light chain ABR3QQYLYHPASEQ ID NO: 1071heavy chain ABR1YTFTSYVMHSEQ ID NO: 1072heavy chain ABR2WIGYVNPFNDGTKYSEQ ID NO: 1073heavy chain ABR3RQAWGYPGFQHSEQ ID NO: 1074light chain ABR1ESVEYYGTSLVQSEQ ID NO: 1075light chain ABR2LLIYAASSVDSSEQ ID NO: 1076light chain ABR3QQSRRVPYSEQ ID NO: 1077heavy chain ABR1YTFTSYDVHSEQ ID NO: 1078heavy chain ABR2WMGWLHADTGITKFSEQ ID NO: 1079heavy chain ABR3RERIQLWFDYSEQ ID NO: 1080light chain ABR1QGISSWLASEQ ID NO: 1081light chain ABR2SLIYAASSLQSSEQ ID NO: 1082light chain ABR3QQYNSYPYSEQ ID NO: 1083heavy chain ABR1DTFSTYAISSEQ ID NO: 1084heavy chain ABR2WMGGIIPIFGKAHYSEQ ID NO: 1085heavy chain ABR3RKFHFVSGSPFGMDVSEQ ID NO: 1086light chain ABR1QSVSSYLASEQ ID NO: 1087light chain ABR2LLIYDASNRATSEQ ID NO: 1088light chain ABR3QQRSNWPSEQ ID NO: 1089heavy chain ABR1FTFSSYIMMSEQ ID NO: 1090heavy chain ABR2WVSSIYPSGGITFYSEQ ID NO: 1091heavy chain ABR3RIKLGTVTTVDYSEQ ID NO: 1092light chain ABR1SSDVGGYNYVSSEQ ID NO: 1093light chain ABR2LMIYDVSNRPSSEQ ID NO: 1094light chain ABR3SSYTSSSTRSEQ ID NO: 1095heavy chain CDR1GFNIKDYFLHSEQ ID NO: 1096heavy chain CDR2WINPDNGNTVYDKFQGSEQ ID NO: 1097heavy chain CDR3RDYTYEKAALDYSEQ ID NO: 1098light chain CDR1RASGNIYNYLASEQ ID NO: 1099light chain CDR2DAKTLADSEQ ID NO: 1100light chain CDR3QHFWSLPFTSEQ IDSLT-1A-Cys5-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDSGNO: 1101(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ IDSLT-1A-Cys7-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1102(inactivated)CGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ IDSLT-1A-Cys8-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1103(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHCGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ IDSLT-1A-Cys9-D1KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1104(inactivated)IGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNvariant 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLCGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASEQ IDcell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNO: 1105protein 4SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ IDcell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNO: 1106protein 5SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ IDcell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNO: 1107protein 6SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ IDcell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNO: 1108protein 7SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ IDStxA-Lys(1)KEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1109TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDStxA-Lys(11)AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1110TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDStxA-Lys(274)AEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1111TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDStxA1-Lys(1)KEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1112TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ IDStxA-Lys(11)KTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDNO: 1113with amino-VRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFterminalSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTtruncationSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDStxA1-Lys(11)KTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDNO: 1114with amino-VRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFterminalSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTtruncationSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ IDStxA-Lys(274)EFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTNO: 1115with amino-GDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNterminalNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMtruncation variantQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEAL1RFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDStxA1-Lys(274)TYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVNO: 1116with amino-RGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSterminalHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTStruncation variantYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR2TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDSLT-1A-Lys(1)KEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1117SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDSLT-1A-Lys(11)AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1118SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDST-1A-Lys(274)AEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1119SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDSLT-1A1-Lys(1)KEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGNO: 1120SGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ IDSLT-1A-Lys(11)KTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDNO: 1121with amino-VRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFterminalSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTtruncation variantSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGF1RTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNAILWDSSTLGAILMRRTISSSEQ IDSLT-1A1-Lys(11)KTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDNO: 1122with amino-VRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFterminalSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTtruncation variantSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGF2RTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARSEQ IDSLT-1A-Lys(274)EFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSNO: 1123with amino-GDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNterminalNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMtruncation variantQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEAL1RFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDSLT-1A-Lys(274)TYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVNO: 1124with amino-RGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSterminalHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTStruncation variantYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR2TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSSEQ IDSLT-2A-Lys(11)DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1125VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIAVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(255)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1126VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKQATECQIVGDRAAIAVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(257)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1127VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQKTECQIVGDRAAIAVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(270)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1128VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIKVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(288)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1129VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIAVNNVLWEANTIAALLNRKPQDLTEPNQSEQ IDSLT-2A1-Lys(11)DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1130VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSEQ IDSLT-2A-Lys(255)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1131with amino-RGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHterminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKQATECQIVGDRAAIAVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(257)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1132with amino-RGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHterminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQKTECQIVGDRAAIAVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(270)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1133with amino-RGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHterminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIKVNNVLWEANTIAALLNRAPQDLTEPNQSEQ IDSLT-2A-Lys(288)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1134with amino-RGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHterminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIAVNNVLWEANTIAALLNRKPQDLTEPNQSEQ IDSLT-2A-Lys(255)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1135with carboxy-VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTEterminalTNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMtruncationQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKSEQ IDSLT-2A-Lys(257)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1136with carboxy-VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTEterminalTNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMtruncationQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQKSEQ IDSLT-2A-Lys(270)DEFTVDFSSQASYVDSLNSIRSAISTPLGNISQGGVSVSVINHNO: 1137with carboxy-VLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTEterminalTNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMtruncationQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIKSEQ IDSLT-2A-Lys(255)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1138with amino- andRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHcarboxy- terminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationsDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKSEQ IDSLT-2A-Lys(257)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1139with amino- andRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHcarboxy- terminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationsDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQKSEQ IDSLT-2A-Lys(270)SYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVNO: 1140with amino- andRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHcarboxy- terminalISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLtruncationsDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQAQATECQIVGDRAAIKSEQ IDIA-SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSLLMIDNO: 1141Cys5-D1::scFv2SGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS | 679,579 |
11857629 | With reference to the Figures, features that are the same across the Figures are denoted with the same reference numbers. DETAILED DESCRIPTION OF THE INVENTION In some embodiments, formulations of the present invention are designed to block or thwart the effects caused by intentional or unintentional over-ingestion of drug products. Under normal dosing conditions the inventive formulations may allow for the complete and/or bioequivalent oral delivery of the desired drug dose. However when excess doses are ingested, either intentionally or unintentionally, the inventive formulations may work to either slow or block the release and subsequent absorption of the excessive doses. Thus, in the case of intentional over-ingestion where a drug abuser would consume excess doses of an abused drug to experience a euphoric effect, the effect would be significantly reduced for the inventive formulations compared to doses which freely release the excess drug of abuse. In this way, the inventive formulation may work as a deterrent from abusing the inventive formulations for the purpose of achieving the euphoric effect. Yet the patient who uses the invention as directed will receive the desired therapeutic treatment. In general, and as described in more detail herein, pharmaceutical formulations of the present invention may be designed with one or more components to control release and/or absorption of an active pharmaceutical ingredient. In some embodiments, a pharmaceutical formulation may be designed with a pH modifying feature and/or a pH dependent solubility feature. A pH modifying feature may impact release and/or absorption of an active ingredient by modifying the pH of the gastric environment based on whether the pharmaceutical composition is taken at an appropriate dosage amount or in excess. A pH modifying feature may be provided by inclusion of one or more buffering and/or antacid ingredients in the pharmaceutical composition. A pH dependent solubility feature may impact release and/or absorption of an active ingredient by containing or releasing the active pharmaceutical ingredient, depending on the pH of the gastric environment. A pH dependent solubility feature may be provided by inclusion of one or more acid soluble ingredients in the pharmaceutical composition. Components Active Pharmaceutical Ingredients Any drug, therapeutically acceptable drug salt, drug derivative, drug analog, drug homologue, or polymorph can be used in the present invention. Suitable drugs for use with the present invention can be found in the Physician's Desk Reference, 59th Edition, the content of which is hereby incorporated by reference. In one embodiment, the drug is an orally administered drug. In certain embodiments, drugs susceptible to abuse are used. Drugs commonly susceptible to abuse include psychoactive drugs and analgesics, including but not limited to opioids, opiates, stimulants, tranquilizers, sedatives, anxiolytics, narcotics and drugs that can cause psychological and/or physical dependence. In one embodiment, the drug for use in the present invention can include amphetamines, amphetamine-like compounds, benzodiazepines, and methyl phenidate or combinations thereof. In another embodiment, the present invention can include any of the resolved isomers of the drugs described herein, and/or salts thereof. A drug for use in the present invention which can be susceptible to abuse can be one or more of the following: alfentanil, amphetamines, buprenorphine, butorphanol, carfentanil, codeine, dezocine, diacetylmorphine, dihydrocodeine, dihydromorphine, diphenoxylate, diprenorphine, etorphine, fentanyl, hydrocodone, hydromorphone, β-hydroxy-3-methylfentanyl, levo-α-acetylmethadol, levorphanol, lofentanil, meperidine, methadone, methylphenidate, morphine, nalbuphine, nalmefene, oxycodone, oxymorphone, pentazocine, pethidine, propoxyphene, remifentanil, sufentanil, tilidine, and tramodol, salts, derivatives, analogs, homologues, polymorphs thereof, and mixtures of any of the foregoing. In another embodiment a drug for use with the present invention which can be susceptible to abuse includes one or more of the following: dextromethorphan (3-Methoxy-17-methy-9a, 13a, 14a-morphinan hydrobromide monohydrate), N-{1-[2-(4-ethyl-5-oxo-2-tetrazolin-1-yl)-ethyl]-4-methoxymethyl-4-piperidyl} propionanilide (alfentanil), 5,5-diallyl barbituric acid (allobarbital), allylprodine, alpha-prodine, 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]-benzodiazepine (alprazolam), 2-diethylaminopropiophenone (amfepramone), (±)-α-methyl phenethylamine (amphetamine), 2-(α-methylphenethyl-amino)-2-phenyl acetonitrile (amphetaminil), 5-ethyl-5-isopentyl barbituric acid (amobarbital), anileridine, apocodeine, 5,5-diethyl barbituric acid (barbital), benzylmorphine, bezitramide, 7-bromo-5-(2-pyridyl)-1H-1,4-benzodiazepin-2(3H)-one (bromazepam), 2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]-triazolo[4,3-a][1,4]diazepine (brotizolam), 17-cyclopropylmethyl-4,5a-epoxy-7a[(S)-1-hydroxy-1,2,2-trimethylpropyl]-6-methoxy-6,14-endo-ethanomorphinan-3-ol (buprenorphine), 5-butyl-5-ethyl barbituric acid (butobarbital), butorphanol, (7-chloro-1,3-dihydro-1-methyl-2-oxo-5-phenyl-2H-1,4-benzodiazepin-3-yl)-dimethyl carbamate (camazepam), (1S,2S)-2-amino-1-phenyl-1-propanol (cathine/D-norpseudoephedrine), 7-chloro-N-methyl-5-phenyl-3H-1,4-benzodiazepin-2-ylamine-4 oxide (chlordiazepoxide), 7-chloro-1-methyl-5-phenyl-1H-1,5-benzodiazepine-2,4(3H,5H)-dione (clobazam), 5-(2-chlorophenyl)-7-nitro-1H-1,4-benzodiazepin-2(3H)-one (clonazepam), clonitazene, 7-chloro-2,3-dihydro-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-carboxylic acid (clorazepate), 5-(2-chlorophenyl)-7-ethyl-1-methyl-1H-thieno[2,3-e][1,4]-diazepin-2(3H)-one (clotiazepam), 10-chloro-11b-(2-chlorophenyl)-2,3,7,11b-tetrahydrooxazolo[3,2-d][1,4]benzodiazepin-6(5H)-one (cloxazolam), (−)-methyl-[3β-benzoyloxy-2β(1αH,5αH)-tropane carboxylate (cocaine), 4,5α-epoxy-3-methoxy-17-methyl-7-morphinen-6α-ol (codeine), 5-(1-cyclohexenyl)-5-ethyl barbituric acid (cyclobarbital), cyclorphan, cyprenorphine, 7-chloro-5-(2-chlorophenyl)-1H-1,4-benzodiazepin-2(3H)-one (delorazepam), desomorphine, dextromoramide, (+)-(1-benzyl-3-dimethylamino-2-methyl-1-phenylpropyl) propionate (dextropropoxyphene), dezocine, diampromide, diamorphone, 7-chloro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2(3H)-one (diazepam), 4,5α-epoxy-3-methoxy-17-methyl-6α-morphinanol (dihydrocodeine), 4,5α-epoxy-17-methyl-3,6a-morphinandiol(dihydromorphine), dimenoxadol, dimephetamol [sic-Tr.Ed.], dimethyl thiambutene, dioxaphetyl butyrate, dipipanone, (6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol (dronabinol), eptazocine, 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (estazolam), ethoheptazine, ethyl methyl thiambutene, ethyl-[7-chloro-5-(2-fluorophenyl)-2,3-dihydro-2-oxo-1H-1,4-benzodiazepin-3-carboxylate] (ethyl loflazepate), 4,5α-epoxy-3-ethoxy-17-methyl-7-morphinen-6α-ol (ethylmorphine), etonitrazene, 4,5α-epoxy-7α-(1-hydroxy-1-methylbutyl)-6-methoxy-17-methyl-6,14-endo-etheno-morphinan-3-ol (etorphine), N-ethyl-3-phenyl-8,9,10-trinorbornan-2-ylamine (fencamfamine), 7-[2-(α-methylphenethylamino)-ethyl] theophylline (fenethylline), 3-(α-methylphenethylamino) propionitrile (fenproporex), N-(1-phenethyl-4-piperidyl) propionanilide (fentanyl), 7-chloro-5-(2-fluorophenyl)-1-methyl-1H-1,4-benzodiazepin-2(3H)-one (fludiazepam), 5-(2-fluorophenyl)-1-methyl-7-nitro-1H-1,4-benzodiazepin-2-(3H)-one (flunitrazepam), 7-chloro-1-(2-diethylaminoethyl)-5-(2-fluorophenyl)-1H-1,4-benzodiazepin-2(3H)-one (flurazepam), 7-chloro-5-phenyl-1-(2,2,2-trifluoroethyl)-1H-1,4-benzodiazepin-2(3H)-one (halazepam), 10-bromo-11b-(2-fluorophenyl)-2,3,7,11b-tetrahydro[1,3]oxazolo[3,2-d][1,4]benzodiazepin-6(5H)-one (haloxazolam), heroin, 4,5α-epoxy-3-methoxy-17-methyl-6-morphinanone (hydrocodone), 4,5α-epoxy-3-hydroxy-17-methyl-6-morphinanone (hydromorphone), hydroxypethidine, isomethadone, hydroxymethyl morphinan, 11-chloro-8,12b-dihydro-2,8-dimethyl-12b-phenyl-4H-[1,3]oxazino[3,2-d][1,4]benzodiazepin-4,7(6H)-dione (ketazolam), 1-[4-(3-hydroxyphenyl)-1-methyl-4-piperidyl]-1-propanone (ketobemidone), (3S,6S)-6-dimethylamino-4,4-diphenylheptan-3-yl acetate (levacetylmethadol (LAAM)), (−)-6-dimethylamino-4,4-diphenyl-3-heptanone (levomethadone), (−)-17-methyl-3-morphinanol (levorphanol), levophenacyl morphan, lofentanil, 6-(2-chlorophenyl)-2-(4-methyl-1-piperazinylmethylene)-8-nitro-2H-imidazo[1,2a][1,4]benzodiazepin-1(4H)-one (loprazolam), 7-chloro-5-(2-chlorophenyl)-3-hydroxy-1H-1,4-benzodiazepin-2(3H)-one (lorazepam), 7-chloro-5-(2-chlorophenyl)-3-hydroxy-1-methyl-1H-1,4-benzodiazepin-2(3H)-one (lormetazepam), 5-(4-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol (mazindol), 7-chloro-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepine (medazepam), N-(3-chloropropyl)-α-methylphenetylamine (mefenorex), meperidine, 2-methyl-2-propyl trimethylene dicarbamate (meprobamate), meptazinol, metazocine, methylmorphine, N,α-dimethylphenethylamine (methamphetamine), (±)-6-dimethylamino-4,4-diphenyl-3-heptanone (methadone), 2-methyl-3-o-tolyl-4(3H)-quinazolinone (methaqualone), methyl-[2-phenyl-2-(2-piperidyl)acetate] (methyl phenidate), 5-ethyl-1-methyl-5-phenyl barbituric acid (methyl phenobarbital), 3,3-diethyl-5-methyl-2,4-piperidinedione (methyprylon), metopon, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine (midazolam), 2-(benzhydrylsulfinyl) acetamide (modafinil), 4,5α-epoxy-17-methyl-7-morphinene-3,6α-diol (morphine), myrophine, (±)-trans-3-(1,1-dimethylheptyl)-7,8,10,10α-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9(6αH)-one (nabilone), nalbuphen, nalorphine, narceine, nicomorphine, 1-methyl-7-nitro-5-phenyl-1H-1,4-benzodiazepin-2(3H)-one (nimetazepam), 7-nitro-5-phenyl-1H-1,4-benzodiazepin-2(3H)-one (nitrazepam), 7-chloro-5-phenyl-1H-1,4-benzodiazepin-2-(3H)-one (nordazepam), norlevorphanol, 6-dimethylamino-4,4-diphenyl-3-hexanone (normethadone), normorphine, norpipanone, the coagulated juice of the plants belonging to the species Papaver somniferum (opium), 7-chloro-3-hydroxy-5-phenyl-1H-1,4-benzodiazepin-2-(3H)-one (oxazepam), (cis-trans)-10-chloro-2,3,7,11b-tetrahydro-2-methyl-11b-phenyloxazolo[3,2-d][1,4]benzodiazepin-6-(5H)-one (oxazolam), 4,5α-epoxy-14-hydroxy-3-methoxy-17-methyl-6-morphinanone (oxycodone), oxymorphone, plants and plant parts of the plants belonging to the species Papaver somniferum (including the subspecies setigerum) (Papaver somniferum), papaveretum, 2-imino-5-phenyl-4-oxazolidinone (pernoline), 1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2,6-methano-3-benzazocin-8-ol (pentazocine), 5-ethyl-5-(1-methylbutyl) barbituric acid (pentobarbital), ethyl-(1-methyl-4-phenyl-4-piperidine-carboxylate) (pethidine), phenadoxone, phenomorphan, phenazocine, phenoperidine, piminodine, pholcodeine, 3-methyl-2-phenyl morpholine (phenmetrazine), 5-ethyl-5-phenyl barbituric acid (phenobarbital), α,α-dimethyl phenethylamine (phentermine), 7-chloro-5-phenyl-1-(2-propinyl)-1H-1,4-benzodiazepin-2(3H)-one (pinazepam), α-(2-piperidyl)benzhydryl alcohol (pipradol), 1′-(3-cyano-3,3-diphenylpropyl)[1,4′-bipiperidine]-4′-carboxamide (piritramide), 7-chloro-1-(cyclopropylmethyl)-5-phenyl-1H-1,4-benzodiazepin-2(3H)-one (prazepam), profadol, proheptazine, promedol, properidine, propoxyphene, N-(1-methyl-2-piperidinoethyl)-N-(2-pyridyl) propionamide, methyl-{3-[4-methoxycarbonyl-4-(N-phenylpropaneamido)piperidino]propanoate} (remifentanil), 5-sec.-butyl-5-ethyl barbituric acid (secbutabarbital), 5-allyl-5-(1-methylbutyl) barbituric acid (secobarbital), N-{4-methoxymethyl-1-[2-(2-thienyl)ethyl]-4-piperidyl} propionanilide (sufentanil), 7-chloro-2-hydroxy-methyl-5-phenyl-1H-1,4-benzodiazepin-2-(3H)-one (temazepam), 7-chloro-5-(1-cyclohexenyl)-1-methyl-1H-1,4-benzodiazepin-2(3H)-one (tetrazepam), ethyl-(2-dimethylamino-1-phenyl-3-cyclohexane-1-carboxylate) (tilidine (cis and trans)), tramadol, 8-chloro-6-(2-chlorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (triazolam), 5-(1-methylbutyl)-5-vinyl barbituric acid (vinylbital), (1R*,2R*)-3-(3-dimethylamino-1-ethyl-2-methyl-propyl) phenol, (1R,2R,4S)-2-[dimethylamino)methyl-4-(p-fluorobenzyloxy)-1-(m-methoxyphenyl) cyclohexanol, each optionally in the form of corresponding stereoisomeric compounds as well as corresponding derivatives, especially esters or ethers, and all being physiologically compatible compounds, especially salts and solvates. In one embodiment, a pharmaceutical composition of the present invention includes one or more opioids such as hydrocodone, hydromorphone, morphine and oxycodone and/or salts thereof, as the therapeutically active ingredient. Typically when processed into a suitable dosage form, as described in more detail below, the drug can be present in such dosage forms in an amount normally prescribed, typically about 0.5 to about 25 percent on a dry weight basis, based on the total weight of the formulation. With respect to analgesics in unit dose form, such drugs may be present in a pharmaceutically acceptable amount; standard doses of such drugs are generally known in the art and are disclosed, for example, in the United States Pharmacopeia and National Formulary (USP 36-NF 31). Rockville, MD: United States Pharmacopeia Convention; 2013, which is incorporated by reference herein in its entirety. In some embodiments, such drugs may be present in an amount of about 5, 25, 50, 75, 100, 125, 150, 175 or 200 mg. In some embodiments, the drug can be present in an amount from about 5 to about 500 mg or about 5 to about 200 mg. In some embodiments, a dosage form contains an appropriate amount of drug to provide a therapeutic effect. In some embodiments, a pharmaceutically active ingredient may include a drug having a narrow therapeutic index. Drugs having a narrow therapeutic index may include but are not limited to aminophylline, carbamazepine, clindamycin, clonidine, digoxin, disopyramide, dyphylinne, guanthidine, isoetharine mesylate, isoproterenol, levothyroxine, lithium carbonate, metaproterenol, minoxidil, oxytriphylline, phenytoin, pasosin, primidone, procainamide, quinidine gluconate, theophylline, valproic acid, valproate sodium and warfarin sodium and the like. With respect to drugs having a narrow therapeutic dose in unit dose form, such drugs may be present in a pharmaceutically acceptable amount; standard doses of such drugs are generally known in the art and are disclosed, for example, in the United States Pharmacopeia and National Formulary (USP 36-NF 31). Rockville, MD: United States Pharmacopeia Convention; 2013, which is incorporated by reference herein in its entirety. In some embodiments, such drugs may be present in an amount of about 0.025, 0.05, 0.1, 0.2, 0.3, 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7.5, 10, 25, 50, 75, 100, 125, 150, 175, 200, and 250 mg. In some embodiments, the drug can be present in an amount of from about 0.01 to about 1000 mg or about 0.05 to about 500 mg. In some embodiments, a dosage form contains an appropriate amount of drug to provide a therapeutic effect. Component Soluble in Acidic Solutions In some embodiments, pharmaceutical compositions of the present invention include one or more components which are soluble in acidic solutions. Acidic solutions may be considered those having a pH of about 1 to about 4. In some embodiments, the acid soluble component is less soluble in slightly acidic, neutral, and/or basic solutions, i.e., those having a pH of greater than about 4. In some embodiments, an acid soluble component is included in a pharmaceutical composition in the form of particle matrix with an active pharmaceutical ingredient. The acid soluble ingredient may be included in the pharmaceutical composition in an amount sufficient to form this matrix. In some embodiments, the active ingredient is sequestered within the acid soluble component. The acid soluble component may impact release of the active pharmaceutical ingredient depending on the pH of the environment, which is raised or maintained by the buffering and/or antacid ingredient as a function of the amount of the pharmaceutical composition ingested: when the pharmaceutical composition is ingested in an appropriate dosage amount, the pH buffering ingredient is not present in an amount to alter or sufficiently raise the gastrointestinal pH, and the acid soluble component dissolves and releases the active pharmaceutical ingredient; when the pharmaceutical component is ingested in an excess amount, the pH buffering ingredient is present in an amount to raise the gastrointestinal pH, thereby preventing the acid soluble ingredient from dissolving and releasing the active pharmaceutical ingredient. In some embodiments, an acid soluble component is included in the pharmaceutical composition an amount of about 1 wt % to about 50 wt %; about 1 wt % to about 48 wt %; about 1 wt % to about 46 wt %; about 1 wt % to about 44 wt %; about 1 wt % to about 42 wt %; about 1 wt % to about 40 wt %; about 2 wt % to about 38 wt %; about 4 wt % to about 36 wt %; about 6 wt % to about 34 wt %; about 8 wt % to about 32 wt %; about 10 wt % to about 30 wt %; about 12 wt % to about 28 wt %; about 14 wt % to about 26 wt %; about 16 wt % to about 24 wt %; about 18 wt % to about 22 wt %; about 1 wt %; about 2 wt %; about 4 wt %; about 6 wt %; about 8 wt %; about 10 wt %; about 12 wt %; about 14 wt %; about 16 wt %; about 18 wt %; about 20 wt %; about 22 wt %; about 24 wt %; about 26 wt %; about 28 wt %; about 30 wt %; about 32 wt %; about 34 wt %; about 36 wt %; about 38 wt %; about 40 wt %; about 42 wt %; about 44 wt %; about 46 wt %; about 48 wt %; or about 50 wt %. Examples of suitable acid soluble components include calcium carbonate, chitosan, cationic copolymers of dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate such as, for example, Eudragit® E PO Eudragit® E100 and Eudragit® E 12.5, di and tribasic calcium phosphate, and magnesium hydroxide. Buffering and/or Antacid Ingredients In some embodiments, pharmaceutical compositions of the present invention include one or more buffering and/or antacid ingredients. Such ingredient may result in an elevation in stomach pH if the pharmaceutical composition is consumed in adequate amounts. In some embodiments, such ingredient may result in rapid and sustained elevation of stomach pH to a pH of greater than about 4 when the pharmaceutical composition is consumed in adequate amounts. In some embodiments, a buffering and/or antacid ingredient may be included in an amount such that stomach pH is not affected when the pharmaceutical composition is taken in appropriate therapeutic amounts, but such that stomach pH may be elevated when the pharmaceutical composition is ingested in excess amounts. In some embodiments, a buffering and/or antacid ingredient is included in the pharmaceutical composition in an amount of about 45 wt % to about 95 wt %; about 50 wt % to about 90 wt %; about 55 wt % to about 85 wt %; about 60 wt % to about 80 wt %; about 65 wt % to about 75 wt %; about 45 wt %; about 50 wt %; about 55 wt %; about 60 wt %; about 65 wt %; about 70 wt %; about 75 wt %; about 80 wt %; about 85 wt %; about 90 wt %; or about 95 wt %. Examples of suitable buffering and/or antacid ingredients include but are not limited to aluminum hydroxide, bismuth aluminate, bismuth carbonate, bismuth subcarbonate, bismuth subgallate, bismuth subnitrate, calcium carbonate, calcium phosphate, dibasic calcium phosphate, dihydroxyaluminum aminoacetate, dihydroxyaluminum sodium carbonate, glycine, magnesium glycinate, magnesium hydroxide, magnesium oxide, potassium bicarbonate, sodium bicarbonate, sodium potassium tartrate, tribasic sodium phosphate and tricalcium phosphate. In some embodiments, one ingredient may act as both an acid soluble ingredient and a buffering and/or antacid ingredient. Examples of such suitable ingredients include calcium carbonate, di and tribasic calcium phosphate, and magnesium hydroxide. Additional Ingredients The present invention can also optionally include other ingredients to enhance dosage form manufacture from a pharmaceutical composition of the present invention and/or alter the release profile of a dosage form including a pharmaceutical composition of the present invention. Some embodiments of the present invention include one or more pharmaceutically acceptable fillers/diluents. In one embodiment, Avicel PH (Microcrystalline cellulose) is a filler used in the formulation. The Avicel PH can have an average particle size ranging from 20 to about 200 μm, preferably about 100 μm. The density ranges from 1.512-1.668 g/cm3. The Avicel PH should have molecular weight of about 36,000. Avicel PH effectiveness is optimal when it is present in an amount of from about 10 to 65 percent, by weight on a solid basis, of the formulation. Typical fillers can be present in amounts from 10 to 65 percent by weight on a dry weight basis of the total composition. Other ingredients can include sugars and/or polyols, Lactose having a particle size of about 20 to about 400 microns and a density of about 0.3 to about 0.9 g/ml can also be included. In some embodiments of the invention, the fillers which can be present at about 10 to 65 percent by weight on a dry weight basis, also function as binders in that they not only impart cohesive properties to the material within the formulation, but can also increase the bulk weight of a directly compressible formulation (as described below) to achieve an acceptable formulation weight for direct compression. In some embodiments, additional fillers need not provide the same level of cohesive properties as the binders selected, but can be capable of contributing to formulation homogeneity and resist segregation from the formulation once blended. Further, preferred fillers do not have a detrimental effect on the flowability of the composition or dissolution profile of the formed tablets. In one embodiment, the present invention can include one or more pharmaceutically acceptable disintegrants. Such disintegrants are known to a skilled artisan. In the present invention, disintegrants can include, but are not limited to, sodium starch glycolate (Explotab®) having a particle size of about 104 microns and a density of about 0.756 g/ml, starch (e.g., Starch 21) having a particle size of about 2 to about 32 microns and a density of about 0.462 g/ml, Crospovidone® having a particle size of about 400 microns and a density of about 1.22 g/ml, and croscarmellose sodium (Ac-Di-Sol) having a particle size of about 37 to about 73.7 microns and a density of about 0.529 g/ml. The disintegrant selected should contribute to the compressibility, flowability and homogeneity of the formulation. Further the disintegrant can minimize segregation and provide an immediate release profile to the formulation. In some embodiments, the disintegrant (s) are present in an amount from about 2 to about 25 percent by weight on a solid basis of the directly compressible formulation. Furthermore, antacids added to the formulations may aid in tablet disintegration when the tablet is introduced to a low pH environment through the effervescence of the antacid ingredient, thus potentially reducing the requirement for additional disintegrants. In one embodiment, the present invention can include one or more pharmaceutically acceptable glidants, including but not limited to colloidal silicon dioxide. In one embodiment, colloidal silicon dioxide (Cab-O-Sil®) having a density of about 0.029 to about 0.040 g/ml can be used to improve the flow characteristics of the formulation. Such glidants can be provided in an amount of from about 0.1 to about 1 percent by weight of the formulation on a solid basis. It will be understood, based on this invention, however, that while colloidal silicon dioxide is one particular glidant, other glidants having similar properties which are known or to be developed could be used provided they are compatible with other excipients and the active ingredient in the formulation and which do not significantly affect the flowability, homogeneity and compressibility of the formulation. In one embodiment, the present invention can include one or more pharmaceutically acceptable lubricants, including but not limited to magnesium stearate. In one embodiment, the magnesium stearate has a particle size of about 450 to about 550 microns and a density of about 1.00 to about 1.80 g/ml. In one embodiment, magnesium stearate can contribute to reducing friction between a die wall and a pharmaceutical composition of the present invention during compression and can ease the ejection of the tablets, thereby facilitating processing. In some embodiments, the lubricant resists adhesion to punches and dies and/or aid in the flow of the powder in a hopper and/or into a die. In an embodiment of the present invention, magnesium stearate having a particle size of from about 5 to about 50 microns and a density of from about 0.1 to about 1.1 g/ml is used in a pharmaceutical composition. In certain embodiments, a lubricant should make up from about 0.1 to about 2 percent by weight of the formulation on a solids basis. Suitable lubricants are stable and do not polymerize within the formulation once combined. Other lubricants known in the art or to be developed which exhibit acceptable or comparable properties include stearic acid, hydrogenated oils, sodium stearyl fumarate, polyethylene glycols, and Lubritab®. In certain embodiments, the most important criteria for selection of the excipients are that the excipients should achieve good content uniformity and release the active ingredient as desired. The excipients, by having excellent binding properties, and homogeneity, as well as good compressibility, cohesiveness and flowability in blended form, minimize segregation of powders in the hopper during compression. Controlled Drug Release Dosage Forms As described herein, pharmaceutical formulations of the present invention may be formulated to slow or block the release and subsequent absorption of excessive doses of an active pharmaceutical ingredient. In some embodiments, a pharmaceutical formulation may be designed with a pH modifying feature and/or a pH dependent solubility feature. A pH modifying feature may impact release and/or absorption of an active ingredient by modifying the pH of the gastric environment based on whether the pharmaceutical composition is taken an appropriate dosage amount or in excess. A pH modifying feature may be provided by inclusion of one or more buffering and/or antacid ingredients in the pharmaceutical composition. A pH dependent solubility feature may impact release and/or absorption of an active ingredient by containing or releasing the active pharmaceutical ingredient, depending on the pH of the gastrointestinal environment. A pH dependent solubility feature may be provided by inclusion of one or more pH soluble ingredients in the pharmaceutical composition. In some embodiments, the pharmaceutical composition may be formulated such that when the composition is taken in appropriate amounts, a pH modifying feature has minimal impact (i.e., the pH of the gastric environment is not substantially modified or is maintained at a desirable level) and a pH dependent solubility feature has a maximal impact (i.e., the active pharmaceutical ingredient is released), thereby allowing release and/or absorption of the active ingredient. However, when the pharmaceutical composition is ingested in excess, in some embodiments the composition is formulated such that the pH modifying feature has a maximal impact (i.e., the pH of the gastric environment is raised) and the pH dependent solubility feature has a minimal impact (i.e., the acid solubility ingredient is not soluble and therefore does not dissolve), thereby thwarting release and/or absorption of the active ingredient. In some embodiment, a pharmaceutical composition may be prepared by intimately mixing the active pharmaceutical ingredient with an acid soluble ingredient(s) by any suitable process (i.e. dry or wet granulation, hot melt extrusion etc) such that a particulate matrix is formed in a particulate form. The release of the drug from this matrix may then be controlled by the immediate pH environment surrounding the matrix when the pharmaceutical composition is ingested. In a low pH environment (i.e., pH 1-4), the matrix may be likely to dissolve and release the drug rapidly; however, in a higher pH environment (i.e., pH>4) the matrix is likely to be insoluble and the release of drug will be retarded and potentially incomplete, thereby diminishing the level of the drug absorbed. In some embodiments, for a single dosage unit, the required amount of acid soluble drug matrix is further mixed with buffering and/or antacid ingredient(s) in a quantity sufficient such that when the single dose unit is ingested, the buffering and/or antacid ingredient(s) will neutralize stomach pH to a point that the stomach pH remains in a range between pH 1-4. The acid soluble drug matrix/antacid/buffer blend may be formed into an oral solid dose form such as a tablet or capsule but not limited to said dosage forms. As a result, a pharmaceutical composition may be formulated having a pH modifying feature and a pH dependant solubility feature such that, under normal dosing conditions (i.e., one or two tablets), when a single dose is ingested, the buffer/antacid ingredient(s) neutralize a portion of the stomach acid, however the stomach acid remains in a range between pH1-4. Under these conditions, the acid soluble drug matrix is soluble in the acidic stomach environment and the drug may be rapidly released in the stomach and absorbed into the bloodstream. Under conditions where excess doses are ingested, intentionally or unintentionally, (i.e., three tablets or greater), the quantity of buffers and/or antacid ingredient(s) from over-ingestion may now be sufficient to cause a rapid and sustainable increase in stomach pH (>pH 4). Thus the acid soluble drug matrix may be less soluble in the higher pH stomach environment and the release of drug from the matrix may be suppressed. In some embodiments, the suppression of drug from the acid soluble matrix is further aided by gastro-intestinal transit, which may transfer the acid soluble matrix particle into the intestine and lower gastro-intestinal tract which have biologically controlled high pH environments (i.e., pH 5.5-8). The overall suppression of drug release from over ingestion results in a pharmacokinetic profile with an increase in Tmax and a decrease in Cmax when compared to an equal oral dose which releases drug in normal stomach pH (i.e., pH 1-4). Suitable formulations and dosage forms of the present invention include but are not limited to powders, caplets, pills, suppositories, gels, soft gelatin capsules, capsules and compressed tablets manufactured from a pharmaceutical composition of the present invention. The dosage forms can be any shape, including regular or irregular shape depending upon the needs of the artisan. Compressed tablets including the pharmaceutical compositions of the present invention can be direct compression tablets or non-direct compression tablets. In one embodiment, a dosage form of the present invention can be made by wet granulation, and dry granulation (e.g., slugging or roller compaction). The method of preparation and type of excipients are selected to give the tablet formulation desired physical characteristics that allow for the rapid compression of the tablets. After compression, the tablets must have a number of additional attributes such as appearance, hardness, disintegrating ability, and an acceptable dissolution profile. Choice of fillers and other excipients typically depend on the chemical and physical properties of the drug, behavior of the mixture during processing, and the properties of the final tablets. Adjustment of such parameters is understood to be within the general understanding of one skilled in the relevant art. Suitable fillers and excipients are described in more detail above. The manufacture of a dosage form of the present invention can involve direct compression and wet and dry granulation methods, including slugging and roller compaction. In some embodiments, one or more component may be sequestered, as described in U.S. Patent Application Publication No. 2012/0202839 which is incorporated by reference herein in its entirety. The present invention can be used to manufacture immediate release, and controlled drug release formulations. Controlled release formulations can include delayed release, bi-modal and tri-modal release, extended and sustained release oral solid dosage preparations. As used herein, the term “about” is understood to mean±10% of the value referenced. For example, “about 45%” is understood to literally mean 40.5% to 49.5%. As used herein, the term “bioequivalence” is understood to mean one or more of Cmax, Tmax, or area under the concentration curve “AUC” of a drug is within 75% to 120% of the same marker for a referenced drug. Certain aspects of the present invention may be better understood as illustrated by the following examples, which are meant by way of illustration and not limitation. Example 1 Two dissolution vessels were prepared: one vessel contained 25 mEq of HCl and a single commercially available calcium carbonate antacid tablet (Tums®), and the other vessel contained 25 mEq of HCl and five calcium carbonate antacid tablets. The pH in each vessel was monitored and the results are shown inFIG.1. For the single tablet, the tablet is completely reacted in about 30 minutes and the dissolution of the single tablet showed no change in pH. However, the introduction of five tablets results in a relatively rapid rise in 10 minutes to a pH greater than 4.5 and after 90 minutes, a considerable amount of non-dissolved solid was observed in the vessel. Therefore, it has been shown that calcium carbonate at a low level (500 mg) could completely dissolve with little effect on pH but with excess amounts of calcium carbonate a rapid pH increase occurs creating a high pH environment, in which calcium carbonate exhibits low solubility. Calcium carbonate has shown a capability as a pH modifier and pH dependent solubility. Example 2 A challenge faced in designing a self-regulating dosage form is to institute regulation (i.e., slower or incomplete release) at elevated pH without compromising the desired rapid release rate associated with immediate release tablets when a single dose is taken. Calcium carbonate was evaluated both in direct blend matrix tablets and dry granulation tablets containing alprazolam, where the granule contained calcium carbonate to control drug release and calcium carbonate outside the granule to effect pH change. Both approaches resulted in slower alprazolam release in single tablets at higher pH (approx. pH 6) compared to low pH (pH1), however, in this case the release separation was not as high as desired (FIG.2). However, the results demonstrate that a granule may be used to control drug release depending on environment. The granules may consist of the drug along with a functional component that inhibits erosion or disintintegration at elevated pH such that slower and/or incomplete release of the drug is observed. The extragranular portion of the tablet may primarily contain the pH controlling agents, which need to be released and react quickly. Example 3 Eudragit® E PO (EPO) is a cationic copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate. Technical literature indicates that this polymer is soluble in acid, up to pH 5; above pH 5 it swells rather than dissolves. Dry granulations containing 5% alprazolam in EPO polymer were prepared and size fractions collected at −16 mesh and +20 mesh (16/20) and at −20 mesh and +30 mesh (20/30). Dissolution on the granulation size fractions were performed at both low pH (pH 1.5) where EPO is soluble and at high pH (pH 6) were EPO is less soluble. The results are shown inFIG.3. At low pH, irrespective of the particle size, a rapid and complete release of alprazolam occurs within 15 minutes. However at high pH, the release of alprazolam is significantly slower and incomplete for both size fractions but slightly elevated for the smaller fraction. It must be noted that this dissolution simulation represents a static pH condition at the potential pH extremes if the tablet is taken as directed (pH 1) and in excess (pH 6). The next example examines a pH modifying system which doesn't affect pH when taken as directed but will rapidly increase pH when over-ingested. Example 4 Subsequent trials to use calcium carbonate as the primary pH modifying agent resulted in a relatively rapid release of alprazolam in 15 minutes from 5% alprazolam/EPO granules (60%). Although the pH change to pH 5 previously seen for calcium carbonate in 10 minutes (FIG.1) may have been perceived fast, given that release of alprazolam is also rapid and completed in 15 minutes, calcium carbonate may not affect a pH change rapidly enough for the alprazolam/EPO granules. In earlier experimentation, sodium bicarbonate had been shown to have a more rapid pH effect, raising acid media from a pH 1 to a pH 6 in less than 2 minutes. Thus, sodium bicarbonate was added to a prototype formulation primarily to control rapid pH elevation and calcium carbonate for a more sustained control of elevated pH. A representative formulation for the invention is shown in the table below: ComponentWt %Wt (mg)Alprazolam (5%) in3.19520.00Eudragit E PO (20/30)Sodium bicarbonate79.87500.0Calcium carbonate DC15.97100.0Magnesium stearate0.9586.0Total100626.0 The prototype formulation was tableted and a dynamic test was performed where the pH modifying agents were contained in the tablets and dynamically reacted in 0.55 N HCl media (about pH 1.6). Multiple tablet doses both with and without self-regulation as well as a single dose with self-regulation were tested and compared. Dissolution media pH and drug release were monitored. As shown inFIG.4, a rapid rise in pH in the prototype multiple tablets was observed with a rise to pH 6 occurring in less than two minutes. Thus, a rapid rise in pH can be affected by the pH modifying agents contained in the tablet. Furthermore, as shown inFIG.5, a single tablet releases alprazolam in 15 minutes showing that the immediate release characteristic of a single tablet is unaffected by the on-board, self-regulating system. However, multiple tablets with self-regulating show approximately a single dose released in 15 minute with a delayed release of the excess alprazolam over approximately 2 hours. By comparison, multiple tablets without self-regulating show that the entire alprazolam dose (approximately 9 mg) is released in approximately 15 minutes, whereas only 20% alprazolam is released in multiple tablets with self-regulating at the same time point. Clearly, prototype self-regulating alprazolam tablets have been shown to release a single dose of alprazolam as intended but multiple tablets show a retardation of the release of excess doses compared to excess doses without self-regulating. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention shown in the specific embodiments without departing from the spirit and scope of the invention as broadly described. Further, each and every reference cited above is hereby incorporated by reference as if fully set forth herein. | 39,859 |
11857630 | DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE DISCLOSURE 1. General Description As described herein, the present disclosure describes a composition which comprises SN-38 and a poly(amino acid) block copolymer as depicted in Formula I: The present disclosure also describes methods for the manufacture of compositions comprising SN-38 and the compound represented by Formula I. Such compositions are pharmaceutically acceptable drug products suitable for administration to human patients. The present disclosure also describes methods for the treatment of cancers comprising administration of a composition comprising SN-38 and a compound represented by Formula I. 2. Definitions The following are definitions of various terms used herein to describe the present disclosure and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. These definitions apply to the terms as they are used throughout this specification unless otherwise indicated in specific instances, either individually or as part of a larger group. It is understood that the terms “SN-38”, “7-ethyl-10-hydroxycamptothecin” refer to (19S)-10,19-diethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] henicosa-1(21),2,4(9),5,7,10,15(20-heptaene-14,18-dione, and any salts, solvates, or hydrates thereof. It is understood that the terms “TFS-3”, “poly(sarcosine)235-block-poly(d-phenylalanine10-co-tyrosine30)”, “PSar235-P(dPhe10/Tyr30)”, “poly[Sar235]-block-poly-[D-Phe10-co-L-Tyr30]”, and a copolymer represented Formula I, all represent the same compound and can be used interchangeably. It is understood that the terms “TYN-38” refers to a formulation comprising SN-38, TFS-3, and trehalose wherein the SN-38 is about 10% weight loading of the formulation. As used herein, the term “block copolymer” refers to a polymer comprising two or more poly(amino acid) portions. As described herein, one or more of the amino acid blocks may be “mixed blocks”, meaning that these blocks can contain a mixture of amino acid monomers thereby creating block copolymers of the present disclosure. One skilled in the art will recognize that a monomer repeat unit is defined by parentheses depicted around the repeating monomer unit. The number (or letter representing a numerical range) on the lower right of the parentheses represents the number of monomer units that are present in the polymer chain. In the case where only one monomer represents the block (e.g. a homopolymer), the block will be denoted solely by the parentheses. In the case of a mixed block, multiple monomers comprise a single, continuous block. It will be understood that brackets will define a portion or block. For example, one block may consist of four individual monomers, each defined by their own individual set of parentheses and number of repeat units present. All four sets of parentheses will be enclosed by a set of brackets, denoting that all four of these monomers combine in random, or near random, order to comprise the mixed block. For clarity, the randomly mixed block of [BCADDCBADABCDABC] would be represented in shorthand by [(A)4(B)4(C)4(D)4]. As used herein, “copolymer” refers to a polymer comprising two or more poly(amino acid portions). As used herein, “weight loading” refers to the ratio of a drug to the total drug product formulation which can include, but is not limited to, drugs, excipients and copolymers. Weight loading is expressed as a weight percentage (% w/w), for example; 20 mg of a drug in a total formulation further comprising 90 mg of a cryoprotectant and 90 mg of a copolymer would be expressed as 10% weight loading, (20/(20+90+90)=10%). As used herein, “feed ratio” refers to the ratio of drug combined with a copolymer during the manufacturing of a drug product. Feed ratio is expressed as a weight percentage (% w/w), for example; 100 mg of a drug combined with 500 mg of a copolymer (independent of other components) would be expressed as a feed ratio of 20% (100/500=20%). Feed ratio is independent of other components present in the drug product. Thus, a 10% feed ratio may result in a drug product containing 5% drug by weight when other components of the drug product are taken into account. Representative feed ratios include from about 1% to about 50%, from about 5% to about 50%, from about 10% to about 50%, from about 10% to about 40%, from about 15% to about 25%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or about 40%. As used herein, “high shear mix” or “high shear mixing”, refers to dispersing a combination of components into a continuous phase which would normally be immiscible via emulsification, sonication, or microfluidizing. As used herein, “unit dosage form” or “unit dose form” refers to a physically discrete unit of a formulation appropriate for the subject to be treated. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgement. The specific effective dose level for any particular subject or organism will depend on a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active agent employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active agent employed; duration of treatment, drugs/and or additional therapies used in combination or coincidental with specific compound(s) employed and like factors well known in the medical arts. As used herein, a “drug product” means a therapeutic agent, and one or more excipients selected from, but not limited to, tonicity agents, cryoprotectants, multiblock copolymers, stabilizing agents, antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. As appreciated by those skilled in the art, the amounts of each excipient will depend on the therapeutic agent, the route of administration, the desired biological endpoint, the target cell or tissue. As used herein, a “cryoprotectant” or “cryoprotective agent” refers to compounds which either prevent freezing or prevent damage, or alteration to other compounds related to freezing. This includes, but is not limited to: sugars, monosaccharides, disaccharides, polyalcohols, amino acids, glycine, polyvinyl pyrrolidine, polyethylene glycol, mannitol, sorbitol, sucrose, glucose, raffinose, sucralose, lactose, trehalose, dextran, and dextrose. As used herein, a “therapeutically effective amount” means an amount of a substance (e.g. a therapeutic agent, composition, and/or formulation) that elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, slow the progression of, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, slows the progression of, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a “therapeutically effective amount” is at least a minimal amount of a compound, or composition containing a compound, which is sufficient for treating one or more symptoms of a disease or disorder associated with proliferative diseases, such as cancer. The term “subject”, as used herein, means a mammal and includes human and animal subjects, such as domestic animals (e.g. horses, dogs, cats, etc.). The terms “treat” or “treating,” as used herein, refers to partially or completely alleviating, inhibiting, delaying onset of, ameliorating, slowing the progression of and/or relieving a disease or disorder, or one or more symptoms of the disease or disorder. As used herein, the terms “treatment,” “treat,” and “treating” refer to partially or completely alleviating, inhibiting, delaying onset of, ameliorating, slowing the progression of and/or relieving a disease or disorder, or one or more symptoms of the disease or disorder, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In some embodiments, the term “treating” includes slowing or halting the progression of a disease or disorder. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g. in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Thus, in some embodiments, the term “treating” includes preventing relapse or recurrence of a disease or disorder. “Metastatic,” used herein to describe cancer, refers to cancer that has spread from the part of the body where it started to other parts of the body. “Locally advanced,” used herein to describe cancer, refers to cancer that has grown outside the organ it started in but has not yet spread to distant parts of the body. A subject is said to have “failed” a therapy, or the term “failure” in the context of a previous treatment, as used herein means the subject relapses from the therapy, or is resistant or refractory to the therapy (e.g., progresses following or while on the therapy). For example, treatment of a subject having breast cancer that has not metastasized or advanced locally may not prevent the breast cancer from metastasizing or advancing locally. If the treatment does not prevent the breast cancer from metastasizing or advancing locally, and the breast cancer metastasizes and/or advances locally, the subject is said to have failed the treatment because the subject's cancer progressed following or while on the treatment. In another example, a subject previously diagnosed with metastatic or locally advanced breast cancer may be treated with a therapy for such cancer, but fail to respond to the therapy. This subject, too, is said to have failed the therapy because the subject is resistant or refractory to the therapy. Similarly, a subject that experiences remission following a therapy, but subsequently relapses, is considered to have failed the prior therapy. “Prior therapy,” as used herein, refers to any therapy given before the referenced therapy for a disease or condition. When a prior therapy includes drug(s), the referenced or subsequent therapy comprises one or more drugs that are different from the drug(s) of the prior therapy. In some embodiments, the subsequent therapy is a second-line therapy (i.e., the second therapy given for a disease or condition). In some embodiments, the subsequent therapy is a third-line therapy (i.e., the third therapy given for a disease or condition). In some embodiments, the subsequent therapy is a fourth-line therapy (i.e., the fourth therapy given for a disease or condition). The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered intraperitoneally or intravenously. Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, refers to variations of ±20% or in some instances 10%, or in some instances ±5%, or in some instances ±2%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the present disclosures. 3. Description of Exemplary Embodiments 3.1 Drug Product In some embodiments the present disclosure provides a drug product comprising a formulation or composition described herein. In some embodiments the present disclosure provides a composition comprising SN-38, and TFS-3. In some embodiments the present disclosure provides a composition comprising SN-38, TFS-3, and a cryoprotectant. In a preferred embodiment, said cryoprotectant is trehalose. The weight loading of SN-38 in the drug product of the present disclosure can have effects on reconstitution properties, stability, and manufacturing. In some embodiments the disclosure is directed to drug products with SN-38 weight loadings from about 0.1% to about 30%. In other embodiments the present disclosure is directed to drug products with SN-38 weight loadings from about 5% to about 15%. One embodiment of the present disclosure provides a composition comprising SN-38, TFS-3, and a cryoprotectant,wherein:the SN-38 is about 1% by weight to about 30% by weight of the composition,the TFS-3 is about 10% by weight to about 90% by weight of the composition,and the cryoprotectant is about 10% by weight to about 90% by weight of the composition. One embodiment of the present disclosure is directed to a composition comprising SN-38, TFS-3, and a cryoprotectant,wherein:the SN-38 is about 5% by weight to about 15% by weight of the composition,the TFS-3 is about 30% by weight to about 60% by weight of the composition,and the cryoprotectant is about 30% by weight to about 60% by weight of the composition. One embodiment of the present disclosure is directed to a composition comprising SN-38, TFS-3, and trehalose,wherein:the SN-38 is about 1% by weight to about 30% by weight of the composition,the TFS-3 is about 10% by weight to about 90% by weight of the composition,and the trehalose is about 10% by weight to about 90% by weight of the composition. One embodiment of the present disclosure is directed to a composition comprising SN-38, TFS-3, and trehalose,wherein:the SN-38 is about 5% by weight to about 15% by weight of the composition,the TFS-3 is about 30% by weight to about 60% by weight of the composition,and the trehalose is about 30% by weight to about 60% by weight of the composition. 3.2 Unit Dosage Form In some embodiments the present disclosure provides a unit dosage form comprising a formulation or composition described herein. In some embodiments, the present disclosure is directed to pharmaceutical packs and/or kits comprising compositions described herein, or a unit dosage form comprising a provided composition, and a container (e.g. foil, or plastic package, or other suitable container). Optionally instructions for use are additionally provided in such kits. Composition of the present disclosure can be provided as a unit dosage form. In some embodiments, a vial comprising SN-38 and TFS-3 is a unit dosage form. In some embodiments, a vial comprising SN-38, TFS-3, and a cryoprotectant is a unit dosage form. In a preferred embodiment, a vial comprising SN-38, TFS-3, and trehalose is a unit dosage form. One embodiment of the present disclosure provides a composition comprising SN-38, TFS-3, and a cryoprotectant,wherein:the SN-38 is present in about 6 mg to about 150 mg,the TFS-3 is present in about 27 mg to about 675 mg,and the cryoprotectant is present in about 27 mg to about 675 mg. One embodiment of the present disclosure provides a composition comprising SN-38, TFS-3, and a cryoprotectant,wherein:the SN-38 is present in about 15 mg to about 60 mg,the TFS-3 is present in about 67 mg to about 270 mg,and the cryoprotectant is present in about 67 mg to about 270 mg. One embodiment of the present disclosure provides a composition comprising SN-38, TFS-3, and trehalose,wherein:the SN-38 is present in about 6 mg to about 150 mg of the composition,the TFS-3 is present in about 27 mg to about 675 mg of the composition,and the trehalose is present in about 27 mg to about 675 mg of the composition. One embodiment of the present disclosure provides a composition comprising SN-38, TFS-3, and trehalose,wherein:the SN-38 is present in about 15 mg to about 60 mg,the TFS-3 is present in about 67 mg to about 270 mg,and the trehalose is present in about 67 mg to about 270 mg. In some embodiments, the present disclosure can be provided as a unit dosage form. For example, a vial comprising SN-38, TFS-3, and trehalose is a unit dosage form that may be provided. In Some embodiments the unit dosage form is selected from those in Table 2: TABLE 2Pharmaceutical Components of Unit Dosage FormComponentFunctionAmount/vialSN-38Active27-33 mgTFS-3Excipient108-162 mgTrehaloseCryoprotectant108-162 mg In a preferred embodiment, the unit dosage form is depicted in Table 3: TABLE 3Pharmaceutical Components of Unit Dosage FormComponentFunctionWeight %Amount/vialSN-38Active10%30 mgTFS-3Excipient45%135 mgTrehaloseCryoprotectant45%135 mg In some embodiments, the unit dosage forms of the present disclosure are provided in a sealed container. In some embodiments, the unit dosage forms of the disclosure are provided as lyophilized powders. In some embodiments, the unit dosage forms of the disclosure are provided as an infusion solution. In some embodiments, the infusion solution comprises a vehicle selected from water, 1,3-butanediol, Ringer's solution or an isotonic sodium chloride solution. In some embodiments, the unit dosage forms contemplated by the disclosure are provided as a kit. The kit may comprise a first and a second container, wherein the first container comprises a composition as described herein, and the second container comprises a vehicle as described herein. In some embodiments, the first container comprises a composition as described herein as a lyophilized dry powder. The kits of the disclosure may allow for the dissolution of the lyophilized compositions described herein immediately prior to the administration of those compositions to a subject in need thereof. In some embodiments, the compositions of the disclosure are formulated with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in the compositions of the disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol and wool fat. The compositions of the disclosure may be formulated for administration in any convenient way for use in human medicine. The compositions of the disclosure may be formulated for a variety of administration methods. Administration methods contemplated by the disclosure include topical, systemic, or local administration. For example, therapeutic compositions of the disclosure may be formulated for parenteral administration (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques), administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, sublingual, transdermal, or nasal administration. The compositions described herein may be formulated as part of an implant or device, or formulated for slow or extended release. In certain embodiments of the disclosure, the compositions are formulated for oral administration, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and the like. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), the compositions of the disclosure may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. In some embodiments, the drug products of this disclosure are formulated as liquid dosage forms for oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixers. The liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyline glycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters or sorbitan and mixtures thereof. The oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. In certain embodiments, the compositions of the disclosure are formulated for parenteral administration. As an example, the compositions of the disclosure can be formulated for parenteral administration by further including one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use. The compositions for parenteral administration may contain antioxidants, buffers, bacteriostats, and/or solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and non-aqueous vehicles which may be employed in the pharmaceutical compositions of the disclosure include water, Ringer's solution, an isotonic salt solution, ethanol, polyols (such as 1,3-butanediol, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In a preferred embodiment, the compositions of the disclosure are intended for parenteral administration, and further comprise a vehicle selected from water, 1,3-butanediol, Ringer's solution or an isotonic sodium chloride solution. As described herein, the compositions of the disclosure may be administered for slow, controlled or extended release. The term “extended release” is widely recognized in the art of pharmaceutical sciences and is used herein to refer to a controlled release of an active compound or agent from a dosage form to an environment over (throughout or during) an extended period of time, e.g. greater than or equal to one hour. An extended release dosage form will release drug at substantially constant rate over an extended period of time or a substantially constant amount of drug will be released incrementally over an extended period of time. The term “extended release” used herein includes the terms “controlled release,” “prolonged release,” “sustained release,” “delayed release,” or “slow release” as these terms are used in the pharmaceutical sciences. In some embodiments, the extended release dosage is administered in the form of a patch or a pump. 3.3 Process of Manufacturing In certain embodiments, the present disclosure provides methods for preparing drug products comprising SN-38 and TFS-3. In one aspect, the disclosure is directed to a method for preparing a sterile, lyophilized drug product comprising SN-38 and TFS-3. This drug product would be suitable for administration to a patient. One embodiment of the disclosure provides a method for preparing a sterile, lyophilized drug product comprising SN-38, TFS-3, and a cryoprotective agent. The general method for providing said drug product comprises the steps of preparing a solution of a cryoprotectant and TFS-3 in water. Preparing a solution of SN-38 in an organic solvent. Adding said SN-38 solution to said solution of a cryoprotectant and TFS-3 while shear mixing with a homogenizer to produce a homogenous emulsion. Processing said homogenous emulsion through a high shear mixer (e.g. microfluidizer). Processing the high shear mixer extruded solution via tangential flow filtration against an aqueous solution of cryoprotectant. Sterile filtering the resulting solution (e.g. aseptic filtration), filing of vials under sterile conditions, and lyophilization under sterile conditions. Suitable cryoprotective agents include, but are not limited to: sugars, monosaccharides, disaccharides, polyalcohols, amino acids, glycine, polyvinyl pyrrolidine, polyethylene glycol, mannitol, sorbitol, sucrose, glucose, raffinose, sucralose, lactose, trehalose, dextran, and dextrose. In a preferred embodiment the cryoprotectant is trehalose. In some embodiments, the disclosure is directed to a method of preparing a unit dosage form comprising:a) dissolving SN-38, or a pharmaceutically acceptable salt thereof, a copolymer of Formula I and, optionally, a cryoprotectant, in an aqueous solution, thereby forming a mixed solution;b) processing the mixed solution through a high shear mixer, thereby forming a high shear mixed solution; andc) optionally lyophilizing the high shear mixed solution. In some embodiments, the disclosure is directed to a method of preparing a unit dosage form comprising:a) dissolving SN-38, or a pharmaceutically acceptable salt thereof, in an organic solvent, thereby forming an SN-38 solution;b) dissolving a copolymer of Formula I and, optionally, a cryoprotectant, in an aqueous solution, thereby forming a copolymer solution;c) mixing the SN-38 solution and the copolymer solution, thereby forming a mixed solution;d) processing the mixed solution through a high shear mixer, thereby forming a high shear mixer solution;e) filtering the high shear mixer solution, thereby forming a filtered solution; andf) optionally lyophilizing the filtered solution. In some embodiments, the disclosure is directed to a method of preparing a unit dosage form comprising:a) dissolving SN-38, or a pharmaceutically acceptable salt thereof, in an organic solvent, thereby forming an SN-38 solution;b) dissolving a copolymer of Formula I and, optionally, a cryoprotectant, in an aqueous solution, thereby forming a copolymer solution;c) mixing the SN-38 solution and the copolymer solution, thereby forming a mixed solution;d) processing the mixed solution through a high shear mixer, thereby forming a high shear mixer solution;e) processing the high shear mixer solution with a diafiltration system, thereby forming a diafiltered solution;f) filtering the diafiltered solution, thereby forming a filtered solution; andg) optionally lyophilizing the filtered solution. 3.4 Methods of Use Compositions comprising irinotecan, a pro-drug of SN-38, are known to be useful for the treatment of patients with cancer, alone or in combination with other therapeutic agents and/or therapies. Such patients include those who have previously been treated for cancer, and those who have not previously been treated for cancer. The compositions of the present disclosure comprise SN-38 and TFS-3 and are useful in the treatment of a variety of cancers and other proliferative diseases. The compositions of the present disclosure are useful in the treatment a cancer including, but not limited to, the following: multiple myeloma, breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach (gastric), skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung, bone, colon, thyroid, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma (including uveal melanoma) sarcoma, bladder carcinoma, liver carcinoma (e.g., hepatocellular carcinoma (HCC)) and biliary passages, kidney carcinoma, myeloid disorders, lymphoid disorders, Hodgkin's disease, hairy cells, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, colorectal carcinoma, large intestine, rectum, brain and central nervous system, endometrial, multiple myeloma (MM), prostate, acute myeloid leukemia (AML), and leukemia. In a preferred embodiment, the caner is colorectal. In a preferred embodiment the cancer is non-small cell lung carcinoma (NSCLC). In a preferred embodiment the cancer is small cell lung carcinoma (SCLC). In a preferred embodiment the cancer is adenocarcinoma of the pancreas. In a preferred embodiment the cancer is ovarian. In a preferred embodiment the cancer is gastric. In a preferred embodiment the cancer is esophageal. In a preferred embodiment the cancer is breast. In some embodiments the cancer is a locally advanced cancer. In some embodiments the cancer is metastatic. In some embodiments the cancer is reoccurring. In some embodiments the cancer is relapsed. In some embodiments the cancer is refractory. In a preferred embodiment the compositions of the disclosure are useful in combination with 5-fluorouracil and leucovorin for the treatment of metastatic colorectal cancer. In a preferred embodiment the compositions of the disclosure are useful as a single agent for the treatment of metastatic colorectal cancer after failure of a 5-fluorouracil-based chemotherapy. In a preferred embodiment the compositions of the disclosure are useful in combination with one or more therapeutic agents for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. In some embodiments, the therapeutic agent is selected from 5-fluorouracil or leucovorin. The present disclosure provides compositions comprising a multiblock copolymer of Formula I and SN-38 that may be administered to a patient in need thereof. Routes of administration include, but are not limited to, parenterally, orally, sublingually, buccally, rectally, vaginally, by the ocular route, by the otic route, nasally, inhalation, nebulization, cutaneously, subcutaneously, topically, systemically, or transdermally. In a preferred embodiment, the route of administration is intravenous. In another preferred embodiment the route of administration is via a central venous catheter. In another preferred embodiment the route of administration is via a peripheral venous catheter. In some embodiments, the present disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer), wherein the method comprises administering to a patient in need thereof a pharmaceutically acceptable composition comprising a copolymer of Formula I and SN-38 wherein the treatment is metronomic. In some embodiments, the present disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer) in a subject in need thereof, wherein the method comprises:a) measuring the UGT1A1 genotype of the subject;b) identifying if the subject has a UGT1A1 *1/*1, UGT1A1 * 1/*28, or UGT1A1 *28/*28 genotype; andc) administering SN-38 to the subject an amount appropriate to the UGT1A1 genotype. In some embodiments, SN-38 is administered to the subject as a pharmaceutically acceptable composition as described herein, comprising a copolymer of Formula I and SN-38 at a dose specific for the genotype of the patient. In some embodiments, the present disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer), wherein the method comprises administering to a patient in need thereof a pharmaceutically acceptable composition comprising a copolymer of Formula I and SN-38 wherein the treatment is given in a single, or on a repeating dosing schedule. In some embodiments, the composition comprising a copolymer of Formula I and SN-38 is administered at least one of 1×, 2×, 3×, 4×, 5×, 6×, or 7× a week. In some embodiments, the composition comprising a copolymer of Formula I and SN-38 is administered at an interval of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21, days 22 days. 23 days, 24 days, 25 days, 26 days, 27 days, 28 days. In some embodiments, the composition comprising a copolymer of Formula I and SN-38 is administered over a period of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 36 months. In a preferred embodiment the composition comprising a copolymer of Formula I and SN-38 is administered on day 1, and day 15 of a 28-day cycle. In another preferred embodiment the composition comprising a copolymer of Formula I and SN-38 is administered on day 1, day 8, and day 15 of a 28-day cycle. In another preferred embodiment the composition comprising a copolymer of Formula I and SN-38 is administered on day 1 of a 21-day cycle. In some embodiments, the disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer), wherein the method comprises administering to a patient in need thereof a pharmaceutically acceptable composition comprising a copolymer of Formula I and SN-38 wherein the administration is performed over about 10 to about 90 minutes. In a preferred embodiment, the administration is performed over about 30 minutes. In another preferred embodiment, the administration is performed over about 60 minutes. In some embodiments, the disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer), wherein the method comprises administering to a patient in need thereof a pharmaceutically acceptable composition comprising a copolymer of Formula I and SN-38 wherein the dose of SN-38 is about 5 to about 100 mg/m2body surface area. In a preferred embodiment the dose of SN-38 is about 20 to about 50 mg/m2body surface area. In some embodiments, the disclosure is directed to a method for treating, stabilizing, or lessening the severity or progression of one or more proliferative diseases (e.g. cancer), wherein the method comprises administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a copolymer of Formula I and SN-38 in combination with one or more therapeutic agents. In some embodiments, the composition comprising a copolymer of Formula I and SN-38 with one or more therapeutic agents are administered simultaneously. In some embodiments, the composition comprising a copolymer of Formula I and SN-28 with one or more therapeutic agents are administered sequentially. In some embodiments, the therapeutic agent is an antimetabolite. In a preferred embodiment, the antimetabolite is 5-fluorouracil. In another preferred embodiment, the antimetabolite is leucovorin. In a preferred embodiment, 5-fluorouracil is administered at about 100 to about 2400 mg/m2body surface area. In another preferred embodiment, leucovorin is administered at about 20 to about 400 mg/m2body surface area In some embodiments, the present disclosure provides an infusion solution comprising about 0.01 mg/mL to about 150 mg/mL SN-38. EXEMPLIFICATION In order for the disclosure to be more fully understood, the following examples are set forth. It will be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosure in any manner. Analytical Methods The following analytical methods were utilized to characterize the compounds of the present disclosure. SN-38 HPLC Method—Assay and identity of SN-38 was determined by high pressure liquid chromatography with UV detection at 265 nm. The column utilized was a Phenomenex Gemini® 5 μm C18 (110 Å, 250×4.6 mm) at ambient temperature. The mobile phase consisted of a 70:30 (v/v) mixture of 10 mM sodium phosphate with 0.1% (v/v) triethylamine, pH 3.5 and acetonitrile. SN-38 drug product samples and standards were prepared by dissolving the material in a 7:3 (v/v) mixture of acetonitrile and DMSO. Separation was achieved with a flow rate of 1.5 mL/min for a total run time of 8 minutes. SN-38 Weigh Loading Analysis—Weight loading was determined by comparing a standard curve of SN-38 to a known concentration of drug product by HPLC analysis. Standards were prepared by dissolving SN-38 in a 7:3 (v/v) mixture of acetonitrile and DMSO at concentrations of 50, 100, 200, 300, and 400 μg/mL. SN-38 drug product samples were prepared by dissolving the material in a 7:3 (v/v) mixture of acetonitrile and DMSO at a concentration between 1-4 mg/mL depending on the weight loading. The amount of SN-38 in the drug product is then converted to weight percentage of the total based on the known quantity of drug product. Example 1—Preparation of SN-38 Drug Product with 15% SN-38 Feed Trehalose (8.0 g) was dissolved in 400 mL of water before the addition of 2.0 g of TFS-3 (Sar235[D-Phe10-co-Tyr30]) to produce a solution of 20 mg/mL trehalose and 5 mg/mL TFS-3. The resulting solution was stirred for 1 hour before filtering through a 0.5 μm polypropylene filter. Separately, a solution of SN-38 was prepared by dissolving 281 mg in 3.75 mL of DMSO, with the assistance of heat, to produce a stock solution of 75 mg/mL. While shear mixing 375 mL of the polymer/trehalose solution with a homogenizer at 10,000 RPM, the SN-38 stock solution was added, and the mixing was continued for 1 minute. The resulting homogenous emulsion was processed with two passes through a microfluidizer with an inlet pressure of 100 PSI and an operating pressure of approximately 25,000 PSI through an auxiliary processing chamber followed by a 50 μm X interaction chamber with the outlet tube cooled in an ice-water bath. The extruded solution was then diafiltered against 2.5 L of 20 mg/mL trehalose using a tangential flow filtration system equipped with a mPES hollow fiber filter (10 kDa MWCO, 790 cm2surface area) at a flow rate of 300 mL/min. The solution was then concentrated to ˜¼ the original volume such that the final polymer concentration was ˜20 mg/mL. The formulation solution was then filtered through a 0.2 μm PES filter with a surface area of 20 cm2. The filtered solution was frozen at −80° C. and lyophilized for 2 days. This yielded the drug formulation as a fragmented, slightly yellow cake with an SN-38 weight loading of 5.73%. Example 2—Preparation of SN-38 Drug Product with 20% SN-38 Feed Using the general method of Example 1 with the following exception: a total of 5.0 mL of the SN-38 solution (75 mg/mL) was homogenized with 375 mL of the polymer/trehalose solution. This yielded the drug product as a fragmented, slightly yellow cake with an SN-38 weight loading of 7.54% Example 3—Preparation of SN-38 Drug Product with 25% SN-38 Feed Using the general method of Example 1 with the following exception: a total of 6.25 mL of the SN-38 solution (75 mg/mL) was homogenized with 375 mL of the polymer/trehalose solution. This yielded the drug product as a fragmented, slightly yellow cake with an SN-38 weight loading of 9.35% Example 4—Preparation of SN-38 Drug Product with 30% SN-38 Feed Using the general method of Example 1 with the following exception: a total of 7.5 mL of the SN-38 solution (75 mg/mL) was homogenized with 375 mL of the polymer/trehalose solution. This yielded the drug product as a fragmented, slightly yellow cake with an SN-38 weight loading of 11.64%. Example 5—Preparation of SN-38 Drug Product with 40% SN-38 Feed Using the general method of Example 1 with the following exception: a total of 10.0 mL of the SN-38 solution (75 mg/mL) was homogenized with 375 mL of the polymer/trehalose solution. This yielded the drug product as a fragmented, yellow cake with an SN-38 weight loading of 14.80% Example 6—Preparation of Sar235-b-p-[D-Phe10-co-L-Tyr30] (TFS-3) A jacketed round-bottom flask equipped to a circulating isopropanol/water bath was cooled to 20° C. prior to the addition of sarcosine N-carboxyanhydride (15.0 g, 130.5 mmol, 235 equiv.), followed by N,N-dimethylformamide (75 mL). The mixture was stirred for <30 seconds before the addition of neopentylamine (1.85 mL of 300 mM in DMF, 48.4 mg, 0.555 mmol, 1 equiv.). The reaction vessel was wrapped in aluminum foil to prevent exposure to light. After 15-20 mins, the reactions started to change from the initial clear and colorless solution to a light orange color that continues to intensify as the reaction proceeds. IR was used to monitor the reaction progression via disappearance of the Sar NCA carbonyl stretches at ˜1850 and 1778 cm−1, with the latter being the preferred wavenumber to monitor. The next day, after a total of 22 h the reaction was complete. The circulating bath temperature was increased to 25° C. prior to the addition of D-phenylalanine N-carboxyanhydride (1.06 g, 5.55 mmol, 10 equiv.) and L-tyrosine N-carboxyanhydride (3.45 g, 16.7 mmol, 30 equiv.). Additional DMF (˜5 mL) was used to rinse down the sides of the funnel and reaction vessel. Significant CO2gas formation was observed shortly after the reaction was initiated. IR was used to monitor the reaction progression via disappearance of the D-Phe NCA and L-Tyr NCA carbonyl stretches at ˜1847 and 1786 cm−1, with the latter being the preferred wavenumber to monitor. As the reaction proceeds, the color changed from a clear bright orange to a clear yellow-orange solution that was apparent after only a few hours. The reaction was complete after a total of 30 h. The reaction mixture (total of ˜100 mL) was transferred to a beaker and fitted with an overhead stirrer. While vigorously stirring, ethyl acetate (400 mL, 4 volumes) was added to precipitate the product. The solids were collected via filtration into a medium fritted glass funnel, and then the semi-dry material was transferred back to the original precipitation beaker along with additional EtOAc (200 mL, 2 volumes) and slurried with vigorous stirring for 20 mins. The solids were collected in the same glass funnel and washed with additional EtOAc (100 mL, 1 volume) once more. The product was dried in a vacuum oven at 90-100° C. for 2 days to yield 11.3 g (87.9%) of the title compound as a fine off-white powder. 1H NMR (DMSO-d6) δ 9.3-9.0 (28H), 8.5-7.8 (45H), 7.4-6.4 (170H), 4.6-3.6 (784H), 3.2-2.5 (1326H), 1.9 (5H), 1.2-1.1 (6H), 0.9-0.8 (14H); GPC (DMF, 50 mM LiBr) Mn=18.1 kDa, Mp=19.3 kDa, PDI=1.07. | 45,409 |
11857631 | DETAILED DESCRIPTION The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein, the term “polymer composition” is a composition comprising one or more polymers. As a class, “polymers” includes homopolymers, heteropolymers, co-polymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas co-polymers contain more than one type of monomer. As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, are meant to be open ended. The terms “a” and “an” are intended to refer to one or more. As used herein, the term “patient” or “subject” refers to members of the animal kingdom including but not limited to human beings. A “coacervate” refers to herein as a reversible aggregation of compositions in a liquid, for example, as described herein, for example, resulting from the aggregation of oppositely-charged polyionic compositions. Exemplary coacervates are illustrated in the examples below with the aggregation of the polycation, polyanion, and active agent(s), as described herein, for example with the aggregation of PEAD, heparin, and PRP, or a combination of FGF-2 and SDF-1α. A “complex” is a non-covalent aggregation of two or more compositions. The term “alkyl” refers to both branched and straight-chain saturated aliphatic hydrocarbon groups. These groups can have a stated number of carbon atoms, expressed as Cx-y, where x and y typically are integers. For example, C5-10, includes C5, C6, C7, C8, C9, and C10. Alkyl groups include, without limitation: methyl, ethyl, propyl, isopropyl, n-, s- and t-butyl, n- and s-pentyl, hexyl, heptyl, octyl, etc. Alkenes comprise one or more double bonds and alkynes comprise one or more triple bonds. These groups include groups that have two or more points of attachment (e.g., alkylene). Cycloalkyl groups are saturated ring groups, such as cyclopropyl, cyclobutyl, or cyclopentyl. As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo. A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain terminal groups or atoms are incorporated into the polymer backbone or are excised. A polymer is said to comprise a specific type of linkage, such as an ester, or urethane linkage, if that linkage is present in the polymer. According to one aspect of the invention, a composition is provided comprising a complex, e.g. a coacervate of a polyanionic polymer, a polycationic polymer, and platelet-rich plasma or serum, including concentrations thereof, according to any aspect described herein. The platelet-rich plasma or serum, including concentrations thereof, is mixed with the polyanionic polymer composition to form a complex, and the resulting complex is then mixed with a polycationic polymer composition to form a composition, e.g., a coacervate. The charges of the polycation and polyanion are generally approximately equal to form a charge-neutral complex, e.g., coacervate. Suitable polyanionic polymers include as a class sulfated and/or sulfamated polymer or oligomers, such as sulfated polysaccharides or sulfated glycosoaminoglycans. Sulfated and/or sulfamated polymer or oligomers include sulfated and/or sulfamated polysaccharides. Synthetic and natural sulfated and/or sulfamated polysaccharides or oligosaccharides include, for example and without limitation, sulfated glycosaminoglycans or sulfated galactans, ulvans and fucans (Jiao, G., et al. Chemical Structures and Bioactivities of Sulfated Polysaccharides from Marine Algae (2011) Mar. Drugs 9:196-223). Non-limiting examples of sulfated and/or sulfamated polysaccharides include, pentosan polysulfates, dermatan sulfates, keratan sulfates, chondroitin sulfates, sulfated agarans (e.g., porphyrans), and carageenans. In another aspect, the sulfated and/or sulfamated polymer or oligomer is a sulfated and/or sulfamated synthetic polymer, such as a polyurethane, polyester, polyurea, polyamide-ester, polyether, polycarbonate, polyamide, or polyolefin, or copolymers thereof, as are broadly-known in the polymer arts. By “sulfated”, it is meant that the polymer comprises a plurality of pendant sulfate (—OSO3) groups, though many such compositions also are “sulfamated”—comprising a plurality of pendant sulfamate (—NSO3) groups. Examples of suitable polysaccharides include, without limitation: a sulfated polysaccharide, a sulfamated polysaccharides, a sulfated and/or sulfamated polydisaccharide, a sulfated glycosaminoglycan, heparin, and heparan sulfate. “Platelet-rich plasma” or “PRP” in its broadest sense is blood plasma with an enriched platelet content, where “enriched” is in reference to normal blood of a patient. Typically platelet content is enriched at least two-fold, and often at least five-fold or ten-fold. Platelet-rich plasma is typically prepared by centrifugation of anti-coagulase-treated blood obtained from one or more patients, and can be autologous. Four forms of PRP are commonly-available: Pure Platelet-Rich Plasma (P-PRP) or leucocyte-poor PRP products are preparations without leucocytes and with a low-density fibrin network after activation; Leucocyte-PRP (L-PRP) products are preparations with leucocytes and with a low-density fibrin network after activation. This is the most common commercial PRP product; Pure platelet-rich fibrin (P-PRF) or leucocyte-poor platelet-rich fibrin preparations are without leucocytes and with a high-density fibrin network; and Leucocyte- and platelet-rich fibrin (L-PRF) or second-generation PRP products are preparations with leucocytes and with a high-density fibrin network (Dhurat et al. Principles and Methods of Preparation of Platelet-Rich Plasma: A Review and Author's Perspective.Journal of Cutaneous and Aesthetic Surgery.2014; 7(4):189-197). A number of methods are broadly-known for preparation of PRP. In one instance, blood is collected in tubes containing anticoagulants. A platelet layer, a buffy coat layer, and a red blood cell (RBC) layer are produced. For production of P-PRP, the platelet layer ad only the superficial buffy coat layer are transferred to a clean tube. For preparation of L-PRP, the platelet layer and buffy coat layer are transferred to a clean tube. The second tube is spun in a centrifuge resulting in a soft platelet pellet at the bottom of the tube. A portion, e.g. ⅔, of the platelet-poor top volume is removed, and the platelet pellet is then dispersed, e.g. homogenized, in the remaining plasma. An alternative method is known as the “buffy coat” method in which whole blood is centrifuged at a high speed to form a tighter buffy coat as compared to the PRP method above. PRP, and in general platelets, can be activated by addition of calcium and thrombin as is broadly-known, or by any other useful means. “Serum” is blood that is allowed to coagulate, and the clot and cellular constituents are then removed. Fractions of serum or platelet-rich plasma may be employed. By “fractions”, it is meant a portion of the serum or platelet-rich plasma prepared by any suitable method, including by precipitation, solvent extraction, filtration, centrifugation, or any other suitable method—so long as the fractionated product is not reduced to a single purified compound, such as a single protein, glycoprotein, polysaccharide, or other composition found in the platelet-rich plasma or serum. Concentrates are solutions in which a portion of the solvent, e.g. water in the case of a blood product, is removed thereby increasing the concentration of compounds present in the solution, such as proteins, glycoproteins, polysaccharides, or other desirable compositions found in the platelet-rich plasma or serum. FGF-2 is Fibroblast growth factor 2 (HGNC: 3676, Entrez Gene: 2247, Ensembl: ENSG00000138685, OMIM: 134920, UniProtKB: P09038), having the sequence, for example: (SEQ ID NO: 1)MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS SDF-1α, Stromal Cell-Derived Factor 1, is the geane product of CXCL12 gene in humans (HGNC: 10672, Entrez Gene: 6387, Ensembl: ENSG00000107562, OMIM: 600835, UniProtKB: P48061), and having an exemplary sequence: (SEQ ID NO: 2)MNAKVVVVLV LVLTALCLSD GKPVSLSYRC PCRFFESHVARANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQEYLEKALNKR FKM. TIMP-3 is TIMP is Tissue Inhibitor of Metalloproteinase 3 (HGNC: 10672, Entrez Gene: 6387, Ensembl: ENSG00000107562, OMIM: 600835, UniProtKB: P48061), having an exemplary amino acid sequence: (SEQ ID NO: 3)MTPWLGLIVLLGSWSLGDWGAEACTCSPSHPQDAFCNSDIVIRAKVVGKKLVKEGPFGTLVYTIKQMKMYRGFTKMPHVQYIHTEASESLCGLKLEVNKYQYLLTGRVYDGKMYTGLCNFVERWDQLTLSQRKGLNYRYHLGCNCKIKSCYYLPCFVTSKNECLWTDMLSNFGYPGYQSKHYACIRQKGGYCSWYRGWAPPDKSIINATDP. Certain polymers described herein, such as heparin and PEAD, are said to be bioerodible or biodegradable. By that, it is meant that the polymer, once implanted and placed in contact with bodily fluids and tissues, or subjected to other environmental conditions, such as composting, will degrade either partially or completely through chemical reactions, typically and often preferably over a time period of hours, days, weeks or months. Non-limiting examples of such chemical reactions include acid/base reactions, hydrolysis reactions, and enzyme catalyzed bond scission. Certain polymers described herein contain labile ester linkages. The polymer or polymers may be selected so that it degrades over a time period. Non-limiting examples of useful in situ degradation rates include between 12 hours and 5 years, and increments of hours, days, weeks, months or years therebetween. A drug delivery composition is provided, comprising, a coacervate of a polycationic polymer, a polyanionic polymer, and an active agent. In certain aspects, the polycationic polymer described herein comprises the structure (that is, comprises the moiety: [—OC(O)-B′—CH(OR1)-B-]nor —[OC(O)—B—C(O)O—CH2—CH(O—R1)-CH2—B′-CH2—CH(O—R2)-CH2-]n, in which B and B′ are the same or different and are organic groups, or B′ is not present, including, but not limited to: alkyl, ether, tertiary amine, ester, amide, or alcohol, and can be linear, branched or cyclic, saturated or unsaturated, aliphatic or aromatic, and optionally comprise one or more protected active groups, such as, without limitation, protected amines and acids, and R1 and R2 are the same or different and are hydrogen or a functional group (e.g., as described herein). As seen below, the composition exhibits low polydispersity, with a polydispersity index of less than 3.0, and in many cases less than 2.0. These compositions are described in U.S. Pat. No. 9,023,972, which is incorporated by reference in its entirety. In one aspect, the polycationic polymer is a polymer composition comprising at least one moiety selected from the following in which B and B′ are residues of aspartic acid or glutamic acid, which are optionally further derivatized with an amine-containing group, for example, the amines of the aspartic acid or glutamic acid are further derivatized with lysine or arginine:(a) [—OC(O)—CH(NHY)—CH2—C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(b) [—OC(O)—CH2—CH(NHY)-C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(c) [—OC(O)—CH(NHY)—CH2—CH2—C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, and/or(d) [—OC(O)—CH2—CH2—CH(NHY)-C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, wherein Y is —C(O)—CH(NH3+)—(CH2)3—NH—C(NH2)2+or —C(O)—CH(NH3+)-(CH2)4-(NH3)+, and R1 and R2 are the same or different and are independently selected from the group consisting of hydrogen, a carboxy-containing group, a C1-6alkyl group, an amine-containing group, a quaternary ammonium containing group, and a peptide. The polymers described herein can be functionalized, e.g., at B, B′, R1 and R2, meaning they comprise one or more groups with an activity, such as a biological activity. For example and without limitation, as shown herein, the polymer may be functionalized with an acetylcholine-like group or moiety, a cross-linking agent (cross-linking agents contain at least two reactive groups that are reactive towards numerous groups, including sulfhydryls and amines, and create chemical covalent bonds between two or more molecules, functional groups that can be targeted with cross-linking agents include primary amines, carboxyls, sulfhydryls, carbohydrates and carboxylic acids. A large number of such agents are available commercially from, e.g., Thermo fisher Scientific (Pierce) and Sigma). Other functions that can be provided by or enhanced by addition of functional groups include: increased hydrophobicity, for instance by functionalizing with a superhydrophobic moiety, such as a perfluoroalkane, a perfluoro(alkylsilane), and/or a siloxane; increased hydrophilicity, for instance by functionalizing with polyethylene glycol (PEG); or antimicrobial, for instance, by functionalizing with a quaternary amine. The polymer can be functionalized with a tag, such as a fluorescent tag (e.g., FITC, a cyanine dye, etc.). The polymer can be functionalized by linking to additional synthetic or natural polymers, including, without limitation: synthetic polymers, such as a polymer derived from an alpha-hydroxy acid, a polylactide, a poly(lactide-co-glycolide), a poly(L-lactide-co-caprolactone), a polyglycolic acid, a poly(dl-lactide-co-glycolide), a poly(1-lactide-co-dl-lactide), a polymer comprising a lactone monomer, a polycaprolactone, a polymer comprising carbonate linkages, a polycarbonate, a polyglyconate, a poly(glycolide-co-trimethylene carbonate), a poly(glycolide-co-trimethylene carbonate-co-dioxanone), a polymer comprising urethane linkages, a polyurethane, a poly(ester urethane) urea, a poly(ester urethane) urea elastomer, a polymer comprising ester linkages, a polyalkanoate, a polyhydroxybutyrate, a polyhydroxyvalerate, a polydioxanone, a polygalactin, or natural polymers, such as chitosan, collagen, gelatin, elastin, alginate, cellulose, hyaluronic acid and other glycosaminoglycans. The compositions may be functionalized with organic or inorganic moieties to achieve desired physical attributes (e.g., hardness, elasticity, color, additional chemical reactivity, etc.), or desired functionality. For example, the polymer composition may be derivatized with maleic acid or phosphate. Further to the above, functional groups may vary as indicated above. For example, in certain aspects, R1 and R2 are the same or different and are independently selected from the group consisting of hydrogen, a carboxy-containing group, a C1-6alkyl group, an amine-containing group, a quaternary ammonium containing group, and a peptide. In one aspect, one or more of B, B′, R1 and R2 are charged such that it is possible to bind various water insoluble organic or inorganic compounds to the polymer, such as magnetic inorganic compounds. As above, in one aspect, one or more of B, B′, R1 and R2 are positively charged. In one aspect, one or both of R1 and R2 are functionalized with a phosphate group. In another aspect, the composition is attached non-covalently to a calcium phosphate (including as a group, for example and without limitation: hydroxyapatite, apatite, tricalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate, and calcium dihydrogen phosphate). In yet another embodiment, R1 and R2 are independently one of Ile-Lys-Val-Ala-Val (IKVAV) (SEQ ID NO: 4), Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO: 5), Ala-Gly-Asp (AGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) (SEQ ID NO: 6), Val-Ala-Pro-Gly-Val-Gly (VAPGVG) (SEQ ID NO: 7), APGVGV (SEQ ID NO: 8), PGVGVA (SEQ ID NO: 9), VAP, GVGVA (SEQ ID NO: 10), VAPG (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), VGVA (SEQ ID NO: 13), VAPGV (SEQ ID NO: 14) and GVAPGV (SEQ ID NO: 15). The composition is formed into a coacervate with active agents or polyanionic or polycationic groups for sequestering active agents for controlled delivery in vivo. Drug products comprising the coacervate described herein may be delivered to a patient by any suitable route of delivery (e.g. oral or parenteral), or as an implantable device for slow release of the active agent. In forming the composition (e.g., coacervate), the cationic polycationic polymer is complexed with a polyanionic polymer, such as heparin or heparan sulfate, which is complexed with an active agent, such as a growth factor, small molecule, cytokine, drug, a biologic, a protein or polypeptide, a chemoattractant, a binding reagent, an antibody or antibody fragment, a receptor or a receptor fragment, a ligand, an antigen and/or an epitope, PRP, or a composition obtained from an organism or cultured cells, tissues or organs and containing a native, complex mixture of proteins and/or growth factors. Specific examples of active agents include interleukins (IL), such as IL-2 and IL-12 (e.g., IL-12 p70), and interferons (IFN), such as IFN-γ. In one aspect, the composition comprises a coacervate of a polycationic polymer comprising one or more of moieties (a), (b), (c), and/or (d), as described above, and further comprising heparin or heparin sulfate complexed (that is non-covalently bound) with FGF-2 and SDF-1α. The composition is formed, for example, by mixing in a suitable solvent, such as an aqueous solution, such as water, saline (e.g. normal saline), or PBS, the polyanionic, polycationic, and active agent constituents of the composition. Additional active agents that may be incorporated into the coacervate include, without limitation, anti-inflammatories, such as, without limitation, NSAIDs (non-steroidal anti-inflammatory drugs) such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen sodium salicylamide, anti-inflammatory cytokines, and anti-inflammatory proteins or steroidal anti-inflammatory agents); antibiotics; anticlotting factors such as heparin, Pebac, enoxaparin, aspirin, hirudin, plavix, bivalirudin, prasugrel, idraparinux, warfarin, coumadin, clopidogrel, PPACK, GGACK, tissue plasminogen activator, urokinase, and streptokinase; growth factors. Other active agents include, without limitation: (1) immunosuppressants; glucocorticoids such as hydrocortisone, betamethasone, dexamethasone, flumethasone, isoflupredone, methylprednisolone, prednisone, prednisolone, and triamcinolone acetonide; (2) antiangiogenics such as fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide, etoposide, pegaptanib, lucentis, tryptophanyl-tRNA synthetase, retaane, CA4P, AdPEDF, VEGF-TRAP-EYE, AG-103958, Avastin, JSM6427, TG100801, ATG3, OT-551, endostatin, thalidomide, bevacizumab, neovastat; (3) anti-proliferatives such as sirolimus, paclitaxel, perillyl alcohol, farnesyl transferase inhibitors, FPTIII, L744, anti-proliferative factor, Van 10/4, doxorubicin, 5-FU, Daunomycin, Mitomycin, dexamethasone, azathioprine, chlorambucil, cyclophosphamide, methotrexate, mofetil, vasoactive intestinal polypeptide, and PACAP; (4) antibodies; drugs acting on immunophilins, such as cyclosporine, zotarolimus, everolimus, tacrolimus and sirolimus (rapamycin), interferons, TNF binding proteins; (5) taxanes, such as paclitaxel and docetaxel; statins, such as atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin and rosuvastatin; (6) nitric oxide donors or precursors, such as, without limitation, Angeli's Salt, L-Arginine, Free Base, nitrates, nitrites, Diethylamine NONOate, Diethylamine NONOate/AM, Glyco-SNAP-1, Glyco-SNAP-2, (.+−.)-S-Nitroso-N-acetylpenicillamine, S-Nitrosoglutathione, NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1, NOR-3, SIN-1, Hydrochloride, Sodium Nitroprusside, Dihydrate, Spermine NONOate, Streptozotocin; and (7) antibiotics, such as, without limitation: acyclovir, ofloxacin, ampicillin, amphotericin B, atovaquone, azithromycin, ciprofloxacin, clarithromycin, clindamycin, clofazimine, dapsone, diclazuril, doxycycline, erythromycin, ethambutol, fluconazole, fluoroquinolones, foscarnet, ganciclovir, gentamicin, itraconazole, isoniazid, ketoconazole, levofloxacin, lincomycin, miconazole, neomycin, norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymyxin B, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin, streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine, trimethoprim sulfate, Zn-pyrithione, and silver salts such as chloride, bromide, iodide and periodate. Further examples of additional active agents include: basic fibroblast growth factor (bFGF or FGF-2), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor-beta pleiotrophin protein, midkine protein, platelet-derived growth factor (PDGF) and angiopoietin-1 (Ang-1). Active agents are included in the delivery system described herein, and are administered in amounts effective to achieve a desired end-point, such as angiogenesis, tissue growth, inhibition of tissue growth, repair of tissue (e.g. an infarct) or any other desirable end-point. According to one aspect, complex structures are provided that comprise the coacervate described herein mixed with, distributed within, or otherwise combined with another composition, such as a hydrogel, a polymer, and/or an inorganic substrate, and can be combined with a medical implant or device such as a prosthetic, a dosage form, a woven or non-woven mesh, etc. According to one aspect, the coacervate is combined with a hydrogel, for example by embedding the coacervate in a hydrogel, such as fibrin. Such a structure is useful for providing complex release profiles for active agents, for instance for promoting specific tissue growth or as a timed-release dosage form. In such an aspect, one or more active agents are distributed by any method in the coacervate and in the hydrogel so as to cause a defined degradation and release pattern. One useful aspect would be to embed the coacervate having a first active agent into a hydrogel, having a second active agent, to provide a complex release profile. In any aspect, the active agent(s) can be any effective active agent(s), for example as described above. As an example, factor A is embedded into a hydrogel, e.g. a fibrin gel, for early release and factor B is contained within the coacervate, for delayed release. For each indicated purpose it is noted that appropriate relative amounts of the coacervate and hydrogel may be used, as well as including effective amounts of the active agents for the intended purpose, respectively in the coacervate and hydrogel. Appropriate and effective amounts of each component can be determined in the ordinary course by a person of skill in the art. Therefore, according to one aspect of the invention, a composition is provided comprising a complex, e.g. a coacervate of a polyanionic polymer, a polycationic polymer, and one or more active agents, that is embedded in a hydrogel, such as a fibrin hydrogel, which contains an active agent for faster release than active agent(s) complexed in the coacervate. In one aspect, the active agent is PRP, serum, or a complex mixture of proteins and/or growth factors produced by cells, tissue, or an organism. In one aspect, the active agents complexed in the coacervate are FGF-2 and SDF-1α by first mixing with the polyanionic polymer, followed by mixing with the polycationic polymer to form the coacervate. The charges of the polycation and polyanion are generally approximately equal to form a charge-neutral complex, e.g., coacervate. TIMP-3 and the coacervate are then mixed into a hydrogel composition, e.g. prior to or during formation of the hydrogel. In one aspect, each active agent is present in amounts effective to treat a myocardial infarct, by application of the composition at or near a myocardial infarct, e.g., by injection. Examples of useful active agents and combinations of agents for incorporation into the described coacervate for treatment of a myocardial infarct include: TIMP-3, FGF-2, and SDF-1α. Also described herein is a method of treatment of myocardial infarction, using the combination of TIMP-3 in a hydrogel, and a coacervate as described comprising FGF-2 and SDF-1α. The coacervate composition according to any aspect described herein is delivered in any manner useful and appropriate for treatment of a condition in a patient, such as for treatment of a wound, cardiovascular disease, or an infarct, such as by enteral, parenteral, or topical routes, for example and without limitation by: intravenous (IV), local injection, intramuscular, intracerebral, subcutaneous, oral, inhalation, topical, enema, intravaginal, intrauterine, ocular, or otic routes. Typically, due to the nature of wounds and myocardial infarcts, the composition is typically applied either topically or by injection at or near the site of the wound or infarct, as opposed to systemically. Suitable excipients or carriers are employed for delivery of the coacervate composition, though the excipients are consistent with maintenance of the coacervate complex. Suitable excipients are broadly-known in the pharmaceutical arts, and include: solvents, such as water, phosphate-buffered saline (PBS), saline; buffers; salts; acids; bases; rheology modifiers; chelating agents; colorants; flavorings; penetration enhancers; and preservatives. The coacervate composition is provided in a suitable vessel for storage, distribution and/or use of the composition. In one aspect, the coacervate composition is provided in a tube, a medical syringe, an IV bag. In another aspect, the coacervate composition is delivered to a patient in an amount effective to treat a myocardial infarction, for example by direct injection of the coacervate composition comprising TIMP-3, FGF-2, and SDF-1α into the heart, e.g., the myocardium at or adjacent to an infarct. An “amount effective” for treatment of a condition is an amount of an active agent or dosage form, such as the coacervate composition described herein, effective to achieve a determinable end-point. The “amount effective” is preferably safe—at least to the extent the benefits of treatment outweighs the detriments and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration. In the context of wound healing, the end point may be decreased wound size, in a myocardial infarction, the end point may be improved cardiac output or improvement in the infarcted tissue, or in both instances any other objectively-determinable indicator of improvement in a patient's condition or symptoms. Using the teachings of the present disclosure, a person of ordinary skill in the arts can prepare the coacervate composition described herein, and titrate the effect on any objectively-determinable end-point, for instance first in an animal model and later in humans. As shown in the Examples below, an example of an “amount effective” is indicated. The coacervate composition may be administered continually for a period of time, or at intervals, ranging from hourly, weekly, monthly, or yearly, including increments therebetween, such as from one to six times per day, daily, every other day, weekly, bi-weekly, monthly, bi-monthly, quarterly, etc. An appropriate dosing schedule can be determined by a person of ordinary skill, such as a physician, and can also be tailored to wound or infarct severity in a patient, or improvement in wound healing, cardiac output or infarct repair. In use, according to one aspect, the coacervate composition is delivered to a patient in an amount effective to treat a wound in a patient. For treating a wound, the coacervate is formed in the presence of platelet-rich plasma or serum according to any aspect described herein. The composition is delivered, for example by injection, at or adjacent to a wound, or application as a solution, gel, or as applied to any medical device or wound dressing. In one aspect, the composition is delivered to a patient at or adjacent to a wound by injection, topical application, spraying (spay or aerosol), swabbing, or any effective means of transferring the composition to the wound location. In another aspect, the composition is applied to a wound dressing, such as a bandage, a suture, a surgical mesh, or a non-woven material as are broadly-known in the medical arts. In yet another aspect, the composition is applied to an implanted medical device, such as a prostheses, so that the composition facilitates integration of the device into the local tissue and/or healing of wound surrounding the device as a result of trauma, disease, or the process of inserting the device, such as a heart valve. In another aspect, the coacervate composition is delivered to a patient in an amount effective to treat a cardiovascular disease, such as coronary heart disease, including treatment of ischemic conditions, such as myocardial infarction. In one aspect, the composition is delivered to a patient's myocardium at or adjacent to an infarct, the composition comprising a hydrogel, such as a fibrin hydrogel, comprising TIMP-3, and a complex or coacervate of a polycationic polymer, a polyanionic polymer, FGF-2 and SDF-1α embedded in the hydrogel. The composition and respective amounts of TIMP-3, FGF-2, and SDF-1α are administered in amounts effective to treat the infarct, that is to improve one or more clinically-relevant markers, such as to improve cardiac function parameters such as myocardial elasticity, to reduce infarct size, to increase revascularization of the infarct, to reduce scarring of the myocardium, and/or stimulate repair of the myocardium. Other conditions, such as myocardial reperfusion injury and peripheral artery disease may be treated in the same manner. In another aspect, a composition is provided comprising a coacervate of a polycationic polymer, a polyanionic polymer, and a composition obtained from an organism or cultured cells, tissues or organs and containing a native, complex mixture of proteins and/or growth factors. A “native, complex mixture of proteins and/or growth factors” refers to a composition produced by a living source, and though it may proceed through one or more purification steps, as in the case of PRP as described herein to remove blood cells, activate platelets, and optionally to remove fibrin and to concentrate the proteins, it is not an isolated or purified single constituent, but includes a plurality of compounds and proteins, such as growth factors, essentially in amounts and proportions found in, or produced by the cells or organism. Examples of suitable sources of the native, complex mixture of proteins and/or growth factors include: a bodily fluid such as blood—including plasma or serum, or processed plasma or serum, conditioned medium from a cell, tissue or organ culture, or a cell or tissue lysate or homogenate. As used herein “conditioned media” is media prepared by the culture of cells or other living tissue therein, wherein the cells or living tissue are natural or genetically-modified. The conditioned media includes proteins and other compositions representative of the secretome of the living material grown therein. The cell secretome refers not only to the collection of proteins that contain a signal peptide and are processed via the endoplasmic reticulum and Golgi apparatus through the classical secretion pathway, but encompasses proteins shed from the cell surface and intracellular proteins released through non-classical secretion pathway or exosomes. These secreted proteins may be enzymes, growth factors, cytokines, hormones, and/or other soluble mediators. The medium used to produce the conditioned media by growth of living cells or tissue therein may be any medium suitable for growth of the living cells or tissue. A large variety of media is available commercially, and one of ordinary skill in the art could determine a useful or optimal medium for use in this context. In one aspect, the media is serum-free. Various other fractionation processes, such as precipitation, centrifugation, affinity separation, or filtration may be applied to clean up, to remove harmful compounds, or to otherwise fractionate the conditioned media. The coacervate compositions according to any aspect described herein are formulated into medically- and pharmaceutically-acceptable dosage forms or devices, such as a liquid, a gel, a spray, an aerosol, or a wound dressing or medical device, such as a non-woven, a bandage, a suture, a mesh, a prosthetic, or an implantable/implanted medical device. The composition may comprise any useful excipient, or inactive ingredient, such as water, saline, phosphate-buffered saline, and effective amounts of any non-interfering active agent, such as, without limitation: an antibiotic, an anti-inflammatory, or an analgesic, or any other useful active agent, as are broadly-known in the pharmaceutical arts. A person of ordinary skill in the medical and pharmaceutical arts can readily fashion any of these products. Example 1—Synthesis and Testing of PEAD Synthesis and testing of PEAD, PEAD-heparin, and PEAD FGF2 are described in U.S. Pat. No. 9,023,972, which is incorporated by reference in its entirety. Briefly, for synthesis of PEAD—t-BOC protected aspartic acid (t-BOC Asp), t-BOC protected arginine (t-BOC-Arg) (EMD Chemicals, NJ), ethylene glycol diglycidyl ether (EGDE), trifluoroacetic acid (TFA) (TCI America, OR), anhydrous 1,4-dioxane and tetra-n-butylammonium bromide (TBAB) (Acros organics, Geel, Belgium), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) (Alfa Aesar, MA) and 4-dimethylaminopyridine (DMAP) (Avocado Research Chemicals Ltd, Lancaster, UK) were used for PEAD synthesis without purification. The synthesis of PEAD is performed as follows. EGDE and t-BOC Asp were polymerized in 1,4-dioxane under the catalysis of TBAB. t-BOC protection was later removed by TFA to generate primary amine. t-BOC-Arg was conjugated by DCC/NHS/DMAP coupling followed by the second de-protection to yield PEAD. The chemical structure was confirmed using NMR and FT-IR. The molecular weight of PEAD was measured by PL-GPC 50 Plus-RI equipped with a PD 2020 light scattering detector (Varian, MA). Two MesoPore 300×7.5 mm columns and 0.1% of LiBr in DMF were used as solid phase and mobile phase, respectively. In one example, the weight-average molecular weight (Mw) is 30,337 Da with polydispersity index (PDI) 2.28. Since PEAD is a positively-charged molecule, addition of PEAD into heparin solution should neutralize the negative charge of heparin and forms PEAD/heparin complex. To test the binding ability of PEAD to heparin, zeta potential measurement was performed and the zeta potential of the complex shifted from negatively-charged (−45 mV) at ratio 1 to positively-charged (+23.2 mV) at ratio 10. Continuing adding more PEAD did not change the zeta potential and +23.2 mV is close to the zeta potential of PEAD itself. Data suggested that for the described PEAD preparation after ratio 10 the complex was all covered by PEAD. Besides it also shows at ratio 5 PEAD almost neutralized all negative charges of heparin. From the macroscopic observation, below ratio 5 the addition of PEAD let the heparin solution became more turbid and precipitate was seen after a few minutes. Whereas the ratio was over 5, the addition of PEAD would let the solution become clear again. Further confirming the binding ability, different amounts of PEAD to heparin solutions were mixed and then precipitated by centrifugation. Because the neutralization of the negative-charged heparin favors the formation of precipitate, we measured the amount of heparin left in the supernatant was measured to determine the binding affinity between PEAD and heparin. For this assay, a heparin binding dye, dimethylmethylene blue (DMB) was applied to detect free heparin by measuring the absorption of DMB at 520 nm. The result shows the amount of heparin in the supernatant was gradually lowered with the addition of PEAD. When the ratio of PEAD to heparin is over 3, >90% of heparin was precipitated through centrifugation. At the ratio 5, that would be >99% of heparin. This result has a good correlation with that of zeta potential measurement because both experiments suggest at ratio 5 PEAD and heparin has the maximum interaction. It is understood that a variety of growth factors can bind to heparin with the dissociation constant (Kd) from μM to nM. The loading efficiency of growth factors to PEAD/heparin complex was studied. 100 ng or 500 ng of fibroblast growth factor-2 (FGF-2) plus125I-labeled FGF-2 used as a tracer were mixed with heparin then added into PEAD solution. After staying at room temperature for 2 hr, centrifugation was used to precipitate PEAD/heparin/FGF-2. The amount of unloaded FGF-2 remaining in the supernatant can be determined by a gamma counter. The result showed PEAD/heparin loaded ˜68% of FGF-2 for both high and low amounts of FGF-2. The other growth factor, NGF, the release is faster. The initial burst reached almost 20%. The release sustained till day 20 and reached a plateau corresponding to ˜30% of the loaded NGF. PRP is a blood product containing many therapeutic growth factors. It is used clinically although its true efficacy for wound healing is debated throughout the field due to a lack of systematic studies concerning its use. PRP is theoretically advantageous due to the complex mixture of therapeutic proteins present, but the short half-life of these proteins could render them useless within minutes. Example 2—Full-Thickness Excisional Wound Pig Model Platelet-rich plasma (PRP) is widely used for many clinical indications including wound healing due to the high concentrations of growth factors. However, the short half-life of these therapeutic proteins requires multiple large doses, and their efficacy is highly debated among clinicians. Here we report a method of protecting these proteins and releasing them in a controlled manner via a heparin-based coacervate delivery vehicle to improve wound healing in a porcine model. Platelet-derived proteins incorporated into the coacervate were protected and slowly released over 3 weeks in vitro. In a porcine model, PRP coacervate significantly accelerated the healing response over 10 days, in part by increasing the rate of wound reepithelialization by 35% compared to control. Additionally, PRP coacervate doubled the rate of wound contraction compared to all other treatments, including that of naked PRP proteins. Wounds treated with PRP coacervate exhibited increased collagen alignment and an advanced state of vascularity compared to control treatments. These results suggest that this preparation of PRP accelerates healing of cutaneous wounds only as a controlled release formulation. The coacervate delivery vehicle is a simple and effective tool to improve the therapeutic efficacy of platelet-derived proteins for wound healing. One alternative to mono-therapy is to harness the mixture of growth factors produced by the patient themself (autologous) or from a healthy allogenic donor in the form of platelet-rich plasma (PRP). PRP is a fraction of blood plasma containing many therapeutic growth factors released from alpha-granules upon platelet activation (F. Mussano, et al., Cytokine, chemokine, and growth factor profile of platelet-rich plasma, Platelets, (2016) 1-5). This protein cocktail has high potential to stimulate an accelerated healing response since it contains numerous factors known to play different vital roles in the natural healing progression. Autologous PRP avoids the risk of an immune response during treatment, although allogenic PRP has also been used safely in a clinical setting without serious adverse effects as long as the platelets were removed (Z. Y. Zhang, et al., The potential use of allogeneic platelet-rich plasma for large bone defect treatment: immunogenicity and defect healing efficacy,Cell Transplant,22 (2013) 175-187). PRP is currently approved for use in orthopedic applications and is under investigation for several others including wound healing (Z. Y. Zhang, et al., The potential use of allogeneic platelet-rich plasma for large bone defect treatment: immunogenicity and defect healing efficacy,Cell Transplant,22 (2013) 175-187; V. R. Driver, et al., G. Autologel Diabetic Foot Ulcer Study, A prospective, randomized, controlled trial of autologous platelet-rich plasma gel for the treatment of diabetic foot ulcers,Ostomy/wound management,52 (2006) 68-70, 72, 74 passim; K. K. Middleton, et al., Evaluation of the effects of platelet-rich plasma (PRP) therapy involved in the healing of sports-related soft tissue injuries,Iowa Orthop J,32 (2012); T. D. Vu, et al., An autologous platelet-rich plasma hydrogel compound restores left ventricular structure, function and ameliorates adverse remodeling in a minimally invasive large animal myocardial restoration model: a translational approach: Vu and Pal “Myocardial Repair: PRP, Hydrogel and Supplements”,Biomaterials,45 (2015) 27-35 150-163; and H. S. Yang, et al., Enhanced skin wound healing by a sustained release of growth factors contained in platelet-rich plasma,Exp Mol Med,43 (2011) 622-629). There are many ways to prepare PRP though very few studies have utilized consistent methodology, wound types, or patient demographics, which has led to conflicting data regarding its efficacy. Numerous studies report that PRP improves the wound healing response (V. R. Driver, et al.,Ostomy/wound management,52 (2006) 68-70, 72, 74 passim; H. S. Yang, et al.,Exp Mol Med,43 (2011) 622-629; and M. J. Martinez-Zapata, et al., Autologous platelet-rich plasma for treating chronic wounds,The Cochrane database of systematic reviews,10 (2012) Cd006899), while numerous others found it to have no significant effect on healing outcomes (M. J. Martinez-Zapata, et al.,The Cochrane database of systematic reviews,10 (2012) Cd006899). Since each study involves a different formulation, it is difficult to determine the true potential of PRP as a wound healing therapy. As with individual growth factor therapies, the proteins found in PRP also have short half-lives, limiting their efficacy without repeated administrations (K. Lee, et al., Growth factor delivery-based tissue engineering: general approaches and a review of recent developments,Journal of the Royal Society, Interface/the Royal Society,8 (2011) 153-170 and H. S. Yang, et al.,Exp Mol Med,43 (2011) 622-629). Complex coacervates form when a cationic polymer solution is mixed with an anionic polymer solution that leads to charge neutralization and phase separation of a polymer-rich phase from the bulk water. Here, we use heparin as the anionic polymer. Many therapeutic growth factors such as VEGF, heparin-binding EGF-like growth factor (HB-EGF), and hepatocyte growth factor (HGF) have a natural affinity for heparan sulfate, a glycosaminoglycan (GAG) found in the ECM. Heparin has similar structure and functionality as heparan sulfate, with the ability to protect growth factors from proteolytic degradation and present them to cell receptors in biomimetic fashion. A synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD), interacts with the anionic heparin via polyvalent charge attraction, forming a complex coacervate containing high concentrations of the polymers while the bulk water phase has little polymer left. Thus this system loads heparin-binding growth factors with high efficiency. The final delivery system consists coacervate droplets suspended within the aqueous phase. The droplets range from 10 to 500 nm in diameter and remain stable for at least one month in vivo. Previous studies have shown that this system delivers growth factors for weeks and significantly extends their half-lives. Coacervate delivery of growth factors can improve cardiac function after myocardial infarction and accelerate wound healing, see Example 3. Here we utilize our coacervate system with PRP in a pig model of wound healing. Pig skin is very similar to human skin and thus provides an appropriate pre-clinical indication for potential success in human patients. Since PRP is widely available and inexpensive, its validation as a growth factor source for therapeutic application in a large animal model takes this technology one step closer to clinical translation. Materials and Methods PRP Preparation and Protein Quantification: Patient samples were obtained from the Central Blood Bank of Pittsburgh. Fresh plasma was obtained within four hours of collection from the patient to maintain high bioactivity of growth factors and cytokines. To isolate PRP, the plasma was then centrifuged at 2,000×g for 15 minutes, and the bottom half of the solution was taken as PRP. Thrombin (Sigma-Aldrich, St. Louis, MO) was added at 1,000 U/mL and CaCl2added at 10% (w/v) with gentle agitation to activate the platelets for one hour. Platelets released their therapeutic proteins into the surrounding plasma upon activation, and the fibrin clot was removed by centrifugation. A 3 kDa MWCO centrifugal filter unit (EMD Millipore, Billerica, MA) was used to further concentrate the proteins in solution. Total protein content was measured by Pierce 660 nm protein assay (Thermo Fisher Scientific, Waltham, MA), and individual growth factor concentrations were quantified using sandwich ELISA kits per manufacturer instructions (PeproTech, Rocky Hill, NJ). Concentrated PRP was stored at −80° C. until later use. Platelet-Derived Protein Loading and Release: The loading and release of platelet-derived proteins from the coacervate delivery system over 4 weeks in vitro was measured. Poly(ethylene argininylaspartate diglyceride) (PEAD) was synthesized as previously described (H. Chu, et al., A [polycation:heparin] complex releases growth factors with enhanced bioactivity,Journal of controlled release: official journal of the Controlled Release Society,150 (2011) 157-163), e.g., as described in Example 1. To form the coacervate, 200 uL PRP was combined with 1.6 mg heparin (porcine intestine, Scientific Protein Labs, Waunakee, WI), allowing heparin-binding proteins to bind. The coacervate then self-assembled upon the addition of 8 mg PEAD in a total volume of 800 μL. The coacervate was pelleted by centrifugation at 12,100×g for 10 minutes and initial growth factor loading was determined by measuring the concentration in the supernatant and comparing to the concentration prior to coacervate formation. Fresh 0.9% saline was added to resuspend the coacervate and samples were incubated at 37° C. At predefined time points extending to 21 days, the pelleting procedure was repeated and the supernatant collected for analysis. Fresh saline was then added to resuspend the coacervate. Animals: Two 3-month old female Yorkshire pigs were used in this study. The pigs were fed standard lab diet twice per day with unrestricted access to water and their health was monitored at least twice daily for any signs of pain or distress. Following surgeries, pigs were housed individually to avoid perturbation of the wound sites. Wounding Procedure: All surgical procedures were conducted under supervision of the Division of Laboratory Animal Resources (DLAR) at the University of Pittsburgh. Sedation was induced using an IM injection of ketamine (20 mg/kg) and xylazine (2 mg/kg) and anesthesia was maintained following intubation with 1-3% isofluorane. Twenty-two full-thickness excisional wounds were created on the back of each pig using 2 cm diameter biopsy punches to ensure consistent wound size (Shoney Scientific Inc, Waukesha, WI). The punch was driven into the fat layer underlying the dermis, and scissors were used to cut along the underlying fat and remove the skin section. Constant pressure was applied with sterile gauze to stop bleeding, using hemostatic collagen (Davol Inc, Warwick, RI) or epinephrine as needed. Once the bleeding was stopped, Avitene Ultrafoam (Davol Inc, Warwick, RI) cut to fit the wound was applied, and group-specific treatment solution was added via sterile pipet. Upon addition of these solutions the collagen foam swelled to form a gel and retained the treatments within the wound site. Collagen foams have previously been used in these healing models and are used clinically to facilitate healing (D. Brett, A Review of Collagen and Collagen-based Wound Dressings,Wounds: a compendium of clinical research and practice,20 (2008) 347-356). One of five treatments was applied to each wound: (1) saline, (2) unloaded coacervate, (3) bolus PRP, (4) full PRP coacervate, or (5) the heparin-binding fraction of PRP coacervate (HB-PRP) (n=8-10). Full PRP coacervate was formed by combining 400 μl PRP with 3.2 mg filter-sterilized heparin and 16 mg filter-sterilized PEAD. To isolate the HB-PRP coacervate, the full PRP coacervate was pelleted by centrifugation, the supernatant was aspirated and discarded, and the pellet was resuspended in fresh DI water. Wound treatments were assigned by forced randomization to account for any differences in the skin based on location, and each wound was assumed to be independent of other wounds and treatments (FIG.1). All wounds received 5 mg ciprofloxacin administered topically to prevent infection. Large Tegaderm bandages (3M, St. Paul, MN) were used to cover wound sites followed by Opsite transparent films (Smith & Nephew, London, UK) around the perimeter, forming a watertight dressing. A surgical pad sprayed with silicone-based medical adhesive (Hollister Inc, Libertyville, IL) was then applied on top of the entire wound area to protect the wounds and bandages, followed by a custom-fit jacket (Lomir Biomedical Inc, Malone, NY). Baytril (2.5 mg/kg) was administered IM once per day for seven days following surgery and amoxicillin (7 mg/kg) was administered orally twice per day for the remainder of the study to prevent infection. Carprofen (2 mg/kg) was administered for pain twice daily for five days following surgery. At days 3 and 7, the bandages were changed under brief sedation with ketamine (20 mg/kg) and xylazine (2 mg/kg). Tissue Harvesting and Processing: Ten days after wounding the animals were sacrificed with an overdose of sodium pentobarbital (100 mg/kg) administered intravenously. Wounds were photographed for analysis before explant. No signs of infection were present in any wound. The wounds were harvested along with at least 1 cm of surrounding healthy tissue at the depth of the muscle fascia. Each wound was then cut in half sagittally prior to processing. For histology measurements and immunostaining of cytokeratin, tissues were fixed in 2% (w/v) paraformaldehyde for 2 hours and then transferred to a 30% (w/v) sucrose solution for cryoprotection for 24 hours. Tissues used for immunostaining of von Willebrand Factor (vWF) remained unfixed. All tissue samples were then embedded in optimal cutting temperature (OCT) media and frozen in liquid nitrogen-cooled 2-methylbutane. Tissues were then cryosectioned at 6 um for further analysis. Measuring Overall Wound Contraction: Overall wound size was measured using images taken of the wound during the wounding procedure and after sacrifice. Wound area was measured using an automated filter and measurement macro in ImageJ and compared to the original wound area. Histology: Tissue sections were stained with hematoxylin and eosin (H&E) for gross morphology and qualitative wound healing parameters such as thickness of granulation tissue and the formation of healthy skin structures. Masson's trichrome stain (MTS) was used to determine qualitative collagen deposition and alignment within the granulation tissue. Immunostaining Tissue Sections: Immunofluorescent staining of tissue sections was used to determine the effect of each treatment on angiogenesis and reepithelialization. A rabbit polyclonal von Willebrand Factor (vWF) antibody (1:400 dilution, US Abcam, Cambridge, MA) followed by an Alexa Fluor 594 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA) was used to detect endothelial cells within the tissue. Since healing is delayed in the center of the wound, images were taken at both the wound edge and the center for quantification. The number of vWF+cells was counted automatically using NIS Elements software (Nikon, Tokyo, Japan) and is reported as blood vessels per mm2area. Reepithelialization of a wound reestablishes a functional barrier between the wound and its environment and is essential in preventing infection of the underlying tissue. Reepethelialization was quantified using a rabbit polyclonal cytokeratin antibody (1:100 dilution, US Abcam, Cambridge, MA) followed by an Alexa Fluor 594 goat anti-rabbit antibody. The length of the epidermal tongue was measured and reported as a percentage of the total wound length. All images were taken using a Nikon Eclipse Ti inverted microscope. Statistics: All parameters were tested for significant differences between treatment groups using one-way independent analysis of variance (ANOVA) followed by Gabriel's post hoc testing with a significance value p<0.05. Analysis was performed using SPSS 22.0 software. Results Coacervate system preferentially loads and releases heparin-binding growth factors: PEAD carries two positive charges per repeat unit. The polycation forms a complex coacervate when mixed with anionic heparin, visible as a turbid solution. The natural affinity between many therapeutic growth factors and heparin allows these proteins to preferentially load into the coacervate system. Although only 7% of total PRP proteins were loaded, heparin-binding VEGF and PDGF each exhibit a loading efficiency exceeding 60% (FIG.2A). These proteins exhibited a burst release in the first day, followed by a nearly linear release over the following three weeks (FIG.2B). Full PRP coacervate accelerates pig wound closure: Porcine skin, like that of humans, heals primarily by reepethelialization. Immunofluorescent detection of cytokeratin for dermal epithelial cells showed a significantly increased reepithelialization rate (35% relative to saline) 10 days after wounding compared to saline and delivery vehicle alone (FIG.3). No significant differences existed between other treatment groups. Wounds treated with full PRP coacervate also exhibited a thicker epidermis adjacent to the wound margin compared to other groups. Positive cytokeratin staining was also observed in sebaceous glands and hair follicles of healthy tissue as expected. Full PRP coacervate modulates vascular density: The vascular density of the wounds was measured at day 10 using vWF immunofluorescence to identify endothelial cells. In the wound center, vascular density was significantly higher than in healthy tissue indicating an influx of blood vessels into the wound bed, and no significant differences between treatment groups were observed (FIG.4(A,B)). However, at the wound edges, blood vessel density of wounds treated with full PRP coacervate resembled that of uninjured tissue (FIG.4(A)). Furthermore, the blood vessel density in full PRP coacervate treated wounds was significantly lower than all other treatment groups (FIG.4(A,C)). Since wounds heal more quickly at the edges, these data suggest that wounds treated with full PRP coacervate were at an advanced stage of healing compared to other treatments. PRP coacervate decreases wound size: Wound closure was also analyzed macroscopically to confirm measurements made histologically. Automated measurements of wound area showed a significant decrease in wound size after ten days compared to all other treatments (65% original wound size for full PRP coacervate treated wounds compared to 83% when treated with saline). The remaining treatments did not cause a significant reduction in wound size (FIG.5b). Further, wounds that did not receive full PRP coacervate treatment were visibly deeper and dark red in color, indicating unhealthy granulation tissue formation (FIG.5a)(J. E. Grey, et al., Wound assessment,BMJ,332 (2006) 285-288). The light pink color and raised appearance of granulation tissue seen in the full PRP coacervate wounds indicate healthy granulation. No signs of infection were present in any wound. PRP Coacervate accelerates granulation tissue maturation: Granulation tissue provides a temporary matrix for cells to infiltrate and repair the wound bed after injury. As expected, H&E staining showed high cellularity and vascularity in the granulation tissue of all wounds relative to surrounding healthy tissue (not shown). Collagen deposition by fibroblasts had begun within 10 days in all wounds as seen by MTS (not shown). Upon careful evaluation it was evident that full PRP coacervate resulted in increased deposition of aligned collagen compared to other treatment groups where the collagen fibers were more randomly oriented (FIG.6). The granulation tissue was thin in saline-treated control wounds, extending only partially to the normal skin surface. DISCUSSION The widespread use of PRP as a therapy remains highly debated in medicine. Autologous PRP is typically highly variable because of inherent differences between patients, methods of preparation, and whether it is used as a liquid or a thrombin-induced hydrogel. This leads to inconsistent results between patients and studies when used clinically as an autologous therapy. PRP activated by thrombin can be applied as a gel and has been described as a controlled release system to improve the efficacy of PRP. However, these studies have been restricted to small animal models or large animals models without sufficient characterization of protein release, and most have not been injectable (Y. Yan, et al., Acceleration of Full-thickness Wound Healing in Porcine Model by Autologous Platelet Gel, Wounds: a compendium of clinical research and practice, 19 (2007) 79-86). Provided herein is a systematic study of the effects of PRP in a porcine model and the benefits of controlled release for wound healing. Appropriate controlled delivery systems are crucial to achieve high therapeutic efficacy of growth factors. Short half-lives, adverse off-target effects, and poor spatio-temporal control are common issues of bolus application which can be solved using controlled release systems. One approach is to harness the native properties of ECM molecules which sequester growth factors, prevent their degradation and promote their bioactivity (G. S. Schultz, et al., Interactions between extracellular matrix and growth factors in wound healing,Wound Repair Regen,17 (2009) 153-162). Several different types of delivery platforms have been developed along this theme, utilizing ECM molecules vitronectin, fibronectin, and heparin. In one such platform, vitronectin complexes with insulin-like growth factor (IGF) and epidermal growth factor (EGF) demonstrated wound healing efficacy in large animals and safety in humans. In another approach, synthetic fibronectin-like peptides were developed to deliver several different heparin-binding growth factors and cytokines and evaluated in small animal studies. Our lab has characterized a heparin-based platform which takes the form of liquid coacervate droplets that load and release heparin-binding proteins (H. Chu, et al., A [polycation:heparin] complex releases growth factors with enhanced bioactivity,Journal of controlled release: official journal of the Controlled Release Society,150 (2011) 157-163 and N. R. Johnson, et al., Lysine-based polycation:heparin coacervate for controlled protein delivery, Acta biomaterialia, 10 (2014) 40-46). HB-EGF delivered by the coacervate system accelerated healing in both diabetic and non-diabetic rodent wound models (N. R. Johnson, et al., Controlled delivery of heparin-binding EGF-like growth factor yields fast and comprehensive wound healing,Journal of controlled release: official journal of the Controlled Release Society,166 (2013) 124-129 and N. R. Johnson, et al., Coacervate delivery of HB-EGF accelerates healing of type 2 diabetic wounds,Wound Repair Regen,23 (2015) 591-600). The use of multiple growth factors with the coacervate system has been explored previously to stimulate angiogenesis with VEGF and hepatocyte growth factor (HGF) (H. K. Awada, et al., Dual delivery of vascular endothelial growth factor and hepatocyte growth factor coacervate displays strong angiogenic effects, Macromolecular bioscience, 14 (2014) 679-686). The use of two growth factors exhibited a near-linear release profile over three weeks with no initial burst release. In comparison, both PDGF and VEGF from the PRP coacervate exhibit a 50% burst release within a day of coacervate formation (FIG.2b) followed by a nearly linear release profile over 3 weeks. Interactions between heparin and growth factors are primarily charge-driven. When using small doses of proteins as was done previously, this interaction has negligible effects on the PEAD:heparin charge-based interaction. However, this study utilizes high protein concentrations; as more proteins bind to a heparin molecule, fewer sulfate groups are available to bind the PEAD polycation. Thus the coacervate formed is less stable than those formed with small protein doses. This allows the coacervate to dissociate at a faster rate, which likely explains the high initial release of proteins shown here. Heparin has been used in many growth factor therapies since the heparin:growth factor complex is stable and resistant to proteolysis. Additionally, heparin potentiates the bioactivity of the proteins by facilitating their reactions with cell surface receptors. Its use in our delivery system provides the advantage of preferentially loading heparin-binding growth factors for sustained release. Although less than 10% of the total protein content of PRP was incorporated into the coacervate, heparin-binding proteins such as PDGF and VEGF exhibited a loading efficiency exceeding 60%. To our knowledge this is the first report of a PRP delivery system that is able to load and release heparin-binding growth factors in a sustained manner. We suspect that the non-heparin binding fraction of the platelet released factors provide important acute signaling for healing the tissue. Therefore, we tested the “full PRP coacervate” along with the heparin-binding fraction only, namely the “HB-PRP coacervate”. Prior studies utilizing heparin have largely involved its covalent immobilization to the surface of a polymeric scaffold or within a hydrogel, thereby endowing the biomaterial with growth factor affinity (S. E. Sakiyama-Elbert, Incorporation of heparin into biomaterials,Acta Biomater,10 (2014) 1581-1587). However, covalent linkage may partially or fully inhibit the activity of heparin, reducing growth factor loading efficiency or creating a steric selectivity for heparin-binding protein of certain sizes or conformations. The coacervate platform employs heparin in free-form which maintains its native functionality and consistently provides high loading capacity. Furthermore, the heparin-binding complexes are mobile and able to interact with cell surface receptors and thereby potentiate growth factor bioactivity. In comparison, immobilized heparin may hold growth factors to a surface or deep within a scaffold that cells must infiltrate to access. A system using free heparin amphiphiles has also been reported, but their growth factor release has not been reported beyond 10 days (S. S. Lee, et al., Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds,Biomaterials,34 (2013) 452-459 and K. Rajangam, et al., Heparin binding nanostructures to promote growth of blood vessels, Nano Lett, 6 (2006) 2086-2090). The advantages of the coacervate system are owed to the unique liquid-liquid phase separated structure held together by electrostatic interactions of heparin and the novel cationic polymer PEAD. A porcine model of full-thickness excisional wounds is one of the best pre-clinical wound healing models as pig skin heals by similar mechanisms to human patients. Here we demonstrate that PRP itself is unable to improve wound healing; however the controlled release of its therapeutic proteins can accelerate the wound healing process. Wound vascularization and collagen fiber deposition and alignment in the granulation tissue indicated improved wound maturation in full PRP coacervate-treated wounds. Additionally, smaller gross wound size and enhanced reepithelialization shows that wound closure was also accelerated. Furthermore, we found that removal of non-heparin-binding proteins from PRP after coacervate formation reduced its efficacy. Interestingly, these data suggest that the non-heparin-binding PRP fraction plays a vital role in the healing response. The use of full PRP coacervate essentially creates two stages of release: an initial diffusion of all unincorporated PRP proteins followed by a sustained release of the heparin-binding growth factors. Sequential release of individual growth factors by the coacervate has been demonstrated to improve the therapeutic efficacy (H. K. Awada, et al., Sequential delivery of angiogenic growth factors improves revascularization and heart function after myocardial infarction,Journal of controlled release: official journal of the Controlled Release Society,207 (2015) 7-17 and H. K. Awada, et al. Factorial Design of Experiments to Optimize Multiple Protein Delivery for Cardiac Repair,ACS Biomaterials Science&Engineering,2 (2016) 879-886). The coacervate creates an analogous situation in the present study where high initial concentrations of unincorporated proteins induce a strong acute response followed by the sustained release of heparin-binding proteins which enact long-term effects. The removal of unincorporated proteins (as in the HB-PRP coacerate) reduces efficacy which could be due to a weaker acute response. Angiogenesis is a vital component of the wound healing process. The formation of leaky capillaries allows cells and proteins to infiltrate the damaged tissue and begin healing. A high capillary density was observed in the center of all wounds assessed in this study, indicating the normal angiogenic process was not significantly disturbed. Since wounds heal from the edge, granulation tissue near the wound edge matures faster than the center of the wound where granulation tissue forms last. Therefore we are able to see a progression of the angiogenic process moving from the edge to the interior of the wound. The vessel density of all treatment groups was lower at the wound perimeter compared to the center, indicating that the wounds had progressed through the peak of angiogenesis and were returning to the levels of native skin. The difference arises in wounds treated with full PRP coacervate, where vessel density is similar to other wounds at the center but is significantly lower at the wound margin. This suggests that controlled PRP release accelerates the angiogenic phase of healing, returning vessel density to near-normal levels at a faster rate and exhibiting a more advanced stage of healing. This has significant relevance when comorbidities are present such as peripheral arterial disease or diabetes. This study demonstrates the therapeutic benefits of controlled PRP release for wound healing applications. A quick and simple assembly method allows adaptability for either rapid autologous preparation at the bedside or an allogenic sourced off-the-shelf product. The liquid nature of our coacervate system allows it to be injected directly into the wound bed or incorporated into a substrate for application. The controlled release mechanism allows treatment to be applied once every few weeks rather than daily as seen in traditional protein therapies. Given the ability of this therapy to capture many proteins, this study suggests that the coacervate vehicle can be adapted to other therapeutic applications in large animal models. In conclusion, this Example evaluated the efficacy of traditional PRP treatment and the advantage of a controlled release PRP formulation on cutaneous wound healing. It is shown that the sustained release of PRP proteins is able to significantly improve wound closure within 10 days of wounding, while the clinical standard of naked PRP proteins demonstrate no significant benefit. This is accompanied by a significant alteration in vascularity of the wound edge, returning vessel density to near-healthy levels. These results suggest that the controlled delivery of PRP proteins using the coacervate vehicle encourages accelerated healing of cutaneous wounds in a porcine model. The widespread use of PRP in humans combined with the clinical relevance of the porcine wound healing model emphasize the impact of this study on the translational potential to enhance autologous procedures with an easy-to-use protein delivery platform. Example 3—Functional Recovery of the Infarcted Myocardium by a Single Injection of Three Proteins After a heart attack, the infarcted myocardium undergoes pathological remodeling instead of repair and regeneration. Protein signaling plays a pivotal role in tissue regeneration. With multiple pathologies developing after myocardial infarction (MI), treatment using several complementary proteins is expected to address these range of pathologies more effectively than a single-agent therapy. Three complementary factors are combined in one injection: tissue inhibitor of metalloproteinases 3 (TIMP-3) was embedded in a fibrin gel for signaling in the initial phase of the treatment, while basic fibroblast growth factor (FGF-2) and stromal cell-derived factor 1 alpha (SDF-1α) were embedded in a heparin-based coacervate and distributed within the same fibrin gel to exert their effects over a longer period of time. The spatiotemporally controlled release of these proteins counters excessive inflammation, extracellular matrix (ECM) degradation, and cell death post MI in rats. The contractility of the treated hearts stabilizes and slightly improves after a drop in the first two weeks whereas all the controls kept deteriorating. Accompanying the functional restoration are reductions in dilation, inflammation, fibrosis, and ECM degradation. Revascularization, cardiomyocyte survival, stem cell homing, and preservation of myocardial strain levels likely all contribute to the repair. This study demonstrates the potential of this multifactorial therapeutic approach in MI. Myocardial infarction (MI) affects 7.6 million Americans with approximately 720,000 experiencing a heart attack each year. MI leads to defects in the contractile function of cardiomyocytes and alterations in the extracellular matrix (ECM) and ventricle geometry. As a consequence of the maladaptation, a non-contracting scar tissue forms, a significant portion of which results in congestive heart failure. Current treatments such as reperfusion, β-blockers, and angiotensin converting enzyme (ACE) inhibitors, reduce damage but do not restore function. Therefore, therapies that can prevent or reverse the multiple pathologies caused by MI, regenerate the myocardium, and restore cardiac function are urgently needed. To treat multiple pathologies resulted from MI, we set out to explore the use of multiple therapeutic proteins. Our recent study using a statistical fractional factorial design of experiment focused our effort into the controlled and timed release of a combination of complementary proteins that are relatively distinct in their roles in cardiac function: tissue inhibitor of metalloproteinases 3 (TIMP-3), basic fibroblast growth factor (FGF-2), and stromal cell-derived factor 1 alpha (SDF-1α) (H. K. Awada, et al., Factorial Design of Experiments to Optimize Multiple Protein Delivery for Cardiac Repair.ACS Biomaterials Science&Engineering2, 879-886 (2016). TIMP-3 inhibits the activity of matrix metalloproteinases (MMPs) which cleave ECM components. FGF-2 plays a chief role in formation of neovasculature. SDF-1α is a potent chemotactic factor that can recruit stem cells to the infarct region. TIMP-3 reduces ECM degradation soon after MI. FGF-2 and SDF-1α promote angiogenesis and recruit progenitor cells to the infarct region, which are events that require prolonged signaling. We designed a composite hydrogel comprised of fibrin gel and heparin-based coacervates to achieve the sequential release of TIMP-3 followed by FGF-2 and SDF-1α. To achieve this controlled release, TIMP-3 was encapsulated in fibrin gel to offer early release, while FGF-2 and SDF-1α were encapsulated in heparin-based coacervates and distributed in the same fibrin gel to offer sustained release (FIG.1A). Complex coacervates form spontaneously by electrostatic interactions between the aqueous solutions of a polycation and a polyanion. A synthetic polycation poly(ethylene argininylaspartate diglyceride) (PEAD), heparin, and heparin-binding proteins were used to form protein-loaded coacervates that have been shown to encapsulate proteins with high efficiency and sustain their release in vivo and in vitro. In this study, the efficacy of the spatiotemporal delivery of TIMP-3, FGF-2, and SDF-1α on cardiac function, ventricular dilation and wall thinning, myocardial strain levels, MMP activity, fibrosis, inflammation, cardiomyocyte survival, angiogenesis, stem cell homing, protein signaling, and cell apoptosis. The first report of controlled delivery of complementary proteins mitigates the MI injury and initiates a robust cardiac repair process, giving hope of a higher level of functional and structural recovery of the infarcted heart. Results Sequential protein release: ability of a fibrin gel-coacervate composite to release TIMP-3 early followed by a sustained release of FGF-2 and SDF-1α by an in vitro release assay was tested (FIG.7(A)). The loading efficiencies were 85% for TIMP-3, 97% for FGF-2, and 98% for SDF-1α (FIG.8). By day one, approximately 40% of loaded TIMP-3 was released, reaching 90% total release by one week (FIG.8) translating into relatively higher concentrations of TIMP-3 reaching a maximum of 1 ng/ml during the first week and decreasing thereafter (FIG.7(B)). We observed a longer sustained release for FGF-2 and SDF-1α with concentrations between 0.25-0.5 ng/ml that lasted for >six weeks due to their encapsulation within the coacervates inside the gel (FIG.7(B)). By one week, only 21% of FGF-2 and 28% of SDF-1α were released, reaching 55% and 48% total release respectively by six weeks (FIG.8). Thus the composite coacervate gel achieved quick release of TIMP-3 after MI to reduce ECM degradation and inflammation, while providing FGF-2 and SDF-1α in a sustained manner for triggering a robust neovasculature formation process and stem cell recruitment. Improved cardiac function and reduced ventricular dilation: The effect of spatiotemporal delivery of TIMP-3, FGF-2, and SDF-1α was evaluated in a rat MI model using sham, saline, and free proteins as controls. We evaluated changes in left ventricle (LV) contractility as a measure of heart function was evaluated. Using echocardiography, fractional area change (FAC) was computed from end-systolic area (ESA) and end diastolic area (EDA) values (FIG.9(A)). Sham group maintained an FAC value of approximately 55% at all time points post-MI, significantly higher than all infarct groups (FIG.9(B)). 1-week post-infarction, FAC values of saline, free protein, and controlled release (CR) groups dropped significantly, however, both CR and free proteins had significantly higher FAC than saline (p<0.01). This suggests that the three-proteins significantly improved cardiac function within one week after MI. At two weeks, CR group diverged from the negative controls and improved function significantly (p<0.001). Although free proteins was significantly better than saline (p<0.001) up to five weeks, function in both groups kept dropping. In contrast, functional improvement of CR group continued and displayed increasingly larger differences relative to the controls. At eight weeks, the last time point of the study, CR led to a 48% FAC, which was 87% that of the normal FAC value and represented a 74% improvement over saline. The two control groups, saline and free proteins, no longer showed any statistical difference at eight weeks (p>0.05) (FIG.9(B)). To evaluate the therapy's effect on ventricular dilation, changes in EDA and ESA values were assessed. The CR group showed significant reduction or trend towards lower EDA and ESA after MI compared to saline (FIG.9(C,D)). On the other hand, saline and free protein groups showed progressively higher ESA and EDA at all time points after MI, with no statistical differences between them (p>0.05) (FIG.9(C,D)). The echocardiography results were consistent with MRI measurement at eight weeks. End-systolic volume (ESV) and end-diastolic volume (EDV) were computed and ejection fraction (EF) was calculated (FIG.9(E)). EF in CR group was 58%, which was at 84% of the sham group (69%) and significantly higher (p<0.001) than saline (41%) and free proteins (46%). The two negative controls showed no difference between each other (p>0.05) (FIG.9(F)). Correspondingly, the left ventricle of the CR group was less dilated with a significantly smaller ESV than saline (p<0.01) and not significantly different from sham (FIG.9(G)). CR also showed a trend towards lower EDV compared with saline and free protein groups (FIG.9(H)). Preserved myocardial elasticity: Myocardial strain analysis at eight weeks post-MI to evaluate the changes in the radial strain levels of the myocardium with respect to the various treatments by normalizing the peak strain of the infarcted region to the average peak strain in four non-infarct regions (FIG.10A). The radial strain, defined as the percent change in myocardial wall thickness, was measured. The CR group exhibited a radial myocardial strain similar to that of sham control (p>0.05), and was significantly higher than the saline group (p<0.01) (FIG.10B). The free proteins group is similar to saline (p>0.05) and significantly less than sham (p<0.05)(FIG.10B). This result suggests that controlled delivery of TIMP-3, FGF-2, and SDF-1α protects myocardial elasticity after MI. The prevention of ventricular wall stiffening helps to maintain the heart's ability to contract and dilate properly. Reduced left ventricle wall thinning, MMP activity, and fibrosis: In order to understand tissue level changes that contributed to the functional improvement, ventricular wall thickness, MMP activity, and fibrosis were investigated at two and eight weeks. H&E stained hearts showed increased granulated scar tissue areas with thinner left ventricle walls in the infarct zone and borderzone that exacerbated with time in saline and free proteins groups but to a less extent in CR group (FIG.11(A)). CR significantly prevented ventricular wall thinning at two weeks compared to saline (p<0.05) (FIG.11(B)). In contrast, left ventricle wall thickness decreased considerably in saline and free proteins groups as early as two weeks. At eight weeks, there were no statistical differences in wall thickness between saline, free proteins, and CR; although CR clearly maintained a thicker wall average (FIG.11(B)). CR wall thickness was not different from sham at both time points (p>0.05). At eight weeks, we evaluated the activity of MMPs in the heart samples. MMP-2 andMMP-9 are important players implicated in many cardiovascular diseases and ECM degradation (T. Etoh, et al., Myocardial and interstitial matrix metalloproteinase activity after acute myocardial infarction in pigs.American journal of physiology. Heart and circulatory physiology281, H987-994 (2001)). All infarct groups showed a high level of MMP-2/9 activity (FIG.11(C)). However, CR showed significantly lower MMP activity compared to saline (p<0.01) and also lower activity than free proteins group but not to a significant level (p>0.05) (FIG.11(C)). MMP activity in CR was not statistically different from sham (p>0.05). The enhanced reduction of MMP activity by the CR group is likely due to the controlled delivery of TIMP-3 within the fibrin gel-coacervate composite, where TIMP-3 can form tight complexes with MMP-2 and MMP-9 to prevent their activation, and thereby reducing ECM degradation and ventricular dilation and remodeling. Interstitial fibrosis develops at the infarct region and extends to non-infarct areas due to the excessive and uncontrollable collagen deposition that takes place in later stages after MI. This increased collagen deposition leads to increased stiffness in the myocardium, leading to contractile dysfunction. The extent of fibrosis was assessed using picrosirius red staining which stains collagen fibers (FIG.11(D)). The saline group, and to a lesser degree the free proteins group, showed extensive amount of fibrosis that extended from the infarct to non-infarct regions, while CR showed far less fibrosis that seemed limited to the infarct area at two weeks (FIG.12) and at eight weeks (FIG.11(D)). Collagen deposition was quantified as a positive fraction of the heart area and no statistical differences were found between the infarct groups at two weeks despite a clear reduction in the CR group (p>0.05) (FIG.11(E)). At eight weeks, collagen deposition increased in all infarct groups, but it was found to be significantly less in CR (11%) compared to both saline (23%) (p<0.01) and free proteins (18%) groups (p<0.01) (FIG.11(E)). Sham had significantly less collagen than all groups at both time points. Reduced inflammation: Modulating the inflammatory response after MI in which certain harmful aspects of inflammation are prevented, can be very beneficial for the treatment of the infarcted myocardium. In this study, inflammation by co-staining for F4/80, a pan-macrophage cell surface marker, and CD163, an M2 macrophage marker (FIG.13(A)) were assessed. Non-M2 macrophages, namely M1, promote inflammation, whereas M2 macrophages contribute to tissue repair and anti-inflammation J. M. Lambert, E. F. Lopez, M. L. Lindsey, Macrophage roles following myocardial infarction.International journal of cardiology130, 147-158 (2008). At two weeks post-MI, CR showed a trend towards decreasing the presence of non-M2 macrophages, while they were present in high numbers in saline and free proteins groups (p>0.05) (FIG.13(A,B)). On the other hand, CR significantly increased the presence of the beneficial M2 macrophages compared saline (p<0.01) (FIG.13(A,B)). Saline and free proteins showed no statistical differences in their M2 macrophage numbers (p>0.05) (FIG.13(A,B)). The sham control showed minimal presence of macrophages. The effect of the treatment on the secretion of pro-inflammatory cytokines was then investigated. Tissue lysates were tested at eight weeks for interleukin 1β (IL-1β), interleukin 6 (IL-6), and tissue necrosis factor α (TNF-α) (FIG.14). Quantitative analysis by ELISA showed significantly lower levels of IL-1β in CR and free proteins groups (p<0.05) compared to saline (FIG.14(A)). There was no statistical differences in the levels of IL-6 between the groups (p>0.05) (FIG.14(B)). Finally, CR significantly reduced the levels of TNF-α compared to saline and free proteins (p<0.01), while the free proteins group was statistically indifferent to saline (p>0.05) (FIG.14(C)). These results indicate the efficacy of the controlled release of TIMP-3, FGF-2, and SDF-1α at reducing the detrimental effects of an excessive inflammatory environment post-MI and at promoting tissue healing through polarization toward M2 macrophages. Increased cardiomyocyte survival and reduced apoptosis: The viability of the cardiac muscle is crucial for the proper function of the heart. Cardiomyocytes are responsible for imparting proper and synchronized contraction of the heart. As MI and the pathologies developed afterward trigger massive death of cardiomyocytes, it is beneficial to increase their survival, prevent their apoptosis, and trigger the regeneration of a viable myocardium. A major loss of viable myocardium was observed in the saline group, followed by the free proteins group, then by the CR group that apparently preserved the live cardiomyocytes to a larger extent at two weeks (FIG.15) and at eight weeks (FIG.16(A)). Quantitative analysis of the area fraction of the viable cardiac muscle demonstrated a reduction in the amount of survived cardiomyocytes in all infarct groups at two weeks, with no statistical differences between them (p>0.05) (FIG.16(B)). At eight weeks, the viability of the cardiac muscle was reduced more in the saline group (64% viable muscle), followed by the free proteins group (75%) with no significant differences between them (p>0.05). In contrast, CR was able to maintain the survival of the cardiac muscle (83%) significantly better than saline at eightweeks (p<0.01) (FIG.16(B)). A number of molecular pathways play important roles in promoting survival or inducing apoptosis of cells. The activated (phosphorylated) MAPK/ERK and Akt pathways have been shown to be cardioprotective after ischemia and preventive of apoptosis (A. Kis, et al., Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase.Journal of molecular and cellular cardiology35, 1063-1071 (2003); A. Tsang, et al., Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circulation research 95, 230-232 (2004); and Y. Wang, Mitogen-activated protein kinases in heart development and diseases. Circulation 116, 1413-1423 (2007)). To analyze the effect of our treatment, we quantified the expression levels of cleaved caspase-3, a pro-apoptosis mediator, and pro-survival markers p-ERK1/2 and p-Akt at 8 weeks by western blotting (FIG.16(C)). Among infarcted animals, the CR group had the lowest level of cleaved caspase-3 and the highest levels of p-ERK1/2 and p-Akt (FIG.16(C)). The free proteins group displayed significantly higher p-ERK1/2 expression than saline (p<0.01) (FIG.16(D)). However, CR significantly reduced the expression of cleaved caspase-3 and increased the expression of p-ERK1/2 and p-Akt compared to both saline (p<0.001) and free proteins (p<0.01) groups (FIG.16(D,E,F)). CR was statistically indifferent to sham in all three cases. Taken together, these results demonstrate the effectiveness of the approach described herein at supporting the long-term survival of cardiomyocytes, preventing their apoptosis, and providing overall cardioprotection after MI through activation of the Akt and ERK1/2 signaling pathways and the suppression of caspase-3 mediated apoptosis. Enhanced angiogenesis: The revascularization of the ischemic myocardium is key to tissue regeneration and functional recovery. New blood vessel formation can help restore the blood, nutrient, and oxygen flow to the damaged myocardial regions, and thereby enhance the survival of cardiomyocytes, and reduce the risk of chronic heart failure. To investigate the process of angiogenesis, tissue slices were co-stained for vWF and α-SMA at two weeks (FIG.17) and eight weeks (FIG.18(A)). Angiogenesis was evaluated only in the infarct groups, and not in sham since angiogenesis happens after infarction and not in healthy hearts. A higher number of neovessels was observed in the CR group compared to saline and free proteins (FIG.18(A)). Quantitative analysis of infarct groups showed significantly higher number of vWF-positive vessels in CR compared to saline at two weeks (p<0.05) (FIG.18(B)). At eight weeks, CR showed a significantly higher number of vWF-positive vessels than both saline and free proteins groups (p<0.01) (FIG.18(B)). Co-localization of vWF and α-SMA was used as markers of mature neovessels, and no significant differences was found among the infarct groups at two weeks (p>0.05) (FIG.18(C)). However, at eight weeks, CR showed significantly higher presence of mature neovessels than saline and free proteins groups (p<0.001) (FIG.18(C)). Our results demonstrate the ability of our treatment to induce robust angiogenesis with stable and mature neovasculature. This enhanced revascularization in the CR group is likely due to the sustained presence of the potent angiogenic factor FGF-2 being provided by the heparin-based coacervate within our composite gel. Greater stem cell homing to the myocardium: Stem cells recruited to the infarcted myocardium have the potential to differentiate into functional cells of cardiac lineages such as cardiomyocytes, vascular endothelial, and mural cells. Stem cells can also impart beneficial paracrine effects that activate repair and regeneration signaling (K. Malliaras, et al., Cardiomyocyte proliferation vs progenitor cells in myocardial regeneration: The debate continues.Global cardiology science&practice2013, 303-315). To examine the homing of stem cells to the infarcted myocardium, c-Kit, a stem cell marker was stained for (FIG.19A). At eight weeks after MI, saline and free proteins groups showed no significant differences in the number of c-Kit-positive cells present at the borderzone (p>0.05) (FIG.19B). In contrast, the CR group showed a significantly greater presence of c-Kit-positive cells at the borderzone compared to both saline and free proteins groups (p<0.01) (FIG.19B). The sham control showed very few stem cells in the area where an infarct would have been induced, suggesting their limited presence in absence of an MI injury. These results indicate the efficacy of the controlled release approach at recruiting stem cells to the infarct region to potentially contribute in the repair and regeneration of the myocardium. The enhanced and long-term presence of stem cells in the CR group is likely due to the sustained availability of the powerful chemoattractant SDF-la within the composite gel. Secretion of key signaling proteins: Certain proteins are involved in triggering cardiac repair mechanisms and others are implicated in advancing pathological changes post infarction. Therefore, regulation of the secretion levels of such proteins represents an important aspect of effective therapies. The presence of proteins such as the ones in the complementary combination, TIMP-3, FGF-2, and SDF-1α, described herein likely affect the signaling and secretions levels of other proteins in the heart after MI. To investigate the effect of our treatment on the levels of relevant proteins, tissue lysates were tested for the levels of insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), sonic hedgehog (Shh), and transforming growth factor-β1 (TGF-β1) at eight weeks (FIG.20). Quantitative analysis by ELISA showed significantly higher levels of IGF-I, an anti-apoptotic factor, in CR (p<0.001) and free proteins (p<0.01) groups compared to saline (FIG.20(A)). Moreover, CR significantly increased the levels of VEGF, a potent angiogenic factor, and Shh, a master cardiac morphogen, over saline (p<0.05), while the free proteins group was statistically indifferent to saline (p>0.05) (FIG.20(B,C)). Lastly, CR significantly decreased the levels of TGF-β1, a pro-fibrotic factor, compared to saline (p<0.001) and free proteins (p<0.05) groups (FIG.20(D)). The free proteins group also significantly decreased the levels of TGF-β(p<0.05) (FIG.20(D)). MI results in multiple pathologies and maladaptive remodeling of the heart. Numerous efforts toward cardiac repair and regeneration are underway. Stem cell-related technology can provide new cardiomyocytes via direct reprogramming and paracrine signaling via cell injection. Proteins and nucleic acids can alter the composition of local signaling molecules and enhance repair and regeneration (H. B. Sager, et al., RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction.Science Translational Medicine8, 342ra380-342ra380 (2016). Tissue-engineered patches can combine cells, growth factors, and mechanical signal to provide comprehensive cues to restore structure and functions of the heart (B. M. Ogle, et al., Distilling complexity to advance cardiac tissue engineering.Science Translational Medicine8, 342ps313-342ps313 (2016)). Proper spatial and temporal signals of proteins can benefit all 3 approaches (B. M. Ogle, et al.,Science Translational Medicine8, 342ps313-342ps313 (2016)). In the present example, the concept of sequential delivery of TIMP-3, FGF-2, and SDF-1α on countering the multitude of pathologies post-MI was explored in a rat model. These three proteins impart significant benefit on cardiac function after MI. The results, presented herein, demonstrate the ability of the fibrin gel-coacervate composite to provide early release of TIMP-3 by one week, followed by a sustained release of FGF-2 and SDF-1α that lasted at least six weeks. The efficacy of spatiotemporal release of TIMP-3, FGF-2, and SDF-1α from the fibrin gel-coacervate composite was tested in a rat MI model and was compared to sham, saline, and free proteins groups. The CR group's significant potential to improve cardiac function and trigger repair mechanisms after infarction was demonstrated bringing it close to the normal case of the sham control in many evaluations. In most cases, CR showed significant differences compared to saline group, and to free proteins group in many cases. The free-proteins group, although showing some potential and trends of improvement in different evaluations, was not able to induce significant repair as CR did compared to saline. This was indicative of the importance of controlled and timed release of TIMP-3, FGF-2, and SDF-1α. Many protein therapies fail to prove long-term efficacy for MI treatment because of the shortcomings of proteins applied in free form, including very short-half lives, low retention at the target site, high doses required, and lack of spatiotemporal cues (P. Tayalia, et al., Controlled growth factor delivery for tissue engineering.Advanced materials21, 3269-3285 (2009)). A recent study concluded that bolus injections of a cocktail of four important proteins: FGF-2, SDF-1α, IGF-I, and hepatocyte growth factor (HGF), did not improve cardiac function, reduce infarct size, or promote stable microvasculature (H. Hwang, et al., The combined administration of multiple soluble factors in the repair of chronically infarcted rat myocardium.Journal of cardiovascular pharmacology57, 282-286 (2011)). The study's results might be attributed to the absence of controlled release because without properly protecting the therapeutic proteins and delivering them spatiotemporally, a therapy might prove ineffective at cardiac repair. The delivery approach provided herein offers a solution to these challenges, by protecting the proteins within the fibrin gel-coacervate composite, localizing their presence at target tissue, and releasing them spatiotemporally. The CR group significantly improved the heart contractile function as early as one week after MI and lasted up to eight weeks in comparison to saline and free proteins groups, which had the cardiac function continuously drop over the period tested, measured by echocardiography and further confirmed by cardiac MRI at eight weeks. A cardiac function improvement of 60-75% above non-treated infarcted hearts is reported. This effectively reduced the risk of MI progressing to heart failure. Significant reductions in ventricular dilation, ventricular wall thinning, myocardial stiffness, and MMP activity. These assessments are interrelated and linked to adverse remodeling and early ECM degradation. The reductions we show in these evaluations might be attributed to the vital role of early TIMP-3 release from the delivery system. TIMP-3 is an ECM-bound enzyme that forms tight non-covalent and stable complexes with the non-activated latent form of MMPs (pro-MMP), blocking the MMP's catalytic domain and preventing its access to substrates (R. Visse, et al., Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry.Circulation research92, 827-839 (2003); M. D. Sternlicht, et al., How matrix metalloproteinases regulate cell behavior.Annual review of cell and developmental biology17, 463-516 (2001); and H. Yu, et al., TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix. The Journal of biological chemistry 275, 31226-31232 (2000)). This effectively inhibits activation of MMPs, responsible for cleaving and hydrolyzing many components of the ECM including elastin, fibronectin, collagen, and proteoglycans (R. Visse, et al.,Circulation research92, 827-839 (2003); M. D. Sternlicht, et al.,Annual review of cell and developmental biology17, 463-516 (2001); and T. H. Vu, et al. Matrix metalloproteinases: effectors of development and normal physiology.Genes&development14, 2123-2133 (2000)). This feature of TIMP-3, being able to reduce ECM degradation, likely contributed to mitigating LV adverse remodeling, wall thinning, and dilation; thereby reducing the risk of cardiac rupture and contractile dysfunction. Other studies have shown the importance of TIMP-3 in cardiac diseases. Deficiency in TIMP-3 has been reported to lead to cardiac dilation, dysfunction, rupture, and mortality. Cell-based TIMP-3 gene delivery improved heart function and reduced cardiac expression and activity of MMP-2 and -9 (H. Tian, et al. Inhibiting matrix metalloproteinase by cell-based timp-3 gene transfer effectively treats acute and chronic ischemic cardiomyopathy.Cell transplantation21, 1039-1053 (2012)). TIMP-3 delivered by collagen or hyaluronic gels was able to improve ejection fraction and reduce ventricular dilation and infarct size in rat and pig models (S. R. Eckhouse, et al., Local hydrogel release of recombinant TIMP-3 attenuates adverse left ventricular remodeling after experimental myocardial infarction.Science translational medicine6, 223ra221 (2014) and A. Uchinaka, et al., Tissue inhibitor of metalloproteinase-1 and -3 improves cardiac function in an ischemic cardiomyopathy model rat.Tissue engineering. Part A20, 3073-3084 (2014)). The unregulated and excessive collagen deposition in the infarct, and later non-infarct regions, leads to interstitial fibrosis that increases myocardial stiffness and risk of contractile dysfunction. Fibrosis arises as a result of an imbalance in ECM structure and increased production of collagen by different cells, mainly myofibroblasts. Myofibroblasts contribute to adverse remodeling and are heavily influenced by the signaling of pro-fibrotic factors such as TGF-β. In the present example, it is demonstrated that the spatiotemporal delivery of TIMP-3, FGF-2, and SDF-1α prevented the development of interstitial fibrosis and the expansion of scar and granulation tissue to a large extent. Our CR group proved very effective at decreasing the levels of TGF-β1, a main promoter of fibrosis post-infarction. Therapies that aimed to antagonize TGF-β and reduce fibrosis proved beneficial for the heart recovery (K. E. Porter, et al., Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA.Cardiovascular research61, 745-755 (2004); Y. Sun, et al., Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart.Journal of molecular and cellular cardiology30, 1559-1569 (1998); N. A. Turner, et al., Chronic beta2-adrenergic receptor stimulation increases proliferation of human cardiac fibroblasts via an autocrine mechanism.Cardiovascular research57, 784-792 (2003); C. M. Yu, et al., Effects of combination of angiotensin-converting enzyme inhibitor and angiotensin receptor antagonist on inflammatory cellular infiltration and myocardial interstitial fibrosis after acute myocardial infarction.Journal of the American College of Cardiology38, 1207-1215 (2001)). These results likely helped in the preservation of myocardial elasticity as witnessed in the CR group. Therefore, the efficacy of the spatiotemporal delivery approach at preventing the excessive deposition of fibrillary collagen reduced the risk of stiffening the ventricular wall, its loss of contractile ability, and progression to heart failure. An excessive inflammatory response can have detrimental effects after MI. Large amounts of reactive oxygen species (ROS) produced by inflammatory cells invading the infarcted myocardium can cause massive cell death. The spatiotemporal delivery approach employed herein proved effective at reducing inflammation and promoting tissue repair. The results revealed the CR's reduction of non-M2 macrophages, which contain M1 macrophages that exacerbate inflammation and ECM degradation. An increase in M2 macrophages, which contribute to reconstruction of the ECM and anti-inflammatory effects is also reported herein. TIMP-3, provided by this delivery approach, can exert anti-inflammatory effects by inhibiting TNF-α-converting enzyme (TACE), the enzyme activator of TNF-α. TNF-α is a pro-inflammatory factor which increases in heart failure and is involved in inducing inflammatory cell invasion of the infarcted myocardium, MMP production, and cell apoptosis. It is demonstrated that the CR groups helps reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The strategy reduced the potentially deleterious impact of excessive inflammation by preventing the infiltration of harmful macrophages into the infarcted myocardium or possibly forcing a change in the phenotype of present ones to become of M2 phenotype involved in tissue repair. As MI causes the death of millions of cardiomyocytes and puts millions more at risk, it is an indispensable task to support the survival of cardiomyocytes after MI and prevent their apoptosis. CR showed remarkable ability to preserve the viability of the cardiac muscle, activate pro-survival molecular pathways ERK1/2 and Akt, inhibit apoptosis mediated by caspase-3, and increase expression of anti-apoptotic factor IGF-I. Studies have proved the important role of activating the PI3K/Akt and Ras-Raf-MEK-ERK pathways to inhibit apoptosis and provide cardioprotection (A. Kis, et al., Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase.Journal of molecular and cellular cardiology35, 1063-1071 (2003); A. Tsang, et al., Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway.Circulation research95, 230-232 (2004); Y. Wang, Mitogen-activated protein kinases in heart development and diseases. Circulation 116, 1413-1423 (2007)). The complementary proteins in the system described herein, TIMP-3, FGF-2, and SDF-1α have all been reported to prevent cardiomyocyte apoptosis. Moreover, CR induced higher secretions levels of IGF-I and Shh. IGF-I is a well-studied potent cardioprotective and anti-apoptotic factor that activates the PI3K/Akt pathway and prevents cardiomyocyte apoptosis. Shh also reduces cardiomyocyte apoptosis through increased expression of pro-survival markers and reduced expression of apoptotic markers, as we and other groups have shown. In addition, the CR group improved revascularization of the infarcted myocardium, triggering a robust angiogenesis process that led to the formation of mature neovessels with potential of participating in blood flow and perfusion. The triggers behind the formation of mature neovasculature in the borderzones of the infarct region can be linked mainly to FGF-2 and, to a lesser degree SDF-1α, present in the described protein combination and delivered in a sustained manner by the coacervate. As a strong angiogenic factor, FGF-2 induces endothelial cell proliferation and sprouting leading to the formation of tube-like structures that evolve into neovessels with lumens. Protein signaling results shows that the delivery group upregulates VEGF expression, which is an endothelial-specific factor that is important for angiogenesis and vasodilation All these indications support our finding that spatiotemporal delivery of TIMP-3, FGF-2, and SDF-1α leads to the formation of new, mature, and stable blood vessels, necessary to the repair of MI injury. Stem cell recruitment to the infarct region is another important aspect of an effective therapy because of the potential of stem cells to ultimately differentiate into cardiac and vascular cells and/or support the repair by paracrine signaling, thereby supporting the survival of remaining cardiomyocytes and regeneration of a viable myocardium that replaces the lost damaged one. Our CR group showed significant ability at homing stem cells to the borderzones of the infarct. This is likely due to the sustained bioavailability of SDF-1α provided by the coacervate within our composite. SDF-1α is a powerful chemoattractant that can mobilize different types of progenitor cells such as endothelial progenitor cells (EPCs), hematopoietic stem cells (HSC), mesenchymal stem cells (MSCs), and cardiac stem cells (CSCs) to the infarcted myocardium There are some limitations to this study. Although an ischemia-reperfusion model would have been more clinically-relevant, a permanent ligation MI model was used in the rats of this study in order to induce more severe damages to the myocardium, thus enabling the detection of bigger differences between comparison groups due to different treatments. Due to limited number of animals at the two-week time point, only histology experiments were performed. Important assessments such as MMP activity, although performed at eight weeks, would be evaluated at earlier time points in future experiments. Small animal models as the one employed in this study provide significant insight in protein therapy. However, a large animal model such as a pig MI model is the logical next step because adult pigs present similar anatomy, response to ischemic insult, and expansion of an infarct to humans. Another area of improvement is to achieve catheter delivery to reduce surgical invasiveness. Viscosity and gelation parameters need to be optimized so that gelation doesn't occur while the injectable material is still in the catheter, or too late after injection where the therapeutic cargo would diffuse away from the target site. Materials and Methods Rationale and study design. The approach here was to inject a fibrin-coacervate composite gel loaded with TIMP-3, FGF-2, and SDF-1α for spatiotemporal release in the infarcted hearts of rats. The effects of this therapeutic approach on cardiac function, dilation, and myocardial strain levels were assessed at different time points after MI by multimodality imaging (echocardiography, MRI). Tissue-level changes were evaluated at two and/or eight weeks using histology, immunohistochemistry, western blot, and ELISA. Power analysis. In a recent study, MRI was for assessment of cardiac function in a rat MI model at four weeks (6). From the results of this cardiac assessment, we had a standard deviation (SD) of σ=3%. Based on this value, a power analysis calculation carried out with Minitab statistical software estimates that in order to be able to detect an effect size of 6% (˜2 SD) between EF % of treatment and sham, with a significance value of 5% and a power of 80%, at least n=6 animals per group were required. The treatment assignments were randomized at the time of surgery. A total of 56 rats were used in this study for four groups: Sham, saline, free proteins, and CR with evaluations performed at two weeks (n=17 rats; 4-5 per group) and eight weeks (n=39; 9-10 per group). Sample sizes for each experimental measurement are provided in the figure legend or text, as appropriate. Release assay of complementary proteins: PEAD was synthesized as previously described (H. Chu, et al., Design, synthesis, and biocompatibility of an arginine-based polyester.Biotechnology progress28, 257-264 (2012)). The release assay was performed as previously described and further detailed herein (H. K. Awada, et al. Sequential delivery of angiogenic growth factors improves revascularization and heart function after myocardial infarction.Journal of controlled release: official journal of the Controlled Release Society207, 7-17 (2015)). Rat acute MI model: The induction of MI was performed as previously described (H. K. Awada, et al., Factorial Design of Experiments to Optimize Multiple Protein Delivery for Cardiac Repair.ACS Biomaterials Science&Engineering2, 879-886 (2016); H. K. Awada, et al.Journal of controlled release: official journal of the Controlled Release Society207, 7-17 (2015)) and further detailed herein. Four groups (n=56 rats) were evaluated: sham, saline, free proteins, and CR. Empty vehicle (empty fibrin gel-coacervate composite) was not tested as a control in this study as it has shown no difference to saline in our previous work (H. K. Awada, et al.Journal of controlled release: official journal of the Controlled Release Society207, 7-17 (2015)). Echocardiography and cardiac MRI Echocardiography and cardiac MRI were performed to compute ESA, EDA, FAC, ESV, EDV, and EF as previously described (6, 19) and further detailed in Supplementary Methods. Myocardial strain level measurements. The B-mode frames of LV short-axis view acquired at eight weeks post-MI were analyzed (n=5 rats per group) using a strain analysis algorithm (VevoStrain™, Vevo2100). Five regions of interest (ROI) were selected along the LV mid-wall including one ROI in the anterior lateral (infarcted area) and four ROIs in the anterior medial, septal, posterior, posterior lateral (unaffected areas) walls of the LV. The peak strain in the infarcted area was normalized to the average peak strains of the four ROIs in unaffected LV walls during full cardiac cycles. The radial strain, defined as the percent change in myocardial wall thickness, was computed. Histology: At two weeks (n=4-5 per group) and eight weeks (n=5-7 per group) post-infarction, rats were sacrificed by injecting 2 ml of saturated potassium chloride (KCl) solution (Sigma Aldrich, St. Louis, MO) in the LV to arrest the heart in diastole. Hearts were harvested, fixed in 2% paraformaldehyde (fisher Scientific, Fair Lawn, NJ) for 1-2 hours, deposited in 30% sucrose solution (w/v) overnight, frozen in O.C.T compound (Fisher Healthcare, Houston, TX), and stored at −20° C. Specimens were cryosectioned at 8 μm thickness from apex to the ligation level with 500 μm intervals. Hematoxylin and eosin (H&E) staining was performed for general evaluation. H&E stained slides were selected and the ventricular wall thickness in the infarct zone (n=3-4 per group at two wks, n=4-6 at eight wks) was measured near the mid-section level of the infarct tissue using NIS Elements AR imaging software (Nikon Instruments, Melville, NY). For assessment of interstitial fibrosis, picrosirius red staining was used to stain collagen fibers and image under polarized light. The fraction area of collagen deposition in the cross-sectional area of the whole heart was measured by NIS software near the mid-section level of the infarct tissue (n=3-5 per group at two wks, n=4-7 at eight wks). An object count tool was used to include RGB pixels specific to the stained collagen fibers in the heart area by defining a proper threshold value. Immunohistochemistry: For evaluation of inflammation, a rabbit polyclonal antibody F4/80 (1:100, Santa Cruz Biotechnology, Dallas, TX), a pan-macrophage surface marker, was used followed by an Alexa fluor 594 goat anti-rabbit antibody (1:200, Invitrogen, Carlsbad, CA). Slides were also co-stained by a mouse anti-rat CD163 (1:150, Bio-Rad Laboratories, Hercules, CA), an M2 macrophage phenotype marker, followed by an Alexa fluor 488 goat anti-mouse antibody (1:200, Invitrogen, Carlsbad, CA). Slides were last counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). For quantification near the mid-section level of the infarct tissue, F4/80-positive and CD163-positive cells were counted in two opposite regions of the infarct border zone, averaged, and reported per mm2areas (n=3-4 rats per group at two wks). For evaluation of cardiac muscle viability, a rabbit polyclonal cardiac troponin I (cTnI) antibody (1:200, US Abcam, Cambridge, MA) was used followed by an Alexa fluor 488 goat anti-rabbit antibody (1:200, Invitrogen, Carlsbad, CA). Slides were counterstained with DAPI. The fraction area of viable cardiac muscle in the cross-sectional area of the whole heart was measured by NIS Elements AR software near the mid-section level of the infarct tissue (n=3-5 per group at two wks, n=5-6 at eight wks). An object count tool was used to include RGB pixels specific to the stained viable cardiac muscle in the heart area by defining a proper threshold value. For evaluation of angiogenesis, endothelial cells (ECs) were detected by a rabbit polyclonal von Willebrand factor (vWF) antibody (1:200, US Abcam, Cambridge, MA) followed by an Alexa fluor 594 goat anti-rabbit antibody (1:200). Mural cells were detected by a FITC-conjugated anti-α-smooth muscle actin (α-SMA) monoclonal antibody (1:500, Sigma Aldrich, St. Louis, MO). Slides were last counterstained with DAPI. For quantification near the mid-section level of the infarct tissue, vWF-positive vessels (defined as those with lumen) and α-SMA-positive vessels were counted in two opposite regions of the infarct border zone, averaged, and reported per mm2areas (n=3-4 rats per group at two wks, n=5-6 per group at eight wks). For evaluation of stem cell homing, stem/progenitor cells were detected by a rabbit polyclonal c-Kit antibody (1:100, Santa Cruz Biotechnology, Dallas, TX) followed by an Alexa fluor 488 goat anti-rabbit antibody (1:200). Slides were counterstained with DAPI. For quantification near the mid-section level of the infarct tissue, c-Kit-positive cells were counted in two opposite regions of the infarct border zone, averaged, and reported per mm2areas (n=5 rats per group at eight wks). Molecular markers expression by western blot: Rat hearts (n=15) were harvested and rapidly stored at −80° C. for western blotting. For protein extraction, myocardial specimens weighing approximately 100 mg were excised from the LV generating a composite material comprising a spectrum between normal, infarct, and borderzone tissue. The tissues were then homogenized at 0.2 μg/ml in a modified lysis RIPA buffer (50 mM Tris-HCl, 1% NP-40, 20 mM DTT, 150 mM NaCl, pH=7.4) with protease and phosphatase inhibitors. The complex was then centrifuged at 12,100 g for 10 min, and the supernatant was collected and stored at −80° C. until use. For total protein content, the extracts above were quantified with Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Waltham, MA). The equivalent of 100 μg protein was separated using 11.5% gel and then transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 5% BSA in TBS with 0.05% TWEEN® 20 (polysorbate 20) for 1 h, then incubated with following antibody solutions: AKT, p-AKT, ERK1/2, p-ERK1/2 (all at 1:300, Santa Cruz Biotechnology, Dallas, TX), cleaved caspase-3 (1:1,000, Cell Signaling Technology, Boston, MA), and GAPDH (1:5000, US Abcam, Cambridge, MA). The membranes were washed with TBS three times and incubated with secondary antibodies for 2 h at room temperature. Signals were visualized using the ChemiDic™ XRS+Imaging System (Bio-Rad Laboratories, Hercules, CA), and band densities were quantified using NIH ImageJ (n=3 per group). Myocardial protein secretion levels by ELISA: The tissue lysates acquired in the western blot section (n=3-4 rats per group) were used for detecting the levels of insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), sonic hedgehog (Shh), transforming growth factor-β1 (TGF-β1), interleukin 1β (IL-1β), IL-6, and tissue necrosis factor-α (TNF-α) in the LV myocardium. Sandwich ELISA kits (PeproTech, Rocky Hill, NJ) were used per the manufacturer's instructions with lysate dilutions for VEGF (1:20), IGF-I (1:50), IL-1β(1:15), IL-6 (1:15), and TNF-α (1:15). For Shh and TGF-β1, indirect ELISAs were run using rabbit polyclonal antibodies against Shh and TGF-β1 (both at 1:30, Santa Cruz Biotechnology, Dallas, TX) followed by a secondary biotinylated goat anti-rabbit IgG (1:100, Santa Cruz Biotechnology, Dallas, TX). Lysates were diluted 1:15 for Shh and 1:25 for TGF-01. The absorbance at 450/540 nm was measured by a SynergyMX plate reader. Results were corrected to account for differences in total protein content of samples. MMP-2/9 activity assay: The tissue lysates acquired in the western blot section (n=3-4 rats per group) were used for detecting the activity of MMP-2/9 in the LV myocardium. The Calbiochem InnoZyme™ Gelatinase activity assay fluorogenic kit (EMD Millipore, Billerica, MA) was followed per the manufacturer's instructions. Briefly, lysate samples (diluted 1:2 in activation buffer) were incubated with a fluorogenic substrate solution that is highly selective for MMP-2 and MMP-9. Gelatinases in the sample lysates of the myocardium cleave the substrate, resulting in an increase in fluorescent signal measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm by a SynergyMX plate reader. The gelatinase control, activated similarly, was used at serial dilutions to create a standard curve for converting the fluorescence values of MMP activity to concentrations (ng/ml). Statistical analysis: Results are presented as means±standard deviations (SD). GraphPad Prism 5.0 software (La Jolla, CA) was used for statistical analysis. Statistical differences between groups were analyzed by one-way ANOVA (multiple groups) or two-way repeated ANOVA (repeated echocardiographic measurements) with 95% confidence interval. Bonferroni multiple comparison test was performed for ANOVA post-hoc analysis. Statistical significance was set at p<0.05. Release assay of complementary proteins: The release assay was performed using 100 ng of each of TIMP-3 (R&D Systems, Minneapolis, MN), FGF-2, and SDF-1α (PeproTech, Rocky Hill, NJ). All solutions were prepared in 0.9% sterile saline. FGF-2 and SDF-1α coacervates were made and mixed with a fibrinogen solution containing TIMP-3, followed by thrombin (Sigma-Aldrich, St. Louis, MO) to induce gelation, resulting in a 100 μl fibrin gel-coacervate composite. FGF-2 and SDF-1α coacervates were made by mixing 1 μl of 100 ng/μl for each of FGF-2 and SDF-1α with 2 μl of 5 mg/ml heparin first (Scientific Protein Labs, Waunakee, WI), then with 2 μl of 25 mg/ml of PEAD at PEAD:heparin:protein mass ratio of 250:50:1. This formed 6 μl of FGF-2/SDF-1α coacervates. Fibrin gel-coacervate composite was made by mixing 8241 of 20 mg/ml fibrinogen (Sigma-Aldrich, St. Louis, MO), 1 μl of 5 mg/ml heparin, 1 μl of 100 ng/μl of TIMP-3; then the 6 μl FGF-2/SDF-1α coacervates were added, followed by 5 μl of 1 mg/ml aprotonin (Sigma-Aldrich, St. Louis, MO). Lastly, 5 μl of 1 mg/ml thrombin (Sigma-Aldrich, St. Louis, MO) was added to induce gelation, resulting in a 100 μl fibrin gel-coacervate composite. A 100p1 of 0.9% saline was deposited on top of the gel composite to be collected at 1 h, 16 h, 1, 4, 7, 14, 28, and 42 days. The samples (n=3) were incubated at 37° C. After centrifugation at 12,100 g for 10 min, supernatant was collected and stored at −80° C. to detect amount of released proteins by sandwich enzyme-linked immunosorbent assay (ELISA) kits (PeproTech, Rocky Hill, NJ) (R&D Systems, Minneapolis, MN). The absorbance at 450/540 nm was measured by a SynergyMX plate reader (Biotek, Winooski, VT). Standard solutions (n=3) that contained 100 ng of each of the proteins in free form in 100p1 of 0.9% saline were prepared to create standard curves and determine total release. Detailed method of rat MI model: Six to seven week old (175-225 g) male Sprague-Dawley rats (Charles River Labs, Wilmington, MA) were anesthetized first then maintained with 2% isoflurane at 0.3 L/min (Butler Schein, Dublin, OH), intubated, and connected to a mechanical ventilator to support breathing during surgery. The body temperature was maintained at 37° C. by a hot pad. The ventral side was shaved and a small incision was made through the skin. Forceps, scissors, and q-tips were used to dissect through the skin, muscles, and ribs. Once the heart was visible, the pericardium was torn. MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery using a 6-0 polypropylene suture (Ethicon, Bridgewater, NJ). Infarct was confirmed by macroscopic observation of a change in color from bright red to light pink in the area below the ligation suture. Five minutes after the induction of MI, different treatment and control solutions were injected intramyocardially at three equidistant points around the infarct zone using a 31-gauge needle (BD, Franklin Lakes, NJ). Four groups (n=56 rats) were evaluated: sham, saline, free proteins, and delivered proteins. Empty vehicle (empty fibrin gel-coacervate composite) was not tested as a control in this study as it has shown no difference to saline in our previous work. The sham group (n=13) underwent the surgery in which the heart was exposed and pericardium was torn, then chest was closed and rat recovered. The saline group (n=14) underwent the surgery in which MI was induced and 100 μl of 0.9% sterile saline was injected around the infarct region. The free proteins group (n=14) underwent the surgery in which MI was induced and 100 μl of 0.9% sterile saline containing 3 μg each of free TIMP-3, FGF-2, and SDF-1α was injected around the infarct region. The delivered proteins group (n=15) underwent the surgery in which MI was induced and 100 μl of fibrin gel-coacervate composite was injected around the infarct region. The fibrin gel-coacervate composite was prepared briefly as follows: 18 μl coacervate solution containing 3 μg each of FGF-2 and SDF-1α, 67p1 of 20 mg/ml fibrinogen, 6 μl of solution containing heparin and 3 μg of TIMP-3, 5 μl of 1 mg/ml aprotonin (Sigma-Aldrich, St. Louis, MO). Lastly, 4 μl of 1.5 mg/ml thrombin (Sigma-Aldrich, St. Louis, MO) was added and the total solution was injected shortly before gelation occurred, approximately 40 seconds after mixing. All solutions were prepared in 0.9% sterile saline. The chest was closed and the rat was allowed to recover. At multiple time points, rats were imaged using echocardiography. At eight weeks, a subset was imaged using cardiac MRI. After 2 (n=17) or 8 weeks (n=39), animals were sacrificed and hearts were harvested for histological, immunohistochemical, and western blot evaluations. Echocardiography: At pre-MI, one, two, five and eight weeks post-MI, rats (n=9-10 per group) were anesthetized then maintained with 1-1.5% isoflurane gas throughout the echocardiographic study. Rats were placed in the supine position, immobilized on a heated stage equipped with echocardiography, and the hair in the abdomen was removed. The body temperature was maintained at 37° C. Short-axis videos of the LV by B-mode were obtained using a high-resolution in small animal imaging system (Vevo 2100, Visual Sonics, Ontario, Canada) equipped with a high-frequency linear probe (MS400, 30 MHz) (FUJIFILM VisualSonics, Canada). End-systolic (ESA) and end-diastolic (EDA) areas were measured using NIH ImageJ and fractional area change (FAC) was calculated as: [(EDA-ESA)/EDA]×100%. Percent improvements of one group over another were calculated as the difference between the % drops in FAC values of the first and second groups divided by the higher % drop of the two groups. Cardiac MRI: Cardiac MRI was used to measure LV volumes and ejection fraction (EF) from infarcted rat hearts at eight weeks (n=5-8 per group). MRI was preformed using a Bruker Biospec 4.7-Tesla 40-cm scanner equipped with a 12-cm shielded gradient set, a 72 mm transmit RF coil (Bruker Biospin, Billerica MA), and a four-channel rat cardiac receive array (Rapid MR International, Columbus, OH). Rats were induced with isoflurane, intubated, and ventilated at 1 mL/100 g of body weight and maintained at 2% isoflurane in 2:1 O2:N2O gas mixture at 60 BPM. During the MRI procedure rats were continually monitored and rectal temperature was maintained at 37° C. with warm air (SA Instruments, Stony Brook, NY). Following pilot scans, rats were imaged using a self-gated cine FLASH sequence (IntraGate) with the following parameters: TR/TE=9.0/3.0 ms, 40×40 mm FOV, 256×256 matrix, FA=10°, and 200 repetitions. 10-12 slices were collected to cover the area between the heart apex to the mitral valves with 1.5 mm slice thickness with common navigator slice. End-systolic and end-diastolic phases were identified for each subject and the LV cavity manually traced using NIH ImageJ to determine LV end-systolic (ESV) and end-diastolic (EDV) volumes. These volumes were used to compute ejection fraction as EF %=[(EDV−ESV)/EDV]×100%. Percent improvements of one group over another were calculated as the difference between the % drops in EF values of the first and second groups divided by the higher % drop of the two groups. The following numbered clauses provide illustrative examples of various aspects of the invention. 1. A composition comprising a coacervate of a polycationic polymer, a polyanionic polymer, and platelet-rich plasma and/or serum, or a fraction or concentrate thereof. 2. The composition of clause 1, comprising platelet-rich plasma and wherein platelets of the platelet-rich plasma are activated to produce a fibrin clot, and the fibrin clot is optionally removed from the platelet-rich plasma. 3. The composition of clause 1, comprising pure platelet-rich fibrin (P-PRF) or leukocyte-rich PRF, with or without the fibrin clot removed. 4. The composition of clause 2 or 3, wherein the fibrin clot is removed and the protein content of the PRP is concentrated (for example, as compared to the solution phase of activated PRP, in which the fibrin is removed, but prior to concentration, e.g., by use of a centrifugal filter unit). 5. A composition comprising a coacervate of a polycationic polymer, a polyanionic polymer, and a composition obtained from an organism or cultured cells, tissues or organs and containing a (complex, e.g.) mixture of proteins and/or growth factors produced by the organism or cultured cells, tissues or organs (that is, the composition is obtained from a living source, and though it may proceed through one or more fractionation and/or purification steps not limited to activation or fractionation, e.g., precipitation, chromatography, and/or affinity separation, as in the case of activated PRP as decribed herein in which the platelets of the PRP are activated, and optionally, the resultant clot is removed and the composition is optionally concentrated, it is not an isolated or purified single constituent, but includes costituents, such as, for example, a plurality (at least two, e.g., three or more or four or more) of proteins and/or growth factors, essentially in relative amounts and/or a ratio found in, or produced by the cells or organism). 6. The composition of clause 5, wherein the composition containing a complex mixture of proteins and/or growth factors is prepared from a bodily fluid of an organism, a cell or tissue lysate, or conditioned media in which cells or a tissue is grown. 7. The composition of clause 5, wherein the cells, tissues, or organism are genetically-modified. 8. A composition comprising:a. a hydrogel comprising TIMP-3; andb. a complex or coacervate of a polycationic polymer, a polyanionic polymer, FGF-2 and SDF-1α embedded in the hydrogel. 9. The composition of any one of clauses 1-8, wherein the polyanionic polymer is a sulfated or sulfamated polysaccharide. 10. The composition of any one of clauses 1-8, wherein the polyanionic is a heparin or heparan sulfate. 11. The composition of any one of clauses 1-10, wherein the polycationic polymer is a polymer composition comprising at least one moiety selected from the following:(a) [—OC(O)—CH(NHY)—CH2—C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(b) [—OC(O)—CH2—CH(NHY)-C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(c) [—OC(O)—CH(NHY)—CH2—CH2—C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, and/or(d) [—OC(O)—CH2—CH2—CH(NHY)-C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, wherein Y is —C(O)—CH(NH3+)—(CH2)3—NH—C(NH2)2+or —C(O)—CH(NH3+)—(CH2)4—(NH3)+, and R1 and R2 are the same or different and are independently selected from the group consisting of hydrogen, a carboxy-containing group, a C1-6alkyl group, an amine-containing group, a quaternary ammonium containing group, and a peptide. 12. The composition of clause 11, wherein the polycationic polymer has a polydispersity index of less than 3.0. 13. The composition of clause 11, wherien R1 and R2 are selected from the group consisting of Ile-Lys-Val-Ala-Val (IKVAV) (SEQ ID NO: 4), Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO: 5), Ala-Gly-Asp (AGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) (SEQ ID NO: 6), Val-Ala-Pro-Gly-Val-Gly (VAPGVG) (SEQ ID NO: 7), APGVGV (SEQ ID NO: 8), PGVGVA (SEQ ID NO: 9), VAP, GVGVA (SEQ ID NO: 10), VAPG (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), VGVA (SEQ ID NO: 13), VAPGV (SEQ ID NO: 14) and GVAPGV (SEQ ID NO: 15). 14. The composition of clause 11, wherein Y is —C(O)—CH(NH3+)—(CH2)4—(NH3)+. 15. The composition of clause 11, wherein Y is —C(O)—CH(NH3+)—(CH2)3—NH—C(NH2)2+. 16. The composition of any one of clauses 1-15, wherein the ratio of the polycationic polymer to the polyanionic polymer in the composition results in a neutral, negative, or positive charge in the coacervate. 17. The composition of clause 8, wherein the hydrogel comprises fibrin. 18. The composition of any one of clauses 8-17, wherein the amounts of TIMP-3, FGF-2 and/or SDF-1α in the compositions are amounts effective to treat a myocardial infarct in a patient (that is, an amount effective to improve one or more clinically-relevant measures of the myocardial infarct in the patient. 19. A method of treating a patient having a myocardial infarct, comprising administering the composition of any one of clauses 8-18 to a patient at or near a site of a myocardial infarct in the patient, in an amount effective to treat a myocardial infarct in a patient. 20. A method of treating a wound in a patient, comprising administering a composition according to any one of clauses 1-7 at or adjacent to a wound in the patient, in an amount effective to treat a wound in a patient. 21. The method of clause 20, wherein the composition is administered to the patient more than once. 22. A method of preparing a therapeutic composition for use in treating a wound in a patient, comprising:a. mixing a polyanionic polymer with platelet-rich plasma, serum, a fraction thereof, a concentrate thereof, or platelet-rich plasma (PRP) in which the platelets have been activated to produce a fibrin clot; andb. mixing the PRP and polyanionic polymer mixture with a polycationic polymer. 23. The method of clause 22, wherein the polyanionic polymer is a sulfated or sulamated polysaccharide, such as heparin or heparan sulfate. 24. The method of clause 22 or 23, wherein the polycationic polymer is a polymer composition comprising at least one moiety selected from the following:(a) [—OC(O)—CH(NHY)—CH2—C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(b) [—OC(O)—CH2—CH(NHY)-C(O)O—CH2—CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n,(c) [—OC(O)—CH(NHY)—CH2—CH2—C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, and/or(d) [—OC(O)—CH2—CH2—CH(NHY)-C(O)O—CH2-CH(O—R1)-CH2—O—CH2—CH2—O—CH2—CH(O—R2)-CH2-]n, wherein Y is —C(O)—CH(NH3+)—(CH2)3—NH—C(NH2)2+or —C(O)—CH(NH3+)—(CH2)4—(NH3)+, and R1 and R2 are the same or different and are independently selected from the group consisting of hydrogen, a carboxy-containing group, a C1-6alkyl group, an amine-containing group, a quaternary ammonium containing group, and a peptide. 25. The method of clause 24, wherein the polycationic polymer has a polydispersity index of less than 3.0. 26. The method of clause 24, wherein R1 and R2 are selected from the group consisting of Ile-Lys-Val-Ala-Val (IKVAV) (SEQ ID NO: 4), Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO: 5), Ala-Gly-Asp (AGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) (SEQ ID NO: 6), Val-Ala-Pro-Gly-Val-Gly (VAPGVG) (SEQ ID NO: 7), APGVGV (SEQ ID NO: 8), PGVGVA (SEQ ID NO: 9), VAP, GVGVA (SEQ ID NO: 10), VAPG (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), VGVA (SEQ ID NO: 13), VAPGV (SEQ ID NO: 14) and GVAPGV (SEQ ID NO: 15). 27. The method of clause 24, wherein Y is —C(O)—CH(NH3+)—(CH2)4—(NH3)+. 28. The method of clause 24, wherein Y is —C(O)—CH(NH3+)—(CH2)3—NH—C(NH2)2+. 29. The method of any one of clauses 22-28, wherein the ratio of the polycationic polymer to the polyanionic polymer in the composition results in a neutral, negative, or positive charge in the coacervate. 30. The method of any one of clauses 22-29, wherein the platelet-rich plasma, serum, a fraction thereof, a concentrate thereof, or platelet-rich plasma (PRP) in which the platelets have been activated to produce a fibrin clot is autologous to a patient to be treated. 31. The method of any one of clauses 22-30, wherein the platelet-rich plasma, serum, a fraction thereof, a concentrate thereof, or PRP in which the platelets have been activated to produce a fibrin clot PRP in which the platelets have been activated is PRP in which the platelets have been activated to produce a fibrin clot PRP in which the platelets have been activated that is further processed to remove the fibrin clot from the PRP prior to mixing with the polyanionic polymer. 32. The method of clause 31, further comprising concentrating the proteins in the PRP. 33. The method of any one of clauses 22-32, further comprising applying the therapeutic composition to a medical device or wound dressing. 34. The method of clause 33, wherein the medical device or wound dressing is a bandage, suture, surgical mesh, limb or joint prosthesis, or a non-woven material. 35. A medical device or wound dressing comprising a composition according to any one of clauses 1-7. 36. The medical device or wound dressing of clause 35, wherein the medical device or wound dressing is a bandage, suture, surgical mesh, limb or joint prosthesis, or a non-woven material. While the present invention is described with reference to several distinct embodiments, those skilled in the art may make modifications and alterations without departing from the scope and spirit. Accordingly, the above detailed description is intended to be illustrative rather than restrictive. | 136,264 |
11857632 | DETAILED DESCRIPTION Crystalline nanocellulose offers high protein stability, good release profile and high drug loading, with increased bioavailability via additives like cyclodextrin. The CNC content can be tailored to control diffusion kinetics. There are numerous chemical functionalities that can be introduced via the hydroxyl groups to provide appropriate surface chemistry. Embodiments described herein also utilize the electrostatic interactions between the proteins and the negative sulfate esters on CNC formed during acid hydrolysis. CNC self-assembles to form a fibrous network with a high surface area to volume ratio translating to large binding area. The protein drug electrostatically binds to the nanocellulose and releases over >24 h, controlled by diffusion. The nanocellulose maintains the protein structure, prevents aggregation, and limits environmental exposure. Described below are methods for making unique tunable hydrogels suitable for sustained delivery of several different drug types. Diabetes affects >9% of the United States population and is treated by insulin protein therapy to reduce blood glucose levels. Generally, the insulin is sourced from porcine or bovine pancreas. This is administered intravenously and significantly reduces morbidity and mortality. However, approximately 60% of patients fail to achieve long-term glycemic control, which may be in part due to low patient compliance. Transdermal or oral delivery has the highest patient adherence. Insulin oral delivery systems with high loading, sustained release, and good bioavailability are being developed; there are several technologies now in the clinical trial stage. (Fonte P, Araujo F, Reis S, Sarmento B. “Oral Insulin Delivery: How Far Are We?”J Diabetes Sci Technol,2013, 7(2), pp. 520-31). However, many of these developing technologies involve synthetic petroleum-based polymers or they are nanoparticle formulations with unknown physiological side effects. Many of the drug delivery systems also incorporate absorption enhancers that are not protein specific which increases the risk of absorption of toxins or allergens along with the therapeutic compound. Enzyme inhibitors are also used, but prolonged use may reach high toxicity and may increase the absorption of other proteins that would otherwise be degraded. (Muheem, A., Ibid.; Renukuntla J, Vadlapudi A D, Patel A, Boddu S H S, Mitra A K. “Approaches for Enhancing Oral Bioavailability of Peptides and Proteins”,International Journal of Pharmaceutics,2013; 447(1-2); pp. 75-93) The leading oral delivery systems being developed for general protein drugs are the GI-MAPS system (Eudragit®) and SNAC carrier microemulsion (Emisphere™) (Bruno, B J., Ibid.). GI-MAPS is a gastrointestinal mucoadhesive patch system within an enteric coating that has demonstrated 6-23% bioavailability of a model protein. GI-MAPS system is a multi layer composition containing materials sourced from fossil fuels. (Eiamtrakarn S, Itoh Y, Kishimoto J, Yoshikawa Y, Shibata N, Murakami M, et al. “Gastrointestinal Mucoadhesive Patch System (GI-MAPS) for Oral Administration of G-CSF, a Model Protein”,Biomaterials,2002, 23(1), pp. 145-52). SNAC is a microemulsion system of (n-(8-[2-hydroxylbenzoyl]amino)caprylic acid) with protein solubilized in the interior that transports the protein drug across the epithelial membrane where the complex dissociates. SNAC delivery demonstrates 5-10% bioavailability but releases the protein as a fast release bolus and not as controlled release for basal insulin levels over prolonged time. (Kidron M, Dinh S, Menachem Y, Abbas R, Variano B, Goldberg M, et al. “A Novel Per-oral Insulin Formulation: Proof of Concept Study in Non-diabetic Subjects”,Diabetic Medicine,2004, 21(4), pp. 354-357). In contrast to the technologies addressed above, the embodiments set forth herein provide a manufacturing process using materials sourced from a bio-feedstock to produce drug carriers with predicted high bioavailability, controlled release time, and superior protein stabilization. In one or more embodiment, CNC delivery systems set forth herein use hydrogels for delivery of pyridostigmine bromide (PB). Pyridostigmine bromide is the standard pretreatment against the lethal effects of organophosphorus (OP) nerve agent (e.g., soman) exposure, currently the sole Food and Drug Administration (FDA)-approved pretreatment medication to reduce the number of daily administrations of fielded medication with logistical and operational mobility improvements. These embodiments address the Joint Chemical and Biological Defense (CBD) program seeking improved formulations of PB that are functional in austere environments and have sustained release with prolonged drug efficacy. The acute toxicity of nerve agents is attributed to the irreversible binding and inactivation of the enzyme acetylcholinesterase (AChE) leading to accumulation of synaptic acetylcholine (ACh). This leads to persistent and excessive stimulation of acetylcholine receptors resulting in acute cholinergic toxicity. Even if patients receive treatment after OP exposure, most of them suffer from wide ranging physiological sequelae, including seizures, neurodegeneration and psychological deficits. The prophylactically administered PB acts as a more readily reversible and “temporary” inhibitor of AChE due to its carbamylation of the active site serine. This precludes OP from longer-term and potentially irreversible inactivation of AChE by phosphorylation, thereby mitigating the risks. Thus, PB effectively creates a safety net by providing a pool of transiently bound AChE that is essentially protected and therefore unavailable for the irreversible binding of nerve agents. The recommended PB dose is one 30 mg tablet every 8 hours beginning several hours prior to nerve agent exposure. In humans, this dosing of PB inhibits 20-40% of red blood cell cholinesterase (i.e., AChE). This level of inhibition protects animals from the lethal effects of soman and other OPs. The PB dosages, referred to as Soman Nerve Agent Pretreatment Pyridostigmine (SNAPP), are issued in blister packs of 21 tablets. NSN 6505-01-178-7903 is one pack of 10 blister packs. Prior to issue, these light- and temperature-sensitive hygroscopic tablets must be stored at between 2-8° C. Once issued to a soldier, the PB in the blister pack is usable for up to three months after which it must be discarded. Storage of this tablet formulation at controlled room temperature (25° C.) reduces shelf life. Therefore, an improved and stable formulation is needed to withstand higher temperatures, for example desert temperatures. Based on the current state of military operations and unrest worldwide, there is a need for a nerve agent pretreatment that can be administered less frequently than SNAPP. Such improved formulations could also be administered as a pretreatment for first responders and health care providers following a suspected nerve agent attack or during mass casualty decontamination. As demonstrated by the capability set forth herein to exhibit sustained release of insulin, a large hydrophobic protein, with a molecular weight less than 50,000 Da, and PB, a hydrophilic small molecule with a molecular weight less than 500 Da, the CNC-based hydrogels described herein provide a tunable excipient for a wide variety of drug types. The description below outlines the compositions and methods for an oral drug delivery system for sustained delivery of proteins, drugs, and hydrophilic small molecules. The delivery system utilizes porous nano-crystalline or nano-fibrillar cellulose networks to form hydrogels that can be loaded via diffusion or in situ loading during gelation. An example of nanocellulose hydrogel crosslinking is shown inFIG.1. Epoxy-terminated crosslinkers, such as epichlorohydrin, can be activated in an alkaline environment and react with available hydroxyl groups. The reaction of epichlorohydrin with cellulose sulfate is activated by NaOH or other bases to make cellulose hydrogels. Then cellulose hydrogels are then reacted with available OH−or O−groups to crosslink with other cellulose groups. The result is a highly porous network suitable for holding large proteins. Alternatively, metal ions can also be used to form ion complexation to form very tight hydrogel networks with lower porosity, suitable for smaller drugs. The general processes and results for making hydrogels by chemical crosslinking is shownFIGS.2A and2B); the use of metal ion complexation is illustrated inFIGS.3A and3B. With reference toFIGS.2A and2BCNC is hydrated via stirring and heating (FIG.2A), resulting in a viscous suspension (FIG.2B). Then a basic solution is added, reducing viscosity. Once the solution is homogenous, a chemical crosslinker is added and the mixture is poured into a crystallization dish and let stand overnight. The hydrogel will then hold its shape. The hydrogel is then washed and cut into pieces, such as 16 mm diameter cylinders, which can be removed from the crystallization dish for subsequent processing. With reference toFIGS.3A,3B and3Cunmodified-CNF (UM-CNF) is hydrated under high-powered sonication (FIG.3A) resulting in a clear solution shown inFIG.3B. A metal ion salt, such as Al(NO3)3, is then dissolved in water and added to the UM-CNF mixture which results in rapid gelation, shown inFIG.3C. The hydrogel is then washed and cut into smaller pieces, such as uniform hydrogel cylinders, for later processing. The properties of chemically crosslinked hydrogels can be tuned by controlling the following parameters: cellulose source, degree of sulfation or other functionalization, percentage of cellulose, pH, crosslinker selection, amount of crosslinker, and mixing (speed and duration). The properties of metal-ion crosslinked gels can be tuned by metal-ion selection. Microfeatures of the cellulose before (FIGS.4and5) and after gelation of (FIGS.6and7) of sulfonated CNCs via chemical crosslinking was visualized using scanning electron microscopy (SEM). During gelation, chemical crosslinking will react with available hydroxyl groups or with functionalized groups to crosslink the cellulose. The sulfonated CNCs started out as micron-sized particles. The micrograph of CNC gel showed porous networks of CNC sheets, which is indicative of porous crosslinked hydrogels. Microfeatures of the TEMPO-oxidized CNF hydrogels before (FIGS.8and9) and after gelation (FIGS.10and11) with metal-ions is shown inFIGS.8-11. The TEMPO-oxidized CNF starts as low-density fibrous clumps. The CNF gel showed tighter packed sheets compared to its initial form, which is not unexpected given that its crosslinking occurs via ionic interactions as opposed to physical chemical bonds. It is important to note that the structures of the gels may not have been maintained during SEM because the gels had to be lyophilized prior to evaluation by SEM. To determine what degradation or swelling may occur in these nanocellulose hydrogels, each sample was weighed and measured before and after soaking in PBS or DI for seven days.FIG.12Ashows a sample of the cellulose sulfate hydrogel fromFIG.2BandFIGS.12B and12Cshow the result of soaking the sample ofFIG.12Ain two different fluids.FIG.12Bshows the hydrogel after soaking in PBS for 7 days;FIG.12Cshows the hydrogel after soaking in deionized water for 7 days. In PBS, CNC hydrogels were fairly stable, maintaining their shape. However, the CNC hydrogels in DI became more brittle, which can be seen in the loss of shape (FIGS.12A-12C). Conversely, TEMPO-oxidized CNF hydrogels exhibit no change in structural integrity in PBS or DI (FIGS.13A-13C).FIG.13Ashows the TEMPO-oxidized CNF hydrogel fromFIG.3Cin two different fluids andFIGS.13B and13Cshow the result of soaking the sample ofFIG.13Ain two different fluids.FIG.13Bshows the hydrogel after soaking in PBS for 7 days;FIG.13Cshows the hydrogel after soaking in deionized water for 7 days. Each hydrogel was weighed in air and water to obtain density via Archimedes principle. The density of the hydrogel can be calculated using the following equation: ρ=MairMair-Mwater(ρ0-ρL)+ρL(Eq.1) where ρ=density of the sample, Mair=mass of the sample in air, Mwater=mass of the sample in water, ρ0=density of water, and ρL=density of air. A decrease in density can occur if hydrogels swell, increase in water weight, or degrade. By evaluating changes to the hydrogel using density instead of mass, errors induced by handling and damage to the hydrogel are mitigated.FIG.14is a bar graph showing the density measurements of chemically crosslinked CNC hydrogels and metal ion complexed TEMPO-oxidized CNF hydrogels prior to soaking in fluids and after soaking in PBS or DI water for seven days. Values are average±standard error. Generally, the average density was slightly lower after the hydrogels soaked in DI water for seven days, for all hydrogel types. Changes in density were not statistically significant, which indicates that the hydrogels are stable in PBS and DI water. They exhibit no signs of swelling. However, the brittleness of sulfated CNC after exposure to DI water may be related to pH. Due to its low ionic strength, DI water has no buffering capabilities. Carbon dioxide from the atmosphere readily dissolves in DI water, forming carbonic acid and giving the DI water a pH of about 5.5. The slightly acidic nature may negatively impact the structure of cellulose because cellulose is digested by acid. Insulin was selected as a relevant large hydrophobic protein to evaluate loading and release in PBS. Insulin was loaded by soaking hydrogels in solutions containing 5 mg/mL, 8 mg/mL, or 11 mg/mL insulin. Aliquots of the supernatant solution were collected at specified timepoints and evaluated using a Coomassie Blue Bradford Protein Assay in order to quantify the amount of insulin present. As the hydrogel encapsulates the insulin, the amount of insulin in the solution should decrease.FIGS.15A and15Bare graphs illustrating supernatant insulin concentrations during encapsulation (FIG.15A) and corresponding insulin loading efficiency (FIG.15B) for sulfated CNC hydrogel.FIGS.15A and15Bshows results for the loading of insulin in sulfated CNC hydrogel. At each insulin concentration, insulin within the solution steadily decreases, which suggests that insulin was incorporated into the hydrogel. Insulin encapsulation efficiency was calculated as E=ci-cfci*100%(Eq.2) where E is the encapsulation efficiency, Ciis the initial insulin concentration in solution, and Cfis the concentration of insulin measured in solution at each timepoint. After 24 hours, the encapsulation efficiency was 98.15±1.85% for 5 mg/mL, 76.51±1.60% for 8 mg/mL, and 73.53±2.79% for 11 mg/mL. This indicates that the hydrogels contain roughly 4.9 mg/mL, 6.12 mg/mL, and 8.09 mg/mL of insulin for 5 mg/mL, 8 mg/mL, and 11 mg/mL initial concentrations, respectively. FIG.16is a graph comparing the insulin release profile for sulfated CNC hydrogels made with no mixing, 10 sec mixing at 650 rpm, 1 min mixing at 400 rpm, 1 min mixing at 650 rpm, 5 min mixing at 650 rpm and 10 min mixing at 650 rpm. The insulin concentration selected for further testing was 8 mg/mL. The effect of stirring speed and duration after addition of crosslinker is shown inFIG.16. Increasing mixing speed and/or duration result in an increased release rate. This suggests that increased mixing speed and/or duration results in an increase in porosity and permeability of the hydrogel. This is significant because the hydrogel can be optimized for delivery of drugs or proteins of varied sizes. This also allows for tunable release profiles to optimize release in therapeutic concentrations. FIGS.17A and17Bare graphs comparing insulin encapsulation (FIG.17A) and cumulative insulin release (FIG.17B) for TEMPO-oxidized CNF hydrogels made with aluminum nitrate (Al(NO3)3), calcium chloride (CaCl2)), or hydrochloric acid (HCl). The effects of metal ion selection for TEMPO-oxidized CNF hydrogels are shown inFIGS.17A and17B. As a trivalent cation Al3+creates hydrogels with this highest strength and lowest porosity and permeability. As a result, CNF hydrogels made with aluminum nitrate (Al(NO3)3) exhibit the lowest release rate for insulin. The divalent Ca2+exhibited loading and release rate between Al3+and H+, as expected. HCl resulted in the mechanically weakest hydrogel. While CNF gels made with HCl loaded the fastest, they also released the fastest. By selecting ion crosslinkers by size and valency, hydrogel strength and permeability can be tuned, making this network highly favorable for encapsulation of a variety of drugs. The onset of thermal changes of the sulfated cellulose hydrogels has been determined by differential scanning calorimetry (DSC). Each sample was scanned from −25° C. to 100° C. at a rate of 5° C./min. The DSC measurements were made on the cellulose, the cellulose hydrogel and the freeze-dried hydrogel samples (with and without loading PB in the hydrogels). About 5-15 mg of sample was cut (excess water was wiped off with a Kimwipe from the hydrogels), placed in an aluminum pan and sealed. The sample pan was heated alongside an empty pan as a reference. FIGS.18A,18B and18Cshows DSC thermographs for cellulose sulfate, cellulose sulfate hydrogels, and freeze-dried cellulose sulfate gels, respectively. The DSC was performed from −25° C. to 100° C.FIG.18Ais the DSC for commercial cellulose powder;FIG.18Bis the DSC for hydrogel made with commercial cellulose powder andFIG.18Cis the DSC for gel fromFIG.18Bafter freeze drying. The cellulose source,FIG.18A, did not show any peaks (endothermic changes) in the tested temperature range. The cellulose hydrogel,FIG.18B, showed an endothermic phase transition around 0° C. which relates to the phase change of the free water present in the interstitial space of the hydrogel. Further, the negative slope from 50° C. to 70° C. corresponds to the breaking of the hydrogen bonds between water molecules (of cellulose-bound water) and the cellulose. FIGS.19A and19Bare graphs of Differential Scanning calorimetry tests performed from −25° C. to 100° C.FIG.19Ais the DSC for PB powder andFIG.19Bis the DSC for freeze-dried gel with PB encapsulated therein.FIG.19Ashows the thermograph of PB. Because of the hygroscopic nature of PB, there is a peak at 0° C. corresponding to the phase change of the water molecules. The rate of hydration of PB changes with increasing temperature, which corresponds to the soft peak between 25° C. and 50° C. The thermal scans on the freeze-dried hydrogels, both without PB (FIG.18C) and PB loaded (FIG.19B), did not show any endothermic changes (peaks). Therefore, it was concluded that the freeze-dried form of the hydrogel is stable (i.e., there is no structural change, bond breaking) in the tested temperature range. To further improve the hydrogel release kinetics, the structure of the hydrogel was modified to change the hydrogel structure and mechanical properties by changing the concentration of the NaOH solution added to the hydrogel. An increase in alkalinity increased the qualitative mechanical strength of the hydrogel, and a decrease in alkalinity decreased the mechanical strength of the hydrogel. It is believed that this plays an important role in the encapsulation and the release properties of PB, as the mechanical strength of the hydrogel affects the pore sizes in the hydrogel. By tuning the pore size and gel crosslinking, the release profile of entrapped drug molecules can be tuned. To observe this effect, hydrogels were made using three different concentrations of NaOH: 50 mM, 0.1 M, and 1 M.FIGS.20A and20Bare graphs showing the dependence on NaOH concentration on PB release from hydrogels over a period of time whereFIG.20Aillustrates the total amount of PB released after an allotted time andFIG.20Billustrates the rates of release at different time points for the different concentrations of NaOH hydrogels. The 50 mM and 0.1 M hydrogels behaved similarly in both loading and releasing due the fact that the pH difference is only about 0.3 (from pH 12.7 to pH 13). However, a change from 0.1 M to 1 M resulted in a pH increase of 1 (pH 13 to pH 14).FIGS.20A and20Billustrate the release of PB in the different hydrogels. The hydrogel with 1 M NaOH had largest quantity of encapsulated PB, however there was no significant difference in release kinetics between each of the samples. While the release rate for hydrogels made with 1 M NaOH was slightly slower, all the encapsulated PB was released after 24 hours, as with the other hydrogels. However, the slightly slower release rate for hydrogels made with 1 M NaOH indicates that further optimization and increase in NaOH concentration may yield hydrogels with a more desirable PB release profile. By modifying the hydrogel formation protocol to include the addition of β-Cyclodextrin during the synthesis of the hydrogel, productivity increased tremendously. This change enabled the hydrogel to load PB much faster. Lyophilization was used on the β-cyclodextrin infused hydrogels to increase the hydrophilic nature of cellulose by dehydrating it. The hydrogels absorb the liquid, PBS in this case, and loads PB into the hydrogel. The liquid level is reduced; however, the amount of PB in the liquid is still the same as prior to adding to the hydrogel. 2 mg/mL PB in PBS was used in order to preserve the amount of available materials. FIGS.21A and21Bare graphs showing the release profile of PB whereFIG.21Ashows the kinetic release profile of PB from hydrogel into a PBS solution andFIG.21Bshows the corresponding rate of release—for the same versus time.FIGS.21A and21B illustrates the release of PB in two ways.FIG.21Ashows that the release rate initially is fast, but slows significantly after the first hour, and then slowly reaches maximum PB released between 8.5-24 hours.FIG.21Bshows the derivative of the graph inFIG.21A. This derivative shows the rate of release versus time, displaying effectively an inverse of the graph inFIG.21A. The current protocol displays an exponential decay release rate profile, which means that the only near-linear portions are in the beginning and at the end of the release. The release of PB into an acidic environment, such as the gastrointestinal system, was tested to determine pH stability of the hydrogel and the release rate. Since stomach acid is composed of HCl, we chose to use HCl dilutions in PBS to vary the pH of the solution. We chose to evaluate the performance at three different pH's, pH 1, pH 3, and pH 7, to simulate performance in the normal acidity range of the human stomach versus performance in pure PBS. The performance of the CNC-PB samples are shown inFIG.22showing the effect of pH on PB release from a CNC hydrogels. A range of acidic pH was evaluated to mimic effects of stomach acid. No significant differences were observed. The results displayed very similar trends across the board. At each of the evaluated pH's, the hydrogels behaved similarly, which is beneficial as the desired application for the drug system is oral ingestion. This test also shows the sturdiness of the hydrogels in an acidic environment. The lack of ability in humans to enzymatically breakdown cellulose means that the cellulose retains most of its mechanical integrity during the drug release in acidic environments. To illustrate that the hydrogel and PB were stable at high temperatures, samples were held at elevated temperatures for an extended period.FIG.23is a graph showing concentrations of normalized PB release from CNC hydrogels at several temperatures. The hydrogels were placed in closed scintillation vial and the vial was wrapped with Teflon tape and Parafilm to seal the vials. The vials were then placed in an incubator at 40° C. for 5 days. After the 5 days, one sample was removed, and the release profile was determined. The other vials were left in the incubator and exposed to 50° C. for another 6 days. After 6 days at 50° C., one sample was removed, and the incubator was set to 60° C. for the final sample.FIG.23shows the normalized release profiles. All the elevated temperature samples displayed the same release profile; however, all the hydrogels that were exposed to an elevated temperature had a faster release profile than the room temperature sample. A radiometric assay was done to evaluate acetylcholinesterase (AChE) Inhibition with both free PB and CNC-PB.FIG.24is a graph showing the radiometric AChE assay results of in-vitro AChE activity after exposure to bare PB and CNC-PB.FIG.24illustrates time-dependent inhibition of mouse blood AChE activity by free PB and encapsulated PB. It was found that AChE activity was significantly different among the groups tested (PBS, free PB and hydrogel encapsulated PB). When compared to free PB, the PB loaded into the hydrogel (CNC-PB) showed significantly longer inhibitory effect (p<0.001) but lower intensity of inhibition (p<0.001). There was also no significant interaction between inhibitory effect and time (p=0.3597). While multiple comparisons indicated AChE, activity was lower with both the PB and PB-loaded hydrogel (CNC-PB) compared to PBS at all time-points, there were no significant time-dependent differences comparing the two different pyridostigmine conditions (PB vs CNC-PB). This data shows a trend of in-vitro differences in the timing of AChE inhibition when either free pyridostigmine or pyridostigmine in CNC was added to a tissue (mouse blood) and incubated at 37° C. While limited by the number of replicates (n=3 independent assays) and by the assay conditions (i.e., the enzyme reaction lasted for 8 minutes during which pyridostigmine could have been released from the hydrogel to act as free pyridostigmine), the data suggest that inhibition was delayed and prolonged by PB in the hydrogel. When these data were evaluated using non-linear curve fitting with a 2-phase exponential decay (i.e., to elucidate the initial more rapid inhibition of AChE vs the apparent later inhibition), some insight into the relative effects of PB source (free vs hydrogel) were obtained. The R2values for the two nonlinear curves were 0.88 and 0.65 for free pyridostigmine and CNC-hydrogel, respectively. When half-life for early and later decay phases between free and hydrogel-PB were compared (T1/2early phase: PB=13.1 min, CNC hydrogel=4.9 min; T1/2late phase: PB=17.3 min, CNC hydrogel=55 min.), it was noted that both the fast (early) and slow (late) phase of inhibition was different. Of interest the late phase was prolonged from 17.3 minutes with free pyridostigmine to 55 minutes with the encapsulated PB. This suggests that PB was being released from the hydrogel in its interaction at 37° C. with the tissue in vitro to prolong AChE inhibition. Performance of free-PB and CNC-encapsulated PB was evaluated in-vivo through time-dependent inhibition by free vs. CNC-encapsulated PB by first estimating maximum tolerated (i.e., non-lethal) doses (MTDs) for each. Signs of cholinergic malfunction include salivation, lacrimation, urination, and defecation, which is often given the acronym, SLUD. Mice were observed and SLUD signs were used to rank reaction on the following scale: 0=no signs; 1=very slight signs such as possible fasciculations, piloerection; 2=slight toxicity including mild fasciculations, salivation or lacrimation; 3=moderate signs such as moderate head and neck area tremors, tail “twitching” and more obvious fasciculations and/or SLUD signs; 4=severe signs, notable whole body tremors, prostrate/reduced ambulation, extensive fasciculations, and/or SLUD signs, occasional choreatic movements; and 5=death. MTDs were estimated using the up-and-down method, treating 1-3 mice at a time and observing functional cholinergic toxicity signs and lethality out to 24 hours. Young adult male CD1 mice (about 35-40 g at time of dosing) were purchased from Charles River and acclimated to the AAALAC-accredited facility at OSU for at least one week before conducting toxicity studies. A solution of free PB was made in PBS at 13.33 mg/ml and used for all PB dosing, adjusting dosing volume accordingly for each dose. Contents of a vial of lyophilized CNC gel (65 mg wet weight) containing 40±5 mg pyridostigmine were first disrupted by mortar and pestle and then rehydrated by adding 3 ml PBS to obtain a putative PB concentration of 13.33 mg/ml. An aliquot of the freshly prepared gel was administered by gavage tube, adjusting dosing volume as with the free pyridostigmine. All gavage treatments were finished within 1-3 minutes of hydrating the gel. Doses evaluated in pyridostigmine and/or pyridostigmine-CNC gel were 20 mg/kg, 39.5 mg/kg, 51.3 mg/kg, 66.7 mg/kg, 86.7 mg/kg and 112.7 mg/kg. One mouse was first treated with the highest dose of pyridostigmine (112.7 mg/kg). It showed severe signs and death within one minute. A second mouse was treated with 51.3 mg/kg showed minimal to moderate signs at 18-20 minutes, but then overt whole-body tremors were noted within a half hour and clonic seizures and death occurred at about 50 minutes. A third mouse treated with 39.5 mg/kg showed very slight to slight signs of a reaction for about the first 20 minutes, but then clear signs of toxicity (scores of ≥3) around 30 minutes after dosing, peaking with a functional score of 4 at 3-4 hours and then prostration, clonic seizures and death at the 270-minute time-point. Accordingly, lethality was noted with 112.7, 51.3 and 39.5 mg/kg PB doses. A fourth mouse was then treated with 20 mg/kg pyridostigmine. Very slight to slight signs of toxicity were noted for the first half hour, followed by moderate signs (score=3) at the 45 min and 2-hour time-points. By 2.5 hours after dosing, no obvious functional signs were noted aside from lethargy. Thus, 20 mg/kg was defined as the MTD for free pyridostigmine. Using the CNC-gel, a fifth mouse was treated with 20 mg/kg PB in gel. This mouse showed no signs for the first hour after dosing and continued to be sign free through 24 hours. Another mouse was treated with 86.7 mg/kg PB in gel. Within 60 minutes after dosing, this mouse showed obvious fasciculations, tail twitching and fine head and neck tremors (score=3). By 120 minutes, whole body tremors, tail twitching, SLUD signs and “puffy face” (suggesting congestion perhaps of salivary tracts), were noted. By 180 minutes, a fine whole-body tremor and prostrate positioning were noted. Interestingly, by 270 minutes, no classic cholinergic signs were noted (score=0), but the animal appeared in “poor” overall condition with dried secretions around the eyes and slow movement around the cage. He survived until the end of the observation period (24 hours). Another mouse was given 66.7 mg/kg PB as CNC-gel. Marked whole body fasciculations were noted by one hour after dosing, along with mild tremors. At 120 minutes, the mouse showed fine tremors and tail twitching (score=3). By 180 minutes, fine, whole body tremors were noted along with apparent exhaustion/prostration. At the 267-minute time-point, clonic seizures were noted followed shortly by death. Another mouse was then given 51.3 mg/kg PB in gel. By one hour, only very slight signs including mild fasciculations were noted (score=1). At 120 minutes, lesser fasciculations were noted but minor occasional tail twitching was observed (score 1-2). At 180 minutes, a score of 0.5 was given based on infrequent fasciculations, while by 170 minutes, no cholinergic signs were noted (score of 0.) FIGS.25A and25Bare graphs showing the toxicity score from mice MTD studies described above.FIG.25Ashows a PB dosage study andFIG.25Bshows a CNC-PB dosage MTD study. Functional signs of toxicity in all mice are shown in whereFIG.25Ashows functional scores after pyridostigmine dosing, whileFIG.25Bshows time-dependent responses after CNC gel dosing. Pertinent observations for the in-vivo studies are that the MTDs for free pyridostigmine and CNC gel containing pyridostigmine were 20 mg/kg and 51.3 mg/kg, respectively. Our experience with anticholinesterases suggests that approximately 0.3-0.5 MTD doses will be non-lethal but elicit substantial blood AChE inhibition, suitable for kinetic evaluations. AChE inhibition over time when dosed with 0.5×MTD (FIG.26A) and 0.3×MTD (FIG.26BandFIG.26C) shows improved longevity of pyridostigmine activity when encapsulated in sulfated CNC hydrogels. Hydrogels made from sulfated CNCs and/or TEMPO-oxidized CNFs can also be tuned to provide an interconnected pore network suitable for 3D cell culture and guided tissue regeneration (Bačáková L1, Novotná K, Pařízek M. “Polysaccharides as cell carriers for tissue engineering: the use of cellulose in vascular wall reconstruction”.Physiological Research.2014, 63). The diameters of mammalian cells are typically in the range of 5 to 100+μm. (Diekjürgen D, Grainger D W. “Polysaccharide matrices used in 3D in-vitro cell culture systems.Biomaterials.2017, 141, pp 96-115). It is known that the porosity and permeability of the CNC and CNF hydrogels can be tuned by altering environment pH, amount of CNC or CNF, and the amount and type of crosslinker as well as mechanical stimulation during formulation. A variety of freeze-thaw and freeze-drying protocols can also be used to change the pore size to make hydrated or freeze-dried CNC or CNF constructs for in-vitro cell culture. Additionally, constructs for guided tissue regeneration can also be produced. 3D cell culture has been shown to improve cell culture and function in-vitro because of the increased area for cell attachment and closer mimicry of native extracellular environments. (Wang J, Zhao L, Zhang A, Huang Y, Tavakoli J, Tang Y. “Novel Bacterial Cellulose/Gelatin Hydrogels as 3D Scaffolds for Tumor Cell Culture.”,Polymers.2018, 10, p. 581). It is thus concluded that hydrogels from the sulfated CNCs and TEMPO-oxidized CNFs provide a robust and tunable excipient for a variety of drugs as demonstrated through controlled release of pyridostigmine bromide and insulin. | 34,173 |
11857633 | DETAILED DESCRIPTION OF THE INVENTION Definitions While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose. The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/−10% of the specified value. In embodiments, about means the specified value. The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10means one to ten carbons). Alkyl is not cyclized. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds (e.g. alkene, alkyne). Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—). The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, —CH2CH2CH2CH2—. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Heteroalkyl is not cyclized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CHO—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, —O—CH3, —O—CH2—CH3, and —CN. Up to two or three heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3and —CH2—O—Si(CH3)3. Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)2R′— represents both —C(O)2R′— and —R′C(O)2—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO2R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —NR′R″ or the like, it will be understood that the terms heteroalkyl and —NR′R″ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —NR′R″ or the like. The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Cycloalkyl and heterocycloalkyl are non-aromatic. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. The term “acyl” means, unless otherwise stated, —C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The examples above may be substituted or unsubstituted and divalent radicals of each heteroaryl example above are non-limiting examples of heteroarylene. A fused ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein. In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section or Drawings. The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom. Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —NR′NR″R′″, —ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO2, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), triphosphate (or derivatives thereof), in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R, R′, R″, R′″, and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ group when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF3and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like). Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —NR′NR″R′″, —ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), triphosphate (or derivatives thereof), in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″, R′″, and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ groups when more than one of these groups is present. Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)—(CRR′)q—U—, wherein T and U are independently —NR—, —O—, —CRR′—, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′—, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CRR′)s—X′—(C″R″R′″)d—, where s and d are independently integers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituents R, R′, R″, and R′″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). A “substituent group,” as used herein, means a group selected from the following moieties:(A) oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O) NH2, —NHSO2H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, —NHSO2CH3, —N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), and(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), substituted with at least one substituent selected from:(i) oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O) NH2, —NHSO2H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, —NHSO2CH3, —N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), and(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), substituted with at least one substituent selected from:(a) oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O) NH2, —NHSO2H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, —NHSO2CH3, —N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), and(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), or triphosphate (or derivatives thereof), substituted with at least one substituent selected from: oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O) NH2, —NHSO2H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, —NHSO2CH3, —N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, monophosphate (or derivatives thereof), diphosphate (or derivatives thereof), and triphosphate (or derivatives thereof). A “size-limited substituent” or “size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. A “lower substituent” or “lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. It will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this invention. The symbol “” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula. A “chemical linker” or “linker” as provided herein is a covalent linker, a non-covalent linker, a peptide linker (a linker including a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene or any combination thereof. Thus, a chemical linker as provided herein may include a plurality of chemical moieties, wherein each of the plurality of moieties can be chemically different. The terms “a” or “an,” as used in herein means one or more. In addition, the phrase “substituted with a[n],” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is “substituted with an unsubstituted C1-C20alkyl, or unsubstituted 2 to 20 membered heteroalkyl,” the group may contain one or more unsubstituted C1-C20alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus, a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R1substituents are present, each R1substituent may be distinguished as R1A, R1B, R1C, R1Detc., wherein each of R1A, R1B, R1C, R1D, etc. is defined within the scope of the definition of R1and optionally differently. Descriptions of compounds of the present invention are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. “Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like. Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction. The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICALAPPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) or locked nucleic acids (LNA) as known in the art), including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATEMODIFICATIONS INANTISENSERESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both. Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism. An “antisense nucleic acid” as referred to herein is a nucleic acid (e.g., DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid (e.g., STAT3) and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA), altering transcript splicing (e.g. single stranded morpholino oligo), or interfering with the endogenous activity of the target nucleic acid. See, e.g., Weintraub,Scientific American,262:40 (1990). Typically, synthetic antisense nucleic acids (e.g. oligonucleotides) are generally between 15 and 25 bases in length. Thus, antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid (e.g., STAT3). In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid (e.g., STAT3) in vitro. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid (e.g., STAT3) in a cell. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid (e.g., STAT3) in an organism. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid (e.g., STAT3) under physiological conditions. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, e.g., phosphorothioate, methylphosphonate, and -anomeric sugar-phosphate, backbone modified nucleotides. In the cell, the antisense nucleic acids hybridize to the corresponding RNA (e.g., STAT3) forming a double-stranded molecule. The antisense nucleic acids interfere with the endogenous behavior of the RNA (e.g., STAT3) and inhibit its function relative to the absence of the antisense nucleic acid. Furthermore, the double-stranded molecule may be degraded via the RNAi pathway. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura,Anal. Biochem.,172:289, (1988)). Further, antisense molecules which bind directly to the DNA may be used. Antisense nucleic acids may be single or double stranded nucleic acids. Non-limiting examples of antisense nucleic acids include siRNAs (including their derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA), micro RNAs (miRNA), saRNAs (small activating RNAs) and small nucleolar RNAs (snoRNA) or certain of their derivatives or pre-cursors. MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs. miRNAs function as post-transcriptional regulators of gene expression, which are integral to almost all known biological processes, including cell growth, proliferation and differentiation as well as organismal metabolism and development. In the rapidly growing field of miRNAs, many miRNAs have been identified to be functionally associated with promoting either disease progression or cancer cell differentiation favoring an improved therapeutic prognosis. However, while miRNA sequences are easy to produce, a “gymnotic” administration of man-made unmodified RNA sequences fails to provide the desired therapeutic benefit. The term “polymeric” refers to a molecule including repeating subunits (e.g., polymerized monomers). For example, polymeric molecules may be based upon polyethylene glycol (PEG), poly[amino(1-oxo-1,6-hexanediyl)], poly(oxy-1,2-ethanediyloxycarbonyl-1,4-phenylenecarbonyl), tetraethylene glycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), or poly(p-xylylene). See, for example, “Chemistry of Protein Conjugation and Cross-Linking” Shan S. Wong CRC Press, Boca Raton, Fla., USA, 1993; “BioConjugate Techniques” Greg T. Hermanson Academic Press, San Diego, Calif., USA, 1996; “Catalog of Polyethylene Glycol and Derivatives for Advanced PEGylation, 2004” Nektar Therapeutics Inc, Huntsville, Ala., USA, which are incorporated by reference in their entirety for all purposes. The term “polymerizable monomer” is used in accordance with its meaning in the art of polymer chemistry and refers to a compound that may covalently bind chemically to other monomer molecules (such as other polymerizable monomers that are the same or different) to form a polymer. The term “block copolymer” is used in accordance with its ordinary meaning and refers to two or more portions (e.g., blocks) of polymerized monomers linked by a covalent bond. In embodiments, a block copolymer is a repeating pattern of polymers. In embodiments, the block copolymer includes two or more monomers in a periodic (e.g., repeating pattern) sequence. For example, a diblock copolymer has the formula: -B-B-B-B-B-B-A-A-A-A-A-, where ‘B’ is a first subunit and ‘A’ is a second subunit covalently bound together. A triblock copolymer therefore is a copolymer with three distinct blocks, two of which may be the same (e.g., -A-A-A-A-A-B-B-B-B-B-B-A-A-A-A-A-) or all three are different (e.g., -A-A-A-A-A-B-B-B-B-B-B-C-C-C-C-C-) where ‘A’ is a first subunit, ‘B’ is a second subunit, and ‘C’ is a third subunit, covalently bound together. The term “amphiphilic polymer” as used herein refers to a polymer containing both hydrophilic and hydrophobic portions. In embodiments, the hydrophilic to hydrophobic portions are present in a 1 to 1 mass ratio. In embodiments, the hydrophilic to hydrophobic portions are present in a 1 to 2 mass ratio. In embodiments, the hydrophilic to hydrophobic portions are present in a 1 to 5 mass ratio. In embodiments, the hydrophilic to hydrophobic portions are present in a 2 to 1 mass ratio. In embodiments, the hydrophilic to hydrophobic portions are present in a 5 to 1 mass ratio. An amphiphilic polymer may be a diblock or triblock copolymer. In embodiments, the amphiphilic polymer may include two hydrophilic portions (e.g., blocks) and one hydrophobic portion (e.g., block). In embodiments, the hydrophilic block to hydrophobic to hydrophilic ratio is 1 to 1 to 1. In embodiments, the hydrophilic block to hydrophobic to hydrophilic ratio is 1.8 to 1 to 1.8. In embodiments, the hydrophilic block to hydrophobic to hydrophilic ratio is 2 to 1 to 2. In embodiments, the hydrophilic block to hydrophobic to hydrophilic ratio is 1 to 1 to 2. The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanidine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region). A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5′-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence. The term “phosphorothioate nucleic acid” refers to a nucleic acid in which one or more internucleotide linkages are through a phosphorothioate moiety (thiophosphate) moiety. The phosphorothioate moiety may be a monothiophosphate (—P(O)3(S)3−—) or a dithiophosphate (—P(O)2(S)23−—). In embodiments of all the aspects provided herein, the phosphorothioate moiety is a monothiophosphate (—P(O)3(S)3−—). That is, in embodiments of all the aspects provided herein, the phosphorothioate nucleic acid is a monothiophosphate nucleic acid. In embodiments, one or more of the nucleosides of a phosphorothioate nucleic acid are linked through a phosphorothioate moiety (e.g. monothiophosphate) moiety, and the remaining nucleosides are linked through a phosphodiester moiety (—P(O)43−—). In embodiments, one or more of the nucleosides of a phosphorothioate nucleic acid are linked through a phosphorothioate moiety (e.g. monothiophosphate) moiety, and the remaining nucleosides are linked through a methylphosphonate linkage. In embodiments, all the nucleosides of a phosphorothioate nucleic acid are linked through a phosphorothioate moiety (e.g. a monothiophosphate) moiety. Phosphorothioate oligonucleotides (phosphorothioate nucleic acids) are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Phosphorothioate nucleic acids may also be longer in lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. As described above, in certain embodiments. the phosphorothioate nucleic acids herein contain one or more phosphodiester bonds. In other embodiments, the phosphorothioate nucleic acids include alternate backbones (e.g., mimics or analogs of phosphodiesters as known in the art, such as, boranophosphate, methylphosphonate, phosphoramidate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press). The phosphorothioate nucleic acids may also include one or more nucleic acid analog monomers known in the art, such as, peptide nucleic acid monomer or polymer, locked nucleic acid monomer or polymer, morpholino monomer or polymer, glycol nucleic acid monomer or polymer, or threose nucleic acid monomer or polymer. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and nonribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. Phosphorothioate nucleic acids and phosphorothioate polymer backbones can be linear or branched. For example, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like. As used herein, a “phosphorothioate polymer backbone” is a chemical polymer with at least two phosphorothioate linkages (e.g. monothiophosphate) (e.g. linking together sugar subunits, cyclic subunits or alkyl subunits). The phosphorothioate polymer backbone may be a phosphorothioate sugar polymer (i.e., a polymer composed of abasic sugar-phosphate modules), which is a phosphorothioate nucleic acid in which one or more (or all) of the chain of pentose sugars lack the bases (nucleobases) normally present in a nucleic acid. The phosphorothioate polymer backbone can include two or more phosphorothioate linkages. The phosphorothioate polymer backbone can include 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more linkages and can contain up to about 100 phosphorothioate linkages. Phosphorothioate polymer backbones may also contain a larger number of linkages, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, and the like. The phosphorothioate nucleic acids and phosphorothioate polymer backbones may be partially or completely phosphorothioated. For example, 50% or more of the internucleotide linkages of a phosphorothioate nucleic acid can be phosphorothioate linkages. Optionally, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the internucleotide linkages of a phosphorothioate nucleic acid are phosphorothioate linkages. Optionally, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the internucleotide linkages of a phosphorothioate nucleic acid are phosphorothioate linkages. Optionally, 75%, 80%, 85%, 90%, 95%, or 99% of the internucleotide linkages of a phosphorothioate nucleic acid are phosphorothioate linkages. Optionally, 90%, 95%, or 99% of the internucleotide linkages of a phosphorothioate nucleic acid are phosphorothioate linkages. In embodiments, the remaining internucleotide linkages are phosphodiester linkages. In embodiments, the remaining internucleotide linkages are methylphosphonate linkages. Optionally, 100% of the internucleotide linkages of the phosphorothioate nucleic acids are phosphorothioate linkages. Similarly, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkages in a phosphorothioate polymer backbone can be phosphorothioate linkages. Optionally, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkages in a phosphorothioate polymer backbone can be phosphorothioate linkages. Optionally, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkages in a phosphorothioate polymer backbone can be phosphorothioate linkages. Optionally, 90%, 95%, or 99%, of the intersugar linkages in a phosphorothioate polymer backbone can be phosphorothioate linkages. In embodiments, the remaining internucleotide linkages are phosphodiester linkages. In embodiments, the remaining internucleotide linkages are methylphosphonate linkages. Optionally, 100% of the intersugar linkages of the phosphorothioate polymer backbone are phosphorothioate linkages. A “labeled nucleic acid or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the nucleic acid may be detected by detecting the presence of the detectable label bound to the nucleic acid. Alternatively, a method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin. In embodiments, the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable label, as disclosed herein and generally known in the art. In embodiments, the phosphorothioate nucleic acid or phosphorothioate polymer backbone is connected to a detectable label through a chemical linker. A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. The phosphorothioate nucleic acids and phosphorothioate polymer backbones provided herein can include one or more reactive moieties, e.g., a covalent reactive moiety. A reactive moiety may be attached to the remainder of the phosphorothioate nucleic acids and phosphorothioate polymer backbones using any appropriate linker, such as a polymer linker known in the art or alternatively a polyethylene glycol linker or equivalent. The linker may, in embodiments, include (i.e. be attached to) a detectable label as described herein. As used herein, the term “covalent reactive moiety” refers to a chemical moiety capable of chemically reactive with an amino acid of a non-cell penetrating protein, as described herein, to form a covalent bond and, thus, a conjugate as provided herein. The term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene. The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton,Proteins(1984)). The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein “corresponds” to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein (e.g., STAT3) in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., STAT3) the identity and location of residues corresponding to specific positions of said protein are identified in other protein sequences aligning to said protein. For example, a selected residue in a selected protein corresponds to lysine at position 685 when the selected residue occupies the same essential spatial or other structural relationship as a lysine at position 685. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with lysine 685 is said to correspond to lysine 685. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the lysine at position 685, and the overall structures compared. In this case, an amino acid that occupies the same essential position as lysine 685 in the structural model is said to correspond to the lysine 685 residue. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. The terms “STAT3,” “STAT3 protein,” “STAT3 peptide” as referred to herein include any of the recombinant or naturally-occurring forms of the Signal transducer and activator of transcription 3 (STAT3) protein or variants or homologs thereof that maintain STAT3 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to STAT3). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 10, 20, 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring STAT3 polypeptide. In embodiments, the STAT3 peptide is substantially identical to the protein identified by the UniProt reference number P40763 or a variant or homolog having substantial identity thereto. In embodiments, the STAT3 peptide includes the sequence of SEQ ID NO:8. In embodiments, the STAT3 peptide is the sequence of SEQ ID NO:8. The term “IL-6” or “Interleukin 6” as referred to herein includes any of the recombinant or naturally-occurring forms of the IL-6 protein or variants or homologs thereof that maintain IL-6 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL-6). IL-6 is a member of the interleukin family and acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-6 polypeptide. In embodiments, the IL-6 protein is substantially identical to the protein identified by the UniProt reference number P05231 or a variant or homolog having substantial identity thereto. As used herein, the terms “cell-penetrating” or “cell-penetration” refer to the ability of a molecule (e.g. a protein) to pass from the extracellular environment into a cell in a significant or effective amount. Thus, a cell-penetrating conjugate is a molecule that passes from the extracellular environment, through the membrane, and into a cell. As used herein, the terms “non-cell penetrating” or “non-cell penetration” refers to the inability of a molecule to pass from the extracellular environment into a cell in a significant or effective amount. Thus, non-cell penetrating nucleic acids or ribonucleic acid compounds generally are not capable of passing from the extracellular environment, through the cell membrane, and into a cell in order to achieve a significant biological effect on a population of cells, organ or organism. The term does not exclude the possibility that one or more of the small number of nucleic acids or ribonucleic acid compounds may enter the cell. However, the term refers to molecules that are generally not able to enter a cell from the extracellular environment to a significant degree. Examples of non-cell penetrating molecules and substances include, but are not limited to, large molecules such as, for example, high molecular weight proteins, nucleic acids or ribonucleic acid compounds. Nucleic acids or ribonucleic acid compounds can be determined to be non-cell penetrating using methods known to those of skill in the art. By way of example, a nucleic acid or ribonucleic acid compound can be fluorescently labeled and the ability of the nucleic acid or ribonucleic acid compound to pass from the extracellular environment into the cell can be determined in vitro by flow cytometric analysis or confocal microscopy. In some embodiments, a “non-cell penetrating nucleic acid or ribonucleic acid compound” refers to a nucleic acid or ribonucleic acid compound that penetrates a cell at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10,000 or 100,000 fold less than the same nucleic acid or ribonucleic acid compound attached to a phosphorothioate nucleic acid or phosphorothioate polymer backbone. In some embodiments, a “non-cell penetrating nucleic acid or ribonucleic acid compound” refers to a nucleic acid or ribonucleic acid compound that does not measurably penetrate a cell. As used herein, the term “intracellular” means inside a cell. As used herein, an “intracellular target” is a target, e.g., nucleic acid, polypeptide or other molecule (e.g., carbohydrate) that is located inside of a cell and is a target to which the non-cell penetrating nucleic acids or ribonucleic acid compounds provided herein bind. Binding can be direct or indirect. Optionally, the non-cell penetrating nucleic acid or ribonucleic acid compound selectively binds the intracellular target. By selectively binds, selectively binding, or specifically binding refers to the agent (e.g., a non-cell penetrating nucleic acid or ribonucleic acid compound) binding one agent (e.g., intracellular target) to the partial or complete exclusion of other agents. By binding is meant a detectable binding at least about 1.5 times the background of the assay method. For selective or specific binding such a detectable binding can be detected for a given agent but not a control agent. Alternatively, or additionally, the detection of binding can be determined by assaying the presence of down-stream molecules or events. As used herein, the term “conjugate” refers to the association between atoms or molecules. The association can be direct or indirect. For example, a conjugate between a nucleic acid and a protein or nucleic acid or ribonucleic acid compound can be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). Optionally, conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the phosphorothioate nucleic acid and phosphorothioate backbone polymer are non-covalently attached to the nucleic acid or ribonucleic acid compound through a non-covalent chemical reaction between a component of the phosphorothioate nucleic acid and phosphorothioate backbone polymer (e.g., a monothiophosphate) and a component of the nucleic acid or ribonucleic acid compound. A “cell” as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g.,Spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization. The term “activating,” as used herein, refers to an nucleic acid conjugate capable of detectably increasing the expression or activity of a given gene or protein (e.g., p53). The activating nucleic acid conjugate can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the activating nucleic acid. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the activating nucleic acid conjugate. As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to an nucleic acid conjugate interaction means negatively affecting (e.g. decreasing) the activity or function of a protein (e.g., STAT3) relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. Thus, in embodiments, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a non-cell penetrating nucleic acid compound (e.g., let7a-3p (SEQ ID NO:1), let7a-5p (SEQ ID NO:2), miR17-3p (SEQ ID NO:3), miR17-5p (SEQ ID NO:4), miR218-5p (SEQ ID NO:5)) as described herein and an intracellular target (e.g., STAT3 (SEQ ID NO:8)). “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. an autoimmune disease, inflammatory autoimmune disease, cancer, infectious disease, immune disease, or other disease) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophagelike synoviocytes, etc). One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant. The terms “subject,” “patient,” “individual,” etc. are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice. The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). The disease may be an autoimmune, inflammatory, cancer, infectious, metabolic, developmental, cardiovascular, liver, intestinal, endocrine, neurological, or other disease. As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma (cutaneous T-cell lymphoma), sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma. Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells, cancer of the hepatic stellate cells, or prostate cancer. The term “leukemia” refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia. The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound, pharmaceutical composition, or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma. The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma. The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lobular carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum. As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast. As used herein, “treating” or “treatment of” a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination. The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection. As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “therapeutically effective dose or amount” as used herein is meant a dose that produces effects for which it is administered (e.g. treating or preventing a disease). The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease. As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. Nucleic Acid Conjugates Provided herein are, inter alia, nucleic acid conjugates including a non-cell penetrating ribonucleic acid compound attached at its 3′ end to a phosphorothioate polymer. The ribonucleic acid compounds conjugated to phophorothioate polymers at their 3′ end exhibit surprising biostability and can be delivered intracellulary with high efficiency. Upon entry into a cell the non-cell penetrating ribonucleic acid compounds provided herein may target and modify the activity of intracellular molecules involved in disease pathology thereby improving disease outcome. The nucleic acid conjugates provided herein including embodiments thereof are useful, inter alia, for the treatment of cancer, inflammatory disease, and pain. In an aspect is provided a nucleic acid conjugate including: (i) a non-cell penetrating ribonucleic acid compound including the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; (ii) a phosphorothioate polymer; and (iii) a chemical linker attaching the phosphorothioate polymer to the 3′ end of the non-cell penetrating ribonucleic acid compound; wherein the phosphorothioate polymer enhances intracellular delivery of the non-cell penetrating nucleic acid compound. In embodiments, the non-cell penetrating ribonucleic acid compound includes the sequence of SEQ ID NO: 1. In embodiments, the non-cell penetrating ribonucleic acid compound includes the sequence of SEQ ID NO:2. In embodiments, the non-cell penetrating ribonucleic acid compound includes the sequence of SEQ ID NO:3. In embodiments, the non-cell penetrating ribonucleic acid compound includes the sequence of SEQ ID NO:4. In embodiments, the non-cell penetrating ribonucleic acid compound includes the sequence of SEQ ID NO:5. In embodiments, the non-cell penetrating ribonucleic acid compound is the sequence of SEQ ID NO:1. In embodiments, the non-cell penetrating ribonucleic acid compound is the sequence of SEQ ID NO:2. In embodiments, the non-cell penetrating ribonucleic acid compound is the sequence of SEQ ID NO:3. In embodiments, the non-cell penetrating ribonucleic acid compound is the sequence of SEQ ID NO:4. In embodiments, the non-cell penetrating ribonucleic acid compound is the sequence of SEQ ID NO:5. In embodiments, the non-cell penetrating ribonucleic acid compound is a micro RNA (miRNA). In embodiments, the non-cell penetrating ribonucleic acid compound is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 10 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 20 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 40 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 50 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 60 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 70 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 80 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is more than about 90 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 10 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 10 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 20 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 20 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 21 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 21 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 22 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 22 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 23 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 23 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 24 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 24 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 26 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 26 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 27 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 27 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 28 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 28 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 29 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 29 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 40 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 40 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 50 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 50 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 60 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 60 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 70 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 70 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 80 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 80 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is about 90 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is 90 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 21 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 21 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 22 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 22 to 30 in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 23 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 23 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 24 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 24 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 25 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 25 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 26 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 26 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 27 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 27 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 28 to about 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 28 to 30 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 29 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 29 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 28 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 28 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 27 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 27 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 26 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 26 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 24 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 24 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 23 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 23 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 20 to about 22 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 20 to 22 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 21 to about 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 21 to 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 22 to about 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 22 to 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from about 23 to about 25 residues in length. In embodiments, the non-cell penetrating ribonucleic acid compound is from 23 to 25 residues in length. In embodiments, the phosphorothioate polymer is a phosphorothioate nucleic acid or an abasic sugar-phosphorothioated polymer. An “abasic sugar-phosphate polymer” as provided herein refers to a polymer including abasic sugar moieties (i.e. a moiety including a ribose or deoxyribose aromatic ring that does not have a base attached to it, which is not substituted with a base), wherein the abasic sugar moieties are covalently linked to other abasic sugar moieties or to sugar moieties substituted with a base and wherein the moieties are connected through a phosphodiester bond or a phosphorothioate bond. In embodiments, the phosphorothioate polymer is a phosphorothioate nucleic acid. In embodiments, the phosphorothioate polymer is an abasic sugar-phosphorothioated polymer. In embodiments, the phosphorothioate polymer is a phosphorothioate deoxyribonucleic acid. In embodiments, the phosphorothioate polymer is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more residues in length. In embodiments, the phosphorothioate polymer is about 10 residues in length. In embodiments, the phosphorothioate polymer is 10 residues in length. In embodiments, the phosphorothioate polymer is more than about 10 residues in length. In embodiments, the phosphorothioate polymer is more than 10 residues in length. In embodiments, the phosphorothioate polymer is about 20 residues in length. In embodiments, the phosphorothioate polymer is 20 residues in length. In embodiments, the phosphorothioate polymer is more than about 20 residues in length. In embodiments, the phosphorothioate polymer is more than 20 residues in length. In embodiments, the phosphorothioate polymer is about 30 residues in length. In embodiments, the phosphorothioate polymer is 30 residues in length. In embodiments, the phosphorothioate polymer is more than about 30 residues in length. In embodiments, the phosphorothioate polymer is more than 30 residues in length. In embodiments, the phosphorothioate polymer is about 40 residues in length. In embodiments, the phosphorothioate polymer is 40 residues in length. In embodiments, the phosphorothioate polymer is more than about 40 residues in length. In embodiments, the phosphorothioate polymer is more than 40 residues in length. In embodiments, the phosphorothioate polymer is about 50 residues in length. In embodiments, the phosphorothioate polymer is 50 residues in length. In embodiments, the phosphorothioate polymer is more than about 50 residues in length. In embodiments, the phosphorothioate polymer is more than 50 residues in length. In embodiments, the phosphorothioate polymer is about 60 residues in length. In embodiments, the phosphorothioate polymer is 60 residues in length. In embodiments, the phosphorothioate polymer is more than about 60 residues in length. In embodiments, the phosphorothioate polymer is more than 60 residues in length. In embodiments, the phosphorothioate polymer is about 70 residues in length. In embodiments, the phosphorothioate polymer is 70 residues in length. In embodiments, the phosphorothioate polymer is more than about 70 residues in length. In embodiments, the phosphorothioate polymer is more than 70 residues in length. In embodiments, the phosphorothioate polymer is about 80 residues in length. In embodiments, the phosphorothioate polymer is 80 residues in length. In embodiments, the phosphorothioate polymer is more than about 80 residues in length. In embodiments, the phosphorothioate polymer is more than 80 residues in length. In embodiments, the phosphorothioate polymer is about 90 residues in length. In embodiments, the phosphorothioate polymer is 90 residues in length. In embodiments, the phosphorothioate polymer is more than about 90 residues in length. In embodiments, the phosphorothioate polymer is more than 90 residues in length. In embodiments, the phosphorothioate polymer is about 100 residues in length. In embodiments, the phosphorothioate polymer is 100 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 11 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 11 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 12 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 12 to 30 in length. In embodiments, the phosphorothioate polymer is from about 13 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 13 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 14 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 14 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 15 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 15 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 16 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 16 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 17 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 17 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 18 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 18 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 19 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 19 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 20 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 20 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 21 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 21 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 22 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 22 to 30 in length. In embodiments, the phosphorothioate polymer is from about 23 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 23 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 24 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 24 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 25 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 25 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 26 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 26 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 27 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 27 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 28 to about 30 residues in length. In embodiments, the phosphorothioate polymer is from 28 to 30 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 29 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 29 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 28 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 28 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 27 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 27 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 26 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 26 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 25 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 25 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 24 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 24 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 23 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 23 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 22 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 22 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 21 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 21 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 20 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 20 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 19 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 19 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 18 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 18 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 17 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 17 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 16 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 16 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 15 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 15 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 14 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 14 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 13 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 13 residues in length. In embodiments, the phosphorothioate polymer is from about 10 to about 12 residues in length. In embodiments, the phosphorothioate polymer is from 10 to 12 residues in length. In embodiments, the phosphorothioate polymer is about 20 residues in length. In embodiments, the phosphorothioate polymer is 20 residues in length. In embodiments, the phosphorothioate polymer includes the sequence of SEQ ID NO:6 or SEQ ID NO:7. In embodiments, the phosphorothioate polymer includes the sequence of SEQ ID NO:6. In embodiments, the phosphorothioate polymer is SEQ ID NO:6. In embodiments, the phosphorothioate polymer includes the sequence of SEQ ID NO:7 In embodiments, the phosphorothioate polymer is SEQ ID NO:7. In embodiments, the phosphorothioate polymer is single-stranded. In embodiments, the phosphorothioate nucleic acid is a single-stranded phosphorothioate nucleic acid. In embodiments, the abasic sugar-phosphorothioated polymer is a single-stranded abasic sugar-phosphorothioated polymer. The conjugates provided herein including embodiments thereof may include a ribonucleic acid compound attached to a phosphorothioate polymer through a chemical linker. In embodiments, the chemical linker is a covalent linker. In embodiments, the chemical linker is a covalent linker. In embodiments, the linker includes the structure of formula: In formula (I), R1is hydrogen, halogen, —CF3, —CN, —CCl3, —COOH, —CH2COOH, —CONH2, —OH, —SH, —NO2, —NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkyl, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkyl, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkyl, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkyl, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted aryl, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroaryl. In embodiments, R1is hydrogen, halogen, —CF3, —CN, —CCl3, —COOH, —CH2COOH, —CONH2, —OH, —SH, —NO2, —NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, the linker is -L1-L2-L3-L4-L5-L6-L7-. In embodiments, L1is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L2is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L3is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L4is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L5is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L6is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, L7is a bond, —NH—N═CH—, —S(O)2—, —NR2—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted alkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted cycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted arylene, or substituted (e.g., substituted with substituent group(s), size-limited substituent group(s), or lower substituent group(s)) or unsubstituted heteroarylene. In embodiments, the linker is a non-immunogenic linker. In embodiments, the conjugate includes a detectable moiety. In embodiments, the detectable moiety is attached to the non-cell penetrating ribonucleic acid compound. In embodiments, the detectable moiety is attached to the phosphorothioate polymer. In embodiments, the detectable moiety forms part of the linker. In embodiments, the detectable moiety is covalently attached to the linker. The non-cell penetrating ribonucleic acid compounds provided herein including embodiments thereof are useful for the treatment of cancer, inflammatory disease, and/or pain by modifying the activity of intracellular molecules. In embodiments, the conjugate as provided herein including embodiments thereof is bound to an intracellular target. Thus, in embodiments, the non-cell penetrating ribonucleic acid compound inhibits the activity or expression of an intracellular target. In embodiments, the non-cell penetrating ribonucleic acid compound inhibits the activity of an intracellular target. In embodiments, the non-cell penetrating ribonucleic acid compound inhibits the expression of an intracellular target. In embodiments, the intracellular target is a signaling molecule or transcription factor. In embodiments, the intracellular target is a signaling molecule. In embodiments, the intracellular target is a transcription factor. In embodiments, the signaling molecule is a phosphatase or kinase. In embodiments, the signaling molecule is a phosphatase. In embodiments, the signaling molecule is a kinase. In embodiments, the intracellular target is a transcription factor. In embodiments, the intracellular target is STAT3. In embodiments, the intracellular target is a STAT3 protein including the amino acid sequence of SEQ ID NO:8. In embodiments, the intracellular target is the amino acid sequence of SEQ ID NO:8. [ In embodiments, the non-cell penetrating ribonucleic acid compound includes a ribonucleic acid compound which inhibits STAT3 activity relative to a standard control. In embodiments, the non-cell penetrating ribonucleic acid compound is a ribonucleic acid compound which inhibits STAT3 activity relative to a standard control. In embodiments, the non-cell penetrating ribonucleic acid compound includes a ribonucleic acid compound, which inhibits expression of a STAT3 target gene relative to a standard control. In embodiments, the non-cell penetrating ribonucleic acid compound is a ribonucleic acid compound, which inhibits expression of a STAT3 target gene relative to a standard control. In embodiments, the STAT3 target gene is an oncogene. In embodiments, the STAT3 target gene comprises Bcl-xL or IL-6. In embodiments, the STAT3 target gene comprises Bcl-xL. In embodiments, the STAT3 target gene comprises IL-6. In embodiments, the STAT3 target gene is Bcl-xL or IL-6. In embodiments, the STAT3 target gene is Bcl-xL. In embodiments, the STAT3 target gene is IL-6. In an aspect is provided a cell including a nucleic acid conjugate as described herein including embodiments thereof. In embodiments, the cell is a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a brain cancer cell, a pancreatic cancer cell, a melanoma cell, a colon cancer cell, a gastric cancer cell, a head-and-neck cancer cell, a liver cancer cell, a lung cancer cell, a cervical cancer cell, a sarcoma cell, a leukemia cell, a lymphoma cell, a multiple myeloma cell or a metastatic lung cancer cell. In embodiments, the cell is a breast cancer cell. In embodiments, the cell is a prostate cancer cell. In embodiments, the cell is an ovarian cancer cell. In embodiments, the cell is a brain cancer cell. In embodiments, the cell is a pancreatic cancer cell. In embodiments, the cell is a melanoma cell. In embodiments, the cell is a colon cancer cell. In embodiments, the cell is a gastric cancer cell. In embodiments, the cell is a head-and-neck cancer cell. In embodiments, the cell is a liver cancer cell. In embodiments, the cell is a lung cancer cell. In embodiments, the cell is a cervical cancer cell. In embodiments, the cell is a sarcoma cell. In embodiments, the cell is a leukemia cell. In embodiments, the cell is a lymphoma cell. In embodiments, the cell is a multiple myeloma cell. In embodiments, the cell is a metastatic lung cancer cell. Pharmaceutical Compositions The conjugates provided herein including embodiments thereof are further contemplated as forming part of a pharmaceutical composition. Therefore, in an aspect is provided a pharmaceutical composition including the nucleic acid conjugate as described herein including embodiments thereof and a pharmaceutically acceptable carrier. Pharmaceutical compositions provided by the present invention include compositions wherein the active ingredient (e.g. compositions described herein, including embodiments thereof) is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, the conjugates described herein will contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule, and/or reducing, eliminating, or slowing the progression of disease symptoms. Determination of a therapeutically effective amount of a conjugate of the invention is within the capabilities of those skilled in the art. The compositions for administration will commonly include an agent as described herein dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs. Solutions of the active compounds as free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions can be delivered via intranasal or inhalable solutions or sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines. Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In some embodiments, oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such compositions is such that a suitable dosage can be obtained. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. Aqueous solutions, in particular, sterile aqueous media, are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium. Vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients, can be used to prepare sterile powders for reconstitution of sterile injectable solutions. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated. DMSO can be used as solvent for extremely rapid penetration, delivering high concentrations of the active agents to a small area. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Thus, the composition can be in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. Thus, the compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g. symptoms of cancer and severity of such symptoms), kind of concurrent treatment, complications from the disease being treated or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art. For any composition (e.g., the conjugates provided herein including embodiments thereof) described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. As is well known in the art, effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan. Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state. Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention. The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration. Methods of Treating Cancer The conjugates as provided herein including embodiments thereof are useful, inter alia, for the treatment of cancer. Thus, in an aspect, a method for treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof, thereby treating the cancer in the subject. In embodiments, the cancer is breast cancer, prostate cancer, ovarian cancer, brain cancer, pancreatic cancer, melanoma, colon cancer, gastric cancer, head-and-neck cancer, liver cancer, lung cancer, cervical cancer, sarcoma, leukemia, lymphoma, multiple myeloma. In embodiments, the cancer is breast cancer. In embodiments, the cancer is prostate cancer. In embodiments, the cancer is ovarian cancer. In embodiments, the cancer is brain cancer. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is melanoma. In embodiments, the cancer is colon cancer. In embodiments, the cancer is gastric cancer. In embodiments, the cancer is head-and-neck cancer. In embodiments, the cancer is liver cancer. In embodiments, the cancer is lung cancer. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is sarcoma. In embodiments, the cancer is leukemia. In embodiments, the cancer is lymphoma. In embodiments, the cancer is multiple myeloma. In embodiments, the cancer is metastatic lung cancer. In embodiments, the method includes decreasing in the subject an expression level of BIRC5 or BclXL relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of BIRC5 and BclXL relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of BIRC5 relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of BclXL relative to a standard control. In embodiments, the standard control is an expression level of BIRC5 or BclXL detected in the absence of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof. The term “BIRC5,” also known as “survivin”, as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), homologs or variants thereof that maintain BIRC5 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to BIRC5). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring BIRC5 polypeptide. In embodiments, the BIRC5 gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000089685 or a variant having substantial identity thereto. The expression level of BRIC5 may be determined by detecting levels of BIRC5 mRNA or protein using methods known in the art. In embodiments, the BIRC5 mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000301633.8, homolog or functional fragment thereof. In embodiments, the BIRC5 protein is the amino acid sequence as identified by Uniprot reference number 015392, homolog or functional fragment thereof. The term “Bcl-xL,” also known as BCL2L1, as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding B-cell lymphoma-extra large (BclXL), homologs or variants thereof that maintain Bcl-xL activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Bcl-xL). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Bcl-XL polypeptide. In embodiments, the BclXL gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000171552 or a variant having substantial identity thereto. The expression level of BclXL may be determined by detecting levels of BclXL mRNA or protein using methods known in the art. In embodiments, the BclXL mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000307677.4, homolog or functional fragment thereof. In embodiments, the BIRC5 protein is the amino acid sequence as identified by Uniprot reference number Q07817, homolog or functional fragment thereof. Methods of Increasing P53 in a Cell The conjugates provided herein including embodiments thereof are further contemplated as a means of increasing p53 in a cell. A “p53 protein” or “p53” as referred to herein includes any of the recombinant or naturally-occurring forms of the tumor protein p53 (p53) or variants or homologs thereof that maintain p53 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared top53). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring p53 protein. In embodiments, the p53 protein is substantially identical to the protein identified by the UniProt reference number P04637 or a variant or homolog having substantial identity thereto. In an aspect, a method of increasing expression of p53 in a cancer cell is provided, the method including contacting a cancer cell with an effective amount of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof, thereby increasing expression of p53 in the cancer cell. Methods of Inhibiting Tumor Vascularization The conjugates provided herein including embodiments thereof are useful for the treatment of cancer through inhibition of tumor vascularization. Thus, in another aspect, a method of inhibiting tumor vascularization in a subject in need thereof is provided, the method including administering to the subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof, thereby inhibiting tumor vascularization in the subject. Methods of Treating an Inflammatory Disease The conjugates provided herein including embodiments thereof are also useful for the treatment of inflammatory disease. Therefore, in another aspect, a method of treating an inflammatory disease in a subject in need thereof is provided, the method including administering to the subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof, thereby treating an inflammatory disease in the subject. As used herein, the term “inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (e.g., an increased level of inflammation compared to a control such as a healthy person not suffering from a disease). Examples of inflammatory diseases include traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis. In embodiments, the method includes decreasing in the subject an expression level of FGA, IL1B or SERPINA3 relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of FGA, IL1B and SERPINA3 relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of FGA relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of IL1B relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of SERPINA3 relative to a standard control. In embodiments, the standard control is an expression level of FGA, IL1B or SERPINA3 detected in the absence of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof. The term “FGA” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding fibrinogen alpha chain (FGA), homologs or variants thereof that maintain FGA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to FGA). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring FGA polypeptide. In embodiments, the FGA gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000171560 or a variant having substantial identity thereto. The expression level of FGA may be determined by detecting levels of FGA mRNA or protein using methods known in the art. In embodiments, the FGA mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000302053.7, homolog or functional fragment thereof. In embodiments, the FGA protein is the amino acid sequence as identified by Uniprot reference number P02671, homolog or functional fragment thereof. The term “IL1B” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding interleukin 1 beta (IL1B), homologs or variants thereof that maintain IL1B activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL1B). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL1B polypeptide. In embodiments, the IL1B gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000125538 or a variant having substantial identity thereto. The expression level of IL1B may be determined by detecting levels of IL1B mRNA or protein using methods known in the art. In embodiments, the IL1B mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000263341.6, homolog or functional fragment thereof. In embodiments, the IL1B protein is the amino acid sequence as identified by Uniprot reference number P01584, homolog or functional fragment thereof. The term “SERPINA3” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding alpha 1-antichymotrypsin, homologs or variants thereof that maintain alpha 1-antichymotrypsin activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to alpha 1-antichymotrypsin). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring alpha 1-antichymotrypsin polypeptide. In embodiments, the SERPINA3 gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000196136 or a variant having substantial identity thereto. The expression level of SERPINA3 may be determined by detecting levels of SERPINA3 mRNA or protein using methods known in the art. In embodiments, the SERPINA mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000467132.5, homolog or functional fragment thereof. In embodiments, the alpha 1-antichymotrypsin protein is the amino acid sequence as identified by Uniprot reference number P01011, homolog or functional fragment thereof. Non-Opioid-Based Methods of Treating Pain Opioids are a class of compound among the most widely used for treatment of pain. Opioid drugs produce effects by interacting with opioid receptors. Opioids have opium- or morphine-like properties allowing them to act as opioid receptor agonists. However, opioids have other pharmacological effects including drowsiness, respiratory depression, and constipation, as well as abuse potential and tolerance. The negative side-effects of opioid use have spurred a need for non-opioid-based pain treatments. Provided herein are, inter alia, non-opioid-based methods for treating pain in a subject in need thereof. In an aspect, a method of treating pain in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof, thereby treating pain in the subject. The pain may emanate from a wide variety of sources or be derived from a wide variety of causes. Thus, the pain may be nociceptive pain (e.g., trauma, procedural, cut, sprains, bone fractures, burns, bumps, bruises), neuropathic pain (e.g., post herpetic neuralgia, reflex sympathetic dystrophy/causalgia, cancer pain, pain induced by treatment of cancer, HIV/AIDS or hepatitis, diabetes, phantom limb pain, entrapment neuropathy, chronic alcohol use, exposure to other toxins, vitamin deficiencies and idiopathic), inflammatory pain (e.g., arthritis, colitis, carditis, pulmonits, nephritis, myositis, vasculitis, endometriosis, neuritis, dermatitis and pain associated with other inflammatory conditions), chronic widespread pain (e.g., fibromyalgia, migraine, irritable bowel syndrome, syndrome X, interstitial bladder syndrome, chronic fatigue syndrome, post-traumatic stress disorder, pain associated with psychiatric illnesses such as anxiety and depression and stress-related pain conditions, and secondary to inflammatory or neuropathic pain syndromes) or mixed etiology (i.e., combinations of two or more of the above four categories). In embodiments, the nucleic acid conjugates useful for treating pain in the methods provided herein does not mediate its analgesic effect through opioid receptors. In embodiments, the nucleic acid conjugate useful for treating pain in the methods provided herein does not have opium- or morphine-like properties. In embodiments, the nucleic acid conjugate useful for treating pain in the methods provided herein is not an opioid receptor ligand. In embodiments, the nucleic acid conjugate useful for treating pain in the methods provided herein does not bind to an opioid receptor. In embodiments, the nucleic acid conjugate useful for treating pain in the methods provided herein is not an opioid receptor agonist. In embodiments, the method includes decreasing in the subject an expression level of PTGS1, PTGS2, CALCA or SST relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of PTGS1, PTGS2, CALCA and SST relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of PTG51 relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of PTG52 relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of CALCA relative to a standard control. In embodiments, the method includes decreasing in the subject an expression level of SST relative to a standard control. In embodiments, the standard control is an expression level of PTGS1, PTGS2, CALCA or SST detected in the absence of a cell penetrating nucleic acid conjugate as described herein including embodiments thereof. The term “PTGS1” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding prostaglandin-endoperoxide synthase 1 (PTGS1), also known as COX-1, homologs or variants thereof that maintain PTGS1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PTGS1). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PTGS1 polypeptide. In embodiments, the PTGS1 gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000095303 or a variant having substantial identity thereto. The expression level of PTGS1 may be determined by detecting levels of PTGS1 mRNA or protein using methods known in the art. In embodiments, the PTGS1 mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000540753.5, homolog or functional fragment thereof. In embodiments, the PTGS1 protein is the amino acid sequence as identified by Uniprot reference number P23219, homolog or functional fragment thereof. The term “PTGS2” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding prostaglandin-endoperoxide synthase 2 (PTGS2), also known as COX-2, homologs or variants thereof that maintain PTGS2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PTGS2). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PTGS2 polypeptide. In embodiments, the PTGS2 gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000073756 or a variant having substantial identity thereto. The expression level of PTGS2 may be determined by detecting levels of PTGS2 mRNA or protein using methods known in the art. In embodiments, the PTGS2 mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000367468.9, homolog or functional fragment thereof. In embodiments, the PTGS2 protein is the amino acid sequence as identified by Uniprot reference number P35354, homolog or functional fragment thereof. The term “SST” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding somatostatin (SST), homologs or variants thereof that maintain SST activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to SST). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring SST polypeptide. In embodiments, the SST gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000157005 or a variant having substantial identity thereto. The expression level of SST may be determined by detecting levels of SST mRNA or protein using methods known in the art. In embodiments, the SST mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000287641.3, homolog or functional fragment thereof. In embodiments, the SST protein is the amino acid sequence as identified by Uniprot reference number P61278, homolog or functional fragment thereof. The term “CALCA” as referred to herein includes any of the recombinant or naturally-occurring forms of the gene encoding calcitonin gene-related peptide (CGRP), homologs or variants thereof that maintain CALCA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CALCA). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CALCA polypeptide. In embodiments, the CALCA gene is substantially identical to the nucleic acid identified by the ENSEMBLE reference number ENSG00000110680 or a variant having substantial identity thereto. The expression level of CALCA may be determined by detecting levels of CALCA mRNA or protein using methods known in the art. In embodiments, the CALCA mRNA is the nucleic acid sequence as identified by the Ensembl ID: ENST00000361010.7, homolog or functional fragment thereof. In embodiments, the CALCA protein is the amino acid sequence as identified by Uniprot reference numbers P01258, P06881, homolog or functional fragment thereof. Methods of Decreasing IL-6 Signaling in a Cell The conjugates provided herein including embodiments thereof are useful for decreasing IL-6 signaling in a cell. Thus, in an aspect, a method of inhibiting IL-6 signaling in a cell is provided, the method including contacting a cell with an effective amount of a cell penetrating nucleic acid conjugate as provided herein including embodiments thereof, thereby inhibiting IL-6 signaling in the cell. Methods of Delivering a Non-Cell Penetrating Nucleic Acid into a Cell The conjugates provided herein including embodiments thereof are useful for delivering non-cell penetrating nucleic acids into a cell. In an aspect is provided a method of delivering a non-cell penetrating nucleic acid into a cell, the method including contacting a cell with the cell penetrating nucleic acid conjugate as provided herein including embodiments thereof, thereby delivering the non-cell penetrating nucleic acid into the cell. EXAMPLES Here, Applicants describe a gymnotic administration of miRNAs, for example, let7a-3p (SEQ ID NO:1), let7a-5p (SEQ ID NO:2), miR17-3p (SEQ ID NO:3), miR17-5p (SEQ ID NO:4), miR218-5p (SEQ ID NO:5), by protecting the operating miRNA sequences through extending the miRNAs on their 3′ ends with phosphorothioated ssDNA oligonucleotides or phosphorothioated single-stranded abasic sugar-phosphate backbone polymers. Applicants show that attachment of phosphorothioated ssDNA oligonucleotides or phosphorothioated single-stranded abasic sugar-phosphate backbone polymers to miRNAs on the 3′ ends of the miRNAs facilitates intracellular delivery of miRNAs into tumor cells and subsequent target of their target genes as well as protection of miRNAs from enzymatic degradation in serum. Example 1: Production of Cell Internalizing Nucleic Acid Compounds Via Covalent Linkage to Phosphorothioated SSDNA Oligonucleotides miRNAs with naturally occurring sequences were fused covalently to phosphorothioated ssDNA (PS) 20meric oligo to facilitate cellular internalization targeting intracellular molecular targets. A non-phosphorothioated, phosphodiester ssDNA oligo (PO) extension of the miRNAs was employed as a non-internalizing control. Applicants modified naturally occurring miRNAs, for example, let7a-3p (SEQ ID NO:1) (FIG.1), let7a-5p (SEQ ID NO:2) (FIG.3), miR17-3p (SEQ ID NO:3) (FIG.5), miR17-5p (SEQ ID NO:4) (FIG.7), and miR218-5p (SEQ ID NO:5) (FIG.9) by attaching a phosphorothioated ssDNA (PS) 20meric oligo to the 3′ end of the miRNAs via a chemical linker. Examples of a phosphorothioated ssDNA (PS) 20meric oligo include, but are not limited to, SEQ ID NO:6 (TCCATGAGCTTCCTGATGCT) and SEQ ID NO:7 (AGCATCAGGAAGCTCATGGA). Applicants designed that the modification by ssDNA oligo avoids any C/G or G/C motifs, because it is known that CpG oligodeoxynucleotides (CpG-ODN) involve undesired Toll-like receptor (TLR) engagement and subsequent intracellular signaling. Applicants used an alkyl chain harboring a fluorophore as a linker to track the conjugate molecule. Example 2: Modified MIRNA Elongated with Phosphorothioated SSDNA Oligonucleotides Undergo Cellular Internalization Once miRNAs were modified by elongation and fluorescently marked to enable intracellular tracking of modified miRNAs, Applicants assessed cellular internalization of PS-modified miRNAs by flow cytometry including PO-modified miRNA as negative non-internalizing controls. Human multiple myeloma cells MM.1 S were incubated either for 30 min or for 48 hrs with modified miRNA as indicated and analyzed by flow cytometry to assess cellular load of cells with modified miRNA. For modified let7a-3p miRNA (FIGS.2A and2B) and modified let7a-5p miRNA (FIGS.4A and4B), 10 μg/ml was used for both 30 min and 48 hr incubation. For miR17-3p miRNA (FIGS.6A and6B), modified miR17-5p miRNA (FIGS.8A and8B) and modified miR218-5p miRNA (FIGS.10A and10B), 20 μg/ml was used for 30 min incubation and 10 μg/ml was used for 48 hr incubation, respectively. Example 3: Modified MIRNA Elongated with Phosphorothioated SSDNA Oligonucleotides Reducing STAT3 Target Gene Expression Human multiple myeloma cancer cells are known to undergo increased cell division through IL-6-triggered STAT3 signaling. Numerous studies have shown that let7a-3p miRNA (SEQ ID NO:1), let7a-5p miRNA (SEQ ID NO:2), miR17-3p miRNA (SEQ ID NO:3), miR17-5p miRNA (SEQ ID NO:4), or miR218-5p miRNA (SEQ ID NO:5) inhibits the activity of transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Human multiple myeloma cells MM.1S were incubated for 48 hrs daily with 10 μg/ml modified miRNA as indicated and expression of the STAT3 target genes was analyzed by RT-PCR. As shown inFIGS.2C,4C,6C,8C and10C, incubation with PS-modified let7a-3p miRNA (SEQ ID NO:1), let7a-5p miRNA (SEQ ID NO:2), miR17-3p miRNA (SEQ ID NO:3), miR17-5p miRNA (SEQ ID NO:4), or miR218-5p miRNA (SEQ ID NO:5) inhibited expression of STAT3 target genes, for example, oncogenic Bcl-xL and/or IL-6 genes. Example 4: Production of Cell Internalizing Nucleic Acid Compounds Via Covalent Linkage to Phosphorothioated Single-Stranded Abasic Sugar-Phosphate Backbone Polymers miRNAs with naturally occurring sequences were fused covalently to a phosphorothioated single-stranded abasic sugar-phosphate backbone (PS) 20meric polymer to facilitate cellular internalization targeting intracellular molecular targets. A non-phosphorothioated, phosphodiester single-stranded abasic sugar-phosphate backbone polymer (PO) extension of the miRNAs was employed as a non-internalizing control. Applicants modified naturally occurring miRNAs, for example, let7a-5p (SEQ ID NO:2) by attaching a phosphorothioated single-stranded abasic sugar-phosphate backbone (PS) 20meric polymer to the 3′ end of the miRNAs via a chemical linker (FIG.11A). Applicants designed the modification based on that phosphorothioated ssDNA oligo enables successful intracellular delivery, but bases (nucleic acids) may not be required to facilitate intracellular delivery of the conjugate and thus may be excluded.FIG.11Bschematically shows the abasic sugar-phosphate module (referred as “D”) lacking a base (a nucleic acid) in comparison to basic “spacers.” Applicants used an alkyl chain harboring a fluorophore as a linker to track the conjugate molecule. Example 5: Modified MIRNA Elongated with Phosphorothioated Single-Stranded Abasic Sugar-Phosphate Backbone Polymers Undergo Cellular Internalization Once miRNAs were modified by elongation and fluorescently marked to enable intracellular tracking of modified miRNAs, Applicants assessed cellular internalization of PS polymer-modified miRNAs by flow cytometry including PO polymer-modified miRNA as negative non-internalizing controls. Human multiple myeloma cells MM.1S were incubated for 30 min (FIG.12A) with 20 μg/ml polymer-modified let7a-5p miRNA as indicated and analyzed by flow cytometry to assess cellular load of cells with modified miRNA. Example 6: Modified MIRNA Elongated with Phosphorothioated Single-Stranded Abasic Sugar-Phosphate Backbone Polymers Reducing Stat3 Target Gene Expression Human multiple myeloma cancer cells are known to undergo increased cell division through IL-6-triggered STAT3 signaling. Numerous studies have shown that let7a-5p miRNA (SEQ ID NO:2) inhibits the activity of Signal Transducer and Activator of Transcription 3 (STAT3). Human multiple myeloma cells MM.1S were incubated for 48 hrs daily with 10 μg/ml polymer-modified let7a-5p miRNA as indicated and expression of the STAT3 target gene, oncogenic Bcl-xL gene, was analyzed by RT-PCR. As shown inFIG.12B, incubation with PS polymer-modified let7a-5p miRNA inhibited expression of Bcl-xL gene. REFERENCES Herrmann, A., Nachaev, S., Lahtz, C., Armstrong, B., Kowolik, C., Kortylewski, M., Jove, R., and Hua, Y. (2014). STAT3 nuclear egress requires exportin 7 via engaging lysine acetylation. MOJ Cell Sci Report 1(1): 00004. DOI: 10.15406/mojcsr.2014.01.00004. P EMBODIMENTS P Embodiment 1. A cell penetrating nucleic acid conjugate comprising:(i) a non-cell penetrating ribonucleic acid compound comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;(ii) a phosphorothioate polymer; and(iii) a chemical linker attaching said phosphorothioate polymer to the 3′ end of said non-cell penetrating ribonucleic acid compound; wherein said phosphorothioate polymer enhances intracellular delivery of the non-cell penetrating nucleic acid compound. P Embodiment 2. The conjugate of P Embodiment 1, wherein said non-cell penetrating ribonucleic acid compound is a micro RNA (miRNA). P Embodiment 3. The conjugate of P Embodiment 1 or 2, wherein said non-cell penetrating ribonucleic acid compound is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more residues in length. P Embodiment 4. The conjugate of any one of P Embodiments 1-3, wherein said non-cell penetrating ribonucleic acid compound is from about 20 to about 30 residues in length. P Embodiment 5. The conjugate of any one of P Embodiments 1-4, wherein said non-cell penetrating ribonucleic acid compound is from about 20 to about 25 residues in length. P Embodiment 6. The conjugate of any one of P Embodiments 1-5, wherein said non-cell penetrating ribonucleic acid compound is 21, 22 or 23 residues in length. P Embodiment 7. The conjugate of any one of P Embodiments 1-6, wherein said phosphorothioate polymer is a phosphorothioate nucleic acid or an abasic sugar-phosphorothioated polymer. P Embodiment 8. The conjugate of any one of P Embodiments 1-7, wherein said phosphorothioate polymer is a phosphorothioate deoxyribonucleic acid. P Embodiment 9. The conjugate of any one of P Embodiments 1-8, wherein said phosphorothioate polymer is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more residues in length. P Embodiment 10. The conjugate of any one of P Embodiments 1-9, wherein said phosphorothioate polymer is from about 10 to about 30 residues in length. P Embodiment 11. The conjugate of any one of P Embodiments 1-10, wherein said phosphorothioate polymer is about 20 residues in length. P Embodiment 12. The conjugate of any one of P Embodiments 1-11, wherein said phosphorothioate polymer comprises the sequence of SEQ ID NO:6 or SEQ ID NO:7. P Embodiment 13. The conjugate of any one of P Embodiments 1-12, wherein said phosphorothioate polymer is single-stranded. P Embodiment 14. The conjugate of any one of P Embodiments 1-13, wherein said chemical linker is a covalent linker. P Embodiment 15. The conjugate of any one of P Embodiments 1-14, wherein said chemical linker is a non-immunogenic linker. P Embodiment 16. The conjugate of any one of P Embodiments 1-15, wherein said conjugate further comprises a detectable moiety. P Embodiment 17. The conjugate of P Embodiment 16, wherein said detectable moiety is attached to said non-cell penetrating ribonucleic acid compound. P Embodiment 18. The conjugate of P Embodiment 16, wherein said detectable moiety is attached to said phosphorothioate polymer. P Embodiment 19. The conjugate of any one of P Embodiments 1-18, wherein said non-cell penetrating ribonucleic acid compound inhibits STAT3 activity relative to a standard control. P Embodiment 20. The conjugate of any one of P Embodiments 1-19, wherein said non-cell penetrating ribonucleic acid compound inhibits expression of a STAT3 target gene relative to a standard control. P Embodiment 21. The conjugate of P Embodiment 20, wherein said STAT3 target gene is an oncogene. P Embodiment 22. The conjugate of P Embodiment 20, wherein said STAT3 target gene is Bcl-xL or IL-6. P Embodiment 23. A cell comprising a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22. P Embodiment 24. A pharmaceutical composition comprising a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22 and a pharmaceutically acceptable carrier. P Embodiment 25. A method of treating cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby treating said cancer in said subject. P Embodiment 26. The method of P Embodiment 25, wherein said cancer is breast cancer, prostate cancer, ovarian cancer, brain cancer, pancreatic cancer, melanoma, colon cancer, gastric cancer, head-and-neck cancer, liver cancer, lung cancer, cervical cancer, sarcoma, leukemia, lymphoma, multiple myeloma. P Embodiment 27. The method of P Embodiment 26, said method comprising decreasing in said subject an expression level of BIRC5 or Bcl-xL relative to a standard control. P Embodiment 28. A method of increasing expression of p53 in a cancer cell, said method comprising contacting a cancer cell with an effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby increasing expression of p53 in said cancer cell. P Embodiment 29. A method of inhibiting tumor vascularization in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby inhibiting tumor vascularization in said subject. P Embodiment 30. A method of treating an inflammatory disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby treating an inflammatory disease in said subject. P Embodiment 31. The method of P Embodiment 30, said method comprising decreasing in said subject an expression level of FGA, IL1B or SERPINA3 relative to a standard control. P Embodiment 32. A method of treating pain in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby treating pain in said subject. P Embodiment 33. The method of P Embodiment 32, said method comprising decreasing in said subject an expression level of PTGS1, PTGS2, CALCA or SST relative to a standard control. P Embodiment 34. A method of inhibiting IL-6 signaling in a cell, said method comprising contacting a cell with an effective amount of a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby inhibiting IL-6 signaling in said cell. P Embodiment 35. A method of delivering a non-cell penetrating nucleic acid into a cell, said method comprising contacting a cell with a cell penetrating nucleic acid conjugate of any one of P Embodiments 1-22, thereby delivering said non-cell penetrating nucleic acid into said cell. INFORMAL SEQUENCE LISTINGlet7a-3p miRNA (SEQ ID NO: 1):CUAUACAAUCUACUGUCUUUClet7a-5p miRNA (SEQ ID NO: 2):UGAGGUAGUAGGUUGUAUAGUUmiR17-3p miRNA (SEQ ID NO: 3):ACUGCAGUGAAGGCACUUGUAGmiR17-5p miRNA (SEQ ID NO: 4):CAAAGUGCUUACAGUGCAGGUAGmiR218-5p miRNA (SEQ ID NO: 5):UUGUGCUUGAUCUAACCAUGUphosphorothioate polymer (SEQ ID NO: 6):TCCATGAGCTTCCTGATGCTphosphorothioate polymer (SEQ ID NO: 7):AGCATCAGGAAGCTCATGGASTAT3 polypeptide (SEQ ID NO: 8):MAQWNQLQQLDTRYLEQLHQLYSDSFPMELRQFLAPWIESQDWAYAASKESHATLVFHNLLGEIDQQYSRFLQESNVLYQHNLRRIKQFLQSRYLEKPMEIARIVARCLWEESRLLQTAATAAQQGGQANHPTAAVVTEKQQMLEQHLQDVRKRVQDLEQKMKVVENLQDDFDFNYKTLKSQGDMQDLNGNNQSVTRQKMQQLEQMLTALDQMRRSIVSELAGLLSAMEYVQKTLTDEELADWKRRQQIACIGGPPNICLDRLENWITSLAESQLQTRQQIKKLEELQQKVSYKGDPIVQHRPMLEERIVELFRNLMKSAFVVERQPCMPMHPDRPLVIKTGVQFTTKVRLLVKFPELNYQLKIKVCIDKDSGDVAALRGSRKFNILGTNTKVMNMEESNNGSLSAEFKHLTLREQRCGNGGRANCDASLIVTEELHLITFETEVYHQGLKIDLETHSLPVVVISNICQMPNAWASILWYNMLTNNPKNVNFFTKPPIGTWDQVAEVLSWQFSSTTKRGLSIEQLTTLAEKLLGPGVNYSGCQITWAKFCKENMAGKGFSFWVWLDNIIDLVKKYILALWNEGYIMGFISKERERAILSTKPPGTFLLRFSESSKEGGVTFTWVEKDISGKTQIQSVEPYTKQQLNNMSFAEIIMGYKIMDATNILVSPLVYLYPDIPKEEAFGKYCRPESQEHPEADPGSAAPYLKTKFICVTPTTCSNTIDLPMSPRTLDSLMQFGNNGEGAEPSAGGQFESLTFDMELTSECATSPM | 174,556 |
11857634 | DETAILED DESCRIPTION It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described example embodiments. Thus, the following more detailed description of the example embodiments, as represented in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely representative of example embodiments. Reference throughout this specification to “one embodiment” or “an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation. As used herein and in the appended claims, the singular forms “a,” “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a therapeutic compound” includes a plurality of such therapeutic compounds and equivalents thereof known to those skilled in the art, and so forth, and reference to “the therapeutic compound” is a reference to one or more such therapeutic compounds and equivalents thereof known to those skilled in the art, and so forth. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, and each separate value as well as intermediate ranges are incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text. As used herein, the term “polymer” refers to a chemical compound that is made of a plurality of small molecules or monomers that are arranged in a repeating structure to form a larger molecule. Polymers may occur naturally or be formed synthetically. The use of the term “polymer” encompasses homopolymers as well as copolymers. The term “copolymer” is used herein to include any polymer having two or more different monomers. Copolymers may, for example, include alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, graft copolymers etc. Examples of polymers include, for example, polyalkylene oxides. As used herein, the term “pendant” refers to a group or moiety attached to a backbone chain of a long molecule such as a polymer as described above. Pendant group may be either (1) short chain or low molecular weight groups or (2) long chain or high molecular groups such as polymers. Pendant groups are sometime referred to as side groups. Long chain pendant groups or high molecular weight pendant groups are sometimes referred to as “pendant chains” or “side chains”. In a number of embodiments, systems, formulations, methods and compositions hereof are provide for co-delivery of small molecule therapeutic agents or drugs (for example, chemotherapeutic therapeutic agents or drugs) and nucleic acid-based therapeutic agents or drugs simultaneously.FIGS.1and2illustrated schematically amphiphilic polymers hereof. The amphiphilic polymer may, for example, be formed via radical polymerization to have a hydrophobic polymer backbone. The hydrophobic polymer backbone may, for example, be formed via a free radical polymerization or via a reversible-deactivation radical polymerization or RDRP (formerly referred to a controlled radical polymerization or CRP). Reversible-Deactivation Radical Polymerization (RDRP) procedures include, for example, Nitroxide Mediated Polymerization (NMP), Atom Transfer Radical Polymerization (ATRP), and Reversible Addition Fragmentation Transfer (RAFT) and others (including cobalt mediated transfer) that have evolved over the last two decades. RDRP provide access to polymer and copolymers comprising radically polymerizable/copolymerizable monomers with predefined molecular weights, compositions, architectures and narrow/controlled molecular weight distributions. Because RDRP processes can provide compositionally homogeneous well-defined polymers, with predicted molecular weight, narrow/designed molecular weight distribution, and high degrees of α- and ω-chain end-functionalization, they have been the subject of much study, as reported in several review articles and ACS symposia. See, for example, Qiu, J.; Charleux, B.; Matyjaszewski, K.,Prog. Polym. Sci.2001, 26, 2083; Davis, K. A.; Matyjaszewski, K.Adv. Polym. Sci.2002, 159, 1; Matyjaszewski, K., Ed. Controlled Radical Polymerization; ACS: Washington, D.C., 1998; ACS Symposium Series 685. Matyjaszewski, K., Ed.; Controlled/Living Radical Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACS Symposium Series 768; and Matyjaszewski, K., Davis, T. P., Eds. Handbook of Radical Polymerization; Wiley: Hoboken, 2002, the disclosures of which are incorporated herein by reference. The hydrophobic polymer backbone may be formed via radical polymerization of radically polymerizable monomers (including conventional of free radical polymerization as well as RDRP). Such monomers may include pendant groups as described above prior to polymerization. Alternatively, such pendant groups may be attached after polymerization. Representative monomers for use herein include styrene, acrylic acid, methacrylic acid, acrylonitrile, vinyl monomers and their derivatives. In a number of embodiments, the degree of polymerization for hydrophobic polymers hereof is, for example, less than 500. In a number of embodiments, the polymer further includes a first plurality of pendant groups (C) attached to the hydrophobic polymer backbone and including at least one cationic group and a second plurality of pendant groups attached to the hydrophobic polymer backbone and including at least one hydrophilic polymer (P). Representative examples of pendant groups (C) are set forth inFIGS.3A and3B. Pendant group hereof may also include both at least one cationic group and at least one hydrophilic polymer. In a number of embodiments, at least one of the first plurality of pendant groups and the second plurality of pendant groups is attached to the hydrophobic polymer backbone via a linking moiety. The linking moiety may include at least one group interactive via π-π stacking. The first plurality of pendant groups may, for example, be attached to the hydrophobic polymeric backbone via a first linking group (L1). The first linking group (L1) may, for example, include at least a first group which is interactive via π-π stacking. Representative examples of first linking group (L1) are illustrated inFIG.3C. The second plurality of pendant groups (L2) may also be attached to the hydrophobic polymer backbone via a second linking group. The second linking group (L2) may, for example, independently include at least a second group interactive via π-π stacking. Representative examples of the second linking group (L1) are illustrated inFIG.3D, wherein linker groups (L2) are attached to a hydrophilic polymer P in the form of polyethylene glycol of PEG. The first linking group (L1) and/or the second linking group (L2) may, for example, include an aromatic group. In general, aromatic groups are cyclic molecules including resonance bonds that exhibit increased stability compared to other geometric or connective arrangements with the same set of atoms. Aromatic groups include, for example, benzyl and naphthyl groups. In a number of embodiments hereof, aromatic groups hereof are benzyl groups. The at least one cationic group may, for example, include an inherently cationic group or a group which forms a cation in the formulations hereof and/or in vivo (for example, an amine group which forms a cation in vivo). The amine group may be an acyclic amine group, a cyclic amine group or a heterocyclic amine group. The at least one cationic group may, for example, be selected from the group consisting of a metformin group, a morpholine group, a piperazine group, a pyrrolidine group, a piperidine group, a thiomorpholine, a thiomorpholine oxide, a thiomorpholine dioxide, imidazole, guanidine or creatine. In a number of embodiments, the at least one cationic group is selected from the group consisting of a metformin group, a morpholine group, a piperazine group or creatine. The cationic amine groups described herein may be substituted or unsubstituted. The hydrophilic oligomer or the hydrophilic polymer may, for example, be selected from the group consisting of a polyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline, a polysaccharide and a polypeptide. In a number of embodiments, the at least one hydrophilic polymer is a polyalkylene oxide. The polyalkylene oxide may, for example, be a polyethylene glycol. A polyethylene glycol or other hydrophilic polymer hereof may, for example, have a molecular weight of at least 500 Da. In a number of embodiments, the polyethylene glycol of other hydrophilic polymer hereof has a molecular weight in the range of 200 Da to 10 KDa or a range of 500 to 5 KDa. Pendant groups hereof (such as the second plurality of pendant groups) which include a hydrophilic polymer may, for example, be attached to the hydrophobic polymer backbone via a linking moiety that is labile under in vivo conditions (for example, under acidic pH conditions). The labile bond may, for example, be sensitive to conditions in a target region (for example, sensitive to or labile under acidic conditions in the region of a tumor). An acid-labile bond may, for example, include a carboxydimethyl maleate, a hydrazine, an imine, an acetal, an oxime, a silyl ether, a cis-asonityl or another acid-labile bond or linkage. Use of a labile bond that is sensitive to acidic conditions may be used to cleave the hydrophilic polymer/oligomer in, for example, an acidic tumor environment. Examples of other suitable labile bonds include disulfide bonds, hypoxia sensitive bonds and glucose-sensitive bonds. A formulation hereof may, for example, include a plurality of polymers (as described above) which are formed via radical polymerization to have a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and including at least one cationic group and a second plurality of pendant groups attached to the hydrophobic polymer backbone and including at least one hydrophilic polymer. The formulation may further include a first therapeutic compound and a second therapeutic compound, wherein the second therapeutic compound is different from the first therapeutic compound and includes a nucleic acid. The first therapeutic compound may, for example, be a hydrophobic compound that does not include a nucleic acid. The first therapeutic compound may, for example, be a small molecule therapeutic compound. Such small molecule therapeutic compounds may, for example, have a molecular weight below 2.0 kDa, below 1.5 kDa or below 1.0 kDa. The second therapeutic compound may, for example, include RNA or DNA. In a number of embodiments, the second therapeutic compound include includes a gene or siRNA. In general, any nucleic acid including negative charges may be used in the formulation hereof including naturally occurring and synthetic nucleic acids (for example, RNA, DNA, locked DNA etc.). In formulations for delivery of compounds in vivo, a plurality of polymers hereof may assemble/self-assemble into structures. A plurality of polymers hereof may, for example, form micelles. Without limitation to any mechanism, the plurality of nucleic acid compounds may interact with cationic groups of the first plurality of pendant groups to stabilize such micelles. FIG.4sets forth an idealized schematic representation of a nucleic acid-base therapeutic agent, compound or drug (for example, a gene, a plasmid, siRNA etc.) and a small-molecule therapeutic agent, compound or drug loading onto a micelle carrier structure formed with the polymers hereof.FIG.4further illustrates multifunctional nanocarriers hereof as co-delivery platforms for cells such as cancer cells. As used herein a therapeutic agent or drug is a biologically active substance which has an effect on the body (for example, a medicinal or therapeutic effect, an intoxicating effect, a performance enhancing effect or another effect). A therapeutic agent may, for example, be an antibody, an antibiotic, an antiviral, an antimycotic, an anticancer agent, an immunosuppressant, a chemotherapeutic agent, an anti-rejection agent, an analgesic agent, or an anti-inflammatory agent. Small molecule drugs suitable for use herein include, but are not limited to JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporin A, a protoporphyrin, GW4064 (3-(2,6-Dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, curcumin, indomethacin, tamoxifen. NLG-919 (an indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, fluorouracil (5-FU), a c-Myc inhibitor such as 10058-F4 (5-[(4-Ethylphenyl)methylene]-2-thioxo-4-thiazolidinone) or doxorubicin. Without limitation to any mechanism, and with reference toFIG.4, it is hypothesized that an inwardly oriented hydrophobic domain is created during micelle formation via the hydrophobic backbone of the polymers hereof, which may orient via intrachain hydrophobic interactions to assume a folded conformation. Pendant aromatic groups, when present, increase hydrophobicity and assist in forming the hydrophobic domain. It was further hypothesized that an outwardly oriented hydrophilic domain was formed by the hydrophilic polymer side chains. Cationic groups likely assume a conformation to orient toward the hydrophilic domain near the interface of the hydrophobic and hydrophilic domains or intermediate therebetween. In a number of embodiments, delivery systems prior to the present invention were designed for delivery of either small molecule drugs or nucleic acid-based therapeutics. In the present studies, we have developed a simple micellar system that is highly effective in codelivery of small molecules drugs and nucleic acids (for example, siRNA, genes, plasmids, etc.). In addition to providing a platform for new combination therapies that involves both small molecule drug therapeutics and nucleic acid therapeutics, the carrier systems hereof also resolve an issue of instability that is associated with most micellar carrier systems. Micelles are formed through the self-assembly of amphiphilic monomers, which is a reversible and dynamic process. Micelles tend to fall apart when they are diluted in the blood upon systemic administration. This instability can be further aggravated by lipid exchange as a result of interactions with lipoproteins in the blood. A number of strategies have been reported to create cross-linking bonds between amphiphilic molecules to stabilize the micelles. Some of the approaches involve a complicated procedure and/or modification of the structure of the amphiphilic molecules. In the systems hereof, multivalent charge-charge interactions between the cationic groups of the amphiphilic polymer molecules and nucleic acids serve as a simple approach to create interactive, non-covalent crosslinks between amphiphilic polymeric molecules of the micelles hereof. Moreover, π-π interactions stacking between the amphiphilic polymer molecules and nucleic acids may additionally or alternatively be used to create interactive, non-covalent crosslinks between amphiphilic polymeric molecules of the micelles hereof π-π interactions or stacking between the groups of amphiphilic polymers forming micelles and numerous compounds such as drugs are, for example, discussed in U.S. Pat. Nos. 10,172,795 and 9,855,341 and U.S. Patent Publication No. 2018/0214563, the disclosure of which are incorporated herein by reference. As a result, micelles hereof that are co-loaded with small molecules and nucleic acid-based molecules are more stable than free micelles or the micelles that are loaded with small molecules alone. In several representative embodiment, a multifunctional delivery system hereof based on an amphiphilic polymer with morpholine attached to the pendant side chains (POEG-st-Pmor; wherein POEG represents polyoxyethylene glycol and mor represents morpholine) for codelivery of small molecule chemotherapy drugs and plasmid DNA. The hydrophobic anticancer drugs may, for example, be incorporated into the hydrophobic core through hydrophobic-hydrophobic interaction and π-π stacking. Morpholine is incorporated into the polymer to introduce positive charges to form complexes with nucleic acids as described above. The positively charged morpholine groups in the polymers may also facilitate the accumulation of the carrier in the lung as a result of the interaction of positively charged tertiary amine with negatively charged cell membrane in the lung. In a number of representative studies, doxorubicin (DOX) and IL-36γ plasmid were selected as a model/representative drug and model/representative nucleic acid (DNA), respectively for study of combination therapies hereof. DOX is a first-line chemotherapeutic drug in the treatment of a broad range of cancers including breast, ovary, bladder, and lung cancers, and breast cancer metastasis. Cytokines are reported to have a synergistic antitumor effect in combination with conventional antitumor treatments such as chemotherapy. Interleukin 36 (α, β, γ) belongs to IL-1 family of cytokines, and the three isoforms share the same receptor complex. IL-36γ is reported to promote the differentiation of type 1 effector lymphocytes, including CD8+, NK, and γδT cells in vitro. The tumoral expression of IL-36γ exerts strong antitumor immune responses in vivo and transforms the tumor microenvironment in favor of tumor eradication. Codelivery of DOX and IL-36γ plasmid via the multifunctional carrier hereof represents a simple and effective approach for the treatment of lung metastasis. The biophysical properties of the nanocarrier co-loaded with DOX and IL-36γ plasmid was first examined. The efficiency of delivery and transfection was then examined both in vitro and in vivo. Finally, the antitumor effect of DOX+IL-36γ plasmid/polymer as well as the underlying mechanism was investigated. The synthesis scheme for POEG-st-Pmor polymer was shown inFIG.5. First, VBMor monomer was synthesized by reacting vinylbenzyl chloride with morpholine. The structure of VBMor monomer was confirmed by 1H NMR. Then, POEG-st-Pmor copolymer was prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization of OEG500 monomer and VBMor monomer. The structures and molecular weight of POEG-st-Pmor polymer were characterized by 1H NMR and gel permeation chromatography (GPC). The average degree of polymerization (DP) of the OEG500 monomer was calculated to be 10 according to the conversion of OEG500 monomer at the end of the polymerization (conversation: 70%). The DP of the VBMor monomer was determined to be 60 by comparing intensities. The average molecular weight Mn of POEG-st-Pmor polymer determined by GPC was 9260, and the polydispersity is 1.13 as determined by gel permeation chromatography, which indicated the successful synthesis of POEG-st-Pmor copolymer with well-defined structure. Although a narrow polydispersity (for example, below 1.75, below 1.5 or below 1.25) may result in formation of more uniform micelles, it is not clear whether in vivo performance of formulation hereof is improved with narrower polydispersity. POEG-st-Pmor micelles were prepared via a dialysis method. As shown inFIGS.6A and6B, POEG-st-Pmor micelles had sizes around 200 nm as tested with a zetasizer. Spherical structure was observed by TEM. The size tested by TEM is smaller than that by DLS, which may be a result the different principles of analysis and the shrinkage of dried micellar nanoparticles during TEM measurement. The critical micelle concentration (CMC) of the polymer was determined using nile red as a fluorescence probe. POEG-st-Pmor has a low CMC around 0.04 mg/ml. The DOX-loaded POEG-st-Pmor micelles were similarly prepared as blank micelles. POEG-st-Pmor carrier could load DOX at a carrier/drug mass ratio starting from 10:1 with sizes ranging from 160 to 190 nm (FIGS.6A and6B, Table 1, setting forth Biophysical characteristics of blank micelles and the micelles co-loaded with IL-36γ plasmid and DOX) and the formulation could remain stable for two weeks at room temperature. Then we tested whether the POEG-st-Pmor could form stable complexes with plasmid DNA. A gel retardation assay was performed to assess the pDNA binding ability of the pMor-based polymer. Plasmid DNA/carrier complexes were fabricated at N/P ratios from 0.1:1 to 30:1. As known to those skilled in the art, the N/P ratio refers to the number of cationic carrier nitrogens/to DNA phosphates. Complete complexation of plasmid DNA by POEG-st-Pmor polymer was achieved at an N/P ratio of 5/1 or greater. To further study the interaction between DNA and carrier, a competitive binding gel-shift assay with dextran sulfate was performed. Substantial amounts of DNA were released from control PEI/DNA complexes at an S/P ratio (molar ratio between the sulfur from dextran sulfate and the phosphate from pDNA) as low as 15/1. In contrast, no obvious release of DNA was observed for POEG-st-Pmor/DNA complexes at an S/P ratio as high as 40/1. TABLE 1MassZetaratioN/PSizepotentialMicelles(mg:mg)ratio(nm)(mV)StabilityPOEG-st-Pmor——184 ± 3.526.4 ± 1.1—IL-36γ/—2095.0 ± 1.810.3 ± 0.1—POEG-st-Pror10:1—178 ± 2.127.1 ± 0.72weeksDOX/20:1—174 ± 1.524.7 ± 0.94weeksPOEG-st-Pmor30:1—162 ± 4.126.01 ± 0.71monthDOX+IL-36γ/20:12084.4 ± 1.710.4 ± 0.21monthPOEG-st-Pmor*The colloidal stability was followed at room temperature by measuring the size and observing precipitates. The surface zeta potential of the POEG-st-Pmor blank micelles was 26.4 mV before the addition of IL-36γ plasmid (FIGS.7A and7Band Table 1). At the N/P ratios of below 1, the complexes were negatively charged and the particle sizes were similar to the sizes of the micelles alone. There was a significant increase in the sizes of the complexes at an N/P ratio of 3. At this ratio, the particle charges were close to neutral. Further increases in the N/P ratios led to a significant decrease in the particle sizes and the particles became more positively charged with continuous increases in the N/P ratios. Specifically, when the micelles were mixed with IL-36γ plasmid at an N/P ratio of 20:1, the average size of the complexes decreased to 70-80 nm (Table 1,FIG.7A). Nonetheless, DNA/micelle complexes were less positively charged compared to free micelles with a surface zeta potential of 10.3 mV (Table 1). We then went on to further explore the possibility of co-delivery of pDNA and DOX by POEG-st-Pmor micelles. As shown inFIG.6Aand Table 1, the size distribution and zeta-potential were not significantly affected when DOX was loaded into the pDNA/polymer complexes at a drug concentration of 1 mg/mL and a carrier/drug ratio of 20/1 (w/w). The colloidal stability of micelle/DNA complexes was tested in BSA solution (30 mg/mL). PEI/DNA complexes were used as a control (N/P=20, zeta potential=18.3 mV). As shown inFIG.7C, exposure of PEI/DNA complexes to BSA led to a rapid increase in the particle sizes. At 5 h post-incubation, the sizes of PEI/DNA complexes increased from 149 to 261 nm. It is also apparent that POEG-st-Pmor/DNA complexes were resistant to BSA-induced aggregation and showed minimal changes in sizes throughout the entire 18 h of observation. The profile of DOX release from the DOX-loaded micelles was examined by a dialysis method with DOX.HCl solution as a control (FIG.7D). Free DOX was rapidly diffused out of the dialysis tubing with 80% of DOX found in the dialysate in the first 4 hours. However, DOX formulated in POEG-st-Pmor micelles showed a slow kinetics of release with less than 25% of DOX released outside of dialysis bag at the first 4 hours, and only 35% of DOX released at 24 h. The micelles co-loaded with DOX and plasmid exhibited an even slower DOX release profile compared to micelles loaded with DOX alone at later time points. The polymer concentration inside the dialysis bag was far above the CMC value and the micelles were stable throughout the entire course of release study. Most delivery systems developed so far are designed for delivery of either small molecule drugs or nucleic acid-based therapeutics. The readily formulized micellar systems hereof are highly effective in codelivery of both small molecules and nucleic acids (for example, plasmid DNA). Various mechanisms are likely to be involved in the formation of free micelles and drug- or drug/gene-loaded micelles. While the hydrophobic interaction and π-π stacking drive the formation of a compact particle, the positive charge-mediated repulsion may impose a negative force on the formation of such particle. Loading of DOX into the micelles led to a small decrease in particle sizes (180 vs 160 nm): this may, for example, be a result of formation of a more compacted particle, which is facilitated by the carrier-DOX interactions. The particle size was more dramatically reduced following the complexation of the micelles with plasmid DNA in the presence or absence of DOX: the size decreased from 180 nm to about 100 nm. The interaction of large-sized plasmid DNA with the positively charged micelles results in a non-covalent cross-linking of micelles. In addition, charge neutralization of DNA is known to induce DNA condensation. Together, these characteristics drive the formation of a more compact hydrophobic core and thus significantly decreased particle size. The significantly increased particle size at an N/P ratio of 3/1 may, for example, be a result of a status of neutral surface, which drives the aggregation of the nanomicelles. On the other hand, the excess surface positive charges at N/P ratios of above 3/1 help to maintain the colloidal stability of the “condensed” nanomicelles through charge-mediated repulsion. Another advantage provided by the nanomicellar formulation hereof is improved stability. As described above, micelles are formed through the self-assembly of amphiphilic monomers, which is a reversible and dynamic process. Once again, micelles tend to fall apart when they are diluted in the blood upon systemic administration. This instability can be further aggravated by lipid exchange as a result of interactions with lipoproteins in the blood. In the systems hereof, the multivalent charge-charge interactions between the cationic polymer and nucleic acid (plasmid DNA, in the current example) serve as a simple approach to “cross-link” the micelles. As a result, micelles that are co-loaded with small molecules and plasmid DNA are likely to be more stable than free micelles or the micelles that are loaded with small molecule alone. This observation is supported by the data from DOX release studies. DOX formulated in the co-loaded nanoparticles exhibited a slower kinetics of release compared to the formulation that is loaded with DOX only (FIG.7D). The in vitro cytotoxicity of DOX+IL36γ plasmid/micelle complexes was evaluated with 4T1.2 breast cancer cells via MTT assay. Cells received various treatments for 72 h and the final concentrations of DOX ranged from 4 to 1000 ng/mL (FIG.8A). The free DOX and the DOX-loaded micelles showed a dose-dependent cell killing profile. The DOX/POEG-st-Pmor micelles had lower IC50s (60 ng/ml) compared to free DOX (130 ng/ml) (FIG.8A). Incorporation of IL36γ plasmid into DOX-loaded micelles led to slightly increased cytotoxicity on 4T1.2 cells. POEG-st-Pmor alone showed minimal cytotoxicity to 4T1.2 cells at the polymer concentration as high as 20 μg/mL (FIG.8B). The cellular uptake of DOX-loaded POEG-st-Pmor micelles was investigated by confocal laser scanning microscopy with free DOX as a control. At 2 h of incubation, DOX-loaded POEG-st-Pmor micelles showed more DOX cellular uptake compared to free DOX at the same DOX concentration. The signals for free DOX were largely found in the nucleus while the signals for the micellar DOX were mainly located outside the nucleus. This may, for example, be a result of different cellular uptake routes of free DOX and DOX micelles. Using EGFP as a reporter gene, we investigated if POEG-st-Pmor carrier could effectively deliver EGFP plasmid into 4T1.2 cultured cells and tumor tissues, leading to expression of biologically active protein. The studies showed that 4T1.2 tumor cells were effectively transfected with EGFP plasmid complexed with POEG-st-Pmor micelles. 4T1.2 cells were also effectively transfected with branched PEI, a control carrier. In studies of the in vivo EGFP expression in tumor, lung and liver tissues, there were significantly more EGFP signals in tumors with POEG-st-Pmor formulation compared to linear PEI. In agreement with previous reports, lungs could be effectively transfected by linear PEI. However, more and stronger signals of EGFP were observed in lungs transfected with POEG-st-Pmor formulation. The liver was hardly transfected with either the formulation hereof or the control linear PEI. These results indicate that POEG-st-Pmor polymer is suitable for in vivo gene delivery to both lungs and distant solid tumors. The complexes of DNA with POEG-st-Pmor polymer were significantly more stable than PEI/DNA complexes following exposure to BSA (30 mg/mL). This may, for example, be a result of the dynamic shielding of the complexes by PEG despite the fact that both complexes remain positively charged. The improved stability of the complexes in the presence of serum proteins may contribute to the efficient delivery of DNA to distant subcutaneous or s.c. tumors. A more effective accumulation in the lung may, for example, be a result of the interaction of tertiary amine moiety with negatively charged cell membrane in the lung. Amine-containing basic compounds have been reported to be predominantly accumulated in the lung due to the specific binding to acidic phospholipids on the cell membrane, which is abundantly distributed in lung tissue. Therefore the carriers hereof are suitable for codelivery of nucleic acid therapeutics and small molecule drugs to both distant solid tumors and lung metastatic lesions. POEG-st-Pmor was more effective than PEI in transfecting either lungs or distant s.c. tumor tissues in vivo. The higher in vivo transfection efficiency of POEG-st-Pmor polymer may, for example, be a result of the enhanced stability of DNA/POEG-st-Pmor complexes in blood circulation due to the PEG shielding. It is also possible that POEG-st-Pmor form more stable complexes with DNA compared to PEI. Single or double-strand DNA may bind to nanoparticles through electrostatic, π-π stacking and hydrophobic interactions or even central cavity insertion. In addition to charge-charge interaction, the hydrophobic backbone of the polymers hereof and the pendant benzene rings can further interact with the base π-systems of nucleic acids through π-π stacking and hydrophobic interactions. This observation was supported by results from competitive binding gel-shift assay studies showing that the pDNA could hardly be released from POEG-st-Pmor micelles by dextran sulfate at an S/P ratio as high as 60. In contrast, substantial amounts of DNA were readily released from DNA/PEI complexes by dextran sulfate at an S/P ratio as low as 15/1. A mouse model of breast cancer lung metastasis (4T1.2) was generated in female Balb/c mice and various treatments were given to each group of mice via tail vein injection (FIG.9A). The carrier alone did not show therapeutic activity compared to control group. Free DOX showed a significant inhibition of lung metastasis and the antitumor activity was further enhanced for micelles co-loaded with DOX and IL-36γ plasmid compared to free DOX, DOX+control plasmid/micelles and IL-36γ plasmid/micelles. The H&E (haemotoxylin and eosin) staining of lung tissues showed clear tumor cell infiltration in all of the groups except the co-delivery group. The group with more tumor nodules had more lung weights (FIGS.9B and9C). Body weights were also monitored during the treatment period. No significant decrease of body weight was observed, indicating the safety of the formulation (FIG.9D). POEG-st-Pmor alone was not active in the 4T1.2 lung metastasis model. Free DOX or delivery of IL-36γ alone via POEG-st-Pmor polymer showed a significant activity in inhibiting the lung metastasis. Combination of the two led to a further improvement in antitumor activity as shown by both smallest number of tumor nodules in the lung and the lowest weights of the tumor-bearing lungs (FIGS.9B and9C). Following demonstration of the significant antitumor activity of DOX+IL-36γ plasmid/POEG-st-Pmor, we examined the immune cell infiltration in the tumor-bearing lungs to elucidate a role of immune response in the overall antitumor activity. As shown inFIGS.10A and10B, there was a significant increase of cytotoxic CD4+T and CD8+T cells in the lung tissues treated with free DOX, IL-36γ plasmid/POEG-st-Pmor or the combination of both compared to untreated control group. Although there was no significant difference in the total number of T cells between DOX+IL-36γ plasmid/POEG-st-Pmor and DOX+control plasmid/POEG-st-Pmor, the numbers of IFN-γ+CD4+and IFN-γ+CD8+T cells were significantly increased in the combination treatment group compared to either of the other treatment groups (FIGS.10C and10D). We also examined the CD11+Gr-1+immunosuppressive myeloid-derived suppressor cells (MDSCs) in lung tissues. The numbers of MDSCs were significantly decreased in all treatment groups except the carrier alone group (FIG.10E). Surprisingly, there was a significant increase in the number of Foxp3+CD4+T cells (regulatory T cells (Treg)) in the mice treated with IL-36γ plasmid, alone or in combination with DOX (FIG.10F). DOX is known to induce immunogenic cell death of tumor cells and enhance the recruitment of T cells, which was consistent with flow studies (FIGS.10A and10B). On the other hand, IL-36γ is effective in promoting the function of T cells through enhancing the production of IFN-γ as shown inFIGS.10C and10D. The synergistic effect between DOX and IL-36γ (s10C and10D) may play an important role in the overall antitumor activity. In another representative embodiment hereof, a co-delivery system (a nanomicellar carrier) for a representative combination of DOX and miR-34a (an oncogenic microRNA or mRNA) was developed which included the natural and endogenous cationic molecule creatine. An important function of creatine is to facilitate recycling of ATP primarily in muscle and brain tissues. It is, for example, widely used by athletes as an ergogenic aid to enhance anaerobic exercise performance. Typically, creatine is produced endogenously or obtained through the diet at a rate of 1 g per day in young adults. Excess amounts of creatine could be metabolized to creatinine and excreted through kidney. MicroRNAs, sometimes referred to a miRNAs or miRs, are short endogenous non-coding RNAs, responsible for post-transcriptional regulation of many target genes that are involved in cancer cell proliferation and tumor progression. As a result of the imperfect complementarity with target mRNAs, miRs are capable of regulating a broad set of genes simultaneously, which benefits the treatment of cancer as a heterogenic disease. Therefore, there has been growing interest in developing miR-based therapies. In particular, expression level of tumor suppressive miR-34a is usually downregulated in cancerous tissues, which could be reintroduced into cancerous tissues to achieve replacement therapy. Recent studies have revealed that introduction of exogenous miR-34a into cancer cells induced cell apoptosis and inhibited cell proliferation and migration through targeting Bcl-2, CD44, SIRT1, Rac1, Fra-1, Notch-1, and various cyclins. In addition, miR-34a has been reported to sensitize breast cancer cells to first line chemotherapies, including doxorubicin (DOX), paclitaxel, and 5-FU. Moreover, a liposome-formulated miR-34a, namely “MRX34”, has entered Phase I clinical trials for the treatment of unresectable primary liver cancer. A significant limitation for miR-based therapy is that the source of miRs has been limited to synthetic RNAs with artificial modifications, which raises concerns over the stability, cost, specificity and safety of these RNA-based therapeutics. To improve the miR-based therapy, a miR-34a prodrug, pre-miR-34a fused to a transfer RNA (tRNA), namely tRNA-mir-34a has been developed. Wang, W. P. et al. Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.The Journal of pharmacology and experimental therapeutics354, 131-141 (2015), the disclosure of which is incorporated herein by reference. These bioengineered miRs are produced and folded inEscherichia coliin large scale with high yield, which have been shown to effectively capture the function and safety properties of natural RNAs and therefore represent a new class of more affordable and biocompatible miR-based agents for research and therapy. Indeed, this tRNA-carried pre-miR-34a was selectively processed into mature miR-34a in human carcinoma cells, resulting in reduced expression of the target genes and consequently the inhibition of cancer cell proliferation in vitro and in vivo. The miR-34a prodrug tRNA/miR-34a, which is converted into mature miR-34a upon intracellular delivery, represents an improvement on DOX/miR-34a combination therapy. Currently, miRNA replacement therapy is limited to the use of synthetic RNAs that are chemically modified to improve their stability. Such modifications may alter the folding, biologic activities and, potentially more importantly, the safety profiles (such as the proinflammatory cytokine response). In contrast, the bioengineered miR-34a prodrug is folded in living cells, which may better capture the functions and safety properties of natural miR-34a. However, the application of tRNA-mir-34a alone only exhibited modest effect against human lung cancer or hepatocarcinoma cells. The nanocarrier system hereof provide for a combination of tRNA-mir-34a and small-molecule chemotherapy (for example, DOX) to work synergistically to increase the efficacy of treatment while minimizing the toxicity associated with each treatment alone. In a number of representative embodiments, a multi-functional delivery system hereof was formed using an amphiphilic polymer (POEG-PCre) with a naturally occurring cationic molecule creatine attached to a pendent side chain. The carrier or delivery system is suitable for the co-delivery of bioengineered a nucleic acid such as tRNA-mir-34a and one or more small molecules such as chemodrugs. Similar to the POEG-st-Pmor polymer describe above, the backbone, the POEG-PCre polymer (POEG-PVBC) includes hydrophobic alkyl main chain and pendent benzyl rings and forms the core of micelles to load hydrophobic molecules such as anticancer drugs through hydrophobic-hydrophobic interaction and π-π stacking. Creatine was post-conjugated to the backbone to introduce positive charges to form complexes with the negatively charged nucleic acids. In addition, the cationic creatine groups in the polymer can also facilitate accumulation of the nanoparticles in, for example, the lung and the subsequent interaction with target cells. The lung is the first capillary bed encountered by cationic NPs after intravenous (i.v.) injection and may be the most effective target organ. It also has been reported that lung tissue is endowed with a much higher polyamine active uptake system than any other major organs, which may benefit the targeted delivery of the creatine-based carrier systems hereof with amine groups to the lungs. The excess amount of positive charges on the nanocarrier was shielded by PEG to improve in vivo stability. The co-delivery of, for example, tRNA-mir-34a and DOX via the multi-functional nanocarriers hereof may provide a safe and effective approach for the treatment of, for example, metastatic TNBC. The biodegradability of the polymer and the nontoxic nature of creatine may provide for an excellent safety profile of the nanocarrier system as further discussed below. The biophysical properties of the nanocarrier co-loaded with DOX and tRNA-mir-34a were studied. The efficiency of delivery and transfection was also examined both in vitro and in vivo. Further, the antitumor effect of DOX+tRNA-mir-34a/polymer as well as the underlying mechanism was investigated. The POEG-PCre polymer was synthesized by RAFT co-polymerization of OEG500 monomer and VBC monomer, followed by conjugation with creatine as illustrated inFIG.11. The structure of the POEG-PVBC and POEG-PCre were characterized via1H nuclear magnetic resonance (NMR) spectra. The units of OEG and VBC were calculated to be 25 and 118, respectively, according to the monomer conversion. The conjugated units of creatine were calculated to be 47 by comparing the characteristic signals at 4.12-4.8, 5.07, and 5.95-7.88 ppm. POEG-PCre micelles were prepared by dialysis. The POEG-PCre copolymer self-assembled into spherical nanoparticles with a size around 180 nm as indicated by DLS and TEM (FIG.12A). The CMC of POEG-PCre copolymer, determined using nile red as a fluorescence probe, was as low as approximately 0.05 mg/mL. The low CMC indicated the stability of POEG-PCre micelles upon dilution in the blood stream after i.v. injection. The DOX-loaded POEG-PCre micelles were similarly prepared. DOX could be incorporated into POEG-PCre micelles at a carrier/drug mass ratio of 5:1 or higher. A carrier/drug ratio of 10:1 was chosen for the subsequent studies because of the relatively high drug loading capacity and the excellent colloidal stability (stable for one month and two months at RT and 4° C., respectively). DOX-loaded POEG-PCre micelles were comparable to blank POEG-PCre micelles in size and morphology. The surface zeta potential of POEG-PCre micelles was approximately +40 mV (FIG.12B). Whether the cationic micelles could form stable complexes with tRNA-mir-34a was tested via gel retardation assay. POEG-PCre micelles were mixed with tRNA-mir-34a at various N/P ratios from 1:1 to 40:1. Complete complexation of tRNA-mir-34a by POEG-PCre polymer was achieved at an N/P ratio of 5:1 or greater. Accordingly, at N/P ratios of below 5, the net charges of nanocomplexes were negative with the particle sizes similar to that of blank micelles. At an N/P ratio of 5, a significant increase in particle size was observed with particle charges close to neutral. Further increases in N/P ratios led to continuous increases in zeta potentials and decreases in particle sizes, suggesting gradual condensation of nucleic acid by POEG-PCre cationic polymer. To form more compact POEG-PCre/tRNA-mir-34a complexes as well as to overcome the neutralization by serum proteins, an N/P ratio of 20 was chosen for all subsequent studies. At this N/P ratio, the average size of the nanocomplexes decreased to around 120 nm. In addition, DOX loading had negligible effect on the size distribution, zeta-potential and nucleic acid binding ability of POEG-PCre micelles. In a number of embodiments of formulations hereof, the N/P ratio may, for example, be 1:1 to 100:1, 1:1 to 40:1, or 5:1 to 20:1. Suitable ranges of N/P ratio are readily determined for particular compositions hereof using the methods described herein. The binding ability of POEG-PCre to plasmid or siRNA was also investigated. Compared to complexation with tRNA-mir-34a, POEG-PCre polymer could form stable complexes with plasmid at a lower N/P ratio of 2.5:1. However, POEG-PCre polymer was not effective in complexing with siRNA at an N/P ratio as high as 80:1, suggesting that the POEG-PCre carrier was unique in forming complexes with nucleic acids with appropriate sizes and secondary structures including tRNA-mirs. In a number of embodiments of formulation based on POEG-PCRE, the nucleic acid may, for example, include approximately 19 to 20,000 base pairs for double stranded molecules or 19 or more base pairs for single stranded molecules. However, one may readily adjust the polymer composition hereof to interact via charge-charge interactions with negatively charged nucleic acids of virtually any size/composition. Ranges of sizes of single or double stranded nucleic acids for use in forming formulations hereof are readily determined using the methods described herein for any polymer composition hereof. To further investigate the interaction between POEG-PCre nanocarrier and tRNA-mirs, a competitive binding gel-shift assay with dextran sulfate was performed. At an N/P ratio of 5:1, substantial amounts of tRNA-mir-34a began to be released from POEG-PCre nanocarrier or PEI at an S/P ratio (molar ratio between the sulfur from dextran sulfate and the phosphate from tRNA-mir-34a) of 5 or greater. When the N/P ratio reached 10:1 or higher, tRNA-mir-34a could not be replaced by dextran sulfate even at an S/P ratio as high as 80. In contrast, an obvious release of tRNA-mir-34a was observed from PEI carrier at the corresponding N/P ratios. These data suggest that, in addition to electrostatic interaction as seen with PEI, other mechanisms of interactions, such as π-π stacking between the pendent benzyl rings of our polymer and the base π-systems of nucleic acids, might be conductive to a more stable nanocomplexing system. The release kinetics of DOX from DOX-loaded POEG-PCre micelles was investigated by dialysis against DPBS of pH 7.4 at 37° C. As shown inFIG.12C, more than 80% of free DOX was diffused out of dialysis bag (MWCO=3500) in the first 4 h. To the contrary, only 16% of DOX was released from DOX/POEG-PCre micelles in first 4 h, and less than 40% of DOX was released for an extended period of 96 h. POEG-PCre micelles co-loaded with DOX and tRNA-mir-34a exhibited a comparable but slightly slower release profile compared with micelles loaded with DOX alone. The stability of the nanocomplexes was studied in various solutions that mimic commonly used buffer, routine cell culture medium or serum, respectively, such as DPBS of pH 7.4, DMEM medium with 10% FBS or 30 mg/mL of BSA. As shown inFIG.12D through12F, no aggregation of nanoparticles was observed in all tested solutions and all of the nanocomplexes showed minimal changes in sizes for up to 48 h. To monitor intracellular co-delivery of DOX and tRNA-mir-34a, tRNA-mir-34a was labeled with MFP488 fluorescent dye to visualize the cell uptake of tRNA-mir-34a along with red fluorescence of DOX. 4T1.2 cells were incubated for 4 h with micelle complexes carrying DOX and/or MFP488-labeled tRNA-mir-34a and then observed under a confocal microscope. Naked tRNA-mir-34a without any transfection agent was used as a negative control. DOX/POEG-PCre showed more DOX fluorescence signal compared to free DOX at the same dose of DOX. The DOX+tRNA-mir-34aMFP488co-loaded POEG-PCre micelles showed even more uptake of DOX than that of the DOX/POEG-PCre micelles, which may, for example, be a result of a more condensed structure following complexation with tRNA-mir-34a and, therefore, higher endocytosis efficiency. Overlay of DOX (red) and tRNA-mir-34aMFP488(green) generated the yellow signals in the merged channel, suggesting the co-localization of DOX and tRNA-mir-34aMFP488inside tumor cells. In addition, fluorescence signals of both DOX and tRNA-mir-34aMFP488were present in the perinuclear regions of the cytoplasm in nanocomplexes treated groups, while the DOX fluorescence signals in free DOX treated group were largely found in the nucleus overlapping with Hoechst staining (blue). These data suggest the different cellular uptake routes of free DOX and DOX-loaded micelles, through passive diffusion and endocytosis, respectively. The processing of tRNA-mir-34a into mature tumor suppressor miR-34a was evaluated by quantitative RT-PCR. As shown inFIG.13A, there was over a 200-fold increase in the amount of mature miR-34a in 4T1.2 cells following treatment with tRNA-mir-34a/POEG-PCre nanocomplexes. Significantly greater amounts of mature miR-34a (˜1200-fold increase) were found in cells treated with the DOX+tRNA-mir-34a co-loaded POEG-PCre micelles. This is not likely a result of the differences in the amounts of tRNA-mir-34a delivered intracellularly as comparable levels of MFP488 fluorescence were found for the two groups. Consistently,FIG.13Bshows that the amounts of chimeric ncRNA scaffold inside 4T1.2 cells were similar after treatment with either POEG-PCre/tRNA-mir or POEG-PCre/DOX+tRNA-mir, suggesting comparable efficiency of delivery of the two formulations. It might, however, be a result of a more efficient processing of tRNA-mir-34a in cells following co-delivery of DOX. Treatment with free DOX or DOX-loaded POEG-PCre micelles led to the induction of Dicer, an RNase III enzyme involved in pre-miRs cleavage, in a dose dependent manner. As a result, the level of pre-miR-34a in DOX+tRNA-mir-34a/POEG-PCre-treated cells was significantly lower than that in cells treated with tRNA-mir-34a/POEG-PCre, suggesting a more complete processing of pre-miR-34a into mature miR-34a as a result of the co-delivered DOX (FIG.13C). Similar trends were observed in MDA-MB-231 cells (FIGS.13Athrough C). The expression of Bcl-2 was then evaluated at both the transcript and protein levels after intracellular delivery of tRNA-mir-34a. tRNA-MSA (Sephadex aptamer tagged methionyl-tRNA), the sole tRNA scaffold, was used as a control. As shown inFIG.13D, the mRNA expression levels of Bcl-2 in cells treated with blank POEG-PCre micelles, tRNA-MSA/POEG-PCre micelles, free DOX, DOX/POEG-PCre micelles or DOX+tRNA-MSA/POEG-PCre micelles were similar to that of untreated control cells, indicating that POEG-PCre carrier, tRNA-MSA as well as DOX had minimal regulatory effects on the expression of Bcl-2. In contrast, significant down-regulation of Bcl-2 expression was observed in the tumor cells treated with POEG-PCre/tRNA-mir-34a or the co-loaded formulation. Consistently, the downregulation of Bcl-2 was also confirmed at the protein level by Western blotting. Down-regulation of Bcl-2 was more dramatic in cells treated with the co-loaded formulation compared to cells treated with POEG-PCre/tRNA-mir-34a. Similar results were found in MDA-MB-231 cells. The in vitro cytotoxicity of DOX+tRNA-mir-34a co-loaded POEG-PCre micelles was evaluated by MTT assay. Tumor cells were treated with various formulations for 48 h, with the doses of DOX, tRNA-mir, and POEG-PCre carrier ranging from 7.8 to 1,000 ng/mL, 0.78 nM to 100 nM, and 78 ng/mL to 10000 ng/mL, respectively. As shown inFIGS.14A and14B, no obvious cell growth inhibition was noticed in cells treated with POEG-PCre nanocarrier alone or tRNA-MSA loaded POEG-PCre micelles, even when the concentration of the polymer reached 10 μg/mL. tRNA-mir-34a/POEG-PCre micelles showed a moderate level of cytotoxicity at high concentrations. Free DOX inhibited the proliferation of tumor cells in a dose dependent manner and the cytotoxicity of DOX/POEG-PCre was comparable to that of free DOX. Incorporation of tRNA-MSA into DOX/POEG-PCre micelles showed minimal impact on the overall cytotoxicity of the co-loaded formulation. In contrast, incorporation of tRNA-mir-34a into DOX loaded POEG-PCre micelles led to a significant improvement in the cytotoxicity of nanocomplexes on 4T1.2 cells, indicating the synergistic effect between co-delivered tRNA-mir-34a and DOX via our POEG-PCre system (FIG.14A). A similar trend was also observed in MDA-MB-231 breast cancer cells (FIG.14B). The enhanced cytotoxicity of DOX+tRNA-mir-34a-co-loaded nanocomplexes was assessed via apoptosis assay. Cell apoptosis was evaluated quantitatively by Annexin V/propidium iodide flow cytometry. Few apoptotic cells were detected in control cells or cells treated with POEG-PCre carrier alone or tRNA-MSA loaded nanocomplexes, which was consistent with MTT assay. An increase in cell apoptosis (˜21%) was observed in cells treated with tRNA-mir-34a loaded nanoparticles, suggesting that increased expression of mature miR-34a after delivery of tRNA-mir-34a into tumor cells can trigger the apoptosis of breast cancer cells, which are consistent with the tumor suppressor functions of miR-34a. Free DOX, DOX/POEG-PCre and DOX+tRNA-MSA POEG-PCre were comparable in inducing cell apoptosis to a significantly higher level (˜31%), which was consistent with previous reports that DOX kills cancer cells through triggering cell apoptosis. Importantly, a significantly higher percentage of necrosis (˜13%), in addition to much greater degree of late apoptosis (˜20%) and comparable level of early apoptosis was obtained in cells treated with DOX+tRNA-mir-34a co-loaded formulation, again suggesting a potent synergy between tRNA-mir-34a and DOX in inducing apoptosis and necrosis. Besides cancer cell viability, cell migration represents a critical process for tumor progression and metastasis. MiR-34a has been reported to be functional as an anti-metastatic miRNA by directly targeting CD44 and NOTCH-1. Therefore, the effect of tRNA-mir-34a and DOX co-loaded nanocomplexes on the cell migration was investigated using the wound closure assay. The wounded area was substantially repopulated by highly aggressive 4T1.2 cancer cells after 24 h in groups with no treatment or when treated with POEG-PCre nanocarrier alone or tRNA-MSA loaded nanocomplexes. Significant inhibition (˜75%) of cell migration was observed in cells treated with tRNA-mir-34a/POEG-PCre. The three DOX-containing formulations (free DOX, DOX/POEG-PCre or DOX+tRNA-MSA/POEG-PCre) were comparable in potency with a modest inhibition of ˜40%. In contrast, the DOX/tRNA-mir-34a-co-loaded group was most effective in inhibiting the migration of cancer cells; the denuded area was well retained with only 4.9% of repopulated cells. To gain insight into the biodistribution of nanocomplexes, the fluorescence signals of DOX and MFP488-labeled tRNA-mir-34a in tissue sections were examined by confocal microscopy. Substantial amounts of fluorescent signals of both DOX and MFP488 were detected in lungs treated with DOX+tRNA-mir-34aMFP488co-loaded micelles. The yellow color in the merged channel indicated the co-localization of DOX and tRNA-mir-34a in lung tissues. Compared to the lungs, substantially lower levels of fluorescent signals were observed in other major organs, suggesting that POEG-PCre nanocarrier is particularly suitable for targeted co-delivery of DOX and tRNA-mir-34a to lungs, which may benefit the prevention and treatment of breast cancer lung metastasis. The in vivo anti-tumor efficacy of DOX+tRNA-mir-34a co-loaded nanocomplexes was evaluated in a breast cancer lung metastasis model, generated by tail-vein injection of 4T1.2 tumor cells into female Balb/c mice. Five days after tumor inoculation, various treatments were applied to each group of mice (n=5) via i.v. administration every 3 d for 3 times. The tumor burdens in the lungs were analyzed 5 days after the last treatment. As shown inFIGS.15A and15B, tRNA-MSA loaded in POEG-PCre micelles showed negligible therapeutic effect compared to the control group. However, tRNA-mir-34a/POEG-PCre nanocomplexes exhibited a moderate inhibition of lung metastasis (P<0.05). Dramatic reduction in the number of tumor nodules in lungs was found in groups treated with free DOX, DOX/POEG-PCre or DOX+tRNA-MSA/POEG-PCre micelles. More importantly, POEG-PCre micelles co-loaded with DOX and tRNA-mir-34a was significantly superior to all other treatments in the inhibition of 4T1.2 tumor metastasis. No significant decrease of body weight was observed, indicating the safety of the formulation (FIG.15C). FIG.15Dshows that the levels of mature miR-34a were significantly and selectively unregulated in tumors treated with tRNA-mir-34a loaded micelles. Consistent with in vitro data, codelivery of tRNA-mir-34a and DOX via POEG-PCre micelles led to a further increase in the amounts of mature miR-34a which may be a result of the upregulation of Dicer expression by DOX-containing formulations in vivo.FIGS.15E and15Fillustrate that the expression levels of Bcl-2 in tumor tissues were significantly suppressed at both mRNA and protein levels following treatment with tRNA-mir-34a, particularly the co-loaded formulation. To further confirm the anticancer efficacy, H&E staining and immunohistochemical analysis of Ki67 were performed in tumor-bearing lungs. Staining of lung tissues showed clearly infiltration of tumor cells (with large nuclei) in all of the groups except the DOX/tRNA-mir-34a co-delivery group. In addition, DOX/tRNA-mir-34a co-delivery group showed the lowest number of Ki67-positive tumor cells. Together, the above data clearly suggest a synergy between DOX and tRNA-mir-34a in the overall antitumor activity in the lung metastasis model. Compared to control or monotherapy, co-delivery of DOX and tRNA-mir-34a did not cause loss of body weight (FIG.15C) or any other signs of stress such as hunched posture and labored movement. In addition, blood chemistry tests were performed to evaluate the effect of our formulations on liver and kidney functions. None of the measured blood biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine were significantly altered by our therapies, indicating the absence of hepatic and renal toxicity. Moreover, the H & E staining of major organs was carried out to further evaluate the potential toxicity of different treatments. Further, there were signs of myocytolysis and myofibrillolysis with fibrils dearrangement in the heart sections in free DOX group. In addition, hepatocellular vacuolation was found in this group, suggesting possible cardiac and liver toxicities despite no significant alterations in ALT and AST levels. In contrast, no significant pathological changes were observed in the major organs in the groups treated with single-loaded or co-loaded nanocomplexes. These results suggested that POEG-PCre based formulations were well tolerated at the doses tested. FIGS.16A and16Bshow that DOX treatment led to a significant increase in the numbers of CD4 and CD8 cells in the tumor-bearing lungs. DOX treatment also led to a trend of increase in the numbers of functional CD8 (IFN-γ+ CD8) cells (FIG.16C). However, the above changes were much more dramatic following the tRNA-mir-34a/DOX combination treatment (FIGS.16A-C). Treatment with tRNA-mir-34a alone or tRNA-mir-34a/DOX combination reduced the numbers of PD1+ CD8 cells (FIG.16D) and T-regulatory cells (Tregs) (FIG.16E).FIG.16Fshows that the numbers of MDSC were significantly decreased following the tRNA-mir-34a/DOX combination treatment. There was a trend of decreases in the numbers of MDSC following the treatment with DOX alone. The studies hereof thus show that POEG-PCre represents a simple nanocarrier for effective co-loading of small molecule drugs and nucleic acids-based therapeutics. POEG-PCre readily self-assembles to form a hydrophobic core that allows effective loading of hydrophobic drugs such as DOX. At the same time, the multiple positive charges from creatine at the interface of the POEG-PCre micelles facilitate the interaction with the negatively charged nucleic acids. As described above, the multivalent charge-charge interactions between the cationic polymer and nucleic acids may also serve as a simple approach to “cross-link” and stabilize the micelles. As a result, the size of tRNA-mir-34a complexed micelles (˜120 nm) was much smaller than that of blank micelles or DOX-loaded micelles (˜180 nm). In addition, DOX/tRNA-mir-34a-coloaded nanoparticles exhibited a slightly slower release kinetics compared to that of micelles loaded with DOX only. Complexation of cationic polymers with large-sized nucleic acids such as plasmid DNA is known to lead to formation of compact particles, which involves structure arrangement and condensation of nucleic acids. On the other hand, cationic polymers tend to form loose complexes with short oligonucleotides as a result of their small lengths. Accordingly, the POEG-PCre polymers hereof readily formed stable complexes with plasmid DNA at N/P ratios above 2.5 but failed to do so with short siRNA at N/P ratio as high as 80. In a number of embodiments, nucleic acids with 100 or more nucleotides (nt) are used. Interestingly, tRNA-mir-34a appears to have an appropriate size (˜200 nt) to form stable complexes with POEG-PCre polymer. The POEG-PCre/tRNA-mir-34a complexes were more stable than PEI/tRNA-mir-34a complexes as evidenced by the fact that tRNA-mir-34a in POEG-PCre/tRNA-mir-34a complexes was more resistant to the replacement by the negatively charged dextran sulphate compared to PEI/tRNA-mir-34a. This observation may be a result of additional mechanisms of interactions between POEG-PCre and nucleic acids in addition to charge interactions. Similar to other polymers hereof, the backbone of POEG-PCre includes hydrophobic alkyl main chains and pendent benzyl rings that can interact with the base π-systems of nucleic acids through hydrophobic interaction and π-π stacking. The enhanced interaction of the polymers hereof with nucleic acids may help to improve the stability of polymer/nucleic acid complexes in the blood circulation. POEG-PCre was highly effective in mediating codelivery of the representative combination of tRNA-mir-34a and DOX to cultured tumor cells in vitro and pulmonary circulation in vivo. Creatine is a derivative of the guanidinium cation. Positively charged guanidinium groups are able to form electrostatic association and bidentate hydrogen bond with anionic cell surface phosphates, carboxylates and/or sulfates to initiate the event of cellular entry. This feature has been widely utilized to design several new guanidinium-rich transporter scaffolds to improve the performance of cell penetration for small molecules, peptides and genes. The efficient intracellular delivery of tRNA-mir-34a and DOX may benefit from this multivalent effect. Following systemic administration, the creatine-based polymer may take advantage of the lung-enriched polyamine transporters to guide the targeted accumulation of nanocomplexes to lung tissues. Indeed, a more predominant distribution of the nanocomplexes in the lungs was observed as described above, which should be beneficial for the prevention and treatment of lung metastasis. Codelivery of DOX and tRNA-mir-34a via POEG-PCre-based nanoparticles led to an enhanced combinational effect both in vitro and in vivo. Codelivery of DOX facilitates the processing of tRNA-mir-34a, resulting in increased levels of mature miR-34a (P<0.001). The underlying mechanism is not clearly understood. However, the data suggest that DOX induced the expression of RNase III enzyme Dicer. It has been reported that Dicer is required for activating the DNA damage response when double-strand DNA breaks and non-coding RNA is synthesized at the site of DNA damage. Therefore, DOX may enhance the expression of Dicer through the DNA damage response. Without limitation to any mechanism, the improved antitumor activity of DOX/tRNA-mir-34a-based therapy is likely attributed to various factors. Overexpression of miR-34a is effective in inhibiting the proliferation of cancer cells and inducing apoptosis. In addition, miR-34a sensitizes cancer cells to chemotherapeutic agents such as DOX. The data further suggest a role of immune response in the overall antitumor activity of DOX/tRNA-mir-34a combination therapy. DOX was reported to trigger immunogenic cell death by promoting tumor infiltration of IL-17-secreting γδ T cells and enhancing the proliferation and activation of IFNγ-secreting CD8+ T cells in tumor draining lymph nodes. Similar results were shown in our studies with a lung metastatic tumor model. However, treatment with DOX+tRNA-mir-34a co-loaded nanocomplexes resulted in even greater numbers of tumor-infiltrating CD4+, CD8+ and CD8+ IFN-γ+CD8+T cells, which may be attributed to the effect of miR-34a on the down-regulation of PD-L1 expression. In addition, the co-treatment led to significantly reduced numbers of MDSCs that are highly immunosuppressive and play an important role in suppressing the antitumor immunity. The combination therapy enabled by the nanocarriers hereof thus helps to create an active tumor immune microenvironment that likely contributes to the overall antitumor activity. In another representative example, PMet-P(cdmPEG2K) polymeric micelles based on polymetformin (PMet) with an intratumor, pH-responsive PEG deshielding functionality was developed for co-loading and tumor-targeted co-delivery of a small molecule therapeutic agent (for example, DOX) and a nucleic-acid-base therapeutic (for example, plasmid encoding interleukin 12 or IL-12 cytokine gene) for a combined chemoimmunotherapy. The positive charge shielding performance of PEG on the PMet-P(cdmPEG2K) micellar surface enhanced the serum stability of micelles and micelleplexes after intravenous injection. IL-12/DOX co-loaded micelleplexes exhibited enhanced cell proliferation inhibition effects than DOX-loaded micelles, and displayed a higher cytotoxicity at acidic extracellular microenvironment of tumor (pH 6.8). The PMet-P(cdmPEG2K) micelles showed the significantly improved EGFP/luciferase reporter plasmid expression and Cy3-siRNA transfection efficiency, and DOX intracellular uptake in 4T1.2 cells at pH 6.8. Moreover, PMet-P(cdmPEG2K) micelles showed excellent EGFP pDNA transfection in an aggressive murine breast cancer (4T1.2) model, which suggested potent in vivo gene delivery for targeted tumor therapy application. PMet-P(cdmPEG2K) micelles co-loaded with DOX and IL-12 cytokine gene was more efficient in tumor growth inhibition, compared to DOX loaded micelles and IL-12 gene loaded micelleplexes. This intratumor pH-responsive deshielding micellar system exhibited significant potential for effective combination of immunotherapy based on plasmid encoding IL-12 cytokine gene and traditional DOX chemotherapy. As described above, cytokine-based therapy has been emerging as a promising strategy for various cancer therapies because of the direct anti-proliferative activity against cancer cells or indirect anti-tumor activity by stimulating the immune system. Among various cytokines, IL-12 is considered to be promising immunostimulatory cytokine with potent anti-tumor activity. It can activate cytotoxic T lymphocytes, natural killer (NK) cells and induce the secretion of IFN-γ. Although recombinant IL-12 protein has demonstrated effective therapeutic effect against several tumor models, severe adverse effects after systemic administration limit its application. As an alternative approach, IL-12 based gene therapy has been reported to cause lower side effects through localized expression of IL-12 protein in tumor cells compared to recombinant IL 12 protein therapy. Unfortunately, only moderate antitumor efficacy has been achieved. The combination strategy of IL-12 based gene therapy with standard cytotoxic chemotherapy holds considerable promise to further improve the therapeutic efficacy. As described above, DOX is a first-line chemotherapeutic agent which has been widely utilized for the treatment of a variety of malignant tumor. In addition, it has been reported that DOX can induce immunogenic cell death and activate antitumor T cell immune responses, leading to synergistic antitumor effect when combined with immunostimulatory agents, including IL-12. However, similar to other small molecule therapeutic/nucleic-acid-base therapeutic combinations, the simultaneous and efficient delivery of DOX and IL-12 gene in vivo is particularly challenging as a result of the differences in physicochemical properties of these two types of agents. Therefore, a drug delivery system capable of co-delivering DOX and IL-12 gene simultaneously with high efficiency for cancer therapy is very desirable. PEGylation of cationic polymeric micelles is an effective way to sterically stabilize micelles and minimize the nonspecific interaction in vivo, thereby prolonging the circulation time and facilitating tumoral accumulation via enhanced permeation and retention (EPR) effects. However, PEGylation may significantly reduce their cellular uptake in tumor tissues, significantly limiting gene transfection and drug delivery efficiency in vivo. PEG shielding/deshielding strategies may be used PEGylated polymers for delivery of genes or drugs. Carboxydimethyl maleate (cdm) is an acid-labile linker which can be cleaved at the acidic tumor extracellular pH (pH=6.5-6.8). Based on the differences in pH between normal tissues (pH=7.4) and tumor tissues (pH=6.5-7.2), conjugation of the cationic polymers with PEG chains via cdm linker is an efficient strategy to construct pH-responsive PEG deshielding carriers for improved cell internalization and targeted gene/drug delivery in tumor tissues. Metformin (Met, dimethybiguanide)-based and cdm-linked cationic micelles PMet-P(cdmPEG2K) with PEG deshielding characteristics was developed for co-loading and tumor-targeted co-delivery of, for example, DOX and a nucleic acid (plasmid encoding IL-12 gene). Metformin is a commonly used antidiabetic drug for type II diabetes treatment. Increasing evidence has also demonstrated that Met shows potent antitumor efficacy against various types of cancers, which may be attributable to the activation of adenosine monophosphate-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) [23-26]. Moreover, the biguanide group of Met can be used as an ideal cationic motif for constructing gene delivery carriers. PMet-P(cdmPEG2K) polymers can self-assemble into micelles under aqueous conditions for DOX encapsulation. The resultant DOX-loaded PMet-P(cdmPEG2K) micelles further complex with IL-12 pDNA through electrostatic interaction. After intravenous injection, IL-12/DOX PMet-P(cdmPEG2K) micelleplexes may prolong circulation in the blood via the PEG shielding and accumulate preferentially in tumor tissue by enhanced permeability and retention (EPR) effect. Once reaching the tumor extracellular acidic pH environment (pH 6.8), IL-12/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes may undergo acid hydrolysis of cdm moieties to deshield PEG shell and expose the positive charges, facilitating DOX endocytosis and IL-12 pDNA transfection in tumor cells for an efficient chemoimmunotherapy combination. DOX kills tumor cells by blocking the cell cycle progression and apoptosis induction. In addition, IL-12 induces the increase in numbers of CD8+T cells and NK cells, as well as the Treg suppression, to relieve the immunosuppression and enhance the antitumor function of the immune system. Overall, DOX/IL-12 pDNA co-loaded PMet-P(cdmPEG2K) micelleplexes generate a synergistic antitumor effect of the chemo-immunotherapy combination treatment. The PMet-P(cdmPEG2K) polymer was synthesized as illustrated inFIG.17. PEG2Kwas first reacted with pH-labile cdm to obtain PEG2K-cdm (compound 1) according to previous published literature methodology. Compound 3 was synthesized by reversible addition fragment chain transfer (RAFT) polymerization of 4-vinylbenzyl chloride and Boc-protected 4-vinylbenzylamine monomers (compound 2). NMR confirmed that Compound 3 included 85% of 4-vinylbenzyl chloride and 15% of Boc-protected 4-vinylbenzylamine moieties. MET was then conjugated on compound 3 to obtain compound 4. The Boc-groups of the synthesized compound 4 was deprotected, and amine-bearing compound 5 was obtained and further conjugated with PEG2K-cdm through the reaction with cdm anhydride residue. The amide bond of PMet-P(cdmPEG2K) was tumor extracellular acid labile and PEG2Kwould be cleaved to expose cationic PMet micelles at tumor sites, facilitating tumor targeted co-delivery of drugs and genes for efficient chemoimmunotherapy combination. The chemical structure of PMet-P(cdmPEG2K) polymer was confirmed by1H NMR. The average unit numbers of MET per PMet-P(cdmPEG2K) molecule, calculated was about 42 by the relative intensity ratio of the methyl protons (b, N—CH3) of MET to the ethylene protons of the benzene ring. The unit number of PEG2Kchain was calculated from the relative intensity ratio of the protons (—OCH3) of PEG2Kchain to the ethylene protons (—CH═CH—) of the benzene ring, showing around 12 units of PEG2Kwere conjugated per PMet-P(cdmPEG2K) molecule. MET conjugating content on PMet-P(cdmPEG2K) polymeric prodrug calculated was about 14% (w/w), exhibiting an excellent MET loading capacity of PMet-P(cdmPEG2K) polymeric prodrug. Met-P(cdmPEG2K) polymer had a lower CMC value of 28.6 μg/mL, which suggested the self-assembling behavior of PMet-P(cdmPEG2K). As a carrier, micelles of the polymer could maintain stability upon dilution in bloodstream after intravenous injection. DOX loaded PMet-P(cdmPEG2K) micelles were prepared by thin film method and the cationic PMet-P(cdmPEG2K) micelles loading with DOX complexed with IL-12 pDNA through electrostatic interaction to prepare the DOX/IL-12 co-loaded micelleplexes. Gel retardation assay was utilized to confirm the micelleplexes formation of blank and DOX-loaded micelles complexed with IL-12 pDNA. The inhibition migration of condensed IL-12 pDNA in agarose gel was observed, indicating a complete formation of IL-12 loaded PMet-P(cdmPEG2K) micelleplexes and IL-12/DOX PMet-P(cdmPEG2K) co-loaded micelleplexes were achieved at and above N/P ratio of 5. It also indicated the DOX encapsulation in the micellar core had little influence on the gene condensing capacity of micelles, as a result of their similar zeta potentials. In addition, DOX encapsulated in IL-12/DOX co-loaded micelleplexes and DOX loaded PMet-P(cdmPEG2K) micelles exhibited a series of opposite migration bands from IL-12 as the N/P ratio increased, owing to the positively charged DOX molecules migrated from micellar cores, which further confirmed that DOX and IL-12 were co-encapsulated in PMet-P(cdmPEG2K) micellar system. Furthermore, the IL-12 pDNA condensing capacities of PMet-P(cdmPEG2K) micelles was also evaluated by dynamic light scattering. The average particle size and zeta potential of IL-12 and IL-12/DOX co-loaded micelleplexes at different N/P ratios. The blank and DOX-loaded PMet-P(cdmPEG2K) micelles showed an average size of 154 nm and 178 nm (see Table 2 below). PMet-P(cdmPEG2K) micelles were able to condense IL-12 pDNA efficiently into a more compact micelleplex structure with a smaller particle size of 100-125 nm and a narrow size distribution above a N/P ratio of 5, which could be favorable for EPR effect mediated tumor tissue accumulation and gene/drug co-delivery via a particle size-dependent endocytosis approach. The more compactable micelleplexes might be a result of the charge neutralization and non-covalent cross-linking of PMet-P(cdmPEG2K) micelles by IL-12 pDNA. Zeta potential of micelles is an important parameter related to the gene condensing ability and in vivo delivery performance. In the case of zeta potential results, blank and DOX-loaded micelles exhibited similar zeta potential of 18.2 and 17.1 mV (Table 2), respectively. The lower zeta potential is a result of the partial shielding by PEG2Kon cationic shell of PMet micelles. The zeta potential of IL-12 and IL-12/DOX PMet-P(cdmPEG2K) micelleplexes reversed from negative charge to positive charge when the N/P ratio increased to 5, indicating the formation of micelleplexes system. The above particle size and zeta potential results of IL-12 and IL-12/DOX co-loaded micelleplexes were consistent with the gel retardation assay results. Table 2 also shows the physicochemical properties of IL-12 and IL-12/DOX PMet-P(cdmPEG2K) micelleplexes at N/P ratio of 20, at which ratio the in vivo gene transfection and therapeutic efficacy were evaluated. IL-12 and IL-12/DOX PMet-P(cdmPEG2K) micelleplexes showed an average size of 103 and 102 nm, and a zeta potential of 10.3 and 10.4 mV, respectively (Table 2). The micelles and micelleplexes observed by TEM all showed compact and spherical morphology. The observed size from TEM was smaller than that from DLS, which was a result of the collapse of micelles during TEM sample preparation processes. In addition, PMet-P(cdmPEG2K) micelles exhibited excellent encapsulation performance for DOX, with DLC and DLE of 9.53% and 99.9%, respectively (Table 1). TABLE 2MassZetaratioN/PSizepotentialDLCDLEStabilityMicelles(mg:mg)ratio(nm)(mV)(%)(%)(d)PMet-——15418.2———P(cdmPEG2K)IL-12 PMet-—2010310.3——10P(cdmPEG2K)DOX PMet-10:1—17817.195.39.537P(cdmPEG2K)20:1—17414.798.24.91730:1—16216.099.93.338IL-12/DOX—2010210.495.39.536PMet-P(cdmPEG2K) To verify that the PEG shell could be deshielded from PMet-P(cdmPEG2K) micelleplexes at extracellular tumor pH, the pH sensitivity of IL-12 loaded and IL-12/DOX co-loaded micelleplexes was investigated by monitoring the pH-dependent zeta potential in HEPES buffer at pH 7.4 and 6.8. The surface charge of micelleplexes increased significantly from 10.7 mV to 29.8 mV after 4 h incubation at pH 6.8. On the contrary, the zeta potential of micelleplexes showed no significant change after 4 h incubation at pH 7.4. The zeta potential changes may, for example, be attributable to partial detachment of PEG shell from IL-12 and IL-12/DOX PMet-P(cdmPEG2K) micelleplexes, and the re-exposure of cationic amino groups on the surface of micelleplexes after cdm linker cleavage under tumor extracellular pH (pH=6.8). The increased positive charges in response to tumor extracellular pH would facilitate the internalization of PMet-P(cdmPEG2K) micelleplexes by tumor cells and generate excellent gene transfection and drug delivery performances. PEGylation of micelles can minimize opsonin adhesion by serum components in blood, thus potentially prolonging the blood circulation time and increasing the tumor tissue accumulation by EPR effect following systemic administration in vivo. To evaluate the blood protein adsorption and interaction, the serum stability of micelles and micelleplex systems were investigated through monitoring the size changes at different time intervals after co-incubation with bovine serum albumin (BSA, 1 mg/mL). “Gold-standard” transfection agent PEI25Kcomplexed with IL-12 gene was investigated as a control for comparison. PMet-P(cdmPEG2K) micelles and micelleplex groups showed a slightly increased size after 24 h incubation, but IL-12/PEI25Kpolyplexes formed a significantly larger aggregates in a short time period after incubation with BSA. These results showed that the positive charge shielding effect of PEG on the PMet-P(cdmPEG2K) micellar surface enhanced the serum stability of micelles and micelleplexes after intravenous injection in vivo. MET amounts released from PMet-P(cdmPEG2K) were analyzed for evaluation of the MET cleavage efficiency of polymeric prodrug. The MET achieved highest released amounts from prodrug micelles at 48 h incubation. The cleavage mechanism may, for example, involve the enzymatic hydrolysis of carbon-carbon bonds between MET and compound 3. However, blank micelles and IL-12 loaded PMet-P(cdmPEG2K) micelleplexes did not exhibit notable tumor cell proliferation inhibition activity against 4T1.2 cells after 72 h incubation. It is possible that the intracellular enzyme in cultured tumor cells was not effective for hydrolyzing MET completely from PMet-P(cdmPEG2K) prodrug carrier, which was not sufficient for generating effective antitumor activity. However, the in vivo enzyme condition at the tumor site is different from and more complicated than that of cultured tumor cells. A more effective MET release from the PMet-P(cdmPEG2K) polymeric prodrug may be expected, and that release may generate a more effective anti-tumor activity, displaying synergistic antitumor effect with DOX in vivo. Cytotoxicities results indicated significant anti-tumor activity of PMet-P(cdmPEG2K) micelleplexes co-delivering IL-12 pDNA and DOX. To further illustrate that the chemotherapeutic agent and gene co-encapsulated into PMet-P(cdmPEG2K) micelles could be co-delivered simultaneously into tumor cells, the intracellular co-delivery of DOX and FAM-labeled siRNA by PMet-P(cdmPEG2K) micelles was observed by confocal laser scanning microscopy (CLSM). FAM-siRNA loaded micelleplexes and DOX-loaded micelles only showed green fluorescence signal of FAM-siRNA and red fluorescence signal of DOX, respectively. However, both red fluorescence signal of DOX and green fluorescence signal of FAM-siRNA was observed in double fluorescence-labeled FAM-siRNA/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes. For FAM-siRNA/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes, DOX fluorescence signals were mainly appeared in nuclei, and FAM-siRNA fluorescence signals were found to be localized largely in perinuclear region of cells. It was demonstrated that DOX and FAM-siRNA could be co-loaded and co-delivered into same 4T1.2 cells simultaneously by PMet-P(cdmPEG2K) micellar system. Intracellular uptake and distribution of DOX-loaded micelles and IL-12/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes was investigated by CLSM. After 4 h incubation, a much stronger intracellular fluorescence signal of DOX loaded micelles and IL-12/DOX co-loaded micelleplexes was observed at pH 6.8 than those at pH 7.4, and DOX fluorescence was mainly localized in the nuclei. PEG deshielding in tumor extracellular pH (pH=6.8) re-exposed the positive charge of micelles and facilitated adherence to negatively charged cellular membranes, promoting the internalization and DOX release of micelles. In contrast, the internalization of DOX solution was not significantly affected by pH, which showed slightly stronger fluorescence signal compared with DOX loaded micelles formulations because of the rapid passive diffusion of free DOX. In addition, compared to DOX-loaded micelles, the smaller size of IL-12/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes facilitated the endocytosis pathway, and showed an increased cellular uptake. The intracellular uptake and trafficking results indicated the PEG deshielding property of PMet-P(cdmPEG2K) micelleplexes under tumor intracellular condition led to the increased intracellular DOX and IL-12 pDNA co-internalization, which would improve the therapeutic efficacy of the combined chemoimmunotherapy. To evaluate the potential application of PMet-P(cdmPEG2K) micelles for gene delivery, in vitro gene transfection efficiency was evaluated against 4T1.2 cells by loading EGFP or luciferase-encoding pDNA. To investigate the optimized N/P ratio of PMet-P(cdmPEG2K) micelles for gene delivery and expression, the transfection efficiency of PMet-P(cdmPEG2K) micelles were quantitative performed with luciferase reported gene (luc-pDNA) at various N/P ratios firstly. Luc pDNA/PEI25Kpolyplexes was used as the positive control for comparison. The luc pDNA/PEI25Kpolyplexes showed the best luciferase gene expression level at N/P ratio of 20. The luciferase expression of luc pDNA/PMet-P(cdmPEG2K) micelleplexes were significantly enhanced with increasing N/P ratios, reaching the highest efficiency at N/P ratio of 20. Further increase of the N/P ratio up to 40 showed decreased transfection efficiency for luc pDNA/PMet-P(cdmPEG2K) micelleplexes. At pH 7.4, PEI25Kexhibited a superior luc-pDNA transfection efficiency, which was about 10-fold higher than that of PMet-P(cdmPEG2K) micelles. However, at pH 6.8, the luc-pDNA/PEI polyplexes exhibited a marked decreased in luciferase expression level. To the contrary, an increased transfection level and a significantly higher expression level was founded in luc-pDNA/PMet-P(cdmPEG2K) micelleplexes compared to incubation at pH 7.4. The gene expression efficiency was also qualitative evaluated using a plasmid encoding EGFP report gene. The fluorescence images showed similar results to the measured data of luciferase pDNA expression. The gene transfection results showed that deshielding of the PEG layer of PMet-P(cdmPEG2K) micelles under tumor extracellular pH (pHe 6.8) conditions led to more EGFP or luciferase encoding gene being delivered and transfected into the cultured cells, further improving the EGFP/luc pDNA expression. With significant EGFP/luc encoding gene delivery and transfection performance, PMet-P(cdmPEG2K) micelles at N/P ratio of 20 were further studied as a co-delivery system for DOX and IL-12 pDNA for immunotherapy and chemotherapy combination via in vitro and in vivo experiments. To evaluate the potential efficiency of PMet-P(cdmPEG2K) micelles for tumor acidity-targeting gene delivery in vivo, the transfection and GFP expression efficiency was examined in liver, tumor and lung after 24 h intravenous injection of PEI25Kand PMet-P(cdmPEG2K) micelles carrying EGFP pDNA in 4T1.2 tumor-bearing mice. The mice only treated with Hoechst 33342 and the mice treated with EGFP pDNA in saline solution via hydrodynamic injection at an EGFP pDNA dose of 25 μg/mice were used as negative and positive control, respectively. The negative control group did not show any detectable green fluorescence in all organs. Bright GFP fluorescence spots were presented in liver tissues, and negligible fluorescence signal was observed in tumor and lung of the positive control group treated with naked EGFP pDNA through hydrodynamic injection. Liver tissues of mice treated with EGFP/PMet-P(cdmPEG2K) micelleplexes showed very few fluorescence spots, while much more GFP fluorescence signals were observed in EGFP/PEI25Kpolyplexes groups in liver. The shielding PEG on the surface of PMet-P(cdmPEG2K) micelles could prevent plasma opsonins absorption and avoid the uptake of reticuloendothelial system (RES), thus prolonging the blood circulation of micelleplexes and decreasing accumulation in liver tissue. Weak GFP fluorescence was detected in the tumor for the EGFP/PEI25Kpolyplexes treated mice. As a comparison, EGFP/PMet-P(cdmPEG2K) micelleplexes induced an evidently higher level of GFP expression in tumor. The prolonged blood circulation promoted the tumor accumulation of PMet-P(cdmPEG2K) micelleplexes via EPR effects, and the PEG deshielding micelleplexes under extracellular tumor acid environment could further efficiently deliver EGFP pDNA to the tumor cells and release the loaded pDNA for GFP expression. Much more and intense GFP expression was observed in the lung of the mice treated with EGFP/PEI25Kpolyplexes. The positively charged EGFP/PEI25Kpolyplexes may, for example, be primarily delivered to the lung of mice in vivo. In contrast, a decreased GFP expression in lung was observed for PMet-P(cdmPEG2K) micelleplexes, which might be contributed to the efficient positive charged shielding effect of micelleplexes by PEG layer. The in vivo EGFP pDNA transfection results indicated potent in vivo gene delivery of PMet-P(cdmPEG2K) micelles for targeted tumor therapy application. It is expected that IL-12 pDNA would exhibit significant IL-12 cytokine expression performance after intravenous injection into mice carried by PMet-P(cdmPEG2K) micelles, generating potent immunostimulatory effect for tumor immunotherapy, and producing synergistic effect with DOX for chemoimmunotherapy combination. Combination of cytotoxic drugs and immunostimulatory agents such as cytokines in chemoimmunotherapy provides a novel approach for cancer therapy. To evaluate the chemoimmunotherapy efficiency of IL-12-encoding pDNA and DOX co-loaded and co-delivered by PMet-P(cdmPEG2K) micelles, the in vivo therapeutic efficacy was studied using 4T1.2 tumor bearing mice. Compared to the saline group, all the treatment groups exhibited tumor growth inhibition activity. Moreover, the co-delivery of DOX with IL-12 pDNA using PMet-P(cdmPEG2K) micelles suppressed tumor growth more efficiently than the delivery of either DOX or IL-12 by PMet-P(cdmPEG2K) micelles. The tumor growth inhibition rate for the treated groups of IL-12 micelleplexes, DOX-loaded micelles and IL-12/DOX co-loaded micelleplexes were calculated to be 28.7%, 41.0% and 66.5%, respectively, after 18 days past the first injection. The superior tumor growth inhibition activity of IL-12/DOX co-loaded micelleplexes suggested a significant synergistic/combined antitumor effect was achieved for DOX and IL-12 pDNA co-delivered by PMet-P(cdmPEG2K) micelles. The evaluation of systemic toxicity is essential for systemic drug delivery of micellar systems. The body weight of mice in different treatment groups was also monitored after first injection. The body weight increased obviously as time prolonged for saline and IL-12 loaded micelleplexes treated groups, which might be a result of the mice of those two groups bearing larger tumor size. However, the mice treated with DOX loaded micelles and IL-12/DOX co-loaded micelleplexes displayed a slight body weight increase. These results indicate that PMet-P(cdmPEG2K) micelles were well-tolerated for DOX and IL-12 pDNA co-delivery in vivo. Although DOX was loaded into micelle formulations hereof in a number of representative embodiments, numerous small molecule compounds such as drugs may be used in the present formulations. Table 3 below, for example, provides a summary of several of the small molecule drugs that have been loaded into PMet-P(cdmPEG5K) formulations hereof. TABLE 3MassParticleDLCMicelles *RatioSize (nm)(%)Pmet-P(cdm PEG5K)142Pmet-P(cdm PEG5K):Doxrubicin10:11339.09%Pmet-P(cdm PEG5K):Paclitaxel10:11409.09%Pmet-P(cdm PEG5K):Docetaxel10:11449.09%Pmet-P(cdm PEG5K):Erlotinib10:11469.09%Pmet-P(cdm PEG5K):Imatinib10:11389.09%Pmet-P(cdm PEG5K):Curcumin5:110416.6%Pmet-P(cdmPEG5K):10058-F420:11374.80%* Micelles were complexed with IL 12 plasmid at a N/P ratio of 20:1 Nanocarriers hereof exhibit the ability to safely co-deliver both small molecule drugs and nucleic acid-based therapeutics. As described above, most chemotherapeutic drugs are poorly water-soluble, while nucleic-acid based therapeutics are polyanionic molecules with high water-solubility, instability and high molecular weight. Most reported carriers are designed for delivery of either small molecule drugs or nucleic acid therapeutics alone. The few carriers which have been described for codelivery of the two different types of therapeutics often involved complicated preparation process. Representative Experimental Procedures Materials and Reagents. Doxorubicin (>99%) was purchased from LC Laboratories (MA, USA). Dicyclohexylcarbodiimide (DCC) was purchased from Alfa Aesar (MA, USA). 4-(Dimethylamino) pyridine (DMAP) was purchased from Calbiochem-Novabiochem Corporation (CA, USA). Creatine, vinylbenzyl chloride, 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid, oligo(ethylene glycol) methacry-late OEGMA (average Mn=500), 2,2-Azobis(isobutyronitrile) (AIBN), trypsin-EDTA solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyl tetrazolium bromide (MTT), metformin hydrochloride, monomethoxy PEG2K, branched and linear polyethyleneimine (PEI, MW=25 kDa), triton X-100, hoechst 33342, lysotracker Green DND-26, FAM-labeled siRNA, AF647-labeled siRNA and Dulbecco's Modified Eagle's Medium (DMEM) were all purchased from Sigma-Aldrich (MO, U.S.A.). Opti-MEM medium was purchased from Invitrogen (Carlsbad, USA). AIBN was purified by recrystallization in anhydrous ethanol. Fetal bovine serum (FBS), TRIzol lysis reagent and penicillin-streptomycin solution were purchased from Invitrogen (New York, U.S.A.). Vinylbenzylamine, 2-propionic-3-methylmaleic anhydride (cdm) were obtained from TCI America (Portland, Oreg. USA). QuantiTect Reverse Transcription Kit was purchased from Qiagen (MD, U.S.A). All solvents used in this study were HPLC grade. EGFP expression plasmid pEGFP-N2and luciferase expression plasmid luc-pDNA were propagated in competentEscherichia coliDH5α cells. IL-12 pDNA and control pDNA were supplied by Shulin Li's Lab. The IL-12 pDNA construct was obtained from Valentis, Inc. The control pDNA used for in vivo study consisted of a deletion of the IL-12 pDNA from the IL-12 construct. All endotoxin-free pDNA were prepared using the endotoxin free Plasmid Maxiprep Kit according to the manufacturer's instructions. Synthesis of VBMor monomer. Vinylbenzyl chloride (167.2 mg, 1.1 mM), morpholine (95.8 mg, 1.1 mM) and K2CO3(0.69 g, 5 mM) were dissolved in 6 mL DMF and stirred at 50° C. for 6 h. After cooling down to room temperature, 20 mL water was added to the mixture, followed by three times extraction with 50 mL CH2Cl2. After evaporation of CH2Cl2, the crude product was purified by column chromatography with petroleum ether/ethyl acetate (v/v, 4/1˜2/1) as the elution liquid. VBMor monomer was obtained with a 71% yield. Synthesis of POEG-st-Pmor polymer. VBMor monomer (228.8 mg, 1.13 mmol), OEG500 (100 mg, 0.20 mmol), AIBN (1 mg, 0.0062 mmol), 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (4 mg, 0.014 mmol), and 1 mL of dried tetrahydrofuran were added into a Schlenk tube, and deoxygenated by free-pump-thawing for three times. Then the mixture was filled with N2and immersed into an oil bath thermostated at 80° C. to start the polymerization. After 24 h, the reaction was quenched by immersing the tube into liquid nitrogen and the mixture was precipitated in hexane for 3 times. The product POEG-st-Pmor was obtained after vacuum drying. Preparation and characterization of IL-36γ Plasmid/DOX-co-formulated micelles. DOX-loaded POEG-st-Pmor micelles were prepared by the dialysis method. Briefly, 10 mg of polymer was dissolved in 5 mL of DMSO and mixed with 100 μL of DOX DMSO solution (10 mg/ml). To remove free DOX from the DOX-incorporated micelles, the solution was dialyzed against PBS using dialysis membrane with a MW cutoff of 3,500. The solution was lyophilized and resolubilized in 1 mL PBS. Drug-free micelles were similarly prepared. For plasmid DNA complexation, polymeric micelles were diluted to different concentrations in water and mixed with plasmid DNA solution to obtain the desired N/P ratios. This mixture was allowed to incubate at RT for 20 min prior to further characterization. In vitro characterization of POEG-st-Pmor polymer. The structure and molecular weight of POEG-st-Pmor polymer was characterized by1H NMR and gel permeation chromatography (GPC) similarly conducted as previously reported. The particle size and zeta potential of POEG-st-Pmor polymer were determined by dynamic light scattering (Nano-ZS 90, Malvern Instruments, Malvern, UK). The morphology of POEG-st-Pmor polymers was observed under a transmission electron microscope (TEM). The micelles were placed on a copper grid covered with nitrocellulose. The samples were negatively stained with phosphotungstic acid and dried at room temperature before measurement. Drug loading capacity (DLC) and drug loading efficiency (DLE) were determined as described before. The amount of DOX loaded in the micelles was determined by high performance liquid chromatography (HPLC, Shimadzu LC-20AD, Japan). The DLC of DOX/micelles was calculated using the equation: DLC=Drug incorporated/(input polymer+Drug)×100% Critical micelle concentration (POEG-st-Pmor). The critical micellar concentration (CMC) was determined using Nile Red as a fluorescence probe. Micelles of various concentrations (0.0001 to 1 mg/mL) were first prepared. Two microliter of a Nile Red solution in acetone (0.97 mg/mL) were then added to each sample and acetone was evaporated prior to fluorescence measurements using a microplate reader. Fluorescence from emission wavelength ranging from 560 to 750 nm was recorded with an excitation wavelength of 550 nm. In vitro drug release study for POEG-st-Pmor micelles. The in vitro DOX release kinetics for the POEG-st-Pmor micelles was determined by a dialysis method. Briefly, 0.5 ml of DOX-loaded micelles and micelles co-loaded with DOX and IL-36γ plasmid at a DOX concentration of 0.5 mg/mL were placed into a dialysis bag (MW cutoff 3,500), respectively. The dialysis bag was incubated in 100 mL PBS with gentle shaking at 37° C. Two ml of PBS solution outside of the dialysis bag was collected at different time points and equal amount of fresh PBS was added back. The concentrations of released DOX were determined by HPLC. Gel retardation assay. Plasmid/polymer complexes were prepared at different N/P ratios, ranging from 0.1 to 20 (plasmid DNA concentration was fixed at 5 mg/ml). The resulting complexes were then electrophoresed on a 1% agarose gel in TAE buffer at 120 mV for 30 min, and visualized using a UV illuminator with ethidium bromide staining. Free plasmid DNA was used as a control. Cell culture and animals. The murine breast cancer cell line 4T1.2 was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. in 5% CO2atmosphere. Female BALB/c mice (4-6 weeks, Charles River, Davis, Calif.) were housed under pathogen-free conditions according to AAALAC guidelines. The mice related experiments were performed following institutional guidelines and approved by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh. In vitro cytotoxicity. The cytotoxicity of DOX-formulated POEG-st-Pmor micelles, IL-36γ plasmid-complexed micelles and DOX+IL-36γ plasmid-co-loaded micelles were assessed in 4T1.2 breast cancer cells and compared to free DOX. Briefly, 4T1.2 cells (2,000 cells/well) were seeded in 96-well plates for overnight and were treated with various concentrations of DOX formulations for 72 h. MTT solution was added to each well and MTT formazan was solubilized by DMSO after 2 h of incubation. The absorbance in each well was measured by a microplate reader at a wavelength of 570 nm. Cell viability was calculated as [(ODtreat−ODblank)/(ODcontrol−ODblank)×100%]. The cytotoxicity of POEG-st-Pmor micelles alone was similarly tested in 4T1.2 cells as described above. Stability of the micelles in BSA. BSA was used to simulate the blood physiological environment to investigate the stability of POEG-st-Pmor micelle complexes under the mimicked physiological conditions. Plasmid DNA/micelle complexes and plasmid DNA+DOX/micelle complexes were prepared as described above and incubated with BSA (30 mg/ml). pDNA/PEI complexes were used as a control. Sizes of complexes were followed at different time points as an indication of stability. In vitro plasmid transfection. 4T1.2 cells were seeded in a 96-well plate and incubated for 24 h until cells were 80% confluent. Cells were then transfected with EGFP plasmid/POEG-st-Pmor complexes (N/P=20) and EGFP plasmid/PEI (N/P=20) complexes in serum-free opti-DMEM medium. After 4 h incubation, transfection medium was removed and 100 μL of fresh complete medium were added to each well. PBS group was used as a control. After 48 h, the transfected cells were observed under a fluorescence microscope (OLYMPUS America, Melville, N.Y.). In vivo fluorescence imaging. Female Balb/C mice bearing 4T1.2 tumor (˜400 mm3) in the mammary fat pad were used to investigate the biodistribution and in vivo transfection efficiency of our micellar carriers. The in vivo transfection efficiency of POEG-st-Pmor micellar carriers was evaluated with EGFP plasmid as a reporter gene. Linear PEI was used to as control. Various formulations were i.v. injected into tumor-bearing mice at a dose of 50 μg plasmid per mouse. One day later, mice were injected with 1 μg of Hoechst and sacrificed one hour later. The fluorescence signal of EGFP in the cryosections was examined under a confocal microscope. Mouse model of breast cancer lung metastasis. Female Balb/c mice were injected with 2×1054 T1.2 cells through the tail vein. Five days after tumor cell injection, mice were randomly divided into 6 groups. POEG-st-Pmor was chosen as a representative carrier system for codelivery of IL-36γ plasmid and DOX. Animals were treated intravenously with free POEG-st-Pmor micelles, IL-36γ plasmid/POEG-st-Pmor micelles, DOX+control plasmid/POEG-st-Pmor micelles and DOX+IL-36γ plasmid/POEG-st-Pmor micelles every three days for three times. The PBS treatment group was used as control. DOX dosage was 5 mg/kg and plasmid dosage was 50 μg per mouse. Lung tissues were harvested and weighted 11 days after the first injection. Pulmonary metastases were enumerated by intra-tracheal injection of India ink solution. India ink-injected lungs were washed in Feket's solution (300 ml 70% EtOH, 30 ml 37% formaldehyde and 5 ml glacial acetic acid) and white tumor nodules against a dark blue lung background were counted. Histopathological analysis. The lung tissues were harvested and fixed in 10% formalin after the above treatments. The fixed samples were then embedded in paraffin and the tissue sections were stained with hematoxylin/eosin and analyzed for the presence of metastases under microscope. The total number of metastases per lung section was counted in different treatment groups. Analysis of tumor-infiltrating lymphocytes and myeloid-derived suppressor cells. Lung tissues were collected in serum free RPMI medium and cut mechanically with scissors. Liberase TL (0.3 mg/ml) and DNase I (0.3 mg/ml) were used to digest the lung tissues and tumor nodules. Tissues were further grinded and filtered through a 40-mm cell strainer. TILs and MDSC cells were further purified and stained with fluorescence-labeled antibody for flow cytometry analysis using a FACS flow cytometer. Synthesis of POEG-st-PVBC polymer, OEG500 (550 mg, 1.1 mmol), VBC monomer (600 μL, 4.27 mmol), 4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid (8 mg, 0.0286 mmol), AIBN (3 mg, 0.0186 mmol), and 1 mL of dried tetrahydrofuran were added into a Schlenk tube, and deoxygenated by free-pump-thawing for three times. Then the mixture was filled with N2and immersed into an oil bath thermostated at 82° C. to start the polymerization. After 16 h, the reaction was quenched and the mixture was precipitated in hexane for 3 times. The product POEG-PVBC was obtained after vacuum drying. Conversion of OEG500 monomer was 66.0% and conversion of VBC monomer was 80.0%. Synthesis of POEG-st-PCre polymer. The POEG-PVBC polymer (270 mg) and creatine (1 g) were mixed in 15 mL anhydrous DMF with K2CO3(1 g). After stirring at 80° C. for 36 h, the reaction mixture was cooled down and transferred into a dialysis bag (MWCO=3500 Da). After dialysis against a dilute hydrochloric acid solution for 1 day and deionized water for 3 days, the solution in the dialysis bag was centrifuged at 4,500 rpm for 12 min and the supernatant was lyophilized to give the POEG-PCre polymer.1H NMR spectra were examined on a 600.0 MHz Bruker spectrometer using DMSO-d6as the solvent. Preparation of DOX+tRNA-mir-34a co-formulated DOX solution was first prepared by dissolving DOX.HCl in DMSO containing triethylamine (5 equiv) overnight to remove HCl. DOX-loaded POEG-PCre micelles were prepared by the dialysis method. Briefly, 2 mg of polymer was dissolved in 200 uL DMSO and mixed with 20 uL of DOX solution (10 mg/mL). The mixture was then dialyzed against distilled deionized (DD) water using a dialysis bag (MWCO=3500) overnight to remove the unloaded free DOX. Drug-free micelles were similarly prepared without adding DOX solution. For tRNA-mir-34a complexation, polymeric micelles were diluted to different concentrations in DD water and mixed with the equivalent volume of tRNA-mir-34a (in 10% of glucose) to obtain the desired N/P ratios. This nanocomplexes was allowed to incubate at RT for 20 min prior to further characterization. In vitro studies were performed with freshly prepared nanocomplexes. For in vivo studies, micelles and tRNA-mir-34a were first mixed in DD water for 20 min, and then lyophilized with glucose as a cryoprotectant. Synthesis of polymetformin (PMet) polymer. 4-Vinylbenzyl chloride (1.27 g), compound 2 (354 mg) ofFIG.17, AIBN (3 mg), 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid (24 mg), and 1 mL of dried tetrahydrofuran were added into a Schlenk tube, and deoxygenated by free-pump-thawing for three times. Then the mixture was filled with N2and immersed into an oil bath thermostated at 90° C. to start the polymerization. After 24 h, the reaction was quenched by immersing the tube into liquid nitrogen and the mixture was dialyzed against DMSO and distilled water for 2 days, respectively. The compound 3 with 85% of vinylbenzyl chloride and 15% of Boc-protected 4-vinylbenzylamine was obtained after precipitation. Metformin hydrochloride (1.65 g), compound 3 (150 mg) and N, N-diisopropylethylamine (1.35 mL) were added in DMSO (4.8 mL), and then the mixture was stirred for 48 h at 110° C. Reaction mixture was dialyzed against 0.5% hydrochloride solution and distilled water for 2 days, respectively. PMet with 15% Boc-protected 4-vinylbenzylamine (Boc-PMet) (compound 4) was obtained after lyophilization. The Boc-PMet (compound 4) polymer was deprotected at room temperature in DMSO/TFA (1/1, v/v) mixture for 2 h, and then dialyzed against distilled water for 2 days. The Boc-deprotected PMet product with 15% free amino group (compound 5) was obtained after lyophilization. Synthesis of PMet-P(cdmPEG2K) polymer. PMet-P(cdmPEG2K) was synthesized by a ring opening reaction of PEG2K-cdm and pMet polymers. Boc-deprotected PMet (compound 5 ofFIG.17, 100 mg) and PEG2K-cdm (compound 1, 303 mg) were dissolved in 4 mL of DMSO and stirred at 37° C. for 24 h. The mixture was dialyzed against DMSO and distilled water for 2 days, respectively. The final products of PMet-P(cdmPEG2K) polymer was obtained after lyophilization.1H NMR spectrum was analyzed on a Varian-400 FT-NMR spectrometer at 400 MHz with DMSO-d6and CDCl3as the solvent. Preparation of micelles with PMet-P(cdmPEG2K) polymer. Blank and DOX loaded PMet-P(cdmPEG2K) micelles were prepared by thin film hydration method. Briefly, DOX (5 mg/mL in 1:1(v/v) of DCM/methanol) and (10 mg/mL in DCM) at designated mass ratios were mixed in a glass tube, and organic solvent was removed through a gentle stream of nitrogen, followed by drying in vacuum for 1 h. The obtained thin-film of PMet-P(cdmPEG2K)/DOX mixture was hydrated in HEPES buffer solution (10 mM, pH 7.4), forming a clear solution of DOX-loaded PMet-P(cdmPEG2K) micelles. The blank micelles were prepared by the same procedure as described above except for no DOX adding. For IL-12 loaded micelleplexes and IL-12/DOX co-loaded micelleplexes preparation, desired amounts of IL-12 pDNA mixed with equal volume of blank micelles or DOX-loaded PMet-P(cdmPEG2K) micelles (the DLC (w/w) was about 9%) solutions at various N/P ratios (the ratios of the number of amino groups in PMet-P(cdmPEG2K) to the number of phosphate groups in IL-12 pDNA), and the resultant mixture was further incubated at room temperature for 20 min. The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope. | 108,827 |
11857635 | DETAILED DESCRIPTION OF EMBODIMENTS Unless otherwise defined, all technical and scientific terms used in the disclosure have the same meaning as those commonly understood by those skilled in the art to which the disclosure relates. For example, “polypeptide and protein drugs” refer to polypeptide and protein substances used for prevention, treatment and diagnosis, wherein polypeptides may be compounds formed by linking α-amino acids together with peptide bonds, and may also be intermediate products of protein hydrolysis; and N polypeptide chains are wound and tangled according to a certain spatial structure to form proteins. The polypeptide and protein drugs may be classified into amino acid and derivatives drugs thereof, polypeptide and protein drugs, enzyme and coenzyme drugs, nucleic acid and degradation products and derivatives drugs thereof, carbohydrate drugs, lipid drugs, cell growth factors and other biological products drugs. The IL-2 described in the disclosure may be natural, recombinant protein (such as recombinant human interleukin 2) or mutant with natural IL-2 function (such as “IL-2-C125A/L18M/L19S” described in doctoral dissertation of Liu Yan “Clone of Recombinant Human Interleukin-2 (IL-2) Mutant and Expression and Purification in Pasteur Pichia Pastoris System”), and also includes products obtained by tissue culture, protein synthesis and cell culture (natural, recombinant cells or mutants). Methods for extracting and separating the natural, recombination IL-2 or mutants are well known to those skilled in the art. The English abbreviations and representative meanings thereof in the disclosure are as follows: IL-2 is interleukin 2; rhIL-2 is recombinant human interleukin 2; HSV is herpes simplex virus; HIV is human immunodeficiency virus; HBV is hepatitis B virus; HCV is hepatitis C virus; and EBv is Epstein-barr virus. The following clearly and completely describes the technical solutions of the disclosure with reference to the examples of the disclosure. Apparently, the described examples are merely some but not all of the examples of the disclosure. Based on the examples in the disclosure, all other examples obtained by those of ordinary skills in the art without going through any creative work shall fall within the scope of protection of the disclosure. The compound raw materials used in the disclosure can be commercially available or prepared according to the disclosed preparing method, which does not limit the scope of the disclosure. The polyethylene glycol and the derivative thereof used in the examples are provided by Beijing JENKEM Technology Co., Ltd. Unless otherwise specified, the molecular weights are all 20K. Other reagents are commercially available. Example 1: Synthesis of Linking Chain (L) BOC-amino acid (92.2 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 23.8 g, 115.3 mmol) were added to dichloromethane (500 mL), cooled in an ice-water bath, and then p-hydroxybenzylalcohol (11.4 g, 92.2 mmol) was added. After addition, the ice bath was removed, and the mixture was reacted overnight at room temperature. The mixture was filtered, and a filter cake was washed with ethyl acetate, a filtrate was evaporated to dryness to obtain a crude product, and the crude product is purified through column chromatography to obtain a product 1. 1a: 19.7 g, in 76.0% yield. 1HNMR: (CDCl3): 8.75 (s, 1H), 7.22 (d, 2H), 7.05 (d, 2H), 4.87 (s, 2H), 3.74 (s, 2H), 1.52 (s, 9H). 1b: 20.3 g, in 74.8% yield. 1HNMR: (CDCl3): 8.74 (s, 1H), 7.21 (d, 2H), 7.05 (d, 2H), 4.88 (s, 2H), 3.77 (m, 1H), 1.51 (s, 9H), 1.27 (d, 3H). 1c: 21.6 g, in 72.5% yield. 1HNMR: (CDCl3): 8.75 (s, 1H), 7.22 (d, 2H), 7.05 (d, 2H), 4.87 (s, 2H), 3.61 (d, 1H), 2.82 (m, 1H), 1.52 (s, 9H), 1.06 (d, 6H). Compound 1 (39.1 mmol) was dissolved in dichloromethane (250 mL), added with trifluoroacetic acid (50 mL), and then the mixture was stirred overnight at room temperature after addition, and concentrated. Dichloromethane was added to the residues and then evaporated to dryness. This process was repeated three times, and ethyl ether was finally to precipitate and filter to obtain a product L. L1: 11.1 g, in 96.7% yield. L2: 11.6 g, in 97.1% yield. L3: 12.7 g, in 96.3% yield. Example 2: Synthesis of Conjugate (mPEG-L-40K) of Monomethoxy Polyethylene Glycol Acetic Acid and Linking Chain Monomethoxy polyethylene glycol-acetic acid (mPEG-CM, 40K, 5 g, 0.125 mmol), compound L (0.25 mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 16.9 mg, 0.125 mmol) were added to a reaction flask, dissolved with dichloromethane, and then diisopropylethylamine (45.2 mg, 0.35 mmol) was added. The mixture was stirred evenly, cooled in an ice-bath, and then EDCI (47.9 mg, 0.25 mmol) was added in batches. After the addition of EDCI, the reaction system was naturally warmed up to room temperature and reacted overnight. After the reaction solution was concentrated the next day, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and dried to obtain a product mPEG-L. mPEG-L1 (40K): 4.6 g, in 92.4% yield. mPEG-L2 (40K): 4.5 g, in 90.8% yield. mPEG-L3 (40K): 4.7 g, in 93.7% yield. Example 3: Preparation of Linking Chain L5 A synthesis route of the linking chain L5 was as follows: Synthesis of Compound (2): 3,4-dihydroxy benzaldehyde (10 g, 72.5 mmol) was dissolved in acetonitrile (150 mL), and added with sodium bicarbonate (8 g, 94.3 mmol), and then the mixture was warmed up to 60° C. Benzyl bromide (12.4 g, 72.5 mmol) was added in the mixture, and then the mixture was warmed up to 80° C. and stirred overnight. The acetonitrile was removed by concentration, and 10% aqueous hydrochloric acid solution (200 mL) was added into the residues, then the mixture was extracted with ethyl acetate (150 mL*3), combined and dried, filtered and concentrated, and the residues were purified through column chromatography to obtain 10 g of off-white solid (in 60% yield).1H NMR: (CDCl3): δ9.82 (s, 1H), 7.48-7.40 (m, 7H), 7.05 (m, 1H), 6.02 (s, 1H), 5.21 (s, 2H). Synthesis of Compound (3): Compound (2) (5 g, 21.9 mmol) was dissolved in DMF (80 mL), and added with potassium carbonate (7.6 g, 54.75 mmol) and potassium iodide (0.73 g 4.38 mmol), and then the mixture was stirred for 10 minutes. ATN-1 (9 g, 28.47 mmol) was added in the mixture and then the mixture was warmed up to 70° C. and stirred overnight. Post-treatment was carried out to add saturated solution of ammonium chloride (400 mL) into the reaction solution, then the mixture was extracted with ethyl acetate (150 mL*3), combined and dried, filtered and concentrated, and the residues were purified through column chromatography to obtain 5 g of white solid (in 61% yield).1H NMR: (CDCl3): δ9.85 (s, 1H), 7.47-7.36 (m, 7H), 7.03 (m, 1H), 5.24 (s, 2H), 5.07 (s, 1H), 4.17 (m, 2H), 3.59 (m, 2H), 1.46 (s, 9H). Synthesis of Compound (4): Compound (3) (5 g, 13.5 mmol) was dissolved in tetrahydrofuran (100 mL), and added with sodium borohydride (0.77 g, 20.25 mmol) at room temperature, then the mixture was stirred for 2 hours at room temperature and then cooled to 0° C. Acetic acid (2 mL) was added to quench the reaction, a solvent was removed by concentration under reduced pressure, and the residues were purified through column chromatography to obtain 4 g of colorless oily matter (in 80% yield).1H NMR: (DMSO-d6): δ7.38-7.05 (m, 8H), 6.80 (m, 1H), 5.09 (s, 2H), 5.06 (m, 1H), 4.40 (m, 2H), 3.98 (m, 2H), 3.31 (m, 2H), 1.38 (s, 9H). Synthesis of Compound (5): Compound (4) (1 g, 2.7 mmol) was dissolved in methanol (8 mL), and added with Pd/C (10%, 0.3 g), then hydrogen was introduced to react overnight. The mixture was filtered, a filtrate was concentrated, and the residues were purified through column chromatography to obtain 0.6 g of product (in 78% yield).1H NMR: (DMSO-d6): δ8.37 (s, 1H), 7.09 (s, 1H), 6.71 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.89 (m, 2H), 3.31 (m, 2H), 1.38 (s, 9H). Synthesis of Compound (6): Compound (5) (0.6 g, 2.1 mmol) was dissolved in acetone (7 mL), and added with potassium carbonate (0.58 g, 4.2 mmol), then the system was cooled to 0° C., acetyl oxide (235 mg, 2.3 mmol) was added, and the mixture was slowly warmed up to room temperature and reacted for 3 hours. Post-treatment was carried out to add an aqueous solution of ammonium chloride (30 mL), then the mixture was extracted with ethyl acetate, combined and concentrated, and purified through column chromatography to obtain 0.5 g of product (in 73% yield).1H NMR: (DMSO-d6): δ7.12 (s, 1H), 6.89 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.89 (m, 2H), 3.31 (m, 2H), 2.14 (s, 3H), 1.38 (s, 9H). Synthesis of Compound (L5): Compound (6) (0.5 g, 1.5 mmol) was dissolved in dichloromethane (7 mL), and added with trifluoroacetic acid (4 mL) to stir for 1 hour at room temperature. A solvent was removed by concentration, and ethyl ether was added into the residues to dissolve out solids, then the solids were filtered and dried to obtain 0.3 g of solid product in 86% yield.1H NMR: (DMSO-d6): δ8.30 (s, 1H), 6.89 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.85 (m, 2H), 3.29 (m, 2H), 2.12 (s, 3H). Example 4: Synthesis of Conjugate (mPEG-L5-NHS-20K) of Monomethoxy Polyethylene Glycol Acetic Acid and Linking Chain Synthesis of mPEG-L5: mPEG (20K)-CM-NHS (2 g, 0.1 mmol) was dissolved in anhydrous methylene chloride, and subcooled to 0° C. DIPEA (78 mg, 0.6 mmol) was added, and then compound L5 was added. The mixture was slowly warmed up to room temperature and stirred overnight. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain a product mPEG-L5 (1.8 g, 90%).1H NMR: (DMSO-d6): δ8.10 (s, 1H), 6.85 (s, 1H), 6.72 (d, 1H), 6.65 (d, 1H), 5.34 (s, 2H), 4.20 (m, 2H), 3.65 (m, 1800H), 3.51 (m, 2H), 3.24 (s, 3H), 2.78 (m, 4H), 2.12 (s, 3H). Synthesis of mPEG-L5-NHS: Compound mPEG-L5 (1 g, 0.05 mmol) was added into a reaction flask, dissolved with dichloromethane (6 mL), and cooled under the protection of N2, then succinimide carbonate (19.0 mg, 0.075 mmol) was added, and the mixture was stirred and dissolved. After that, DIPEA (12.9 mg, 0.1 mmol) was added, and a cold bath was removed after the addition of DIPEA, and the mixture was reacted overnight at room temperature. The reaction solution was concentrated, and the residues were crystallized with isopropyl alcohol to obtain a product mPEG-L5-NHS. 1H NMR: (DMSO-d6): δ8.10 (s, 1H), 6.85 (s, 1H), 6.72 (d, 1H), 6.65 (d, 1H), 4.65 (s, 2H), 4.15 (m, 2H), 3.65 (m, 1800H), 3.29 (m, 2H), 3.24 (s, 3H), 2.12 (s, 3H). Example 5: Synthesis of Y-PEG-L5-NHS-20K Refer to Example 4 for a preparing method of U-PEG-L5-NHS-20K. Example 6: Synthesis of U-PEG-L5-NHS-20K Refer to Example 4 for a preparing method of U-PEG-L5-NHS-20K. Example 7: Synthesis of 8Arm-PEG-L5-NHS-20K Refer to Example 4 for a preparing method of 8arm-PEG-L5-NHS-20K. Example 8: Synthesis of mPEG-L5-rhIL-2 (20K) Under the purging of nitrogen, mPEG-L5-NHS (20K, prepared in Example 4) stored at −20° C. was warmed up to room temperature. An mPEG-L5-NHS (20K) stock solution (200 mg/mL) was prepared in DMSO and added into a rhIL-2 solution (reaction grouping, reaction conditions such as a molar ratio of reactant mPEG-L5-NHS to rhil-2 (hereinafter referred to as a molar ratio of PEG to rhIL-2 molar ratio) and a specific reaction pH were shown in Table 1). A final concentration of rhlL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer solution (1 M, pH 9.0) was added to the mixture respectively to reach a final concentration of 20 mM, and then the mixture was reacted at room temperature for 2 hours to provide conjugates. After 2 hours, 1 M of glycine (pH 6.0) was added respectively to a final concentration of 100 mM to terminate the reaction. Coupling conditions of different reaction groups were determined by RP-HPLC for the terminated reaction system, and a coupling efficiency and a coupling degree were analyzed and evaluated according to migration conditions of the conjugates (left shift represented a high coupling degree), an RP-HPLC spectrogram was shown inFIG.1, and it could be seen fromFIG.1that both the coupling degrees and the coupling efficiencies of the reaction group 3 were greater than that of a reaction group 4/greater than that of a reaction group 5/greater than that of a reaction group 2/greater than that of a reaction group 6/greater than that of a reaction group 1. SEC+MALS analysis reactions were further carried out in the reaction group 3 to evaluate the coupling degree, and the results were shown inFIG.2. Coupling degree results of the conjugate mPEG-L5-rhIL-2 (20K) prepared in this example were shown in Table 1. TABLE 1SEC-MALS Coupling Degree Result Table of The Conjugate Preparedin Example 8ReactionReactionReactionReactionReactionReactiongroup 1group 2group 3group 4group 5group 6Molar10:120:130:130:130:130:1ratio offedmaterialsReaction9.89.89.88.07.26.0pHCoupling1, 2, 31, 2, 34 to 73 to1, 2, 31, 2, 3degreeRatioGreaterGreaterGreaterGreaterGreaterGreaterthanthanthanthanthanthan90%90%90%90%70%80% The products obtained in the above table may be divided into three categories: mPEG-L5-rhL-2 with a coupling degree of 4 to 7, mPEG-L5-rhL-2 with a coupling degree of 3 to 5, and mPEG-L5-rhL-2 with a coupling degree of 1 to 3. The optimal content may be controlled to be greater than 90%. Then the reaction-terminated mixtures were diluted with purified water respectively to provide an electroconductibility smaller than 0.5 ms/cm (25° C.). A pH value was adjusted to 4.0 with glacial acetic acid, and then the mixture was purified through column chromatography, as shown inFIG.3. Analysis was carried out with reference to RP-HPLC (FIG.4), wherein PEG was enriched in a penetration peak, mPEG-L5-rhIL-2 was eluted in 0 to 50% B (0 to 0.5 M NaCl gradient), elution peaks (A3 to A8) were collected sectionally, and it was observed that the coupling degree was distributed from high to low. Example 9: Synthesis of mPEG-L5-rhIL-2 (40K) Refer to Example 8 for a preparing method of mPEG-L5-rhIL-2 (40K). Example 10: Synthesis of Y-PEG-L5-rhIL-2 (20K) Refer to Example 8 for a preparing method of Y-PEG-L5-rhIL-2 (20K). Example 11: Synthesis of U-PEG-L5-rhIL-2 (20K) Refer to Example 8 for a preparing method of U-PEG-L5-rhIL-2 (20K). Example 12: Synthesis of 8Arm-PEG-L5-rhIL-2 (20K) Refer to Example 8 for a preparing method of 8arm-PEG-L5-rhIL-2 (20K). Example 13: Synthesis of mPEG-L1-rhIL-2 (20K) Under the purging of nitrogen, mPEG-L1-NHS (20K, refer to Example 2 for a preparing method of mPEG-L1-NHS (20K) stored at −20° C. was warmed up to room temperature. An mPEG-L1-NHS (20K) stock solution (200 mg/mL) was prepared in DMSO and added into a rhIL-2 solution (a molar ratio of PEG to rIL-2 was respectively 1:1, 3:1, 10:1, and 30:1). A final concentration of rhIL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer solution (1 M, pH 9.0) was added to the mixture respectively to reach a final concentration of 20 mM, and then the mixture was reacted at room temperature for 2 hours to provide conjugates. After 2 hours, 1 M of glycine (pH 6.0) was added respectively to a final concentration of 100 mM to terminate the reaction. Then the reaction-terminated mixtures were diluted with purified water respectively to provide an electroconductibility smaller than 0.5 ms/cm (25° C.). A pH value was adjusted to 4.0 with glacial acetic acid, and then the mixture was purified through column chromatography. Example 14: Pharmacodynamics Research of IL-2 Modified by the Disclosure PEG and rIL-2 were coupled under the reaction conditions of the reaction group 3 in Example 8. The coupled mixtures were purified by ion exchange chromatography, and a PEG-rIL-2 conjugate was separated for a pharmacodynamic experiment. A subcutaneous melanoma B16 model of a C57BL/6 mouse was used to evaluate a tumor inhibitory effect of an mPEG-L5-rhIL-2 conjugate (hereinafter referred to as PEG-rhIL-2). An experimental process is as follows: 106B16 cells were implanted in a middle of a back of each C57BL/6 mouse of 5 to 6 weeks old. After the tumor grows to a measurable size, the experimental mice were randomly grouped with each group including 6 mice, and test compounds rhIL-2, PEG-rhIL-2 and solvent control were applied to the mice in different dose concentrations and dose schemes. Body weight and tumor volume were measured every other day. The grouping of the efficacy experiments was shown in Table 2, and the experimental results were shown in Table 3 andFIG.5. TABLE 2Grouping of Efficacy ExperimentsDoseAdministrationGroupingconcentrationrouteFrequencySolvent controlN/AIVInjected oncegrouprhIL-2 group1 mg/KgIVOnce a day, andinjected for 5 daysPEG-rhIL-21 mg/KgIVInjected oncegroup TABLE 3Efficacy Experimental ResultsTumor volume/mm3Time/daySolvent control grouprhIL-2 groupPEG-rhIL-2 group110010210431501801475300260225788047028091260650310 According to the data, on the 9tday of administration, compared with the tumor volume (1260 mm3) of the solvent control group, the tumor volume (310 mm3) of the rhIL-2 group modified by the disclosure was greatly reduced, and compared with the tumor volume (650 mm3) of the unmodified rhIL-2 group, the anti-tumor effect of the mPEG-L5-rhL-2 group modified by the disclosure was increased by 27%, and the administration frequency of the mPEG-L5-rhL-2 group was greatly reduced. As an anti-tumor drug, the drug modified by the disclosure has the advantages of reducing an administration frequency, and greatly improving a bioavailability of the drug and a patient compliance. Example 15: Preparation of Monomethoxy Polyethylene Glycol-Adriamycin Conjugate (mPEG-L-Dox (40K)) Compound mPEG-L (0.075 mmol, prepared in Example 2) was added into a reaction flask, dissolved with dichloromethane (30 mL), and cooled under the protection of N2, then succinimide carbonate (23.0 mg, 0.09 mmol) was added, and the mixture was stirred and dissolved. After that, triethylamine (10.1 mg, 0.1 mmol) was added, and a cold bath was removed after the addition of triethylamine, and the mixture was reacted overnight at room temperature. The reaction solution was concentrated, and the residues were crystallized with isopropyl alcohol to obtain a product mPEG-L-NHS. mPEG-L1-NHS (40K): 2.6 g, in 88.5% yield. mPEG-L2-NHS (40K): 2.7 g, in 89.2% yield. mPEG-L3-NHS (40K): 2.6 g, in 87.9% yield. Compound mPEG-L-NHS (0.06 mmol, prepared in last step) was dissolved in dichloromethane (25 mL), and cooled under the protection of N2, then diisopropylethylamine (12.9 mg, 0.1 mmol) was added, and the mixture was stirred and dissolved. After that, doxorubicin hydrochloride (52.2 mg, 0.09 mmol) was added, and after the addition of hydrochloricacid adriamycin, the mixture was reacted at room temperature for 5 hours. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain a red solid product. mPEG-L1-Dox (40K): 2.0 g, in 84.9% yield.1H NMR: (DMSO-d6): δ8.84 (s, 1H), 8.68 (s, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d, 3H), 5.52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.61 (s, 2H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m, 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H). mPEG-L2-Dox (40K): 2.1 g, in 85.7% yield.1H NMR: (DMSO-d6): δ8.82 (s, 1H), 8.67 (s, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d, 3H), 5.52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.59 (d, 1H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m, 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H), 1.58 (d, 3H). mPEG-L3-Dox (40K): 2.1 g, in 84.6% yield.1H NMR: (DMSO-d6): δ8.83 (s, 1H), 8.65 (s, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d, 3H), 5.52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.54 (d, 1H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m, 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H), 1.34 (d, 3H), 1.16 (d, 6H). Example 16: Preparation of Conjugate (PEG-L3 (20K)) of Polyethylene Glycol Acetic Acid and Linking Chain L3 Polyethylene glycol-acetic acid (PEG-CM, 20K, 5 g, 0.25 mmol), compound L3 (168.5 mg, 0.5 mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 67.6 mg, 0.5 mmol) were added to a reaction flask, dissolved with dichloromethane, and then diisopropylethylamine (193.6 mg, 1.5 mmol) was added. The mixture was stirred evenly, cooled in an ice-bath, and then EDCI (191.7 mg, 1 mmol) was added in batches. After the addition of EDCI, the reaction system was naturally warmed up to room temperature and reacted overnight. After the reaction solution was concentrated the next day, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain 4.8 g of product PEG-L3 (20K) in 96% yield. Example 17: Preparation of Conjugate (PEG-L3-Dox (20K)) of Polyethylene Glycol Acetic Acid and Linking Chain L3 Compound PEG-L3 (2 g, 0.1 mmol, prepared in Example 16) was added into a reaction flask, dissolved with dichloromethane (40 mL), and cooled under the protection of N2, then succinimide carbonate (51.2 mg, 0.2 mmol) was added, and the mixture was stirred and dissolved. After that, triethylamine (30.3 mg, 0.3 mmol) was added, and a cold bath was removed after the addition of triethylamine, and the mixture was reacted overnight at room temperature. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain 1.8 g of product PEG-L3-NHS (20K) in 88.5% yield. Compound PEG-L3-NHS (1.6 g, 0.08 mmol, prepared in last step) was dissolved in dichloromethane (30 mL), and cooled under the protection of N2, then diisopropylethylamine (19.4 mg, 0.15 mmol) was added, and the mixture was stirred evenly. After that, hydrochloricacid adriamycin (69.6 mg, 0.12 mmol) was added, and after the addition of hydrochloricacid adriamycin, the mixture was reacted at room temperature for 5 hours. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain 1.3 g of red solid product PEG-L3-Dox (20K) in 81.2% yield.1H NMR: (DMSO-d6): δ8.81 (s, 2H), 8.68 (s, 2H), 7.62 (m, 2H), 7.53 (d, 2H), 7.33 (d, 8H), 7.16 (d, 4H), 5.52 (s, 4H), 5.12 (t, 2H), 4.83 (s, 4H), 4.54 (d, 2H), 4.32 (t, 2H), 4.06 (m, 2H), 3.96 (s, 6H), 3.71 (m, 2H), 3.67 (m, 1800H), 3.41 (s, 4H), 3.27 (m, 2H), 2.97 (m, 2H), 2.39 (s, 4H), 2.28 (d, 4H), 2.05 (d, 4H), 1.34 (d, 6H), 1.16 (d, 12H). Example 18: Preparation of 4Arm-PEG-L3 (20K) 4arm-PEG-acetic acid (20K, 5 g, 0.025 mmol), compound L3 (674 mg, 2.0 mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 135.1 mg, 1 mmol) were added to a reaction flask, dissolved with dichloromethane, and then diisopropylethylamine (129.1 mg, 1.0 mmol) was added. The mixture was stirred evenly, cooled in an ice-bath, and then EDCI (191.7 mg, 1 mmol) was added in batches. After the addition of EDCI, the reaction system was naturally warmed up to room temperature and reacted overnight. After the reaction solution was concentrated the next day, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and dried to obtain 4.7 g of product 4arm-PEG-L3 (20K) in 94.0% yield. Example 19: Preparation of 4Arm-PEG-L3-Dox (20K) Compound 4arm-PEG-L3 (2 g, 0.1 mmol, prepared in Example 18) was added into a reaction flask, dissolved with dichloromethane (40 mL), and cooled under the protection of N2, then succinimide carbonate (1.02 g, 0.4 mmol) was added, and the mixture was stirred and dissolved. After that, triethylamine (60.6 mg, 0.6 mmol) was added, and a cold bath was removed after the addition of triethylamine, and the mixture was reacted overnight at room temperature. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol to obtain 1.8 g of product 4arm-PEG-L3-NHS (20K) in 90.0% yield. Compound 4arm-PEG-L3-NHS (1.5 g, 0.075 mmol, prepared in last step) was dissolved in dichloromethane (30 mL), and cooled under the protection of N2, then diisopropylethylamine (77.5 mg, 0.6 mmol) was added, and the mixture was stirred evenly. After that, hydrochloricacid adriamycin (261 mg, 0.45 mmol) was added, and after the addition of hydrochloricacid adriamycin, the mixture was reacted at room temperature for 5 hours. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain 1.3 g of red solid product 4arm-PEG-L3-Dox (20K) in 86.7% yield.1H NMR: (DMSO-d6): δ8.82 (s, 4H), 8.67 (s, 4H), 7.62 (m, 4H), 7.53 (d, 4H), 7.33 (d, 8H), 7.16 (d, 12H), 5.51 (s, 8H), 5.12 (t, 4H), 4.83 (s, 8H), 4.55 (t, 4H), 4.33 (m, 12H), 4.06 (m, 4H), 3.95 (s, 12H), 3.67 (m, 1800H), 3.55 (t, 8H), 3.47 (s, 4H), 3.28 (m, 4H), 3.08 (m, 4H), 2.27 (d, 8H), 2.04 (m, 8H), 1.68 (t, 8H), 1.42 (d, 12H), 1.16 (d, 24H). Example 20: Preparation of 8Arm-PEG-L4-NHS (20K) 8-arm-PEG-N3(20K, 2 g, 0.1 mmol), compound L4 (prepared using the method in Chinese patent application CN201510354709.6, 220 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added into N,N-dimethylformamide (20 mL), quickly stirred to dissolve the mixture, then an aqueous solution (4.4 mL 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol) was added to react overnight at room temperature, and the mixture was deposited with isopropyl alcohol to obtain 1.8 g of product. Compound 8arm-PEG-L4 (2 g, 0.1 mmol, prepared in last step) was added into a reaction flask, dissolved with dichloromethane (40 mL), and cooled under the protection of N2, then succinimide carbonate (1.02 g, 0.4 mmol) was added, and the mixture was stirred and dissolved. After that, triethylamine (60.6 mg, 0.6 mmol) was added, and a cold bath was removed after the addition of triethylamine, and the mixture was reacted overnight at room temperature. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol to obtain 1.7 g of product 8arm-PEG-L4-NHS (20K) in 85% yield. Example 21: Preparation of 8Arm-PEG-L4-Dox (20K) Compound 8arm-PEG-L4-NHS (20K, 1.5 g, 0.075 mmol, prepared in Example 20) was dissolved in dichloromethane (30 mL), and cooled under the protection of N2, then diisopropylethylamine (77.5 mg, 0.6 mmol) was added, and the mixture was stirred evenly. After that, hydrochloricacid adriamycin (261 mg, 0.45 mmol) was added, and after the addition of hydrochloricacid adriamycin, the mixture was reacted at room temperature for 5 hours. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain 1.4 g of red solid product 8arm-PEG-L4-Dox (20K) in 93.3% yield. Example 22: Preparation of 4Arm PEG-L5-Dox (20K) Synthesis of 4Arm PEG-L5 4arm PEG (20K)-CM-NHS (2 g, 0.1 mmol) was dissolved in anhydrous methylene chloride, and subcooled to 0° C. DIPEA (78 mg, 0.6 mmol) was added, and then compound L5 (prepared in Example 3) was added. The mixture was slowly warmed up to room temperature and stirred overnight. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain a product 4arm PEG-L5 (1.8 g, 90%).1H NMR: (DMSO-d6): δ8.11 (s, 4H), 6.86 (s, 4H), 6.74 (d, 4H), 6.65 (d, 4H), 5.35 (s, 8H), 4.20 (m, 8H), 3.66 (m, 1800H), 3.52 (m, 4H), 3.24 (s, 6H), 2.78 (m, 8H), 2.13 (s, 6H). Synthesis of 4Arm PEG-L5-NHS Compound 4arm PEG-L5 (1.5 g, 0.075 mmol, prepared in last step) was added into a reaction flask, dissolved with dichloromethane (30 mL), and cooled under the protection of N2, then succinimide carbonate (68.0 mg, 0.03 mmol) was added, and the mixture was stirred and dissolved. After that, DIPEA (51.6 mg, 0.4 mmol) was added, and a cold bath was removed after the addition of DIPEA, and the mixture was reacted overnight at room temperature. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol to obtain a product 4armPEG-L5-NHS (1.4 g, 93%). 1H NMR: (DMSO-d6): δ8.12 (s, 4H), 6.85 (s, 4H), 6.72 (d, 4H), 6.65 (d, 4H), 4.65 (s, 8H), 4.15 (m, 8H), 3.65 (m, 1800H), 3.29 (m, 8H), 3.24 (s, 12H), 2.12 (s, 9H). Synthesis of 4Arm PEG-L5-Dox Compound 4arm PEG-NHS (1 g, 0.05 mmol, prepared in last step) was added in a reaction flask, dissolved with dichloromethane (20 mL), and cooled under the protection of N2, then diisopropylethylamine was added, and the mixture was stirred evenly. After that, hydrochloricacid adriamycin (348.8 mg, 0.3 mmol) was added, and after the addition of hydrochloricacid adriamycin, the mixture was reacted overnight at room temperature. After the reaction solution was concentrated, the residues were crystallized with isopropyl alcohol, subjected to suction filtration and then dried to obtain a brownish red solid product 4arm PEG-L5-Dox (0.84 g, 84%). 1H NMR: (DMSO-d6): δ8.81 (s, 4H), 8.67 (s, 4H), 7.63 (m, 4H), 7.53 (d, 4H), 7.34 (d, 8H), 7.17 (d, 12H), 5.52 (s, 8H), 5.12 (t, 4H), 4.83 (s, 8H), 4.55 (t, 4H), 4.33 (m, 12H), 4.06 (m, 4H), 3.95 (s, 12H), 3.67 (m, 1800H), 3.55 (t, 8H), 3.47 (s, 4H), 3.28 (m, 4H), 3.09 (m, 4H), 2.26 (d, 8H), 2.05 (m, 8H), 1.66 (t, 8H), 1.43 (d, 12H), 1.17 (d, 24H). Example 23: Research on Tumor Growth Inhibition in HCT116 Tumor Heterotopic Transplantation Mouse Model Experimental Purpose: The experiment was carried out to detect a tumor growth inhibition effect of a drug on the HCT116 tumor heterotopic transplantation mouse model. Drug Grouping: G1: solvent; G2: positive drug adriamycin (commercially available); and G3: 4arm PEG-L5-Dox (prepared in Example 22). Experimental Method: Cell Culture: An original culture solution was removed by suction after HCT116 cells (commercially available) were received, and 10 mL of fresh culture solution was added. The cells were collected and passaged when 90% of the cells were converged. After the culture solution was removed, 10 ml of EDTA/PBS solution was added and placed at room temperature for 5 minutes, and then the EDTA/PBS solution was removed by suction. 3 ml of 0.25% pancreatin (37° C.) was evenly mixed and spread on cell surfaces, and then immediately removed by suction. The culture flask with no medium was put into an incubator until the cells were separated from a wall of the culture flask (5 minutes). 20 mL of DMEM containing 10% FBS was added, and gently blown and beaten to blend the cells. The cells was counted with a counting plate and diluted with DMEM containing 10% FBS. 40 mL of cell suspension was transferred to a 150 mm cell culture dish and then put into an incubator. The cell suspension was changed every other day, and when 80% of the cells were converged, the cells were passaged using the same method above. Preparation of Cell Suspension for Inoculation: The cell suspension was changed in advance when it was detected that 80% of the cells were converged on the inoculation day of the cells. After 3 hours, the culture solution was removed by a pipette, 20 ml of EDTA/PBS solution was added in each dish and placed at room temperature for 5 minutes, and then the EDTA/PBS solution was removed by suction. 3 ml of 0.25% pancreatin (37° C.) was evenly mixed and spread on cell surfaces, and then immediately removed by suction. The culture flask with no medium was put into an incubator until the cells were separated from a wall of the culture flask (5 minutes). 20 mL of DMEM containing 10% FBS was added, and gently blown and beaten to blend the cells. The supernatant was removed after the cell suspension was transferred to a centrifuge tube and centrifuged at a room temperature for 5 minutes, and then serum-free DMEM was added, and gently blended. The mixture was centrifuged again, the supernatant was removed, and a small amount of ice-cold PBS was added, and gently blended. The cells were counted with a counting plate and diluted with PBS to a concentration of 2*107cells/mL, and then the cells were placed on ice until inoculation. Tumor Inoculation: HCT116 cells were subcutaneously inoculated in a right front axilla of a mouse. Each mouse was inoculated with 200 μL of PBS cell suspension, and a total of 30 mice were inoculated. Tumor volumes were measured twice a week after inoculation, and when an average tumor volume reached 100 to 200 mm3, 18 mice with similar tumor sizes were selected and randomly divided into 3 groups with 6 mice in each group. The mice were administrated within 24 hours after grouping, and the mice were weighted before each administration to adjust a dosage. The mice were continuously observed and measured after last administration, the body weights and the tumor volumes were measured twice a week, and the tumor volume (mm3) was calculated according to a formula that V=0.5(a*b2), wherein a represented a length diameter and b represented a width diameter. When the average tumor volume of the mice in a certain group was greater than 2000 mm3, the mice in the group were sacrificed. Differences of the tumor volumes among the groups were compared by a t test, and P<0.05 was regarded as a statistically significant difference. Experimental Results: The experimental results are shown inFIG.6, both the positive drug adriamycin and the compound 4armPEG-L5-Dox have obvious anti-tumor effects, and the compound 4arm PEG-L5-Dox has a better effect than that of the adriamycin, and the effect gap is gradually increased with the prolonging of an administration time, which is probably related to a fact that the adriamycin in the compound 4arm PEG-L5-Dox uses polyethylene glycol as a carrier, so that the drug can stay at a tumor site for a longer time and can achieve the effects of sustained-release and controlled-release. Weight losses of the mice during the administration are acceptable, and it is valuable to further research the compound. The “coupling degree” in the example of the disclosure refers to a number of PEG molecules coupled to each IL-2 molecule (i.e., the n value in the general formula IX of the disclosure), for example, mPEG-L5-rhIL-2 with a coupling degree of 4 refers to coupling 4 mPEG-L5 chain segments to each rhIL-2 molecule; mPEG-L5-rhIL-2 with a coupling degree of 4 to 7 refers to a mixture containing mPEG-L5-rhIL-2 with a coupling degree of 4, mPEG-L5-rhIL-2 with a coupling degree of 5, mPEG-L5-rhIL-2 with a coupling degree of 6, and mPEG-L5-rhIL-2 with a coupling degree of 7. The DN structure in the reaction formulae of Examples 17, 19, 21 and 22 of the disclosure is Those described above are merely preferred examples of the disclosure, but are not intended to limit the disclosure. Any modifications and equivalent substitutions made without departing from the principle of the disclosure shall all fall within the scope of protection of the disclosure. | 35,321 |
11857636 | DETAILED DESCRIPTION OF THE INVENTION In a general sense, the invention is based on the recognition that a drug can be released from trans-cyclooctene derivatives satisfying formula (1a) upon cyclooaddition with compatible dienes, such as tetrazine derivatives. The dienophiles of formula (1a) have the advantage that they react (and effectuate drug release) with substantially any diene. Without wishing to be bound by theory, the inventors believe that the molecular structure of the retro Diels-Alder adduct is such that a spontaneous elimination reaction within this rDA adduct releases the drug. Particularly, the inventors believe that appropriately modified rDA components lead to rDA adducts wherein the bond to the drug on the dienophile is destabilized by the presence of a lone electron pair on the diene. The general concept of using the retro-Diels Alder reaction in Prodrug activation is illustrated inFIG.1. InFIG.1, “TCO” stands for trans-cyclooctene. The term trans-cyclooctene is used here as possibly including one or more hetero-atoms, and particularly refers to a structure satisfying formula (1a). In a broad sense, the inventors have found that—other than the attempts made on the basis of the Staudinger reaction—the selection of a TCO as the trigger moiety for a prodrug, provides a versatile tool to render drug (active) moieties into prodrug (activatable) moieties, wherein the activation occurs through a powerful, abiotic, bio-orthogonal reaction of the dienophile (Trigger) with the diene (Activator), viz the aforementioned retro Diels-Alder reaction, and wherein the Prodrug is a Drug-dienophile conjugate. It will be understood that inFIG.1in the retro Diels-Alder adduct as well as in the end product, the indicated TCO group and the indicated diene group are the residues of, respectively, the TCO and diene groups after these groups have been converted in the retro Diels-Alder reaction. A requirement for the successful application of an abiotic bio-orthogonal chemical reaction is that the two participating functional groups have finely tuned reactivity so that interference with coexisting functionality is avoided. Ideally, the reactive partners would be abiotic, reactive under physiological conditions, and reactive only with each other while ignoring their cellular/physiological surroundings (bio-orthogonal). The demands on selectivity imposed by a biological environment preclude the use of most conventional reactions. The inverse electron demand Diels Alder reaction, however, has proven utility in animals at low concentrations and semi-equimolar conditions (R. Rossin et al,Angewandte Chemie Int Ed25 2010, 49, 3375-3378). The reaction partners subject to this invention are strained trans-cyclooctene (TCO) derivatives and suitable dienes, such as tetrazine derivatives. The cycloaddition reaction between a TCO and a tetrazine affords an intermediate, which then rearranges by expulsion of dinitrogen in a retro-Diels-Alder cycloaddition to form a dihydropyridazine conjugate. This and its tautomers is the retro Diels-Alder adduct. The present inventors have come to the non-obvious insight, that the structure of the TCO of formula (1a), par excellence, is suitable to provoke the release of a drug linked to it, as a result of the reaction involving the double bond available in the TCO dienophile, and a diene. The features believed to enable this are (a) the nature of the rDA reaction, which involves a rearrangement of double bonds, which can be put to use in provoking an elimination cascade; (b) the nature of the rDA adduct that bears a dihydro pyridazine group that is non-aromatic (or another non-aromatic group) and that can rearrange by an elimination reaction to form conjugated double bonds or to form an (e.g. pyridazine) aromatic group, (c) the nature of the rDA adduct that may bear a dihydro pyridazine group that is weakly basic and that may therefore catalyze elimination reactions. In a broad sense, the invention puts to use the recognition that the rDA reaction, using a dienophile of formula (1a), as well as the rDA adduct embody a versatile platform for enabling provoked drug release in a bioorthogonal reaction. The fact that the reaction is bio-orthogonal, and that many structural options exist for the reaction pairs, will be clear to the skilled person. E.g., the rDA reaction is known in the art of pre-targeted medicine. Reference is made to, e.g., WO 2010/119382, WO 2010/119389, and WO 2010/051530. Whilst the invention presents an entirely different use of the reaction, it will be understood that the various structural possibilities available for the rDA reaction pairs as used in pre-targeting, are also available in the field of the present invention. The dienophile trigger moiety used in the present invention comprises a trans-cyclooctene ring, the ring optionally including one or more hetero-atoms. Hereinafter this eight-membered ring moiety will be defined as a trans-cyclooctene moiety, for the sake of legibility, or abbreviated as “TCO” moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated drug upon reaction. The skilled person is familiar with the fact that the dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity. Thus, in general, the invention is not limited to strictly drug-substituted trans-cyclooctene. The person skilled in organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same endocyclic double bond as the trans-cyclooctene, but which may have one or more heteroatoms elsewhere in the ring. I.e., the invention generally pertains to eight-membered non-aromatic cyclic alkenylene moieties, preferably a cyclooctene moiety, and more preferably a trans-cyclooctene moiety, comprising a conjugated drug. Other than is the case with e.g. medicinally active substances, where the in vivo action is often changed with minor structural changes, the present invention first and foremost requires the right chemical reactivity combined with an appropriate design of the drug-conjugate. Thus, the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienophiles. It should be noted that, depending on the choice of nomenclature, the TCO dienophile may also be denoted E-cyclooctene. With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z-isomer. In the present invention, any substituted variants of the invention, whether or not formally “E” or “Z,” or “cis” or “trans” isomers, will be considered derivatives of unsubstituted trans-cyclooctene, or unsubstituted E-cyclooctene. The terms “trans-cyclooctene” (TCO) as well as E-cyclooctene are used interchangeably and are maintained for all dienophiles according to the present invention, also in the event that substituents would formally require the opposite nomenclature. I.e., the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below are in the E (entgegen) or trans position. The present invention will further be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where an indefinite or definite article is used when referring to a singular noun e.g. “a” or “an”, “the”, this includes a plural of that noun unless something else is specifically stated. It is furthermore to be noticed that the term “comprising”, used in the description and in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B. In several chemical formulae below reference is made to “alkyl” and “aryl.” In this respect “alkyl”, each independently, indicates an aliphatic, straight, branched, saturated, unsaturated and/or or cyclic hydrocarbyl group of up to ten carbon atoms, possibly including 1-10 heteroatoms such as O, N, or S, and “aryl”, each independently, indicates an aromatic or heteroaromatic group of up to twenty carbon atoms, that possibly is substituted, and that possibly includes 1-10 heteroatoms such as O, N, P or S. “Aryl” groups also include “alkylaryl” or “arylalkyl” groups (simple example: benzyl groups). The number of carbon atoms that an “alkyl”, “aryl”, “alkylaryl” and “arylalkyl” contains can be indicated by a designation preceding such terms (i.e. C1-10alkyl means that said alkyl may contain from 1 to 10 carbon atoms). Certain compounds of the invention possess chiral centers and/or tautomers, and all enantiomers, diasteriomers and tautomers, as well as mixtures thereof are within the scope of the invention. In several formulae, groups or substituents are indicated with reference to letters such as “A”, “B”, “X”, “Y”, and various (numbered) “R” groups. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise. In all embodiments of the invention as described herein, alkyl is preferably lower alkyl (C1-4alkyl), and each aryl preferably is phenyl. Earlier work (R. Rossin et al,Angewandte Chemie Int Ed2010, 49, 3375-3378) demonstrated the utility of the inverse-electron-demand Diels Alder reaction for pretargeted radioimmunoimaging. This particular cycloaddition example occurred between a (3,6)-di-(2-pyridyl)-s-tetrazine derivative and a E-cyclooctene, followed by a retro Diels Alder reaction in which the product and nitrogen is formed. Because the trans cyclooctene derivative does not contain electron withdrawing groups as in the classical Diels Alder reaction, this type of Diels Alder reaction is distinguished from the classical one, and frequently referred to as an “inverse electron demand Diels Alder reaction”. In the following text the sequence of both reaction steps, i.e. the initial Diels-Alder cyclo-addition (typically an inverse electron demand Diels Alder cyclo-addition) and the subsequent retro Diels Alder reaction will be referred to in shorthand as “retro Diels Alder reaction.” Retro Diels-Alder Reaction The Retro Diels-Alder coupling chemistry generally involves a pair of reactants that couple to form an unstable intermediate, which intermediate eliminates a small molecule (depending on the starting compounds this may be e.g. N2, CO2, RCN), as the sole by-product through a retro Diels-Alder reaction to form the retro Diels-Alder adduct. The paired reactants comprise, as one reactant (i.e. one Bio-orthogonal Reactive Group), a suitable diene, such as a derivative of tetrazine, e.g. an electron-deficient tetrazine and, as the other reactant (i.e. the other Bio-orthogonal Reactive Group), a suitable dienophile, such as a strained cyclooctene (TCO). The exceptionally fast reaction of e.g. electron-deficient (substituted) tetrazines with a TCO moiety results in a ligation intermediate that rearranges to a dihydropyridazine retro Diels-Alder adduct by eliminating N2as the sole by-product in a [4+2] Retro Diels-Alder cycloaddition. In aqueous environment, the initially formed 4,5-dihydropyridazine product may tautomerize to a 1,4-dihydropyridazine product. The two reactive species are abiotic and do not undergo fast metabolism or side reactions in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media. Thus, the compounds and the method of the invention can be used in a living organism. Moreover, the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein. References on the Inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include: Thalhammer, F; Wallfahrer, U; Sauer, J, Tetrahedron Letters, 1990, 31 (47), 6851-6854; Wijnen, J W; Zavarise, S; Engberts, J B F N, Journal Of Organic Chemistry, 1996, 61, 2001-2005; Blackman, M L; Royzen, M; Fox, J M, Journal Of The American Chemical Society, 2008, 130 (41), 13518-19), R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch, R. C. M. Vulders, l. Verel, J. Lub, M. S. Robillard, Angew Chem Int Ed 2010, 49, 3375, N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder, Angew Chem Int Ed 2009, 48, 7013, and Devaraj et al., Angew.Chem.Int.Ed., 2009, 48, 1-5. It will be understood that, in a broad sense, according to the invention the aforementioned retro Diels-Alder coupling and subsequent drug activation chemistry can be applied to basically any pair of molecules, groups, or moieties that are capable of being used in Prodrug therapy. I.e. one of such a pair will comprise a drug linked to a dienophile (the Trigger). The other one will be a complementary diene for use in reaction with said dienophile. Trigger The Prodrug comprises a Drug denoted as DDlinked, directly or indirectly, to a Trigger moiety denoted as TR, wherein the Trigger moiety is a dienophile. The dienophile, in a broad sense, is an eight-membered non-aromatic cyclic alkenylene moiety (preferably a cyclooctene moiety, and more preferably a trans-cyclooctene moiety). Optionally, the trans-cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position. The person skilled in organic chemistry will understand that the term “fixed in substantially the same plane” refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings. E.g., the at least two exocyclic bonds can be the two bonds of a double bond to an oxygen (i.e. C═O). The at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane. Examples of the latter include strained rings such as cyclopropyl and cyclobutyl. Without wishing to be bound by theory, the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the retro-Diels-Alder reaction. In this invention, the TCO satisfies the following formula (1a): A and P each independently are CRa2or CRaXD, provided that at least one is CRaXD. XDis (O—C(O)p-(LD)n-(DD), S—C(O)-(LD)n-(DD), O—C(S)-(LD)n-(DD), S—C(S)-(LD)n-(DD), O—S(O)-(LD)n-(DD), wherein p=0 or 1, (LD)nis an optional linker, with n=0 or 1, preferably linked to TRvia S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched. DDis one or more therapeutic moieties or drugs, preferably linked via S, N, NH, or O, wherein these atoms are part of the therapeutic moiety. Preferably, XDis (O—C(O))p-(LD)n-(DD), where p=0 or 1, preferably 1, and n=0 or 1. It is preferred that when DDis bound to TRor LDvia NH, this NH is a primary amine (—NH2) residue from DD, and when DDis bound via N, this N is a secondary amine (—NH—) residue from DD. Similarly, it is preferred that when DDis bound via O or S, said O or S are, respectively, a hydroxyl (—OH) residue or a sulfhydryl (—SH) residue from DD. It is further preferred that said S, N, NH, or O moieties comprised in DDare bound to an aliphatic or aromatic carbon of DD. It is preferred that when LDis bound to TRvia NH, this NH is a primary amine (—NH2) residue from LD, and when LDis bound via N, this N is a secondary amine (—NH—) residue from LD. Similarly, it is preferred that when LDis bound via O or S, said O or S are, respectively, a hydroxyl (—OH) residue or a sulfhydryl (—SH) residue from LD. It is further preferred that said S, N, NH, or O moieties comprised in LDare bound to an aliphatic or aromatic carbon of LD. Where reference is made in the invention to a linker LDthis can be self-immolative or not, or a combination thereof, and which may consist of multiple self-immolative units. By way of further clarification, if p=0 and n=0, the drug species DDdirectly constitutes the leaving group of the elimination reaction, and if p=0 and n=1, the self-immolative linker constitutes the leaving group of the elimination. The position and ways of attachment of linkers LDand drugs DDare known to the skilled person (see for example Papot et al,Anti-Cancer Agents in Medicinal Chemistry,2008, 8, 618-637). Nevertheless, typical but non-limiting examples of self-immolative linkers LDare benzyl-derivatives, such as those drawn below. On the right, an example of a self-immolative linker with multiple units is shown; this linker will degrade not only into CO2and one unit of 4-aminobenzyl alcohol, but also into one 1,3-dimethylimidazolidin-2-one unit. In an interesting embodiment, Y,Z,X,Q each independently are selected from the group consisting of CRa2, C═CRa2, C═O, C═S, C═NRb, S, SO, SO2, O, NRb, and SiRc2, with at most three of Y, Z, X, and Q being selected from the group consisting of C═CRa2, C═O, C═S, and C═NRb, wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O—O, O—NRb, S—NRb, O—S, O—S(O, O—S(O)2, and S—S, and such that Si is only adjacent to CRa2or O. In a preferred embodiment, the TCO of formula (1a) is an all-carbon ring. In another preferred embodiment, the TCO of formula (1a) is a heterocyclic carbon ring, having of one to three oxygen atoms in the ring, and preferably a single oxygen atom. In another interesting embodiment, one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CRa═CRa, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5-or 6-membered ring, of a conjugated 7-membered ring, or of CRa═CRa; when not part of a fused ring P and A are independently CRa2or CRaXD, provided that at least one is CRaXD; when part of a fused ring P and A are independently CRaor CXD, provided that at least one is CXD; the remaining groups (Y,Z,X,Q) being independently from each other CRa2, C═CRa2, C═O, C═S, C═NRb, S, SO, SO2, O, NRb, SiRc2, such that at most 1 group is C═CRa2, C═O, C═S, C═NRb, and no adjacent pairs of atoms are present selected from the group consisting of O—O, O—NRb, S—NRb, O—S, O—S(O), O—S(O)2, and S—S, and such that Si, if present, is adjacent to CRa2or O, and the CRa2=CRa2bond, if present, is adjacent to CRa2or C═CRa2groups; T, G each independently denotes H, or a substituent selected from the group consisting of alkyl, F, Cl, Br, or I. In some embodiments fused rings are present that result in two exocyclic bonds being fixed in substantially the same plane. These are selected from fused 3-membered rings, fused 4-membered rings, fused bicyclic 7-membered rings, fused aromatic 5-membered rings, fused aromatic 6-membered rings, and fused planar conjugated 7-membered rings as defined below: Fused 3-membered rings are: Therein E, G are part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are CRaor CXD, and such that CXDcan only be present in A and P. E-G is CRa-CRaor CRa-CXD, and D is CRa2C═O, C═S, C═NRb, NRb, O, S; or E-G is CRa-N or CXD-N, and D is CRa2, C═O, C═S, C═NRb, NRbO, or S. Fused 4-membered rings are: E-G is part of the above mentioned8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C, CRaor CXD, and such that CXDcan only be present in A and P. E, G are CRa, CXDor N, and D,M independently from each other are CRa2, C═O, C═S, C═NRb, C═CRa2, S, SO, SO2, O, NRbbut no adjacent O—O or S—S groups; or E-D is C═CRaand G is N, CRa, CXDand M is CRa2, S, SO, SO2, O, NRb; or E-D is C═N and G is N, CRa, CXDand M is CRa2, S, SO, SO2, O; or D-M is CRa═CRaand E, G each independently are CRa, CXDor N; or D-M is CRa═N and E is CRa, CXD, N, and G is CRaor CXD; or E is C, G is CRa, CXDor N, and D,M are CRa2, S, SO, SO2, O, NRb, or at most one of C═O, C═S, C═NRb, C═CRa2, but no adjacent O—O or S—S groups; or E and G are C, and D and M independently from each other are CRa2, S, SO, SO2, O, NRbbut no adjacent O—O, or S—S groups. Fused bicyclic 7-membered rings are: E-G is part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C, CRaor CXD, and such that CXDcan only be present in A and P; E,G are C, CRa, CXDor N; K, L are CRa; D,M form a CRa═CRaor CRa═N, or D,M independently from each other are CRa2, C═O, C═S, C═NRb, C═CRa2, S, SO, SO2, O, NRbbut no adjacent O—O, S—S, N—S groups; J is CRa2, C═O, C═S, C═NRb, C═CRa2, S, SO, SO2, O, NRb; at most 2 N groups; or E, G are C, CRa, CXD; K is N and L is CRa; D,M form a CRa═CRabond or D,M independently from each other are CRa2, C═O, C═S, C═NRb, C═CRa2, NRbbut no adjacent O—O, S—S, N—S groups; J is CRa2, C═O, C═S, C═NRb, C═CRa2, S, SO, SO2, O, NRb; at most 2 N groups; or E,G are C, CRa, CXD; K and L are N; D,M, J independently from each other are CRa2, C═O, C═S, C═NRb, C═CRa2groups; Fused aromatic 5-membered rings are E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ. E and G are C; one of the groups L, K, or M are0, NRb, S and the remaining two groups are independently from each other CRaor N; or E is C and G is N; L, K, M are independently from each other CRaor N. Fused aromatic 6-membered rings are: E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ. E, G is C; L, K, D , M are independently from each other CRaor N Fused planar conjugated 7-membered rings are E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ E, G is C; L, K, D, M are CRa; J is S, O, CRa2, NRb. each Raas above-indicated can independently be H, alkyl, aryl, OR′, SR′, S(═O)R′″, S(═O)2R′″, S(═O)2NR′R″, Si—R′″, Si—O—R′″, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, F, Cl, Br, I, N3, SO2H, SO3H, SO4H, PO3H, PO4H, NO, NO2, CN, OCN, SCN, NCO, NCS, CF3, CF2—R′, NR′R″, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═S)O—R′, C(═O)S—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, OC(═O)NR′—R′″, SC(═O)NR′—R′″, OC(═S)NR′—R′″, SC(═S)NR′—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, CR′NR″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Each Rbas above indicated is independently selected from the group consisting of H, alkyl, aryl, O-aryl, O-alkyl, OH, C(═O)NR′R″ with R′ and R″ each independently being H, aryl or alkyl, R′CO-alkyl with R′ being H, alkyl, and aryl; Each Rcas above indicated is independently selected from the group consisting of H, alkyl, aryl, O-alkyl, O-aryl, OH; wherein two or more Ra,b,cmoieties together may form a ring; Preferably, each Rais selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(═O)NR′R″, NR′C(═O)—R′″, with R′ and R″ each independently being H, aryl or alkyl, and with R′″ independently being alkyl or aryl. In all of the above embodiments, optionally one of A, P, Q, Y, X, and Z, or the substituents or fused rings of which they are part, or the self-immolative linker LD, or the drug DD, is bound, optionally via a spacer or spacers SP, to one or more targeting agents TTor masking moieties MM. The synthesis of TCO's as described above is well available to the skilled person. This expressly also holds for TCO's having one or more heteroatoms in the strained cycloalkene rings. References in this regard include Cere et al.Journal of Organic Chemistry1980, 45, 261 and Prevost et al.Journal of the American Chemical Society2009, 131, 14182. In a preferred embodiment, the trans-cyclooctene moiety satisfies formula (1b): wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each Raindependently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR′, SR′, S(═O)R′″, S(═O)2R′″, S(═O)2NR′R″, Si—R′″, Si—O—R′″, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, F, Cl, Br, I, N3, SO2H, SO3H, SO4H, PO3H, PO4H, NO, NO2, CN, OCN, SCN, NCO, NCS, CF3, CF2—R′, NR′R″, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═S)O—R′, C(═O)S—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, OC(═O)NR′—R′″, SC(═O)NR′—R′″, OC(═S)NR′—R′″, SC(═S)NR′—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, CR′NR″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Each Rdas above indicated is independently selected from the group consisting of H, alkyl, aryl, OR′, SR′, S(═O)R′″, S(═O)2R′″, Si—R′″, Si—O—R′″, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, F, Cl, Br, I, N3, SO2H, SO3H, PO3H, NO, NO2, CN, CF3, CF2—R′, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═S)O—R′, C(═O)S—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, CR′NR″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; wherein two Ra,dmoieties together may form a ring; with optionally one Ra,dcomprised in a linker moiety, optionally via a spacer SP, to a targeting agent TTor a masking moiety MM, and wherein T and G each independently denote H, or a substituent selected from the group consisting of alkyl, F, Cl, Br, and I, and XDis as defined above for formula (1a). Preferably, each Raand each Rdis selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(═O)NR′R″, NR′C(═O)—R′″, with R′ and R″ each independently being H, aryl or alkyl, and with R′″ independently being alkyl or aryl. In the foregoing dienophiles, it is preferred that the at least two exocyclic bonds fixed in the same plane are selected from the group consisting of (a) the single bonds of a fused cyclobutyl ring, (b) the hybridized bonds of a fused aromatic ring, (c) an exocyclic double bond to an oxygen, and (d) an exocyclic double bond to a carbon. The TCO, containing one or two XDmoieties, may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents, such as XD, on the TCO. In this respect, reference is made to Whitham et al.J. Chem. Soc.(C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of trans-cyclo-oct-2-en-ol, identified as (1RS, 2RS) and (1SR, 2RS), respectively. In these isomers the OH substituent is either in the equatorial or axial position. In a preferred embodiment, for prodrug structures where the can be either in the axial or the equatorial position, the XDis in the axial position. Preferred dienophiles, which are optimally selected for drug release believed to proceed via a cascade elimination mechanism, are selected from the following structures: Use of TCO as a Carrier The invention also pertains to the use of a trans-cyclooctene satisfying formula (1a), in all its embodiments, as a carrier for a therapeutic compound. The trans-cyclooctene is to be read as a TCO in a broad sense, as discussed above, preferably an all-carbon ring or including one or two hetero-atoms. A therapeutic compound is a drug or other compound or moiety intended to have therapeutic application. The use of TCO as a carrier according to this aspect of the invention does not relate to the therapeutic activity of the therapeutic compound. In fact, also if the therapeutic compound is a drug substance intended to be developed as a drug, many of which will fail in practice, the application of TCO as a carrier still is useful in testing the drug. In this sense, the TCO in its capacity of a carrier is to be regarded in the same manner as a pharmaceutical excipient, serving as a carrier when introducing a drug into a subject. The use of a TCO as a carrier has the benefit that it enables the administration, to a subject, of a drug carried by a moiety that is open to a bioorthogonal reaction, with a diene, particularly a tetrazine. This provides a powerful tool not only to affect the fate of the drug carried into the body, but also to follow its fate (e.g. by allowing a labeled diene to react with it), or to change its fate (e.g. by allowing pK modifying agents to bind with it). This is all based on the possibility to let a diene react with the TCO in the above-discussed rDA reaction. The carrier is preferably reacted with an Activator as discussed below, so as to provoke the release of the therapeutic compound from the TCO, as amply discussed herein. Activator The Activator comprises a Bio-orthogonal Reactive Group, wherein this Bio-orthogonal Reactive Group of the Activator is a diene. This diene reacts with the other Bio-orthogonal Reactive Group, the Trigger, and that is a dienophile (vide supra). The diene of the Activator is selected so as to be capable of reacting with the dienophile of the Trigger by undergoing a Diels-Alder cycloaddition followed by a retro Diels-Alder reaction, giving the Retro Diels-Alder adduct. This intermediate adduct then releases the drug or drugs, where this drug release can be caused by various circumstances or conditions that relate to the specific molecular structure of the retro Diels-Alder adduct. Without wishing to be bound by theory, the inventors believe that the Activator is selected such as to provoke drug release via an elimination or cascade elimination (via an intramolecular elimination reaction within the Retro Diels-Alder adduct). This elimination reaction can be a simple one step reaction, or it can be a multiple step reaction that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end product or to the next intermediate structure. When several steps are involved, one can speak of a cascade reaction. In any case, whether it be a simple or a cascade process, the result of the elimination reaction is that the drug gets released from the retro Diels-Alder adduct. Without wishing to be bound by theory, the design of both components (i.e. the diene Activator and the dienophile Trigger) is such that the distribution of electrons within the retro Diels-Alder adduct is unfavorable, so that a rearrangement of these electrons must occur. This situation initiates the intramolecular (cascade) elimination reaction to take place, and it therefore induces the release of the drug or drugs. Occurrence of the elimination reaction in and drug release from the Prodrug is not efficient or cannot take place prior to the Retro Diels-Alder reaction, as the Prodrug itself is relatively stable as such. Elimination can only take place after the Activator and the Prodrug have reacted and have been assembled in the retro Diels-Alder adduct. Without wishing to be bound by theory, the above two examples illustrate how the unfavorable distribution of electrons within the retro Diels-Alder adduct can be relieved by an elimination reaction, thereby releasing the drug. In one scenario, the elimination process produces end product A, where this product has a conjugation of double bonds that was not present in the retro Diels-Alder adduct yet. Species A may tautomerize to end product B, or may rearrange to form end product C. Then, the non-aromatic dihydro pyridazine ring in the retro Diels-Alder adduct has been converted to the aromatic pyridazine ring in the end product C. The skilled person will understand that the distribution of electrons in the retro Diels-Alder adduct is generally unfavorable relative to the distribution of the electrons in the end products, either species A or B or C. Thus, the formation of a species stabler than the retro Diels-Alder adduct is the driving force for the (cascade) elimination reaction. In any case, and in whatever way the process is viewed, the drug species (here the amine ‘drug-NH2’) is effectively expelled from the retro Diels-Alder adduct, while it does not get expelled from the Prodrug alone. Below scheme depicts a possible alternative release mechanism for the cascade elimination, in addition to the two discussed above. Without wishing to be bound by theory, the below examples illustrates how the unfavorable distribution of electrons within the retro Diels-Alder adduct may be relieved by an elimination reaction, thereby releasing the drug. This process may evolve via various tauromerisations that are all equilibria. Here, the rDA reaction produces tautomers A and B, which can interchange into one and other. Tautomer B can lead to the elimination into product C and thereafter into D. The Activator is a diene. The person skilled in the art is aware of the wealth of dienes that are reactive in the Retro Diels-Alder reaction. The diene comprised in the Activator can be part of a ring structure that comprises a third double bond, such as a tetrazine (which is a preferred Activator according to the invention). Generally, the Activator is a molecule comprising a heterocyclic moiety comprising at least 2 conjugated double bonds. Preferred dienes are given below, with reference to formulae (2)-(4). In formula (2) R1is selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, OR′, SR′, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R″, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N+O−, N+R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C═O, and Y is CR with R being selected from the group consisting of H, alkyl, aryl, C(═O)OR′, C(═O)SR′, C(═S)OR′, C(═S)SR′, C(═O)NR′R″ with R′ and R″ each independently being H, aryl or alkyl. A diene particularly suitable as a reaction partner for cyclooctene is given in formula (3), wherein R1and R2each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO2, OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C═O, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O)2R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, CC(═S)NR′R″, R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N+O−. Another diene particularly suitable as a reaction partner for cyclooctene is diene (4), wherein R1and R2each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO, NO2, OR′, SR′, CN, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2OR′, PO3R′R″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N+O−; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O)2R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, CC(═S)NR′R″, R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N+O−. According to the invention, particularly useful dienes are 1,2-diazine, 1,2,4-triazine and 1,2,4,5-tetrazine derivatives, as given in formulas (5), (6) and (7), respectively. The 1,2-diazine is given in (5), wherein R1and R2each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO2, OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; X and Y each independently are selected from the group consisting of O, N-alkyl, N-aryl, C═O, CN-alkyl, CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O)2R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, CC(═S)NR′R″, with R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl, where X-Y may be a single or a double bond, and where X and Y may be connected in a second ring structure apart from the 6-membered diazine. Preferably, X-Y represents an ester group (X═O and Y═C═O; X-Y is a single bond) or X-Y represents a cycloalkane group (X═CR′ and Y═CR″; X-Y is a single bond; R′ and R″ are connected), preferably a cyclopropane ring, so that R′ and R″ are connected to each other at the first carbon atom outside the 1,2-diazine ring. The 1,2,4-triazine is given in (6), wherein R1and R2each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO2, OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; X is selected from the group consisting of CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O)2R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, CC(═S)NR′R″, R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl. The 1,2,4,5-tetrazine is given in (7), wherein R1and R2each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO, NO2, OR′, SR′, CN, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2OR′, PO3R′R″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl. Electron-deficient 1,2-diazines (5), 1,2,4-triazines (6) or 1,2,4,5-tetrazines (7) are especially interesting as such dienes are generally more reactive towards dienophiles. Di- tri- or tetra-azines are electron deficient when they are substituted with groups or moieties that do not generally hold as electron-donating, or with groups that are electron-withdrawing. For example, R1and/or R2may denote a substituent selected from the group consisting of H, alkyl, NO2, F, Cl, CF3, CN, COOR, CONHR, CONR2, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl or phenyl, optionally substituted with one or more electron-withdrawing groups such as NO2, F, Cl, CF3, CN, COOR, CONHR, CONR, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, Ar, wherein R is H or C1-C6alkyl, and Ar stands for an aromatic group, particularly phenyl, pyridyl, or naphthyl. The 1,2,4,5-tetrazines of formula (7) are most preferred as Activator dienes, as these molecules are most reactive in retro Diels-Alder reactions with dienophiles, such as the preferred TCO dienophiles, even when the R1and/or R2groups are not necessarily electron withdrawing, and even when R1and/or R2are in fact electron donating. Electron donating groups are for example OH, OR′, SH, SR′, NH2, NHR′, NR′R″, NHC(═O)R″, NR′C(═O)R″, NHC(═S)R″, NR′C(═S)R″, NHSO2R″, NR′SO2R″ with R′ and R″ each independently being alkyl or aryl groups. Examples of other electron donating groups are phenyl groups with attached to them one or more of the electron donating groups as mentioned in the list above, especially when substituted in the 2-, 4- and/or 6-position(s) of the phenyl group. According to the invention, 1,2,4,5-tetrazines with two electron withdrawing residues, or those with one electron withdrawing residue and one residue that is neither electron withdrawing nor donating, are called electron deficient. In a similar way, 1,2,4,5-tetrazines with two electron donating residues, or those with one electron donating residue and one residue that is neither electron withdrawing nor donating, are called electron sufficient. 1,2,4,5-Tetrazines with two residues that are both neither electron withdrawing nor donating, or those that have one electron withdrawing residue and one electron donating residue, are neither electron deficient nor electron sufficient. The 1,2,4,5-tetrazines can be asymmetric or symmetric in nature, i.e. the R1and R2groups in formula (7) may be different groups or may be identical groups, respectively. Symmetric 1,2,4,5-tetrazines are more convenient as these Activators are more easily accessible via synthetic procedures. We have tested several 1,2,4,5-tetrazines with respect to their ability as Activator to release a model drug compound (e.g. benzyl amine) from a Prodrug via an elimination (cascade) process, and we have found that tetrazines that are electron deficient, electron sufficient or neither electron deficient nor electron sufficient are capable to induce the drug release. Furthermore, both symmetric as well as asymmetric tetrazines were effective. Electron deficient 1,2,4,5 tetrazines and 1,2,4,5-tetrazines that are neither electron deficient nor electron sufficient are generally more reactive in retro Diels-Alder reactions with dienophiles (such as TCOs), so these two classes of 1,2,4,5-tetrazines are preferred over electron sufficient 1,2,4,5-tetrazines, even though the latter are also capable of inducing drug release in Prodrugs. In the following paragraphs specific examples of 1,2,4,5-tetrazine Activators according to the second embodiment of this invention will be highlighted by defining the R1and R2residues in formula (7). Symmetric electron deficient 1,2,4,5-tetrazines with electron withdrawing residues are for example those with R1=R2=H, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,4-pyrimidyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,3,4-triazyl or 2,3,5-triazyl. Other examples are those with R1=R2=phenyl with COOH or COOMe carboxylate, or with CN nitrile, or with CONH2, CONHCH3or CON(CH3)2amide, or with SO3H or SO3Na sulfonate, or with SO2NH2, SO2NHCH3or SO2N(CH3)2sulfonamide, or with PO3H2or PO3Na2phosphonate substituents in the 2-, 3- or 4-position of the phenyl group, or in the 3- and 5-positions, or in the 2- and 4-positions, or in the 2,- and 6-positions of the phenyl group. Other substitution patterns are also possible, including the use of different substituents, as long as the tetrazine remains symmetric. See below for some examples of these structures. Symmetric electron sufficient 1,2,4,5-tetrazines with electron donating residues are for example those with R1=R2=OH, OR′, SH, SR′, NH2, NHR′, NR′2, NH—CO-R′, NH—SO—R′, NH—SO2—R′, 2-pyrryl, 3-pyrryl, 2-thiophene, 3-thiophene, where R′ represents a methyl, ethyl, phenyl or tolyl group. Other examples are those with R1=R2=phenyl with OH, OR′, SH, SR′, NH2, NHR′, NR′2, NH—CO—R′, NR″—CO—R′, NH—SO—R′ or NH—SO2—R′ substituent(s), where R′ represents a methyl, ethyl, phenyl or tolyl group, where R″ represents a methyl or ethyl group, and where the substitution is done on the 2- or 3- or 4- or 2- and 3- or 2- and 4- or 2- and 5- or 2- and 6- or 3- and 4- or 3- and 5- or 3-, 4- and 5-position(s). See below for some examples of these structures. Symmetric 1,2,4,5-tetrazines with neither electron withdrawing nor electron donating residues are for example those with R1=R2=phenyl, methyl, ethyl, (iso)propyl, 2,4-imidazyl, 2,5-imidazyl, 2,3-pyrazyl or 3,4-pyrazyl. Other examples are those where R1=R2=a hetero(aromatic) cycle such as a oxazole, isoxazole, thiazole or oxazoline cycle. Other examples are those where R1=R2=a phenyl with one electron withdrawing substituent selected from COOH, COOMe, CN, CONH2, CONHCH3, CON(CH3)2, SO3H, SO3Na, SO2NH2, SO2NHCH3, SO2N(CH3)2, PO3H2or PO3Na2and one electron donating substituent selected from OH, OR′, SH, SR′, NH2, NHR′, NR′2, NH—CO—R′, NR″—CO—R′, NH—SO—R′ or NH—SO2—R′ substituent(s), where R′ represents a methyl, ethyl, phenyl or tolyl group and where R″ represents a methyl or ethyl group. Substitutions can be done on the 2- and 3-, 2- and 4-, 2,- and 5-, 2- and 6, 3- and 4-, and the 3- and 5-positions. Yet other examples are those where R1=R2=a pyridyl or pyrimidyl moiety with one electron donating substituent selected from OH, OR′, SH, SR′, NH2, NHR′, NR′2, NH—CO—R′, NR″—CO—R′, NH—SO—R′ or NH—SO2—R′ substituents, where R′ represents a methyl, ethyl, phenyl or tolyl group and where R″ represents a methyl or ethyl group. See below for some examples. In case asymmetric 1,2,4,5-tetrazines are considered, one can choose any combination of given R1and R2residues that have been highlighted and listed above for the symmetric tetrazines according to formula (7), provided of course that R1and R2are different. Preferred asymmetric 1,2,4,5-tetrazines are those where at least one of the residues R1or R2is electron withdrawing in nature. Find below some example structures drawn. Further Considerations Regarding the Activator Preferred Activators are 1,2-diazines, 1,2,4-triazines and 1,2,4,5-tetrazines, particularly 1,2,4,5-tetrazines, are the preferred diene Activators. In the below, some relevant features of the Activator will be highlighted, where it will also become apparent that there are plentiful options for designing the right Activator formulation for every specific application. According to the invention, the Activator, e.g. a 1,2,4,5-tetrazine, has useful and beneficial pharmacological and pharmaco-kinetic properties, implying that the Activator is non-toxic or at least sufficiently low in toxicity, produces metabolites that are also sufficiently low in toxicity, is sufficiently soluble in physiological solutions, can be applied in aqueous or other formulations that are routinely used in pharmaceutics, and has the right log D value where this value reflects the hydrophilic/hydrophobic balance of the Activator molecule at physiological pH. As is known in the art, log D values can be negative (hydrophilic molecules) or positive (hydrophobic molecules), where the lower or the higher the log D values become, the more hydrophilic or the more hydrophobic the molecules are, respectively. Log D values can be predicted fairly adequately for most molecules, and log D values of Activators can be tuned by adding or removing polar or apolar groups in their designs. Find below some Activator designs with their corresponding calculated log D values (at pH=7.4). Note that addition of methyl, cycloalkylene, pyridine, amine, alcohol or sulfonate groups or deletion of phenyl groups modifies the log D value, and that a very broad range of log D values is accessible. The given log D numbers have been calculated from a weighed method, with equal importance of the ‘VG’ (Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K., J. Chem. Inf. Comput. Sci., 1989, 29, 163-172), ‘KLOP’ (according to Klopman, G.; Li, Ju-Yun.; Wang, S.; Dimayuga, M.: J.Chem.Inf.Comput.Sci., 1994, 34, 752) and ‘PHYS’ (according to the PHYSPROP© database) methods, based on an aqueous solution in 0.1 M in Na+/K+Cl−. The Activator according to the invention has an appropriate reactivity towards the Prodrug, and this can be regulated by making the diene, particularly the 1,2,4,5-tetrazines, sufficiently electron deficient. Sufficient reactivity will ensure a fast retro Diels-Alder reaction with the Prodrug as soon as it has been reached by the Activator. The Activator according to the invention has a good bio-availability, implying that it is available inside the (human) body for executing its intended purpose: effectively reaching the Prodrug at the Primary Target. Accordingly, the Activator does not stick significantly to blood components or to tissue that is non-targeted. The Activator may be designed to bind to albumin proteins that are present in the blood (so as to increase the blood circulation time, as is known in the art), but it should at the same time be released effectively from the blood stream to be able to reach the Prodrug. Accordingly, blood binding and blood releasing should then be balanced adequately. The blood circulation time of the Activator can also be increased by increasing the molecular weight of the Activator, e.g. by attaching polyethylene glycol (PEG) groups to the Activator (‘pegylation’). Alternatively, the PKPD of the activator may be modulated by conjugating the activator to another moiety such as a polymer, protein, (short) peptide, carbohydrate. The Activator according to the invention may be multimeric, so that multiple diene moieties may be attached to a molecular scaffold, particularly to e.g. multifunctional molecules, carbohydrates, polymers, dendrimers, proteins or peptides, where these scaffolds are preferably water soluble. Examples of scaffolds that can be used are (multifunctional) polyethylene glycols, poly (propylene imine) (PPI) dendrimers, PAMAM dendrimers, glycol based dendrimers, heparin derivatives, hyaluronic acid derivatives or serum albumine proteins such as HSA. Depending on the position of the Prodrug (e.g. inside the cell or outside the cell; specific organ that is targeted) the Activator is designed to be able to effectively reach this Prodrug. Therefore, the Activator can for example be tailored by varying its log D value, its reactivity or its charge. The Activator may even be engineered with a targeting agent (e.g. a protein, a peptide and/or a sugar moiety), so that the Primary Target can be reached actively instead of passively. In case a targeting agent is applied, it is preferred that it is a simple moiety (i.e. a short peptide or a simple sugar). According to the invention, a mixture of different Activators can be applied. This may be relevant for regulation of the release profile of the drug. The Activator that according to the invention will cause and regulate drug release at the Primary Target may additionally be modified with moieties giving extra function(s) to the Activator, either for in-vitro and/or for in-vivo studies or applications. For example, the Activator may be modified with dye moieties or fluorescent moieties (see e.g. S. Hilderbrand et al., Bioconjugate Chem., 2008, 19, 2297-2299 for 3-(4-benzylamino)-1,2,4,5-tetrazine that is amidated with the near-infrared (NIR) fluorophore VT680), or they may be functionalized with imaging probes, where these probes may be useful in imaging modalities, such as the nuclear imaging techniques PET or SPECT. In this way, the Activator will not only initiate drug release, but can also be localized inside the (human) body, and can thus be used to localize the Prodrug inside the (human) body. Consequently, the position and amount of drug release can be monitored. For example, the Activator can be modified with DOTA (or DTPA) ligands, where these ligands are ideally suited for complexation with111In3+-ions for nuclear imaging. In other examples, the Activator may be linked to123I or18F moieties, that are well established for use in SPECT or PET imaging, respectively. Furthermore, when used in combination with e.g. beta-emitting isotopes, such as Lu-177, or Y-90, prodrug activation can be combined with localized radiotherapy in a pretargeted format. Preferred activators are: The 1,2,4,5-tetrazine given in Formula (8a) and (8b), wherein each R1and each R2independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2—R′, NO2, OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O)2R′″, S(═O)2NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl. Other preferred activators are: The Activator can have a link to a desired moiety such as a peptide, protein, carbohydrate, PEG, or polymer. Preferably, these Activators satisfy one of the following formulae: Prodrug A Prodrug is a conjugate of the Drug DDand the Trigger TRand thus comprises a Drug that is capable of therapeutic action after its release from the Trigger. Such a Prodrug may optionally have specificity for disease targets. The general formula of the Prodrug is shown below in Formula (9a) and (9b). The moiety YMcan either be a targeting agent TTor a masking moiety MM; SPis spacer; TRis Trigger, LDis linker, and DDis drug. For applications where drugs are released from a targeting agent: YMis a targeting agent TT; Formula (9a): k=1; m,r≥1; t,n≥0. Formula (9b): k=1; m,n,r≥1; t≥0. For applications where masked drugs are unmasked: YMis a masking moiety MM; Formula (9a) and (9b): r=1; m≥1; k,n,t≥0. Although it has been omitted for the sake of clarity in the above formula, DDcan further comprise TTand/or MM, optionally via SP. Drugs that can be used in a Prodrug relevant to this invention include but are not limited to: antibodies, antibody derivatives, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids, steroids, organic drug compounds, toxins, hormones, viruses, whole cells, phage. Typical drugs for which the invention is suitable include, but are not limited to: bi-specific and trispecific mAb fragments, immunotoxins, comprising eg ricin A, diphtheria toxin, cholera toxin. Other embodiments use auristatins, maytansines, calicheamicin, Duocarmycins, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs; antiproliferative/antitumor agents, antibiotics, cytokines, anti-inflammatory agents, anti-viral agents, antihypertensive agents, chemosensitizing and radiosensitizing agents. In other embodiments the released Drug DDis itself a prodrug designed to release a further drug DD. Drugs optionally include a membrane translocation moiety (adamantine, poly-lysine/argine, TAT) and/or a targeting agent (against eg a tumor cell receptor) optionally linked through a stable or labile linker. Exemplary drugs for use as conjugates to the TCO derivative and to be released upon retro Diels Alder reaction with the Activator include but are not limited to: cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs . Exemplary classes of cytotoxic agents include the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, anti-tubulin agents, topoisomerases inhibitors, platinum-based drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols. Particularly useful members of those classes include, for example, duocarmycin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil DNA minor groove binders, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, and their analogues. Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A (U.S. Pat. No. 4,486,414), dolastatin B (U.S. Pat. No. 4,486,414), dolastatin 10 (U.S. Pat. Nos. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat. No. 4,986,988), dolastatin 14 (U.S. Pat. No. 5,138,036), dolastatin 15 (U.S. Pat. No. 4,879,278), dolastatin 16 (U.S. Pat. No. 6,239,104), dolastatin 17 (U.S. Pat. No. 6,239,104), and dolastatin 18 (U.S. Pat. No. 6,239,104), each patent incorporated herein by reference in their entirety. In exemplary embodiments of the invention, the drug moiety is a mytomycin, vinca alkaloid, taxol, anthracycline, a calicheamicin, maytansinoid or an auristatin. It will be understood that chemical modifications may also be made to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention. Drugs containing an amine functional group for coupling to the TCO include mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabine, dolastatins (including auristatins) and derivatives thereof. Drugs containing a hydroxyl function group for coupling to the TCO include etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one (U.S. Pat. No. 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof. Drugs containing a sulfhydryl functional group for coupling to the TCO include esperamicin and 6-mecaptopurine, and derivatives thereof. It will be understood that the drugs can optionally be attached to the TCO derivative through a linker LDor a self-immolative linker LD, or a combination thereof, and which may consist of multiple (self-immolative, or non immolative) units. It will further be understood that one ore more targeting agents TTor masking moieties MMmay optionally be attached to the Drug DD, Trigger TR, or Linker LD, optionally via a spacer or spacers SP. Several drugs may be replaced by an imageable label to measure drug targeting and release. According to a further particular embodiment of the invention, the Prodrug is selected so as to target and or address a disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. According to one embodiment, the Prodrug and/or the Activator can be multimeric compounds, comprising a plurality of Drugs and/or bioorthogonal reactive moieties. These multimeric compounds can be polymers, dendrimers, liposomes, polymer particles, or other polymeric constructs. In the Prodrug, the Drug DDand the Trigger TR—the TCO derivative—can be directly linked to each other. They can also be bound to each other via a linker or a self-immolative linker LD. It will be understood that the invention encompasses any conceivable manner in which the dienophile Trigger is attached to the Drug. The same holds for the attachment of an optional targeting agent TTor masking moiety MMto the Prodrug. Methods of affecting conjugation to these drugs, e.g. through reactive amino acids such as lysine or cysteine in the case of proteins, are known to the skilled person. It will be understood that the drug moiety is linked to the TCO in such a way that the drug is eventually capable of being released after formation of the retro Diels-Alder adduct. Generally, this means that the bond between the drug and the TCO, or in the event of a linker, the bond between the TCO and the linker LD, or in the event of a self-immolative linker LD, the bond between the linker and the TCO and between the drug and the linker, should be cleavable. Predominantly, the drug and the optional linker is linked via a hetero-atom, preferably via O, N, NH, or S. The cleavable bond is preferably selected from the group consisting of carbamate, thiocarbamate, carbonate, ether, ester, amine, amide, thioether, thioester, sulfoxide, and sulfonamide bonds. Thus, in the invention, linker concepts can be applied analogously to those known to the skilled person. Most reported prodrugs consist of three components: a trigger, a linker, and a parent drug, optionally a targeting molecule is attached to either the linker or the trigger. The trigger, which can e.g. be a substrate for a site-specific enzyme, or pH labile group, is often connected to the parent drug via a self-elimination linker. This linker is incorporated to facilitate enzymatic cleavage of the trigger, increasing active site accessibility and decreasing steric hindrance from the attached drug. Also the linker facilitates the straightforward use of a broad range of prodrugs in combination with the same trigger. Furthermore, the linker modulates prodrug stability, pharmacokinetics, organ distribution, enzyme recognition, and release kinetics. After trigger activation/removal, the linker must spontaneously eliminate to release the parent drug. Depending on the attached drug the linker or parts thereof can remain on the drug without impairing its action. The general concept is depicted inFIG.2. Two types of self-elimination linkers can be distinguished a) the electronic cascade linker b) the cyclization linker. The most prominent example of a cascade linker is the 1,6 elimination spacer shown in Scheme 1 in a β-glucuronide prodrug of anticancer agent 9-aminocamptothecin. After unmasking of the aromatic hydroxyl function by the enzyme β-glucuronidase (present in certain necrotic tumor areas), this group becomes electron-donating and initiates an electronic cascade that leads to expulsion of the leaving group, which releases the free drug after elimination of CO2. This cascade, based on a quinone-methide rearrangement, can also be initiated by the lone pair of an unmasked amine or thiol instead of the hydroxyl. The formed quinone-methide species is trapped by water to form a phenol derivative. Some other trigger-linker concepts are depicted in Scheme 2. The trigger in A is activated by plasmatic esterases. Hydrolysis of the tert-butyl ester affords the free aromatic hydroxyl group, which starts the quinone-methide cascade. This construct has been targeted by conjugation to an antibody (R). In B, the hydrolysis of cephalosporins by beta-lactamase enzymes is used as a trigger. Hydrolysis of the lactam ring can lead expulsion of the drug substituent depending on its leaving group nature. Drugs have been conjugated via an ester, amide, sulfide, amine and carbamate link. Two examples of aromatic cyclization-based linkers are C and D. In C cleavage by penicillin G-amidase leads to intramolecular attack of the amine on the carbonyl, releasing the drug. D shows a phosphatase-sensitive prodrug. Cleavage of the phosphate by human alkaline phosphatase affords a hydroxyl that reacts to a lactam by releasing the drug. In E an example is shown of a prodrug that it triggered by the reduction of a nitro group to an amine. This reduction can be performed by nitroreductase in the presence of NADPH. Furthermore, a number of heterocyclic nitro constructs are known (F) that are reduced in hypoxic (tumor) tissue and, hence, can initiate a cascade without the assistance of an enzyme. Other triggers used in prodrug therapy are sensitive to plasmin, tyrosine hydroxylase (highly expressed in neuroblastoma), tyrosinase or cathepsin B. The Combination of and Reaction Between the TCO-Trigger and the Activator The drug, whether or not via a linker, is preferably attached to a carbon atom that is adjacent to the double bond in the TCO ring. Hereunder, some nonlimiting combinations of TCO Prodrugs and tetrazine Activators illustrate the possibilities for cascade elimination induced drug release from the retro Diels-Alder adduct. Note that in cases of release of amine functional drugs these can be e.g. primary or secondary amine, aniline, imidazole or pyrrole type of drugs, so that the drug may be varying in leaving group character. Release of drugs with other functionalities may also be possible (e.g. thiol functinalized drugs), in case corresponding hydrolytically stable TCO Prodrugs are applied. The drawn fused ring products may or may not tautomerize to other more favorable tautomers. The above example of urethane (or carbamate) substituted TCOs gives release of an amine functional drug from the adduct. The tetrazine Activator is symmetric and electron deficient. The above examples of urethane (or carbamate) substituted TCOs gives release of an amine functional drug from the adduct. The tetrazine Activator is asymmetric and electron deficient. Note that use of an asymmetric tetrazine leads to formation of retro Diels-Alder adduct regiomers, apart from the stereo-isomers that are already formed when symmetric tetrazine are employed. The above example of urethane (or carbamate) TCOs gives release of an amine functional drug from the adduct. The tetrazine Activator is symmetric and electron sufficient. In a preferred embodiment, the drug is provided in the form of an antibody-toxin conjugate. The conjugate is provided with a TCO moiety as identified above, so as to enable bio-orthogonal chemically activated toxin release. In another embodiment, the drug is a bi- or trispecific antibody derivative that serves to bind to tumor cells and recruit and activate T-cells, the T-cell binding function of which is inactivated by being linked to a TCO moiety as described above. The latter, again, serving to enable bio-orthogonal chemically activated drug activation. Targeting The kits and method of the invention are very suitable for use in targeted delivery of drugs. A “primary target” as used in the present invention relates to a target for a targeting agent for therapy. For example, a primary target can be any molecule, which is present in an organism, tissue or cell. Targets include cell surface targets, e.g. receptors, glycoproteins; structural proteins, e.g. amyloid plaques; abundant extracullular targets such as stroma, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and/or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof. Examples of primary targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up regulated or down-regulated in a certain disorder. According to a particular embodiment of the present invention, the primary target is a protein such as a (internalizing or non-internalizing) receptor. According to the present invention, the primary target can be selected from any suitable targets within the human or animal body or on a pathogen or parasite, e.g. a group comprising cells such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as Golgi bodies or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, antibodies, proteins, carbohydrates, monosaccharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)angiogenesis marker, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opoid receptors, GPIIb/IIIa receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin, VEGF/EGF and VEGF/EGF receptors, TAG72, CEA, CD19, CD20, CD22, CD40, CD45, CD74, CD79, CD105, CD138, CD174, CD227, CD326, CD340, MUC1, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-B, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinase (MMP), P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, herpes simplex virus tyrosine kinase, human tyrosine kinase. In order to allow specific targeting of the above-listed primary targets, the targeting agent TTcan comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, VHH, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin, peptide mimetics, carbohydrates, monosacharides, polysaccharides, viruses, whole cells, drugs, polymers, liposomes, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids. Examples of organic compounds envisaged within the context of the present invention are, or are derived from, estrogens, e.g. estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol. In a preferred embodiment, the targeting agent TTis an antibody. According to a particular embodiment of the present invention, the primary target is a receptor and a targeting agent is employed, which is capable of specific binding to the primary target. Suitable targeting agents include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands. Other examples of targeting agents of protein nature include interferons, e.g. alpha, beta, and gamma interferon, interleukins, and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Alternative examples of targeting agents include DNA, RNA, PNA and LNA which are e.g. complementary to the primary target. According to a further particular embodiment of the invention, the primary target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting primary targets with tissue-, cell- or disease- specific expression. For example, membrane folic acid receptors mediate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types. Masking Moieties Masking moieties MMcan be a protein, peptide, polymer, polyethylene glycol, carbohydrate, organic construct, that further shield the bound drug DDor Prodrug. This shielding can be based on eg steric hindrance, but it can also be based on a non covalent interaction with the drug DD. Such masking moiety may also be used to affect the in vivo properties (eg blood clearance; recognition by the immunesystem) of the drug DDor Prodrug. Spacers Spacers SPinclude but are not limited to polyethylene glycol (PEG) chains varying from 2 to 200, particularly 3 to 113 and preferably 5-50 repeating units. Other examples are biopolymer fragments, such as oligo- or polypeptides or polylactides. Further preferred examples are shown in Example 11. Administration In the context of the invention, the Prodrug is usually administered first, and it will take a certain time period before the Prodrug has reached the Primary Target. This time period may differ from one application to the other and may be minutes, days or weeks. After the time period of choice has elapsed, the Activator is administered, will find and react with the Prodrug and will thus activate Drug release at the Primary Target. The compositions of the invention can be administered via different routes including intravenous injection, intraperatonial, oral administration, rectal administration and inhalation. Formulations suitable for these different types of administrations are known to the skilled person. Prodrugs or Activators according to the invention can be administered together with a pharmaceutically acceptable carrier. A suitable pharmaceutical carrier as used herein relates to a carrier suitable for medical or veterinary purposes, not being toxic or otherwise unacceptable. Such carriers are well known in the art and include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. It will be understood that the chemical entities administered, viz. the prodrug and the activator, can be in a modified form that does not alter the chemical functionality of said chemical entity, such as salts, hydrates, or solvates thereof. After administration of the Prodrug, and before the administration of the Activator, it is preferred to remove excess Prodrug by means of a Clearing Agent in cases when prodrug activation in circulation is undesired and when natural prodrug clearance is insufficient. A Clearing Agent is an agent, compound, or moiety that is administered to a subject for the purpose of binding to, or complexing with, an administered agent (in this case the Prodrug) of which excess is to be removed from circulation. The Clearing Agent is capable of being directed to removal from circulation. The latter is generally achieved through liver receptor-based mechanisms, although other ways of secretion from circulation exist, as are known to the skilled person. In the invention, the Clearing Agent for removing circulating Prodrug, preferably comprises a diene moiety, e.g. as discussed above, capable of reacting to the TCO moiety of the Prodrug. EXAMPLES The following examples demonstrate the invention or aspects of the invention, and do not serve to define or limit the scope of the invention or its claims. Methods.1H-NMR and13C-NMR spectra were recorded on a Varian Mercury (400 MHz for1H-NMR and 100 MHz for13C-NMR) spectrometer at 298 K. Chemical shifts are reported in ppm downfield from TMS at room temperature. Abbreviations used for splitting patterns are s=singlet, t=triplet, q=quartet, m=multiplet and br=broad. IR spectra were recorded on a Perkin Elmer 1600 FT-IR (UATR). LC-MS was performed using a Shimadzu LC-10 AD VP series HPLC coupled to a diode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an Ion-Trap (LCQ Fleet, Thermo Scientific). Analyses were performed using a Alltech Alltima HP C183 μ column using an injection volume of 1-4 μL, a flow rate of 0.2 mL min−1and typically a gradient (5% to 100% in 10 min, held at 100% for a further 3 min) of CH3CN in H2O (both containing 0.1% formic acid) at 25° C. Preparative RP-HPLC (CH3CN/H2O with 0.1% formic acid) was performed using a Shimadzu SCL-10A VP coupled to two Shimadzu LC-8A pumps and a Shimadzu SPD-10AV VP UV-vis detector on a Phenomenex Gemini 5 μ C18110A column. Size exclusion (SEC) HPLC was carried out on an Agilent 1200 system equipped with a Gabi radioactive detector. The samples were loaded on a Superdex-200 10/300 GL column (GE Healthcare Life Sciences) and eluted with 10 mM phosphate buffer, pH 7.4, at 0.35-0.5 mL/min. The UV wavelength was preset at 260 and 280 nm. The concentration of antibody solutions was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) from the absorbance at 322 nm and 280 nm, respectively. Materials. All reagents, chemicals, materials and solvents were obtained from commercial sources, and were used as received: Biosolve, Merck and Cambridge Isotope Laboratories for (deuterated) solvents; and Aldrich, Acros, ABCR, Merck and Fluka for chemicals, materials and reagents. All solvents were of AR quality. 4-(t-Butyldimethylsilyloxymethyl)-2,6-dimethylphenol was synthesized according to a literature procedure (Y. H. Choe, C. D. Conover, D. Wu, M. Royzen, Y. Gervacio, V. Borowski, M. Mehlig, R. B. Greenwald,J. Controlled Release2002, 79, 55-70). Doxorubicine hydrochloride was obtained from Avachem Scientific. Example 1 Synthesis of Tetrazine Activators General Procedures Apart from the tetrazines described in detail below, a series of other tetrazines have been prepared. Pinner-type reactions have been used, where the appropriate nitriles have been reacted with hydrazine to make the dihydro 1,2,4,5-tetrazine intermediates. Instead of nitriles, amidines have also been used as reactants, as is known in the art. The use of sulfur in this reaction is also known, as in some cases this aids the formation of the dihydro 1,2,4,5-tetrazine. Oxidation of this intermediate results in the tetrazine diene Activators. The below reactions describe some of the prepared tetrazines, and illustrate some of the possibilities (e.g. use of solvent, concentrations, temperature, equivalents of reactants, options for oxidation, etc.) to make and isolate tetrazines. Other methods known in the art may also be used to prepare tetrazines or other Activators. Synthesis of 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (2) 2-Cyanopyridine (10.00 g, 96.0 mmol) and hydrazine hydrate (15.1 g; 300 mmol) were stirred overnight at 90° C. in an inert atmosphere. The turbid mixture was cooled to room temperature, filtered, and the residue was subsequently washed with water (20 mL) and ethanol (20 mL), and dried in vacuo to yield the crude dihydrotetrazine 1 as an orange solid (7.35 g; 65%). The dihydrotetrazine (1, 100 mg; 0.419 mmol) was suspended in acetic acid (3 mL), and sodium nitrite (87 mg; 1.26 mmol) was added. An immediate color change from orange to dark red was observed, and the oxidized product was isolated by filtration. The residue was washed with water (10 mL) and dried in vacuo to yield the title compound as a purple solid (2, 92 mg; 93%). 1H NMR (CDCl3): δ=9.00 (d, 2H), 8.76 (d, 2H), 8.02 (t, 2H), 7.60 (dd, 2H) ppm.13C NMR (CDCl3): δ=163.9, 151.1, 150.1, 137.5, 126.6, 124.5 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=237.00 (M+H+), λmax=296 and 528 nm. Synthesis of 3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5) 2-Cyanopyridine (5.00 g, 48.0 mmol), 5-amino-2-cyanopyridine (5.72 g; 48.0 mmol) and hydrazine hydrate (15.1 g; 300 mmol) were stirred overnight at 90° C. in an inert atmosphere. The turbid mixture was cooled to room temperature, filtered, and the residue was subsequently washed with water (20 mL) and ethanol (20 mL), and dried in vacuo. The orange solid was suspended in acetone (200 mL), impregnated onto silica gel (20 gram), and chromatographed using a gradient (0% to 70%) of acetone and heptane, to yield dihydrotetrazine 3 as an orange solid (1.46 g; 12% yield). The dihydrotetrazine (3, 90 mg; 0.355 mmol) was dissolved in THF (1 mL), and acetic anhydride (54.4 mg; 0.533 mmol) was added. The solution was heated to reflux in an inert atmosphere for 18 hr. The orange precipitate was isolated by filtration, and washed with THF (3 mL) to give the acetamide of the dihydrotetrazine (4, 90 mg; 86% yield). Acetamide 4 (50 mg, 0.169 mmol) was suspended in acetic acid (1 mL), and sodium nitrite (35 mg; 0.508 mmol) was added. An immediate color change from orange to dark red was observed, and the oxidized product was isolated by filtration. The residue was washed with water (5 mL) and dried in vacuo to yield the title compound as a purple solid (5, 42 mg; 84%). 1H NMR (DMSO-d6): δ=9.03 (d, 1H), 8.93 (d, 1H), 8.61 (dd, 2H), 8.42 (dd, 1H), 8.16 (dt, 1H), 7.73 (dd, 1H), 2.17 (s, 3H) ppm.13C NMR (DMSO-d6): δ=169.5, 163.0, 162.8, 150.6, 150.2, 143.8, 141.2, 138.5, 137.8, 126.6, 126.1, 124.9, 124.2, 24.1 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=293.9 (M+H+), λmax=323 and 529 nm. Synthesis of 3-(2-pyridyl)-6-methyl-1,2,4,5-tetrazine (7) 2-Cyanopyridine (500 mg, 4.8 mmol), acetamidine hydrochloride (2.00 g, 21.2 mmol) and sulfur (155 mg, 4.8 mmol) were stirred in ethanol (5 mL) under an inert atmosphere of argon. Hydrazine hydrate (2.76 g; 55.2 mmol) was added and the mixture was stirred overnight at 20° C. The turbid mixture was filtered and the filtrate was evaporated to dryness, to yield 2.9 g of orange colored crude product 6. Subsequently, 6 (800 mg) was suspended in a mixture of THF (3 mL) and acetic acid (4 mL). A solution of NaNO2(2.0 g; 29.0 mmol) in water (3 mL) was added at 0° C. Instantaneous coloration to a red/purple suspension was observed. After 5 minutes of stirring at 0° C., chloroform and water were added. The purple chloroform layer was washed twice with water and then concentrated. The solid residue was stirred in a 1:1 mixture of chloroform and hexane, and then filtered. The filtrate was concentrated and the crude product was purified by silica column chromatography applying chloroform/acetone mixtures as eluent, yielding pure product (7, 48 mg, 21% yield overall, as calculated from 2-cyanopyridine). 1H NMR (CDCl3): δ=8.96 (d, 1H), 8.65 (d, 1H), 7.99 (t, 1H), 7.56 (dd, 1H), 3.17 (s, 3H) ppm.13C NMR (CDCl3): δ=168.1, 163.6, 150.9, 150.3, 137.4, 126.3, 123.9, 21.4 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=174.3 (M+H+), λmax=274 and 524 nm. Synthesis of 3,6-bis(2-aminophenyl)-1,2,4,5-tetrazine (9) 2-Aminobenzonitrile (1.00 g; 8.46 mmol) was dissolved in ethanol (3 mL) and hydrazine hydrate (2.06 g; 41.2 mmol) was added. The mixture was cooled to 0° C. and sulfur (0.17 g; 5.30 mmol) was added. Stirring was continued for 15 min, and subsequently the mixture was heated at 90° C. After 3 hr, the yellow precipitate was isolated by filtration, washed with ethanol (10 mL), and subsequently triturated twice with chloroform (2 times 10 mL), to yield the yellow intermediate 8 (343 mg, 30%). Intermediate 8 (105 mg; 0.394 mmol) was dissolved in ethanol (15 mL), and oxygen was bubbled through this solution at 50° C. Within minutes, the color changed from yellow to dark orange/red, and a precipitate was formed. After 2 hr, the precipitate was filtered, washed with ethanol and dried to give the product 9 as dark red crystals (89 mg, 86%). 1H NMR (DMSO-d6): δ=8.39 (d, 2H), 7.32 (t, 2H), 7.04 (s, 4H), 6.93 (d, 2H), 6.75 (t, 2H) ppm.13C NMR (DMSO-d6): δ=162.7, 149.6, 133.0, 129.0, 117.1, 115.8, 111.6 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=265.4 (M+H+), λmax=237, 293, 403 and 535 nm. Synthesis of 3,6-bis(4-hydroxyphenyl)-1,2,4,5-tetrazine (11) 4-Hydroxybenzonitrile (1.06 g; 8.90 mmol) was dissolved in hydrazine hydrate (3.09 g; 61.7 mmol), and the mixture was heated to 90° C. for 16 hr. The yellow precipitate was filtered and washed with water (25 mL) and ethanol (10 mL), to yield crude intermediate 10 as a yellow powder (870 mg; 62%). The intermediate (10, 173 mg; 0.645 mmol) was suspended in ethanol (10 mL), and oxygen was bubbled through this mixture at 50° C. Within minutes, the color changed from yellow to dark orange/red. After 6 hr, the precipitate was filtered, washed with ethanol and dried, to give the product 11 as dark red crystals (136 mg, 80%). 1H NMR (DMSO-d6): δ=10.35 (br. s, 2H), 8.36 (d, 4H), 7.02 (d, 4H) ppm.13C NMR (DMSO-d6): δ=162.6, 161.5, 129.2, 122.6, 116.3 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=267.1 (M+H+), λmax=235, 330 and 535 nm. Synthesis of 3,6-bis(4-aminophenyl)-1,2,4,5-tetrazine (13) 4-Aminobenzonitrile (1.00 g; 8.46 mmol) was dissolved in ethanol (3 mL), and subsequently hydrazine hydrate (2.12 g; 42.2 mmol) and sulfur (0.176 g; 5.5 mmol) were added. The mixture was heated at 90° C. for 90 min, and the yellow precipitate was isolated by filtration, washed with ethanol (10 mL), and subsequently triturated with acetone (12 mL) to yield the yellow intermediate 12 (190 mg, 17%). Intermediate 12 (50 mg; 0.188 mmol) was dissolved in DMSO (1 mL), and oxygen was bubbled through this solution at 20° C. After 5 hr, the reaction mixture was poured in brine (13 mL), and the red precipitate was filtered off, washed with water (10 mL), and dried in vacuo. The red powder was further purified by trituration with acetone (15 mL), to yield product 13 as a red solid (13.7 mg, 27%). 1H NMR (DMSO-d6): δ=8.17 (d, 2H), 7.75 (d, 2H), 6.02 (s, 4H) ppm.13C NMR (DMSO-d6): δ=162.3, 152.8, 128.5, 118.3, 113.8 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=265.2 (M+H+), λmax=241, 370 and 530 nm. Synthesis of 3,6-bis(3-aminophenyl)-1,2,4,5-tetrazine (15) 3-Aminobenzonitrile (1.00 g; 8.460 mmol) was dissolved in hydrazine hydrate (2.50 mL; 51.4 mmol), and the mixture was heated to 90° C. for 3 days. Water (5 mL) was added, and the yellow precipitate was filtered off and washed with water (15 mL) and ethanol (10 mL), to yield the crude intermediate 14 as a orange powder (910 mg; 81%). Intermediate 14 (50 mg; 0.188 mmol) was suspended in ethanol (4 mL), and oxygen was bubbled through this mixture at 50° C. Within minutes, the color changed from yellow to red. After 16 hr, the precipitate was filtered off, and washed with ethanol, to give the product 15 as a red powder (31 mg, 62%). 1H NMR (DMSO-d6): δ=7.77 (s, 2H), 7.66 (d, 2H), 7.30 (t, 2H), 6.85 (d, 2H), 5.53 (s, 4H) ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=265.2 (M+H+), λmax=240, 296 and 527 nm. Synthesis of 3,6-bis(aminomethyl)-1,2,4,5-tetrazine (20) Boc-amino acetonitrile (1.00 g; 6.40 mmol) was dissolved in methanol (10 mL) and sodium methoxide (0.145 mL 25% in MeOH; 0.64 mmol) was added. The mixture was stirred at 20° C. for 18 hr, and subsequently ammonium chloride (0.34 g; 6.40 mmol) was added, and the mixture was stirred at 20° C. for 3 days. The solution was precipitated in diethyl ether (40 mL), and the precipitate was collected by filtration, washed, and dried to yield the amidine hydrochloride 17. The amidine hydrochloride (17, 241 mg; 1.15 mmol) was dissolved in hydrazine hydrate (3 mL; 61.9 mmol), and the solution was stirred at 20° C. for 16 hr. Then it was diluted with water (10 mL), and the precipitate was collected by centrifugation, and dried. The colorless solid was dissolved in acetic acid (1.5 mL) and sodium nitrite (28 mg; 0.41 mmol) was added. The pink mixture was stirred for 15 min and subsequently chloroform (15 mL) and saturated sodium bicarbonate (30 mL) were added. The organic layer was isolated and washed with water (15 mL), dried over sodium sulfate, and evaporated to dryness, to yield the Boc-protected tetrazine as a pink solid (19, 70 mg; 35%). This compound (12 mg; 0.035 mmol) was dissolved in chloroform (1 mL), and TFA (1 mL) was added. The mixture was stirred for 15 min, and the precipitated in diethyl ether (15 mL). The pink precipitate was filtered off, washed, and dried to give the title compound as its TFA salt (20, 10 mg, 78%). 1H NMR (D2O): δ=5.06 (s, 4H) ppm.13C NMR (D2O): δ=164.5, 41.1 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=141 (M+H+), λmax=267 and 517 nm. Synthesis of 2,2′,2″-(10-(2-oxo-2-(6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)hexylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (27) and 2,2′,2″-(10-(2-oxo-2-(11-oxo-11-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)undecylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (28) 5-Amino-2-cyanopyridine 21 (1.02 g; 8.60 mmol), N-Boc-6-amino-hexanoic acid 22 (0.99 g; 4.30 mmol), DCC (1.77 g; 8.60 mmol), DMAP (1.05 g; 8.60 mmol), and PPTS (0.37 g; 1.47 mmol) were suspended in chloroform (15 mL). The mixture was stirred at room temperature for 18 hr, and then evaporated to dryness, and stirred in acetonitrile (20 mL). The precipitate was removed by filtration, and the filtrate was evaporated to dryness, dissolved in chloroform (20 mL), and washed with respectively aqueous citric acid (15 mL 0.5 M), aqueous potassium hydrogencarbonate (15 mL, 1 M), and water (15 mL). The organic phase was dried over sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (silica, hexane/ethylacetate=1:1) to yield the product 23 as a white solid (0.95 g; 61%). MS (ESI, m/z): Calcd for C17H25N4O3+([M+H]+): 333.19, Found: 333.17. Tert-butyl 6-(6-cyanopyridin-3-ylamino)-6-oxohexylcarbamate 23 (0.70 g; 2.1 mmol), 2-cyanopyridine (0.87 g; 8.4 mmol), hydrazine hydrate (1.25 g; 20 mmol) were dissolved in ethanol (2 mL), and sulfur (0.22 g; 7 mmol) was added. The mixture was stirred at 70° C. under an inert atmosphere of argon for 2 hr, and then at 50° C. for 16 hr. The orange suspension was diluted with chloroform (10 mL), and the resulting solution was washed with water (2 times 15 mL). The organic phase was dried over sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (silica, chloroform/acetone=4:1) to yield the product 24 as an orange solid (0.65 g; 66%). MS (ESI, m/z): Calcd for C23H31N8O3+([M+H]+): 467.25, Found: 467.33. Tert-butyl 6-oxo-6-(6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)hexylcarbamate 24 (0.30 g; 0.64 mmol) was dissolved in THF (1.5 mL), and acetic acid (2 mL) was added. Sodium nitrite (0.25 g; 3.62 mmol) was dissolved in water (1 mL) and added dropwise. The red solution was poured in aqueous potassium hydrogencarbonate (50 mL; 1 M), and the product was extracted with chloroform (50 mL). The organic layer was washed with water (50 mL), and dried over sodium sulfate and evaporated to dryness, to yield the product 25 as a purple solid (0.25 g; 83%). MS (ESI, m/z): Calcd for C23H29N8O3+([M+H]+): 465.23, Found: 465.42. tert-Butyl 6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)hexylcarbamate 25 (66 mg; 0.14 mmol) was dissolved in chloroform (6 mL), and TFA (6 mL) was added. The solution was stirred at room temperature for 2 hr, and subsequently evaporated to dryness, to yield the product 26 as its TFA salt (52 mg; 100%). MS (ESI, m/z): Calcd for C18H21NaO+([M+H]+): 365.19, Found: 365.33. 6-Amino-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)hexanamide 26 (52 mg; 0.14 mmol) was dissolved in DMF (2.5 mL), and DIPEA was added (320 mg; 2.0 mmol). N-Hydroxysuccinimide activated DOTA (161 mg; 0.2 mmol) was added, and the mixture was stirred at room temperature for 5 hr. The solution was evaporated to dryness, and the crude product was dissolved in a mixture of acetonitrile and water, and purified by preparative RP-HPLC. After lyophilisation the pure product 27 was obtained as a pink fluffy solid (80 mg, 76% yield). 1H-NMR (30% acetonitrile-d3in D2O): δ=8.90 (m, 2H, ArH), 8.68 (d, 1H, ArH), 8.60 (dd, 1H, ArH), 8.31 (m, 1H, ArH), 8.24 (t, 1H, ArH), 7.82 (t, 1H, ArH), 3.80 (br s, 6H, NCH2COOH), 3.72 (br s, 2H, NCH2CONH),3.34-3.23 (br m, 18H, NCH2CH2N, CH2NHCO), 2.49 (t, 2H, NHCOCH2), 1.70 (m, 2H, NHCOCH2CH2), 1.59 (m, 2H, CH2CH2NHCO), 1.41 (m, 2H, CH2CH2CH2NHCO) ppm.13C-NMR (30% acetonitrile-d3in D2O): δ=175.5, 171.5 (br), 162.6, 162.5, 150.1, 148.1, 142.9, 141.6, 139.6, 138.4, 128.0, 127.9, 125.4, 124.8, 55.4, 54.3 (br), 49.4 (br), 39.4, 36.5, 28.2, 25.9, 24.6 ppm. ESI-MS: m/z for C34H47N22O8+([M+H]+): 751.37; Obs. [M+H]+751.58, [M+Na]+773.50, [M+2H]2+376.42, [M+3H]3+251.33. FT-IR (ATR): v=3263, 3094, 2941, 2862, 1667, 1637, 1582, 1540, 1460, 1431, 1395, 1324, 1296, 1272, 1251, 1226, 1198, 1128, 1087, 1060, 1020, 992, 977, 920, 860, 831, 798, 782, 742, 718, 679, 663 cm−1. For 28, a procedure was used comparable to the described synthesis of 2,2′,2″-(10-(2-oxo-2-(6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)hexylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (27). After lyophilisation the pure product 28 was obtained as a pink fluffy solid (90 mg, 78% yield). 1H-NMR (DMSO-d6): δ=10.65 (s, 1H, NH), 9.06 (d, 1H, ArH), 8.93 (d, 1H, ArH), 8.61 (t, 2H, ArH), 8.44 (dd, 1H, ArH), 8.16 (t, 2H, ArH, NH), 7.73 (dd, 1H, ArH), 3.51 (br s, 6H, NCH2COOH), 3.28 (br s, 2H, NCH2CONH), 3.06 (q, 2H, CH2NHCO), 3.34-3.23 (br m, 16H, NCH2CH2N), 2.43 (t, 2H, NHCOCH2), 1.64 (m, 2H, NHCOCH2CH2), 1.42 (m, 2H, CH2CH2NHCO), 1.38-1.22 (m, 12H, CH2) ppm.13C-NMR (DMSO-d6): δ=173.0, 171.0 (br), 169.1 (br), 163.5, 163.2, 151.0, 150.6, 144.2, 141.7, 139.1, 138.2, 127.0, 126.5, 125.3, 124.6, 57.3 (br), 55.2 (br), 50.7, 39.0, 36.8, 29.5, 29.4, 29.3, 29.19, 29.17, 29.1, 26.9, 25.3 ppm. ESI-MS: m/z Calcd for C39H57N12O8+([M+H]+): 821.44; Obs. [M+Na]+843.58, [M+H]+821.58, [M+2H]2+411.42, [M+3H]3+274.67. FT-IR (ATR): v=3261, 3067, 2925, 2851, 1633, 1583, 1541, 1458, 1433, 1394, 1324, 1298, 1270, 1249, 1228, 1200, 1165, 1128, 1088, 1059, 1016, 991, 920, 885, 860, 832, 798, 782, 764, 742, 719, 687, 661 cm−1. DOTA-Tetrazine Activator 29 The tetrazine 29 above has been described in detail in Robillard et al., Angew. Chem., 2010, 122, 3447-3450. It also serves as an example a structure that can be used as an Activator according to this invention. The amide function on one of the 2-pyridyl groups of the 1,2,4,5-tetrazine moiety is an electron donating group, while both pyridine groups can be viewed as electron withdrawing. The tetrazine can therefore be seen as slightly electron deficient. Activator 29 displays suitable and favorable pharmacological properties: 29 is rather stable in PBS solution with little degradation within 2 hr and most of the material still intact after overnight incubation; it has a 10 min blood clearance half-life in mice; its partial volume of distribution (Vd) in mice corresponds to the total extracellular water compartment, as it does not significantly enter cells. Activator 29 contains a DOTA ligand, and such ligands are instrumental in a variety of imaging modalities (e.g. MRI, SPECT). Consequently, Activator 29 is not only suitable for drug release, but it can simultaneously be used for imaging purposes. In fact, Activator 29 has been employed as a SPECT/CT imaging probe after complexation with111In3+. See Robillard et al., Angew. Chem., 2010, 122, 3447-3450 for further details. Note that the amino-1,2,4,5-tetrazine moieties comprised in activators 27-29 can be used for conjugation to a range additional functional groups such as sugars, PEG, polymers, peptides (such as RGD or c-RGD), proteins, fluorescent molecules or dye molecules. Example 2 Synthesis of (E)-cyclooctene Model Prodrugs and Prodrugs Synthesis of (E)-cyclooct-2-enol (31), (E)-cyclooct-2-en-1-yl benzylcarbamate (32), and (E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33). Synthesis of (E)-cyclooct-2-enol (31) A solution of (Z)-cyclooct-2-enol 30 (2.36 g, 14.0 mmol) and methyl benzoate (1.8 mL, 1.94 g, 14.3 mmol, 1.0 eq) in diethyl ether/heptanes 1:2 (500 mL) was irradiated for 32 hr, while it was continuously lead through a column filled with silica/silver nitrate 10:1 (41 g), silica (0.5 cm) and sand (0.5 cm). The column was placed in the dark during the irradiation. The column was eluted with dichloromethane (250 mL) to give unreacted starting material. The silica was stirred with dichloromethane/12.5% aqueous ammonia 1:1 (3×100 mL). The combined organic layers were dried over sodium sulfate, filtered and evaporated in vacuo to give crude product 31 as a grey oil. The oil was purified by column chromatography (silica, eluents pentane/diethyl ether 10% to 50%) to give (E)-cyclooct-2-enol 31 (major isomer, second fraction, 440 mg, 3.49 mmol, 24.9%) as a colorless oil and (E)-cyclooct-2-enol 31 (minor isomer, first fraction, 325 mg, 2.58 mmol, 18.4%) as a colorless oil. The major diastereoisomer is identical to the (1RS,2RS)-trans-cyclooct-2-en-1-ol prepared via a different route by G. H. Whitham, M. Wright, J. Chem. Soc. (C) 1971, 883. The minor diastereoisomer is identical to the (1SR,2RS)-trans-cyclooct-2-en-1-ol prepared via a different route by G. H. Whitham, M. Wright, J. Chem. Soc. (C) 1971, 886. Minor isomer:1H-NMR (CDCl3, 300 MHz) δ=0.71-0.82 (m, 1H), 1.05-1.17 (m, 1H), 1.43-1.72 (m, 4H), 1.80-2.09 (m, 4H), 2.45-2.52 (m, 1H), 4.61 (s, 1H), 5.54-5.61 (m, 1H), 5.90-6.00 (m, 1H) ppm.13C-NMR (CDCl3, 75 MHz) δ=23.35, 29.42, 36.08, 36.27, 43.40, 71.40, 130.78, 135.39 ppm. Major isomer.1H-NMR (CDCl3, 300 MHz) δ=0.64-0.90 (m, 2H), 1.31-1.51 (m, 2H), 1.66-1.95 (m, 4H), 2.06-2.14 (m, 1H), 2.22-2.37 (m, 1H), 2.78 (br, 1H), 4.15-4.23 (m, 1H), 5.45-5.65 (m, 2H) ppm.13C-NMR (CDCl3, 75 MHz) δ=27.83, 29.28, 30.52, 35.58, 36.05, 44.48, 131.86, 136.00 ppm. Note: Reference is made to Whitham et al J. Chem. Soc. (C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of trans-cyclo-oct-2-en-ol, identified as (1RS, 2RS) and (1SR, 2RS), respectively. In these isomers the OH substituent is either in the equatorial or axial position. The above mentioned major and minor isomer refer respectively to the equatorial and axial isomer. Throughout the following examples major/equatorial and minor/axial are used interchangeably for trans-cyclo-oct-2-en-ol derivatives, and this characterization is based on the aforementioned characterization of the parent compound trans-cyclo-oct-2-en-ol. Synthesis of (E)-cyclooct-2-en-1-yl benzylcarbamate (major isomer) (32) To a solution of (E)-cyclooct-2-enol 31 (major isomer 100 mg, 0.792 mmol) in dichloromethane (6 mL) were added benzyl isocyanate (101 μL, 110 mg, 0.826 mmol, 1.04 eq) and a drop of triethylamine. The flask was covered with aluminum foil and the solution was stirred under a nitrogen atmosphere at room temperature overnight. Evaporation of the reaction mixture gave mainly starting material. Benzyl isocyanate (200 μL, 220 mg, 1.65 mmol, 2.08 eq) and a drop of triethylamine in dichloromethane (6 mL) were added and the solution was stirred overnight at room temperature, at 50° C. for 1 hr and at 25-30° C. over the weekend. The volatiles were removed by bulb-to-bulb distillation (50° C., 2 hr). The residue was purified by column chromatography to give carbamate 32 (101 mg, 0.389 mmol, 49.2%) as a white solid. 1H-NMR (CDCl3, 300 MHz) δ=0.81-0.86 (m, 2H), 1.35-1.55 (m, 2H), 1.82-1.99 (m, 4H), 2.21-2.30 (m, 1H), 2.38-2.47 (m, 1H), 4.36 (d, 5.8 Hz, 2H), 4.96 (br, 1H), 5.08-5.20 (m, 1H), 5.48-5.57 (m, 1H), 5.71-5.82 (m, 1H), 7.26-7.36 (M, 5H) ppm.13C-NMR (CDCl3, 75 MHz) δ=27.69, 29.25, 35.68, 35.76, 35.83, 41.32, 44.53, 78.33, 100.02, 127.65, 127.78, 128.86, 132.03, 133.31, 138.88 ppm. Synthesis of (E)-cyclooct-2-en-1-yl benzylcarbamate (minor isomer) (32). To a solution of (E)-cyclooct-2-enol 31 (minor isomer 100 mg, 0.792 mmol) in dichloromethane (6 mL) were added benzyl isocyanate (101 μL, 110 mg, 0.826 mmol, 1.04 eq) and a drop of triethylamine. The flask was covered with aluminum foil and the solution was stirred under a nitrogen atmosphere at room temperature overnight. Evaporation of the reaction mixture gave mainly starting material. Benzyl isocyanate (200 μL, 220 mg, 1.65 mmol, 2.08 eq) and a drop of triethylamine in dichloromethane (6 mL) were added and the solution was stirred overnight at room temperature, at 50° C. for 1 hr and at 25-30° C. over the weekend. The volatiles were removed by bulb-to-bulb distillation (50° C., 2 hr). The residue was purified by column chromatography to give carbamate 32 (43 mg, 0.166 mmol, 20.9%) as a white solid. 1H-NMR (CDCl3, 300 MHz) δ=0.74-0.93 (m, 2H), 1.01-1.14 (m, 1H), 1.41-1.57 (m, 1H), 1.62-1.76, 2H), 1.84-2.12 (m, 3H), 2.46-2.49 (m, 1H), 4.40 (d, J=6.0 Hz, 2H), 5.05 (br, 1H), 5.40 (s, 1H), 5.52-5.59 (m, 1H), 5.79-5.89 (m, 1H), 7.31-7.36 (m, 5H) ppm.13C-NMR (CDCl3, 75 MHz) δ=24.34, 29.33, 36.13, 36.20, 40.97, 45.30, 74.33, 127.67, 127.85, 128.87, 131.72, 131.99, 138.87, 156.11 ppm. Synthesis of (E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (major isomer) (33). To a solution of (E)-cyclooct-2-enol 31 (major isomer 260 mg, 2.06 mmol) in dichloromethane (12 mL) were added 3,5-dimethylphenyl isocyanate (305 318 mg, 2.16 mmol, 1.05 eq) in dichloromethane (3 mL) and a few drops of triethylamine. The flask was covered with aluminum foil and the solution was stirred under a nitrogen atmosphere at 29° C. for 4 nights. Evaporation of the reaction mixture gave 0.57 g off-white solid. The residue was purified by column chromatography (silica, 30 mL eluens ethyl acetate/heptanes 5 to 10%) to give partially purified carbamate 33 (94 mg). The product was further purified by column chromatography (silica, 30 mL eluens ethyl acetate/heptanes 5%) to give carbamate 33 (72 mg, 0.263 mmol, 12.8% yield, contains ca 10% Z-isomer) as a white solid. 1H-NMR (CDCl3, 300 MHz) δ=0.79-0.98 (m, 2H), 1.28-2.02 (m, 4H), 1.80-2.07 (m, 3H), 2.30 (s, 6H), 2.42-2.50 (m, 1H), 5.13-5.22 (m, 1H), 5.55-5.87 (m, 2H), 6.49 (br, 1H), 6.71 (s, 1H), 7.04 (s, 2H) ppm.13C-NMR (CDCl3, 75 MHz) δ=21.61, 27.67, 29.24, 35.70, 35.84, 41.21, 79.34, 116.59, 125.22, 131.83, 133.51, 138.11, 138.50, 153.43 ppm. Synthesis of (E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (minor isomer) (33) To a solution of (E)-cyclooct-2-enol 31 (minor isomer, contains also Z isomer, 260 mg, 2.06 mmol) in dichloromethane (12 mL) were added 3,5-dimethylphenyl isocyanate (305 μL, 318 mg, 2.16 mmol, 1.05 eq) in dichloromethane (3 mL) and a few drops of triethylamine. The flask was covered with aluminum foil and the solution was stirred under a nitrogen atmosphere at 30° C. for 2 nights and at 50° C. overnight. Evaporation of the reaction mixture gave 0.54 g yellow solid. The residue was purified by column chromatography (silica, 40 mL eluens ethyl acetate/heptanes 5%) to give partially purified carbamate 33 (20 mg). The product was further purified in vacuo (0.08 mbar) at 40° C. for 3 hr and at room temperature overnight to give carbamate 33 (11 mg, 0.040 mmol, 2.0%) as a light yellow semi-solid. 1H-NMR (CDCl3, 300 MHz) δ=0.78-0.90 (m, 1H), 1.07-2.18 (m, 8H), 2.30 (s, 6H), 2.45-2.53 (m, 1H), 5.42 (s, 1H), 5.56-5.62 (m, 1H), 5.83-5.94 (m, 1H), 6.60 (s, 1H), 6.71 (s, 1H), 7.03 (s, 2H) ppm.13C-NMR (CDCl3, 75 MHz) δ=21.64, 24.42, 29.43, 36.77, 40.19, 74.46, 116.47, 118.77, 125.35, 131.34, 132.31, 138.00, 138.91 ppm. Synthesis of (E)-cyclooct-2-en-1-yl (4-nitrophenyl) carbonate (34) A solution of minor (E)-cyclooct-2-enol 31 (304 mg, 2.41 mmol) in 15 mL dichloromethane was cooled in ice. 4-(N,N-Dimethylamino)pyridine (1.16 g, 9.50 mmol) was added, followed by 4-nitrophenylchloroformate (0.90 g, 4.46 mmol). The solution was stirred overnight, then poured on a 20 g silica column. Elution was performed with dichloromethane, then with dichloromethane containing 5% TBME. The product fractions were combined and rotary evaporated to yield minor-34 as a solidifying oil (338 mg, 1.16 mmol, 48%). In a similar fashion, from major (E)-cyclooct-2-enol 31 (259 mg, 2.06 mmol) in 10 mL dichloromethane, with 4-(N,N-dimethylamino)pyridine (1.11 g, 9.09 mmol) and 4-nitrophenylchloroformate (0.85 g, 4.22 mmol), the major-34 was obtained as a solidifying oil (234 mg, 0.80 mmol, 39%). 1H-NMR of minor 34 (CDCl3): δ=0.9 (m, 1H), 1.25 (m, 1H), 1.5-2.2 (m, 6H), 2.25 (dd, 1H), 2.6 (m, 1H), 5.45 (s, 1H), 5.6 (dd, 1H), 6.0 (m, 1H), 7.4 (d, 2H), 8.3 (d, 2H) ppm.13C-NMR (CDCl3): δ=24.0, 29.0, 36.0, 36.0, 40.6 (all CH2), 79.0, 122.0, 125.8, 129.8, 133.2 (all CH), 145.4, 10 151.8, 156.0 (C and C═O) ppm. 1H-NMR of major 34 (CDCl3): δ=0.8-1.0 (m, 2H), 1.4-2.1 (m, 6H), 2.35 (m, 1H), 2.45 (m, 1H), 5.2 (m, 1H), 5.65 (m, 1H), 5.85 (m, 1H), 7.4 (d, 2H), 8.3 (d, 2H) ppm.13C-NMR (CDCl3): δ=27.8, 29.0, 35.8, 36.0, 40.4 (all CH2), 83.0, 121.8, 125.0, 130.4, 134.4 (all CH), 145.8, 152.0, 156.0 (C and C═O) ppm. Synthesis of (E)-cyclooct-2-en-1-yl (4-(hydroxymethyl)phenyl)carbamate (35) The PNP-derivative 34 derived from the minor alcohol 31 (136 mg, 0.467 mmol) was dissolved in 7.5 g THF. Diisopropylethylamine (182 mg, 1.41 mmol) was added, followed by 1-hydroxybenzotriazole (24 mg, 0.178 mmol) and 4-aminobenzylalcohol (94 mg, 0.76 mmol). The mixture was stirred in the dark at ca. 30° C. for 6 days. The solvent was removed by rotary evaporation and the residue was chromatographed on 20 g silica, using dichloromethane with gradually increasing amounts of TBME as the eluent. The product eluted with ca. 5% TBME. Rotary evaporation of the product fractions left the product minor-35 as a viscous oil (112 mg, 0.407 mmol, 87%). In a similar fashion, the PNP-derivative 34 derived from the major alcohol 31 (145 mg, 0.498 mmol) in 6.0 g THF, was reacted with diisopropylethylamine (210 mg, 1.63 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol) and 4-aminobenzylalcohol (128 mg, 1.04 mmol) for 3 days at ca. 30° C. Rotary evaporation and chromatography yielded the product major-35 as a viscous oil (110 mg, 0.40 mmol, 80%). 1H-NMR of minor-35 (CDCl3): δ=0.8 (m, 1H), 1.1 (m, 1H), 1.45 (m, 1H), 1.6-2.2 (m, 6H), 2.4 (m, 1H), 4.6 (s, 2H), 5.4 (s, 1H), 5.55 (dd, 1H), 5.85 (m, 1H), 7.15 (bs, 1H), 7.2-7.4 (AB, 4H) ppm.13C-NMR (CDCl3): δ=24.2, 29.0, 36.0, 36.0, 41.0, 65.0 (all CH2), 75.0, 119.0, 128.0, 131.0, 132.6 (all CH), 136.0, 138.0, 153.6 (C and C═O) ppm. 1H-NMR of the major-35 (CDCl3): δ=0.8-1.0 (m, 2H), 1.4-2.1 (m, 6H), 2.3 (m, 1H), 2.45 (m, 1H), 4.65 (s, 2H), 5.2 (m, 1H), 5.6 (m, 1H), 5.8 (m, 1H), 6.6 (bs, 1H), 7.45-7.65 (AB, 4H) ppm.13C-NMR (CDCl3): δ=27.4, 29.2, 35.8, 36.0, 41.2, 65.0 (all CH2), 79.8, 119.0, 128.2, 132.0, 134.0 (all CH), 136.0, 137.8, 153.6 (C and C═O) ppm. Synthesis of minor (E)-ethyl2-(4-(((cyclooct-2-en-1-yloxy)carbonyl)amino)phenyl)-2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)acetate (37) The PNP-derivative 34 derived from the minor alcohol 31 (300 mg, 1.03 mmol) was dissolved in 10.3 g THF. Diisopropylethylamine (362 mg, 2.80 mmol) was added, followed by 1-hydroxybenzotriazole (75 mg, 0.556 mmol) and ethyl 2-(4-aminophenyl)-2-hydroxyacetate (325 mg, 1.67 mmol, prepared as described in WO 2009109998). The mixture was stirred in the dark at ca. 30° C. for 6 days. The solvent was removed by rotary evaporation and the residue was chromatographed on 21 g silica, using dichloromethane with gradually increasing amounts of TBME as the eluent. The product eluted with ca. 5% TBME. Rotary evaporation of the product fractions afforded minor (E)-ethyl 2-(4-(((cyclooct-2-en-1-yloxy)carbonyl)amino)phenyl)-2-hydroxyacetate (36) as a viscous oil (350 mg, 1.01 mmol, 99%). 1H-NMR (CDCl3): δ=0.8 (m, 1H), 1.1 (m, 1H), 1.2 (t, 3H), 1.4-2.2 (m, 7H), 2.5 (m, 1H), 4.1-4.3 (2q, 2H), 5.1 (s, 1H), 5.45 (s, 1H), 5.55 (dd, 1H), 5.85 (m, 1H), 6.7 (bs, 1H), 7.3-7.45 (AB, 4H) ppm. The product 36 obtained above (80 mg, 0.23 mmol) was dissolved in 4.1 g acetonitrile. Diisopropylethylamine (215 mg, 1.67 mmol) was added, followed by N,N′-disuccinimidyl carbonate (217 mg, 0.85 mmol). The solution was stirred for 2 days at ca. 30° C. The solvent was removed by rotary evaporation and the residue was chromatographed on 16 g silica, using dichloromethane with gradually increasing amounts of TBME as the eluent. The product eluted with ca. 20% TBME. Rotary evaporation of the product fractions afforded the product minor-(E)-ethyl 2-(4-(((cyclooct-2-en-1-yloxy)carbonyl)amino)phenyl)-2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)acetate (37) as a viscous oil (60 mg, 0.123 mmol, 53%). 1H-NMR (CDCl3): δ=0.8 (m, 1H), 1.1 (m, 1H), 1.2 (t, 3H), 1.4-2.2 (m, 7H), 2.5 (m, 1H), 2.6 (s, 4H), 4.15-4.3 (2q, 2H), 5.4 (s, 1H), 5.55 (dd, 1H), 5.8 (s) and 5.85 (m) (2H), 6.7 (bs, 1H), 7.35-7.5 (AB, 4H) ppm. Synthesis of (E)-cyclooctene Doxorubicin Prodrug (38) The PNP-derivative 34 derived from the minor alcohol 31 (20 mg, 0.0687 mmol) was dissolved in 3.0 g DMF. Diisopropylethylamine (80 mg, 0.62 mmol) was added, followed by doxorubicin hydrochloride (45 mg, 0.0776 mmol). The mixture was stirred in the dark at ca. 30° C. for 3 days. The solvent was removed under high vacuum and the residue was chromatographed on 17 g silica, using dichloromethane with gradually increasing amounts of methanol as the eluent. Rotary evaporation of the product fractions left a residue which was stirred with 5 mL TBME. After addition of 15 mL heptane and filtration minor-38 was obtained (27 mg, 0.039 mmol, 50%). The filtrate contained an additional amount of product. In a similar fashion, from the PNP-derivative 34 derived from the major alcohol 31 (22 mg, 0.0756 mmol) in 7.2 g DMF, after reaction with diisopropylethylamine (80 mg, 0.62 mmol) and doxorubicin hydrochloride (47.7 mg, 0.0822 mmol), followed by removal of the solvent under high vacuum, chromatography and TBME/heptane treatment major-38 was obtained (21 mg, 0.030 mmol, 30%). The filtrate contained an additional amount of product. 1H-NMR of minor-38 (CDCl3): δ=0.7-2.0 (m) and 1.35 (d) (18H), 2.2 (m, 2H), 2.4 (m, 2H), 3.0-3.4 (dd, 2H), 3.65 (s, 1H), 3.9 (m, 1H), 4.1 (s+m, 4H), 4.8 (s, 1H), 5.05 (m, 1H), 5.2-5.85 (m, 2H), 7.4 (d, 1H), 7.8 (t, 1H), 8.05 (d, 1H) ppm. 1H-NMR of major-38 (CDCl3): δ=0.7-2.0 (m) and 1.35 (d) (18H), 2.2 (m, 2H), 2.4 (m, 2H), 3.0-3.4 (dd, 2H), 3.65 (s, 1H), 3.9 (m, 1H), 4.1 (s+m, 4H), 4.8 (s, 1H), 5.0 (m, 1H), 5.3-5.8 (m, 2H), 7.4 (d, 1H), 7.8 (t, 1H), 8.05 (d, 1H) ppm. MS: 694.3 (M−1). Synthesis of (E)-cyclooctene-doxorubicin Prodrug 46 n-Butyllithium (97 mL, 2.5 N in hexanes, 0.242 mol) was added to diisopropylamine (23.66 g, 0.234 mol) in 100 mL THF at temperatures below −20° C. The solution was cooled and cyclooct-2-enone (39, 23.07 g, 0.185 mol), dissolved in 60 mL THF, was added over a 20 min period at −65 to −80° C. The solution was then stirred for 1 hr at −67 to −72° C. Ethyl bromoacetate (45.4 g, 0.272 mol), dissolved in 40 mL THF, was added over a 25 min period at −63 to −75° C. The resulting mixture was stirred for 3 hr at −55 to −70° C. Heptane (50 mL) was added at −60° C., followed by the addition of a solution of 40 g ammonium chloride in 100 mL water (with cooling), allowing the temperature to rise from −70° C. to −30° C. The cooling-bath was removed and the mixture was stirred for an additional 30 min, whereby the temperature raised to −15° C. The mixture was poured in 200 mL TBME and 50 mL water, the layers were separated and the organic layer was washed with 50 mL water. The successive aqueous layers were extracted with 250 mL TBME. The organic layers were dried and rotary evaporated. The excess of ethyl bromoacetate was removed under high vacuum by warming in a Kugelrohr apparatus. The residue comprising (Z)-ethyl 2-(2-oxocyclooct-3-en-1-yl)acetate (40) was used as such in the next step. 1H-NMR (CDCl3): δ=1.25 (t, 3H), 1.4-2.6 (m, 9H), 2.9 (2d, 1H), 3.55 (m, 1H), 4.15 (q, 2H), 6.05-6.5 (m, 2H) ppm. A solution of the crude ester 40 in a mixture of 180 mL THF and 20 mL methanol was cooled in ice. Phosphotungstic acid (250 mg) was added, followed by the portion-wise addition of sodium borohydride (4.0 g, 0.105 mol) over a 30 min period, at temperatures below 7° C. The mixture was stirred for 90 min in ice, then 250 mL water and 250 mL toluene were added. The layers were separated and the organic layer was washed with 50 mL water. The successive aqueous layers were extracted with 250 mL toluene. The organic layers were dried and rotary evaporated. Crude 41 did not produce well-defined fractions, therefore all material was combined and hydrolyzed by refluxing for 2 hr with 25 mL 50% sodium hydroxide solution in 200 mL ethanol (another 25 mL water being added during the process). Most of the ethanol was removed by rotary evaporation. Some water was added to the residue. The mixture was extracted with 2×200 mL toluene. The organic layers were washed with 50 mL water. Toluene (200 mL) was added to the combined aqueous layers, which were acidified with concentrated hydrochloric acid. The layers were separated and the organic layer was washed with 20 mL water. The successive aqueous layers were extracted with 200 mL toluene. The 2 organic layers were dried and rotary evaporated. Kugelrohr distillation yielded the lactone 42 as a mixture of 2 isomers in a ca. 2:1 ratio (7.33 g, 44.1 mmol, 24% based on cyclooct-2-enone). 1H-NMR (CDCl3): δ=1.2-2.6 (m, 10H), 2.6-2.8 (m, 1H), 4.95 (m, 0.35 H), 5.35 (m, 0.65H), 5.6 (m, 1H), 5.85 (m, 1H) ppm.13C-NMR (CDCl3): δ=24.1, 25.2, 27.0, 28.0, 29.2, 29.6, 34.4, 36.8 (all CH2), 43.5, 47.2, 80.8, 81.9 (all CH), 126.4, 129.6, 130.2, 134.2 (all CH), 176.4 (C═O), 177.0 (C═O) ppm. The lactone 42 obtained above (7.33 g, 44.1 mmol) was mixed with 10.0 g methyl benzoate and ca. 500 mL heptane/ether (ca. 4:1). The mixture was irradiated for 36 hr while the solution was continuously flushed through a 69 g silver nitrate impregnated silica column (containing ca. 6.9 g silver nitrate). The column material was then flushed with 250 mL portions of heptane/TBME in the ratios 3:1, 2:1, 1:1, 1:2 and then with 400 mL TBME. The first two fraction contained only methyl benzoate. The last 3 fractions were washed with 200 mL 10% ammonia, dried and rotary evaporated. After removal of most of the methyl benzoate under high vacuum, the combined residue weighed 800 mg (a mixture of the Z and E isomer, as well as methyl benzoate). The remaining column material was stirred with TBME and ammonia, then filtered and the layers were separated. The solid was treated twice more with the aqueous layer and TBME, then filtered and the layers were separated. The organic layers were dried and rotary evaporated to yield 3.70 g of 43 as a ca. 4:1 mixture of isomers, each isomer probably consisting of 2 E-isomers (22.29 mmol, 51%). 1H-NMR (CDCl3): δ=0.8-2.75 (m, 10.6H), 3.0 (m, 0.4H), 4.45 (t, 0.2H), 5.0 (m, 0.8H), 5.6 (dd, 0.5H), 5.65 (m, 0.5H), 5.8 (m, 0.5H), 6.05 (m, 0.5H) ppm. The recovered major isomer (see experiment below) has the following data: 1H-NMR (CDCl3): δ=0.8-2.75 (m, 10.6H), 3.0 (m, 0.4H), t, 0.2H), 4.95 (m, 1H), 5.6 (dd, 0.8H), 5.65 (m, 0.3H), 5.8 (m, 0.3H), 6.05 (m, 0.6H) ppm. 13C-NMR (CDCl3): δ=21.6, 25.8, 30.0, 30.4, 33.0, 34.8, 35.4, 36.0, 38.0 (all CH2), 46.0, 47.0, 80.8, 84.0 (all CH), 128.2, 131.4, 133.0, 134.0 (all CH), 177.2 (C═O), 177.4 (C═O) ppm. The ratio of the signals indicates a ca. 2:1 isomer ratio. Diisopropylethylamine (5.91 g, 45.8 mmol) was added to a solution of the lactone 43 (865 mg, 5.21 mmol) in 15 mL dichloromethane, followed by the addition of beta-alanine ethyl ester hydrochloride (1.38 g, 8.98 mmol). The mixture was stirred for 16 days at room temperature, then rotary evaporated at 55° C. The residue was chromatographed on 50 g silica using dichloromethane as the eluent. This yielded the starting lactone 43 (the major E-isomer, which by C-NMR appeared to be a mixture of 2 isomers). Further elution with dichloromethane containing increasing amounts of methanol gave the amide 44. The product was taken up in 75 mL TBME and washed with 5 g citric acid in 25 mL water and with 2×10 mL water. The successive aqueous layers were extracted with 50 mL TBME. The combined organic layers were dried and rotary evaporated to yield amide 44 (360 mg, 1.27 mmol, 24%), consisting of a mixture of isomers. 1H-NMR (CDCl3): δ=0.8-2.7 (m), 1.25 (t), 2.45 (t) (16H), 3.5 (q, 2H), 3.9 (t, 0.5H), 4.15 (q, 2H), 4.35 (m, 0.5H), 5.5-5.9 (m, 2H), 6.2-6.5 (2 bt, 1H) ppm. 13C-NMR (CDCl3) (signals of a fraction which was much enriched in 1 set of isomers): δ=4.3 (CH3), 22.4, 27.8, 29.9, 33.0, 34.0, 34.1, 34.2, 34.5, 35.3, 35.3, 35.5, 35.7, 36.1, 36.2, 41.7 (all CH2), 46.2 (CH), 51.6 (CH), 60.9 (CH2), 77.1, 80.2, 131.2, 131.7, 134.2, 135.6 (all CH), 172.7, 173.9, 175.1 (all C═O) ppm. The amide 44 (115 mg, 0.406 mmol, mainly 1 set of isomers) was dissolved in 4.4 g acetonitrile. Diisopropylethylamine (370 mg, 2.87 mmol) was added, followed by N,N′-disuccinimidyl carbonate (355 mg, 1.38 mmol). The solution was stirred for 2 days at ca. 30° C. The solvent was removed by rotary evaporation and the residue was chromatographed on 16 g silica, using dichloromethane with gradually increasing amounts of TBME as the eluent. The product eluted with ca. 20% TBME. Rotary evaporation of the product fractions afforded the NHS carbonate 45 as a viscous oil (150 mg, 0.353 mmol, 87%). 1H-NMR (CDCl3): δ=0.8-2.6 (m), 1.25 (t), 2.55 (t) (16H), 2.85 (q, 4H), 3.5 (q, 2H), 4.15 (q, 2H), 4.95 (t, 0.8H), 5.2 (dd, 0.2H), 5.55-6.0 (m, 2H), 6.4 (bt, 1H) ppm. The NHS-carbonate 45 obtained above (150 mg, 0.353 mmol) was dissolved in 7.56 g DMF. Diisopropylethylamine (132 mg, 1.02 mmol) was added, followed by doxorubicin hydrochloride (66 mg, 0.114 mmol). The mixture was stirred in the dark at room temperature for 3 days. The solvent was removed under high vacuum and the residue was chromatographed on 13 g silica, using dichloromethane with gradually increasing amounts of methanol as the eluent. Rotary evaporation of the product fractions afforded 112 mg of prodrug 46. 1H-NMR (CDCl3, only relevant signals given): δ=1.25 (t), 3.2 (m), 3.5 (m), 4.05 (s), 4.15 (q), 4.8 (s), 5.2-5.8 (m), 6.15 (m), 6.25 (m), 7.4 (d), 7.8 (t), 8.0 (d) ppm. Optionally prodrug 46 may be conjugated to an antibody by converting the ester functionality to a carboxylic acid, which may then be converted into an NHS ester for lysine conjugation. Synthesis of minor-(E)-cyclooct-2-en-1-yl (2,5-dioxopyrrolidin-1-yl) carbonate (47) N,N′-disuccinimidyl carbonate (372 mg, 1.45 mmol) is added to a stirred mixture of minor alcohol 31 (77 mg, 0.61 mmol), 3.33 g acetonitrile and diisopropylethylamine (410 mg, 3.18 mmol). The mixture was stirred at 25° C. for 3 d, adding an additional 120 mg N,N′-disuccinimidyl carbonate after 2 days. The solution was chromatographed on 15 g silica using dichloromethane and then dichloromethane containing a small amount TBME as the eluent. The product fractions were rotary evaporated to yield the product 47 as a solid (62 mg, 0.23 mmol, 38%). 1H-NMR (CDCl3): δ=0.8 (m, 1H), 1.15 (m, 1H), 1.45-2.15 (m, 6H), 2.2 (dd, 1H), 2.55 (m, 1H), 2.8 (s, 4H), 5.4 (s, 1H), 5.5 (d, 1H), 6.0 (m, 1H) ppm. Example 3 Stability and Reactivity of Tetrazine Activators Hydrolytic Stability Tests of Tetrazines 10 μL of a solution of the specific tetrazine in DMSO (25 mM) was diluted with PBS buffer (3 mL) (or a mixture of PBS and acetonitrile in case the aqueous solubility was too low). This solution was filtered and, the decrease of the absorption band at 525 nm was monitored using UV spectroscopy. The rate of hydrolysis and half-life time was determined from these data. Reactivity of Tetrazines Towards trans-cyclooct-4-ene-1-ol (Axial Isomer) A competition experiment was performed to determine the reactivity ratio of a specific tetrazine and 3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5) (that was chosen as the reference tetrazine), in the inverse-electron demand Diels-Alder reaction with trans-cyclooct-4-ene-1-ol (“minor” isomer with OH in axial position, see: Whitham et al.J. Chem. Soc.(C), 1971, 883-896)). To acetonitrile (0.100 mL) was added 5 μL of a solution of the specific tetrazine in DMSO (25 mM) and 5 μL of a solution of the reference tetrazine in DMSO (25 mM). This mixture was diluted with water (0.9 mL), and the absolute amounts of both tetrazines was determined by HPLC-MS/PDA analysis. Subsequently, a solution of trans-cyclooct-4-ene-1-ol (axial isomer) in DMSO (25 μL 2.5 mM) was slowly added, and the mixture was stirred for 5 min. Again, the absolute amounts of both tetrazines was determined by HPLC-MS/PDA analysis, and conversions for both tetrazines was calculated. From these conversions, the reactivity ratio (R=k2,TCO/k2,Ref) of both tetrazines was calculated using the mathematical procedure from Ingold and Shaw (J. Chem. Soc.,1927, 2918-2926). The table below demonstrates how the reactivity and stability profile of tetrazines can be tailored to certain specifications by varying the substituents. stability in PBS at 20° C.Reactivity ratiotetrazinet1/2(hr)(R = k2,TZ/k2,Ref)441.173400.4801241.6>300*<0.01*1151.073.6*5.3*35*1.84*3.22.71170.950.681.5>1500.192.40.83>300*<0.01*1830.77>300*<0.01*>300*<0.01*41.76>300*<0.01*>300*<0.01*2.73.0610.32.82300.253000.180.422>300*<0.01*n.d.1.2>300*<0.01*>300*<0.01*>300*<0.01*16n.d.*This value was determined in a 50:50 mixture of PBS and acetonitrile. Example 4 Stability and Reactivity of Trans-Cyclooctene Model Prodrugs and Prodrugs Stability 10 μL of a solution of the specific trans-cyclooctene derivative in dioxane (25 mM) was diluted with PBS buffer (3 mL), and this solution was stored at 20° C. in the dark. The fate of the TCO compound was monitored by HPLC-MS analysis, and an estimation of the half-life time was made. Reactivity of Trans-Cyclooctene Derivatives Towards bis(2-pyridyl)-1,2,4,5-tetrazine: Second-Order Rate Constant Determination The kinetics of the inverse-electron demand Diels-Alder reaction of a trans-cyclooctene derivative with 3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5), performed in acetonitrile at 20° C., was determined using UV-visible spectroscopy. A cuvette was filled with acetonitrile (3 mL) and equilibrated at 20° C. 3-(5-Acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5, 2.50×10−7mol) was added, followed by the trans-cyclooctene derivative (2.50×10−7mol). The decay of the absorption at λ=540 nm was monitored, and from this curve the second-order rate constant, k2, was determined assuming second order rate kinetics. Reactivity of Trans-Cyclooctene Derivatives Towards bis(2-pyridyl)-1,2,4,5-tetrazine: Competition Experiment A competition experiment was performed to determine the reactivity ratio of a specific trans-cyclooctene derivative and trans-cyclooct-4-ene-1-ol (axial isomer) (that was chosen as the reference trans-cyclooctene), in the inverse-electron demand Diels-Alder reaction with bis(2-pyridyl)-1,2,4,5-tetrazine (2). To acetonitrile (0.05 mL) was added a solution of the specific trans-cyclooctene derivative in dioxane (5 μL 25 mM; 1.25×10−7mol) and a solution of the reference trans-cyclooctene in dioxane (5 μL 25 mM; 1.25×10−7mol). This mixture was diluted with water (0.45 mL). Subsequently, a solution of bis(2-pyridyl)-1,2,4,5-tetrazine (2, 6.25×10−8mol) in a mixture of acetonitrile (0.05 mL) and water (0.45 mL) was slowly added while stirring vigorously. After addition, the mixture was stirred for an additional 5 min. The conversion of both trans-cyclooctene derivatives was determined by HPLC-MS/PDA analysis, and from these conversions, the reactivity ratio (R=k2,TCO/k2,Ref) of the specific trans-cyclooctene derivative was calculated using the mathematical procedure from Ingold and Shaw (J. Chem. Soc.,1927, 2918-2926). stability in PBSrate contant*reactivity ratio**trans-cyclooctene derivativeat 20° C., t1/2k2(M−1s−1)(R = k2,TCO/k2,Ref)>3 days5771minor isomer>>20 days0.2632 major isomer>>20 days400.06732 minor isomer>>20 days25.733 minor isomer>>20 days0.1533 major isomer>>20 days38 minor>>20 days38 major*determined by UV-visible spectroscopy in acetonitrile at 20° C.**determined by a competition experiment Example 5 Activation of Model Prodrugs The example demonstrates the Inverse Electron Demand Diels-Alder reaction of 1,2,4,5-tetrazines and a model trans-cyclooctene prodrug, and subsequent elimination of the model drug (e.g. benzylamine). General Procedure: 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2) and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2, 5.91×10−5g; 2.5×10−7mol) was dissolved in 0.2 mL acetonitrile, and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32, the isomer with the carbamate in the axial position; 6.48×10−5g; 2.50×10−7mol) was added. After 5 min, the reaction mixture was diluted with water (0.8 mL) and stirred at 20° C. for 24 hr. HPLC-MS analysis of the mixture proved the formation of the elimination product (the rDA adduct without the aminobenzyl carbamate) with m/z=+317 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 6-Methyl-3-(4-butanamido-2-pyridinyl)-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) According to above general procedure, both title compounds were reacted and analysis by HPLC-MS demonstrated the formation of the elimination product with m/z=+339 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 6-Phenyl-3-(4-aminophenyl)-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) According to above general procedure, both title compounds were reacted and analysis by HPLC-MS demonstrated the formation of the elimination product with m/z=+330 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 6-Phenyl-3-(3-aminophenyl)-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) According to above general procedure, both title compounds were reacted and analysis by HPLC-MS demonstrated the formation of the elimination product with m/z=+330 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 6-H-3-(4-Aminomethylphenyl)-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) According to above general procedure, both title compounds were reacted and analysis by HPLC-MS demonstrated the formation of the elimination product with m/z=+268 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 3,6-Diphenyl-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1 -yl benzylcarbamate (32) According to above general procedure, both title compounds were reacted and analysis by HPLC-MS demonstrated the formation of the elimination product with m/z=+315 Da (M+H+), and release of benzylamine (m/z=+108 Da: M+H+). 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (9) and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (3.34 mg; 1.26×10−5mol) was dissolved in 0.5 mL DMSO-d6, and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32; 3.28 mg; 1.26×10−5mol) was added. After 5 min, the reaction mixture was diluted with D2O (0.2 mL) and stirred at 20° C. for 24 hr.1H-NMR of the reaction mixture indicated the formation of benzylamine: δ=3.86 ppm (s, 2H, PhCH2NH2). HPLC-MS analysis of this mixture demonstrated the formation of the elimination product (tr=5.45 min: m/z=+345 Da (M+H+)), and release of benzylamine: (tr=0.88 min: m/z=+108 Da: (M+H+)). 3,6-Bis(4-hydroxyphenyl)-1,2,4,5-tetrazine (11) and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32) 3,6-Bis(4-hydroxyphenyl)-1,2,4,5-tetrazine (11, 6.65×10−5g; 2.50×10−7mol) was dissolved in 0.5 mL acetonitrile, and minor-(E)-cyclooct-2-en-1-yl benzylcarbamate (32; 6.48×10−5g; 2.50×10−7mol) was added. After 2 min, the reaction mixture was diluted with water (0.5 mL) and stirred at 20° C. for 5 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+347 Da (M+H+), and release of benzylamine: m/z=+108 Da (M+H+). 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (9) and minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33) 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (9, 6.60×10−5g; 2.50×10−7mol) was dissolved in acetonitrile (0.3 mL) and this mixture was diluted with PBS buffer (0.7 mL). Next, minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33, the isomer with the carbamate in the axial position; 6.84×10−5g; 2.50×10−7mol) was added. The solution was stirred at 20° C. for 20 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+345 Da (M+H+), and release of 3,5-dimethylaniline: m/z=+122 Da (M+H+). 3,6-Bis(4-hydroxyphenyl)-1,2,4,5-tetrazine (11) and minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33) 3,6-Bis(4-hydroxyphenyl)-1,2,4,5-tetrazine (11, 6.65×10−5g; 2.50×10−7mol) was dissolved in acetonitrile (0.2 mL) and this mixture was diluted with PBS buffer (0.8 mL). Next, minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33; 6.84×10−5g; 2.50×10−7mol) was added. The solution was stirred at 20° C. for 20 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+347 Da (M+H+), and release of 3,5-dimethylaniline: m/z=+122 Da (M+H+). 3,6-Diphenyl-1,2,4,5-tetrazine and minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl) carbamate (33) 3,6-Diphenyl-1,2,4,5-tetrazine (5.85×10−5g; 2.50×10−7mol) was dissolved in acetonitrile (0.3 mL) and this mixture was diluted with PBS buffer (0.7 mL). Next, minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33; 6.84×10−5g; 2.50×10−7mol) was added. The solution was stirred at 20° C. for 20 hr. HPLC-MS analysis of the mixture proved the formation of the elimination product with m/z=+315 Da (M+H+), and release of 3,5-dimethylaniline: m/z=+122 Da (M+H+). 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7) and minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl) carbamate (33) 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7, 4.33×10−5g; 2.50×10−7mol) was dissolved in PBS buffer (1 mL). Next, minor-(E)-cyclooct-2-en-1-yl (3,5-dimethylphenyl)carbamate (33; 6.84×10−5g; 2.50×10−7mol) was added. The solution was stirred at 20° C. for 20 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+254 Da (M+H+), and release of 3,5-dimethylaniline: m/z=+122 Da (M+H+). Example 6 Activation of Doxorubicin Prodrugs 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7) and minor-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38) 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7, 4.33×10−6g; 2.50×10−8mol) was dissolved in PBS buffer (1 mL) (c=25 μM). Next, minor-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38, the isomer with the carbamate in the axial position; 1.74×10−5g; 2.50×10−8mol) was added. The solution was stirred at 20° C. for 4 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+254 Da (M+H+), and release of doxorubicin (69% yield): m/z=+544 Da (M+H+) and λmax=478 nm. Comparable results were obtained at concentrations of 2.5 and 1.0 μM. 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7) and major-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38) 3-(2-Pyridyl)-6-methyl-1,2,4,5-tetrazine (7, 4.33×10−6g; 2.50×10−8mol) was dissolved in PBS buffer (1 mL) (c=25 μM). Next, major-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38, the isomer with the carbamate in the equatorial position; 1.74×10−5g; 2.50×10−8mol) was added. The solution was stirred at 20° C. for 16 hr. HPLC-MS analysis of the mixture showed a conversion of the DA-reaction of 40%, and demonstrated the formation of the elimination product with m/z=+254 Da (M+H+), and release of doxorubicin (20% yield): m/z=+544 Da (M+H+) and λmax=478 nm. 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (9) and minor-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38) 3,6-Bis(2-aminophenyl)-1,2,4,5-tetrazine (9, 2.64×10−6g; 1.00×10−8mol) was dissolved in acetonitrile (0.1 mL). This mixture was diluted with PBS buffer (0.9 mL). Next, minor-(E)-cyclooct-2-en-1-yl doxorubicin carbamate (38; 6.96×10−6g; 1.00×10−8mol) was added. The solution was stirred at 20° C. for 18 hr. HPLC-MS analysis of the mixture demonstrated the formation of the elimination product with m/z=+345 Da (M+H+), and release of doxorubicin (90% yield): m/z=+544 Da (M+H+) and λmax=478 nm. Example 7 Cell Proliferation Assay with Doxorubicin Prodrug Minor-38 and Tetrazine 7 A431 squamous carcinoma cells were maintained in a humidified CO2(5%) incubator at 37° C. in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 0.05% glutamax (Invitrogen) in the presence of penicillin and streptomycin. The cells were plated in 96-well plates (Nunc) at a 2500 cells/well density 24 hr prior to the experiment. Doxorubicin (Dox) and the prodrug minor-38 (1 mM in DMSO) and the tetrazine 7 (10 mM in PBS) were serially diluted in pre-warmed culture medium immediately before the experiment and added to the wells (200 μl final volume per well). The prodrug was either added alone or in combination with 10 μM or 1.5 mol eq. tetrazine 7 (with respect to the prodrug). After 72 hr incubation at 37° C., cell proliferation was assessed by an MTT assay. Briefly, methylthiazolyldiphenyltetrazolium bromide (MTT) was dissolved in PBS at 5 mg/ml, filtered through 0.22 μm and 25 μl was added to each well. After 120 min incubation at 37° C., the medium was gently aspirated. The formed formazan crystals were dissolved in 100 μl DMSO and the absorbance was measured with a plate reader (BMG Labtech) at 560 nm. IC50values (±standard error; see Table) were derived from the normalized cell growth curves shown inFIG.3, generated with GraphPad Prism (version 5.01). The cell proliferation assay shows that, while tetrazine 7 is non-toxic (IC50>100 μM) and the prodrug 38 is slightly toxic (IC50=3.017±0.486 μM), the combination of these two components results in higher toxicity on A431 cells (0.137±0.012 μM and 0.278±0.022 μM IC50when using serial dilutions or a constant amount of tetrazine 7, respectively). This confirms that doxorubicin is released following the retro Diels-Alder reaction between the trans-cyclooctene of the prodrug and the tetrazine. IC50Values for Doxorubicin (Dox), Prodrug 38 with and Without Activation by Tetrazine 7, and Tetrazine 7 Alone, Determined in A431 Cell Line CompoundIC50(μM)Dox0.020 ± 0.002Prodrug 383.017 ± 0.486Prodrug 38 + tetrazine 7 (1.5 eq.)0.137 ± 0.012Prodrug 38 + tetrazine 7 (10 μM)0.278 ± 0.022Tetrazine 7>100 Example 8 Antibody Masking by Modification with (E)-cyclooct-2-en-1-yl NHS Carbonate 47, and Subsequent Antibody Activation by Reaction with Tetrazine Activator Antibody Conjugation with Minor-(E)-cyclooct-2-en-1-yl NHS Carbonate 47 A solution of CC49 (8 mg/mL, 62.5 μL) in PBS was added with 6.2 μL DMF and the pH was adjusted to 9 with 1 M sodium carbonate buffer. Subsequently, minor-(E)-cyclooct-2-en-1-yl NHS carbonate 47 freshly dissolved in dry DMF was added (5 μg/μL, 40 mol eq. with respect to CC49) and the resulting solution was incubated for 3 hr at room temperature, under gentle shaking and in the dark. After incubation the reaction mixture was diluted to 500 μL with PBS and unreacted 47 was eliminated by means of a Zeba desalting spin column (40 kDa MW cut-off, Pierce) pre-equilibrated with PBS. The concentration of the obtained mAb solution was measured by UV-Vis (Nanodrop) and the purity and integrity of the product were assessed by SDS-PAGE. The conjugation yield was determined with a tetrazine titration. The DOTA-tetrazine derivative 29 was radiolabeled with carrier-added177Lu as previously described (Rossin et al.,Angew Chem Int Ed,2010, 49, 3375-3378). The TCO-modified mAb (25 μg) was reacted with a known excess of177Lu-DOTA-tetrazine in PBS (50 μL). After 10 min incubation at 37° C., the reaction mix was added with non-reducing sample buffer and analyzed by SDS-PAGE. After gel electrophoresis, the radioactivity distribution in each lane was assessed with phosphor imager. The reaction yields between177Lu-DOTA-tetrazine and the CC49-TCO construct was estimated from the intensity of the radioactive mAb band with respect to the total radioactivity in the lane. With this procedure an average of 20 TCO moieties per CC49 molecule was found (50% conjugation yield). CC49 and CC49-TCO(47) Radiolabeling The unmodified CC49 was radiolabeled with125I with the Bolton-Hunter procedure according to the manufacturer instruction. Briefly, ca. 40 MBq sodium [125I]iodide was diluted with 50 μL PBS and added with 1 μL Bolton-Hunter reagent (SHPP, Pierce) solution in DMSO (0.1 μg/μL) and 25 μL chloramine-T (Sigma-Aldrich) solution in PBS (4 mg/mL). The solution was mixed for 10-20 sec, then 5 μL DMF and 100 μL toluene were added. After vortexing, the organic phase containing125I-SHPP was transferred into a glass vial and dried at room temperature under a gentle stream of N2. 30 μg CC49 in PBS (50 μL) were then added to the125I-SHPP coated glass vial and the pH was adjusted to 9 with 1M sodium carbonate buffer pH 9.6. The vial was incubated at room temperature under gentle agitation for ca. 60 min then the125I-mAb labeling yield was evaluated with radio-ITLC (47%). The crude125I-mAb was purified through Zeba Desalting spin columns (40 kDa MW cut-off, Pierce) pre-equilibrated with saline solution and the radiochemical purity of the obtained125I-labeled CC49 was greater than 98%, as determined by radio-ITLC and radio-HPLC. The CC49 carrying 20 TCO(47) moieties per molecules was reacted with DOTA-tetrazine 29 (1 mol eq. with respect to mAb) which was previously radiolabeled with non-carrier-added177Lu as described (Rossin et al.,Angew Chem Int Ed,2010, 49, 3375-3378). After 10 min incubation 91% radiochemical purity for the177Lu-labeled CC49-TCO(47) by radio-HPLC and the reaction mixture was used without further purification. Antibody Activation Experiments In this example we show that by over-modifying CC49 with TCO 47 we can significantly reduce the ability of the mAb to bind its target and that by reacting the over-modified CC49-TCO construct with tetrazine 7 the target binding capability is restored. The mAb re-activation upon reaction with the tetrazine indicates TCO release following the electronic cascade mediated elimination mechanism. The capability of CC49 constructs to bind their target was evaluated by using an immunoreactivity assay modified from a previously described method (Lewis et al., Bioconjug Chem, 2006, 17, 485-492). Briefly, the radiolabeled mAb constructs (1 μg) were reacted with a 20-fold molar excess of bovine submaxillary mucin type I-S (BSM; Sigma-Aldrich) in 1% BSA solution (100 μL). After 10 min incubation at 37° C. the mixtures were analyzed by radio-HPLC using a Superdex-200 column (GE Healthcare Biosciences) eluted with PBS at 0.35 mL/min. In these conditions non-TCO-modified125I-CC49 eluted from the column in a broad peak with a 39 min retention time, shown inFIG.4A. As expected, after incubation with BSM the125I activity eluted from the column in a peak corresponding to a higher MW species (25 min retention time), confirming the binding of125I-CC49 to BSM, 100% immunoreactivity, shown inFIG.4B. When the177Lu-labeled CC49 carrying 20 TCO 47 moieties per molecule was analyzed by radio-HPLC, the mAb eluted from the column in two broad unresolved peaks with 31 min and 36 min retention times, accounting for 43% and 57% of the total mAb-related activity, respectively, shown inFIG.5A. This behavior suggests over-modification of CC49 with TCO groups. In fact, the change of MW after conjugation is relatively small and not likely to cause a 3 min change in retention time (from 39 to 36 min) between CC49 and CC49-TCO. Therefore, the shorter retention in the column is more likely due to conformational changes caused by the 20 TCO moieties attached to the mAb. Also, the broad peak eluting from the column at 31 min is a sign of mAb aggregation. As a consequence, after incubating the177Lu-labeled CC49-TCO with BSM, only a small amount (ca. 20% of the total) of177Lu activity was associated with a high MW species in the radio-chromatogram, shown inFIG.5B. The ca. 20% residual immunoreactivity confirms that the over-modified CC49-TCO(47) has lost its target binding capability. Subsequently, the177Lu-labeled CC49-TCO(47) was reacted with a large excess of tetrazine 7 (500-fold molar excess with respect to TCO) in PBS at 37° C. At various time points (1 hr, 4 hr and 24 hr) an aliquot of the reaction mixture (containing 1 μg mAb) was withdrawn, incubated with BSM and analyzed by radio-HPLC. As short as 1 hr after addition of tetrazine 7, the radio-chromatogram showed the disappearance of the radioactive peak attributed to CC49-TCO aggregates, a significant reduction of the peak at 36 min and the formation of an intense peak due to the formation of a177Lu-CC49-TCO-BSM adduct, where Rt=24 min; 72% of the total mAb-related activity; shown inFIG.5C. A further slight increase in peak area was observed with time (76% after 24 hr incubation of CC49-TCO with tetrazine 7). The rapid increase in CC49 immunoreactivity following retro Diels-Alder cycloaddition between TCO 47 and tetrazine 7 is indicative of TCO release as a result of the electronic cascade mediated elimination mechanism. Example 9 Exemplary General Synthesis Routes and Key Intermediates for the Preparation of TCO Based Triggers The brackets around LDand SPsignify that they are optional. The TTfeatured in this example can optionally be replaced by MM. Example 10 Structures of Exemplary LDMoieties The linkers LDare so-called self-immolative linkers, meaning that upon reaction of the trigger with the activator the linker will degrade via intramolecular reactions thereby releasing the drug DD. Some of the above also contain a SP. Example 11 Structures of Exemplary SPMoieties Note that the maleimide, active ester and bromo acetamide groups are active groups to which targeting moieties TTand masking moieties MM, optionally via further spacers SP, can be coupled. Maleimides and bromo acetamide groups typically react with thiols, while active esters are typically suitable for coupling to primary or secondary amines. Example 12 Structures of TCO Triggers with Depicted Exemplary LDMoieties and which Function Via the Cascade Elimination Mechanism The TTfeatured in this example can optionally be replaced by MM. Example 13 Structures of TCO Triggers with Depicted Exemplary LDand/or SPMoieties and which Function Via the Cascade Elimination Mechanism Trigger conjugated to TTvia amine or thiol of TT. The TTfeatured in this example can optionally be replaced by MM. Example 14 Structures of TCO Triggers with Depicted Exemplary LDand/or SPMoieties and Which Function Via the Cascade Elimination Mechanism Trigger conjugated to TTvia amine or thiol of TT. The TTfeatured in this example can optionally be replaced by MM. Example 15 Structures of Antibody-Drug Conjugates, Which Function Via the Cascade Elimination Mechanism Auristatin E (MMAE) toxin is attached via a self immolative linker LDto a TCO trigger and, in cases via SP, to a targeting antibody or fragment (conjugated through cysteine or lysine residue). Ab=antibody or antibody fragment; q=Ab modification # and is typically between 1 and 10. Example 16 Structures of Antibody-Drug Conjugates, Which Function Via the Cascade Elimination Mechanism Auristatin E (MMAE) toxin is attached to a TCO trigger and via SPto a targeting antibody or fragment (conjugated through cysteine or lysine residue). Ab=antibody or antibody fragment; q=Ab modification # and is typically between 1 and 10. Example 17 Structures of Antibody-Drug Conjugates, Which Function Via the Cascade Elimination Mechanism Maytansine toxin is attached via a self immolative linker LDto a TCO trigger and, in cases via SP, to a targeting antibody or fragment (conjugated through cysteine or lysine residue). Ab=antibody or antibody fragment; q=Ab modification ratio and is typically between 1 and 10. Example 18 Structures of Trigger-Drug Constructs That can be Conjugated to a Targeting Agent TTeg Via an Amine or Thiol Moiety, and Which Function Via the Cascade Elimination Mechanism Auristatin E (MMAE) toxin is attached via a self immolative linker LDto a TCO trigger and, in cases via SP, to a reactive moiety for TTconjugation. Example 19 Structures of Trigger-Drug Constructs That can be Conjugated to a Targeting Agent TTeg Via an Amine or Thiol Moiety, and Which Function Via the Cascade Elimination Mechanism Auristatin E (MMAE) toxin is attached to a TCO trigger and via SPto a reactive moiety for TTconjugation. Example 20 Structures of Trigger-Drug Constructs That can be Conjugated to a Targeting Agent TTeg Via an Amine or Thiol Moiety, and Which Function Via the Cascade Elimination Mechanism Maytansine toxin is attached via a self immolative linker LDto a TCO trigger TRand, in cases via SP, to a reactive moiety for TTconjugation. Example 21 Activation of Tumor Bound CC49-Auristatin E Conjugate CC49 as mAb or mAb fragment binds the non-internalizaling pan-solid tumor marker TAG72. After Prodrug administration, tumor binding and clearance from blood, the Activator is injected. The reaction of the Activator with the TCO trigger in the Prodrug results in release of Auristatin E from CC49 (antibody, or antibody fragment), allowing it to penetrate the cancer cell inside which it has its anticancer action. Example 22 Activation of Tumor-Bound T-Cell Engaging Triabody FIG.6shows the structure of an embodiment of a triabody prodrug, including a tumor-binding moiety, a CD3 T-cell engaging moiety, and a CD28 T-cell co-stimulatory moiety. The anti-CD28 domain can be blocked by a Masking Moiety M.sup.M. After Prodrug administration, tumor binding and clearance from blood, the Activator is injected. The reaction of the Activator with the TCO trigger in the Prodrug results in release of the Masking Moiety from the anti-CD28 domain, enabling CD28 co-stimulation of T-cells, and boosting the T-cell mediated anticancer effect, while avoiding off target toxicity. The triabody comprises a tumor-binding moiety, a CD3 T-cell engaging moiety, and a CD28 T-cell co-stimulatory moiety. As the CD3 and CD28 combined in one molecule will result in unacceptable toxic effect off target, the anti-CD28 domain is blocked by a Masking Moiety MM, a peptide resembling the CD28 binding domain and which has affinity for the anti-CD28 moiety. This peptide is linked through a further peptide or a PEG chain SPto the TCO trigger which is itself conjugated to a site specifically engineered cysteine. After Prodrug administration, tumor binding and clearance from blood, the Activator is injected. The reaction of the Activator with the TCO trigger in the Prodrug results in release of the Masking Moiety from the anti-CD28 domain enabling CD28 co-stimulation of T-cells, boosting the T-cell mediated anticancer effect, while avoiding off target toxicity. | 148,813 |
11857637 | DEFINITIONS The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings. “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3—), ethyl (CH3CH2—), n-propyl (CH3CH2CH2—), isopropyl ((CH3)2CH—), n-butyl (CH3CH2CH2CH2—), isobutyl ((CH3)2CHCH2—), sec-butyl ((CH3)(CH3CH2)CH—), t-butyl ((CH3)3C—), n-pentyl (CH3CH2CH2CH2CH2—), and neopentyl ((CH3)3CCH2—). The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1carbon atom) have been optionally replaced with a heteroatom such as —O—, —N—, —S—, —S(O)n— (where n is 0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-aryl, —SO2-heteroaryl, and —NRaRb, wherein R′ and R″ may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from —O—, —NR10—, —NR10C(O)—, —C(O)NR10— and the like. This term includes, by way of example, methylene (—CH2—), ethylene (—CH2CH2—), n-propylene (—CH2CH2CH2—), iso-propylene (—CH2CH(CH3)—), (—C(CH3)2CH2CH2—), (—C(CH3)2CH2C(O)—), (—C(CH3)2CH2C(O)NH—), (—CH(CH3)CH2—), and the like. “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below. The term “alkane” refers to alkyl group and alkylene group, as defined herein. The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R′NHR″— where R′ is alkyl group as defined herein and R″ is alkylene, alkenylene or alkynylene group as defined herein. The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. “Alkoxy” refers to the group —O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. The term “substituted alkoxy” refers to the groups substituted alkyl-O—, substituted alkenyl-O—, substituted cycloalkyl-O—, substituted cycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein. The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy is defined herein. The term “haloalkoxy” refers to the groups alkyl-O— wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like. The term “haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like. The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers. The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl. “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (—C═CH), and propargyl (—CH2C═CH). The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl, and —SO2-heteroaryl. “Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like. “Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substituted alkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—, substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substituted heteroaryl-C(O)—, heterocyclyl-C(O)—, and substituted heterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)— “Acylamino” refers to the groups —NR20C(O)alkyl, —NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, —NR20C(O)substituted cycloalkyl, —NR20C(O)cycloalkenyl, —NR20C(O)substituted cycloalkenyl, —NR20C(O)alkenyl, —NR20C(O)substituted alkenyl, —NR20C(O)alkynyl, —NR20C(O)substituted alkynyl, —NR20C(O)aryl, —NR20C(O)substituted aryl, —NR20C(O)heteroaryl, —NR20C(O)substituted heteroaryl, —NR20C(O)heterocyclic, and —NR20C(O)substituted heterocyclic, wherein R20is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. “Aminocarbonyl” or the term “aminoacyl” refers to the group —C(O)NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. “Aminocarbonylamino” refers to the group —NR21C(O)NR22R23where R21, R22, and R23are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group. The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein. The term “acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—, aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein. “Aminosulfonyl” refers to the group —SO2NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein. “Sulfonylamino” refers to the group —NR21SO2R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. “Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl, —SO2-heteroaryl and trihalomethyl. “Aryloxy” refers to the group —O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein. “Amino” refers to the group —NH2. The term “substituted amino” refers to the group —NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen. The term “azido” refers to the group —N3. “Carboxyl,” “carboxy” or “carboxylate” refers to —CO2H or salts thereof. “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substituted alkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl, —C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. “(Carboxyl ester)oxy” or “carbonate” refers to the groups —O—C(O)O— alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substituted alkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O— substituted cycloalkenyl, —O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. “Cyano” or “nitrile” refers to the group —CN. “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like. The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl. “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds. The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl. “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond. “Cycloalkoxy” refers to —O-cycloalkyl. “Cycloalkenyloxy” refers to —O-cycloalkenyl. “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo. “Hydroxy” or “hydroxyl” refers to the group —OH. “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl and —SO2-heteroaryl, and trihalomethyl. The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like. “Heteroaryloxy” refers to —O-heteroaryl. “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, —S(O)—, or —SO2— moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein. Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like. Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl, —SO2-heteroaryl, and fused heterocycle. “Heterocyclyloxy” refers to the group —O-heterocyclyl. The term “heterocyclylthio” refers to the group heterocyclic-S—. The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein. The term “hydroxyamino” refers to the group —NHOH. “Nitro” refers to the group —NO2. “Oxo” refers to the atom (═O). “Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2—, phenyl-SO2—, and 4-methylphenyl-SO2—. “Sulfonyloxy” refers to the group —OSO2-alkyl, OSO2-substituted alkyl, OSO2-alkenyl, OSO2-substituted alkenyl, OSO2-cycloalkyl, OSO2-substituted cycloalkyl, OSO2-cycloalkenyl, OSO2-substituted cylcoalkenyl, OSO2-aryl, OSO2-substituted aryl, OSO2-heteroaryl, OSO2-substituted heteroaryl, OSO2-heterocyclic, and OSO2substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein. “Thiol” refers to the group —SH. “Thioxo” or the term “thioketo” refers to the atom (═S). “Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to —S(O)—. The sulfoxide may exist as one or more stereoisomers. The term “substituted thioalkoxy” refers to the group —S-substituted alkyl. The term “thioaryloxy” refers to the group aryl-S— wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein. The term “thioheteroaryloxy” refers to the group heteroaryl-S— wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein. The term “thioheterocyclooxy” refers to the group heterocyclyl-S— wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein. In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below. In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with ═O, ═NR70, ═N—OR70, ═N2or ═S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, —R60, halo, ═O, —OR70, —SR70, —NR80R80, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO2, ═N2, —N3, —SO2R70, —SO2O M+, —SO2OR70, —OSO2R70, —OSO2O−M+, —OSO2OR70, —P(O)(O−)2(M+)2, —P(O)(OR70)O−M+, —P(O)(OR70)2, —C(O)R70, —C(S)R70, —C(NR70)R70, —C(O)O−M+, —C(O)OR70, —C(S)OR70, —C(O)NR80R80, —C(NR70)NR80R80, —OC(O)R70, —OC(S)R70, —OC(O)O−M+, —OC(O)OR70, —OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70CO2−M+, —NR70CO2R70, —NR70C(S)OR70, —NR70C(O)NR80R80, —NR70C(NR70)R70and —NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80's, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of 0, N and S, of which N may have —H or C1-C3alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, —NR80R80is meant to include —NH2, —NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl. In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, —R60, halo, —O−M+, —OR70, —SR70, —S−M+, —NR80R80, trihalomethyl, —CF3, —CN, —OCN, —SCN, —NO, —NO2, —N3, —SO2R70, —SO3−M+, —SO3R70, —OSO2R70, —OSO3−M+, —OSO3R70, —PO3−2(M+)2, —P(O)(OR70)O−M+, —P(O)(OR70)2, —C(O)R70, —C(S)R70, —C(NR70)R70, —CO2−M+, —CO2R70, —C(S)OR70, —C(O)NR80R80, —C(NR70)NR80R80, —OC(O)R70, —OC(S)R70, —OCO2−M+, —OCO2R70, —OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70CO2−M+, —NR70CO2R70, —NR70C(S)OR70, —NR70C(O)NR80R80, —NR70C(NR70)R70and —NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not —O−M+, —OR70, —SR70, or —S−M+. In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, —R60, —O−M+, —OR70, —SR70, —S−M+, —NR80R80, trihalomethyl, —CF3, —CN, —NO, —NO2, —S(O)2R70, —S(O)2O−M+, —S(O)2OR70, —OS(O)2R70, —OS(O)2O−M+, —OS(O)2OR70, —P(O)(O−)2(M+)2, —P(O)(OR70)O−M+, —P(O)(OR70)(OR70), —C(O)R70, —C(S)R70, —C(NR70)R70, —C(O)OR70, —C(S)OR70, —C(O)NR80R80, —C(NR70)NR80R80, —OC(O)R70, —OC(S)R70, —OC(O)OR70, —OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70C(O)OR70, —NR70C(S)OR70, —NR70C(O)NR80R80, —NR70C(NR70)R70and —NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined. In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent. It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—. As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds. The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like. The term “salt thereof” means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate. “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. “Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible. It will be appreciated that the term “or a salt or solvate or stereoisomer thereof” is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound. “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor. “Patient” refers to human and non-human subjects, especially mammalian subjects. The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless specifically indicated otherwise, “polypeptide,” “peptide,” and “protein” can include genetically coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); immunologically tagged proteins; and the like. “Native amino acid sequence” or “parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include a modified amino acid residue. The terms “amino acid analog,” “unnatural amino acid,” and the like may be used interchangeably, and include amino acid-like compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids, and α-amino acids, and the like. The terms “amino acid side chain” or “side chain of an amino acid” and the like may be used to refer to the substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and/or conjugates described herein. The term “carbohydrate” and the like may be used to refer to monomers units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides. The term “carbohydrate derivative” includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates. The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like. An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody. The term “natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies. The term “humanized antibody” or “humanized immunoglobulin” refers to a non-human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a framework region, a constant region or a CDR, for example) that have been substituted with a correspondingly positioned amino acid from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98/45331 and WO 98/45332. In particular embodiments, a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art. The term “chimeric antibodies” refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used. An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, 1991). The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. Throughout the present disclosure, the numbering of the residues in an immunoglobulin heavy chain and in an immunoglobulin light chain is that as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. A “parent Ig polypeptide” is a polypeptide comprising an amino acid sequence which lacks an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions). In the context of an Ig polypeptide, the term “constant region” is well understood in the art, and refers to a C-terminal region of an Ig heavy chain, or an Ig light chain. An Ig heavy chain constant region includes CH1, CH2, and CH3 domains (and CH4 domains, where the heavy chain is a μ or an ε heavy chain). In a native Ig heavy chain, the CH1, CH2, CH3 (and, if present, CH4) domains begin immediately after (C-terminal to) the heavy chain variable (VH) region, and are each from about 100 amino acids to about 130 amino acids in length. In a native Ig light chain, the constant region begins begin immediately after (C-terminal to) the light chain variable (VL) region, and is about 100 amino acids to 120 amino acids in length. As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. TABLE 1CDR DefinitionsKabat1Chothia2MacCallum3VHCDR131-3526-3230-35VHCDR250-6553-5547-58VHCDR395-10296-10193-101VLCDR124-3426-3230-36VLCDR250-5650-5246-55VLCDR389-9791-9689-961Residue numbering follows the nomenclature of Kabat et al., supra2Residue numbering follows the nomenclature of Chothia et al., supra3Residue numbering follows the nomenclature of MacCallum et al., supra By “genetically-encodable” as used in reference to an amino acid sequence of polypeptide, peptide or protein means that the amino acid sequence is composed of amino acid residues that are capable of production by transcription and translation of a nucleic acid encoding the amino acid sequence, where transcription and/or translation may occur in a cell or in a cell-free in vitro transcription/translation system. The term “control sequences” refers to DNA sequences that facilitate expression of an operably linked coding sequence in a particular expression system, e.g. mammalian cell, bacterial cell, cell-free synthesis, etc. The control sequences that are suitable for prokaryote systems, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate the initiation of translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking is accomplished by ligation or through amplification reactions. Synthetic oligonucleotide adaptors or linkers may be used for linking sequences in accordance with conventional practice. The term “expression cassette” as used herein refers to a segment of nucleic acid, usually DNA, that can be inserted into a nucleic acid (e.g., by use of restriction sites compatible with ligation into a construct of interest or by homologous recombination into a construct of interest or into a host cell genome). In general, the nucleic acid segment comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. Expression cassettes can also comprise elements that facilitate expression of a polynucleotide encoding a polypeptide of interest in a host cell, e.g., a mammalian host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like. As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. As used herein, the term “substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated. The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells. By “reactive partner” is meant a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include a cysteine or serine of a sulfatase motif and Formylglycine Generating Enzyme (FGE), which react to form a reaction product of a converted aldehyde tag containing a formylglycine (FGly) in lieu of cysteine or serine in the motif. Other exemplary reactive partners include an aldehyde of an fGly residue of a converted aldehyde tag (e.g., a reactive aldehyde group) and an “aldehyde-reactive reactive partner”, which comprises an aldehyde-reactive group and a moiety of interest, and which reacts to form a reaction product of a modified aldehyde tagged polypeptide having the moiety of interest conjugated to the modified polypeptide through a modified fGly residue. “N-terminus” refers to the terminal amino acid residue of a polypeptide having a free amine group, which amine group in non-N-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide. “C-terminus” refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, which carboxyl group in non-C-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide. By “internal site” as used in referenced to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or at the C-terminus. Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION The present disclosure provides anti-CD25 antibody-maytansine conjugate structures. The disclosure also encompasses methods of production of such conjugates, as well as methods of using the same. Embodiments of each are described in more detail in the sections below. Antibody-Drug Conjugates The present disclosure provides conjugates, e.g., antibody-drug conjugates. By “conjugate” is meant a first moiety (e.g., an antibody) is stably associated with a second moiety (e.g., a drug). For example, a maytansine conjugate includes a maytansine (e.g., a maytansine active agent moiety) stably associated with another moiety (e.g., the antibody). By “stably associated” is meant that a moiety is bound to another moiety or structure under standard conditions. In certain embodiments, the first and second moieties are bound to each other through one or more covalent bonds. In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide conjugated to a second moiety. In certain embodiments, the moiety conjugated to the polypeptide can be any of a variety of moieties of interest such as, but not limited to, a detectable label, a drug, a water-soluble polymer, or a moiety for immobilization of the polypeptide to a membrane or a surface. In certain embodiments, the conjugate is a maytansine conjugate, where a polypeptide is conjugated to a maytansine or a maytansine active agent moiety. “Maytansine”, “maytansine moiety”, “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof. A maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein. The moiety of interest can be conjugated to the polypeptide at any desired site of the polypeptide. Thus, the present disclosure provides, for example, a modified polypeptide having a moiety conjugated at a site at or near the C-terminus of the polypeptide. Other examples include a modified polypeptide having a moiety conjugated at a position at or near the N-terminus of the polypeptide. Examples also include a modified polypeptide having a moiety conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible where the modified polypeptide is conjugated to two or more moieties. In certain embodiments, a conjugate of the present disclosure includes a maytansine conjugated to an amino acid reside of a polypeptide at the α-carbon of an amino acid residue. Stated another way, a maytansine conjugate includes a polypeptide where the side chain of one or more amino acid residues in the polypeptide have been modified to be attached to a maytansine (e.g., attached to a maytansine through a linker as described herein). For example, a maytansine conjugate includes a polypeptide where the α-carbon of one or more amino acid residues in the polypeptide has been modified to be attached to a maytansine (e.g., attached to a maytansine through a linker as described herein). Embodiments of the present disclosure include conjugates where a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 or more moieties. The moieties may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example where a polypeptide is conjugated to a first moiety at a first amino acid residue and conjugated to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to first and second moieties at a first amino acid residue and conjugated to third and fourth moieties at a second amino acid residue, etc. The one or more amino acid residues of the polypeptide that are conjugated to the one or more moieties may be naturally occurring amino acids, unnatural amino acids, or combinations thereof. For instance, the conjugate may include a moiety conjugated to a naturally occurring amino acid residue of the polypeptide. In other instances, the conjugate may include a moiety conjugated to an unnatural amino acid residue of the polypeptide. One or more moieties may be conjugated to the polypeptide at a single natural or unnatural amino acid residue as described above. One or more natural or unnatural amino acid residues in the polypeptide may be conjugated to the moiety or moieties as described herein. For example, two (or more) amino acid residues (e.g., natural or unnatural amino acid residues) in the polypeptide may each be conjugated to one or two moieties, such that multiple sites in the polypeptide are modified. As described herein, a polypeptide may be conjugated to one or more moieties. In certain embodiments, the moiety of interest is a chemical entity, such as a drug or a detectable label. For example, a drug (e.g., maytansine) may be conjugated to the polypeptide, or in other embodiments, a detectable label may be conjugated to the polypeptide. Thus, for instance, embodiments of the present disclosure include, but are not limited to, the following: a conjugate of a polypeptide and a drug; a conjugate of a polypeptide and a detectable label; a conjugate of two or more drugs and a polypeptide; a conjugate of two or more detectable labels and a polypeptide; and the like. In certain embodiments, the polypeptide and the moiety of interest are conjugated through a coupling moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bonded) to the coupling moiety, thus indirectly binding the polypeptide and the moiety of interest (e.g., a drug, such as maytansine) together through the coupling moiety. In some cases, the coupling moiety includes a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound. For instance, a general scheme for coupling a moiety of interest (e.g., a maytansine) to a polypeptide through a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety is shown in the general reaction scheme below. Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl coupling moiety are also referred to herein as a hydrazino-iso-Pictet-Spengler (HIPS) coupling moiety and an aza-hydrazino-iso-Pictet-Spengler (azaHIPS) coupling moiety, respectively. In the reaction scheme above, R is the moiety of interest (e.g., maytansine) that is conjugated to the polypeptide. As shown in the reaction scheme above, a polypeptide that includes a 2-formylglycine residue (fGly) is reacted with a drug (e.g., maytansine) that has been modified to include a coupling moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety) to produce a polypeptide conjugate attached to the coupling moiety, thus attaching the maytansine to the polypeptide through the coupling moiety. As described herein, the moiety can be any of a variety of moieties such as, but not limited to, chemical entity, such as a detectable label, or a drug (e.g., a maytansinoid). R′ and R″ may each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z may be CR11, NR12, N, O or S, where R11and R12are each independently selected from any of the substituents described for R′ and R″ above. Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties may be modified to be attached (e.g., covalently attached) to a linker. As such, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety attached to a drug (e.g., maytansine) through a linker. Various embodiments of the linker that may couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety to the drug (e.g., maytansine) are described in detail herein. In certain embodiments, the polypeptide may be conjugated to a moiety of interest, where the polypeptide is modified before conjugation to the moiety of interest. Modification of the polypeptide may produce a modified polypeptide that contains one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide may be modified at one or more amino acid residues to provide one or more reactive groups suitable for conjugation to the moiety of interest (e.g., a moiety that includes a coupling moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above). For example, the polypeptide may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an “aldehyde tag” or “ald-tag”, which as used herein refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C/S)TPSR) (SEQ ID NO: 154) that has been converted by action of a formylglycine generating enzyme (FGE) to contain a 2-formylglycine residue (referred to herein as “FGly”). The FGly residue generated by an FGE may also be referred to as a “formylglycine”. Stated differently, the term “aldehyde tag” is used herein to refer to an amino acid sequence that includes a “converted” sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to FGly by action of an FGE, e.g., L(FGly)TPSR) (SEQ ID NO: 144). A converted sulfatase motif may be derived from an amino acid sequence that includes an “unconverted” sulfatase motif (i.e., a sulfatase motif in which the cysteine or serine residue has not been converted to FGly by an FGE, but is capable of being converted, e.g., an unconverted sulfatase motif with the sequence: L(C/S)TPSR) (SEQ ID NO: 154). By “conversion” as used in the context of action of a formylglycine generating enzyme (FGE) on a sulfatase motif refers to biochemical modification of a cysteine or serine residue in a sulfatase motif to a formylglycine (FGly) residue (e.g., Cys to FGly, or Ser to FGly). Additional aspects of aldehyde tags and uses thereof in site-specific protein modification are described in U.S. Pat. Nos. 7,985,783 and 8,729,232, the disclosures of each of which are incorporated herein by reference. In some cases, the modified polypeptide containing the FGly residue may be conjugated to the moiety of interest by reaction of the FGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety, as described above). For example, an FGly-containing polypeptide may be contacted with a reactive partner-containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some instances, the reactive partner-containing drug may include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above. For example, a maytansine may be modified to include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety. In some cases, the maytansine is attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl, such as covalently attached to a a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl through a linker, as described in detail herein. In certain embodiments, a conjugate of the present disclosure includes a polypeptide (e.g., an antibody, such as an anti-CD25 antibody) having at least one modified amino acid residue. The modified amino acid residue of the polypeptide may be coupled to a drug (e.g., maytansine) containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above. In certain embodiments, the modified amino acid residue of the polypeptide (e.g., anti-CD25 antibody) may be derived from a cysteine or serine residue that has been converted to an FGly residue as described above. In certain embodiments, the FGly residue is conjugated to a drug containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above to provide a conjugate of the present disclosure where the drug is conjugated to the polypeptide through the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety. As used herein, the term FGly′ refers to the modified amino acid residue of the polypeptide (e.g., anti-CD25 antibody) that is coupled to the moiety of interest (e.g., a drug, such as a maytansine). In certain embodiments, the conjugate includes at least one modified amino acid residue of the formula (I) described herein. For instance, the conjugate may include at least one modified amino acid residue with a side chain of the formula (I): whereinZ is CR4or N;R1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;R2and R3are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R2and R3are optionally cyclically linked to form a 5 or 6-membered heterocyclyl;each R4is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;L is a linker comprising -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-, wherein a, b, c and d are each independently 0 or 1, where the sum of a, b, c and d is 1 to 4;T1, T2, T3and T4are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, (EDA)w, (PEG)n, (AA)p, —(CR13OH)h—, piperidin-4-amino (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12;V1, V2, V3and V4are each independently selected from the group consisting of a covalent bond, —CO—, —NR15—, —NR15(CH2)q—, —NR15(C6H4)—, —CONR15—, —NR15CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO2—, —SO2NR15—, —NR15SO2— and —P(O)OH—, wherein q is an integer from 1 to 6;each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl;each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; W1is a maytansinoid; andW2is an anti-CD25 antibody. In certain embodiments, Z is CR4or N. In certain embodiments, Z is CR4. In certain embodiments, Z is N. In certain embodiments, R1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R1is hydrogen. In certain embodiments, R1is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R1is methyl. In certain embodiments, R1is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R1is alkynyl or substituted alkynyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R1is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R1is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R1is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-5substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3_s cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R1is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl. In certain embodiments, R2and R3are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R2and R3are optionally cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R2is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R2is hydrogen. In certain embodiments, R2is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R2is methyl. In certain embodiments, R2is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R2is alkynyl or substituted alkynyl. In certain embodiments, R2is alkoxy or substituted alkoxy. In certain embodiments, R2is amino or substituted amino. In certain embodiments, R2is carboxyl or carboxyl ester. In certain embodiments, R2is acyl or acyloxy. In certain embodiments, R2is acyl amino or amino acyl. In certain embodiments, R2is alkylamide or substituted alkylamide. In certain embodiments, R2is sulfonyl. In certain embodiments, R2is thioalkoxy or substituted thioalkoxy. In certain embodiments, R2is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R2is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R2is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R2is heterocyclyl or substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl. In certain embodiments, R3is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R3is hydrogen. In certain embodiments, R3is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R3is methyl. In certain embodiments, R3is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R3is alkynyl or substituted alkynyl. In certain embodiments, R3is alkoxy or substituted alkoxy. In certain embodiments, R3is amino or substituted amino. In certain embodiments, R3is carboxyl or carboxyl ester. In certain embodiments, R3is acyl or acyloxy. In certain embodiments, R3is acyl amino or amino acyl. In certain embodiments, R3is alkylamide or substituted alkylamide. In certain embodiments, R3is sulfonyl. In certain embodiments, R3is thioalkoxy or substituted thioalkoxy. In certain embodiments, R3is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R3is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R3is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R3is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl. In certain embodiments, R2and R3are optionally cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 6-membered heterocyclyl. In certain embodiments, each R4is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. The various possibilities for each R4are described in more detail as follows. In certain embodiments, R4is hydrogen. In certain embodiments, each R4is hydrogen. In certain embodiments, R4is halogen, such as F, Cl, Br or I. In certain embodiments, R4is F. In certain embodiments, R4is Cl. In certain embodiments, R4is Br. In certain embodiments, R4is I. In certain embodiments, R4is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R4is methyl. In certain embodiments, R4is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R4is alkynyl or substituted alkynyl. In certain embodiments, R4is alkoxy or substituted alkoxy. In certain embodiments, R4is amino or substituted amino. In certain embodiments, R4is carboxyl or carboxyl ester. In certain embodiments, R4is acyl or acyloxy. In certain embodiments, R4is acyl amino or amino acyl. In certain embodiments, R4is alkylamide or substituted alkylamide. In certain embodiments, R4is sulfonyl. In certain embodiments, R4is thioalkoxy or substituted thioalkoxy. In certain embodiments, R4is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, R4is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R4is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5cycloalkyl or C3-8substituted cycloalkyl. In certain embodiments, R4is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl. In certain embodiments, W1is a maytansinoid. Further description of the maytansinoid is found in the disclosure herein. In certain embodiments, W2is an anti-CD25 antibody. Further description of anti-CD25 antibodies that find use in the subject conjugates is found in the disclosure herein. In certain embodiments, the compounds of formula (I) include a linker, L. The linker may be utilized to bind a coupling moiety to one or more moieties of interest and/or one or more polypeptides. In some embodiments, the linker binds a coupling moiety to either a polypeptide or a chemical entity. The linker may be bound (e.g., covalently bonded) to the coupling moiety (e.g., as described herein) at any convenient position. For example, the linker may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety to a drug (e.g., a maytansine). The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety may be used to conjugate the linker (and thus the drug, e.g., maytansine) to a polypeptide, such as an anti-CD25 antibody. For example, the coupling moiety may be used to conjugate the linker (and thus the drug, e.g., maytansine) to a modified amino acid residue of the polypeptide, such as an FGly reside of an anti-CD25 antibody. In certain embodiments, L attaches the coupling moiety to W1, and thus the coupling moiety is indirectly bonded to W1through the linker L. As described above, W1is a maytansinoid, and thus L attaches the coupling moiety to a maytansinoid, e.g., the coupling moiety is indirectly bonded to the maytansinoid through the linker, L. Any convenient linkers may be utilized in the subject conjugates and compounds. In certain embodiments, L includes a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, L includes an alkyl or substituted alkyl group. In certain embodiments, L includes an alkenyl or substituted alkenyl group. In certain embodiments, L includes an alkynyl or substituted alkynyl group. In certain embodiments, L includes an alkoxy or substituted alkoxy group. In certain embodiments, L includes an amino or substituted amino group. In certain embodiments, L includes a carboxyl or carboxyl ester group. In certain embodiments, L includes an acyl amino group. In certain embodiments, L includes an alkylamide or substituted alkylamide group. In certain embodiments, L includes an aryl or substituted aryl group. In certain embodiments, L includes a heteroaryl or substituted heteroaryl group. In certain embodiments, L includes a cycloalkyl or substituted cycloalkyl group. In certain embodiments, L includes a heterocyclyl or substituted heterocyclyl group. In certain embodiments, L includes a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is a polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below. In some embodiments, L is a linker described by the formula -(L1)a-(L2)b-(L3)c-(L4)a-, wherein L1, L2, L3and L4are each independently a linker unit, and a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4. In certain embodiments, the sum of a, b, c and d is 1. In certain embodiments, the sum of a, b, c and d is 2. In certain embodiments, the sum of a, b, c and d is 3. In certain embodiments, the sum of a, b, c and d is 4. In certain embodiments, a, b, c and d are each 1. In certain embodiments, a, b and c are each 1 and d is 0. In certain embodiments, a and b are each 1 and c and d are each 0. In certain embodiments, a is 1 and b, c and d are each 0. In certain embodiments, L1is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, L2, if present, is attached to W1. In certain embodiments, L3, if present, is attached to W1. In certain embodiments, L4, if present, is attached to W1. Any convenient linker units may be utilized in the subject linkers. Linker units of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L1, L2, L3and L4(if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine). In some embodiments, L1(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L1comprises a polyethylene glycol. In some embodiments, L1comprises a modified polyethylene glycol. In some embodiments, Li comprises an amino acid residue. In some embodiments, L1comprises an alkyl group or a substituted alkyl. In some embodiments, L1comprises an aryl group or a substituted aryl group. In some embodiments, L1comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L2(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L2comprises a polyethylene glycol. In some embodiments, L2comprises a modified polyethylene glycol. In some embodiments, L2comprises an amino acid residue. In some embodiments, L2comprises an alkyl group or a substituted alkyl. In some embodiments, L2comprises an aryl group or a substituted aryl group. In some embodiments, L2comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L3(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L3comprises a polyethylene glycol. In some embodiments, L3comprises a modified polyethylene glycol. In some embodiments, L3comprises an amino acid residue. In some embodiments, L3comprises an alkyl group or a substituted alkyl. In some embodiments, L3comprises an aryl group or a substituted aryl group. In some embodiments, L3comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L4(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L4comprises a polyethylene glycol. In some embodiments, L4comprises a modified polyethylene glycol. In some embodiments, L4comprises an amino acid residue. In some embodiments, L4comprises an alkyl group or a substituted alkyl. In some embodiments, L4comprises an aryl group or a substituted aryl group. In some embodiments, L4comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L is a linker comprising -(L1)a-(L2)b-(L3)c-(L4)d-, where:(L1)a- is -(T1-V1)a-;(L2)b- is -(T2-V2)b-;(L3)c- is -(T3-V3)c-; and(L4)d- is -(T4-V4)d-,wherein T1, T2, T3and T4, if present, are tether groups;V1, V2, V3and V4, if present, are covalent bonds or linking functional groups; anda, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4. As described above, in certain embodiments, L1is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). As such, in certain embodiments, T1is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, V1is attached to W1(the maytansinoid). In certain embodiments, L2, if present, is attached to W1. As such, in certain embodiments, T2, if present, is attached to W1, or V2, if present, is attached to W1. In certain embodiments, L3, if present, is attached to W1. As such, in certain embodiments, T3, if present, is attached to W1, or V3, if present, is attached to W1. In certain embodiments, L4, if present, is attached to W1. As such, in certain embodiments, T4, if present, is attached to W1, or V4, if present, is attached to W1. Regarding the tether groups, T1, T2, T3and T4, any convenient tether groups may be utilized in the subject linkers. In some embodiments, T1, T2, T3and T4each comprise one or more groups independently selected from a (C1-C12)alkyl, a substituted (C1-C12)alkyl, an (EDA)w, (PEG)n, (AA)p, —(CR13OH)h—, piperidin-4-amino (4AP), an acetal group, a disulfide, a hydrazine, and an ester, where w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12. In certain embodiments, when the sum of a, b, c and d is 2 and one of T1-V1, T2-V2, T3-V3, or T4-V4is (PEG)n-CO, then n is not 6. For example, in some instances, the linker may have the following structure: where n is not 6. In certain embodiments, when the sum of a, b, c and d is 2 and one of T1-V1, T2-V2, T3-V3, or T4-V4is (C1-C12)alkyl-NR15, then (C1-C12)alkyl is not a C5-alkyl. For example, in some instances, the linker may have the following structure: where g is not 4. In certain embodiments, the tether group (e.g., T1, T2, T3and/or T4) includes a (C1-C12)alkyl or a substituted (C1-C12)alkyl. In certain embodiments, (C1-C12)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, (C1-C12)alkyl may be an alkyl or substituted alkyl, such as C1-C12alkyl, or C1-C10alkyl, or C1-C6alkyl, or C1-C3alkyl. In some instances, (C1-C12)alkyl is a C2-alkyl. For example, (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12alkylene, or C1-C10alkylene, or C1-C6alkylene, or C1-C3alkylene. In some instances, (C1-C12)alkyl is a C2-alkylene. In certain embodiments, substituted (C1-C12)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C1-C12alkyl, or substituted C1-C10alkyl, or substituted C1-C6alkyl, or substituted C1-C3alkyl. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkyl. For example, substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C1-C12alkylene, or substituted C1-C10alkylene, or substituted C1-C6alkylene, or substituted C1-C3alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkylene. In certain embodiments, the tether group (e.g., T1, T2, T3and/or T4) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether. In certain embodiments, (EDA)wincludes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6). The linked ethylene diamine (EDA) moieties may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the EDA moiety is described by the structure: where y is an integer from 1 to 6, r is 0 or 1, and each R12is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5 or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl. In certain embodiments, any two adjacent R12groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R12groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R12groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH). In certain embodiments, the tether group includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidin-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the 4AP moiety is described by the structure: where R12is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R12is a polyethylene glycol moiety. In certain embodiments, R12is a carboxy modified polyethylene glycol. In certain embodiments, R12includes a polyethylene glycol moiety described by the formula: (PEG)k, which may be represented by the structure: where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2. In certain embodiments, R17is selected from OH, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R17is COOH. In certain embodiments, a tether group (e.g., T1, T2, T3and/or T4) includes (PEG)n, where (PEG)nis a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG)nis described by the structure: where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, n is 2. In some instances, n is 3. In some instances, n is 6. In some instances, n is 12. In certain embodiments, a tether group (e.g., T1, T2, T3and/or T4) includes (AA)p, where AA is an amino acid residue. Any convenient amino acids may be utilized. Amino acids of interest include but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as a non-naturally occurring alpha-amino acid or a non-naturally occurring beta-amino acid, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, a tether group (e.g., T1, T2, T3and/or T4) includes a moiety described by the formula —(CR13OH)h—, where h is 0 or n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R13is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R13is hydrogen. In certain embodiments, R13is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R13is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R13is alkynyl or substituted alkynyl. In certain embodiments, R13is alkoxy or substituted alkoxy. In certain embodiments, R13is amino or substituted amino. In certain embodiments, R13is carboxyl or carboxyl ester. In certain embodiments, R13is acyl or acyloxy. In certain embodiments, R13is acyl amino or amino acyl. In certain embodiments, R13is alkylamide or substituted alkylamide. In certain embodiments, R13is sulfonyl. In certain embodiments, R13is thioalkoxy or substituted thioalkoxy. In certain embodiments, R13is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R13is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R13is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R13is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl. In certain embodiments, R13is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13. Regarding the linking functional groups, V1, V2, V3and V4, any convenient linking functional groups may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like. In some embodiments, V1, V2, V3and V4are each independently selected from a covalent bond, —CO—, —NR15—, —NR15(CH2)q—, —NR15(C6H4)—, —CONR15—, —NR15CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO2—, —SO2NR15—, —NR15SO2— and —P(O)OH—, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In some embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. The various possibilities for each R15are described in more detail as follows. In certain embodiments, R15is hydrogen. In certain embodiments, each R15is hydrogen. In certain embodiments, R15is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R15is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R15is alkynyl or substituted alkynyl. In certain embodiments, R15is alkoxy or substituted alkoxy. In certain embodiments, R15is amino or substituted amino. In certain embodiments, R15is carboxyl or carboxyl ester. In certain embodiments, R15is acyl or acyloxy. In certain embodiments, R15is acyl amino or amino acyl. In certain embodiments, R15is alkylamide or substituted alkylamide. In certain embodiments, R15is sulfonyl. In certain embodiments, R15is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R15is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R15is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R15is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5heterocyclyl or C3-8substituted heterocyclyl. In certain embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, the hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents are as described above for R15. In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes an ester. As described above, in some embodiments, L is a linker comprising -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-, where a, b, c and d are each independently 0 or 1, where the sum of a, b, c and dis 1 to 4. In some embodiments, in the subject linker:T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;T2, T3and T4are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, (EDA)w, (PEG)n, (AA)p, —(CR13OH)h—, 4-amino-piperidine (4AP), an acetal group, a disulfide, a hydrazine, and an ester; andV1, V2, V3and V4are each independently selected from a covalent bond, —CO—, —NR15—, —NR15(CH2)q—, —NR15(C6H4)—, —CONR15—, —NR15CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO2—, —SO2NR15—, —NR15SO2— and —P(O)OH—, wherein q is an integer from 1 to 6; wherein:(PEG)nis n where n is an integer from 1 to 30;EDA is an ethylene diamine moiety having the following structure: where y is an integer from 1 to 6 and r is 0 or 1;4-amino-piperidine (4AP) is AA is an amino acid residue, where p is an integer from 1 to 20; andeach R15and R12is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; andR13is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In certain embodiments, T1, T2, T3and T4and V1, V2, V3and V4are selected from the following table, e.g., one row of the following table: TABLE 2T1V1T2V2T3V3T4V4(C1-C12)alkyl—CONR15—(PEG)n—CO—————(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—CO———(C1-C12)alkyl—CO—(AA)p—————(C1-C12)alkyl—CONR15—(PEG)n—NR15—————(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—NR15———(C1-C12)alkyl—CO—(EDA)w—CO—————(C1-C12)alkyl—CONR15—(C1-C12)alkyl—NR15—————(C1-C12)alkyl—CONR15—(PEG)n—CO—(EDA)w———(C1-C12)alkyl—CO—(EDA)w—————(C1-C12)alkyl—CO—(EDA)w—CO—(CR13OH)h—CONR15—(C1-C12)alkyl—CO—(C1-C12)alkyl—CO—(AA)p—NR15—(C1-C12)alkyl—CO———(C1-C12)alkyl—CONR15—(PEG)n—CO—(AA)p———(C1-C12)alkyl—CO—(EDA)w—CO—(CR13OH)h—CO—(AA)p—(C1-C12)alkyl—CO—(AA)p—NR15—(C1-C12)alkyl—CO—(AA)p—(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—CO—(AA)p—(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—SO2—(AA)p—(C1-C12)alkyl—CO—(EDA)w—CO—(CR13OH)h—CONR15—(PEG)n—CO—(C1-C12)alkyl—CO—(CR13OH)h—CO—————(C1-C12)alkyl—CONR15—substituted (C1-—NR15—(PEG)n—CO———C12)alkyl(C1-C12)alkyl—SO2—(C1-C12)alkyl—CO—————(C1-C12)alkyl—CONR15—(C1-C12)alkyl—(CR13OH)h—CONR15———(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—CO—(AA)p—NR15—(C1-C12)alkyl—CO—(AA)p—NR15—(PEG)n—P(O)OH—(AA)p—(C1-C12)alkyl—CO—(EDA)w—(AA)p———(C1-C12)alkyl—CONR15—(C1-C12)alkyl—NR15———CO———(C1-C12)alkyl—CONR15—(C1-C12)alkyl—NR15———CO—(C1-C12)alkyl—NR15—(C1-C12)alkyl—CO—4AP—CO—(C1-C12)alkyl—CO—(AA)p—(C1-C12)alkyl—CO—4AP—CO—(C1-C12)alkyl—CO——— In certain embodiments, L is a linker comprising -(L1)a-(L2)b-(L3)c-(L4)d-, where -(Li)a- is -(T1-V1)a-; -(L2)b- is -(T2-V2)b-; -(L3)c- is -(T3-V3)c-; and -(L4)d- is -(T4-V4)d-. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (PEG)n, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is (CR13OH)h, V3is —CONR15—, T4is (C1-C12)alkyl and V4is —CO—. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (C1-C12)alkyl, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (PEG)n, V2is —CO—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is absent, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (PEG)n, V2is —NR15—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (PEG)n, V3is —NR15—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (C1-C12)alkyl, V2is —NR15—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (PEG)n, V2is —CO—, T3is (EDA)w, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is absent, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (PEG)n, V2is —CO—, T3is (AA)p, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is (CR13OH)h, V3is —CO—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (C1-C12)alkyl, V3is —CO—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (PEG)n, V3is —CO—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR11—, T3is (PEG)n, V3is —SO2—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is (CR13OH)h, V3is —CONR15—, T4is (PEG)nand V4is —CO—. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (CR13OH)h, V2is —CO—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is substituted (C1-C12)alkyl, V2is —NR15—, T3is (PEG)n, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —SO2—, T2is (C1-C12)alkyl, V2is —CO—, T3is absent, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (C1-C12)alkyl, V2is absent, T3is (CR13OH)h, V3is —CONR15—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (PEG)n, V3is —CO—, T4is (AA)pand V4is —NR15—. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (AA)p, V2is —NR15—, T3is (PEG)n, V3is —P(O)OH—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is absent, T3is (AA)p, V3is absent, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is (CR13OH)h, V3is —CONR15—, T4is (C1-C12)alkyl and V4is —CO(AA)p-. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (C1-C12)alkyl, V2is —NR15—, T3is absent, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CONR15—, T2is (C1-C12)alkyl, V2is —NR15—, T3is absent, V3is —CO—, T4is (C1-C12)alkyl and V4is —NR15—. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is (EDA)w, V2is —CO—, T3is (CR13OH)h, V3is —CONR15—, T4is (PEG)nand V4is —CO(AA)p-. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is 4AP, V2is —CO—, T3is (C1-C12)alkyl, V3is —CO—, T4is (AA)pand V4is absent. In certain embodiments, T1is (C1-C12)alkyl, V1is —CO—, T2is 4AP, V2is —CO—, T3is (C1-C12)alkyl, V3is —CO—, T4is absent and V4is absent. In certain embodiments, the linker is described by one of the following structures: In certain embodiments of the linker structures depicted above, each f is independently 0 or an integer from 1 to 12; each y is independently 0 or an integer from 1 to 20; each n is independently 0 or an integer from 1 to 30; each p is independently 0 or an integer from 1 to 20; each h is independently 0 or an integer from 1 to 12; each R is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and each R′ is independently H, a sidechain of an amino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments of the linker structures depicted above, each f is independently 0, 1, 2, 3, 4, 5 or 6; each y is independently 0, 1, 2, 3, 4, 5 or 6; each n is independently 0, 1, 2, 3, 4, 5 or 6; each p is independently 0, 1, 2, 3, 4, 5 or 6; and each h is independently 0, 1, 2, 3, 4, 5 or 6. In certain embodiments of the linker structures depicted above, each R is independently H, methyl or —(CH2)m—OH where m is 1, 2, 3 or 4 (e.g., 2). In certain embodiments of the linker, L, T1is (C1-C12)alkyl, V1is —CO—, T2is 4AP, V2is —CO—, T3is (C1-C12)alkyl, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is ethylene, V1is —CO—, T2is 4AP, V2is —CO—, T3is ethylene, V3is —CO—, T4is absent and V4is absent. In certain embodiments, T1is ethylene, V1is —CO—, T2is 4AP, V2is —CO—, T3is ethylene, V3is —CO—, T4is absent and V4is absent, where T2(e.g., 4AP) has the following structure: whereinR12is a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol). In certain embodiments, the linker, L, includes the following structure: whereineach f is independently an integer from 1 to 12; andn is an integer from 1 to 30. In certain embodiments, f is 1. In certain embodiments, f is 2. In certain embodiments, one f is 2 and one f is 1. In certain embodiments, n is 1. In certain embodiments, the left-hand side of the above linker structure is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety, and the right-hand side of the above linker structure is attached to a maytansine. Any of the chemical entities, linkers and coupling moieties set forth in the structures above may be adapted for use in the subject compounds and conjugates. Additional disclosure related to hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl compounds and methods for producing a conjugate is found in U.S. Application Publication No. 2014/0141025, filed Mar. 11, 2013, and U.S. Application Publication No. 2015/0157736, filed Nov. 26, 2014, the disclosures of each of which are incorporated herein by reference. Anti-CD25 Antibodies As noted above, a subject conjugate can comprise, as substituent W2an anti-CD25 antibody, where the anti-CD25 antibody has been modified to include a 2-formylglycine (FGly) residue. As used herein, amino acids may be referred to by their standard name, their standard three letter abbreviation and/or their standard one letter abbreviation, such as: Alanine or Ala or A; Cysteine or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P; Glutamine or Gln or Q; Arginine or Arg or R; Serine or Ser or S; Threonine or Thr or T; Valine or Val or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y. In some cases, a suitable anti-CD25 antibody specifically binds a CD25 polypeptide, where the epitope comprises amino acid residues within a CD25 antigen. In some embodiments, the anti-CD25 antibody binds an epitope within amino acids 101-137, an epitope within amino acids 106-132, an epitope within amino acids 111-127, an epitope comprising amino acids 116-122, or an epitope consisting of amino acids 116-122, of a CD25 amino acid sequence depicted in Table 3 below. TABLE 3Human CD25 Amino Acid SequenceHuman CD25 AminoELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGAcid SequenceFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSAT(SEQ ID NO: 1)RNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI The CD25 epitope can be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 200 amino acids to about 251 amino acids of the human CD25 amino acid sequence depicted in Table 3. A “CD25 antigen” or “CD25 polypeptide” can comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 200 amino acids to about 251 amino acids of the human CD25 amino acid sequence depicted in Table 3. In some cases, a suitable anti-CD25 antibody exhibits high affinity binding to CD25. For example, in some cases, a suitable anti-CD25 antibody binds to CD25 with an affinity of at least about 10−7M, at least about 10−8M, at least about 10−9M, at least about 10−10M, at least about 10−11M, or at least about 10−12M, or greater than 10−12M. In some cases, a suitable anti-CD25 antibody binds to an epitope present on CD25 with an affinity of from about 10−7M to about 10−8M, from about 10−8M to about 10−9M, from about 10−9M to about 10−10M, from about 10−10M to about 10−11M, or from about 10−11M to about 10−12M, or greater than 10−12M. In some cases, a suitable anti-CD25 antibody competes for binding to an epitope within CD25 with a second anti-CD25 antibody (e.g., basiliximab or daclizumab) and/or binds to the same epitope within CD25, as a second anti-CD25 antibody (e.g., basiliximab or daclizumab). In some cases, an anti-CD25 antibody that competes for binding to an epitope within CD25 with a second anti-CD25 antibody also binds to the same epitope as the second anti-CD25 antibody (e.g., basiliximab or daclizumab). In some cases, an anti-CD25 antibody that competes for binding to an epitope within CD25 with a second anti-CD25 antibody binds to an epitope that is overlapping with the epitope bound by the second anti-CD25 antibody (e.g., basiliximab or daclizumab). In some cases, the anti-CD25 antibody is humanized. In some embodiments, the anti-CD25 antibody is an IgG1 antibody. For example, in certain aspects, the anti-CD25 antibody is an IgG1 kappa antibody. In certain aspects, the anti-CD25 antibody competes with interleukin-2 (IL-2) for binding to CD25. In some embodiments, the anti-CD25 antibody comprises one, two, three, four, five, or all six complementarity determining regions (CDRs) of the anti-CD25 antibody basiliximab. In certain aspects, the anti-CD25 antibody comprises one, two, three, four, five, or all six complementarity determining regions (CDRs) of the anti-CD25 antibody daclizumab. In certain aspects, the anti-CD25 antibody is a FGly′-containing antibody based on basiliximab or daclizumab. For example, in some embodiments, the antibody is a derivative of daclizumab, where the difference between daclizumab and the derivative is the presence of one or more FGly′ residues (and optionally, the associated FGE recognition sequence amino acids) in the derivative. Provided in Table 4 are nucleic acid and amino acid sequences for an example daclizumab-based antibody according to one embodiment. In the amino acid sequences in Table 4, variable regions are underlined and CDRs are shown in bold. In this example daclizumab-based antibody, the italicized residues at the C-terminus of the heavy chain replace a lysine residue at the C-terminus of a standard IgG1 heavy chain. The underlined residues (LCTPSR) (SEQ ID NO: 16) among the italicized residues constitute the aldehyde tag, where the C is converted to an FGly residue by FGE upon expression of the heavy chain. The non-underlined residues among the italicized residues are additional residues that are different from a standard IgG1 heavy chain sequence. TABLE 4Example daclizumab-based antibodyLight chain aminoDIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPacid sequenceKLLIYTTSNLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYC(SEQ ID NO: 2)HQRSTYPLTFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHeavy chain aminoQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPacid sequenceGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMEL(SEQ ID NO: 3)SSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGSLight chain-encodingGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTDNA sequenceGTAGGAGACAGAGTCACCATCACTTGCAGCGCCAGCAGCA(SEQ ID NO: 4)GTATTAGTTACATGCACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACCACCTCCAACTTGGCCAGTGGGGTCCCAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCACCAGAGGAGCACCTATCCCCTGACTTTCGGCCAGGGGACCAAGGTGGAGGTGAAACGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGAHeavy chain-encodingCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCDNA sequenceCTGGGAGCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATAC(SEQ ID NO: 5)ACCTTCACCAGCTACAGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGATACATCAACCCTAGTACCGGTTACACAGAGTACAACCAGAAGTTCAAGGACAAGGCCACCATCACCGCCGACGAGTCCACGAACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCGGAGGAGTGTTCGACTACTGGGGCCAGGGAACCCTGGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCAAGAGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGTCCTTATGTACCCCTTCTAGAGGATCCTGATGA In some embodiments, a suitable anti-CD25 antibody can induce apoptosis in a cell that expresses CD25 on its cell surface. An anti-CD25 antibody suitable for use in a subject conjugate will in some cases inhibit the proliferation of human tumor cells that express on their surface (e.g., overexpress) CD25, where the inhibition occurs in vitro, in vivo, or both in vitro and in vivo. For example, in some cases, an anti-CD25 antibody suitable for use in a subject conjugate inhibits proliferation of human tumor cells that express on their surface (e.g., overexpress) CD25 by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80%, e.g., by at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%. In some embodiments, the anti-CD25 antibody competes for binding to CD25 (e.g., an epitope of CD25 comprising amino acid residues within a CD25 antigen (e.g., an epitope within amino acids 101-137, an epitope within amino acids 106-132, an epitope within amino acids 111-127, an epitope comprising amino acids 116-122, or an epitope consisting of amino acids 116-122, of a CD25 amino acid sequence depicted in Table 3)) with an antibody comprising a heavy chain complementarity determining region (CDR) selected from SYRMH (daclizumab VH CDR1; SEQ ID NO:6), YINPSTGYTEYNQKFKD (daclizumab VH CDR2; SEQ ID NO:7), GGGVFDY (daclizumab VH CDR3; SEQ ID NO:8), and any combination thereof. In some cases, the anti-CD25 antibody is humanized. In certain aspects, the anti-CD25 antibody competes for binding to CD25 (e.g., an epitope of CD25 comprising amino acid residues within a CD25 antigen (e.g., an epitope within amino acids 101-137, an epitope within amino acids 106-132, an epitope within amino acids 111-127, an epitope comprising amino acids 116-122, or an epitope consisting of amino acids 116-122, of a CD25 amino acid sequence depicted in Table 3)) with an antibody comprising a light-chain CDR selected from SASSSISYMH (daclizumab VL CDR1; SEQ ID NO:9), YTSILHS (daclizumab VL CDR2; SEQ ID NO:10), and QQGNTLPWT (daclizumab VL CDR3; SEQ ID NO:11), and any combination thereof. In some cases, the anti-CD25 antibody is humanized. In some embodiments, the anti-CD25 antibody competes for binding to CD25 (e.g., an epitope of CD25 comprising amino acid residues within a CD25 antigen (e.g., an epitope within amino acids 101-137, an epitope within amino acids 106-132, an epitope within amino acids 111-127, an epitope comprising amino acids 116-122, or an epitope consisting of amino acids 116-122, of a CD25 amino acid sequence depicted in Table 3)) with an antibody comprising the following six CDRs: SYRMH (daclizumab VH CDR1; SEQ ID NO:6), YINPSTGYTEYNQKFKD (daclizumab VH CDR2; SEQ ID NO:7), GGGVFDY (daclizumab VH CDR3; SEQ ID NO:8), SASSSISYMH (daclizumab VL CDR1; SEQ ID NO:9), YTSILHS (daclizumab VL CDR2; SEQ ID NO:10), and QQGNTLPWT (daclizumab VL CDR3; SEQ ID NO:11). In some cases, the anti-CD25 antibody is humanized. In some embodiments, the anti-CD25 antibody comprises the following six CDRs: SYRMH (daclizumab VH CDR1; SEQ ID NO:6), YINPSTGYTEYNQKFKD (daclizumab VH CDR2; SEQ ID NO:7), GGGVFDY (daclizumab VH CDR3; SEQ ID NO:8//), SASSSISYMH (daclizumab VL CDR1; SEQ ID NO:9), YTSILHS (daclizumab VL CDR2; SEQ ID NO:10), and QQGNTLPWT (daclizumab VL CDR3; SEQ ID NO:11). In some cases, the anti-CD25 antibody is humanized. In certain aspects, the anti-CD25 antibody comprises VH CDRs present in an anti-CD25 VH region comprising the following amino acid sequence: (daclizumab VH, SEQ ID NO: 12)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSS. In some embodiments, the anti-CD25 antibody comprises VL CDRs present in an anti-CD25 VL region comprising the following amino acid sequence: (daclizumab VL, SEQ ID NO: 13/DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVK. In certain aspects, the anti-CD25 antibody comprises VH CDRs present in (daclizumab VH, SEQ ID NO: 12)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSS and VL CDRs present in DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPAR FSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVK (daclizumab VL, SEQ ID NO:13). Modified Constant Region Sequences As noted above, the amino acid sequence of an anti-CD25 antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (FGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as an FGE-modification site. Sulfatase Motifs A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length. Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acid residues in length. In certain embodiments, polypeptides of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide includes a modification (insertion, addition, deletion, and/or substitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. Where an amino acid sequence native to the polypeptide (e.g., anti-CD25 antibody) contains one or more residues of the desired sulfatase motif, the total number of modifications of residues can be reduced, e.g., by site-specification modification (insertion, addition, deletion, substitution/replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif. In certain embodiments, the extent of modification of the native amino acid sequence of the target anti-CD25 polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target anti-CD25 polypeptide may minimize the impact such modifications may have upon anti-CD25 function and/or structure. It should be noted that while aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a “consensus sulfatase motif”), it will be readily appreciated that longer aldehyde tags are both contemplated and encompassed by the present disclosure and can find use in the compositions and methods of the present disclosure. Aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues, or can be longer and comprise a minimal sulfatase motif which can be flanked at the N- and/or C-terminal sides of the motif by additional amino acid residues. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a native, wild-type Ig heavy chain. An aldehyde tag can be present within a CH1 domain of an Ig heavy chain. An aldehyde tag can be present within a CH2domain of an Ig heavy chain. An aldehyde tag can be present within a CH3domain of an Ig heavy chain. An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region. In certain embodiments, the sulfatase motif used may be described by the formula: X1Z10X2Z20X3Z30(I′) whereZ10is cysteine or serine (which can also be represented by (C/S));Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; andX2and X3independently can be any amino acid, though usually an aliphatic amino acid, a polar, uncharged amino acid, or a sulfur containing amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. The amino acid sequence of an anti-CD25 heavy and/or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X1Z10X2Z20X3Z30, whereZ10is cysteine or serine;Z20is a proline or alanine residue;Z30is an aliphatic amino acid or a basic amino acid;X1is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X1is present;X2and X3are each independently any amino acid,where the sequence is within or adjacent a solvent-accessible loop region of the Ig constant region, and wherein the sequence is not at the C-terminus of the Ig heavy chain. The sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde tagged polypeptide is expressed or an FGE which is to be contacted with the aldehyde tagged polypeptide in a cell-free in vitro method. For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including a human FGE), the sulfatase motif can be of the formula: X1CX2PX3Z30(I″) whereX1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present;X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V or G; andZ30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I. Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO:16), MCTPSR (SEQ ID NO:17), VCTPSR (SEQ ID NO:18), LCSPSR (SEQ ID NO:19), LCAPSR (SEQ ID NO:20), LCVPSR (SEQ ID NO:21), LCGPSR (SEQ ID NO:22), ICTPAR (SEQ ID NO:23), LCTPSK (SEQ ID NO:24), MCTPSK (SEQ ID NO:25), VCTPSK (SEQ ID NO:26), LCSPSK (SEQ ID NO:27), LCAPSK (SEQ ID NO:28), LCVPSK (SEQ ID NO:29), LCGPSK (SEQ ID NO:30), LCTPSA (SEQ ID NO:31), ICTPAA (SEQ ID NO:32), MCTPSA (SEQ ID NO:33), VCTPSA (SEQ ID NO:34), LCSPSA (SEQ ID NO:35), LCAPSA (SEQ ID NO:36), LCVPSA (SEQ ID NO:37), and LCGPSA (SEQ ID NO:38). FGly-Containing Sequences Upon action of FGE on the modified anti-CD25 heavy and/or light chain, the serine or the cysteine in the sulfatase motif is modified to FGly. Thus, the FGly-containing sulfatase motif can be of the formula: X1(FGly)X2Z20X3Z30(I′″)whereFGly is the formylglycine residue;Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; andX2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. As described above, the modified polypeptide containing the FGly residue may be conjugated to a drug (e.g., a maytansinoid) by reaction of the FGly with the drug (e.g., a drug containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety, as described above) to produce an FGly′-containing sulfatase motif. As used herein, the term FGly′ refers to the modified amino acid residue of the sulfatase motif that is coupled to the drug, such as a maytansinoid (e.g., the modified amino acid residue of formula (I)). Thus, the FGly′-containing sulfatase motif can be of the formula: X1(FGly′)X2Z20X3Z30(II) whereFGly′ is the modified amino acid residue of formula (I);Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; andX2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. In certain embodiments, the modified amino acid residue of formula (I) is positioned at a C-terminus of a heavy chain constant region of the anti-CD25 antibody. In some instances, the heavy chain constant region comprises a sequence of the formula (II): X1(FGly′)X2Z20X3Z30(II) whereinFGly′ is the modified amino acid residue of formula (I);Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, XV is present;X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G; andwherein the sequence is C-terminal to the amino acid sequence QKSLSLSPGK (SEQ ID NO: 158), and where the sequence may include 1, 2, 3, 4, 5, or from 5 to 10, amino acids not present in a native, wild-type heavy Ig chain constant region. In certain embodiments, the heavy chain constant region comprises the sequence SLSLSPGSL(FGly′)TPSRGS (SEQ ID NO: 39) at the C-terminus of the Ig heavy chain, e.g., in place of a native SLSLSPGK (SEQ ID NO:40) sequence. In certain embodiments, the modified amino acid residue of formula (I) is positioned in a light chain constant region of the anti-CD25 antibody. In certain embodiments, the light chain constant region comprises a sequence of the formula (II): X1(FGly′)X2Z20X3Z30(II) whereinFGly′ is the modified amino acid residue of formula (I);Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present;X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G; and wherein the sequence is C-terminal to the amino acid sequence KVDNAL (SEQ ID NO:42) and/or is N-terminal to the amino acid sequence QSGNSQ (SEQ ID NO:43). In certain embodiments, the light chain constant region comprises the sequence KVDNAL(FGly′)TPSRQSGNSQ (SEQ ID NO:44). In certain embodiments, the modified amino acid residue of formula (I) is positioned in a heavy chain CH1 region of the anti-CD25 antibody. In certain embodiments, the heavy chain CH1 region comprises a sequence of the formula (II): X1(FGly′)X2Z20X3Z30(II) whereinFGly′ is the modified amino acid residue of formula (I);Z20is either a proline or alanine residue (which can also be represented by (P/A));Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present;X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G; andwherein the sequence is C-terminal to the amino acid sequence SWNSGA (SEQ ID NO:46) and/or is N-terminal to the amino acid sequence GVHTFP (SEQ ID NO:47). In certain embodiments, the heavy chain CH1 region comprises the sequence SWNSGAL(FGly′)TPSRGVHTFP (SEQ ID NO:48). Site of Modification As noted above, the amino acid sequence of an anti-CD25 antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an FGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). The anti-CD25 polypeptides used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as “target Ig polypeptides” or “target anti-CD25 antibodies” or “target anti-CD25 Ig polypeptides.” The site in an anti-CD25 antibody into which a sulfatase motif is introduced can be any convenient site. As noted above, in some instances, the extent of modification of the native amino acid sequence of the target anti-CD25 polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and/or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target anti-CD25 polypeptide may minimize the impact such modifications may have upon anti-CD25 function and/or structure. An anti-CD25 antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions). An Ig constant region can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent-accessible loop region of the Ig constant region. An Ig constant region can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above. In some cases, an aldehyde-tagged anti-CD25 antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain). The aldehyde-tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an FGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4. In some cases, an aldehyde-tagged anti-CD25 antibody comprises an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an FGly-modified anti-CD25 antibody polypeptide. Exemplary constant regions include human gamma 1 and gamma 3 regions. With the exception of the sulfatase motif, a modified constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild type amino acid sequence. In some embodiments the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. As noted above, an isolated aldehyde-tagged anti-CD25 polypeptide can comprise a heavy chain constant region modified to include a sulfatase motif as described above, where the sulfatase motif is in or adjacent a surface-accessible loop region of the anti-CD25 polypeptide heavy chain constant region. In some instances, a target anti-CD25 immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgG1 heavy chain constant region corresponding to one or more of: 1) amino acids 122-127; 2) amino acids 137-143; 3) amino acids 155-158; 4) amino acids 163-170; 5) amino acids 163-183; 6) amino acids 179-183; 7) amino acids 190-192; 8) amino acids 200-202; 9) amino acids 199-202; 10) amino acids 208-212; 11) amino acids 220-241; 12) amino acids 247-251; 13) amino acids 257-261; 14) amino acid 269-277; 15) amino acids 271-277; 16) amino acids 284-285; 17) amino acids 284-292; 18) amino acids 289-291; 19) amino acids 299-303; 20) amino acids 309-313; 21) amino acids 320-322; 22) amino acids 329-335; 23) amino acids 341-349; 24) amino acids 342-348; 25) amino acids 356-365; 26) amino acids 377-381; 27) amino acids 388-394; 28) amino acids 398-407; 29) amino acids 433-451; and 30) amino acids 446-451; wherein the amino acid numbering is based on the amino acid numbering of human IgG1 as depicted inFIG.12B. In some instances, a target anti-CD25 immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgG1 heavy chain constant region corresponding to one or more of: 1) amino acids 1-6; 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71; 8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 127-131; 13) amino acids 137-141; 14) amino acid 149-157; 15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 200-202; 22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-287; 29) amino acids 313-331; and 30) amino acids 324-331; wherein the amino acid numbering is based on the amino acid numbering of human IgG1 as set out in SEQ ID NO:164// (human IgG1 constant region; sequence depicted inFIG.12B). Exemplary surface-accessible loop regions of an IgG1 heavy chain include: 1) ASTKGP (SEQ ID NO:49); 2) KSTSGGT (SEQ ID NO:50); 3) PEPV (SEQ ID NO:51); 4) NSGALTSG (SEQ ID NO:52); 5) NSGALTSGVHTFPAVLQSSGL (SEQ ID NO:53); 6) QSSGL (SEQ ID NO:54); 7) VTV; 8) QTY; 9) TQTY (SEQ ID NO:55); 10) HKPSN (SEQ ID NO:56); 11) EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO:57); 12) FPPKP (SEQ ID NO:58); 13) ISRTP (SEQ ID NO:59); 14) DVSHEDPEV (SEQ ID NO:60); 15) SHEDPEV (SEQ ID NO:61); 16) DG; 17) DGVEVHNAK (SEQ ID NO:62); 18) HNA; 19) QYNST (SEQ ID NO:63); 20) VLTVL (SEQ ID NO:64); 21) GKE; 22) NKALPAP (SEQ ID NO:65); 23) SKAKGQPRE (SEQ ID NO:66); 24) KAKGQPR (SEQ ID NO:67); 25) PPSRKELTKN (SEQ ID NO:68); 26) YPSDI (SEQ ID NO:69); 27) NGQPENN (SEQ ID NO:70); 28) TPPVLDSDGS (SEQ ID NO:71); 29) HEALHNHYTQKSLSLSPGK (SEQ ID NO:72); and 30) SLSPGK (SEQ ID NO:73), as shown inFIGS.12A and12B. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgG2 heavy chain constant region corresponding to one or more of: 1) amino acids 1-6; 2) amino acids 13-24; 3) amino acids 33-37; 4) amino acids 43-54; 5) amino acids 58-63; 6) amino acids 69-71; 7) amino acids 78-80; 8) 87-89; 9) amino acids 95-96; 10) 114-118; 11) 122-126; 12) 134-136; 13) 144-152; 14) 159-167; 15) 175-176; 16) 184-188; 17) 195-197; 18) 204-210; 19) 216-224; 20) 231-233; 21) 237-241; 22) 252-256; 23) 263-269; 24) 273-282; 25) amino acids 299-302; where the amino acid numbering is based on the numbering of the amino acid sequence set forth in SEQ ID NO: 165// (human IgG2; also depicted inFIG.12B). Exemplary surface-accessible loop regions of an IgG2 heavy chain include 1) ASTKGP (SEQ ID NO:49); 2) PCSRSTSESTAA (SEQ ID NO:74); 3) FPEPV (SEQ ID NO:75); 4) SGALTSGVHTFP (SEQ ID NO:76); 5) QSSGLY (SEQ ID NO:77); 6) VTV; 7) TQT; 8) HKP; 9) DK; 10) VAGPS (SEQ ID NO:78); 11) FPPKP (SEQ ID NO:58); 12) RTP; 13) DVSHEDPEV (SEQ ID NO:60); 14) DGVEVHNAK (SEQ ID NO:62); 15) FN; 16) VLTVV (SEQ ID NO:79); 17) GKE; 18) NKGLPAP (SEQ ID NO:80); 19) SKTKGQPRE (SEQ ID NO:81); 20) PPS; 21) MTKNQ (SEQ ID NO:82); 22) YPSDI (SEQ ID NO:69); 23) NGQPENN (SEQ ID NO:70); 24) TPPMLDSDGS (SEQ ID NO:84); 25) GNVF (SEQ ID NO:85); and 26) HEALHNHYTQKSLSLSPGK (SEQ ID NO:72), as shown inFIG.12B. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgG3 heavy chain constant region corresponding to one or more of: 1) amino acids 1-6; 2) amino acids 13-22; 3) amino acids 33-37; 4) amino acids 43-61; 5) amino acid 71; 6) amino acids 78-80; 7) 87-91; 8) amino acids 97-106; 9) 111-115; 10) 147-167; 11) 173-177; 16) 185-187; 13) 195-203; 14) 210-218; 15) 226-227; 16) 238-239; 17) 246-248; 18) 255-261; 19) 267-275; 20) 282-291; 21) amino acids 303-307; 22) amino acids 313-320; 23) amino acids 324-333; 24) amino acids 350-352; 25) amino acids 359-365; and 26) amino acids 372-377; where the amino acid numbering is based on the numbering of the amino acid sequence set forth in SEQ ID NO: 166// (human IgG3; also depicted inFIG.12B). Exemplary surface-accessible loop regions of an IgG3 heavy chain include 1) ASTKGP (SEQ ID NO:49); 2) PCSRSTSGGT (SEQ ID NO:87); 3) FPEPV (SEQ ID NO:75); 4) SGALTSGVHTFPAVLQSSG (SEQ ID NO:88); 5) V; 6) TQT; 7) HKPSN (SEQ ID NO:56); 8) RVELKTPLGD (SEQ ID NO:89); 9) CPRCPKP (SEQ ID NO:90); 10) PKSCDTPPPCPRCPAPELLGG (SEQ ID NO:91); 11) FPPKP (SEQ ID NO:58); 12) RTP; 13) DVSHEDPEV (SEQ ID NO:60); 14) DGVEVHNAK (SEQ ID NO:62); 15) YN; 16) VL; 17) GKE; 18) NKALPAP (SEQ ID NO:65); 19) SKTKGQPRE (SEQ ID NO:81); 20) PPSREEMTKN (SEQ ID NO:92); 21) YPSDI (SEQ ID NO:69); 22) SSGQPENN (SEQ ID NO:93); 23) TPPMLDSDGS (SEQ ID NO:84); 24) GNI; 25) HEALHNR (SEQ ID NO:95); and 26) SLSPGK (SEQ ID NO:40), as shown inFIG.12B. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgG4 heavy chain constant region corresponding to one or more of: 1) amino acids 1-5; 2) amino acids 12-23; 3) amino acids 32-36; 4) amino acids 42-53; 5) amino acids 57-62; 6) amino acids 68-70; 7) amino acids 77-79; 8) amino acids 86-88; 9) amino acids 94-95; 10) amino acids 101-102; 11) amino acids 108-118; 12) amino acids 122-126; 13) amino acids 134-136; 14) amino acids 144-152; 15) amino acids 159-167; 16) amino acids 175-176; 17) amino acids 185-186; 18) amino acids 196-198; 19) amino acids 205-211; 20) amino acids 217-226; 21) amino acids 232-241; 22) amino acids 253-257; 23) amino acids 264-265; 24) 269-270; 25) amino acids 274-283; 26) amino acids 300-303; 27) amino acids 399-417; where the amino acid numbering is based on the numbering of the amino acid sequence set forth in SEQ ID NO: 167// (human IgG4; also depicted inFIG.12B). Exemplary surface-accessible loop regions of an IgG4 heavy chain include 1) STKGP (SEQ ID NO:96); 2) PCSRSTSESTAA (SEQ ID NO:74); 3) FPEPV (SEQ ID NO:75); 4) SGALTSGVHTFP (SEQ ID NO:76); 5) QSSGLY (SEQ ID NO:77); 6) VTV; 7) TKT; 8) HKP; 9) DK; 10) YG; 11) CPAPEFLGGPS (SEQ ID NO:97); 12) FPPKP (SEQ ID NO:58); 13) RTP; 14) DVSQEDPEV (SEQ ID NO:98); 15) DGVEVHNAK (SEQ ID NO:62); 16) FN; 17) VL; 18) GKE; 19) NKGLPSS (SEQ ID NO:99); 20) SKAKGQPREP (SEQ ID NO:100); 21) PPSQEEMTKN (SEQ ID NO:101); 22) YPSDI (SEQ ID NO:69); 23) NG; 24) NN; 25) TPPVLDSDGS (SEQ ID NO:71); 26) GNVF (SEQ ID NO:85); and 27) HEALHNHYTQKSLSLSLGK (SEQ ID NO:102), as shown inFIG.12B. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an IgA heavy chain constant region corresponding to one or more of: 1) amino acids 1-13; 2) amino acids 17-21; 3) amino acids 28-32; 4) amino acids 44-54; 5) amino acids 60-66; 6) amino acids 73-76; 7) amino acids 80-82; 8) amino acids 90-91; 9) amino acids 123-125; 10) amino acids 130-133; 11) amino acids 138-142; 12) amino acids 151-158; 13) amino acids 165-174; 14) amino acids 181-184; 15) amino acids 192-195; 16) amino acid 199; 17) amino acids 209-210; 18) amino acids 222-245; 19) amino acids 252-256; 20) amino acids 266-276; 21) amino acids 293-294; 22) amino acids 301-304; 23) amino acids 317-320; 24) amino acids 329-353; where the amino acid numbering is based on the numbering of the amino acid sequence set forth in SEQ ID NO: 168 (human IgA; also depicted inFIG.12B). Exemplary surface-accessible loop regions of an IgA heavy chain include 1) ASPTSPKVFPLSL (SEQ ID NO:103); 2) QPDGN (SEQ ID NO:104); 3) VQGFFPQEPL (SEQ ID NO:105); 4) SGQGVTARNFP (SEQ ID NO:106); 5) SGDLYTT (SEQ ID NO:107); 6) PATQ (SEQ ID NO:108); 7) GKS; 8) YT; 9) CHP; 10) HRPA (SEQ ID NO:109); 11) LLGSE (SEQ ID NO:110); 12) GLRDASGV (SEQ ID NO:111); 13) SSGKSAVQGP (SEQ ID NO:112); 14) GCYS (SEQ ID NO:113); 15) CAEP (SEQ ID NO:114); 16) PE; 17) SGNTFRPEVHLLPPPSEELALNEL (SEQ ID NO:115); 18) ARGFS (SEQ ID NO:116); 19) QGSQELPREKY (SEQ ID NO:117); 20) AV; 21) AAED (SEQ ID NO:118); 22) HEAL (SEQ ID NO:119); and 23) IDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO:120), as shown inFIG.12B. A sulfatase motif can be provided within or adjacent one or more of these amino acid sequences of such modification sites of an Ig heavy chain. For example, an Ig heavy chain polypeptide can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C-terminal to these modification sites. Alternatively or in addition, an Ig heavy chain polypeptide can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites. In some embodiments, an Ig heavy chain polypeptide may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids. Alternatively or in addition, where a native amino acid sequence provides for one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site. The amino acid sequence of a surface-accessible loop region can thus be modified to provide a sulfatase motif, where the modifications can include insertions, deletions, and/or substitutions. For example, where the modification is in a CH1 domain, the surface-accessible loop region can have the amino acid sequence NSGALTSG (SEQ ID NO:52), and the aldehyde-tagged sequence can be, e.g., NSGALCTPSRG (SEQ ID NO:122), e.g., where the “TS” residues of the NSGALTSG (SEQ ID NO:52) sequence are replaced with “CTPSR,” (SEQ ID NO:123) such that the sulfatase motif has the sequence LCTPSR (SEQ ID NO:16). As another example, where the modification is in a CH2 domain, the surface-accessible loop region can have the amino acid sequence NKALPAP (SEQ ID NO:65), and the aldehyde-tagged sequence can be, e.g., NLCTPSRAP (SEQ ID NO:125), e.g., where the “KAL” residues of the NKALPAP (SEQ ID NO:65) sequence are replaced with “LCTPSR,” (SEQ ID NO:16) such that the sulfatase motif has the sequence LCTPSR (SEQ ID NO:16). As another example, where the modification is in a CH2/CH3 domain, the surface-accessible loop region can have the amino acid sequence KAKGQPR (SEQ ID NO:67), and the aldehyde-tagged sequence can be, e.g., KAKGLCTPSR (SEQ ID NO:126), e.g., where the “GQP” residues of the KAKGQPR (SEQ ID NO:100) sequence are replaced with “LCTPS,” (SEQ ID NO:31) such that the sulfatase motif has the sequence LCTPSR (SEQ ID NO:16). As noted above, an isolated aldehyde-tagged anti-CD25 Ig polypeptide can comprise a light chain constant region modified to include a sulfatase motif as described above, where the sulfatase motif is in or adjacent a surface-accessible loop region of the Ig polypeptide light chain constant region. Illustrative examples of surface-accessible loop regions of a light chain constant region are presented inFIGS.12A and12C. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an Ig light chain constant region corresponding to one or more of: 1) amino acids 130-135; 2) amino acids 141-143; 3) amino acid 150; 4) amino acids 162-166; 5) amino acids 163-166; 6) amino acids 173-180; 7) amino acids 186-194; 8) amino acids 211-212; 9) amino acids 220-225; 10) amino acids 233-236; wherein the amino acid numbering is based on the amino acid numbering of human kappa light chain as depicted inFIG.12C. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of an Ig light chain constant region corresponding to one or more of: 1) amino acids 1-6; 2) amino acids 12-14; 3) amino acid 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51; 7) amino acids 57-65; 8) amino acids 83-83; 9) amino acids 91-96; 10) amino acids 104-107; where the amino acid numbering is based on SEQ ID NO:162 and 170 (human kappa light chain; amino acid sequence depicted inFIG.12C). Exemplary surface-accessible loop regions of an Ig light chain (e.g., a human kappa light chain) include: 1) RTVAAP (SEQ ID NO:127); 2) PPS; 3) Gly (see, e.g., Gly at position 150 of the human kappa light chain sequence depicted inFIG.12C); 4) YPREA (SEQ ID NO:128); 5) PREA (SEQ ID NO:129); 6) DNALQSGN (SEQ ID NO:130); 7) TEQDSKDST (SEQ ID NO:131); 8) HK; 9) HQGLSS (SEQ ID NO:132); and 10) RGEC (SEQ ID NO:133), as shown inFIGS.12A and12C. Exemplary surface-accessible loop regions of an Ig lambda light chain include QPKAAP (SEQ ID NO:134), PPS, NK, DFYPGAV (SEQ ID NO:135), DSSPVKAG (SEQ ID NO:136), TTP, SN, HKS, EG, and APTECS (SEQ ID NO:137), as shown inFIG.12C. In some instances, a target immunoglobulin is modified to include a sulfatase motif as described above, where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions. In certain embodiments, the sulfatase motif is within, or adjacent to, a region of a rat Ig light chain constant region corresponding to one or more of: 1) amino acids 1-6; 2) amino acids 12-14; 3) amino acids 121-22; 4) amino acids 31-37; 5) amino acids 44-51; 6) amino acids 55-57; 7) amino acids 61-62; 8) amino acids 81-83; 9) amino acids 91-92; 10) amino acids 102-105; wherein the amino acid numbering is based on the amino acid numbering of rat light chain as set forth in SEQ ID NO: 173 (sequence depicted inFIG.12C). In some cases, a sulfatase motif is introduced into the CH1 region of an anti-CD25 heavy chain constant region. In some cases, a sulfatase motif is introduced at or near (e.g., within 1 to 10 amino acids of) the C-terminus of an anti-CD25 heavy chain. In some cases, a sulfatase motif is introduced in the light-chain constant region. In some cases, a sulfatase motif is introduced into the CH1 region of an anti-CD25 heavy chain constant region, e.g., within amino acids 121-219 of the IgG1 heavy chain amino acid sequence depicted inFIG.12A. For example, in some cases, a sulfatase motif is introduced into the amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO:138). For example, in some of these embodiments, the amino acid sequence GALTSGVH (SEQ ID NO:139) is modified to GALCTPSRGVH (SEQ ID NO:140), where the sulfatase motif is LCTPSR (SEQ ID NO:16). In some cases, a sulfatase motif is introduced at or near the C-terminus of an anti-CD25 heavy chain, e.g., the sulfatase motifs introduced within 1 amino acid, 2 amino acids (aa), 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa the C-terminus of an anti-CD25 heavy chain. As one non-limiting example, the C-terminal lysine reside of an anti-CD25 heavy chain can be replaced with the amino acid sequence SLCTPSRGS (SEQ ID NO:141). In some cases, a sulfatase motif is introduced into the constant region of a light chain of an anti-CD25 antibody. As one non-limiting example, in some cases, a sulfatase motif is introduced into the constant region of a light chain of an anti-CD25 antibody, where the sulfatase motif is C-terminal to KVDNAL (SEQ ID NO:42), and/or is N-terminal to QSGNSQ (SEQ ID NO:43). For example, in some cases, the sulfatase motif is LCTPSR (SEQ ID NO:16), and the anti-CD25 light chain comprises the amino acid sequence KVDNALLCTPSRQSGNSQ (SEQ ID NO:142). Drugs for Conjugation to a Polypeptide The present disclosure provides drug-polypeptide conjugates. Examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be modified as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agents. In certain embodiments, the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid. In certain embodiments, the drug is a maytansinoid, which as the following structure: whereindicates the point of attachment between the maytansinoid and the linker, L, in formula (I). By “point of attachment” is meant that thesymbol indicates the bond between the N of the maytansinoid and the linker, L, in formula (I). For example, in formula (I), W1is a maytansinoid, such as a maytansinoid of the structure above, whereindicates the point of attachment between the maytansinoid and the linker, L. As described above, in certain embodiments, L is a linker described by the formula -(L1)a-(L2)b-(L3)c-(L4)d-, wherein L1, L2, L3and L4are each independently a linker unit. In certain embodiments, L1is attached to the coupling moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, L2, if present, is attached to W1(the maytansinoid). In certain embodiments, L3, if present, is attached to W1(the maytansinoid). In certain embodiments, L4, if present, is attached to W1(the maytansinoid). As described above, in certain embodiments, the linker -(L1)a-(L2)b-(L3)c-(L4)d- is described by the formula -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-, wherein a, b, c and d are each independently 0 or 1, where the sum of a, b, c and d is 1 to 4. In certain embodiments, as described above, L1is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). As such, in certain embodiments, T1is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, V1is attached to W1(the maytansinoid). In certain embodiments, as described above, L2, if present, is attached to W1(the maytansinoid). As such, in certain embodiments, T2, if present, is attached to W1(the maytansinoid), or V2, if present, is attached to W1(the maytansinoid). In certain embodiments, as described above, L3, if present, is attached to W1(the maytansinoid). As such, in certain embodiments, T3, if present, is attached to W1(the maytansinoid), or V3, if present, is attached to W1(the maytansinoid). In certain embodiments, as described above, L4, if present, is attached to W1(the maytansinoid). As such, in certain embodiments, T4, if present, is attached to W1(the maytansinoid), or V4, if present, is attached to W1(the maytansinoid). Embodiments of the present disclosure include conjugates where a polypeptide (e.g., anti-CD25 antibody) is conjugated to one or more drug moieties (e.g., maytansinoid), such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, or 10 or more drug moieties. The drug moieties may be conjugated to the polypeptide at one or more sites in the polypeptide, as described herein. In certain embodiments, the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 10, or from 0.5 to 10, or from 1 to 10, such as from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 2, such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2. In certain embodiments, the conjugates have an average DAR of 1.5 to 2. In certain embodiments, the conjugates have an average DAR of 1.75 to 1.85. In certain embodiments, the conjugates have an average DAR of 1.8. By average is meant the arithmetic mean. Formulations The conjugates of the present disclosure can be formulated in a variety of different ways. In general, where the conjugate is a polypeptide-drug conjugate, the conjugate is formulated in a manner compatible with the drug conjugated to the polypeptide, the condition to be treated, and the route of administration to be used. In some embodiments, provided is a pharmaceutical composition that includes any of the conjugates of the present disclosure and a pharmaceutically-acceptable excipient. The conjugate (e.g., polypeptide-drug conjugate) can be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, e.g., oral, topical or parenteral administration. Where the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue), the conjugate can be provided as a ready-to-use dosage form, or as a reconstitutable storage-stable powder or liquid composed of pharmaceutically acceptable carriers and excipients. Methods for formulating conjugates can be adapted from those readily available. For example, conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human. Methods of Treatment The polypeptide-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug (i.e., the drug prior to conjugation to the polypeptide). In some embodiments, provided are methods that include administering to a subject an effective amount of any of the conjugates of the present disclosure. In certain aspects, provided are methods of delivering a drug to a target site in a subject, the method including administering to the subject a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject. By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and/or (iii) relief, that is, causing the regression of clinical symptoms. The subject to be treated can be one that is in need of therapy, where the host to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the polypeptide-drug conjugates disclosed herein. Generally, such subjects are “mammals”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). The amount of polypeptide-drug conjugate administered can be initially determined based on guidance of a dose and/or dosage regimen of the parent drug. In general, the polypeptide-drug conjugates can provide for targeted delivery and/or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the polypeptide-drug conjugates can provide for reduced dose and/or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in an polypeptide-drug conjugate of the present disclosure. Furthermore, as noted above, because the polypeptide-drug conjugates can provide for controlled stoichiometry of drug delivery, dosages of polypeptide-drug conjugates can be calculated based on the number of drug molecules provided on a per polypeptide-drug conjugate basis. In some embodiments, multiple doses of a polypeptide-drug conjugate are administered. The frequency of administration of a polypeptide-drug conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, a polypeptide-drug conjugate is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc. Methods of Treating Cancer The present disclosure provides methods that include delivering a conjugate of the present disclosure to an individual having a cancer. The methods are useful for treating a wide variety of cancers, including carcinomas, sarcomas, leukemias, and lymphomas. In the context of cancer, the term “treating” includes one or more (e.g., each) of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with a cancer. Carcinomas that can be treated using a subject method include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, etc. Sarcomas that can be treated using a subject method include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated using a subject method include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Leukemias that can be treated using a subject method include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's B cell lymphoma; and the like. In certain aspects, provided are methods of treating cancer in a subject, such methods including administering to the subject a therapeutically effective amount of a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to treat cancer in the subject. In some embodiments, the cancer is a hematologic malignancy. Hematologic malignancies of interest include, but are not limited to, leukemias, e.g., acute myeloid leukemia (AML). CD25 is up-regulated in refractory AML. In certain aspects, the methods are for treating relapsed and/or refractory AML. In some embodiments, the hematologic malignancy is lymphoma. CD25 is expressed on Hodgkin lymphoma (HL) cells and various T and B cell non-Hodgkin lymphoma (NHL) cells. When the hematologic malignancy is lymphoma, in certain aspects, the lymphoma is Hodgkin lymphoma (HL). When the lymphoma is Hodgkin lymphoma, in certain aspects, the Hodgkin lymphoma is relapsed and/or refractory Hodgkin lymphoma. When the hematologic malignancy is lymphoma, in certain aspects, the lymphoma is Non-Hodgkin lymphoma (NHL). When the lymphoma is Non-Hodgkin lymphoma, in certain aspects, the Non-Hodgkin lymphoma is relapsed and/or refractory Non-Hodgkin lymphoma. Methods of Reducing Inhibitory Signals Derived from T Regulatory Cells in a Subject CD25 is expressed on regulatory T cells. As such, in certain aspects, provided are methods of reducing inhibitory signals derived from T regulatory cells in a subject, such methods including administering to the subject a therapeutically effective amount of any of the conjugates of the present disclosure, where the administering is effective to reduce inhibitory signals derived from T regulatory cells in the subject. The methods of reducing inhibitory signals derived from T regulatory cells find use in a variety of contexts, including but not limited to, as part of an immunomodulatory combination therapy approach by reducing the inhibitory signals derived from the Treg populations and allowing for a productive anti-tumor immune response in a subject having cancer. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. By “average” is meant the arithmetic mean. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. General Synthetic Procedures Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969. During any of the processes for preparation of the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4thedition, Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below. Example 1 A linker containing a 4-amino-piperidine (4AP) group was synthesized according to Scheme 1, shown below. Synthesis of (9H-fluoren-9-yl)methyl 4-oxopiperidine-1-carboxylate (200) To a 100 mL round-bottom flask containing a magnetic stir bar was added piperidin-4-one hydrochloride monohydrate (1.53 g, 10 mmol), Fmoc chloride (2.58 g, 10 mmol), sodium carbonate (3.18 g, 30 mmol), dioxane (20 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc (100 mL) and extracted with water (1×100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting material was dried in vacuo to yield compound 200 as a white solid (3.05 g, 95% yield). 1H NMR (CDCl3) δ 7.78 (d, 2H, J=7.6), 7.59 (d, 2H, J=7.2), 7.43 (t, 2H, J=7.2), 7.37 (t, 2H, J=7.2), 4.60 (d, 2H, J=6.0), 4.28 (t, 2H, J=6.0), 3.72 (br, 2H), 3.63 (br, 2H), 2.39 (br, 2H), 2.28 (br, 2H). MS (ESI) m/z: [M+H]+Caled for C20H20NO3322.4; Found 322.2. Synthesis of (9H-fluoren-9-yl)methyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (201) To a dried scintillation vial containing a magnetic stir bar was added piperidinone 200 (642 mg, 2.0 mmol), H2N-PEG2-CO2t-Bu (560 mg, 2.4 mmol), 4 Å molecular sieves (activated powder, 500 mg), and 1,2-dichloroethane (5 mL). The mixture was stirred for 1 h at room temperature. To the reaction mixture was added sodium triacetoxyborohydride (845 mg, 4.0 mmol). The mixture was stirred for 5 days at room temperature. The resulting mixture was diluted with EtOAc. The organic layer was washed with saturated NaHCO3(1×50 mL), and brine (1×50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to yield compound 201 as an oil, which was carried forward without further purification. Synthesis of 13-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oic acid (202) To a dried scintillation vial containing a magnetic stir bar was added N-Fmoc-piperidine-4-amino-PEG2-CO2t-Bu (201) from the previous step, succinic anhydride (270 mg, 2.7 mmol), and dichloromethane (5 mL). The mixture was stirred for 18 hours at room temperature. The reaction mixture was partitioned between EtOAc and saturated NaHCO3. The aqueous layer was extracted with EtOAc (3×). The aqueous layer was acidified with HCl (1 M) until the pH ˜3. The aqueous layer was extracted (3×) with DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The reaction mixture was purified by C18 flash chromatography (elute 10-100% MeCN/water with 0.1% acetic acid). Product-containing fractions were concentrated under reduced pressure and then azeotroped with toluene (3×50 mL) to remove residual acetic acid to afford 534 mg (42%, 2 steps) of compound 202 as a white solid. 11H NMR (DMSO-d6) δ 11.96 (br, 1H), 7.89 (d, 2H, J=7.2), 7.63 (d, 2H, J=7.2), 7.42 (t, 2H, J=7.2), 7.34 (t, 2H, J=7.2), 4.25-4.55 (m, 3H), 3.70-4.35 (m, 3H), 3.59 (t, 2H, J=6.0), 3.39 (m, 5H), 3.35 (m, 3H), 3.21 (br, 1H), 2.79 (br, 2H), 2.57 (m, 2H), 2.42 (q, 4H, J=6.0), 1.49 (br, 3H), 1.37 (s, 9H). MS (ESI) m/z: [M+H]+Calcd for C35H47N2O9639.3; Found 639.2. Synthesis of (2S)-1-(((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1 (6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-8-(piperidin-4-yl)-11,14-dioxa-3,8-diazaheptadecan-17-oic acid (203) To a solution of ester 202 (227 mg, 0.356 mmol), diisopropylethylamine (174 μL, 1.065 mmol), N-deacetyl maytansine 124 (231 mg, 0.355 mmol) in 2 mL of DMF was added PyAOP (185 mg, 0.355 mmol). The solution was stirred for 30 min. Piperidine (0.5 mL) was added to the reaction mixture and stirred for an additional 20 min. The crude reaction mixture was purified by C18 reverse phase chromatography using a gradient of 0-100% acetonitrile:water affording 203.2 mg (55%, 2 steps) of compound 203. Synthesis of 17-(tert-butyl) 1-((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl) (2S)-8-(1-(3-(2- ((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,3-dimethyl-4,7-dioxo-11,14-dioxa-3,8-diazaheptadecanedioate (204) A solution of piperidine 203 (203.2 mg, 0.194 mmol), ester 12 (126.5 mg, 0.194 mmol), 2,4,6-trimethylpyridine (77 μL, 0.582 mmol), HOAT (26.4 mg, 0.194 mmol) in 1 mL DMF was stirred 30 min. The crude reaction was purified by C18 reverse phase chromatography using a gradient of 0-100% acetonitrile:water with 0.1% formic acid affording 280.5 mg (97% yield) of compound 204. MS (ESI) m/z: [M+H]+Calcd for C81H106ClN8O181513.7; Found 1514.0. Synthesis of (2S)-8-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-(((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oic acid (205) To a solution of compound 204 (108 mg, 0.0714 mmol) in 500 μL anhydrous DCM was added 357 μL of a 1M solution of SnCl4in DCM. The heterogeneous mixture was stirred for 1 h and then purified by C18 reverse phase chromatography using a gradient of 0-100% acetonitrile:water with 0.1% formic acid affording 78.4 mg (75% yield) of compound 205. MS (ESI) m/z: [M−H]−Calcd for C77H96ClN8O181455.7; Found 1455.9. Example 2 A linker containing a 4-amino-piperidine (4AP) group was synthesized according to Scheme 2, shown below. Synthesis of tert-butyl 4-oxopiperidine-1-carboxylate (210) To a 100 mL round-bottom flask containing a magnetic stir bar was added piperidin-4-one hydrochloride monohydrate (1.53 g, 10 mmol), di-tert-butyl dicarbonate (2.39 g, 11 mmol), sodium carbonate (1.22 g, 11.5 mmol), dioxane (10 mL), and water (1 mL). The reaction mixture was stirred at room temperature for 1 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting material was dried in vacuo to yield 1.74 g (87%) of compound 210 as a white solid. 1H NMR (CDCl3) δ 3.73 (t, 4H, J=6.0), 2.46 (t, 4H, J=6.0), 1.51 (s, 9H). MS (ESI) m/z: [M+H]+Calcd for C10H18NO3200.3; Found 200.2. Synthesis of tert-butyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (211) To a dried scintillation vial containing a magnetic stir bar was added tert-butyl 4-oxopiperidine-1-carboxylate (399 mg, 2 mmol), H2N-PEG2-COOt-Bu (550 mg, 2.4 mmol), 4 A molecular sieves (activated powder, 200 mg), and 1,2-dichloroethane (5 mL). The mixture was stirred for 1 h at room temperature. To the reaction mixture was added sodium triacetoxyborohydride (845 mg, 4 mmol). The mixture was stirred for 3 days at room temperature. The resulting mixture was partitioned between EtOAc and saturated aqueous NaHCO3. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford 850 mg of compound 211 as a viscous oil. MS (ESI) m/z: [M+H]+Calcd for C21H41N2O6417.3; Found 417.2. Synthesis of 13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oic acid (212) To a dried scintillation vial containing a magnetic stir bar was added tert-butyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate 211 (220 mg, 0.5 mmol), succinic anhydride (55 mg, 0.55 mmol), 4-(dimethylamino)pyridine (5 mg, 0.04 mmol), and dichloromethane (3 mL). The mixture was stirred for 24 h at room temperature. The reaction mixture was partially purified by flash chromatography (elute 50-100% EtOAc/hexanes) to yield 117 mg of compound 212 as a clear oil, which was carried forward without further characterization. MS (ESI) m/z: [M+H]+Caled for C25H45N2O9517.6; Found 517.5. Synthesis of 17-(tert-butyl) 1-((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl) (2S)-8-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,3-dimethyl-4,7-dioxo-11,14-dioxa-3,8-diazaheptadecanedioate (213) To a dried scintillation vial containing a magnetic stir bar was added 13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oic acid 212 (55 mg, 0.1 mmol), N-deacyl maytansine 124 (65 mg, 0.1 mmol), HATU (43 mg, 0.11 mmol), DMF (1 mL), and dichloromethane (0.5 mL). The mixture was stirred for 8 h at room temperature. The reaction mixture was directly purified by C18 flash chromatography (elute 5-100% MeCN/water) to give 18 mg (16%) of compound 213 as a white film. MS (ESI) m/z: [M+H]+Calcd for C57H87ClN5O71148.6; Found 1148.7. Synthesis of (2S)-1-(((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-8-(piperidin-4-yl)-11,14-dioxa-3,8-diazaheptadecan-17-oic acid (214) To a dried scintillation vial containing a magnetic stir bar was added maytansinoid 213 (31 mg, 0.027 mmol) and dichloromethane (1 mL). The solution was cooled to 0° C. and tin(IV) tetrachloride (1.0 M solution in dichloromethane, 0.3 mL, 0.3 mmol) was added. The reaction mixture was stirred for 1 h at 0° C. The reaction mixture was directly purified by C18 flash chromatography (elute 5-100% MeCN/water) to yield 16 mg (60%) of compound 214 as a white solid (16 mg, 60% yield). MS (ESI) m/z: [M+H]+Calcd for C48H71ClN5O5992.5; Found 992.6. Synthesis of (2S)-8-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-1-(((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oic acid (215) To a dried scintillation vial containing a magnetic stir bar was added maytansinoid 214 (16 mg, 0.016 mmol), (9H-fluoren-9-yl)methyl 1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate (5) (13 mg, 0.02 mmol), DIPEA (8 μL, 0.05 mmol), and DMF (1 mL). The solution was stirred for 18 h at room temperature. The reaction mixture was directly purified by C18 flash chromatography (elute 5-100% MeCN/water) to yield 18 mg (77%) of compound 215 as a white solid. MS (ESI) m/z: [M+H]+Calcd for C77H98ClN8O81457.7; Found 1457.9. Synthesis of (2S)-1-(((14S,16S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-8-(1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-oic acid (216) To a dried scintillation vial containing a magnetic stir bar was added maytansinoid 215 (18 mg, 0.012 mmol), piperidine (20 μL, 0.02 mmol), and DMF (1 mL). The solution was stirred for 20 minutes at room temperature. The reaction mixture was directly purified by C18 flash chromatography (elute 1-60% MeCN/water) to yield 15 mg (98%) of compound 216 (also referred to herein as HIPS-4AP-maytansine or HIPS-4-amino-piperidin-maytansine) as a white solid. MS (ESI) m/z: [M+H]+Calcd for C62H88ClN8O161235.6; Found 1236.0. Example 3 Experimental Procedures General Experiments were performed to create site-specifically conjugated antibody-drug conjugates (ADCs). Site-specific ADC production included the incorporation of formylglycine (FGly), a non-natural amino acid, into the protein sequence. To install FGly (FIG.1), a short consensus sequence, CXPXR, (SEQ ID NO: 174) where X is serine, threonine, alanine, or glycine, was inserted at the desired location in the conserved regions of antibody heavy or light chains using standard molecular biology cloning techniques. This “tagged” construct was produced recombinantly in cells that coexpress the formylglycine-generating enzyme (FGE), which cotranslationally converted the cysteine within the tag into an FGly residue, generating an aldehyde functional group (also referred to herein as an aldehyde tag). The aldehyde functional group served as a chemical handle for bioorthogonal conjugation. A hydrazino-iso-Pictet-Spengler (HIPS) ligation was used to connect the payload (e.g., a drug, such as a cytotoxin (e.g., maytansine)) to FGly, resulting in the formation of a stable, covalent C═C bond between the cytotoxin payload and the antibody. This C═C bond was expected to be stable to physiologically-relevant conditions encountered by the ADC during circulation and FcRn recycling, e.g., proteases, low pH, and reducing reagents. Antibodies bearing the aldehyde tag may be produced at a variety of locations. Experiments were performed to test the effects of inserting the aldehyde tag at the heavy chain C-terminus (CT). Biophysical and functional characteriziaton was performed on the resulting ADCs made by conjugation to maytansine payloads via a HIPS linker. Cloning, Expression, and Purification of Tagged Antibodies The aldehyde tag sequence was inserted at the heavy chain C-terminus (CT) of the anti-CD25 antibody daclizumab using standard molecular biology techniques. For small-scale production, CHO—S cells were transfected with human FGE expression constructs and pools of FGE-overexpressing cells were used for the transient production of antibodies. For larger-scale production, GPEx technology (Catalent, Inc., Somerset, NJ) was used to generate a clonal cell line overexpressing human FGE (GPEx). Then, the FGE clone was used to generate bulk stable pools of antibody-expressing cells. Antibodies were purified from the conditioned medium using a Protein A chromatography (MabSelect, GE Healthcare Life Sciences, Pittsburgh, PA). Purified antibodies were flash frozen and stored at −80° C. until further use. Bioconjugation, Purification, and HPLC Analytics C-terminally aldehyde-tagged aCD25 antibody (daclizumab, 15 mg/mL) was conjugated to a maytansine payload attached to a HIPS-4AP linker (8 mol. equivalents drug:antibody) for 48-72 h at 37° C. in 20 mM sodium citrate, 50 mM NaCl pH 5.0 containing 0.85% DMA. After conjugation, free drug was removed by tangential flow filtration and the ADC was buffer exchanged into 20 mM sodium citrate, 50 mM NaCl pH 5.5. To determine the drug-to-antibody ratio (DAR) of the final product, ADCs were examined by analytical HIC (Tosoh #14947) with mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0, and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate pH 7.0. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC; Tosoh #08541) with a mobile phase of 300 mM NaCl, 25 mM sodium phosphate pH 6.8. Results The anti-CD25 antibody modified to contain the aldehyde tag at the heavy chain C-terminus (CT) was conjugated to a maytansine payload attached to a HIPS-4AP linker. Upon completion, remaining free drug was removed during buffer exchange by tangential flow filtration. These reactions were high yielding, with >90% conjugation efficiency and >80% total yield. The resulting ADCs had drug-to-antibody ratios (DARs) of 1.75-1.84 and were predominately monomeric.FIGS.2-5document DARs from representative reactions as determined by HIC and reversed phase PLRP chromatography, and show the monomeric integrity as determined by SEC. FIG.2shows a hydrophobic interaction column (HIC) trace of an aldehyde-tagged anti-CD25 antibody conjugated at the heavy chain C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker.FIG.2indicates that the DAR was 1.83 as determined by HIC. FIG.3shows a reversed phase chromatography (PLRP) trace of an aldehyde-tagged anti-CD25 antibody conjugated at the heavy chain C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker.FIG.3indicates that the DAR was 1.81 as determined by PLRP. FIG.4shows a graph of analytical size exclusion chromatography (SEC) analysis of an aldehyde-tagged anti-CD25 antibody conjugated at the heavy chain C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker. As shown inFIG.4, analytical SEC indicated 98.2% monomer for the final product. FIG.5shows a graph of intact mass analysis of an aldehyde-tagged anti-CD25 antibody conjugated at the heavy chain C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker. As shown inFIG.5, a linker/payload-related increase in mass for a DAR2 conjugated species was observed. In Vitro Cytotoxicity The CD25-positive leukemia and lymphoma cell lines, SU-DHL-1, HDLM-2, EoL-1, L-540, and Karpas 299, were obtained from the ATCC and DSMZ cell banks, or were tested at Crown Bioscience, Inc. The cells were maintained in RPMI-1640 medium (Cellgro) supplemented with 10 or 20% fetal bovine serum (Invitrogen) and Glutamax (Invitrogen). 24 h prior to plating, cells were passaged to ensure log-phase growth. On the day of plating, 5000 cells/well were seeded onto 96-well plates in 100 μL normal growth medium supplemented with 10 IU penicillin and 10 μg/mL streptomycin (Cellgro). Cells were treated at various concentrations with 20 μL of diluted analytes, and the plates were incubated at 37° C. in an atmosphere of 5% CO2. After 5 d, 100 μL/well of Cell Titer-Glo reagent (Promega) was added, and luminescence was measured using a Molecular Devices SpectraMax M5 plate reader. GraphPad Prism software was used for data analysis. Results The anti-CD25 HIPS-4AP-maytansine conjugate exhibited very potent activity against all five tested cell lines, comparable to free maytansine.FIGS.6-10show the results for the SU-DHL-1 (IC50: 0.24 nM), HDLM-2 (IC50: 0.06 nM), EoL-1 (IC50: 0.07 nM), L-540 (IC50: 0.43 nM), and Karpas 299 (IC50: 0.49 nM) cell lines, respectively. The ADC IC50concentrations ranged from 0.06 to 0.49 nM in these assays, while the free maytansine IC50concentrations ranged from 0.03 to 0.19 nM. Xenograft Studies Female CB17.SCID mice (6/group) were inoculated subcutaneously with 5×106Karpas 299 cells. Treatment began when the tumors reached an average of 205 mm3, at which time the animals were dosed intravenously with vehicle alone or the anti-CD25 HIPS-4AP-maytansine conjugate (10 mg/kg). One group received a single dose of the anti-CD25 HIPS-4AP-maytansine conjugate, while another group was dosed once per week for a total of four doses (qwk×4). The animals were monitored twice weekly for body weight and tumor size. Animals were euthanized when tumors reached 2000 mm3. Results The median time to endpoint for animals in the vehicle control groups was 28 days. At that point, none of the animals that had been dosed with the anti-CD25 HIPS-4AP-maytansine conjugate had detectable tumors. Data is shown inFIG.11. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. | 207,080 |
11857638 | DETAILED DESCRIPTION 1. Definition of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Terms such as “alkyl,” “cycloalkyl,” “alkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “C1-4alkyl,” “C3-8cycloalkyl,” “C1-6alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “C3alkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C1-4,”the members of the group that follows may have any number of carbon atoms falling within the recited range. A “C1-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged. As used herein, the term “alkenyl” refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. As used herein, the term “alkoxy” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy. As used herein, the term “alkyl” refers to a linear or branched saturated hydrocarbon radical. Representative examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, and hexyl. The term “alkylene,” as used herein, means a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH(CH3)CH2—, and —CH2CH(CH3)CH(CH3)CH2—. The term “alkenylene,” as used herein, means a divalent group derived from a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. Representative examples of alkenylene include, but are not limited to —CH═CH—, —CH2CH═CH—, and —CH2CH═CH(CH3)—. As used herein, the term “alkynyl” refers to a straight or branched hydrocarbon radical having one or more carbon-carbon triple bonds. Alkynyl groups of the present invention include, but are not limited to, ethynyl, propynyl, and butynyl. As used herein, the term “amido” refers to —CONH2. As used herein, the term “amino” refers to an —NH2group. As used herein, the term “amino acid” refers to both natural and unnatural amino acids. It also includes protected natural and unnatural amino acids. As used herein, the term “aryl” means phenyl or bicyclic aryl. The bicyclic aryl is naphthyl, dihydronaphthalenyl, tetrahydronaphthalenyl, indanyl, or indenyl. The phenyl and bicyclic aryls are attached to the parent molecular moiety through any carbon atom contained within the phenyl or bicyclic aryl. As used herein, the term “phenylene” means a divalent phenyl radical, e.g., 1,4-phenylene As use herein, the term “azide” or “azido” refers to an —N═N+═N−(i.e., —N3) group. As used herein, the term “derivative” may refer to a compound that is derived from a similar compound by some chemical or physical process. The derivative is a compound of similar chemical structure. The derivative may be a structural analogue. As used herein, the term “carboxy” refers to a —C(O)—OH group. The term “cycloalkyl” as used herein, means a saturated carbocyclic ring system containing zero heteroatoms as ring atoms, and zero double bonds. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl groups of the present invention may contain an alkylene bridge of 1, 2, 3, or 4 carbon atoms, linking two non-adjacent carbon atoms of the group. Examples of such bridged systems include, but are not limited to, bicyclo[2.2.1]heptanyl and bicyclo[2.2.2]octanyl. The cycloalkyl groups described herein can be appended to the parent molecular moiety through any substitutable carbon atom. As used herein, the term “di(alkyl)amino” refers to two independently selected alkyl groups, as defined herein, appended to the parent molecular moiety through an amino group, as defined herein. Representative examples of di(alkyl)amino include, but are not limited to, N,N-dimethylamino, N-ethyl-N-methylamino, and N-isopropyl-N-methylamino. As used herein, the term “halogen” or “halo” refers to a fluoro, chloro, bromo, or iodo radical. As used herein, the term “haloalkoxy” refers to an alkoxy group, as defined herein, substituted by one, two, three, or four halogen atoms. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy. As used herein, the term “haloalkyl” refers to an alkyl group, as defined herein, substituted by one, two, three, or four halogen atoms. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and 4,4,4,-trifluorobutyl. As used herein, the term “hydroxy” refers to an —OH group. The term “linker” refers to a divalent chemical group or combination of divalent chemical groups, where the divalent groups may comprise rings or chains. The divalent chemical group(s) are composed of atoms or combinations of atoms connected by covalent bonds. Linkers (e.g., first and second linker moieties) connect separate chemical moieties such as, for example, connecting an antibody-linking moiety (X1or X2) to the parent molecular moiety. Linkers are connected to an antibody-linking moiety and a parent molecular moiety by covalent bonds. The linker length may vary depending on the particular application. Generally, the first and second linker moieties contain at least a linear arrangement of from 2 to 50 atoms, through a combination of chain(s) and/or ring(s), where the linear arrangement may include side branching and/or substitution. The linkers may include one or more heteroatoms such as oxygen, nitrogen, sulfur, or phosphorus. Linkers may contain oxo groups, amino groups, alkyl groups, halogens and nitro groups. Linkers may also contain aryl groups. Exemplary divalent groups include —C1-12alkylene-, —(C2-6alkylene-O)—C1-6alkylene-, C3-8cycloalkylene; —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR20—, —C(R21)═N—NH—, —CH(CO2H)—, an amino acid moiety, a protected amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene are optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, halo, cyano, and hydroxy; R20and R21at each occurrence are independently hydrogen or C1-4alkyl; and x is an integer from 1 to 20. In some embodiments, a first or second linker moiety contains or consists of at least one of —C1-12alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-, or C3-8cycloalkylene bonded to the parent molecular moiety, and optionally, one or more additional divalent moieties arranged to connect to the antibody linking moiety (X1or X2), the one or more additional divalent moieties being selected from the group consisting of —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR20—, —C(R21)═N—NH—, —CH(CO2H)—, an amino acid moiety, a protected amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene are optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, halo, cyano, and hydroxy; wherein x, R20, and R21are as defined herein. The linkers may be “traceless” or “self-immolative” linkers. The term “traceless linker” or “self-immolative linker” refers to a linker wherein biotransformation of a conjugate results in spontaneous cleavage of the linker from the parent molecular moiety. Antibody linking moieties (X1or X2) are divalent chemical groups bonded to the terminal end of a first or second linker moiety, respectively, and connect to a cell binding moiety (e.g. antibody moiety, small molecule drug moiety, polynucleotide). Exemplary antibody linking moieties include —C(O)CH2—, —NHC(S)—, —NHC(O)—, —C(O)—, —CH(SO3H)—C(O)—, —NH—, or —N(C1-6alkyl)-. Antibody linking moieties may be bonded to Cb through N-terminal cysteine of Cb, through a sulfhydryl group (e.g., cysteine), through an amino (e.g., lysine, dUallylamine), through a carbonyl moiety (derived from a carboxyl in Cb), or through a ring formed by click chemistry (e.g., a triazole). For example, the antibody binding moiety is bonded to in an antibody to form a triazole moiety (e.g., The triazole is formed by [3+2]cycloaddition of an azide to an alkyne, where the azide may originate from X1a/X2aand the alkyne from the antibody, or vice versa. The term “amino acid” refers to naturally occurring and synthetic/unnatural amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. For example, the term “natural amino acid” refers to any one of the common, naturally occurring L-amino acids found in proteins, including glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), cysteine (Cys), and methionine (Met). As used herein, the term “unnatural amino acid” refers to all amino acids that are not natural amino acids as described above. Such amino acids include the D-isomers of any of the naturally occurring amino acids described above. Unnatural amino acids also include homoserine, ornithine, norleucine, and thyroxine. Unnatural amino acids as part of a cell-binding agent may contain an alkyne or azide group capable of undergoing cycloaddition with a corresponding functional group at X1a/X2ato form a triazole. Additional unnatural amino acids are well known to one of ordinary skill in the art. An unnatural amino acid may be a D- or L-isomer. An unnatural amino acid may also be an alpha amino acid or a beta amino acid. An unnatural amino acid may also be a post-translationally modified amino acid such as a phosphorylated serine, threonine or tyrosine, an acylated lysine, or an alkylated lysine or arginine. Many forms of post-translationally modified amino acids are known. One amino acid that may be used in particular is citrulline (cit), which is a precursor to arginine and is involved in the formation of urea in the liver. In some embodiments, L1bor L2bcomprise a peptide sequence cleavable by a protease expressed in tumor tissue. In some embodiments, a peptide sequence is selected from the group consisting of Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly. In particular embodiments, a peptide sequence is Val-Cit or Val-Lys. As used herein, the term “protected amino acid” refers to an amino acid side chain that additionally contains a protected functional group. Protecting groups are well known in the art and are intended to protect such functional groups as amino, hydroxy, thio, or carboxy against undesirable reactions during synthetic procedures. The protecting groups may be removed by a chemical reaction following the synthesis. Examples of protected amino acid side chains include benzyloxymethyl derived from serine, (4-methoxyphenyl)methyl derived from tyrosine, and tert-butylpropanoate derived from glutamate. As used herein, the term “amino acid side chain” refers to the group attached to the co-carbon of an amino acid. It is the characterizing portion of an amino acid and is derived from a corresponding amino acid by elimination of the NH2CHC(O)OH moiety. For example, the amino acid side chain of alanine is methyl, and the amino acid side chain of phenylalanine is phenylmethyl. An amino acid side chain may be a natural amino acid side chain or an unnatural amino acid side chain. In some embodiments, an amino acid side chain may be a protected amino acid side chain. As used herein, the term “amino protecting group” refers to a moiety that prevents chemical reactions from occurring on the nitrogen atom to which that protecting group is attached. An amino protecting group must also be removable by a chemical reaction. Such groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable amino protecting groups include, but are not limited to, carbobenzyloxy (—NHCO—OCH2C6H5or —NH-Cbz); t-butyloxycarbonyl (—NHCO—OC(CH3)3or —NH-Boc); 9-fluorenylmethyloxycarbonyl (—NH—Fmoc), 2,2,2-trichloroethyloxycarbonyl (—NH-Troc), and allyloxycarbonyl (—NH-Alloc). (In each of the above, the —NH— represents the nitrogen from the amino group that is being protected.) As used herein, the term “amino blocking group” refers to a moiety that prevents chemical reactions from occurring on the nitrogen atom to which that blocking group is attached. In contrast to an amino protecting group, an amino blocking group is not intended to be removed by a chemical reaction. Such groups include, for example, acyl groups such as acetyl (—NHCO—CH3) and succinyl (—NH—CO—CH2—CH2—COO—). (In each of the above, the —NH— represents the nitrogen from the amino group that is being blocked.) As used herein, the term “nitrogen protecting group” refers to groups intended to protect an amino group against undesirable reactions during synthetic procedures. Representative nitrogen protecting groups include acetyl, benzoyl, benzyl, benzyloxycarbonyl (Cbz), formyl, phenylsulfonyl, tert-butoxycarbonyl (Boc), tert-butylacetyl, trifluoroacetyl, and triphenylmethyl (trityl). As used herein, the term “oxo” refers to a double bonded oxygen (═O) radical. The term “saccharide” refers to a sugar or other carbohydrate, especially a simple sugar. It includes both the alpha- and the beta-anomers. The saccharide can be a C6-polyhydroxy compound, typically a C6-pentahydroxy, and often a cyclic glycal. It includes the known simple sugars and their derivatives, as well as polysaccharides with two or more monosaccharide residues. The saccharide can include protecting groups on the hydroxyl groups. The hydroxyl groups of the saccharide can be replaced with one or more acetamido, halo or amino groups. Additionally, one or more of the carbon atoms can be oxidized, for example to keto or carbonyl groups. Suitable saccharides include galactose, glucose, glucuronic acid and neurominic acid. As used herein, the term “sulfonyl” refers to S(O)2. A group is “substitutable” if it comprises at least one carbon or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition. If a group is described as being “substituted”, a non-hydrogen radical is in the place of hydrogen radical on a carbon or nitrogen of the group. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen radical is in the place of a hydrogen radical on the alkyl substituent. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen radical may be identical or different (unless otherwise stated). When a group is referred to as “unsubstituted” or not referred to as “substituted” or “optionally substituted”, it means that the group does not have any substituents. If a group is described as being “optionally substituted”, the group may be either (1) not substituted or (2) substituted. If a group is described as being optionally substituted with up to a particular number of non-hydrogen radicals, that group may be either (1) not substituted; or (2) substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the group, whichever is less. If substituents are described as being “independently selected” from a group, each substituent is selected independent of the other. Each substituent, therefore, may be identical to or different from the other substituent(s). A “payload moiety” refers to a payload radical (e.g., -A1, -A2) attached to the parent molecular moiety at a substitutable atom in the payload. In preferred embodiments, payloads have a chemically reactive functional group selected from the group consisting of a primary or secondary amine, hydroxyl, and sulfhydryl. Payloads HA1and HA2preferably are a drug (e.g., cytotoxic drug, antibiotic) or a reporter (e.g., a probe). Non-limiting examples of preferred payloads include a duocarmycin (duocarmycins and duocarmycin analogs and derivatives, CC-1065, CBI-based duocarmycin analogues, MCBI-based duocarmycin analogues, CCBI-based duocarmycin analogues), a doxorubicin (e.g., doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin), a dolastatin (e.g., dolestatin-10), combretastatin, calicheamicin, a maytansine (e.g., maytansine, maytansine analogues, DM-1, DM-4, analogs disclosed in WO2004/103272, which is incorporated herein by reference), an auristatin (e.g., auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethylauristatin E (MMAE)), a pyrrolobenzodiazepine (e.g. SGN-1882, SGN-1996, pyrrolobenzodiazepines disclosed in US2011/0256157 and U.S. Pat. No. 9,242,013, which are incorporated herein by reference), SN-38, 5-benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N8acetyl spermidine and camptothecin. In some embodiments the payload is an antibiotic agent. Suitable antibiotic agents and methods to conjugate to a cell binding agent include those described in WO2014/193722 and WO2014/194247, which are incorporated herein by reference. Other drug conjugates provided by the invention are conjugates of synthetic glucocorticoids (e.g., dexamethasone), kinase inhibitors (e.g., dasatinib), and LXR nuclear receptor agonists, as described by Liu et al, Expert Opinion of Biological Therapy (2016) 16(5), 591-593, which is incorporated herein by reference. As used herein, the term “reporter moiety” refers to a moiety that, under appropriate conditions directly or indirectly generates a detectable signal. Exemplary reporter moieties include, but are not limited to, fluorophores, luminescent molecules, chemiluminescent molecules, dyes, radiolabels, colorimetric molecules, and substrates for enzymes such as luciferases. In some embodiments, a reporter moiety may indirectly generate a detectable signal, for example, when the reporter moiety is a substrate for an enzyme, e.g., a luciferase. The reaction of the enzyme with the substrate then produces a detectable signal such as fluorescence or luminescence. As used herein, the term “bioluminescent reporter moiety” may refer to a moiety that is a substrate for a luciferase. For example, the bioluminescent reporter moiety can be a luciferin, a luciferin derivative, e.g., pre-luciferin, aminoluciferin, quionolyl-luciferin, napthyl luciferin, fluorolucifeirn, chloroluciferin, precursors of luciferin derivatives, a coelenterazine, or a coelenterazine derivative or analog, e.g., furimazine. The luminescent signal generated may be detected using a luminometer. As used herein, the term “fluorescent reporter moiety” may refer to a moiety that fluoresces. For example, the fluorescent reporter moiety may be a fluorophore, such as coumarin, R110, fluoroscein, DDAO, resorufin, cresyl violet, sily xanthene, or carbopyronine. Fluorescence may be detected using a fluorometer. Colorimetric payload release may be detected as an increase in absorbance at a specified wavelength. In certain embodiments, the reporter moiety is a bioluminescent reporter moiety. In certain embodiments, the reporter moiety is a luciferin, a luciferin derivative or analog, a preluciferin or analog, coelenterazine, or a coelenterazine derivative or analog. In some embodiments, the reporter moiety is luciferin, pro-luciferin, aminoluciferin, quionolyl-luciferin, napthyl luciferin, chloroluciferin, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine (“coelenterazine-hh”), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl coelenterazine, in addition to those disclosed in WO 2003/040100, U.S. Patent Publication No. 20080248511, U.S. Patent Publication No. US 20120117667, and U.S. Patent Publication No. US 2015/0307916, the disclosures of which are incorporated by reference herein. In certain embodiments, the reporter moiety is a fluorescent reporter moiety. In certain embodiments, the reporter moiety is a coumarin, R110, fluoroscein, DDAO, resorufin, cresyl violet, sily xanthene, or carbopyronine. In some embodiments, the reporter moiety is rhodamine 123, rhodamine X, Alexa dyes (e.g., Alexa Fluor-350, -430, -488, -and -660), DyLight 594, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), fluorescein, 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-Fam), 5- or 6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET), 5- or 6-carboxy-4′5′2′4′5′7′ hexachlorofluorescein (HEX), 5′ or 6′-carboxy-4′, 5′-dichloro-2,′7′-dimethoxyfluorescein (JOE), 6-JOE, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE) rhodol, fluorescein isothiocyanate, coumarin, 7-amino-4-methylcoumarin, aminocoumarin, hydroxycoumarin, silyl xanthene, or carbopyronine. Payload moieties -A1may be attached to the parent molecule by a substitutable oxygen or sulfur. For example, representative drug payload moieties -A1include Payload moieties -A2may be attached to the parent molecule by a substitutable oxygen, nitrogen, or sulfur atom. For example, representative drug payload moieties -A2include Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exoforms; R-, S-, and mesa-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”). included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including1H,2H (D), and3H (T); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like. A cell binding agent may be of any kind and include peptides and non-peptides. These can include antibodies or a fragment(s) of an antibody that contains at least one binding site, lymphokines, hormones, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance (e.g., a protein, a carbohydrate, nucleic acid, etc.). The cell binding agent may be a nucleotide or polynucleotide including nucleic acids such as DNA and RNA, and those that include dU allylamine nucleotides. The cell binding agent may be, or comprise, a polypeptide. The polypeptide may be a cyclic polypeptide. Other proteins/peptide include ApoB, insulin, transferrin, and siderophores (e.g., for use with antibiotics). Siderophores are described, for example, by de Carvalho et al., Frontiers in Microbiology (June 2014), 5 (290), doi 10.3389/fmicb.2014.00290, Starr et al., J. Med. Chem. (2014) 57, 3845-3855, and Miller et al., Biol. Met. (1991) 4(1), 62-9, which are incorporated herein by reference. The cell binding agent may be a small molecule drug as described in Casi et al., J. Med. Chem. (2015) 58 (22), 8751-8761 and Srinivasarao et al., Nat. Rev. Drug Discovery (2015), 14, 203-219, which are incorporated herein by reference. For example, the small molecule drug may be folate. The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e. “antigen-binding portion”) or a single chain(s) thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2, and CH3, and may be of the mu, delta, gamma, alpha, or epsilon isotype. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, C°, which may be of the kappa or lambda isotype. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The terms “cell binding moiety” and “antibody moiety” refer to a cell binding radical or antibody radical onto which a group (e.g., G) is substituted. For example, a group G in Ab-(G)pis in place of a hydrogen radical on the antibody. Thus, an antibody moiety (or cell binding moiety) includes amino acid side chain heteroatoms (e.g., —NH— from a lysine; —S— from a cysteine) that are bonded to G. In the case of engineered antibodies bearing unnatural amino acid side chains (e.g., that contain an alkyne or azido), the antibody moiety includes those atoms of the conjugate that are derived from the unnatural amino acid side chain. For example, in the case of an alkyne-containing side chain conjugated to an azido by Click chemistry, the resulting alkene-diyl fragment is considered part of the antibody moiety (e.g., The antibody may be a multispecific antibody, a human antibody, a humanized antibody (fully or partially humanized), an animal antibody such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), a recombinant antibody, a chimeric antibody, a single-chain Fv (“scFv”), a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′) fragment, a F(ab′)2 fragment, a disulfide-linked Fv (“sdFv”), and an anti-idiotypic (“anti-Id”) antibody, a dual-domain antibody, a dual variable domain (DVD) or a triple variable domain (TVD) antibody (dual-variable domain immunoglobulin, and functionally active epitope-binding fragment of any of the above. In particular, an antibody includes an immunoglobulin molecule and an immunologically active fragment of an immunoglobulin molecule, namely, a molecule that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. A wide variety of tumor-specific or other disease-specific antigens have been identified and antibodies to those antigens have been used or proposed for use in the treatment of such tumors or other diseases. The antibodies that are known in the art can be used in the conjugates of the invention, in particular, for the treatment of the disease with which the target antigen is associated. Non-limiting examples of target antigens (and their associated diseases) to which an antibody-linker-drug conjugate of the invention can be targeted include: Her2 (breast cancer), CD20 (lymphomas), EGFR (solid tumors), CD22 (lymphomas, including nonHodgkin's lymphoma), CD52 (chronic lymphocytic leukemia), CD33 (acute myelogenous leukemia), CD4 (lymphomas, autoimmune diseases, including rheumatoid arthritis), CD30 (lymphomas, including non-Hodgkin's lymphoma), Muc18 (melanoma), integrins (solid tumors), PSMA (prostate cancer, benign prostatic hyperplasia), CEA (colorectal cancer), CD11a (psoriasis), CD80 (psoriasis), CD23 (asthma), CD40L (immune thromobcytopenic purpura), CTLA4 (T cell lymphomas) and BLys (autoimmune diseases, including systemic lupus erythematosus). Example antibodies are also found in Table 1. TABLE 1AntigensAntibodiesCD30Ch IgG1CD22Hz IgG4CD33Hz IgG4CD19Hz IgG1CD138Ch IgG4CD22Hz IgG1CD79bHz IgG1CD74Hz IgG1HER2Hz IgG1GPNMBHu IgG2PSMAHu IgG1CD56Hz IgG1SLC44A4Hu IgG2CA6Hu IgG1CA-IXHu IgG1MesothelinHu IgG1CD70Hu IgG1CD66e/Hz IgG1CEACAM5Nectin-4Hu IgG1Ch: chimeric;Hz: humanized;Hu: fully human;GPNMB: Glycoprotein NMB;PSMA: prostate specific membrane antigen. The antibody may be a therapeutic antibody such as Trastuzumab (Herceptin), Abciximab (ReoPro), Adalimumab (Humira), Alemtuzumab (Campath), Basiliximab (Simulect), Belimumab (Benlysta), Bevacizumab (Avastin), Brentuximab vedotin (Adcetris), Canakinumab (Ilaris), Cetuximab (Erbitux), Certolizumab pegol (Cimzia), Daclizumab (Zenapax), Denosumab (Prolia, Xgeva), Eculizumab (Soliris), Efalizumab (Raptiva), Gemtuzumab (Mylotarg), Golimumab (Simponi), Ibritumomab tiuxetan (Zevalin), Infliximab (Remicade), Ipilimumab (MDX-101; Yervoy), Muromonab-CD3 (Orthoclone OKT3), Natalizumab (Tysabri), Ofatumumab (Arzerra), Omalizumab (Xolair), Palivizumab (Synagis), Panitumumab (Vectibix), Ranibizumab (Lucentis), Rituximab (Rituxan, Mabthera), Tocilizumab (or Atlizumab) (Actemra and RoActemra), and Tositumomab (Bexxar). 2. Conjugates Disclosed herein are quinone-containing conjugates of formula (I). The intracellular space is a reducing environment that may reduce the quinone moiety. Upon uptake of conjugates of the invention by cells, the quinone may be reduced by intracellular reduction potential to tracelessly release the payload (e.g., antimitotic agent), i.e., meaning no residual linker atoms are left on the payload moiety. As described in US2015/0307916, a quinone-linked cargo molecule (e.g. luciferin, a fluorophore or furimazine) was efficiently released upon entry into cells. Quinone moieties also appear to be stable in a non-reducing environment suggesting they would be stable outside of target cells, for example, in the blood stream. Since quinone reduction followed by traceless elimination of a covalent adduct appears to occur rapidly and efficiently in all the cell types tested so far, and quinone moieties are expected to be stable in the blood stream, quinone-containing conjugates may provide a universal means for efficiently and selectively releasing payloads inside of target cells. Payloads HA1attached by a substitutable oxygen or sulfur atom may be released as shown in Scheme 1 upon intracellular reduction of the quinone moiety. Alternatively, payloads HA1attached by an oxygen or sulfur atom may be released as shown in Scheme 2a upon intracellular reduction of the quinone moiety. Payloads HA2attached by an oxygen, sulfur, or substitutable nitrogen atom via self-immolative substitutable benzyl linker may be released upon intracellular quinone reduction as shown in Scheme 2b. In another variation, an antibody may be attached to one side of the quinone through a second L2linker while payloads may be attached to the other side of the quinone as shown in Schemes 3a-3c. Payloads HA2directly attached by an oxygen, sulfur, or substitutable nitrogen atom may be released as shown in Scheme 3a upon intracellular reduction of the quinone moiety. Similarly, payloads HA1attached by an oxygen or sulfur atom via self-immolative amine-linker may be released upon intracellular quinone reduction as shown in Scheme 3b. Payloads HA2attached by an oxygen, sulfur, or substitutable nitrogen atom via self-immolative unsubstituted or substituted benzyl linker may be released upon intracellular quinone reduction as shown in Scheme 3c. According to one aspect of the invention are conjugates of formula (I). In some embodiments, formula (I) is Ab-(G)p, wherein Ab is an antibody moiety, and G and p are as defined herein. In some embodiments, the cell binding moiety is a small molecule drug moiety, where the parent small molecule drug has affinity for a target receptor on a cell. Representative examples of small molecule drugs and target receptors include folate (folate receptor), somatostatin analogs (somatostatin receptor), and aromatic sulfonamides (carbonic anhydrase IX), as described in Casi et al., J. Med. Chem. (2015) 58 (22), 8751-8761 and Srinivasarao et al., Nat. Rev. Drug Discovery (2015), 14, 203-219. Preferably, p is 1, when Cb is a small molecule drug moiety. In preferred embodiments, the small molecule drug moiety is folate. In some embodiments of formula (I), G is formula (II), (III), or (IV) and A1and/or A2is an anticancer drug. In further embodiments, formula (I) is Ab-(G)p, wherein Ab is an antibody moiety, p is as defined herein, G is formula (II), (III), or (IV), and A1and/or A2is an anticancer drug. In some embodiments, G is formula (II), (III), or (IV) and A1and/or A2is a reporter moiety. In preferred embodiments, G is formula (III) or (IV) and A1and/or A2is a reporter moiety. In further embodiments, formula (I) is Ab-(G)p, wherein Ab is an antibody moiety, p is as defined herein, G is formula (II), (III), or (IV), and A1and/or A2is a reporter moiety, as defined herein. In preferred embodiments, formula (I) is Ab-(G)p, wherein Ab is an antibody moiety, p is as defined herein, G is formula (III) or (IV), and A1and/or A2is a reporter moiety. In other embodiments, Cb is a small molecule drug moiety, p is 1, and A1and/or A2is a reporter moiety, as defined herein. In further embodiments, the small molecule drug moiety is folate. In some embodiments, X1or X2is —C(O)CH2—, —NHC(S)—, —NHC(O)—, —C(O)—, —CH(SO3H)—C(O)—, —NH—, or —N(C1-6alkyl)-. In some embodiments, X1or X2is or —C(O)CH2-bonded to Cb through a sulfhydryl group (e.g., cysteine) in Cb; or X1or X2is —NHC(S)—, —NHC(O)—, —C(O)—, or —CH(SO3H)—C(O)— bonded to Cb through an amino (e.g., lysine) in Cb; or X1or X2is or X1or X2is bonded to Cb through N-terminal cysteine of Cb; or X1or X2is —NH—, or —N(C1-6alkyl)- bonded to Cb through a carbonyl of Cb. In further embodiments, X1or X2is —NH—, or —N(C1-6alkyl)- bonded to folate through a carbonyl derived from a carboxyl of folate (i.e. In some embodiments, a first or second linker moiety contains or consists of at least one of —C1-12alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-, or C3-8cycloalkylene bonded to the parent molecular moiety, and optionally one or more additional divalent moieties covalently linked to the antibody linking moiety (X1or X2), the one or more additional divalent moieties being selected from the group consisting of —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR20—, —C(R21)═N—NH—, —CH(CO2H)—, an amino acid moiety, a protected amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene are optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C14alkoxy, halo, cyano, or hydroxy; wherein x, R20and R21are as defined herein. In some embodiments, L1is L1aor L1a-L1b, wherein L1bis bonded to X1; L1ais —C1-12alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-, or C3-8cycloalkylene; L1bcomprises, or consists of, one or more covalently bonded divalent members, the one or more divalent members being selected from the group consisting of —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR20—, —C(R21)═N—NH—, —CH(CO2H)—, an amino acid moiety, a protected amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene of L1aand/or L1bis optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, halo, cyano, or hydroxy; R20and R21at each occurrence are independently hydrogen or C1-4alkyl; and x is an integer from 1 to 20. In further embodiments included in the foregoing, L1bis —C(O)NR20-L1c-, —NR20C(O)-L1c-, —NR20C(O)O-L1c-, —C(O)-L1c-, —S-L1c-, —S(O)-L1c-, —S(O)2-L1c-, or —NR20-L1c-; and L1cis —C1-6alkylene-, —C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-, C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In further embodiments included in the foregoing, L1bis —C(O)NR20-L1c-, -cit-val-C(O)C1-6alkylene-, —NR20C(O)—, —NR20C(O)-L1c-, —NR20C(O)O-L1c-, -ala-val-C(O)C1-6alkylene-, -cit-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, -ala-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, —S—S—C1-6alkylene-, —NH—N═C(R21)-phenylene-O—C1-6alkylene-, —C1-6alkylene-, —C2-6alkylene-O—, or C3-8cycloalkylene, and L1cis —C1-6alkylene-, —C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-, C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In further embodiments included in the foregoing, L1a-L1bis —C1-12alkylene-C(O)NR20-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-C(O)NR20-L1c-, —C3-8cycloalkylene-C(O)NR20-L1c-, —C1-12alkylene-NR20C(O)-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-NR20C(O)-L1c-, —C3-8cycloalkylene-NR20C(O)-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-cit-val-C(O)C1-6alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-ala-val-C(O)C1-6alkylene-, —C1-12alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-12alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-C3-8cycloalkylene-, or —(C2-6alkylene-O)x—C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-. In further embodiments included in the foregoing —(C2-6alkylene-O)x—C1-6alkylene—in L1ais —(CH2CH2O)x—C1-6alkylene-. In further embodiments included in the foregoing, Lais —(CH2CH2O)1-2—CH2CH2—; L1bis —C(O)NR20-L1c-, -ala-val-C(O)(CH2)5—, -cit-val-C(O)(CH2)5—, or and L1cis —CH2CH2—, —CH2CH2—NR20C(O)—CH2CH2—S—S—CH(CH3)CH2CH2—, or —CH2CH2—NR20C(O)NH—N═C(CH3)-1,4-phenylene-O—CH2CH2CH2—, or In further embodiments included in the foregoing, L1b-X1is —C(O)NR20—C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-C(O)—, —C(O)NR20—C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-CH(SO3H)—C(O)—, —C(O)NR20—C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-C(O)—, or In further embodiments included in the foregoing, L1-X1is In other embodiments included in the foregoing are the following further representative L1-X1: In certain embodiments, x is 2, 3, or 4. In some embodiments, L2is L2aor L2a-L2b, wherein L2bis bonded to X2; L2ais —C2-6alkylene-; L2bcomprises, or consists of, one or more covalently bonded divalent members, the one or more divalent members being selected from the group consisting of —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR30—, —C(R31)═N—N—, —CH(CO2H)—, an amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene of L1bare optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C14alkoxy, halo, cyano, or hydroxy; and R30and R31at each occurrence are independently hydrogen or C1-4alkyl. In further embodiments included in the foregoing, L2bis —C(O)NR30-L2c-, —NR30C(O)-L2c-, —NR30C(O)O-L2c-, —C(O)-L2c-, —O-L2c-, —S-L2c-, —S(O)-L2c-, —S(O)2-L2c-, or —NR30-L2c-; and L2cis —C1-6alkylene-, —C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR30C(O)NH—N═C(R31)-phenylene-O—C1-6alkylene-, —C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In further embodiments included in the foregoing, L2bis —C(O)NR30-L2c-, -cit-val-C(O)C1-6alkylene-, —NR30C(O)—, —NR30C(O)-L2c-, —NR30C(O)O-L2c-, -ala-val-C(O)C1-6alkylene-, -cit-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, -ala-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, —S—S—C1-6alkylene-, —NH—N═C(R31)-phenylene-O—C1-6alkylene-, —C1-6alkylene-, —C2-6alkylene-O—, or C3-8cycloalkylene, and L2cis —C1-6alkylene-, —C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR30C(O)NH—N═C(R31)-phenylene-O—C1-6alkylene-, —C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In further embodiments included in the foregoing, L2b-X2is —C(O)NR30—C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-C(O)—, —C(O)NR30—C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-CH(SO3H)—C(O)—, —C(O)NR30—C1-6alkylene-NR30C(O)NH—N═C(R31)-phenylene-O—C1-6alkylene-C(O)—, or In some preferred embodiments according to formula (I), R9and R10are methyl. In other preferred embodiments according to formula (I), R11, R12, and R13are each methyl. In some embodiments according to formula (I), R14is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R14is —C2-C30-alkylene-CO2H. In certain embodiments, R14is —CH2CO2H; —(CH2)2CO2H; —(CH2)3CO2H; —(CH2)4CO2H; —(CH2)5CO2H; —(CH2)6CO2H; —(CH2)7CO2H; —(CH2)8CO2H; —(CH2)9CO2H; —(CH2)10CO2H; —(CH2)11CO2H; —(CH2)12CO2H; —(CH2)13CO2H; —(CH2)14CO2H; —(CH2)15CO2H; —(CH2)16CO2H; —(CH2)17CO2H; —(CH2)18CO2H; —(CH2)19CO2H; —(CH2)20CO2H; —(CH2)21CO2H; —(CH2)22CO2H; —(CH2)23CO2H; —(CH2)24CO2H; —(CH2)25CO2H; —(CH2)26CO2H; —(CH2)27CO2H; —(CH2)28CO2H; —(CH2)29CO2H; or —(CH2)30CO2H. In certain embodiments, R14is —(CH2)15CO2H. In preferred embodiments, R14is methyl. In some embodiments, R15is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R15is CH2CO2H; (CH2)2CO2H; (CH2)3CO2H; (CH2)4CO2H; (CH2)5CO2H; (CH2)6CO2H; (CH2)7CO2H; (CH2)8CO2H; (CH2)9CO2H; (CH2)10CO2H; (CH2)11CO2H; (CH2)12CO2H; (CH2)13CO2H; (CH2)14CO2H; (CH2)15CO2H; (CH2)16CO2H; (CH2)17CO2H; (CH2)18CO2H; (CH2)19CO2H; (CH2)20CO2H; (CH2)21CO2H; (CH2)22CO2H; (CH2)23CO2H; (CH2)24CO2H; (CH2)25CO2H; (CH2)26CO2H; (CH2)27CO2H; (CH2)28CO2H; (CH2)29CO2H; or (CH2)30CO2H. In certain embodiments, R15is —(CH2)15CO2H. In other preferred embodiments, R17is hydrogen. In preferred embodiments, one of m and n is 1, and the other is 0. In certain embodiments, x is 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In certain embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In preferred embodiments, G is formula (II). In further embodiments formula (II) is formula (IIA), wherein R9, R10, R11, R12, R13, R14, L1, X1, and A1are as defined herein. In some embodiments, L1-X1is In certain embodiments, x is 2, 3, or 4. In other preferred embodiments are conjugates wherein G is formula (IIB) wherein L1, X1, and A1are as defined herein. In other preferred embodiments are conjugates wherein G is formula (IIB-1) wherein x, X1, and A1are as defined herein. In further embodiments according to (IIB-1), x is 2, 3, or 4. In further embodiments according to the foregoing, X1is or —C(O)—. In preferred embodiments, G is formula (III). In further embodiments formula (III) is formula (IIIA), wherein R9, R10, R11, R13, R14, L2, X2, Y, and A1are as defined herein. In other preferred embodiments are conjugates wherein G is formula (IIIB) wherein L2, X2, Y, and A1are as defined herein. In further embodiments formula (III) is formula (IIIC), wherein R9, R10, R11, R13, L2, X2, and A2are as defined herein. In other preferred embodiments are conjugates wherein G is formula (IIID) wherein L2, X2, and A2are as defined herein. In further embodiments formula (III) is formula (IIIE), wherein R9, R10, R11, R13, R14, R16, L2, X2, and A2are as defined herein. In other preferred embodiments are conjugates wherein G is formula (IIIF) wherein L2, X2, and A2are as defined herein. In preferred embodiments, G is formula (IV). In further embodiments formula (IV) is formula (IVA), wherein R9, R10, R11, R12, R13, R16, L1, X1, and A2are as defined herein. In some embodiments, L1-X1is In certain embodiments, x is 2, 3, or 4. In preferred embodiments, G is formula (V), wherein R9, R10, R11, R12, R13, R17, L1, X1, Y, A1, m, and n are as defined herein. In some embodiments, L1-X1is In certain embodiments, x is 2, 3, or 4. In further embodiments, formula (V) is formula (VA), wherein R9, R10, R11, R12, R13, R15, L1, X, and A1are as defined herein. In some embodiments, L1-X1is In certain embodiments, x is 2, 3, or 4. In other preferred embodiments are conjugates wherein G is formula (VA-1) wherein R9, R10, R11, R12, R13, L1, X1, and A1are as defined herein. In other preferred embodiments are conjugates wherein G is formula (VA-2) wherein x, X1, and A1are as defined herein. In further embodiments according to (VA-2), x is 2, 3, or 4. In further embodiments according to the foregoing, X1is or —C(O)—. In some embodiments, p is a number from 1 to 10. In other embodiments, p is a number from 1 to 5. In still other embodiments, p is a number from 2-5. In still other embodiments, p is 3, 4, or 5. In a composition comprising a plurality of conjugates of formula (I), p represents the average number of groups G per antibody, also referred to as a drug-to-antibody ratio (DAR). Thus, in a composition comprising a plurality of conjugates p may have non-integer values (e.g., 3.5). In some embodiments, the DAR is from 2-5. In still other embodiments, the DAR is from 3-5. The preferred DAR may vary according to the particular antibody, payload, linker, and condition to be treated. 3. Conjugation Reagents According to other aspects of the invention are conjugation reagents and their chemical precursors for coupling with an antibody or other cell binding agent. In the conjugation reagents, X1a/X2ais a reactive functional group suitable for coupling with an amino, sulfhydryl, or N-terminal cysteine of an antibody; or a non-natural amino acid comprising an azido- or alkyne-containing side chain in a modified antibody; or X1a/X2ais a protected reactive functional group, or is a chemical precursor to the reactive functional group. Additionally, the conjugation reagents may be used to label non-biological agents, including but not limited to, solid supports/surfaces, nanoparticles, such as [60] fullerene, core-shell nanoparticles, liposome, dendrimer, and gold nanoparticles, and detergents. The detergent may be comprised within a liposome and/or solid-lipid nanoparticle (SLN) that may optionally be used to test drug delivering efficiency. The detergent may be a transfecting reagent such as Lipofectamine 2000 or Fugene 6. In some aspects, the conjugation reagents can be conjugated with a nucleoside, nucleotide, or a polynucleotide. The conjugation reagents of the invention may be conjugated with a nucleoside, nucleotide, or polynucleotide in any way known to one of ordinary skill in the art such as through a phosphoramidite, an activated ester, or a reactive platinum complex. In certain embodiments, the labeling conjugation reagents may be conjugated to an agent using an activated carboxylic acid such as an NHS ester, pentafluorobenzene ester, an anhydride, an acetyl chloride, or using direct carboxylic acid and amine coupling reactions, or click chemistry, or maleimide, or activated carbonate, or phosphoramidite. For example, a conjugation reagent can be attached to dU allylamine phosphoamidite and further incorporated in oligomers of interest by a traditional phosphoamidite chemistry. Alternatively, dU allylamine modified oligomers of interest can be labeled with the conjugation reagent through an activated ester by post labeling. If the labeled oligomers are primers of interest, they can also be used to amplify a sequence of interest through PCR. In other aspects the conjugation reagents include compounds of formulas 7 or 10, or their salts, wherein R9, R10, R11, R12, R13, R14, R17, L1, X1a, G2, m, n′, and A1are as defined herein. In some embodiments, L1is L1aor L1a-L1b, wherein L1bis bonded to X1a; L1ais —C1-12alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-, or C3-8cycloalkylene; L1bcomprises, or consists of, one or more covalently bonded divalent members, the one or more divalent members being selected from —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR20—, —C(R21)═N—NH—, —CH(CO2H)—, an amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene of L1aand/or L1bis optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, halo, cyano, or hydroxy; R20and R21at each occurrence are independently hydrogen or C1-4alkyl; and x is an integer from 1 to 20. In other embodiments included in the foregoing, L1bis —C(O)NR20-L1c-, —NR20C(O)-L1c-, —NR20C(O)O-L1c-, —C(O)-L1c-, —O-L1c-, —S-L1c-, —S(O)-L1c-, —S(O)2-L1c-, or —NR20-L1c-; and L1cis —C1-6alkylene-, —C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-, C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In other embodiments included in the foregoing, L1bis —C(O)NR20-L1c-, -cit-val-C(O)C1-6alkylene-, —NR20C(O)—, —NR20C(O)-L1c-NR20C(O)O-L1c-, -ala-val-C(O)C1-6alkylene-, -cit-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, -ala-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, —S—S—C1-6alkylene-, —NH—N═C(R21)-phenylene-O—C1-6alkylene-, —C1-6alkylene-, —C2-6alkylene-O—, or C3-8cycloalkylene, and L1cis —C1-6alkylene-, —C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-, C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In other embodiments included in the foregoing, L1a-L1bis —C1-12alkylene-C(O)NR20-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-C(O)NR20-L1c-, C3-8cycloalkylene-C(O)NR20-L1c, C1-12alkylene-NR20C(O)-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-NR20C(O)-L1c-, —C3-8cycloalkylene-NR20C(O)-L1c-, —(C2-6alkylene-O)x—C1-6alkylene-cit-val-C(O)C1-6alkylene-, —(C2-6alkylene-O)—C1-6alkylene-ala-val-C(O)C1-6alkylene-, —C1-12alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-2alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —(C2-6alkylene-O)x—C1-6alkylene-C3-8cycloalkylene-, or —(C2-6alkylene-O)x—C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-. In other embodiments included in the foregoing, L1ais —(CH2CH2O)1-2—CH2CH2—; L1bis —C(O)NR20-L1c-, -ala-val-C(O)(CH2)5—, -cit-val-C(O)(CH2)5—, or and L1cis —CH2CH2—, —CH2CH2—NR20C(O)—CH2CH2—S—S—CH(CH3)CH2CH2—, or —CH2CH2—NR20C(O)NH—N═C(CH3)-1,4-phenylene-O—CH2CH2CH2—, or In other embodiments included in the foregoing, L1b-X1ais —C(O)NR20—C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-C(O)—OR40, —C(O)NR20—C1-6alkylene-NR20C(O)—C1-6alkylene-S—S—C1-6alkylene-CH(SO3H)—C(O)—OR40, —C(O)NR20—C1-6alkylene-NR20C(O)NH—N═C(R21)-phenylene-O—C1-6alkylene-C(O)—OR40, or In other embodiments included in the foregoing, L1-X1ais wherein R40is as defined herein. In certain embodiments, R40is hydrogen. In certain embodiments, R40is In certain embodiments, x is 2, 3, or 4. In certain embodiments included in the foregoing, L1-X1ais In some preferred embodiments according to formula (7) or (10), R9and R10are methyl. In other preferred embodiments according to formula (7) or (10), R11, R12, and R13are each methyl. In some embodiments according to formula (7), R14is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R14is —C2-C30-alkylene-CO2H. In certain embodiments, R14is —CH2CO2H; —(CH2)2CO2H; —(CH2)3CO2H; —(CH2)4CO2H; —(CH2)5CO2H; —(CH2)6CO2H; —(CH2)7CO2H; —(CH2)8CO2H; —(CH2)9CO2H; —(CH2)10CO2H; —(CH2)11CO2H; —(CH2)12CO2H; —(CH2)13CO2H; —(CH2)14CO2H; —(CH2)15CO2H; —(CH2)16CO2H; —(CH2)17CO2H; —(CH2)18CO2H; —(CH2)19CO2H; —(CH2)20CO2H; —(CH2)21CO2H; —(CH2)22CO2H; —(CH2)23CO2H; —(CH2)24CO2H; —(CH2)25CO2H; —(CH2)26CO2H; —(CH2)27CO2H; —(CH2)28CO2H; —(CH2)29CO2H; or —(CH2)30CO2H. In certain embodiments, R14is —(CH2)15CO2H. In preferred embodiments, R14is methyl. In some embodiments according to formula (10), G2is Y is NR15; and R15is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R15is —C2-C30-alkylene-CO2H. In certain embodiments, R15is CH2CO2H; (CH2)2CO2H; (CH2)3CO2H; (CH2)4CO2H; (CH2)5CO2H; (CH2)6CO2H; (CH2)7CO2H; (CH2)8CO2H; (CH2)9CO2H; (CH2)10CO2H; (CH2)11CO2H; (CH2)12CO2H; (CH2)13CO2H; (CH2)14CO2H; (CH2)15CO2H; (CH2)16CO2H; (CH2)17CO2H; (CH2)18CO2H; (CH2)19CO2H; (CH2)20CO2H; (CH2)21CO2H; (CH2)22CO2H; (CH2)23CO2H; (CH2)24CO2H; (CH2)25CO2H; (CH2)26CO2H; (CH2)27CO2H; (CH2)28CO2H; (CH2)29CO2H; or (CH2)30CO2H. In certain embodiments, R15is —(CH2)15CO2H. In preferred embodiments, R15is methyl. In other preferred embodiments of formula (7) or (10), R17is hydrogen. In preferred embodiments of formula (7) or (10), m is 0 and n′ is 0. In certain embodiments of formula (7) or (10), x is 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In certain embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the compound of formula (7) has formula (7A), or a salt thereof, wherein R9, R10, R11, R12, R13, R14, R40, x, and A1are as defined above. In certain embodiments, the compound of formula (7) has formula (7B), or a salt thereof, wherein z is an integer selected from 1 to 30; and R9, R10, R11, R12, R13, R14, and A1are as defined above. In certain embodiments, z is 16. In other preferred embodiments are compounds of formula (7C) wherein L1, X1a, and A1are as defined herein. In other preferred embodiments are compounds of formula (7E) wherein x, X1a, and A1are as defined herein. In further embodiments according to (7E), x is 2, 3, or 4. In further embodiments according to the foregoing, X1ais or —C(O)OR40 In certain embodiments, the compound of formula (10) has formula (10A) wherein R9, R10, R11, R12, R13, R17, Y, m′, n, L1, X1a, and A1are as defined herein. In certain embodiments, the compound of formula (10) has formula (10A-a), or a salt thereof, wherein R9, R10, R11, R12, R13, R15, R40, x, and A1are as defined above. In certain embodiments, the compound of formula (10) has formula (10A-b), or a salt thereof, wherein z is an integer selected from 1 to 30; and R9, R10, R11, R12, R13, R15, and A1are as defined above. In certain embodiments, z is 16. In other preferred embodiments are compounds of formula (10A-c) wherein L1, X1a, and A1are as defined herein. In other preferred embodiments are compounds of formula (10A-d) wherein x, X1a, and A1are as defined herein. In further embodiments according to (10A-d), x is 2, 3, or 4. In further embodiments according to the foregoing, X1ais or —C(O)OR40 In certain embodiments, the compound of formula (10) has formula (10B) wherein R9, R10, R11, R12, R13, R16, L1, X1a, and A2are as defined above. In other preferred embodiments are compounds of formula (10B-a), wherein R9, R10, R11, R12, R13, L1, X1a, and A2are as defined above. In other preferred embodiments are compounds of formula (10B-b), wherein R9, R10, R11, R12, R13, R40, z, and A2are as defined above. In other preferred embodiments are compounds of formula (10B-c), wherein x, X1a, and A2are as defined above. In another aspect the conjugation reagents are compounds of formula 12, or their salts, G1is -A2, R9and R10are each independently selected from C1-4alkyl; R11and R13are each independently selected from the group consisting of H, C1-4alkyl, C1-4alkoxy, bromo, chloro and amino; L2is a second linker moiety; Y is O or NR15; R14and R15are each independently H, C1-30alkyl optionally substituted with 1-8 halogens, —C1-30alkylene-OH, —C1-30alkylene-C1-4alkoxy, —C1-30alkylene-COOH, or —C1-30alkylene-amido; R16, at each occurrence, is independently H, halogen, CH3, OCH3, or NO2; R17, at each occurrence, is independently H or C1-4alkyl, or both R17together with the carbon to which they are attached form a cycloalkyl ring having from 3-7 carbons; m is an integer from 0-2; n is an integer from 0-2; X2ais —OSO2R50, OH, —Cl, —Br, —I, —N3, —C≡CH, —CN, COOH, —COOR40, —COJ1, —CH(SO3H)—C(O)OR40, —NCO, —NCS, —C(O)CH2J1, —NH2, or —NH(C1-6alkyl); R40is C1-6alkyl, 4-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, —C(O)—OR41, or R41is C1-6alkyl or phenyl; J1is —Cl, —Br, —I, or —OSO2R50; R50is C1-6alkyl, C1-6haloalkyl, phenyl, 4-methylphenyl, 4-nitrophenyl, or 4-bromophenyl; A1is a payload moiety bonded through a substitutable oxygen or sulfur atom; and A2is a payload moiety bonded through a substitutable oxygen, sulfur, or nitrogen atom. In some embodiments, L2is L2aor L2a-L2b, wherein L2bis bonded to X2a; L2ais —C2-6alkylene-; L2bcomprises, or consists of, one or more covalently bonded divalent members, the one or more divalent members being selected from the group consisting of —C1-6alkylene-, —C2-6alkylene-O—, C3-8cycloalkylene, —C(O)—, —O—, —S—, —S(O)—, —S(O)2—, —NR30—, —C(R31)═N—N—, —CH(CO2H)—, an amino acid moiety, and phenylene; wherein the C3-8cycloalkylene and phenylene of L1bare optionally independently substituted with 1-4 substituents independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, C14alkoxy, halo, cyano, and hydroxy; and R30and R31at each occurrence are independently hydrogen or C1-4alkyl. In further embodiments included in the foregoing, L2bis —C(O)NR30-L2c-, -cit-val-C(O)C1-6alkylene-, —NR30C(O)—, —NR30C(O)-L2c-, —NR30C(O)O-L2c-, -ala-val-C(O)C16alkylene-, -cit-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, -ala-val-C(O)O—C2-6alkylene-O—C1-6alkylene-, —S—S—C1-6alkylene-, —NH—N═C(R31)-phenylene-O—C1-6alkylene-, —C1-6alkylene-, —C2-6alkylene-O—, or C3-8cycloalkylene, and L2c is —C1-6alkylene-, —C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-, —C1-6alkylene-NR30C(O)NH—N═C(R31)-phenylene-O—C1-6alkylene-, —C3-8cycloalkylene, —(C2-6alkylene-O)—C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-, —C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-C3-8cycloalkylene-C1-6alkylene-, —C1-6alkylene-S—S—C1-6alkylene-, or In further embodiments included in the foregoing, L2b-X2ais —C(O)NR30—C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-C(O)—OR40, —C(O)NR30—C1-6alkylene-NR30C(O)—C1-6alkylene-S—S—C1-6alkylene-CH(SO3H)—C(O)—OR40, —C(O)NR30—C1-6alkylene-NR30C(O)NH—N═C(R31)-phenylene-O—C1-6alkylene-C(O)—OR40, or In some embodiments included in the foregoing, L2is L2a-L2b; L2bis —C(O)NR30-L2c- or —C(O)NR30—. In still further embodiments included in the foregoing L2ais —CH2CH2—. In some preferred embodiments according to formula (12), R9and R10are methyl. In other preferred embodiments according to formula (12), R11and R13are each methyl. In some embodiments according to formula (12), R14is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R14is —C2-C30-alkylene-CO2H. In certain embodiments, R14is —CH2CO2H; —(CH2)2CO2H; —(CH2)3CO2H; —(CH2)4CO2H; —(CH2)5CO2H; —(CH2)6CO2H; —(CH2)7CO2H; —(CH2)8CO2H; —(CH2)9CO2H; —(CH2)10CO2H; —(CH2)11CO2H; —(CH2)12CO2H; —(CH2)13CO2H; —(CH2)14CO2H; —(CH2)15CO2H; —(CH2)16CO2H; —(CH2)17CO2H; —(CH2)18CO2H; —(CH2)19CO2H; —(CH2)20CO2H; —(CH2)21CO2H; —(CH2)22CO2H; —(CH2)23CO2H; —(CH2)24CO2H; —(CH2)25CO2H; —(CH2)26CO2H; —(CH2)27CO2H; —(CH2)28CO2H; —(CH2)29CO2H; or —(CH2)30CO2H. In certain embodiments, R14is —(CH2)15CO2H. In preferred embodiments, R14is methyl. In some embodiments, R15is H, C1-6alkyl, —C1-6alkylene-OH, —C1-6alkylene-C1-4alkoxy, —C1-6alkylene-CO2H, or —C1-6alkylene-amide. In certain embodiments, R15is CH2CO2H; (CH2)2CO2H; (CH2)3CO2H; (CH2)4CO2H; (CH2)5CO2H; (CH2)6CO2H; (CH2)7CO2H; (CH2)8CO2H; (CH2)9CO2H; (CH2)10CO2H; (CH2)11CO2H; (CH2)12CO2H; (CH2)13CO2H; (CH2)14CO2H; (CH2)15CO2H; (CH2)16CO2H; (CH2)17CO2H; (CH2)18CO2H; (CH2)19CO2H; (CH2)20CO2H; (CH2)21CO2H; (CH2)22CO2H; (CH2)23CO2H; (CH2)24CO2H; (CH2)25CO2H; (CH2)26CO2H; (CH2)27CO2H; (CH2)28CO2H; (CH2)29CO2H; or (CH2)30CO2H. In certain embodiments, R15is —(CH2)15CO2H. In preferred embodiments, R15is methyl. In other preferred embodiments, R17is hydrogen. In preferred embodiments, one of m and n is 1, and the other is 0. 4. Pharmaceutical Compositions In another preferred embodiment, the present invention provides a pharmaceutical formulation comprising a conjugate of the invention and a pharmaceutically acceptable carrier. The conjugates described herein including pharmaceutically acceptable carriers can be delivered to a patient using parenteral administration, including intramuscular, subcutaneous, and intravenous injections. Preferably, the conjugates of the invention comprising an antibody or antibody fragment as the targeting moiety are administered parenterally, more preferably intravenously. The conjugates may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injection is a preferred method of administration for the compositions of the current invention. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles. The compositions may take the form of liposomes or nanoparticles. Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Suspending, stabilizing and/or dispersing agents may be added such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. Pharmaceutical formulations for parenteral administration include aqueous solutions of the conjugates in water-soluble form. Additionally, suspensions of the conjugates may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. Alternatively, the conjugate may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In addition to the formulations described previously, the conjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection, or a transdermal patch. Thus, for example, the conjugates may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Suitable carriers, diluents, excipients, etc., can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. Compounds and conjugates may be in the form of a salt, such as a pharmaceutically acceptable salt. The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts. Acid addition salts can be formed with an amino group. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Basic addition salts may be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. A preferred pharmaceutical composition is a composition formulated for injection such as intravenous injection and includes about 0.01% to about 100% by weight of the drug conjugate, based upon 100% weight of total pharmaceutical composition. 5. Methods A. Drug Conjugate Methods of Use The present invention provides a number of methods that can be practiced utilizing the compounds and conjugates of the invention. Methods for using the drug conjugate of the current invention include: killing or inhibiting the growth or replication of a tumor cell or cancer cell, treating cancer, treating a pre-cancerous condition, killing or inhibiting the growth or replication of a cell that expresses an auto-immune antibody, treating an autoimmune disease, treating an infectious disease, preventing the multiplication of a tumor cell or cancer cell, preventing cancer, preventing the multiplication of a cell that expresses an autoimmune antibody, preventing an autoimmune disease, preventing an infectious disease, and cell ablation. These methods of use comprise administering to an animal such as a mammal or a human in need thereof an effective amount of an antibody drug conjugate. Preferred drug conjugates for many of the methods of use described herein include antibodies and antibody fragments, which target the particular tumor cell, cancer cell, or other target area. The drug conjugate of the current invention may be useful for treating cancer, autoimmune disease and infectious disease in an animal. Compositions and methods for treating tumors by providing a subject the composition in a pharmaceutically acceptable manner with a pharmaceutically effective amount of a composition of the present invention are provided. The drug conjugates may be particularly useful for the treatment of cancer and for the inhibition of the multiplication of a tumor cell or cancer cell in an animal. Cancer, or a precancerous condition, includes, but is not limited to, a tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of the antibody drug conjugates of the current invention. The conjugate delivers a payload to a tumor cell or cancer cell. In one embodiment, the antibody drug conjugate specifically binds to or associates with a cancer-cell or a tumor-cell-associated antigen. The conjugate can be taken up inside a tumor cell or cancer cell through, for example, receptor-mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, the quinone moiety may be reduced leading to release of the drug as described above. The released drug may freely diffuse and induce cytotoxic activities. The conjugate may bind to, for example, a tumor cell or cancer cell, a tumor cell or cancer cell antigen, which is on the surface of the tumor cell or cancer cell, or a tumor cell or cancer cell antigen, which is an extracellular matrix protein associated with the tumor cell or cancer cell. An antibody may be designed specifically for a particular tumor cell or cancer cell type. Therefore, the type of tumors or cancers that may be effectively treated can be altered by the choice of antibody. Representative examples of precancerous conditions that may be targeted by the conjugate, include, but are not limited to: metaplasia, hyperplasia, dysplasia, colorectal polyps, actinic ketatosis, actinic cheilitis, human papillomaviruses, leukoplakia, lychen planus, and Bowen's disease. Representative examples of cancers or tumors that may be targeted by the conjugate include, but are not limited to: lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, renal cancer, kidney cancer, pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer, and leukemias. It will be readily apparent to the ordinarily skilled artisan that the particular cell binding agent used in the conjugate may be chosen such that it targets the drug to the tumor tissue to be treated with the drug (i.e., a targeting agent specific for a tumor-specific antigen is chosen). Examples of such targeting antibodies are well known in the art with non-limiting examples of: anti-Her2 for treatment of breast cancer, anti-CD20 for treatment of lymphoma, anti-PSMA for treatment of prostate cancer, and antiCD30 for treatment of lymphomas, including non-Hodgkin's lymphoma. In an embodiment, the present invention provides a method of killing a cell. The method includes administering to the cell an amount of a conjugate of the invention sufficient to kill said cell. In an exemplary embodiment, the conjugate is administered to a subject bearing the cell. In a further exemplary embodiment, the administration serves to retard or stop the growth of a tumor that includes the cell (e.g., the cell can be a tumor cell). For the administration to retard the growth, the rate of growth of the cell should be at least 10% less than the rate of growth before administration. Preferably, the rate of growth will be retarded at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely stopped. Autoimmune diseases for which the drug conjugates may be used in treatment include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflannnatory bowel diseases (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)). Diseases of particular interest may include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis. The conjugates of the Invention may be useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The conjugates of the Invention may be used accordingly in a variety of settings for the treatment of an infectious disease in an animal. The conjugates may be used to deliver a drug to a target cell. Without being bound by theory, in one embodiment, the conjugate associates with an antigen on the surface of a target cell, and the conjugate is then taken up inside a target cell through receptor-mediated endocytosis. Once inside the cell, the payload drug may be released. The released drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the drug is cleaved from the conjugate outside the target cell, and the drug subsequently penetrates the cell. In one embodiment, the conjugates kill or inhibit the multiplication of cells that produce a particular infectious disease. Particular types of infectious diseases that can be treated with the conjugates include, but are not limited to, Diptheria, Pertussis, Occult Bacteremia, Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococcal, Peritonitis, Bactermia, Meningitis, Acute Purulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis,Salmonella, Typhoid Fever, Dysentery, Conjuntivitis, Sinusitis, Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus, Necrotizing Enteritis, Actinomycosis, Mixed Anaerobic Infections, Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever, Tuberculosis, Lymphadenitis, Leprosy,Chlamydia, ChlamydiaPneumonia, Trachoma, Inclusion Conjunctivitis, Systemic Fungal Diseases (Histoplarnosis, Coccicidiodomycosis, Blastomycosis, Sporotrichosis, Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis, Mycetoma, Chromomycosis), Rickettsial Diseases (Typhus, Rocky Mountain Spotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses, Rickettsialpox, Q Fever, Bartonellosis), Parasitic Diseases (Malaria, Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis, Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis, Entarnebiasis, Giardiasis. Cryptosporidiasis, Isosporiasis, Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection, Hookworm Infection, Threadworm Infection, Ocular Larva Migrans, Trichinosis, Guinea Worm Disease, Lymphatic F ilariasis, Loiasis, River Blindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch, Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis, Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease, Alveolar Hydatid Disease), Viral Diseases (Measles, Subacute sclerosing panencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease, Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis, Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-Mouth Disease, Bornholm Disease, Genital Herpes, Genital Warts, Aseptic Meningitis, Myocarditis, Pericarditis, Gastroenteritis, Acquired Immunodeficiency Syndrome (AIDS), Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral “Walking” Pneumonia, Acute Febrile Respiratory Disease, Acute pharyngoconjunctival fever, Epidemic keratoconjunctivitis, Herpes Simplex Virus 1 (HSV-1), Herpes Simples Virus 2 (HSV-2), Shingles, Cytomegalic Inclusion Disease, Rabies, Progressive Multifocal Leukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-Jakob Disease, Gerstrnann-Straussler-Scheinker Disease, Tropical Spastic Paraparesis, Western Equine Encephalitis, California Encephalitis, St. Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis, Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, Marburg Virus Infections, Ebola Virus Infections, and Smallpox). Cell ablation uses include destroying or inhibiting: (1) cardiac pacemaker cells responsible for arrhythmias; (2) over-active neurons responsible for neurological disorders; and (3) hyperactive endocrine functions etc. B. Dosages Pharmaceutical compositions suitable for use with the present invention include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response, and the discretion of the attending physician. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein. For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target plasma concentrations will be those concentrations of active compound(s) that are capable of inhibition cell growth or division. In preferred embodiments, the cellular activity is at least 25% inhibited. Target plasma concentrations of active compound(s) that are capable of inducing at least about 50%, 75%, or even 90% or higher inhibition of cellular activity are presently preferred. The percentage of inhibition of cellular activity in the patient can be monitored to assess the appropriateness of the plasma drug concentration achieved, and the dosage can be adjusted upwards or downwards to achieve the desired percentage of inhibition. As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a circulating concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring cellular inhibition and adjusting the dosage upwards or downwards, as described above. A therapeutically effective dose can also be determined from human data for compounds which are known to exhibit similar pharmacological activities. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound as compared with the known compound. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan. In the case of local administration, the systemic circulating concentration of administered compound will not be of particular importance. In such instances, the compound is administered so as to achieve a concentration at the local area effective to achieve the intended result. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of a conjugate to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of a conjugate. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. For other modes of administration, dosage amount and interval can be adjusted individually to provide plasma levels of the administered compound effective for the particular clinical indication being treated. For example, in one embodiment, a compound according to the invention can be administered in relatively high concentrations multiple times per day. Alternatively, it may be more desirable to administer a compound of the invention at minimal effective concentrations and to use a less frequent administration regimen. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease. Utilizing the teachings provided herein, an effective therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent. A compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, such as chemotherapeutics); immune therapy; surgery; and radiation therapy. A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy. Classes of immunooncology agents include PD-1 antagonists. C. Methods of Evaluating Cellular Uptake In some aspects, provided are methods for evaluating cellular uptake of an agent. The methods may comprise contacting a sample comprising a cell with a labeled agent as detailed herein and detecting light emission, whereby the detection of light emission indicates cellular uptake of the agent. The cellular uptake of the agent results in the reduction of the compound and the generation of a released reporter moiety. (1) Fluorescent Reporter Moiety In some embodiments, the reporter moiety may include a fluorescent reporter moiety. The fluorescent reporter moiety may include a fluorophore. Light emission is detected by exposing the sample to a wavelength of light and detecting the fluorescence generated by the released reported moiety. An increase in fluorescence or a change in fluorescence wavelength as compared to the fluorescence or fluorescence wavelength of a control sample indicates cellular uptake of the agent. The fluorophore may be conjugated in such a way as to quench its fluorescence to provide a fluorogenic or profluorescent probe that releases the payload as an unquenched fluorophore by way of its self-immolating property. The control sample may be sample medium only, without cells, a sample with cells but without experimental treatment, or a sample not contacted with labeled agent. Fluorescence may be detected inside or outside the cell. Non-reducible analogues of the disclosed quinone labeling moieties may be used as control labels. (2) Bioluminescent Reporter Moiety In some aspects, the reporter moiety may include a bioluminescent reporter moiety. The bioluminescent reporter moiety may include a prosubstrate for a luciferase. In some embodiments, the cell includes a luciferase. The luciferase may be expressed in the cell. Light emission is detected by detecting luminescence produced by the luciferase utilizing the released reporter moiety. The detection of any light emission may indicate the cellular uptake of the agent. Alternatively, luminescence of the sample may be compared to the luminescence of a control sample, wherein cellular uptake of the agent is indicated if the luminescence of the sample is higher than the luminescence of the control sample. The control sample may be a sample that is not contacted with a labeled agent or a cell type that is incompetent for uptake of the agent. Luminescence may be detected inside or outside the cell. In some embodiments, the cell does not include or express a luciferase, and luciferase is added to the sample. Light emission is detected by detecting luminescence produced by the luciferase utilizing the released reporter moiety that may exit the cell or be present in a cell lysate. The detection of any light emission may indicate the cellular uptake of the agent. Alternatively, luminescence of the sample may be compared to the luminescence of a control sample, wherein cellular uptake of the agent is indicated if the luminescence of the sample is higher than the luminescence of the control sample. The control sample may be sample medium only, without cells, a sample with cells but without experimental treatment, a cell type incompetent for uptake of the agent or a sample not contacted with labeled agent. Luminescence may be detected inside or outside the cell. Non-reducible analogues of the disclosed quinone labeling moieties may be used as control labels. (3) Sample The labeled agents may be used with samples containing biological components. The sample may comprise cells, tissues, or organs in vitro or in vivo. The compounds are generally non-toxic to living cells and other biological components within the concentrations of use. Cells may include eukaryotic cells, e.g., yeast, avian, plant, insect or mammalian cells, including but not limited to human, simian, murine, canine, bovine, equine, feline, ovine, caprine or swine cells, or prokaryotic cells, or cells from two or more different organisms, or cell lysates or supernatants thereof. The cells may not have been genetically modified via recombinant techniques (non-recombinant cells), or may be recombinant cells which are transiently transfected with recombinant DNA and/or the genome of which is stably augmented with a recombinant DNA, or which genome has been modified to disrupt a gene, e.g., disrupt a promoter, intron or open reading frame, or replace one DNA fragment with another. The recombinant DNA or replacement DNA fragment may encode a molecule to be detected by the methods of the invention, a moiety which alters the level or activity of the molecule to be detected, and/or a gene product unrelated to the molecule or moiety that alters the level or activity of the molecule. The cell may or may not express a luciferase. (4) Contact The labeled agents may be combined with the sample in a way that facilitates contact between the compound and the sample components of interest. Typically, the labeled agent or a solution containing the labeled agent is simply added to the sample. The cell uptake levels for selected labeled agents can be monitored with/without treatments that permeabilize the plasma membrane, such as electroporation, shock treatments or high extracellular ATP. Alternatively, selected labeled agents can be physically inserted into cells, e.g., by pressure microinjection, scrape loading, patch clamp methods, or phagocytosis. An additional detection reagent typically produces a detectable response due to the presence of a specific cell component, intracellular substance, or cellular condition, according to methods generally known in the art. When the additional detection reagent has, or yields a product with, spectral properties that differ from those of the subject labeled agents, multi-color applications are possible. This is particularly useful where the additional detection reagent is a dye or dye conjugate having spectral properties that are detectably distinct from those of the labeled agents. In certain embodiments, washing steps are unnecessary when using the disclosed labeling reagents and labeled agents. (5) Light Detection The labeled agents are generally utilized by combining a labeled agent as described above with a sample of interest comprising a cell under conditions selected to yield a detectable optical response or light output. Typically, a specified characteristic of the sample is determined by comparing the optical response with a standard or expected response. The sample may be illuminated at a wavelength selected to elicit the optical response. Alternatively, the light emission from the sample may be measured in a reading device that can measure the light output (luminescence) generated by the luciferase and bioluminescent reporter moiety, e.g., using a luminometer or photomultiplier. The optical response or light output may also be measured over time, for example in the same reaction chamber for a period of seconds, minutes, hours, etc. A detectable optical response means a change in, or occurrence of, an optical signal that is detectable either by observation or instrumentally. Typically, the detectable response is a change in fluorescence or luminescence such as a change in the intensity, excitation or emission wavelength distribution of fluorescence or luminescence, fluorescence or luminescence lifetime, fluorescence or luminescence polarization, or a combination thereof. The degree and/or location of the signal, compared with a standard or expected response, indicates whether, and to what degree, the sample possesses a given characteristic. At any time after or during contact with the labeled agent, the sample is illuminated with a wavelength of light selected to give a detectable optical response and observed with a means for detecting the optical response. Equipment that is useful for illuminating the compounds of the invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes. These illumination sources are optionally integrated into laser scanners, fluorescence microplate readers, standard or minifluorometers, or chromatographic detectors. The optical response or light output may be optionally detected by visual inspection or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes. Where the sample is examined using a flow cytometer, examination of the sample optionally includes sorting portions of the sample according to their fluorescence response. 6. Kits In another aspect, the present invention provides kits containing one or more of the compounds or compositions of the invention and directions for using the compound or composition. In an exemplary embodiment, the invention provides a kit for conjugating a linker arm of the invention or linker arm of the invention with a payload to another molecule. The kit includes the linker or linker plus payload, and directions for attaching the linker to a particular functional group. The kit may also include one or more of a cytotoxic drug, a targeting agent, a detectable label, pharmaceutical salts or buffers. The kit may also include a container and optionally one or more vial, test tube, flask, bottle, or syringe. Other formats for kits will be apparent to those of skill in the art and are within the scope of the present invention. 7. Chemical Synthesis A. Preparation of Conjugation Reagents Conjugation reagents 7 may be prepared as generally illustrated in Scheme 4. Aldehydes 1, wherein P1is a nitrogen protecting group, may be subjected to a reductive amination reaction with amines 2 to provide amines 3. Typical reaction conditions include combining the reactants with a reducing agent (e.g., NaBH4, NaBH3CN, NaH(OAc)3) in a solvent (e.g., ethanol, methanol, tetrahydrofuran, dichloroethane), optionally in the presence of a mild acid (e.g., acetic acid, ammonium hydroxide). Payload moieties -A1may be appended to amines 3 by reaction of a payload HA1with a suitable carbonate (e.g., bis(pentafluorophenyl) dicarbonate), chloroformate (e.g., 4-nitrophenyl chloroformate), phosgene, or triphosgene in an organic solvent (e.g., tetrahydrofuran) in the presence of a base (e.g., triethylamine or diisopropylethyl amine) to form an intermediate carbonate or carbonyl chloride (not shown) that may be reacted with amines 3 to provide carbamates 4. The protecting group P1may be removed using conditions well known in the art. For example, a tert-butoxycarbonyl (Boc) group may be removed with 50% of trifluoroacetic acid in the presence of triisopropyl silane or thioanisole in a solvent such as methylene chloride at room temperature for 30 minutes or longer to provide intermediates 5, which may be coupled with quinone carboxylic acid 6 under a variety of conditions. For example, quinone acid 6 may be converted to a mixed anhydride with a suitable chloroformate (e.g., isobutyl chloroformate) in the presence of a base (e.g., N-methylmorpholine) in a solvent (e.g., tetrahydrofuran) and the mixed anhydride reacted with 5 to provide 7. Alternatively, quinone acid 6 may be directly coupled to an amine linker by standard DCC coupling or DCC/HOBt conditions. Quinone acid 6 may also be activated by converting to acetyl chloride using oxalyl chloride or thionyl chloride, or converting to NHS ester using TSTU under basic conditions, or converting to a pentafluorobenzene ester. Certain compounds of formula 7 may be converted to other compounds of formula 7 by further functional manipulation of the X1agroup. For example, compounds 7.1 wherein X1ais —COOH may be converted to compound 7.2, wherein X1ais by reacting with TSTU under basic condition. Compound 7.2 may be further reacted with in the presence of a base (e.g., diispropylethylamine) in a solvent (e.g., dimethylformamide) to form compounds of formula 7.3, wherein L1bis —C(O)NR20-L1c-, L1cis C1-6alkylene, and X1ais (Scheme 5). Alternatively, the corresponding carboxylic acid 7.1 may be coupled with amines using standard amide bond forming conditions that are well known in the art (e.g., DCC and HOBt; DCC and DMAP). Conjugation reagents include 10A family and 10B family (Scheme 6). Conjugation reagents 10C may be prepared by a general method illustrated in Scheme 7. Similarly, quinone acid 6 may be coupled to protected-diamine intermediate 3a by activating 6 with isobutyl chloroformate or acetyl chloride or other suitable standard coupling conditions to yield quinone amide 8a. Amine protecting group P1, such as Boc, may be removed by TFA in the presence radical scavengers, such as triisopropyl silane or thioanisole to yield quinone diamine compound 9a. Payload HA1may be activated with a suitable carbonate or chloroformate (e.g., bis(pentafluorophenyl) dicarbonate, 4-nitrophenyl chloroformate), phosgene, or triphosgene in an organic solvent (e.g., tetrahydrofuran) in the presence of a base (e.g., triethylamine) to form an intermediate carbonate or carbonyl chloride (not shown) that may be reacted with amines 9a to provide carbamates 10C. Certain compounds of formula 10A may be converted to other compounds of formula 10A by further functional manipulation of the X1agroup. For example, compounds 10A.1 wherein X1ais —COOH, may be converted to compound 10A.2, wherein X1ais by reacting with TSTU under basic condition. Compound 10A.2 may be further reacted with in the presence of a base (e.g., diispropylethylamine) in a solvent (e.g., dimethylformamide) to form compounds of formula 10A.3, wherein L1bis —C(O)NR20-L1c-, L1cis C1-6alkylene, and X1ais (Scheme 8). Alternatively, the corresponding carboxylic acid 10A.1 may be coupled with amines using standard amide bond forming conditions that are well known in the art (e.g., DCC and HOBt; DCC and DMAP). Conjugation reagents 10B family may be prepared by a general method illustrated in Scheme 9. Quinone carboxylic acid 6 may be coupled to aminobenzyl alcohol 8b to generate compound 9b. which can be activated by triphosgene, isobutyl chloroformate, or bis(pentafluorobenzene) carbonate and further conjugated to payload A2moiety. Alternatively, payload HA2may be activated with a suitable carbonate or chloroformate (e.g., bis(pentafluorophenyl) dicarbonate, 4-nitrophenyl chloroformate), phosgene, or triphosgene in an organic solvent (e.g., tetrahydrofuran) in the presence of a base (e.g., triethylamine) to form an intermediate carbonate or carbonyl chloride (not shown) that may be reacted with alcohol 9b to provide carbamates 10B. Conjugation reagents 12 may be prepared as generally illustrated in Scheme 10 and using chemistries disclosed by Zheng et al. in J. Org. Chem. (1999) 64 (1) 156-161 (e.g., Schemes 2-4). For example, a MOM-protected lactone may be transformed to quinone carboxylic acid 11 by oxidation with N-bromosuccinimide in an appropriate solvent (e.g., tetrahydrofuran and water). In the transformation of 11 to 12, G1as may be appended as shown generally in Scheme 11. Carboxylic acid 11 may be coupled with amine 13 using conditions analogous to those used to convert 5 to 7 in Scheme 4. In the transformation of 11 to 12, G1as -A2may be appended as shown generally in Scheme 12. HA2may be reacted with 11 under standard amide coupling conditions for forming an amide, ester, or thioester, depending on whether A2is bonded through a nitrogen, oxygen, or sulfur atom. In the transformation of 11 to 12, G1as may be appended as shown generally in Scheme 13. Aniline 15 may be coupled with carboxylic acid 11 using standard amide bond forming conditions. Alcohol 16 may be converted to an activated carbonate (e.g., 4-nitrophenyl- or pentafluorophenyl-carbonate) or chloroformate and reacted with HA2under conditions analogous to those disclosed in US2008/0279868 to provide 17. B. Preparation of Drug Conjugates Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups, which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) maleimide groups (ii) activated disulfides, (iii) active esters such as NHS (N-hydroxysuccinimide) esters, HOBt (N-hydroxybenzotriazole) esters, haloformates, and acid halides; (iv) alkyl and benzyl halides such as haloacetamides; and (v) carboxyl. Conjugation reagents may also be coupled with an engineered antibody using click chemistry such as the reaction between an alkyne and an azide. See Jain et al., Pharm. Res. (2015) 32: 3526-40, which is hereby incorporated by reference. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541 teaches engineering antibodies by introduction of reactive cysteine amino acids. In general, to prepare conjugates of a partially reduced antibody having an average 2 drugs per antibody, the relevant antibody is reduced using a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) (about 1.8 equivalents) in PBS with 1 mM DTPA, adjusted to pH 8 with 50 mM borate. The solution is incubated at 37° C. for 1 hour, purified using a 50 mL G25 desalting column equilibrated in PBS/1 mM DTPA at 4° C. The thiol concentration, the protein concentration, and the ratio of thiol to antibody can be determined using procedures disclosed in U.S. Pat. No. 7,829,531. Conjugates having an average 4 drugs per antibody can be made using the same methodology, using about 4.2 equivalents of a suitable reducing agent to partially reduce the antibody. The partially reduced antibody samples may be conjugated to a corresponding Drug-Linker compound using about 2.4 and about 4.6 molar equivalents of Drug-Linker compound per antibody to prepare the 2 and 4 drugs per antibody conjugates, respectively. The conjugation reactions may be incubated on ice for 1 hour, quenched with about 20-fold excess of cysteine to drug, and purified by elution over a G25 desalting 25 column at about 4° C. The resulting Drug-Linker-antibody conjugates may be concentrated to about 3 mg/mL, sterile filtered, aliquoted and stored frozen. Alternatively, free thiol groups may be introduced into the antibody through reaction of lysines of the antibody with 2-iminothiolane. Initially, the antibody to be conjugated may be buffer exchanged into 0.1 M phosphate buffer pH 8.0 containing 50 mM NaCl, 2 mM DTPA, pH 8.0 and concentrated to 5-10 mg/mL. Thiolation may be achieved through addition of 2-iminothiolane to the antibody. The amount of 2-iminothiolane to be added is determined in preliminary experiments and varies from antibody to antibody. In the preliminary experiments, a titration of increasing amounts of 2-iminothiolane is added to the antibody, and following incubation with the antibody for one hour at room temperature, the antibody is desalted into 50 mM HEPES buffer pH 6.0 using a Sephadex G-25 column and the number of thiol groups introduced determined rapidly by reaction with dithiodipyridine (DTDP). Reaction of thiol groups with DTDP results in liberation of thiopyridine which is monitored at 324 nm. Samples at a protein concentration of 0.5-1.0 mg/ml may be used. The absorbance at 280 nm is used to accurately determine the concentration of protein in the samples, and then an aliquot of each sample (0.9 mL) is incubated with 0.1 mL DTDP (5 mM stock solution in ethanol) for 10 minutes at room temperature. Blank samples of buffer alone plus DTDP may also be incubated alongside. After 10 minutes, absorbance at 324 nm is measured and the number of thiols present quantitated using an extinction coefficient for thiopyridine of 19800M−1. Typically, a thiolation level of three thiol groups per antibody is desired. For example, this may be achieved through adding a 15 fold molar excess of 2-iminothiolane followed by incubation at room temperature for 1 hour. Antibody to be conjugated is therefore incubated with 2-iminothiolane at the desired molar ratio and then desalted into conjugation buffer (50 mM HEPES buffer pH 6.0 containing 5 mM Glycine, 3% Glycerol and 2 mM DTPA). The thiolated material is maintained on ice whilst the number of thiols introduced is quantitated as described above. After verification of the number of thiols introduced, the drug-linker molecule containing a reactive X1a/X2agroup may be added at a 3-fold molar excess per thiol. The conjugation reaction may be carried out in conjugation buffer also containing a final concentration of 5% ethylene glycol dimethyl ether (or a suitable alternative solvent). The drug-linker stock solution may be dissolved in 90% ethylene glycol dimethyl ether, 10% dimethyl sulfoxide. For addition to antibody, the stock solution may be added directly to the thiolated antibody, which has enough ethylene glycol dimethyl ether added to bring the final concentration to 5%, or pre-diluted in conjugation buffer containing a final concentration of 10% ethylene glycol dimethyl ether, followed by addition to an equal volume of thiolated antibody. The conjugation reaction may be incubated at room temperature for 2 hours with mixing. Following incubation the reaction mix may be centrifuged at 14000 RPM for 15 minutes, and the pH adjusted to 7.2 if purification is not immediate. Purification of conjugate may be achieved through chromatography using a number of methods. Conjugate may be purified using size-exclusion chromatography on a Sephacryl S200 column pre-equilibrated with 50 mM HEPES buffer pH 7.2 containing 5 mM glycine, 50 mM NaCl and 3% glycerol. Chromatography may be carried out at a linear flow rate of 28 cm/h. Fractions containing conjugate are collected, pooled and concentrated. Alternatively, purification may be achieved through ion-exchange chromatography. Conditions vary from antibody to antibody and need to be optimized in each case. For example, antibody-drug conjugate reaction mix may be applied to an SP-Sepharose column pre-equilibrated in 50 mM HEPES, 5 mM Glycine, 3% glycerol, pH 6.0. The antibody conjugate may be eluted using a gradient of 0-1 M NaCl in equilibration buffer. Fractions containing the conjugate may be pooled, the pH adjusted to 7.2, and the sample concentrated as required. To conjugate with amino groups of an antibody (e.g., lysine), a solution of an antibody in aqueous buffer may be incubated with a molar excess of a conjugation reagent bearing a reactive group X1a/X2a. The reaction mixture may be quenched by addition of excess amine (such as ethanolamine, taurine, etc.). The antibody drug conjugate may then be purified by gel filtration. The number of payload molecules bound per antibody molecule can be determined by measuring spectrophotometrically. An average of 1-10 payload molecules/antibody molecule may be linked by this method. In some embodiments, a preferred average number of linked payload molecules is 2-5. In other embodiments, a preferred average number of linked payload molecules is 3-4.5. 8. Examples The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. Example 1 Synthesis of N-[(PEG)4-COO-t-Bu]-N′—Boc-N′-methyl-ethylenediamine To a solution of N-t-BuOOC-(PEG)4-amine (0.557 g, 1.73 mmol) in 20 mL of methanol, (N-methyl)-N-Boc acetaldehyde (0.3 g, 1.73 mmol) was added. The mixture was stirred at room temperature for 3 hours. NaBH4(0.196 g, 5.2 mmol) was added to the mixture at 0° C., and the resultant mixture was stirred at 0° C. for 1 hour and then 1 hour at room temperature. The reaction was quenched by adding 5 mL of water. After removal of solvent, 5 mL of water was added, and the mixture was extracted three times with methylene chloride. The combined organic layer was dried over Na2SO4, and the product was purified by flash silica chromatography using heptane/ethyl acetate to methylene chloride/methanol to give a yield of 64.5% (0.535 g).1H NMR (300 MHz, CD2Cl2) δ ppm: 3.5-3.8 (m, 18H, CH2), 3.45 (br, 2H, CH2), 2.98 (br, 2H, CH2), 2.83 (s, 3H, NCH3), 2.46 (t, 2H, COCH2), 1.42 (s, 18H, CH3); MS-ESI (m/e): 479.6 [M+H]. Using analogous procedures, the following intermediate amines were likewise prepared from appropriate starting materials. TABLE 21H NMR (300 MHz, CD2Cl2) δ ppm: 3.68 (t, 2H, OCH2), 3.56-3.60 (br, 10H, CH2), 3.53 (t, 2H, OCH2), 3.29 (t, 2H, CH2), 2.85 (s, 3H, NCH3), 2.7-2.8 (m, 4H, NCH2), 2.47 (t, 2H, COCH2), 1.44 (s, 18H, CH3); MS-ESI (m/e): 435.5 [M + H].1H NMR (300 MHz, CD2Cl2) δ ppm: 3.67 (t, 2H, OCH2), 3.56-3.60 (br, 6H, OCH2), 3.29 (t, 2H, CH2), 2.85 (s, 3H, NCH3), 2.7- 2.8 (m, 4H, NCH2), 2.46 (t, 2H, COCH2), 1.45 (s, 18H, CH3); MS-ESI (m/e): 391.4 [M + H].MS (m/e) (C16H32N2O4), 317.4[M + H].1H NMR (300 MHz, CD2Cl2) δ 3.25-3.30 (t, J = 9, 2H), 2.83 (s, 3H), 2.69-2.73 (t, J = 6, 2H), 2.56-2.61 (t, J = 9, 2H), 2.16-2.21 (t, J = 6, 2H), 1.46-1.61 (m, 4H), 1.42 (s, 18 H), 1.26-1.38 (m, 2H). MS (m/e) [M + H] (C18H37N2O4) calculated 345.5, observed345.4. Example 2 (Prophetic Example) Synthesis of N-[(PEG)4COO-t-Bu]-N′—Boc-N′-methyl-ethylene-diamine payload carbamate 20 (Prophetic Example) To a mixture of a payload molecule HO-A1a(0.526 mmol) and bis(pentafluorophenyl) dicarbonate (0.276 g, 0.630 mmol) in 10 mL of dry tetrahydrofuran, triethylamine (0.106 mg, 1.05 mmol) may be added at room temperature under argon. The mixture may be stirred for 2-3 minutes, and N-[(PEG)4COO-t-Bu]-N′—Boc-N′-methyl-ethylene diamine (0.553 mg, 1.16 mmol) added. The mixture may be stirred at room temperature for 30 minutes, and the product purified. Synthesis of N-[(PEG)4COOH]—(N′-methyl)-ethylenediamine payload carbamate 21 (Prophetic Example) N-[(PEG)4COO-t-Bu]-N′—Boc-N-methyl-ethylenediamine payload carbamate (0.136 mmol) and triisopropylsilane (50 μL) may be dissolved in 10 mL of methylene chloride and trifluoroacetic acid (1:1 in volume), and the mixture stirred at room temperature for 2 hours. After removal of the solvent, the residue may be dried under high vacuum overnight. Synthesis of 22 (Prophetic Example) To a solution of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (107 mg, 0.428 mmol) and isobutyl chloroformate (58.4 mg, 0.427 mmol) in 10 mL dry tetrahydrofuran, N-methyl morpholine (86.5 mg, 0.855 mmol) may be added at 0° C. The resultant mixture may be stirred 30 minutes at 0° C., and N′-methyl-N-[(PEG)4COOH]ethylene-diamine] payload carbamate 21 in 5 mL of CH2Cl added, and the mixture stirred for 1 hour. The product may be directly purified with flash silica column chromatography. Synthesis of 23 (Prophetic Example) To a solution of 22 (0.314 mmol) and N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (1.57 mmol) in 20 mL acetonitrile and methylene chloride (1:1), diisopropylethylamine (325 mg, 2.51 mmol) may be added at room temperature. The mixture may be stirred for 30 minutes. 120 mL of methylene chloride may be added, and the resultant mixture washed with citric acid (30%) solution and water. The organic layer may be dried over Na2SO4and purified. Synthesis of 25 (Prophetic Example) Approximately 100 mg (392 μmol, 2 equiv.) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione TFA salt may be added to a vial together with 2 mL DMF and 196 μmol (1 equiv.) of the respective NHS ester 24 dissolved in 1 mL DMF. 70 μL diisopropylethylamine (392 μmol, 2 equiv.) may be added and then stirred at room temperature for over an hour. The reaction mixture may be dried down, and then purified by flash column chromatography. Example 3 Antibody Drug Conjugate (Prophetic Lysine Conjugation Example) To demonstrate conjugation of an antibody with the conjugation reagents of the present invention, the monoclonal antibody, Herceptin, may be labeled with 24. Lyophilized Herceptin may be dissolved in 0.1 M Sodium Bicarbonate, pH 8.6 at 10 mg/mL, and then diluted to 1 mg/mL in 100 μL of the same buffer. A 50 mM stock of 24 may be prepared in 100% DMSO. For labeling of the antibody, 24 may be added to 100 μL of diluted Herceptin (0.66 nmol). The sample may then be covered in foil and incubated for 60 minutes at room temperature on a tube rotator. To remove free 24, 100 μL of the labeling reaction may be placed onto an equilibrated G-25 Sequencing MicroSpin Column (Amersham, 200 μL resin) and eluted by spinning for 30 seconds at 3000 rpm. The conjugated Herceptin may be contained in the flow-through. Conjugation efficiency may be determined by spectrophotometry by taking the absorbance of the Herceptin-24 at 280 nm and 320 nm. The degree of labeling may be 2.3 molecules of 24 per one Herceptin antibody molecule. Example 4 Antibody Drug Conjugate (Prophetic Lysine Conjugation Example) A solution of huN901 antibody (2.5 mg/mL) in aqueous buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM ethylenediaminetetraacetic acid disodium salt), pH 6.5, may be incubated with a 6-fold molar excess of conjugation reagent (e.g., 24) in dimethylacetamide (DMA) to give a final DMA concentration of 20%. The reaction may be allowed to proceed for 13 hours at ambient temperature. The reaction mixture may be split into two portions. One portion may be purified by passage over a Sephadex G25 gel filtration column, and the second portion purified over a Sephacryl S300 gel filtration column. In each case the fractions containing monomeric conjugate may be pooled. The concentration of the conjugate may be determined spectrophotometrically. Purification by Sephadex G25 chromatography may give a conjugate containing, on the average, 2.08 payload molecules linked per antibody molecule. Purification by Sephacryl S300 chromatography may give a conjugate containing, on the average, 1.61 payload molecules linked per antibody molecule. Example 5 Antibody Drug Conjugate (Prophetic Thiol Conjugation Example) A solution of PBS/diethylenetriaminepentaacetic acid (2.2 mL) may be added to 4.2 mL of reduced antibody and the resulting solution cooled to 0° C. using an ice bath. In a separate flask, a stock DMSO solution of conjugation reagent (e.g., 25) (8.5 mol conjugation reagent per mol reduced antibody) may be diluted with MeCN. The MeCN solution of conjugation reagent may be rapidly added to the antibody solution, and the reaction mixture stirred using a vortex instrument for 5-10 seconds, returned to the ice bath, and allowed to stir at 0° C. for 1 hour, after which time 218 μL of a cysteine solution (100 mM in PBS/DTPA) may be added to quench the reaction. 60 μL of the quenched reaction mixture may be saved as a “qrm” sample. While the reaction proceeds, three PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) may be placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath). The quenched reaction mixture, which contained the conjugate, may be concentrated to −3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions), which may be pre-cooled to 4° C. in a refrigerator, and the concentrated reaction mixture eluted through the pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate may be collected in a volume of 1.4 mL per column for a total eluted volume of 4.2 mL. The eluted conjugate solution may then be filtered using a sterile 0.2 micron syringe-end filter, 250 μL of conjugate solution set aside for analysis, and the remainder of the conjugate solution frozen in sterile vials. The concentration of conjugate, the number of drug molecules per antibody, the amount of quenched drug-linker and the percent of aggregates may be determined using procedures disclosed in U.S. Pat. No. 7,829,531. Example 6 Antibody Drug Conjugate (Prophetic Thiol Conjugation Example) The conjugation method described herein is based on introduction of free thiol groups to the antibody through reaction of lysines of the antibody with 2-iminothiolane followed by reaction of the drug-linker molecule with an active maleimide group. Initially the antibody to be conjugated may be buffer exchanged into 0.1 M phosphate buffer pH 8.0 containing 50 mM NaCl, 2 mM DTPA, pH 8.0 and concentrated to 5-10 mg/ml. Thiolation may be achieved through addition of 2-iminothiolane to the antibody. The amount of 2-iminothiolane to be added may be determined in preliminary experiments and varies from antibody to antibody. In the preliminary experiments, a titration of increasing amounts of 2-iminothiolane may be added to the antibody, and following incubation with the antibody for one hour at room temperature, the antibody may be desalted into 50 mM HEPES buffer pH 6.0 using a Sephadex G-25 column, and the number of thiol groups introduced determined rapidly by reaction with dithiodipyridine (DTDP). Reaction of thiol groups with DTDP results in liberation of thiopyridine, which is monitored at 324 nm. Samples at a protein concentration of 0.5-1.0 mg/ml may be used. The absorbance at 280 nm may be used to accurately determine the concentration of protein in the samples, and then an aliquot of each sample (0.9 ml) may be incubated with 0.1 ml DTDP (5 mM stock solution in ethanol) for 10 minutes at room temperature. Blank samples of buffer alone plus DTDP may also be incubated alongside. After 10 minutes, absorbance at 324 nm may be measured and the number of thiols present quantitated using an extinction coefficient for thiopyridine of 19800M−1. Typically, a thiolation level of three thiol groups per antibody is desired. For example, this may be achieved through adding a 15 fold molar excess of 2-iminothiolane followed by incubation at room temperature for 1 hour. Antibody to be conjugated may therefore be incubated with 2-iminothiolane at the desired molar ratio and then desalted into conjugation buffer (50 mM HEPES buffer pH 6.0 containing 5 mM Glycine, 3% Glycerol and 2 mM DTPA). The thiolated material may be maintained on ice whilst the number of thiols introduced is quantitated as described above. After verification of the number of thiols introduced, the drug-linker molecule containing an active maleimide group may be added at a 3-fold molar excess per thiol. The conjugation reaction may be carried out in conjugation buffer also containing a final concentration of 5% ethylene glycol dimethyl ether (or a suitable alternative solvent). The drug-linker stock solution may be dissolved in 90% ethylene glycol dimethyl ether, 10% dimethyl sulfoxide. For addition to antibody, the stock solution may be added directly to the thiolated antibody, which has enough ethylene glycol dimethyl ether added to bring the final concentration to 5%, or pre-diluted in conjugation buffer containing a final concentration of 10% ethylene glycol dimethyl ether, followed by addition to an equal volume of thiolated antibody. The conjugation reaction may be incubated at room temperature for 2 hours with mixing. Following incubation the reaction mix may be centrifuged at 14000 RPM for 15 minutes, and the pH adjusted to 7.2 if purification is not immediate. Purification of conjugate may be achieved through chromatography using a number of methods. Conjugate may be purified using size-exclusion chromatography on a Sephacryl S200 column pre-equilibrated with 50 mM HEPES buffer pH 7.2 containing 5 mM glycine, 50 mM NaCl and 3% glycerol. Chromatography may be carried out at a linear flow rate of 28 cm/h. Fractions containing conjugate may be collected, pooled, and concentrated. Alternatively, purification may be achieved through ion-exchange chromatography. Conditions may vary from antibody to antibody and need to be optimized in each case. For example, antibody-drug conjugate reaction mix may be applied to an SP-Sepharose column pre-equilibrated in 50 mM HEPES, 5 mM Glycine, 3% glycerol, pH 6.0. The antibody conjugate may be eluted using a gradient of 0-1M NaCl in equilibration buffer. Fractions containing the conjugate may be pooled, the pH was adjusted to 7.2 and the sample concentrated as required. Example 7 Cellular Release of Luciferin from Herceptin Conjugates of PBI-5508 and PBI-6855 Herceptin conjugates of PBI-5508 (lysine conjugate) and PBI-6855 (thiol conjugate), prepared and conjugated to Herceptin using general methods described herein or in WO2015/116867, displayed the following characteristics: ADC InformationADC NameHerceptin-5508-01Lot #N/AYield68%Total quantity (mg)16.24Concentration2.90(mg/mL)Volume (ml)5.50DAR~5HIC-HPLC3.60%SEC-HPLC4.20%RP-HPLCn/aEndotoxin (EU/mg)n/aICEn/aADC NameHerceptin-6855Lot #N/AYield50%Total quantity (mg)12.60Concentration3.70(mg/mL)Volume (ml)3.40DAR~4HIC-HPLC4.40%SEC-HPLC18.40%RP-HPLCn/aEndotoxin (EU/mg)n/aICEn/aHIC-HPLC indicates the % of unconjugated antibodySEC-HPLC indicates the % of high molecular weight aggregation The cyanobenzothiazole moiety of Herceptin conjugated PBI-5508 and PBI-6855 is converted to a D-luciferin moiety by incubation with D-cysteine. Conditions that release the D-luciferin moiety produce free D-luciferin that can be detected with a luciferin detection reagent that minimally contains ATP, Mg2+, and luciferase that produces light in proportion to the amount of free D-luciferin. Luciferin was released from the Herceptin conjugates upon exposure to cultured cells, consistent with the proposed mechanism of cellular uptake followed by release in the reducing environment inside of cells (FIGS.1and2). The results inFIGS.1and2were obtained following incubations with SKBR2 cells or MCF7 cells. SKBR3 are known as HER2 positive cells because they express human epidermal growth factor receptor 2 (HER2) on the outer surface of their plasma membranes. MCF7 cells are HER2 negative cells because they do not express HER2. The Herceptin antibody binds to HER2 so the Herceptin luciferin-conjugates are expected to bind to and be internalized by the HER positive SKBR3 cells but not by HER2 negative MCF7 cells.FIGS.1and2show that in the presence of Her2 positive SKBR3 cells both conjugates released luciferin such that it accumulated over time. In contrast, medium alone and Her2 negative MCF7 cells released little or no luciferin from the conjugates. Herceptin-5508 or Herceptin-6855 was incubated with the HER2 positive cell line SKB3 or with the HER2 negative cell line MCF7 for 1, 2, 3, 4, or 5 days. 10,000 cells per well were initially plated in a 96 well plate in 50 μL of McCoys medium and 1 hour later the conjugates were added to a concentration of 30 nM each. At each time point shown, the medium was removed to a second plate with an equal volume of a lytic luciferin detection reagent containing ATP and UltraGlo® luciferase (Promega Corporation), and luminescence was read on a plate reading luminometer (medium transfer assay protocol,FIG.1). In this case, luciferin released inside of cells is detected after it diffuses into the medium. The detection reagent was also added to the cells remaining in the original plate, and luminescence was read on a plate reading luminometer (homogeneous assay protocol,FIG.2). In this case, luciferin that remains associated with the cells is detected in the cell lysate. Parallel incubations in McCoys medium with no cells were also performed and luminescence from those wells was measured according to the medium transfer and the homogeneous protocols. An additional set of parallel measurements were taken from wells with medium but without conjugates added, and those background values were subtracted from the conjugate values to give net luminescence. Example 8 Non-Cellular Release of Luciferin from Herceptin Conjugates of PBI-5508 Using a luciferin moiety as a probe payload on antibodies and on one protein that is not an antibody (bovine serum albumin), it was unexpectedly found that conjugation to a protein stabilizes TMQ-linked payload against non-cell mediated release by a reductive mechanism. This property may provide an additional advantage in ADCs where it is critical that payload is not released outside of target cells. The observations are as follows. When D-luciferin is derivatized on the 6′ hydroxyl by addition of the TMQ linker, its activity as a substrate for luciferase is blocked (PBI-4312), but free D-luciferin that is fully active with luciferase is readily released from a luciferin-TMQ derivative when subjected to reducing conditions inside or outside of a cell. However, release of D-luciferin from a protein-TMQ-luciferin conjugate by an extracellular reductive mechanism was substantially diminished compared to luciferin-TMQ. In other words, the protein moiety of the conjugate stabilizes the luciferin payload against reductive release from the TMQ linker in a non-cellular environment. Non-cellular reducing conditions include an aqueous reducing agent solution (e.g. DTT) or a reductase reaction mixture (FIG.3). This was true for two conjugates with a luciferin payload as well as a luciferin-TMQ conjugate with bovine serum albumin (BSA). Whereas the reaction conditions produced substantial amounts of light with PBI-4312 (>900,000 RLU after 1 hour), only minimal signals were produced from similar amounts of the protein conjugates (<800 RLUs after 1 hour). Bovine serum albumin, Praluent (an antibody drug for treating hypercholesterolemia), and Herceptin (an antibody drug for treating Her2 positive breast cancer) were conjugated to PBI-5508 and purified to remove unconjugated reactants. The three conjugate preparations (Herceptin-PBI-5508, Praluent-PBI-5508, BSA-PBI-5508) were then incubated with D-cysteine to convert the CBT moieties to luciferin moieties that would be released as free D-luciferin after reduction of the TMQ moiety. The PBI-5508 conjugates as well as PBI-4312, a luciferin-TMQ derivative that was not conjugated to a protein, were then incubated with a luciferin detection reagent supplemented with a reductase enzyme (human diaphorase) and NADH for 15 minutes or for 1 hour. It was previously shown that these conditions release luciferin, but that luciferin is not released when the reductase/NADH is left out. The luciferin detection reagent contains ATP and UltraGlo luciferase and it produces light in proportion to the amount of free D-Luciferin present in the reaction mixture. The stabilization against extracellular reductive release of a payload from a TMQ linker by tertiary conjugation to an antibody may provide an additional measure safety for conjugates bearing a cytotoxic agent. Any ranges given either in absolute terms or in approximate terms are intended to encompass both, and any definitions used herein are intended to be clarifying and not limiting. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges (including all fractional and whole values) subsumed therein. Furthermore, the invention encompasses any and all possible combinations of some or all of the various embodiments described herein. Any and all patents, patent applications, scientific papers, and other references cited in this application, as well as any references cited therein, are hereby incorporated by reference in their entirety. | 133,964 |
11857639 | DETAILED DESCRIPTION OF THE INVENTION In the following description, it is understood that other embodiments may be utilized and structural and operational changes may be made without departure from the scope of the present embodiments disclosed herein. Definitions As used herein the words “a” and “an” and the like carry the meaning of “one or more.” As used herein, the terms “optional” or “optionally” mean that the subsequently described event(s) can or cannot occur or the subsequently described component(s) may or may not be present (e.g., 0 wt. %). According to a first aspect, the present disclosure relates to a nanomedicinal composition comprising a nanocarrier and a pharmaceutical agent mixture comprising an anti-cancer therapeutic and an antioxidant. The nanocarrier comprises a porous silicate matrix and particles of a magnetic ferrite of formula MFe2O4disposed in the pores of the porous silicate matrix, where M is at least one transition metal selected from the group consisting of Cu, Ni, Co, and Mn. The pharmaceutical agent mixture is disposed in the pores of and/or on a surface of the nanocarrier. In general, any suitable porous silicate matrix known to one of ordinary skill in the art may be used in the nanomedicinal composition. Examples of such suitable porous silica, silicate, or aluminosilicate materials include, but are not limited to, MCM-41, MCM-48, Q-10 silica, hydrophobic silica, mesobeta, mesoZSM-5, SBA-15, KIT-5, KIT-6, mesosilicalite, hierarchical porous silicalite, and SBA-16. For the purposes of this disclosure, “silicate matrix” also includes aluminum-containing silicate materials. Such materials may be known as or referred to as aluminosilicates. Further, the term “silicate matrix” should be understood to include silica itself. Methods of obtaining the various types porous silica, silicate, or aluminosilicate material are well-known in the art [see for example Gobin, Oliver Christian “SBA-16 Materials: Synthesis, Diffusion, and Sorption Properties” Dissertation, Laval University, Ste-Foy, Quebec, Canada, January 2006, in particular section 2.2; and U.S. patent application Ser. No. 15/478,794—both incorporated herein by reference in their entireties]. In some embodiments, the porous silicate matrix is present in an amount of 55 to 85 wt %, preferably 57.5 to 82.5 wt %, preferably 60 to 80 wt %, preferably 62.5 to 77.5 wt %, preferably to 75 wt %, preferably 67.5 to 72.5 wt %, preferably 69 to 71 wt %, preferably 70 wt %, based on a total weight of the nanocarrier. In some embodiments, the porous silicate matrix is at least one selected from the group consisting of MCM-41 and mesosilicalite. MCM-41 (Mobil Composition of Matter No. 41) is a mesoporous silica material with a hierarchical structure from a family of silicate and aluminosilicate solids that were developed by researchers at Mobil Oil Corporation and that can be used as catalysts or catalyst supports. MCM-41 and MCM-48 both comprise an amorphous silica wall and possess long range ordered framework with uniform mesopores. These materials also possess large surface area, which can be up to more than 1,000 m2g−1. The pore diameter of these materials can be controlled to fall within a mesoporous range between 1.5 and 20 nm by adjusting the synthesis conditions and/or by employing surfactants with different chain lengths in their preparation. In embodiments where the porous silicate matrix is MCM-41, the nanocarrier may be referred to as a “MCM-41 nanocarrier”. Silicalite is a polymorph of silica having a structure analogous to zeolite. The term “mesosilicalite” may be used to refer to any silicalite material which contains mesopores. The term “hierarchical silicalite” is used to indicate a silicalite which has at least two types of pore systems with different pore size ranges. For example, a hierarchical silicalite may have a pore size range in the micropore range and a pore size range in the mesopore range. Such a material may be classified as both a mesosilicalite and a hierarchical silicalite. In some embodiments, the hierarchical silicalite includes mesopores of a hexagonal structure and micropores. In some embodiments, the micropores have a microporous volume in the range of 0.05 cc/g to 0.1 cc/g, cc/g to 0.09 cc/g, or 0.07 cc/g to 0.08 cc/g. U.S. patent application Ser. No. 15/478,794 which published as US 2018/0280303—incorporated herein by reference in its entirety, discloses the synthesis of an exemplary silicalite having a particle size in the range of 1-5 nm using Ludox AS-40 and tetrapropylammonium bromide (TPABr) as silica and templating agent, respectively. In embodiments where the porous silicate matrix is mesosilicalite, the nanocarrier may be referred to as a “mesosilicalite nanocarrier”. The hexagonal structure of mesosilicalite may be described as well-ordered and comparable in structure to MCM-41 mesoporous material as described by Kresge et al. (1992), Sayari, et al. (1996), and Moller, et al. (2013) [Kresge C T, Leonowicz M E, Roth W J, Vartuli J C, Beck J S, Nature 359 (1992)710-712; 15; A. Sayari, Chem. Mater. 8 (1996) 1840-1852; and K. Moller, T. Bein, Chem. Soc. Rev., 42 (2013) 3689, each incorporated herein in their entirety]. In some embodiments, the mesopores and micropores for the porous silicate matrix characterize the hierarchical structure of the silicalite, wherein the mesopores form the mesophase and the micropores form the microphase. The relative weight ratios of these two phases approximate the relative weight ratios of the SiMCM-41 and silicalite used in the synthesis. In some embodiments, the hierarchy of the mesophase and microphase in hierarchical silicalite results in improved interaction with materials that can be carried, adsorbed, absorbed and/or otherwise contacted by the porous silicate matrix due to a greater surface area of contact with two phases instead of one phase, and an improved flow, or exchange, of the materials that may be carried into and out of the porous silicate matrix. The presence of micropores and mesopores in the porous silicate matrix may exhibit a unique hysteresis pattern. The pore size distribution of the silicalite typically exhibits two types of pores between 2.4 nm and 3.7 nm, while Q-10 silica and SiMCM-41 each show one type of pore at 15 nm and 2.9 nm, respectively. The porous silicate matrix may comprise two types of materials, a first material having 2D properties and a second material having 3D properties. The first material may be layered under the second material, thus forming a hierarchically structured nanocarrier. In some such embodiments, the amount of mesophase and microphase is calculated based on the weight percentage of composite SiMCM-41/silicalite in comparison to parent silicalite and SiMCM-41. Alternatively, a calibration curve may be constructed from the X-Ray diffraction spectra of mesosilicalite nanocarriers synthesized from different amounts of parent silicalite and SiMCM-41. Then, the amount of mesophase and microphase may be determined from an X-Ray diffraction measurement. In some embodiments of the mesosilicalite nanocarrier, the mesopores are ordered. The ordered structure of the mesopores may be a result of a template employed in the process of preparing the mesosilicalite nanocarrier, described further herein. The template, for example a tetrapropylammonium hydroxide, may assist colloidal silica, described further in the method of preparing the mesosilicalite nanocarrier, to self-order in formation of cylindrical pores which form the hexagonal structure. The ordered structure may allow for improved diffusion of materials into and out of the nanocarrier. This characteristic may make the nanocarriers useful as drug delivery agents. In a preferred embodiment, the porous silicate matrix has a surface area in the range 300 m2/g to 1400 m2/g, more preferably in the range 400 m2/g to 1200 m2/g, and most preferably in the range of 600 m2/g to 1000 m2/g. The preferred porous silicate matrix has at least one type of pores with a diameter in the range of 1 nm to 60 nm, preferably in the range of 1.5 nm to 30 nm, more preferably in the range 2 nm to 10 nm, and most preferably in the range of 3 nm to 7 nm. Also, the preferred porous silicate matrix has a pore volume in the range of 0.11 cc/g to 1.5 cc/g, preferably in the range of 0.15 cc/g to 1.25 cc/g, more preferably in the range of 0.25 cc/g to 1 cc/g, and most preferably in the range of 0.5 cc/g to 0.75 cc/g. In some embodiments, the porous silicate matrix is present in the form of particles. In general, the porous silicate matrix particles can be any shape known to one of ordinary skill in the art. Examples of suitable shapes the porous silicate matrix particles may take include spheres, spheroids, lentoids, ovoids, solid polyhedra such as tetrahedra, cubes, octahedra, icosahedra, dodecahedra, rectangular prisms, triangular prisms (also known as nanotriangles), nanoplatelets, nanodisks, blocks, flakes, discs, granules, angular chunks, and mixtures thereof. Nanorods or nanowires are not a shape that the porous silicate matrix particles are envisioned as having in any embodiments. In some embodiments, the porous silicate matrix particles have uniform shape. Alternatively, the shape may be non-uniform. As used herein, the term “uniform shape” refers to an average consistent shape that differs by no more than 10%, by no more than 5%, by no more than 4%, by no more than 3%, by no more than 2%, by no more than 1% of the distribution of porous silicate matrix particles having a different shape. As used herein, the term “non-uniform shape” refers to an average consistent shape that differs by more than 10% of the distribution of porous silicate matrix particles having a different shape. In one embodiment, the shape is uniform and at least 90% of the porous silicate matrix particles are spherical or substantially circular, and less than 10% are polygonal. In another embodiment, the shape is non-uniform and less than 90% of the porous silicate matrix particles are spherical or substantially circular, and greater than 10% are polygonal. In some embodiments, the porous silicate matrix particles have a mean particle size of 25 to 500 nm, preferably 30 to 450 nm, preferably 40 to 400 nm, preferably 50 to 350 nm, preferably 60 to 300 nm, preferably 70 to 250 nm. In embodiments where the porous silicate matrix particles are spherical, the particle size may refer to a particle diameter. In embodiments where the porous silicate matrix particles are polyhedral, the particle size may refer to the diameter of a circumsphere. In some embodiments, the particle size refers to a mean distance from a particle surface to particle centroid or center of mass. In alternative embodiments, the particle size refers to a maximum distance from a particle surface to a particle centroid or center of mass. In some embodiments where the porous silicate matrix particles have an anisotropic shape such as nanorods, the particle size may refer to a length of the nanorod, a width of the nanorod, or an average of the length and width of the nanorod. In some embodiments, the particle size refers to the diameter of a sphere having an equivalent volume as the particle. In some embodiments, the porous silicate matrix particles are monodisperse, having a coefficient of variation or relative standard deviation, expressed as a percentage and defined as the ratio of the particle size standard deviation (a) to the particle size mean (0 multiplied by 100 of less than 25%, preferably less than 10%, preferably less than 8%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%. In some embodiments, the porous silicate matrix particles of the present disclosure are monodisperse having a particle size distribution ranging from 80% of the average particle size to 120% of the average particle size, preferably 90-110%, preferably 95-105% of the average particle size. In some embodiments, the porous silicate matrix particles are not monodisperse. The nanocarrier also comprises particles of a magnetic ferrite of formula MFe2O4disposed in the pores of the porous silicate matrix, where M is at least one transition metal selected from the group consisting of Cu, Ni, Co, and Mn. In some embodiments, the magnetic ferrite is a mixed metal or doped metal ferrite of formula M1-xAxFe2O4, where A represents a transition metal or rare earth element and 0<x≤0.5. Examples of such mixed meal or doped metal ferrites include Mn0.5Zn0.5Fe2O4(also represented as (MnZn)Fe2O4), Mn0.9Ce0.1Fe2O4, and Mn0.9Co0.1Fe2O4. In preferred embodiments, the magnetic ferrite is copper ferrite of formula CuFe2O4. In some embodiments, the magnetic ferrite crystallizes in the spinel crystal structure. The spinel crystal structure is characterized by a cubic close packed lattice of anions (in this case oxygen anions), in which the cations (M and Fe) occupy some or all of the tetrahedral sites and octahedral sites. In the normal spinel structure, divalent cations occupy tetrahedral holes and trivalent cations occupy octahedral holes. In the inverse spinel structure, the divalent cations occupy octahedral holes while half of the trivalent cations occupy octahedral holes, and the other half of the trivalent cations occupy tetrahedral holes. Intermediate structures between these end members with different cation ordering schemes also exist, including random cation distribution (also known as cation disordered structures). In some embodiments, the magnetic ferrite crystallizes in the normal spinel structure. In alternative embodiments, the magnetic ferrite crystallizes in the inverse spinel structure. In other alternative embodiments, the magnetic ferrite crystallizes in an intermediate spinel structure. In some embodiments, the magnetic ferrite is present in an amount of 15 to 45 wt %, preferably 17.5 to 42.5 wt %, preferably 20 to 40 wt %, preferably 22.5 to 37.5 wt %, preferably 25 to 35 wt %, preferably 27.5 to 32.5 wt %, preferably 29 to 31 wt %, preferably 30 wt %, based on a total weight of the nanocarrier. In general, the magnetic ferrite particles can be any shape known to one of ordinary skill in the art as described above. In some embodiments, the magnetic ferrite has a mean particle size of 0.5 to 50 nm, preferably 0.75 to 25 nm, preferably 1 to 15 nm, preferably 1.5 to 10 nm. The incorporation of the magnetic ferrite particles occupies a portion of the pores present in the porous silicate matrix. In some embodiments, the incorporation of the magnetic ferrite particles reduces a post-ferrite incorporation pore volume of the porous silicate matrix to 5 to 75%, preferably 7.5 to 70%, preferably 10 to 67.5%, preferably 12.5 to 65%, preferably 15 to 62.5% of an initial pore volume of the porous silicate matrix. That is, the nanocarrier has a pore volume of 5 to 75%, preferably 7.5 to 70%, preferably 10 to 67.5%, preferably 12.5 to 65%, preferably 15 to 62.5% of the pore volume of the porous silicate matrix. In some embodiments in which the porous silicate matrix is MCM-41, the nanocarrier has a total pore volume of 0.0385 to 1.35 cc/g, 0.05 to 1.25 cc/g, preferably 0.1 to 1.0 cc/g, preferably 0.15 to 0.90 cc/g, preferably 0.20 to 0.85 cc/g, preferably 0.25 to 0.80 cc/g, preferably 0.30 to 0.75 cc/g, preferably 0.35 to 0.70 cc/g, preferably 0.40 to 0.65 cc/g, preferably 0.45 to 0.60 cc/g, preferably 0.50 to 0.55 cc/g. In some embodiments in which the porous silicate matrix is mesosilicalite, the nanocarrier has a total pore volume of 0.03 to 0.45 cc/g, preferably 0.05 to 0.30 cc/g, preferably 0.075 to 0.20 cc/g, preferably 0.9 to 0.15 cc/g, preferably 0.1 cc/g. In general, the anti-cancer therapeutic may be any suitable anti-cancer therapeutic known to one of ordinary skill in the art. Exemplary anti-cancer therapeutics include, but are not limited to, alkylating antineoplastic agents including busulfan, carmustine, chlorambucil, cyclophosphamide, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, mercaptopurine, procarbazine; antimetabolites including cladribine, cytarabine, fludarabine, gemcitabine, pentostatin, 5-fluorouracil, clofarabine, capecitabine, methotrexate, thioguanine; anti-microtubule agents including etoposide, vinblastine, vincristine, teniposide, docetaxel, paclitaxel, vinorelbine, vindesine; cytotoxic antibiotics including daunorubicin, doxorubicin, idarubicin, mitomycin, actinomycin, epirubicin; topoisomerase inhibitors including irinotecan, mitoxantrone, topotecan, platinum-containing anti-cancer therapeutics, and mixtures thereof. In preferred embodiments of the invention, the anti-cancer therapeutic comprises a platinum (II) complex. Any platinum(II) complexes effective for treatment of cancer can be used including, but not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, strataplatin or mixtures thereof. In some embodiments, the platinum(II) complex is at least one selected from the group consisting of cisplatin, carboplatin, and oxaliplatin. In preferred embodiments, the platinum (II) complex is cisplatin. In alternative preferred embodiments, the anti-cancer therapeutic comprises tamoxifen. In general, the antioxidant may be any suitable antioxidant known to one of ordinary skill in the art. Examples of such antioxidants include, but are not limited to curcumin (and curcumin derivatives known as curcuminoids), Coenzyme Q10, quercetin, rutin, ascorbic acid, gallic acid, edaravone, N-acetylcysteine, alfa-lipoic acid, diosmin, hesperidin, oxerutins, baicalein, tocotrienols, resveratrol or other stilbenoids such as pterostilbene, retinoids and carotenes including Vitamin A, beta carotene, and alpha-carotene, astaxanthin, canthaxanthin, lutein, lycopene, and zeaxanthin, natural phenols including flavonoids, silymarin, xanthones, eugenol, phenolic acids, lipoic acid, acetylcysteine, uric acid, glutathione, and catechin. In some embodiments, the antioxidant is at least one selected from the group consisting of quercetin, rutin, coenzyme Q10, gallic acid, and curcumin. Quercetin has the following chemical structure: Quercetin is a plant flavonol from the flavonoid group. It is found in a wide variety of food sources, but has very low water solubility and bioavailability. Inclusion of quercetin in the nanomedicinal composition of the present invention may overcome these disadvantageous properties of quercetin to increase an amount of quercetin which is delivered. Quercetin may be present in a crystalline or amorphous form or in a mixture of both crystalline and amorphous forms, for example at a ratio of 1-99 wt. %:99-1 wt. %, 10-90 wt. %:90-10 wt. %; 20-80 wt. %:80-wt. %, 30-70 wt. %:70-30 wt. %, 40-60 wt. %:60-40 wt. % or about 50 wt. %:about 50 wt. % (or any intermediate ratio of crystalline:amorphous forms). Rutin has the following structure: Rutin is the glycoside combining the flavonol quercetin and the disaccharide rutinose (α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranose). Rutin may be present in a crystalline or amorphous form or in a mixture of both crystalline and amorphous forms, for example at a ratio of 1-99 wt. %:99-1 wt. %, 10-90 wt. %:90-10 wt. %; 20-80 wt. %:80-20 wt. %, 30-70 wt. %:70-30 wt. %, 40-60 wt. %:60-40 wt. % or about 50 wt. %:about 50 wt. % (or any intermediate ratio of crystalline: amorphous forms). Coenzyme Q10 (CoQ10) conforms to the following chemical structure: CoQ10 is a 1,4-benzoquinone, where Q refers to the quinone chemical group and 10 refers to the number of isoprenyl chemical subunits in its tail. Other forms of Coenzyme Q may be distinguished from CoQ10 by their number of isoprenyl subunits. A CoQ such as CoQ10 may be present in a crystalline or amorphous form or in a mixture of both crystalline and amorphous forms, for example at a ratio of 1-99 wt. %:99-1 wt. %, 10-90 wt. %:90-10 wt. %; 20-80 wt. %:80-20 wt. %, 30-70 wt. %:70-30 20 wt. %, 40-60 wt. %:60-40 wt. % or about 50 wt. %:about 50 wt. % (or any intermediate ratio of crystalline: amorphous forms). Gallic acid has the following structure: Gallic acid is a potent antioxidant against cancers (leukemia, colon and lung cancer cells) and other metabolic disorders. Gallic acid may be present in a crystalline or amorphous form or in a mixture of both crystalline and amorphous forms, for example at a ratio of 1-99 wt. %:99-1 wt. %, 10-90 wt. %:90-10 wt. %; 20-80 wt. %:80-20 wt. %, 30-70 wt. %:70-30 wt. %, 40-60 wt. %: 60-40 wt. % or about 50 wt. %:about 50 wt. % (or any intermediate ratio of crystalline: amorphous forms). Curcumin has the following structure: A curcuminoid is a linear diarylheptanoid. This class of compounds includes curcumin in both its keto and enolate forms as well as curcumin derivatives such as demethoxycurcumin and bisdemethoxycurcumin and their geomentrical isomers and metabolites including sulfate conjugates and glucoronides. Other examples of curcumin derivatives or analogs include those 10 described by Raja, et al., U.S. Pat. No. 9,447,023 B2, Raja, et al., U.S. Pat. No. 9,650,404 B2, Johnson, et al., U.S. Pat. No. 9,556,105 B2 or Vander Jagt, et al., U.S. Pat. No. 9,187,397 B2 (all incorporated by reference); especially for their descriptions of curcuminoid formulas and various chemical species of curcuminoids. In some embodiments of the invention curcumin or another curcuminoid may be included as an antioxidant in the nanomedicinal composition of the present disclosure. Mixtures of curcuminoids are also contemplated such as one isolated from rhizomes of turmeric comprised of Curcumin (75-81%), Demethoxycurcumin (15-19%) and Bisdemethoxycurcumin (2.5-6.5%). The content of any one of a curcuminoid in a mixture may range from about 0 to about 100 wt. %, for example, 10-90 wt. %, 20-80 wt. %, 30-70 wt. %, 40-60 wt %, 50 wt. %, 40 wt. %, 33.3 wt. %, 30 wt. %, 20 wt. %, 10 wt. % or 5 wt % or 1 wt. %. A mixture may contain two, three or more different curcuminoids. Curcumin may be present in a crystalline or amorphous form or in a mixture of both crystalline and amorphous forms, for example at a ratio of 1-99 wt. %:99-1 wt. %, 10-90 wt. %: 90-10 wt. %; 20-80 wt. %:80-20 wt. %, 30-70 wt. %:70-30 wt. %, 40-60 wt. %:60-40 wt. % or about 50 wt. %:about 50 wt. % (or any intermediate ratio of crystalline: amorphous forms). In some embodiments disclosed herein, curcumin will be in an amorphous form to increase its solubility. Curcumin and its derivatives are known for their antimicrobial, anti-oxidative, anti-inflammatory, and anti-cancer properties such as malignancies in the brain or nervous system. Curcumin has also been proposed as an agent to treat oxidative stress, such as oxidative stress in the brain, and for treatment of neurodegenerative disease like Alzheimer's disease (“AD”) or Parkinson's disease (“PD”); Lee, et al., Curr. Neuropharmacol. 2013 July; 11(4):338-378 (incorporated by reference). Curcumin may also be functionalized or prepared as a conjugate with another moiety to modify or improve its pharmacokinetic properties. For example, curcumin can be adsorbed through functionalization to a silane, carboxylic acid, or biotin. Biocompatibility of a curcuminoid/hierarchical aluminosilicate can be increased by the modification with chitosan, or poly (D,L-lactide-co-glycolide), or polyethylene glycol. In preferred embodiments, the antioxidant is curcumin. In some embodiments, a weight ratio of the antioxidant to the anti-cancer therapeutic is 1:1 to 10:1, preferably 2:1 to 9:1, preferably 3:1 to 8:1, preferably 4:1 to 7:1, preferably 5:1 to 6:1. In some embodiments, the pharmaceutical agent mixture is present in the nanomedicinal composition in an amount of 5 to 50 wt %, preferably 10 to 47.5 wt %, preferably 15 to 45 wt %, preferably 17.5 to 42.5 wt %, preferably 20 to 40 wt %, preferably 22.5 to 37.5 wt %, preferably 25 to 35 wt %, preferably 26 to 34 wt %, preferably 27 to 33 wt %, preferably 28 to 31 wt %, preferably 29 to 30 wt %, based on a total weight of nanomedicinal composition. In some embodiments, the nanomedicinal composition comprises a biocompatible coating. Such a biocompatible coating may be disposed upon the nanocarrier and/or the pharmaceutical agent mixture. In general, the biocompatible coating may be any suitable coating known to one of ordinary skill in the art. Examples of such suitable biocompatible coatings include, but are not limited to, agarose, agar, carrageen, alginic acid, alginate, an alginic acid derivative, a hyaluronate derivative, a polyanionic polysaccharide, chitin, chitosan, fibrin, a polyglycolide, a polylactide, a polycaprolactone, a dextran or copolymer thereof, polyvinyl pyrrolidone, a polyacrylate, a wax, a polyethylene-polyoxypropylene-block polymer, wool fat, poly(L-lactic acid), poly(DL-Lactic acid) copoly(lactic/glycolic acid), cellulose, a cellulose derivative, a glycol, polylactide-polyglycolide, polymethyldisiloxane, polycaprolactone, polylactic acid, and ethylene vinyl acetate. In some embodiments, the nanomedicinal composition releases greater than 10 wt %, preferably greater than 12.5 wt %, preferably greater than 15 wt %, preferably greater than 17.5 wt %, preferably greater than 20 wt %, preferably greater than 22.5 wt %, preferably greater than 25 wt %, preferably greater than 27.5 wt %, preferably greater than 30 wt %, preferably greater than 32.5 wt %, preferably greater than 35 wt %, preferably greater than 37.5 wt %, preferably greater than 40 wt % of a total weight of the antioxidant. In some embodiments, this release occurs within 20 hours, preferably within 18 hours, preferably within 16 hours, preferably within 14 hours, preferably within 12 hours of contact with a suitable biological medium. Examples of suitable biological media include, but are not limited to, buffered saline solutions such as phosphate buffered saline, cell culture media such as Minimum Essential Medium (MEM, also known as Eagle's minimal essential medium EMEM), Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), RPMI-1640, Ham's F-10, and F-12; animal tissue, or a subject's body. In some embodiments, the nanomedicinal composition releases greater than 50 wt %, preferably greater than 52.5 wt %, preferably greater than 55 wt %, preferably greater than 57.5 wt %, preferably greater than 60 wt %, preferably greater than 62.5 wt %, preferably greater than 65 wt %, preferably greater than 67.5 wt %, preferably greater than 70 wt %, preferably greater than 72.5 wt %, preferably greater than 75 wt %, preferably greater than 77.5 wt %, preferably greater than 80 wt % of a total weight of the anti-cancer therapeutic. In a preferred aspect of the invention the antioxidant is provided as an outer component of the nanomedicinal composition. As an outer component the antioxidant is mainly located or disposed at an outside surface of particles of the nanomedicinal composition. The position of the antioxidant mainly at the surface or enriched at the surface can be achieved by treating a composition that contains the porous silicate, the magnetic ferrite and the anti-cancer therapeutic with antioxidant particles or, preferably, a solution comprising an antioxidant agent. Subsequent drying of the resultant particles preferentially disposes the antioxidant at an exterior portion of particles of the nanomedicinal composition. In other embodiments the antioxidant is present throughout the nanomedicinal composition and, in addition, is a main component of the exterior surface of the nanomedicinal composition. This structure of the nanomedicinal composition is obtained by preparing a nanomedicine medicinal composition containing the porous silicate, magnetic ferrite, anti-cancer therapeutic, and antioxidant in particulate form then treating the resulting particulate material with a solution of the same antioxidant or a second antioxidant to place the antioxidants at an exterior and/or surface location of the nanomedicinal composition. Placing the antioxidant at a surface position of particles of the nanomedicinal composition provides an important benefit. One aspect that may be positively affected is the release rate of the pharmaceutical agent mixture. The antioxidant may inhibit release of the pharmaceutical agent mixture for a time period while the nanomedicinal composition travels through the vascular system of a patient undergoing treatment. This provides an induction period during which only minor amounts of the pharmaceutical agent mixture are released. When the pharmaceutical agent mixture reaches a target site, such as a tumor, for example by application of a magnetic field to direct the particles to the tumor, the induction period has delayed release of the pharmaceutical agent mixture. Upon arrival at the target site the nanomedicinal composition may be held in place (for example by application of a strong magnetic field) and release the pharmaceutical agent mainly at the target site. The release of the pharmaceutical agent mixture may be facilitated by an acidic pH at a tumor site. Such facilitation may, for example, take the form of an increased rate of release, an increased total amount released, or both. In this aspect of the invention the release of the pharmaceutical agent may be due over a release period of at least 2 hours, preferably at least 4 hours, preferably at least 6 hours, preferably at least 8 hours, preferably at least 10 hours, preferably at least 12 hours, preferably at least 14 hours, preferably at least 16 hours, preferably at least 18 hours, preferably at least 20 hours Initial release rates are preferably 10 wt % of the total amount of pharmaceutical agent in the nanomedicinal composition during the induction period. Upon passage of the induction period and arrival of the nanomedicinal composition at a target site, a major portion of the pharmaceutical agent is released. In some embodiments, the major portion comprises at least 25 wt %, preferably at least 30 wt %, preferably at least 35 wt %, preferably at least 40 wt %, preferably at least 45 wt %, preferably at least 50 wt % of a total amount of pharmaceutical agent released. In some embodiments, the induction period is provided by a coating disposed on the nanomedicinal composition, the coating as described above. In such embodiments, the coating may inhibit the release of the pharmaceutical agent mixture. Removal of the coating by any suitable process, for example by dissolving, degrading, or digesting, may allow the pharmaceutical gent mixture to be released. In some embodiments, the nanomedicinal composition has an antioxidant release rate of 0.1 to 10 wt % per hour, preferably 0.25 to 9 wt % per hour, preferably 0.5 to 7.5 wt % per hour, preferably 0.75 to 5 wt % per hour, preferably 1 to 4 wt % per hour based on a total initial weight of antioxidant. In such embodiments, the antioxidant release rate may be an average antioxidant release rate measured over the release period as described above. In some embodiments, the nanomedicinal composition has an initial antioxidant release rate which is maintained over an initial release period. In such embodiments, the initial release period may be followed by a second release period which has a second antioxidant release rate. The initial antioxidant release rate and/or second antioxidant release rate may be average release rates as described above. In some embodiments, the nanomedicinal composition has an anti-cancer therapeutic release rate of 0.5 to 15 wt % per hour, preferably 1 to 14 wt % per hour, preferably 2.5 to 13 wt % per hour, preferably 5 to 11 wt % per hour, preferably 6 to 10 wt % per hour based on a total initial weight of anti-cancer therapeutic. In such embodiments, the anti-cancer therapeutic release rate may be an average anti-cancer therapeutic release rate measured over the release period as described above. In some embodiments, the nanomedicinal composition has an initial anti-cancer therapeutic release rate which is maintained over an initial release period. In such embodiments, the initial release period may be followed by a second release period which has a second anti-cancer therapeutic release rate. The initial anti-cancer therapeutic release rate and/or second anti-cancer therapeutic release rate may be average release rates as described above. In some embodiments, the initial release period comprises the first 20 hours of release, preferably the first 18 hours of release, preferably the first 16 hours of release, preferably the first 14 hours of release, preferably the first 12 hours of release, preferably the first 10 hours of release. Such “first hours of release” are preferably measured from the initiation of release. The initiation of release may be measured by, for example a delivery of the nanomedicinal composition to a tumor site, the application of a magnet or magnetic field to target the nanomedicinal composition to a tumor site, the application of an alternating magnetic field for magnetic heating, or a pre-determined amount of time after administration. Such a pre-determined time may be any suitable amount of time known to one of ordinary skill in the art, for example, an expected time for delivery of the nanomedicinal composition to the tumor site, an expected circulation time, an expected coating degradation time, or the like. The present disclosure also relates to a method of forming the nanomedicinal composition, the method comprising mixing an M(II) salt and a Fe(III) salt with the porous silicate matrix to form a powdery mixture, calcining the powdery mixture to form the nanocarrier, mixing the nanocarrier and the anti-cancer therapeutic in an aqueous solution thereby forming a therapeutic-containing nanocarrier, and mixing the therapeutic-containing nanocarrier and the antioxidant in an impregnation solution thereby forming the nanomedicinal composition. In general, any suitable M(II) salt known to one of ordinary skill in the art may be used. Examples of such suitable M(II) salts include, but are not limited to halide salts, acetate salts, oxalate salts, formate salts, hydroxide salts, sulfate salts, sulfite salts, phosphate salts, hydrogen phosphate salts, dihydrogen phosphate salts, carbonate salts, bicarbonate salts, and nitrate salts. Preferably, the M(II) salt does not comprise an anion which itself comprises a metal, such as chromate salts, aluminate salts, and arsenate salts. In preferred embodiments, the M(II) salt is a nitrate salt. The M(II) salt may be used in anhydrous form or in hydrate form. In general, any suitable Fe(III) salt known to one of ordinary skill in the art may be used, as described above. In preferred embodiments, the Fe(III) salt is Fe(III) nitrate. The Fe(III) salt may be used in anhydrous form or in hydrate form. In some embodiments, the calcining is performed at a temperature of 700 to 1,000° C., preferably 725 to 975° C., preferably 750 to 950° C., preferably 775 to 925° C., preferably 800 to 900° C., preferably 825 to 875° C., preferably 840 to 860° C., preferably 850° C. The calcining step may be carried out under air, nitrogen, argon or a combination thereof. The mixture of gas may be 60% to 100%, or 70% to 90% nitrogen and 0% to 80%, 10% to 70%, or 30% to 50% argon. In preferred embodiments, the calcining is performed in ambient air. In some embodiments, the calcining is performed for 1 to 12 hours, preferably 1.5 to 11 hours, preferably 2 to 10 hours, preferably 2.5 to 9.5 hours, preferably 3 to 9 hours, preferably 3.5 to 8.5 hours, preferably 4 to 8 hours, preferably 4.5 to 7.5 hours, preferably 5 to 7 hours, preferably 5.5 to 6.5 hours, preferably 6 hours. In some embodiments, the aqueous solution is a saline. In preferred embodiments, the aqueous solution is phosphate buffered saline. In some embodiments, the anti-cancer therapeutic is present in the aqueous solution at a concentration of 1.0 to 20.0 mM. preferably 2.5 to 17.5 mM, preferably 5.0 to 15.0 mM, preferably 7.5 to 12.5 mM, preferably 8.0 to 11.0 mM, preferably 9 to 11.0 mM, preferably 9.5 to 10.5 mM, preferably 10.0 mM. In some embodiments, the nanocarrier is present in the aqueous solution at a concentration of 20 to 100 mg/mL, preferably 30 to 90 mg/mL, preferably 35 to 85 mg/mL, preferably 40 to 80 mg/mL, preferably 45 to 75 mg/mL, preferably 50 to 70 mg/mL, preferably 55 to 65 mg/mL, preferably 60 mg/mL. In some embodiments, the impregnation solution comprises an alcohol solvent. In some embodiments, the alcohol solvent is methanol. In some embodiments, the impregnation solution comprises water. In some embodiments, the impregnation solution comprises glycerol. In some embodiments, the antioxidant is present in the impregnation solution at a concentration of 1.0 to 20.0 mM, preferably 2.5 to 17.5 mM, preferably 5.0 to 15.0 mM, preferably 7.5 to 12.5 mM, preferably 8.0 to 11.0 mM, preferably 9 to 11.0 mM, preferably 9.5 to 10.5 mM, preferably 10.0 mM. In some embodiments, the therapeutic-containing nanocarrier is present in the impregnation solution at a concentration of 2 to 30 mg/mL, preferably 4 to 28 mg/mL, preferably 6 to 26 mg/mL preferably 8 to 24 mg/mL, preferably 10 to 22 mg/mL, preferably 12 to 20 mg/mL, preferably 13 to 19 mg/mL, preferably 14 to 18 mg/mL, preferably 15 to 17 mg/mL, preferably 16 mg/mL. The present disclosure also relates to a method for treating a cancer in a subject, the method comprising administering to a subject in need of therapy a pharmaceutical composition comprising the nanomedicinal composition. The cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, and lung cancer. In preferred embodiments, the cancer is breast cancer. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers. As used herein, a “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism, does not abrogate the biological activity and properties of the administered active ingredient, and/or does not interact in a deleterious manner with the other components of the composition in which it contains. The term “carrier” encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well-known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a pharmaceutical composition will depend upon the intended route of administration for the pharmaceutical composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g. Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which is incorporated herein by reference in its entirety). Examples of physiologically acceptable carriers include antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatine, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or non-ionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). An “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils, and polyethylene glycols. In some embodiments, the pharmaceutically acceptable carrier and/or excipient is at least one selected from the group consisting of a buffer, an inorganic salt, a fatty acid, a vegetable oil, a synthetic fatty ester, a surfactant, and a polymer. Exemplary buffers include, without limitation, phosphate buffers, citrate buffer, acetate buffers, borate buffers, carbonate buffers, bicarbonate buffers, and buffers with other organic acids and salts. Exemplary inorganic salts include, without limitation, calcium carbonate, calcium phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc oxide, zinc sulfate, and magnesium trisilicate. Exemplary fatty acids include, without limitation, an omega-3 fatty acid (e.g., linolenic acid, docosahexaenoic acid, eicosapentaenoic acid) and an omega-6 fatty acid (e.g., linoleic acid, eicosadienoic acid, arachidonic acid). Other fatty acids, such as oleic acid, palmitoleic acid, palmitic acid, stearic acid, and myristic acid, may be included. Exemplary vegetable oils include, without limitation, avocado oil, olive oil, palm oil, coconut oil, rapeseed oil, soybean oil, corn oil, sunflower oil, cottonseed oil, and peanut oil, grape seed oil, hazelnut oil, linseed oil, rice bran oil, safflower oil, sesame oil, brazil nut oil, carapa oil, passion fruit oil, and cocoa butter. Exemplary synthetic fatty esters include, without limitation, methyl, ethyl, isopropyl and butyl esters of fatty acids (e.g., isopropyl palmitate, glyceryl stearate, ethyl oleate, isopropyl myristate, isopropyl isostearate, diisopropyl sebacate, ethyl stearate, di-n-butyl adipate, dipropylene glycol pelargonate), C12-C16 fatty alcohol lactates (e.g., cetyl lactate and lauryl lactate), propylene dipelargonate, 2-ethylhexyl isononoate, 2-ethylhexyl stearate, isopropyl lanolate, 2-ethylhexyl salicylate, cetyl myristate, oleyl myristate, oleyl stearate, oleyl oleate, hexyl laurate, isohexyl laurate, propylene glycol fatty ester, and polyoxyethylene sorbitan fatty ester. As used herein, the term “propylene glycol fatty ester” refers to a monoether or diester, or mixtures thereof, formed between propylene glycol or polypropylene glycol and a fatty acid. The term “polyoxyethylene sorbitan fatty ester” denotes oleate esters of sorbitol and its anhydrides, typically copolymerized with ethylene oxide. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Surfactants that may be present in the compositions of the present disclosure include zwitterionic (amphoteric) surfactants, e.g., phosphatidylcholine, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), anionic surfactants, e.g., sodium lauryl sulfate, sodium octane sulfonate, sodium decane sulfonate, and sodium dodecane sulfonate, non-ionic surfactants, e.g., sorbitan monolaurate, sorbitan monopalmitate, sorbitan trioleate, polysorbates such as polysorbate 20 (Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80), cationic surfactants, e.g., decyltrimethylammonium bromide, dodecyltrimethyl—ammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, and dodecylammonium chloride, and combinations thereof. Exemplary polymers include, without limitation, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), a polyvinyl alcohols, and copolymers, terpolymers, or combinations or mixtures therein. The copolymer/terpolymer may be a random copolymer/terpolymer, or a block copolymer/terpolymer. Depending on the route of administration e.g. oral, parental, or topical, the pharmaceutical composition may be in the form of solid dosage form such as tablets, caplets, capsules, powders, and granules, semi-solid dosage form such as gels, pastes, and suppositories, liquid dosage forms such as suspension, and dispersions, inhalation dosage form such as aerosols, sprays, and powders. Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the active ingredient can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatine, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering ingredients such as sodium citrate, magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such pharmaceutical compositions can also comprise adjuvants, such as wetting ingredients, emulsifying and suspending ingredients, and sweetening, flavoring, and perfuming ingredients. For therapeutic purposes, formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection dispersions or suspensions. The term “parenteral”, as used herein, includes intravenous, intravesical, intraperitoneal, subcutaneous, intramuscular, intralesional, intracranial, intrapulmonal, intracardial, intrasternal, and sublingual injections, or infusion techniques. These dispersions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The active ingredient can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting ingredients and suspending ingredients. The sterile injectable preparation can also be a sterile injectable dispersion or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectable. Dimethylacetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting ingredients such as those discussed above are also useful. Suppositories for rectal administration can be prepared by mixing the active ingredient with a suitable non-irritating excipient, such as cocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug. Such suppositories may be advantageous for treating colorectal cancer, but may be unsuitable for treating other cancers. Administration by inhalation may be advantageous for treating lung cancer, but may be unsuitable for treating other cancers. In other embodiments, the pharmaceutical composition comprising the nanomedicinal composition disclosed herein thereof has different release rates categorized as immediate release and controlled- or sustained-release. The presence of the particles of a magnetic ferrite in the nanomedicinal composition may serve one or more purposes. A first purpose may be to aid targeting the drug to a particular diseased tissue by applying external magnetic field to the diseased tissues, and thereby concentrating the drug in the diseased tissues in need of treatment and minimize the drug contacts with healthy tissues. A second purpose may be that the particles of a magnetic ferrite are magnetic contrasting agent used in magnetic resonance (MM) imaging. Thus, the method of treatment may involve a combination of administering effective amount of the drug to a subject, while observing and targeting the drug to the diseased tissue by an applied external magnetic field. A third purpose may be that the particles of a magnetic ferrite may be used in magnetic heating. Such heating is a response to exposure of the nanocarrier to an alternating magnetic field. Such heating may be useful for a purpose such as increasing the rate of anti-cancer therapeutic and/or antioxidant release, increasing the amount of anti-cancer therapeutic and/or antioxidant released, and/or hyperthermia treatment of cancer. Hyperthermia treatment is a treatment method which involves heating a tissue, tumor, or other area to a temperature above its normal temperature. Such heating may be achieved by the application of an alternating magnetic field to a magnetic material co-located with the desired treatment area, laser heating, microwave heating, or any other suitable heating method known to one of ordinary skill in the art. In some embodiments, the method further comprises exposing the subject to an alternating magnetic field, thereby raising the temperature of the nanomedicinal composition. As used herein, the terms “treat”, “treatment”, and “treating” in the context of the administration of a therapy to a subject in need thereof refers to the reduction or inhibition of the progression and/or duration of a disease (e.g. cancer), the reduction or amelioration of the severity of the disease, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies. “Treating” or “treatment” of the disease includes preventing the disease from occurring in a subject that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development), ameliorating the disease, providing relief from the symptoms or side-effects of the disease (including palliative treatment), and relieving the disease (causing regression of the disease). With regard to the disease, these terms simply mean that one or more of the symptoms of the disease will be reduced. Such terms may refer to one, two, three, or more results following the administration of one, two, three, or more therapies: (1) a stabilization, reduction (e.g. by more than 10%, 20%, 30%, 40%, 50%, preferably by more than 60% of the population of cancer cells and/or tumour size before administration), or elimination of the cancer cells, (2) inhibiting cancerous cell division and/or cancerous cell proliferation, (3) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with a pathology related to or caused in part by unregulated or aberrant cellular division, (4) an increase in disease-free, relapse-free, progression-free, and/or overall survival, duration, or rate, (5) a decrease in hospitalization rate, (6) a decrease in hospitalization length, (7) eradication, removal, or control of primary, regional and/or metastatic cancer, (8) a stabilization or reduction (e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, preferably at least 80% relative to the initial growth rate) in the growth of a tumor or neoplasm, (9) an impairment in the formation of a tumor, (10) a reduction in mortality, (11) an increase in the response rate, the durability of response, or number of patients who respond or are in remission, (12) the size of the tumor is maintained and does not increase or increases by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%, (13) a decrease in the need for surgery (e.g. colectomy, mastectomy), and (14) preventing or reducing (e.g. by more than 10%, more than 30%, preferably by more than 60% of the population of metastasized cancer cells before administration) the metastasis of cancer cells. The term “subject” and “patient” are used interchangeably. As used herein, they refer to any subject for whom or which therapy, including with the pharmaceutical compositions according to the present disclosure is desired. In most embodiments, the subject is a mammal, including but is not limited to a human, a non-human primate such as a chimpanzee, a domestic livestock such as a cattle, a horse, a swine, a pet animal such as a dog, a cat, and a rabbit, and a laboratory subject such as a rodent, e.g. a rat, a mouse, and a guinea pig. In preferred embodiments, the subject is a human. As used herein, a subject in need of therapy includes a subject already with the disease, a subject which does not yet experience or exhibit symptoms of the disease, and a subject predisposed to the disease. In preferred embodiments, the subject is a person who is predisposed to cancer, e.g. a person with a family history of cancer. People who (i) had inflammatory bowel disease, or a genetic syndrome such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (Lynch syndrome), and/or (ii) consumes a low-fiber and high-fat diet are at a higher risk of contracting colon cancer. White women or a person with (i) certain inherited genes (e.g. mutated BRCA1, BRCA2, ATM, TP53, CHEK2, PTEN, CDH1, STK11, and PALB2), (ii) radiation occurred to one's chest, and/or (iii) exposure to diethylstilbestrol (DES) are at a higher risk of contracting breast cancer. People who (i) smoke or regularly breathe in second-hand smoke, (ii) exposed to carcinogens including, but not limited to polycyclic aromatic hydrocarbons (e. g. benzo[a]pyrene, benz[a]anthracene, and methylated derivatives thereof), asbestos, radioactive substances (e.g., uranium, radon), and/or (iii) inhaled chemicals or minerals (e.g., arsenic, beryllium, cadmium, silica, vinyl chloride, nickel compounds, chromium compounds, coal products, mustard gas, and chloromethyl ethers) are at a higher risk of contracting lung cancer. The terms “administer”, “administering”, “administration”, and the like, as used herein, refer to the methods that may be used to enable delivery of the active ingredient and/or the composition to the desired site of biological action. Routes or modes of administration are as set forth herein. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), and rectal administration. Those of ordinary skill in the art are familiar with administration techniques that can be employed with the compounds and methods described herein. In preferred embodiments, the active ingredient and/or the pharmaceutical composition described herein are administered orally. The dosage amount and treatment duration are dependent on factors, such as bioavailability of a drug, administration mode, toxicity of a drug, gender, age, lifestyle, body weight, the use of other drugs and dietary supplements, the disease stage, tolerance and resistance of the body to the administered drug, etc., and then determined and adjusted accordingly. The terms “effective amount”, “therapeutically effective amount”, “pharmaceutically effective amount” or “sufficient amount” refer to that amount of the active ingredient being administered which will relieve to some extent one or more of the symptoms of the disease being treated. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate “effective amount” may differ from one individual to another. An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study. In some embodiments, an effective amount is in the range of 0.1-30 g/kg of the nanomedicinal composition per body weight of the subject. In treating certain cancers, the best approach is often a combination of surgery, radiotherapy, and/or chemotherapy. Therefore, in at least one embodiment, the pharmaceutical composition is employed in conjunction with radiotherapy. In another embodiment, the pharmaceutical composition is employed with surgery. The radiotherapy and/or surgery may be before or after the composition is administered. A treatment method may comprise administering the pharmaceutical composition of the current disclosure as a single dose or multiple individual divided doses and applying a magnetic field to the diseased tissue, wherein the nanomedicinal composition is accumulated and releases the loaded anti-cancer therapeutic and/or antioxidant in or nearby the diseased tissues. In some embodiments, the pharmaceutical composition is administered at various dosages (e.g. a first dose with an effective amount of nanomedicinal composition comprising 200 mg of the anti-cancer therapeutic per kilogram of the subject and a second dose with an effective amount of the nanomedicinal composition comprising 50 mg of the anti-cancer therapeutic per kilogram of the subject). In some embodiments, the interval of time between the administration of the pharmaceutical composition and the administration of one or more additional therapies may be about 1-5 minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, or any period of time in between. Preferably, the pharmaceutical composition is administered once daily for at least 2 days, 5 days, 6 days, or 7 days. In certain embodiments, the pharmaceutical composition and one or more additional therapies are administered less than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, or 5 years apart. The methods for treating cancer and other proliferative disorders described herein inhibit, remove, eradicate, reduce, regress, diminish, arrest or stabilize a cancerous tumor, including at least one of the tumor growth, tumor cell viability, tumor cell division and proliferation, tumor metabolism, blood flow to the tumor and metastasis of the tumor. In some embodiments, the size of a tumor, whether by volume, weight or diameter, is reduced after the treatment by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, relative to the tumor size before treatment. In other embodiments, the size of a tumor after treatment does not reduce but is maintained the same as the tumor size before treatment. Methods of assessing tumor size include, but are not limited to, CT scan, MRI, DCE-MRI and PET scan. In most embodiments of treatment, the method further comprises measuring a concentration of a biomarker and/or detecting a mutation in a biomarker before and/or after the pharmaceutical composition comprising the nanomedicinal composition of the present disclosure is administered. As used herein, the term “biomarker” refers to a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention. Generic cancer biomarkers include circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Exemplary biomarkers for breast cancer include, without limitation, BRCA1, BRCA2, HER-2, estrogen receptor, progesterone receptor, CA 15-3, CA 27.29, CEA, Ki67, cyclin D1, cyclin E, and ERβ. Potentially predictive cancer biomarkers include, without limitation, mutations in genes BRCA1 and BRCA2 for breast cancer and/or ovarian cancer, overexpression of CEA, NSE, CYFRA-21-1, CA-125, and CA-199 for lung cancer, overexpression of TYMS, mutations in genes p53 and KRAS for colon cancer. The mutation in the biomarker may be detected by any suitable procedure known to one of ordinary skill in the art, such as restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR) assay, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), hetero-duplex analysis, protein truncation test (PTT), and oligonucleotide ligation assay (OLA). The procedures to detect the mutation are well-known to those of ordinary skill in the art. The term “sample” used herein refers to any biological sample obtained from the subject in need of therapy including a single cell, multiple cells, fragments of cells, a tissue sample, and/or body fluid. Specifically, the biological sample may include red blood cells, white blood cells, platelets, hepatocytes, epithelial cells, endothelial cells, a skin biopsy, a mucosa biopsy, an aliquot of urine, saliva, whole blood, serum, plasma, lymph. In some embodiments, the biological sample is taken from a tumor. The concentration level of the cancer biomarker in a sample may be measured by an assay, for example an immunoassay. Typical immunoassay methods include, without limitation, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), Western blotting, immunohistochemistry (IHC), immunocytochemistry, immunostaining, and multiple reaction monitoring (MRM) based mass spectrometric immunoassay. The protocol for measuring the concentration of the biomarker and/or detecting the mutation in the biomarker is known to those of ordinary skill, for example by performing the steps outlined in the commercially available assay kit sold by Sigma-Aldrich, Thermo Fisher Scientific, R & D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam, Trevigen, Dojindo Molecular Technologies, Biovision, and Enzo Life Sciences. In some embodiments, the concentration of the biomarker is measured before and after the administration. When the concentration of the biomarker is maintained, the method may further comprise increasing the effective amount of the nanomedicinal composition by at least 5%, at least 10%, or at least 30%, up to 50%, up to 60%, or up to 80% of an initial effective amount nanomedicinal composition that contains in the range of 1-300 mg of the anti-cancer therapeutic per kilogram of the body weight of the subject. The increased effective amount may be in a range of 1.05-540 mg/kg, preferably 15-420 mg/kg, more preferably 25-270 mg/kg. The subject may be administered with the increased dosage for a longer period (e.g. one more week, 2 more weeks, or 2 more months) than the duration prescribed with the initial effective amount. In some embodiments, the mutation in the biomarker is detected before administering the pharmaceutical composition to identify subjects predisposed to the disease. For example, subjects with a BRCA1 germline mutation are at a higher risk of contracting breast cancer, or ovarian cancer. In some embodiments, the biomarkers are measured/detected after each administration. For example, the measurement may be 1-5 minutes, 1-30 minutes, 30-60 minutes, 1-2 hours, 2-12 hours, 12-24 hours, 1-2 days, 1-15 weeks, 15-20 weeks, 20-30 weeks, weeks, 40-50 weeks, 1 year, 2 years, or any period of time in between after the administration. In some embodiments, the administration is stopped once the subject is treated. The examples below are intended to further illustrate protocols for preparing, characterizing, and using the nanomedicinal composition or for treating a cancer using the nanomedicinal composition and are not intended to limit the scope of the claims. Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. Examples Ludox AS-40 (silica source), Tetrapropylammonium bromide (micropore template), Cetyltrimethyl ammonium bromide (mesoporous template), Cu(NO3)2.3H2O, Fe(NO3)3.9H2O, and cisplatin were purchased from Sigma Aldrich. Curcumin was obtained from Molecule on (New Zealand). Q-10 silica with pore diameter of about 18 nm was obtained from Fuji Silysia Chemical Ltd. Spherical hydrophobic silica was purchased from Superior Silica, USA. All the reagents used in in vitro study were of analytical grade. Gibco cell culture products such as DMEM (Dulbecco's Modified Eagle Medium), heat-inactivated fetal bovine serum (HI-FBS), 100× Penicillin Streptomycin, and 100×MEM NEAA (MEM non-essential amino acids) were obtained from Thermo Fisher. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, cat. M2128 was purchased from Sigma-Aldrich. Hoechst 33342 nuclear stain, cat. 62249, and cleaved-Caspase antibody 3 (c-Caspase 3), cat. 9661, were procured from Thermo Scientific and Cell Signaling, respectively. Alexa Fluor 594 goat anti-rabbit secondary antibody, cat. R37117 was obtained from Invitrogen. Preparation of 30% CuFe2O4/Mesosilicalite and 30% CuFe2O4/MCM-41 Mesosilicalite was prepared by disintegrating silicalite crystals in alkaline medium following the top-down approach. A detailed synthesis procedure for mesosilicalite, MCM-41, SBA-16, and mesobeta are provided in Vijaya Ravinayagam; Rabindran Jermy, B., J. Nanopart Res. 2017, 19, 190, incorporated herein by reference in its entirety. The copper ferrite impregnated mesosilicalite and MCM-41 was prepared by dry mixing. Briefly, 0.61 g of copper nitrate trihydrate, 1.01 g of iron nitrate nonahydrate and 1.4 g of predried structured silica was physically mixed for 30 min using mortar pistol. The obtained mixture was calcined at 850° C. for 6 h. Curcumin coating and Cisplatin functionalization over 30% CuFe2O4/Mesosilicalite and 30% CuFe2O4/MCM-41 40 mg of curcumin was dissolved in 10 ml of methanol for 10 min. Then 160 mg of cisplatin loaded mesosilicalite or MCM-41 was added and the mixture was sonicated for 2 min. Then the solvent was evaporated using rotary evaporator. Cisplatin (30 mg) was first added in normal saline solution (10 ml) and stirred to form a clear solution. Then, copper ferrite nanocomposite (600 mg) was added and stirred overnight under ice cold dark environment. The solution was then filtered, washed and dried. The functionalized cisplatin was calculated using UV-visible spectroscopy at 208 nm. Characterization Techniques The phase of support carriers CuFe2O4/mesosilicalite, CuFe2O4/MCM-41, CuFe2O4/SBA-16, CuFe2O4/Hydrophobic silica, CuFe2O4/MesoZ SM-5 and CuFe2O4/Mesobeta was identified using benchtop XRD (Miniflex 600, Rigaku, Japan). The textural features including BET surface area, pore size and pore volume were measured using nitrogen adsorption technique (ASAP-2020 plus, Micromeritics, USA). The ferrite nanoparticle chemical coordination was analyzed using DRS-UV-visible spectroscopy analysis (JASCO, Japan). Vibrating sample magnetometer (LDJ electronics, 9600) was used to determine the magnetic property of CuFe2O4/mesosilicalite and CuFe2O4/MCM-41. The functional groups of curcumin, cisplatin in nanoformulation were determined using FT-IR spectroscopy (Perkin Elmer). The morphological variations of spinel ferrite/mesosilicalite/curcumin/cisplatin were investigated using transmission electron microscopy (TEM, JEM2100F, JEOL). Drug Release Study The release trend of curcumin and cisplatin was investigated using different nanoformulations. Curcumin release was carried out by dissolving 30 mg of curcumin loaded sample in 50 ml of PBS (pH 5.6). 10 ml of solution was withdrawn and replaced with equal volume of fresh solution. The release content was identified at specific wavelength of 428 nm. Prior to the cisplatin release study, the dialysis membrane was activated and then 30 mg of nanoformulation was dispersed in 50 ml of PBS solution (pH 5.6). The cisplatin release was monitored at 37° C. At regular time intervals, 10 ml of solution was withdrawn and cisplatin release was measured using UV-visible spectroscopy. The withdrawn solution was replaced with equal volume of fresh PBS solution. Cell Lines and Cell Culture Setting Human mammary adenocarcinoma (MCF7) and the non-cancerous human foreskin fibroblasts (HFF) cell lines were used to assess the cytotoxic effect of the prepared compounds. Cells were cultured in a DMEM culture medium containing 10% HI-FBS, 1% Penicillin Streptomycin, and 1% MEM NEAA. Cells were maintained in a humidified setting at 37° C. with 5% CO2. For cell viability assay, cells were seeded in a 96-well plate with a density of 20,000 cells/well. While for microscopic images, 50,000 cells/well were plated on an eight-well chamber slide. On the following day, cells were switched to the starve culture medium that contains 0.5% HI-FBS. Cells were maintained for 24 h in the starve medium before the treatment was added. Cell Treatment MCF7 and HFF cells were treated with the subsequent conditions for 48 h: CuFe2O4/Mesosilicalite, Curcumin, Cisplatin, CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, or CuFe2O4/MCM-41/Curcumin/Cisplatin. Treatment concentrations of CuFe2O4/Mesosilicalite, CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposites were: 0.025, 0.05, 0.1, and 0.5 mg/ml. Based on the drug loading experiments, simple calculations were followed to reflect the actual quantity of curcumin and cisplatin that adsorbed on CuFe2O4/Mesosilicalite nanoparticles. According to the loading experiments, 1 mg of CuFe2O4nanoparticles contains 0.25 mg and 0.045 mg of curcumin and cisplatin, respectively. Thus, if CuFe2O4/Mesosilicalite concentration was 0.5 mg/ml, there is 0.125 mg/ml of adsorbed curcumin and 0.0225 mg/ml of functionalized cisplatin. Therefore, the treatment concentrations of curcumin were: 0.00625, 0.0125, 0.025 and 0.125 mg/ml and the treatment concentrations of cisplatin were: 0.001125, 0.00225, 0.0045 and 0.0225 mg/ml. Cell Viability (MTT) and EC50 To assess the compounds' cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a cell viability assay, was used. It measures the cell viability by assessing the mitochondria's ability to convert yellow MTT solution into purple formazan insoluble crystals. Following Mosmann's protocol, 5 mg/ml of MTT powder was dissolved in PBS, and 0.5 mg/ml of MTT working solution was prepared [Mosmann, T., J Immunol Methods, 1983, 65, 55-6317]. Cells were washed with PBS and followed by the addition of 100 ul MTT working solution. All the experimental conditions were run in triplicates (technical repeats) with four biological repeats (n=4). The 96-well plate was incubated for three h at 37° C. An MTT negative control (background control) was included in the experimental setting by adding MTT working solution to wells that contain no cells. After the incubation time, 0.04 N HCl isopropyl alcohol was added to solubilize the formed formazan granules. The difference in color intensity was measured by SYNERGY-neo2 BioTek ELISA reader at 570 nm as a measuring wavelength. The technical triplicate readings of each condition were averaged, and the absorbance from MTT negative control was deducted from these readings. An initial reading was measured before MTT addition at the same wavelength. Thus, to remove undesirable interference in the measurement, the initial reading was subtracted from the final reading. Then, the treatment groups were analyzed by comparing them to the control (untreated cells). Cell viability was calculated using the following equation (1): %CellViability=averagedsamplereadaveragedcontrolread×100(1) The cell viability assay data of the five biological repeats (n=5) were plotted on a graph against their corresponding dose. Afterward, the plotted data were used to extrapolate a best-fit line equation for each compound for MCF7 and HFF cell lines. These equations were used to calculate the half-maximal effective concentration (EC50) of each drug. Immunofluorescent Staining and Microscopic Examination Cells were plated in an 8-well chamber slide at a concentration of 50,000 cells/well. Cells were then treated for 48 h with the following conditions: CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin at a concentration of 0.5 mg/ml. Cells were fixed and stained with the apoptotic marker cleaved Caspase 3 (c-Caspase 3) antibody (1:200, Cell Signaling Technology) and incubated at 4° C. overnight. After PBS washing, Alexa Fluor 594-conjugated secondary antibody (Invitrogen, Thermo Fisher Scientific) was added to the cells at a final concentration of 1:1000 for 1 h at room temperature. Cells were then washed and stained with the nuclear stain Hoechst 33342 (Thermo Fisher Scientific) at a concentration of 2 μg/ml and incubated at room temperature for min. After staining, immunofluorescent images were taken using a confocal fluorescent microscope-Zeiss LSM 700. Bright-field images were captured using an inverted microscope-Nikon Eclipse TS100. Although both light and fluorescent pictures were taken from the same sample, they were not taken from the same field of view. Statistics The cell viability assay was performed in five independent experiments (n=5). Statistical analysis was performed using Prism 9 software (GraphPad, La Jolla, CA). The analysis was performed using one-way ANOVA with Dunnett's post hoc test. Error bars±S.E.M. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 versus control. In case there was no indication of significance, it means that results were non-significant. The data analysis of drug delivery was done using Prism 8 software and SPSS software version 20.0. Results The X-ray diffraction pattern of meso and microphase of two supports were identified at low and high angle of MCM-41 and Mesosilicalite (FIG.1A). The XRD pattern of CuFe2O4impregnated over different structured nanocarriers are shown inFIGS.1A-1C. Conventional MCM-41 showed a typical hexagonal structure with peaks at low angle corresponding to the plane (100), (110) and (200). Mesosilicalite showed the presence of MCM-41 and high silica zeolite peaks at 2 theta value of 7.9° and 8.7° corresponding to plane (101) and (200), respectively (SeeFIG.1B). In the higher angle (20-60°), a broad peak of MCM-41 indicates the presence of amorphous framework bound to hexagonal phase. Mesosilicalite showed the typical characteristics peaks of MFI structure with corresponding to (321), (113), (501), (422) and (313) plane. In case of spinel impregnated MCM-41 and mesosilicalite, the presence of cubic phase of copper spinel appears with intense peak at 35.6° corresponding to (103) plane. A trace of a-Fe2O3appears along with a less intense peak corresponding to CuO at 38.7° (SeeFIG.1A). The characteristics of textural surface, pore volume and average pore size of MCM-41, CuFe2O4/MCM-41, Mesosilicalite, and CuFe2O4/Mesosilicalite are shown inFIGS.2A-2D. The surface area and pore size distribution of CuFe2O4impregnated over different structured materials are shown inFIG.2EandFIG.2F, respectively. Before, spinel ferrite impregnation, MCM-41 showed a typical type IV isotherm indicating the presence of uniform pore size distributions (3.7 nm) with high surface area of 914 m2/g and pore volume of 0.85 cc/g. In case of pure hexagonal mesopores of MCM-41, surprisingly, impregnation of spinel, only slightly reduced the surface area to 885 m2/g, while pore volume reduced to 0.52 cc/g. In case of mesosilicalite, which contains micro and mesopores, the initial surface area of 804 m2/g reduced significantly after impregnation to 74 m2/g. An increase in dual pore sizes from 3.0 nm to 5.9 nm indicates the pore filling effect and formation of external pores in the hierarchical micro and mesopores after spinel ferrite loading. Pore volume reduction from 0.6 cc/g to 0.1 cc/g reflects the pore filling effect. Overall, the result shows unique deposition of spinel occurs over two supports. The textural characteristics vary depending on the structure of different shaped materials. A summary of these characteristics is presented in Table 1. TABLE 1Textural characteristics of 30% CuFe2O4impregnated on different mesostructured materialsBJHBETadsorptionAverageSurfacecumulativePorePoreareasurface areavolumeDiameterSample(m2/g)(m2/g)(cm3/g)(nm)Mesosilicalite8047850.603.0CuFe2O4/70310.105.9MesosilicaliteMCM-4191411160.853.7CuFe2O4/8858570.522.4MCM-41SBA-169885910.692.79CuFe2O4/SBA-161441030.123.4Q-10 silica2482641.2520.1CuFe2O4/2232461.0118.2Q-10 silicaHydrophobic silica84530.199.29CuFe2O4/49370.1412.1Hydrophobic silicaMesobeta533(208)a3410.392.9CuFe2O4/462(157)a2470.353.03MesobetaMesoZSM-57107190.583.28CuFe2O4/67300.095.48MesoZSM-5*amicropore surface area FIG.3shows the diffuse reflectance UV-visible spectra of curcumin, cisplatin, MCM-41, CuFe2O4/MCM-41, CuFe2O4/mesosilicalite, CuFe2O4/MCM-41/curcumin/cisplatin and CuFe2O4/mesosilicalite/curcumin/cisplatin. Curcumin and cisplatin revealed broad absorption between 200-600 nm. The support SiMCM-41 showed the absorption bands at about 210 and 260 nm, indicating the framework coordinated siliceous species. In case of CuFe2O4/MCM-41 and CuFe2O4/mesosilicalite, the spectra showed a weak absorption below 230 nm and a strong and broad absorption between 240-800 nm. The peaks of CuFe2O4/MCM-41 and CuFe2O4/mesosilicalite correlate with cubic spinel exhibits tetrahedral (215 nm) and octahedral (440-700 nm) crystalline coordination sites [Najmoddin, N., et. al., Microporous Mesoporous Mater. 2014, 190, 346-355]. The presence of such peak absorption indicates the dispersion and integrated spinel ferrites over both supports. In the case of the mesosilicalite support, an enhanced intense broad peak shows the presence of higher crystalline mixed phase of oxides due to octahedral coordinated spinel species compared to the MCM-41 support (seeFIG.3). This can be attributed mainly due to presence of micropores, which tends to accommodate spinel species at the external surface of mesosilicalite. After loading of curcumin and cisplatin, the absorption maximum increases significantly over both CuFe2O4/Mesosilicalite and CuFe2O4/MCM-41 nanocomposites. However, in case with CuFe2O4/mesosilicalite/curcumin, a two band abortion at about 400 nm and 500 nm clearly indicates the presence of distributed curcumin and Pt species. However, with CuFe2O4/MCM-41/curcumin, a broadness of absorption peak indicates cohabitation of curcumin and Pt species over MCM-41. Such increase in homogenous expansion behavior clearly indicates the composite formation over mesoporous MCM-41 support than with mesosilicalite. The magnetic characteristics and saturation value of CuFe2O4/MCM-41 and CuFe2O4/Mesosilicalite nanocomposites were analyzed by vibrating sample magnetometer at room temperature (FIG.4). A distribution of cations at different coordination sites of A and B characterize the magnetic property. CuFe2O4/MCM-41 and CuFe2O4/Mesosilicalite showed ferromagnetic property with saturation magnetization value of about 0.9 emu/g. The saturation magnetization value was reported to be related to the magnetic phase concentration. Fe loaded on high surface area MCM-41 was reported to exhibit magnetization of 3.86 emu/g [Kiatphuengporn, S., et. al., Chem. Engg. J. 2016, 306, 866-875]. In a previous study, spinel impregnation over spherical silica with lower surface area of 178 m2/g was found to exhibit similar ferromagnetism with magnetic value of about 7.6 emu/g [Jermy, B. R., et. al., J. Nanotechnol. 2019, 10, 2217-2228]. Further, it has been reported that superparamagnetic effect was due to anti parallel spins of Fe3+species in tetrahedral coordination site. FromFIG.4, it is clear that the support has been shown to influence the magnetic property. The loading of spinel over mesosilicalite support tends to generate different types of nanoclusters at the pore walls (as evidenced by increased pore diameter from 3.0 to 5.9 nm). The generation of small sized nanoclusters reported to generate super paramagnetic Fe3+ions, while larger nanoclusters generates ferromagnetic behavior [Cuello, N. I., et. al., Mater. Sci. Eng.; C 2017, 78, 674-681]. In line with the diffuse reflectance spectra, presence of tetrahedral and octahedral species over high surface area parent mesosilicalite and MCM-41 is proposed to lead the broad hysteresis structure characteristics of ferromagnetism. The observed reduction in saturation magnetization value is mainly attributed due to presence of siloxane layers on copper spinel ferrites. The FT-IR spectra of cisplatin, curcumin, CuFe2O4/mesosilicalite, CuFe2O4/mesosilicalite/curcumin and CuFe2O4/mesosilicalite/curcumin/cisplatin are shown inFIG.5. Drug cisplatin showed a characteristic functional group related to NH group of platinum complex between 400-1800 cm−1. A symmetric and asymmetric bending of NH 2 group was clearly observed between 1300-1600 cm−1, while plane bending of cisplatin can be seen at about 800 cm-1. In the case of curcumin, the presence of carbonyl (C═O), carbon-carbon double bond (C═C), and methylene (CH 2) bending peaks are observed between 1625-1450 cm−1. Both symmetric and asymmetric vibrations corresponding to ether bond (C—O—C) are observed between 1300-1000 cm−1[Bhandari, R., et. al., Mater. Sci. Eng. C. 2016, 67, 59-64]. A peak corresponding to the enolic OH group of curcumin appears clearly at about 960 cm−1. In the case of CuFe2O4/mesosilicalite, a zeolitic peak indicating hybrid formation between mesoporous and microporous zeolite appears at about 550 cm−1. In the case of CuFe2O4/mesosilicalite/curcumin (curcumin loading step 1), there was no distinct peaks of curcumin was observed. A significant reduction in hydroxyl group of enol indicates an effective interaction inside the mesosilicalite pores and curcumin. In the case of CuFe2O4/mesosilicalite/curcumin/cisplatin (cisplatin loading step 2) sample, the loading of cisplatin tends to shows the peaks of curcumin. It indicates that during cisplatin functionalization step, curcumin present inside the pores of mesosilicalite diffuse out and present at the external surface of mesosilicalite. The presence of peak at about 1023 cm−1indicates the C—O—C stretching of C6H5—O—CH3group [Mohan, P. R. K., et. al., Vib. Spectrosc. 2012, 62, 77-84]. Also, the presence of cisplatin peaks reveals an effective functionalization of drug at external surface of mesosilicalite. The morphological features of CuFe2O4/Mesosilicalite/Curcumin/Cisplatin were analyzed using transmission electron microscope at different scale bar of 50 nm and 10 nm (FIGS.6A-6C). The images showed the presence of curcumin at the external surface of MCM-41, where uniformly layered hexagonal pore channels coexisting with curcumin. As detected in XRD and DRS-UV analysis, though the active spinel metal components were not readily distinguishable in TEM analysis, the migration of curcumin can be clearly observed as a surface coating at the external surface of mesosilicalite. The release of curcumin over CuFe2O4impregnated on different structured nanoporous mesosilicalite (hexagonal micro/mesopore), SBA-16 (cubic), MCM-41 (hexagonal mesopore), Q-10 (large pore), hydrophobic silica, mesobeta (BEA large pore) and mesoZSM-5 (MFI) are studied under acidic pH (pH 5.6) condition for 72 h (seeFIG.7). The percentage cumulative release profile of curcumin over hexagonal shaped silica (MCM-41 and mesosilicalite) was found to be superior of about 40-50% compared to cubic shaped SBA-16. CuFe2O4/SBA-16, which contains a 3D pore architecture showed a release of about 25% for 72 h. This indicates the ink shaped pores of SBA-16 (about 3.3 nm) are slightly restricted with spinel ferrite impregnation but showed a sustained release behavior with respect to curcumin. MesoZSM-5 with MFI structure consisting of sinusoidal pores of about 0.56 nm and spherical silica with hydrophobic character showed a release ability of 20%. Q-10 silica with pore size of about 18 nm and mesobeta with medium pore size of 3 nm showed comparatively a less curcumin release of 10%. The release trend shows the structured silica with different pore shapes profoundly affects the release of curcumin. For quick release, hexagonal shaped channel pores of MCM-41 and mesosilicalite can be more readily utilized than cubic shaped pores of SBA-16 and aluminosilicates. Based on the present requirement, cisplatin was loaded on curcumin/CuFe2O4/mesosilicalite and curcumin/CuFe2O4/MCM-41. The presence of micro and mesopores of mesosilicalite was found to favor the high release of cisplatin (88% for 72 h), while mesopores of MCM-41 showed a release of about 63% for 72 h. This suggest that cisplatin tends to functionalize on the external micropores of mesosilicalite, while MCM-41 is able to accommodate the cisplatin inside the mesopores. For different structured mesoporous nanocarriers, data analysis was done using Prism 8 software and SPSS software version 20.0 (Tables 2-5). TABLE 2Descriptive Statistics of Curcumin Release overdifferent CuFe2O4/structured silica based nonoformulations95% ConfidenceStd.Std.Interval for MeanSamplesNMeanDeviationErrorLower BoundUpper BoundMinimumMaximumCuFe2O4/MCM-411240.6719.6165.66328.2053.13197411330.0811.9963.32722.8337.3324221317.157.5482.09312.5921.7232531339.9211.8073.27532.7947.0619514136.383.9481.0954.008.7721251313.155.0801.40910.0816.221186138.692.626.7287.1110.2821171317.235.8901.63413.6720.79328Total10321.4815.8361.56018.3824.571741 - Curcumin/CuFe2O4/Mesosilicalite,2 - Curcumin/CuFe2O4/SBA-16,3 - Curcumin/CuFe2O4/MCM-41,4 - Curcumin/CuFe2O4/Q-10 Silica,5 - Curcumin/CuFe2O4/Hydrophobic silica,6 - Curcumin/CuFe2O4/Mesobeta and7 - Curcumin/CuFe2O4/MesoZSM-5 TABLE 3Curcumin release over different CuFe2O4/structuredsilica based nanoformulations (One way ANOVA)Sum of SquaresdfMean SquareFSig.Between16267.63872323.94823.709.000GroupsWithin9312.0519598.022GroupsTotal25579.689102 TABLE 4Post Hoc tests for mean differencein Curcumin release among differentCuFe2O4/structured silica based nanoformulationsComparision ofCuFe2O4/structuredsilica basedMeanStandardSignificancenanoformulationsDifferenceError(p < 0.05)CuFe2O4/MCM-41723.443.9630.000*1712.853.8830.008*270.083.8831.0003722.693.8830.000*4710.853.8830.036*574.083.8830.829678.543.8830.148*Significant at 0.05 level1-Curcumin/CuFe2O4/Mesosilicalite,2-Curcumin/CuFe2O4/SBA-16,3-Curcumin/CuFe2O4/MCM-41,4-Curcumin/CuFe2O4/Q-10 Silica,5-Curcumin/CuFe2O4/Hydrophobic silica,6-Curcumin/CuFe2O4/Mesobeta and7-Curcumin/CuFe2O4/MesoZSM-5 TABLE 5Pearson correlation of Curcumin release overdifferent CuFe2O4/structured based nanoformulationsCuFe2O4/MCM-411234567CuFe2O4/MCM-41110.341120.5150.937**130.5370.914**0.943**140.744**0.599*0.644*0.666*150.3750.932**0.956**0.917**0.504160.4550.810**0.898**0.881**0.5350.828**170.1570.777**0.5410.5380.1460.617*0.4631**Correlation is significant at the 0.01 level (2-tailed).*Correlation is significant at the 0.05 level (2-tailed).1 - Curcumin/CuFe2O4/Mesosilicalite,2 - Curcumin/CuFe2O4/SBA-16,3 - Curcumin/CuFe2O4/MCM-41,4 - Curcumin/CuFe2O4/Q-10 Silica,5 - Curcumin/CuFe2O4/Hydrophobic silica,6 - Curcumin/CuFe2O4/Mesobeta and7 - Curcumin/CuFe2O4/MesoZSM-5 Table 2 shows the mean and standard deviation of curcumin drug release at pH 5.6. CuFe2O4/MCM-41 showed a high mean score of 40.67, whereas CuFe2O4/Q-10 Silica/Curcumin demonstrated a low mean score of 6.38. The maximum score of curcumin drug release at pH 5.6 was observed in CuFe2O4/MCM-41/Curcumin and CuFe2O4/MesoZSM-5/Curcumin, however the minimum curcumin release was observed in CuFe2O4/Hydrophobic silica/Curcumin. From Table 3, the results showed that there is significant difference between the groups with respect to curcumin release at pH 5.6 (p<0.05). As significant difference was found, Scheffe's post hoc test was used to determine the significant difference between two groups at the same time. It is observed that the mean difference in the curcumin release between the groups such as CuFe2O4/MCM-41 and CuFe2O4/MesoZSM-5; CuFe2O4/Mesosilicalite and CuFe2O4/MesoZSM-5; CuFe2O4/MCM-41 and CuFe2O4/MesoZSM-5; CuFe2O4/Q-10 silica and CuFe2O4/MesoZSM-5 was found to be significant (p<0.05). However, no significant mean difference was observed between the groups such as CuFe2O4/SBA-16 and CuFe2O4/MesoZSM-5; CuFe2O4/Hydrophobic silica and CuFe2O4/MesoZSM-5; CuFe2O4/MesoZSM-5 and CuFe2O4/MesoZSM-5 with respect to the curcumin release (p>0.05). Notably, CuFe2O4/MCM-41 showed a high mean difference score of curcumin release (40.67) with CuFe2O4/MesoZSM-5 when compared other CuFe2O4/structured silica based nanoformulations (See Table 4). Using Pearson correlation, it was inferred that CuFe2O4/MCM-41 has a significant strong and positive correlation with only CuFe2O4/Q-10 silica/Curcumin (p<0.01). The formulation variable CuFe2O4/Mesosilicalite/Curcumin showed a significant strong and positive correlation with the variables such as CuFe2O4/SBA-16/Curcumin, CuFe2O4/MCM-41/Curcumin, CuFe2O4/Hydrophobic silica/Curcumin, CuFe2O4/MesoZSM-5/Curcumin, and CuFe2O4/Mesobeta/Curcumin (p<0.01). Similarly, CuFe2O4/SBA-16/Curcumin described a significant strong and positive correlation with the formulation variables such as CuFe2O4/MCM-41/Curcumin, CuFe2O4/Hydrophobic silica/Curcumin, and CuFe2O4/Me sob eta/Curcumin (p<0.01). Further, CuFe2O4/MCM-41/Curcumin showed a significant strong and positive correlation with CuFe2O4/Hydrophobic silica/Curcumin and CuFe2O4/Mesobeta/Curcumin (p<0.01). A significant strong and positive relationship was observed between CuFe2O4/Hydrophobic silica/Curcumin and CuFe2O4/Mesobeta/Curcumin (p<0.01) (Table 5). On the other hand, CuFe2O4/Q-10 silica/Curcumin described a significant moderate and positive correlation with CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/SBA-16/Curcumin, and CuFe2O4/MCM-41/Curcumin (p<0.05). A significant moderate and positive correlation was also observed between CuFe2O4/MesoZSM-5/Curcumin and CuFe2O4/Hydrophobic silica/Curcumin (p<0.05) (See Table 5). To test the efficiency of the nanocomposites as potential chemotherapeutic agents, their effects on the cell viability of MCF7 (breast cancer) and HFF (human foreskin fibroblast) cell lines were investigated. The cell viability assay, MTT, was performed after 48 h of treatment with the following conditions: CuFe2O4/Mesosilicalite, Curcumin, Cisplatin, CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposites. The results are shown inFIG.8Afor MCF7 cell line and inFIG.8Bfor HFF cell line. Treatment concentrations of CuFe2O4/Mesosilicalite, CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposites were: 0.025, 0.05, 0.1, and 0.5 mg/ml. While treatment concentrations for curcumin were: 0.00625, 0.0125, 0.025 and 0.125 mg/ml and that for the cisplatin group: 0.00225, 0.0045, and 0.0225 mg/ml. As detailed in the Materials and Methods section, treatment concentrations of curcumin and cisplatin were adjusted to reflect the actual concentration adsorbed onto the mesosilicalite nanocomposites. The results were analyzed by comparing data from the treated cells with the control group (untreated cells). Cells treated with CuFe2O4/Mesosilicalite nanocomposites had no effect on either MCF7 or HFF cells suggesting that the CuFe2O4/Mesosilicalite nanocarrier did not interfere with cell viability. Pure curcumin reduced cell viability only at the highest concentration in MCF7; however, it minimally reduced the cell viability in HFF cell lines. As anticipated, pure cisplatin resulted in a reduction in cell viability in both MCF7 and HFF cell lines. Furthermore, cells that were treated with the nanocomposites CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin all resulted in a significant reduction in cell viability in a dose-dependent manner (seeFIGS.8A-8B). Upon close investigation at the third dose of treatment in MCF7 cells, the pure curcumin reduced cell viability to 80.71%, while the nanocomposites that were coated with curcumin either with or without cisplatin significantly reduced the cell viability to 20.98% (group D inFIG.8A), and 8.96% (group E inFIG.8A). In contrast, using the same dose on the non-cancerous cell line HFF, the pure curcumin reduced cell viability to 84.12%, whereas the nanocomposites that were coated with curcumin did not result in a significant reduction in cell viability (72.88%; group D inFIG.8B). Whereas HFF treated with nanocomposites that were coated with curcumin and functionalized with cisplatin resulted in a significant reduction in cell viability of 12.93% (group E inFIG.8B). Interestingly, the CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposite resulted in a significant reduction of cell viability of 24.44% in MCF7 (group F inFIG.8A), and an insignificant reduction in viability of 75.53% in HFF (group F inFIG.8B). Using a drug combination of cisplatin and curcumin nanocomposites will increase the cumulative cytotoxic effects of both compounds, solve the problem of cisplatin drug resistance in tumors, and increase the bioavailability of curcumin. The CuFe2O4/Mesosilicalite/Curcumin/Cisplatin nanocomposite had a stronger effect even at lower concentrations on both cell lines. However, using the MCM-41/mesosilicalite in the cisplatin/curcumin nanocomposite had a significant effect on MCF7, while having a minimal effect on HFF. These results suggest that using MCM-41 with a cisplatin/curcumin drug combination has the potential of affecting cancerous cells while sparing normal ones. To calculate the EC50 of treatment conditions, the data from release profiles (seeFIGS.9A-9H) was used to extrapolate the line equation and calculate the EC50. The calculated EC50 values are compiled in Table 6 for MCF7 cell line and Table 7 for HFF cell line. Treatment with cisplatin resulted in an EC50 of 4.425 and 3.763 μg/ml in MCF7 and HFF, respectively. Coating the CuFe2O4/Mesosilicalite with curcumin resulted in an EC50 of 80.2 μg/ml in MCF7 and 141.0 mg/ml in HFF. Moreover, functionalizing the CuFe2O4/Mesosilicalite with curcumin and cisplatin resulted in an EC50 of 81.23 μg/ml in MCF7 and 76.83 μg/ml in HFF. However, functionalizing curcumin and cisplatin onto CuFe2O4/MCM-41 nanocomposite resulted in an EC50 of 72.51 μg/ml in MCF7 and 154.2 mg/ml in HFF. These results show that while using the mesosilicalite resulted in a similar EC50, using the MCM-41 support resulted in a fold difference in EC50 values between MCF7 and HFF. When comparing between the nanocomposites, these results show that both CuFe2O4/Mesosilicalite/Curcumin/Cisplatin and CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposites are potential novel chemotherapeutic options. However, the results show that CuFe2O4/MCM-41/Curcumin/Cisplatin may be a better candidate due to the wider difference in EC50 (a fold change) between cancerous and non-cancerous cell line. TABLE 6Calculated EC50 values for the nanocarrier against MCF-7 cell line.Log EC50EC50ValueDrug GroupValue(μg/mL)R2Cisplatin0.64594.4250.7444CuFe2O4/Mesosilicalite/1.90480.200.9106CurcuminCuFe2O4/Mesosilicalite/1.91081.230.7990Curcumin/CisplatinCuFe2O4/MCM-41/1.86072.510.9067Curcumin/Cisplatin TABLE 7Calculated EC50 values for the nanocarrier against HFF cell lineLog EC50EC50ValueDrug GroupValue(μg/mL)R2Cisplatin0.57553.7630.6306CuFe2O4/Mesosilicalite/2.149141.00.9415CurcuminCuFe2O4/Mesosilicalite/1.88676.830.8560Curcumin/CisplatinCuFe2O4/MCM-41/2.188154.21.7742Curcumin/Cisplatin Further, the apoptotic effects of treating cells with CuFe2O4/Mesosilicalite/Curcumin, CuFe2O4/Mesosilicalite/Curcumin/Cisplatin, and CuFe2O4/MCM-41/Curcumin/Cisplatin nanocomposites were explored. Cells were viewed under light and fluorescent microscopes. For the latter, cells were stained with the apoptotic marker cleaved-Caspase 3 (c-Caspase 3), 10 and Hoechst, which is a nuclear marker. These images are presented inFIG.10. The images clearly show cells stained (magenta) with c-Caspase 3, which is the activated form of the protein, after treatment with our nanocomposites for 48 h. These results suggest that the nanocomposites significantly reduce cell viability by activating apoptosis. Curcumin has been extensively investigated as a chemotherapeutic agent in breast cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, osteoclastoma, and bladder cancer [Li, W., et. al., Oncol Rep. 2017, 37, 3459-3466; Cao, F., et. al., Int J Clin Exp Pathol. 2015, 8(6), 6037-6045; and Zhang, L., et. al., Int J Oncol. 2018, 53, 515-526]. It has been found to activate INK pathway, induce the generation of reactive oxygen species (ROS), and subsequently apoptosis [Syng-Ai, C., et. al., Mol Cancer Ther. 2004, 3(9), 1101-1108; and Zhu, Y., & Bu, S., Evidence-Based Complementary and Alternative Medicine 2017, 1-13]. However, curcumin has some problems that hinder its full potential such as low solubility, low bioavailability, and rapid elimination from the body [Anand, P., et. al., Mol Pharm. 2007, 4(6), 807-818]. Research is being conducted to increase the bioavailability of curcumin. On the other hand, cisplatin is a well-established chemotherapeutic drug. Unfortunately, it is fraught with several issues such as drug resistance and systemic toxicity [Dasari, S., & Bernard Tchounwou, P., European J. Pharmacol. 2014, 740, 364-378]. The mechanism of action of cisplatin is by activating the INK pathway and inducing oxidative stress, DNA damage, and apoptosis. However, cisplatin will also result in increased levels of Glutathione S transferase (GST), resulting in a reduction in ROS, and resistance to cisplatin. Therefore, a combination treatment of cisplatin and curcumin, which increases ROS, might augment the cytotoxic effect and prevent cisplatin-related drug resistance [Townsend, D. M., & Tew, K. D., Oncogene 2003, 22, 7369-7375]. Moreover, these results show that the structural framework of silica had a significant effect on the cytotoxic effectiveness of nanocomposites. In a previous publication by the inventors of the current disclosure, cisplatin-functionalized cubic spinel CuFe2O4loaded on monodispersed spherical hydrophilic silica (HYPS) nanoparticles was tested on MCF7 breast cancer cells [Jermy, B. R., et. al., J. Nanotechnol. 2019, 10, 2217-2228]. In the present disclosure, two nanoformulations using silicate materials having hexagonally shaped pores: a) the mesosilicalite with zeolite (strong) framework, and b) the MCM-41 with amorphous (weak) framework were used. Both of which were coated with curcumin and functionalized with cisplatin. While the EC50 of the previous spherical shaped silica nanoformulation was equal to 180.89 μg/ml (See Jermy, B. R., et. al., J. Nanotechnol. 2019, 10, 2217-2228) the present hexagonal shaped silica nanocomposites had EC50 values of 81.23 μg/ml (mesosilicalite) and 72.51 μg/ml (MCM-41). These results clearly indicate that changing the structural framework from spherical to hexagonally shaped silica increased the chemotherapeutic efficiency and reduced the EC50 values. This improved effectiveness marks a significant improvement brought about by the change in the silicate matrix. | 101,007 |
11857640 | DETAILED DESCRIPTION The present invention relates to novel adenoviral vectors derived from a chimpanzee adenovirus, AdY25, immunogenic compositions thereof and their use in medicine. AdY25 is a chimpanzee adenovirus which has been sequenced for the first time by the present inventors. The nucleotide sequence is provided in SEQ ID NO. 1. A first aspect of the present invention therefore provides a nucleic acid molecule having the sequence of SEQ ID NO. 1. In one embodiment, the nucleic acid molecule is isolated. The person skilled in the art will appreciate that there are homologues, equivalents and derivatives of all of the nucleic acid sequences described herein. Thus, the invention also encompasses nucleic acid molecules having a sequence substantially identical to the nucleic acid sequences described herein over their entire length. One of skill in the art will appreciate that the present invention can also include variants of those particular nucleic acid molecules which are exemplified herein. These may occur in nature, for example because of strain variation. For example, additions, substitutions and/or deletions are included. One of skill in the art will also appreciate that variation from the particular nucleic acid molecules exemplified herein will be possible in view of the degeneracy of the genetic code. Preferably, the variants have substantial identity to the nucleic acid sequences described herein over their entire length. As used herein, nucleic acid sequences which have “substantial identity” preferably have at least 80%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity with said sequences. Desirably, the term “substantial identity” indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art nucleic acid sequences. When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package). BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention, when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length. The above applied mutatis mutandis to all nucleic acid sequences disclosed in the present application. Preferably, the nucleic acid molecule according to the first aspect has a sequence at least 98% identical to SEQ ID NO.1, more preferably at least 98.6% identical to SEQ ID NO.1. Preferably, the nucleic acid molecule according to the first aspect comprises one or more nucleotide sequences selected from the group consisting of;(a) nucleotides 18302 to 21130 of SEQ ID NO. 1 or a sequence substantially identical thereto;(b) nucleotides 13891 to 15486 of SEQ ID NO. 1 or a sequence substantially identical thereto; and(c) nucleotides 32290 to 33621 of SEQ ID NO. 1 or a sequence substantially identical thereto. These nucleotide sequences encode the (a) hexon, (b) penton and (c) fibre capsid proteins of AdY25, the exterior regions of which determine the properties of the viral vector, including serotype. The nucleic acid molecule according to the first aspect may also comprise one or more nucleotide sequences selected from the group consisting of:(a) a nucleotide sequence encoding a hexon protein comprising the amino acid sequence of SEQ ID NO.2, or a sequence at least 98.2% identical thereto; or a nucleotide sequence encoding a hexon protein having a sequence at least 98.2% identical to the protein encoded by nucleotides 18302 to 21 130 of SEQ ID NO. 1;(b) a nucleotide sequence encoding a penton protein comprising the amino acid sequence of SEQ ID NO.3, or a sequence at least 98.3% identical thereto; or a nucleotide sequence encoding a penton protein having a sequence at least 98.3% identical to the protein encoded by nucleotides 13891 to 15486 of SEQ ID NO. 1; and(c) a nucleotide sequence encoding a fiber protein comprising the amino acid sequence of SEQ ID NO.4 or a sequence at least 99.1% identical thereto; or a nucleotide sequence encoding a fiber protein having a sequence at least 99.1% identical to the protein encoded by nucleotides 32290 to 33621 of SEQ ID NO. 1. Nucleic acid molecules comprising a sequence complementary to the nucleic acid molecule according to the first aspect of the present invention are within the scope of the present invention. Nucleic acid molecules which hybridize only to the nucleic acid molecule according to the first aspect of the present invention are also encompassed by the present application. Thus, the conditions used for hybridisation are sufficiently stringent that only such nucleic acid sequences would remain hybridised. The person skilled in the art would easily be able to determine such conditions. The nucleic acid can be DNA, including cDNA, RNA including mRNA or PNA (peptide nucleic acid) or a mixture thereof. Table 1 provides an overview of the wildtype AdY25 sequences disclosed herein: SEQIDCorresponding nucleotides inNO.DescriptionSEQ ID NO. 11GenomeN/A(nucleotide sequence)2Hexon proteinNucleotides 18302 to 21130 (L3)3Penton proteinNucleotides 13891 to 15486 (L2)4Fibre proteinNucleotides 32290 to 33621 (L5)5E1ANucleotides 577 to 1143 and 1237to 14436EIB 19 KDaNucleotides 1602 to 21657E1B 55 KDaNucleotides 1907 to 34068pIXNucleotides 3491 to 39199IVa2Nucleotides 5587 to 5602 and 3978to 5311 (E2)10PolymeraseNucleotides 13838 to 13846 and5081 to 8662 (E2)11pTPNucleotides 13838 to 13846 and8463 to 10392 (E2)1252/55 kDaNucleotides 10827 to 12017 (L1)13IIIaNucleotides 12041 to 13807 (L1)14VIINucleotides 15493 to 1607415VNucleotides 16119 to 1714116MuNucleotides 17161 to 1739417VINucleotides 17470 to 1820118EndoproteaseNucleotides 21146 to 2177519DNA bindingNucleotides 21852 to 23390protein20100 kDaNucleotides 23419 to 25827 (L4)2122 KDaNucleotides 25544 to 260982233 KDaNucleotides 25544 to 25871 and26041 to 26372 (L4)23pVIIINucleotides 25602 to 26285 (L4)24E3 12.5 KDaNucleotides 27139 to 2745925E3 CRIaINucleotides 27413 to 2805126E3 gp19 KDaNucleotides 28033 to 2856327E3 22.3 KDaNucleotides 29350 to 2997928E3 31 KDaNucleotides 29999 to 3090729E3 10.4 KDaNucleotides 30916 to 3119130E3 15.2 KDaNucleotides 31200 to 3164331E3 14.7 KDaNucleotides 31636 to 3204032E4 Orf 6/7Nucleotides 34688 to 34861 and33716 to 3396533E4 Orf 6Nucleotides 33965 to 3486134E4 Orf 4Nucleotides 34764 to 3513235E4 Orf 3Nucleotides 35141 to 3549436E4 Orf 2Nucleotides 35491 to 3588037E4 Orf 1Nucleotides 35930 to 36304 The genome sequence data has confirmed early serological studies that simian AdY25 is closely related to human group E adenovirus, AdHu44. Alignment of the amino acid sequences of hexon and fibre proteins from different adenoviral serotypes have been used to create the phylogenetic trees inFIGS.1A and1B. These are the major surface-exposed capsid components and are believed to be the primary determinants of vector tropism. Alignment of whole genomic nucleotide sequences of different adenoviral species have been used to create the phylogenetic tree inFIG.2. The genome and the fibre proteins align AdY25 with the group E adenoviruses. However, the hexon proteins align AdY25 with the group D adenoviruses. Merely for the convenience of those of skill in the art, a sample ofE. colistrain DH10B containing bacterial artificial chromosomes (BACs) containing the cloned genome of chimpanzee adenovirus Y25 (pBACe3.6 Y25, cell line name “Y25”) was deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052401. TheE. colicontaining the BAC is a class I genetically modified organism. The genotype ofE. colistrain DH10B is: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu)7697 araD139 galU galK nupG rpsL λ31. Chimpanzee adenovirus Y25 is provisionally classified within the species Human adenovirus E based on the nucleotide sequence of the viral DNA polymerase. The BAC propagates within the bacteria during replication and can be maintained by selection with chloramphenicol. TheE. colistrain DH10B containing the BAC into which the genome is cloned can be propagated in Luria-Bertani broth or agar containing 12.5 μg/mL chloramphenicol at 37° C. Converting the BAC clones of the viral genomes into viruses (“rescue”) can be carried out by the following steps. TheE. colihost is propagated and the BAC DNA is purified from the bacteria according to standard methods. The DNA is linearised with the restriction endonuclease PmeI and transfected into any cell line supporting growth of human adenoviruses (e.g. A549 cells). The resulting adenovirus can then be propagated and purified for use as a vaccine, for example. All of these reagents and cells are publicly available. If the deposition were rescued, the resulting virus would be a wild-type adenovirus. In respect of all designated states to which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited material be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. Rule 32(1) EPC, Rule 13(1) and Schedule 1 of the UK Patent Rules 2007, Regulation 3.25(3) of the Australian Patent Regulations and generally similar provisions mutatis mutandis for any other designated state. Furthermore, merely for the convenience of those of skill in the art, a sample ofE. colistrain DH10B containing bacterial artificial chromosomes (BACs) containing the cloned genome of chimpanzee adenovirus Y25 with deletion of the E1 region (pBACe3.6 Y25delE1, cell line name “Y25delE1”) was deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052402. TheE. colicontaining the BAC is a class I genetically modified organism. The genotype ofE. colistrain DH10B is: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu) 7697 araD139 gal U galK nupG rpsL λ−. Chimpanzee adenovirus Y25 is provisionally classified within the species Human adenovirus E based on the nucleotide sequence of the viral DNA polymerase. The BAC propagates within the bacteria during replication and can be maintained by selection with chloramphenicol. TheE. colistrain DH10B containing the bacterial artificial chromosomes into which the genomes are cloned can be propagated in Luria-Bertani broth or agar containing 12.5 μg/mL chloramphenicol at 37° C. Converting the BAC clones of the viral genomes into viruses (“rescue”) can be carried out by the following steps. TheE. colihost is propagated and the BAC DNA is purified from the bacteria according to standard methods. The DNA is linearised with the restriction endonuclease PmeI and transfected into HEK293 cells (or a similar E1 complementing cell line). The resulting adenovirus can then be propagated and purified for use as a vaccine for example. All of these reagents and cells are publicly available. If the deposition were rescued, the resulting virus would be a class I genetically modified organism. In respect of all designated states to which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited material be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. Rule 32(1) EPC, Rule 13(1) and Schedule 1 of the UK Patent Rules 2007, Regulation 3.25(3) of the Australian Patent Regulations and generally similar provisions mutatis mutandis for any other designated state. A specific embodiment of the first aspect of the present invention provides the complete genomic sequence of a chimpanzee adenovirus referred to herein as AdY25, wherein said genomic sequence comprises or consists of the genomic sequence deposited in a BAC inE. colistrain DH10B by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052401, or the genomic sequence deposited in a BAC inE. colistrain DH10B by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052402. The inventors have discovered that viral vectors based on the newly sequenced AdY25 can be highly effective. A second aspect of the present invention therefore provides an adenovirus vector comprising a capsid derived from chimpanzee adenovirus AdY25, wherein said capsid encapsidates a nucleic acid molecule comprising an exogeneous nucleotide sequence of interest operably linked to expression control sequences which direct the translation, transcription and/or expression thereof in an animal cell and an adenoviral packaging signal sequence. As used herein, the phrase “viral vector” refers to a recombinant virus or a derivative thereof which is capable of introducing genetic material, including recombinant DNA, into a host cell or host organism by means of transduction or non-productive infection. For example, the vector of the present invention may be a gene delivery vector, a vaccine vector, an antisense delivery vector or a gene therapy vector. As used herein, “AdY25” and “Y25” refer to the chimpanzee adenovirus AdY25 or vectors derived therefrom or based thereon. Shorthand terms are used to indicate modifications made to the wildtype virus. For example, “ΔE1” or “delE1” indicates deletion or functional deletion of the E1 locus. The phrase “Ad5E4Orf6” indicates that the viral vector comprises heterologous E4 open reading frame 6 from the Ad5 virus. The vector of the present invention comprises a capsid derived from chimpanzee adenovirus AdY25. Preferably, the capsid comprises the native or wildtype AdY25 capsid proteins, including penton proteins, hexon proteins, fiber proteins and/or scaffolding proteins. However, one of skill in the art will readily appreciate that small modifications can be made to the capsid proteins without adversely altering vector tropism. In a particularly preferred embodiment, the vector capsid comprises one or more capsid proteins selected from the group consisting of:(a) a hexon protein comprising the amino acid sequence of SEQ ID NO. 2 or a sequence substantially identical thereto;(b) a penton protein comprising amino acid sequence of SEQ ID NO. 3 or a sequence substantially identical thereto; and(c) a fibre protein comprising the amino acid sequence of SEQ ID NO. 4 or a sequence substantially identical thereto. One of skill in the art will appreciate that the present invention can include variants of those particular amino acid sequences which are exemplified herein. Particularly preferred are variants having an amino acid sequence similar to that of the parent protein, in which one or more amino acid residues are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein of the present invention. Various amino acids have similar properties, and one or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Variants include naturally occurring and artificial variants. Artificial variants may be generated using mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms. Preferably, the variants have substantial identity to the amino acid sequences exemplified herein. As used herein, amino acid sequences which have “substantial identity” preferably have at least 80%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity with said sequences. Desirably, the term “substantial identity” indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art amino acid sequences. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. The above applied mutatis mutandis to all amino acid sequences disclosed in the present application. Preferably, the hexon protein comprises an amino acid sequence at least 98.2% identical to SEQ ID NO. 2. Preferably, the penton protein comprises an amino acid sequence at least 98.3% identical to SEQ ID NO. 3. Preferably, the fiber protein comprises an amino acid sequence at least 99.1% identical to SEQ ID NO. 4. The nucleotide sequences for the AdY25 hexon, penton and fibre proteins are set out in nucleotides 18302 to 21130 of SEQ ID NO.1 (hexon protein), nucleotides 13891 to 15486 of SEQ ID NO. 1 (penton protein) and nucleotides 32290-33621 of SEQ ID NO.1 (fibre protein). The vector capsid may comprise one or more AdY25 capsid proteins encoded by these nucleotide sequences or sequences substantially identical thereto. The vector according to the second aspect of the present invention may comprise one of the hexon, penton and fibre proteins as described above, any combination of two of said proteins, or all three of said proteins. The vector of the present invention also comprises a nucleic acid molecule. As a minimum, the nucleic acid molecule comprises an exogeneous nucleotide sequence of interest, operably linked to expression control sequences which direct the translation, transcription and/or expression thereof in an animal cell and an adenoviral packaging signal sequence. Preferably, the exogeneous nucleotide sequence encodes a molecule of interest. The molecule of interest may be a protein, polypeptide or nucleic acid molecule of interest. The exogeneous nucleotide sequence may encode one or more, two or more or three or more molecules of interest. Proteins and polypeptides of interest include antigens, molecular adjuvants, immunostimulatory proteins and recombinases. Preferably, the protein or polypeptide of interest is an antigen. In one embodiment, the antigen is a pathogen-derived antigen. Preferably, the pathogen is selected from the group consisting of bacteria, viruses, prions, fungi, protists and helminthes. Preferably, the antigen is derived from the group consisting ofM. tuberculosis, Plasomodiumsp, influenza virus, HIV, Hepatitis C virus, Cytomegalovirus, Human papilloma virus, malaria parasites,leishmaniaparasites or any mycobacterial species. Preferred antigens include TRAP, MSP-1, AMA-1 and CSP fromPlasmodium, influenza virus antigens and ESAT6, TB10.4 85 A and 85B antigens from Mycobacterium tuberculosis. Particularly preferred antigens include Ag85A fromMycobacterium tuberculosisand nucleoprotein (NP) and matrix protein 1 (M1) from influenza A virus, preferably influenza A virus. In an alternative embodiment, the antigen is a self-antigen. Suitable self-antigens include antigens expressed by tumour cells which allow the immune system to differentiate between tumour cells and other cell types. Suitable self-antigens include antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g. foetal antigens). For example, GD2 is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier. However, GD2 is expressed on the surfaces of a wide range of tumour cells including small-cell lung cancer, neuroblastoma, melanomas and osteosarcomas. Other suitable self-antigens include cell-surface receptors that are found on tumour cells but are rare or absent on the surface of healthy cells. Such receptors may be responsible for activating cellular signalling pathways that result in the unregulated growth and division of the tumour cell. For example, ErbB2 is produced at abnormally high levels on the surface of breast cancer tumour cells. Preferably, the self antigen comprises a tumour-associated antigen (TAA). As used herein, the term ‘antigen’ encompasses one or more epitopes from an antigen and includes the parent antigen, and fragments and variants thereof. These fragments and variants retain essentially the same biological activity or function as the parent antigen. Preferably, they retain or improve upon the antigenicity and/or immunogenicity of the parent antigen. Generally, “antigenic” is taken to mean that the protein or polypeptide is capable of being used to raise antibodies or T cells or indeed is capable of inducing an antibody or T cell response in a subject. “Immunogenic” is taken to mean that the protein or polypeptide is capable of eliciting a potent and preferably a protective immune response in a subject. Thus, in the latter case, the protein or polypeptide may be capable of generating an antibody response and a non-antibody based immune response. Preferably, fragments of the antigens comprise at least n consecutive amino acids from the sequence of the parent antigen, wherein n is preferably at least, or more than, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 57, 58, 59, 60, 70, 80, 90 or 100. The fragments preferably include one or more epitopic regions from the parent antigen. Indeed, the fragment may comprise or consist of an epitope from the parent antigen. Alternatively, the fragment may be sufficiently similar to such regions to retain their antigenic/immunogenic properties. The antigens of the present invention include variants such as derivatives, analogues, homologues or functional equivalents of the parent antigen. Particularly preferred are derivatives, analogues, homologues or functional equivalents having an amino acid sequence similar to that of the parent antigen, in which one or more amino acid residues are substituted, deleted or added in any combination. Preferably, these variants retain an antigenic determinant or epitope in common with the parent antigen. Preferably, the derivatives, analogues, homologues, and functional equivalents have an amino acid sequence substantially identical to amino acid sequence of the parent antigen. The exogeneous nucleotide sequence may encode more than one antigen. The viral vector may be designed to express the one or more antigen genes as an epitope string. Preferably, the epitopes in a string of multiple epitopes are linked together without intervening sequences such that unnecessary nucleic acid and/or amino acid material is avoided. The creation of the epitope string is preferably achieved using a recombinant DNA construct that encodes the amino acid sequence of the epitope string, with the DNA encoding the one or more epitopes in the same reading frame. An exemplary antigen, TIPeGFP, comprises an epitope string which includes the following epitopes: E6FP, SIV-gag, PyCD4 and Py3. Alternatively, the antigens may be expressed as separate polypeptides. One or more of the antigens or antigen genes may be truncated at the C-terminus and/or the N-terminus. This may facilitate cloning and construction of the vectored vaccine and/or enhance the immunogenicity or antigenicity of the antigen. Methods for truncation will be known to those of skill in the art. For example, various well-known techniques of genetic engineering can be used to selectively delete the encoding nucleic acid sequence at either end of the antigen gene, and then insert the desired coding sequence into the viral vector. For example, truncations of the candidate protein are created using 3′ and/or 5′ exonuclease strategies selectively to erode the 3′ and/or 5′ ends of the encoding nucleic acid, respectively. Preferably, the wild type gene sequence is truncated such that the expressed antigen is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids relative to the parent antigen. Preferably, the antigen gene is truncated by 10-20 amino acids at the C-terminus relative to the wild type antigen. More preferably, the antigen gene is truncated by 13-18 amino acids, most preferably by 15 amino acids at the C-terminus relative to the wild type antigen. Preferably, the Ag85A antigen is C-terminally truncated in this manner. One or more of the antigen genes may also comprise a leader sequence. The leader sequence may affect processing of the primary transcript to mRNA, translation efficiency, mRNA stability, and may enhance expression and/or immunogenicity of the antigen. Preferably, the leader sequence is tissue plasminogen activator (tPA). Preferably, the tPA leader sequence is positioned N-terminal to the one or more antigens. The leader sequence such as the tPA leaders sequence may be linked to the sequence of the antigen via a peptide linker. Peptide linkers are generally from 2 to about 50 amino acids in length, and can have any sequence, provided that it does not form a secondary structure that would interfere with domain folding of the fusion protein. One or more of the antigen genes may comprise a marker such as the Green Fluorescent Protein (GFP) marker to facilitate detection of the expressed product of the inserted gene sequence. One or more of the antigen genes may comprise a nucleic acid sequence encoding a tag polypeptide that is covalently linked to the antigen upon translation. Preferably the tag polypeptide is selected from the group consisting of a PK tag, a FLAG tag, a MYC tag, a polyhistidine tag or any tag that can be detected by a monoclonal antibody. The nucleic acid sequence encoding the tag polypeptide may be positioned such that, following translation, the tag is located at the C-terminus or the N-terminus of the expressed antigen or may be internal to the expressed antigen. Preferably, the tag is located at the C-terminus of the expressed antigen. In a preferred embodiment, one or more of the antigen genes encode a PK tag. A tag of this type may facilitate detection of antigen expression and clones expressing the antigen, and/or enhance the immunogenicity or antigenicity of the antigen. If a tag polypeptide is used, nucleotides encoding a linker sequence are preferably inserted between the nucleic acid encoding the tag polypeptide and the nucleic acid encoding the expressed antigen. An exemplary linker is IPNPLLGLD (SEQ ID NO.49). In an alternative embodiment, the exogeneous sequence of interest may be non-protein encoding. For example, the exogeneous nucleotide sequence may be an miRNA or immuno stimulatory RNA sequence. The adenoviral vector may comprise one or more exogeneous nucleotide sequences, for example 1, 2 or 3 or more exogeneous nucleotide sequences. Preferably, each exogeneous nucleotide sequence embodies a transgene. The exogeneous nucleotide sequence embodying the transgene can be a gene or a functional part of the gene. The adenoviral vector may comprise one nucleotide sequence encoding a single molecule of interest. Alternatively, the adenoviral vector may comprise one nucleotide sequence or more than one nucleotide sequence encoding more than one molecule of interest. Preferably, the exogeneous nucleotide sequence is located in a nucleic acid molecule that contains other, adenoviral sequences. The exogeneous nucleotide sequence may be inserted into the site of a partially or fully deleted AdY25 gene, for example into the site of an E1 deletion or an E3 deletion. The exogeneous nucleotide sequence may be inserted into an existing AdY25 gene region to disrupt the function of that region. Alternatively, the exogeneous nucleotide sequence may be inserted into a region of the AdY25 genome with no alteration to the function or seqeuence of the surrounding genes. The exogeneous nucleotide sequence or transgene is preferably operably linked to regulatory sequences necessary to drive translation, transcription and/or expression of the exogeneous nucleotide sequence/transgene in a host cell, for example a mammalian cell. As used herein, the phrase “operably linked” means that the regulatory sequences are contiguous with the nucleic acid sequences they regulate or that said regulatory sequences act in trans, or at a distance, to control the regulated nucleic acid sequence. Such regulatory sequences include appropriate expression control sequences such as transcription initiation, termination, enhancer and promoter sequences, efficient RNA processing signals, such as splicing and polyadenylation signals, sequences that enhance translation efficiency and protein stability and sequences promote protein secretion. Additionally they may contain sequences for repression of transgene expression, for example during production in cell lines expression a transactivating receptor. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. Preferably, the promoter is selected from the group consisting of human CMV promoters, simian CMV promoters, murine CMV promoters, ubiquitin, the EF1 promoter, frog EF1 promoter, actin and other mammalian promoters. Most preferred are human CMV promoters and in particular the human CMV major immediate early promoter. The exogeneous nucleotide sequence(s) of interest may be introduced into the viral vector as part of a cassette. As used herein, the term “cassette” refers to a nucleic acid molecule comprising at least one nucleotide sequence to be expressed, along with its transcriptional and translational control sequences to allow the expression of the nucleotide sequence(s) in a host cell, and optionally restriction sites at the 5′ and 3′ ends of the cassette. Because of the restriction endonuclease sites, the cassettes can easily be inserted, removed or replaced with another cassette. Changing the cassette will result in the expression of different sequence(s) by the vector into which the cassette is incorporated. Alternatively, any method known to one of skill in the art could be used to construct, modify or derive said cassette, for example PCR mutagenesis, In-Fusion®, recombineering, Gateway® cloning, site-specific recombination or topoisomerase cloning. The expression control sequences preferably include the adenovirus elements necessary for replication and virion encapsidation. Preferably, the elements flank the exogeneous nucleotide sequence. Preferably, the Y25 vector comprises the 5′ inverted terminal repeat (ITR) sequences of Y25, which function as origins of replication, and 3′ ITR sequences. The packaging signal sequence functions to direct the assembly of the viral vector. As one of skill in the art will appreciate, there are minimum and maximum contraints upon the length of the nucleic acid molecule that can be encapsidated in the viral vector. Therefore, if required, the nucleic acid molecule may also comprise “stuffing”, i.e. extra nucleotide sequence to bring the final vector genome up to the required size. Preferably, the nucleic acid molecule comprises sufficient “stuffing” to ensure that the nucleic acid molecule is about 80% to about 108% of the length of the wild-type nucleic acid molecule. The nucleic acid molecule may also comprise one or more genes or loci from the AdY25 genome. The wildtype AdY25 genome comprises 4 early transcriptional units (E1, E2, E3 and E4), which have mainly regulatory functions and prepare the host cell for viral replication. The genome also comprises 5 late transcriptional units (L1, L2, L3, L4 and L5), which encode structural proteins including the penton (L2), the hexon (L3), the scaffolding protein (L4) and the fiber protein (L5), which are under the control of a single promoter. Each extremity of the genome comprises an Inverted Terminal Repeat (ITR) which is necessary for viral replication. The viral vector of the present invention may comprise the complete native AdY25 genome, into which the exogeneous nucleotide sequence has been inserted. However, one of skill in the art will appreciate that various modifications to the native AdY25 genome are possible, and indeed desirable, when creating a viral vector. One or more native AdY25 genes may be deleted, functionally deleted or modified to optimise the viral vector. As used herein, the phrase “deleted” refers to total deletion of a gene, whilst “functional deletion” refers to a partial deletion of a gene/locus, or some other modification such as a frame shift mutation, which destroys the ability of the adenovirus to express the gene/locus or renders the gene product non-functional. The AdY25 genome may be modified to increase the insert capacity or hinder replication in host cells and/or increase growth and yield of the viral vector in transformed packaging cell lines. One of skill in the art will appreciate that any number of early or late genes can be functionally deleted. Replication of such modified viral vectors will still be possible in transformed cell lines which comprise a complement of the deleted genes. For example, the viral proteins necessary for replication and assembly can be provided in trans by engineered packaging cell lines or by a helper virus. Therefore, in addition to the exogeneous nucleotide sequence, the vector of the present invention may comprise the minimal adenoviral sequences, the adenoviral genome with one or more deletions or functional deletions of particular genes, or the complete native adenoviral genome, into which has been inserted the exogeneous nucleotide sequence. Preferably, the vector of the present invention comprises the native Y25 late transcriptional units (L1-L5) and/or the native Y25 Inverted Terminal Repeats (ITR) or sequences substantially identical thereto. The amino acid sequences of the native L1, L2, L3, L4, L5 loci, and the corresponding nucleic sequences, are set out in Table 1, above. Preferably, one or more of the early transcriptional units are modified, deleted or functionally deleted. In one embodiment, the viral vector is non-replicating or replication-impaired. As used herein, the term “non-replicating” or “replication-impaired” means not capable of replicating to any significant extent in the majority of normal mammalian cells, preferably normal human cells. It is preferred that the viral vector is incapable of causing a productive infection or disease in the human patient. However, the viral vector is preferably capable of stimulating an immune response. Viruses which are non-replicating or replication-impaired may have become so naturally, i.e. they may be isolated as such from nature. Alternatively, the viruses may be rendered non-replicating or replication-impaired artificially, e.g. by breeding in vitro or by genetic manipulation. For example, a gene which is critical for replication may be functionally deleted. Preferably, the adenoviral vector replication is rendered incompetent by functional deletion of a single transcriptional unit which is essential for viral replication. Preferably, the E1 gene/locus is deleted or functionally deleted. The E1 gene/locus may be replaced with a heterologous transgene, for example a nucleotide sequence or expression cassette encoding a protein or polypeptide of interest. The wildtype AdY25 E1 amino acid sequence, and the corresponding nucleic acid sequence, are set out in Table 1, above. As discussed herein, the recombinant adenovirus may be created by generating a molecular clone of AdY25 in a Bacterial Artificial Chromosome (BAC), and the E1 locus is preferably deleted by including an extra homology flank downstream of the adenovirus E1 region to enable simultaneous deletion of E1 during homologous recombination between the AdY25 viral DNA and a linearised BAC “rescue vector”, as described in Example 1. Preferably, the viral vector according to the present invention comprises one or more recombination sites to enable the insertion of one or more transgenes or cassettes comprising the exogeneous nucleotide sequence. Preferably, the recombination sites comprise phage lambda site specific recombination sites. These recombination sites may be introduced at any suitable locus, but are preferably introduced at the Ad E1 locus. Thus, the non-replicating or replication-impaired vector may be prepared by replacing the E1 gene with a nucleotide sequence encoding the protein or polypeptide of interest. Preferably, the recombination sites attR1 and attR2 are introduced at the Ad E1 locus as part of an Invitrogen Gateway® destination cassette as described in Example 1. Preferably, the vector lacks an adenovirus E3 gene/locus. Deletion of the adenovirus E3 region increases the insert capacity of the new vector by approximately 5 kb. Deletion of E3 has little consequence to viral vector yield since this region is not required for virus replication and therefore does not need to be provided in trans in the packaging cell line. The E3 locus may be deleted using GalK recombineering as described in Example 2. The wildtype AdY25 E3 amino acid sequence, and the corresponding nucleic acid sequence, are set out in Table 1, above. In a particularly preferred embodiment of the present invention, both the E1 and E3 loci are deleted from the AdY25 genome. Preferably, the vector of the present invention comprises the native E2 locus. E2 is a transcriptional unit comprising the open reading frames encoding the Polymerase, PTP and IVa2 proteins. The wildtype AdY25 E4 amino acid sequence, and the corresponding nucleotide sequence, are set out in Table 1, above. Preferably, the vector of the present invention comprises a nucleotide sequence encoding E2 or a sequence substantially identical thereto. As stated above, the viral vectors of the present invention may be produced in engineered cell lines containing a complement of any deleted genes required for viral replication. However, replication of viral vectors according to the present invention may be sub-optimal in cells designed to facilitate replication of other serotypes. For example, as shown inFIG.3A, the first generation of AdY25-based vectors comprising the wildtype E4 locus were found to grow inefficiently in HEK293 cells and yield was approximately two logs lower than for comparable AdHu5-based vectors. It is hypothesized that the low yield resulted from suboptimal interaction between the cell-expressed E1 proteins (designed to support propagation of AdHu5 viruses) and vector-encoded E4 gene products. Therefore, the adenoviral vectors according to the present invention preferably further comprise one or more modifications designed to optimise vector growth and yield in transformed cell lines, such as HEK293, expressing the genes functionally deleted in the adenoviral vector according to the present invention. In one embodiment, the native E4 region may be replaced in its entirety with a heterologous E4 region from other serotype(s), which heterologous E4 region preferably increases vector yield and growth in a transformed cell line. For example, the native E4 region may be replaced with the E4 region from AdHu5 to increase vector yield and growth in HEK293. The adenovirus E4 region comprises at least 6 Open Reading Frames (ORFs or Orfs). Thus, in an alternative embodiment, one or more of the ORFs in the E4 region may be replaced with one or more heterologous ORFs from the E4 region of other adenoviral serotype(s), which heterologous ORF(s) preferably increase(s) vector yield and growth in a transformed cell line. Preferably, 1, 2, 3, 4, 5 or 6 ORFs in the E4 region may be replaced 1, 2, 3, 4, 5 or 6 heterologous ORFs from the E4 region of other serotype(s), e.g. AdHu5. Of particular importance for viral replication in HEK293 cells is the gene product of E4Orf6, a multifunctional protein implicated in late viral mRNA splicing and selective export of viral mRNA, viral DNA synthesis and inhibition of apoptosis. Suboptimal interaction between E4Orf6 and the cell-expressed E1B-55K is believed to reduce the yield of AdChOX1 vectors in HEK293 cells. Therefore, the native E4Orf6 region may be replaced with a heterologous E4Orf6 region. For example, the entire native E4 locus may be replaced with the E4Orf6 gene from AdHu5, as described in Example 3. The amino acid sequence of E4Orf6 from AdHu5 is found in SEQ ID NO.40. A corresponding nucleotide sequence is found at nucleotides 28248 to 29132 of SEQ ID NO. 38. In one embodiment, the vector of the present invention comprises the nucleotide sequence of AdHu5E4Orf6 or a sequence substantially identical thereto. As described in Example 3 and shown inFIG.3A, this modification was found to improve viral yield and growth. In a preferred embodiment, more than one ORF in the E4 region is replaced with more than one heterologous ORF from the E4 region of other serotype(s). For example, native E4Orf4−, E4Orf6 and E4Orf7 may be replaced with the E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5. In a particularly preferred embodiment, the recombinant E4 region comprises the E4Orf1, E4Orf2 and E4Orf3 coding regions from AdY25 and the E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5. The amino acid sequence of E4Orf4 from AdHu5 is found in SEQ ID NO. 41. A corresponding nucleotide sequence is found at nucleotides 29053 to 29397 of SEQ ID NO. 38. The amino acid sequence of the E4Orf6 from AdHu5 is found in SEQ ID NO. 40. A corresponding nucleotide sequence is found at nucleotides 28248 to 29132 of SEQ ID NO. 38. The amino acid sequence of the E4Orf6/7 from AdHu5 is found in SEQ ID NO. 39. A corresponding nucleotide sequence is found at nucleotides 28959 to 29132 and 27969 to 28247 of SEQ ID NO. 38. In one embodiment, the vector of the present invention comprises the nucleotide sequences of AdHu5 E4Orf4, E4Orf6 and E4Orf6/7 or sequences substantially identical thereto. In a particularly preferred embodiment of the present invention, the genome of the viral vector according to the present invention lacks the nucleotide sequences which encode the adenovirus E1 and E3 regions, and has the native E4 locus replaced with E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5, and the E4Orf1, E4Orf2 and E4Orf3 coding regions from AdY25. This particularly preferred embodiment is referred to herein interchangeably as “ChAdOX1” or “AdChOX1”. As described in Example 3, and shown inFIG.3A, the modification of the vector in this way was surprisingly found to increase the rate of hexon production and the growth and replication of the virus. An exemplary nucleotide sequence encoding ChAdOX1 is set out in SEQ ID NO. 38. In this embodiment, E1A, E1B 19 kDa and E1B 55 kDa are deleted and replaced with a Gateway® Destination Cassette (nucleotides 592 to 2550 of SEQ ID NO. 38). E3 CRla1, E3 gp19 kDa, E3 22.3 kDa, E3 31 kDa, E3 10.4 kDa, E3 15.2 kDa and E3 14.7 kDa are deleted and replaced with a Pac1 site (nucleotides 26286 to 26293 of SEQ ID NO. 38). The native E4 region is deleted and replaced with E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5, and the E4Orf1, E4Orf2 and E4Orf3 coding regions from AdY25, as described above. The viral vector encoded by SEQ ID NO. 38 also comprises a number of wild-type AdY25 proteins, the nucleotide sequences of which are set out in Table 2, below: Corresponding nucleotides inProteinSEQ ID NO. 38pIX2638 to 3066IVa24734 to 4749 and 3125 to 4458Polymerase12985 to 12993 and 4228 to 7809pTP12985 to 12993 and 7610 to 953952/55 kD9974 to 11164IIIa11188 to 12954Penton13038 to 14633VII14640 to 15221V15266 to 16288Mu16308 to 16541VI16617 to 17348Hexon17449 to 20277Endoprotease20293 to 20922DNA Binding Protein20999 to 22537100 kDa22566 to 2497422K24691 to 2524533K24691 to 25018, 25188 . . . 25519VIII25602 to 26285Fiber26543 to 27874E4Orf329406 to 29759E4Orf229756 to 30145E4Orf130195 to 30569 Preferably, the genome of the viral vector according to the present invention comprises the nucleotide sequence of SEQ ID NO.38 or a sequence substantially identical thereto, into which is inserted the exogeneous nucleotide sequence encoding the protein of interest. As described in Example 5 and shown inFIGS.5A and5B, modification of the E4 region was found to have little impact on immunogenicity of the viral vector, but did improve the rate of viral growth and replication. Therefore, such E4 modifications can be used to enhance the rate of production of the viral vectors, but will not have a negative impact on the immunogenicity of the vectors. Example 4 andFIG.4demonstrate that the immune responses elicited by the AdY25-based vector ChAdOX1 are robust and comparable to those elicited by AdCh63 (also known as ChAd63) and AdCh68 (also known as AdC68, ChAd68, C9 or SAdV-25). However, the humoral immunogenicity of ChAdOX1 was found to be superior to that of AdCh68, as described in Example 7 andFIG.7. One of skill in the art would expect T-cell responses and antibody responses to correlate fully with one another. The superiority of the humoral responses to ChAdOX1 is therefore surprising. The prevalence of vector neutralising antibodies in human sera from the UK and the Gambia was also surprisingly found to be much lower for the AdY25-based vectors than for another chimpanzee adenoviral vector, AdCh63 (see Example 6 andFIGS.6A and6B). This data suggest that vectors based on AdY25 may encounter less pre-existing immunity within the human population, not only in comparison to vectors based on human adenoviruses, but also in comparison to other existing vectors based on chimpanzee adenoviruses. Example 8 andFIGS.8A and8B demonstrate that ChAdOX1 is capable of inducing immune responses againstMycobacterium tuberculosis, whilst Example 9 andFIG.9demonstrate that ChAdOX1 is capable of inducing immune responses against Influenza A. A third aspect of the present invention provides a pharmaceutical or immunogenic composition comprising the viral vector according to the second aspect of the present invention optionally in combination with one or more additional active ingredients, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Preferably, the composition is an immunogenic and/or antigenic composition. The immunogenic and/or antigenic compositions according to the present invention may be prophylactic (to prevent infection), post-exposure (to treat after infection but before disease) or therapeutic (to treat disease). Preferably, the composition is prophylactic or post-exposure. Preferably, the composition is a vaccine. Where the immunogenic composition is for prophylactic use, the subject is preferably an infant, young child, older child or teenager. Where the immunogenic composition is for therapeutic use, the subject is preferably an adult. The composition may comprise one or more additional active agents, such as an anti-inflammatory agent (for example a p38 inhibitor, glutamate receptor antagonist, or a calcium channel antagonist), AMPA receptor antagonist, a chemotherapeutic agent and/or an antiproliferative agent. The composition may also comprise one or more antimicrobial compounds. Examples of suitable antimicrobial compounds include antituberculous chemotherapeutics such as rifampicin, isoniazid, ethambutol and pyrizinamide. Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration). Suitable adjuvants are well known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminium hydroxide), alum plus Bordatella pertussis and immune stimulatory complexes (ISCOMs, typically a matrix of Quil A containing viral proteins). The composition according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration and the compositions adapted accordingly. For oral administration, the composition can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges. A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatine capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule. Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule. Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration. Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser. Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin. Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas. Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections. Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule. The pharmaceutical composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Preferably, the composition is substantially isotonic with humans. Preferably, the pharmaceutical compositions of the present invention deliver an immunogenically or pharmaceutically effective amount of the viral vector to a patient. As used herein ‘immunogenically or pharmaceutically effective amount’ means that the administration of that amount to an individual, either as a single dose or as a series of doses, is effective for prevention or treatment of a disease or condition. In particular, this phrase means that a sufficient amount of the viral vector is delivered to the patient over a suitable timeframe such that a sufficient amount of the antigen is produced by the patient's cells to stimulate an immune response which is effective for prevention or treatment of a disease or condition. This amount varies depending on the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system, the degree of protection desired, the formulation of the vaccine, the doctor's assessment of the medical situation and other relevant factors. In general, a pharmaceutically effective dose comprises 1×107to 1×1012viral particles, preferably 1×1010to 1×1011particles. The immunogenic composition of the present invention may also comprise one or more other viral vectors, preferably other adenoviral vectors. A fourth aspect of the present invention provides the use of the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. In particular, the fourth aspect provides the use of the viral vector or the immunogenic composition of the present invention in medicine. This aspect also provides: i) the viral vector or the immunogenic composition according to the present invention for use in medicine and ii) the use of the viral vector or the immunogenic composition according to the present invention in the manufacture of a medicament for use in medicine. Some exemplary medical uses are described in further detail below. In one embodiment, the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention may be used to deliver a transgene into a host cell. This method preferably comprises the step of administering to said host cell a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. Preferably, the host cell is an animal cell, more preferably a mammalian cell. Preferred mammals include chickens, other poultry, cows, sheep, goats, pigs, wild boar, buffalo, bison, horses, camelids, deer, elephants, badgers, possums, cats, lions, monkeys and humans. Preferably, the host cell is a somatic cell. The host cell may be selected from the group consisting of an antigen-presenting dendritic cell, langerhans cell, macrophage, B cell, lymphocyte, leukocyte, myocyte and fibroblast. This method may be carried out in vitro or in vivo. Where the method is carried out in vitro, the viral vector or immunogenic composition is brought into contact with the host cell under suitable conditions such that transduction or non-productive infection of the host cell with the viral vector is facilitated. In this embodiment, the host cell may comprise an isolated host cell or a sample from an animal subject. Where the method is carried out in vivo, the viral vector or immunogenic composition is preferably administered to the animal subject such that transduction of one or more cells of the subject with the viral vector is facilitated. Preferably, the viral vector or immunogenic composition is administered to the subject by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration. Preferably, the transduction of the host cell with the viral vector of the present invention results in the stable delivery of the exogeneous nucleotide sequence of interest into the host cell. Therefore, in another embodiment, the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention may be used to elicit an immune response in an animal. This method preferably comprises the step of administering to said animal a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. Where the protein or polypeptide of interest is an antigen, expression of the protein or polypeptide in an animal will result in the elicitation of a primary immune response to that antigen, leading to the development of an immunological memory which will provide an enhanced response in the event of a secondary encounter, for example upon infection by the pathogen from which the antigen was derived. Preferably, the animal is a naive animal, i.e. an animal that has not previously been exposed to the pathogen or antigens in question. As well as eliciting an immune response in an animal, the viral vector of the present invention or the immunogenic composition thereof can be used to boost the immune response of an animal previously exposed to the antigen. Therefore, in a further embodiment, the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention may be used to boost an immune response in an animal. This method preferably comprises the step of administering to said animal a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. Preferably, the animal subject has been previously exposed to the antigen in question, or “primed”. For example, the subject may have previously been inoculated or vaccinated with a composition comprising the antigen, or may have previously been infected with the pathogen from which the antigen was derived. The subject may be latently infected with the pathogen from which the antigen was derived. In another embodiment, the vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention may be used to treat or prevent at least one disease in a patient. This method preferably comprising the step of administering to said patient a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. Preferably, the disease is selected from the group consisting of Tuberculosis and other mycobacterial infections, malaria, influenza, HIV/AIDS, Hepatitis C, Cytomegalovirus infection, Human papilloma virus infection, adenoviral infection, leishmaniasis,streptococcusspp.,staphylococcusspp.,meningococcusspp., infection, rift valley fever, foot and mouth disease and chikungunya virus infection. As well as inducing an immune response against the pathogenic organism from which the heterologous antigen is derived, the adenoviral vector of the present invention may also induce an immune response against the adenovirus from which the viral vector is derived. As such, an immune response against AdY25 may be elicited. The immune response induced against AdY25 may also be cross-reactive with other adenoviral serotypes, and as such an immune response against more than one adenovirus may be elicited. The viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention can therefore also be used for treating or preventing an adenoviral disease. This embodiment of the present invention therefore also provides the treatment or prevention of at least one adenoviral disease and at least one non-adenoviral disease in a patient. In a further embodiment, the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention may be used to induce an immune response in an animal that will break tolerance to a self antigen. This method preferably comprises the step of administering to said animal a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. Many tumour cells are tolerated by the patient's immune system, on the grounds that tumour cells are essentially the patient's own cells that are growing, dividing and spreading without proper regulatory control. Thus, cancerous tumours are able to grow unchecked within the patient's body. However, the viral vector of the present invention can be used to stimulate a patient's immune system to attack the tumour cells in a process known as “cancer immunotherapy”. Specifically, the vector of the present invention can be used to ‘train’ the patient's immune system to recognise tumour cells as targets to be destroyed. This can be achieved by including within the viral vector an exogeneous nucleotide sequence encoding a suitable self-antigen. As described previously, suitable self-antigens include antigens expressed by tumour cells which allow the immune system to differentiate between tumour cells and other cell types. Suitable self-antigens include antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g. foetal antigens). For example, GD2 is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier. However, GD2 is expressed on the surfaces of a wide range of tumour cells including small-cell lung cancer, neuroblastoma, melanomas and osteosarcomas. Other suitable self-antigens include cell-surface receptors that are found on tumour cells but are rare or absent on the surface of healthy cells. Such receptors may be responsible for activating cellular signalling pathways that result in the unregulated growth and division of the tumour cell. For example, ErbB2 is produced at abnormally high levels on the surface of breast cancer tumour cells. Thus, the adenoviral vector of the present invention may be used to induce an immune response against a tumour cell, and can therefore be used in the treatment of cancer. The following details apply mutatis mutandis to all of the above uses of the vector and immunogenic composition of the present invention. The treatment and prevention of many diseases, including liver stage malaria, tuberculosis and influenza, are associated with the maintenance of a strong cell-mediated response to infection involving both CD4+ and CD8+ T cells and the ability to respond with Th1-type cytokines, particularly IFN-γ, TNF-α, IL-2 and IL-17. Although many subunit vaccine platforms effectively generate human immunity, the generation of robust cell-mediated immune responses, particularly CD4+ and CD8+ T cell immune responses, has been much more challenging. The viral vector of the present invention preferably stimulates both cellular and humoral immune responses against the encoded antigen. It is also desirable to induce a memory immune response. Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that arise directly from differentiated effector T cells and persist in a uniformly quiescent state. Memory T cells have been shown to be heterogeneous and to comprise at least two subsets, endowed with different migratory capacity and effector function; effector memory T cells (TEM) and central memory T cells (CTM). TEM resemble the effector cells generated in the primary response in that they lack the lymph node-homing receptors L-selectin and CCR7 and express receptors for migration into inflamed tissues. Upon re-encounter with antigen, these TEM can rapidly produce IFN-γ or IL-4 or release pre-stored perform. TCM express L-selectin and CCR7 and lack immediate effector function. These cells have a low activation threshold and, upon restimulation in secondary lymphoid organs, proliferate and differentiate to effectors. Preferably, the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention is capable of eliciting, inducing or boosting an antigen-specific immune response. Preferably, the immune response is a strong T cell immune response, for example a strong CD8+ and CD4+ T cell response. Preferably, the T cell immune response is a protective T cell immune response. Preferably, the T cell immune response is long lasting and persists for at least 1, 2, 5, 10, 15, 20, 25 or more years. Preferably, the immune response induced is a memory T cell immune response. The viral vector of the second aspect of the present invention or immunogenic composition according to the third aspect of the present invention may be administered to the host cell or subject either as a single immunisation or multiple immunisations. Preferably, the viral vector or immunogenic composition thereof are administered as part of a single, double or triple vaccination strategy. They may also be administered as part of a homologous or heterologous prime-boost immunisation regime. The vaccination strategy or immunisation regime may include second or subsequent administrations of the viral vector or immunogenic composition of the present invention. The second administration can be administered over a short time period or over a long time period. The doses may be administered over a period of hours, days, weeks, months or years, for example up to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks or 0.25, 0.5, 0.75, 1, 5, 10, 15, 20, 25, 30, 35 or 40 or more years after the first administration. Preferably, the second administration occurs at least 2 months after the first administration. Preferably, the second administration occurs up to 10 years after the first administration. These time intervals preferably apply mutatis mutandis to the period between any subsequent doses. The viral vector and/or immunogenic composition may be administered alone or in combination with other viral or non-viral DNA/protein vaccines. Preferred examples include MVA, FP9 and other adenoviral vector vaccines. The viral vector and/or immunogenic composition may be administered to the subject by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration. Alternatively, the viral vector and/or immunogenic composition may be administered to an isolated host cell or sample from a subject by contacting the cell(s) with the viral vector or immunogenic composition in vitro under conditions that facilitate the transduction of the host cell with the viral vector. The viral vector and immunogenic composition of the present invention are not limited to the delivery of nucleic acid sequences encoding antigens. Many diseases, including cancer, are associated with one or more deleterious mutant alleles in a patient's genome. Gene therapy is a process involving the insertion of genes into the patient's cells or tissues to replace the deleterious mutant or non-functional allele(s) with ‘normal’ or functional allele(s). Commonly, a functional allele is inserted into a non-specific location within the genome to replace the non-functional allele. Alternatively, the non-functional allele may be swapped for the functional allele through homologous recombination. Subsequent expression of the functional allele within the target cell restores the target cell to a normal state and thus provides a treatment for the disease. The ‘normal’ or functional allele(s) may be inserted into a patient's genome using a viral vector. The present invention therefore also provides the use of the viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention in gene therapy. This method preferably comprises the step of administering to said animal a viral vector according to the second aspect of the present invention or the immunogenic composition according to the third aspect of the present invention. The vector of the present invention may comprise an exogeneous nucleotide sequence encoding the functional or ‘normal’ protein, the non-functional or ‘mutant’ version of which is associated with a disease or condition. Preferably, the target cell is a somatic cell. The subject to be treated is preferably mammalian. Preferred mammals include chickens, other poultry, cows, sheep, goats, pigs, wild boar, buffalo, bison, horses, camelids, deer, elephants, badgers, possums, cats, lions, monkeys and humans. A fifth aspect of the present invention provides a polynucleotide sequence encoding the viral vector according to the second aspect of the present invention. Preferably, the polynucleotide sequence comprises the sequence of SEQ ID NO. 38 or a sequence substantially identical thereto. The polynucleotide may additionally comprise the exogeneous nucleotide sequence of interest. A sixth aspect of the present invention provides a host cell transduced or infected with the viral vector according to the second aspect of the present invention. Following transduction or infection, the host cell will express the exogeneous nucleotide sequence in the nucleic acid molecule to produce the molecule of interest, in addition to any other adenoviral proteins encoded by the nucleic acid molecule. Preferably, the host cell is stably transduced and suitable for viral propagation. The host cell may be an isolated host cell, part of a tissue sample from an organism, or part of a multicellular organism or organ or tissue thereof. Preferably, the host cell is a somatic cell. Preferably, the host cell is not a stem cell, more particularly an embryonic stem cell, more particularly a human embryonic stem cell. The host cell may be selected from the group consisting of an antigen-presenting dendritic cell, langerhans cell, macrophage, B cell, lymphocyte, leukocyte, myocyte and fibroblast. Preferably, the host cell is an animal cell, more preferably a mammalian cell. Preferred mammals include chickens, other poultry, cows, sheep, goats, pigs, wild boar, buffalo, bison, horses, camelids, deer, elephants, badgers, possums, cats, lions, monkeys and humans. The fifth aspect of the present invention also encompasses an animal transduced or infected with the viral vector according to the second aspect of the present invention. Preferably, the animal comprises one or more cells transformed or transfected with the viral vector according to the second aspect of the present invention. Preferably, the animal is a mammal. Preferred mammals include chickens, other poultry, cows, sheep, goats, pigs, wild boar, buffalo, bison, horses, camelids, deer, elephants, badgers, possums, cats, lions, monkeys and humans. In a seventh aspect, the present invention provides a method of producing the viral vector according to the second aspect of the present invention. Preferably, the method comprises the step of incorporating a nucleotide sequence derived from AdY25 into a Bacterial Artificial Chromosome (BAC) to produce an Ad-BAC vector. Unlike plasmid vectors, BACs are present withinE. Coliin single copy conferring increased genetic stability. In addition, the single copy BAC vectors permit very precise modifications to be made to the viral genome by recombineering (recombination mediated genetic engineering). Preferably, incorporation of the nucleotide sequence derived from AdY25 into a Bacterial Artificial Chromosome (BAC) comprises the steps of:constructing a BAC rescue vector comprising regions of homology to the left and right flanks of the viral nucleotide sequence;linearising the BAC rescue vector; andperforming homologous recombination in a host cell between the viral nucleotide sequence and the linearised BAC rescue vector to incorporate the viral nucleotide sequence into the BAC rescue vector. Preferably, the nucleotide sequence incorporated into the BAC rescue vector comprises the sequence of SEQ ID NO. 1 or SEQ ID NO. 38 or a sequence substantially identical thereto. Preferably, the method additionally comprises the step of further modifying the Ad-BAC vector genome. These further modifications may be carried out by GalK recombineering. This technique, pioneered by Saren Warming and colleagues, utilises the GalK gene for both positive and negative selection of recombinant clones6. SW102E. Colicells, in which recombination may be performed, have been specifically engineered to lack the GalK gene which is required for the utilisation of galactose as the sole carbon source. Gene deletion is performed by recombination between the vector genome and a PCR amplified GalK cassette, flanked by 50 bp regions of homology either side of the gene targeted for deletion. Selection on mimimal media containing only galactose should ensure that only recombinants containing the GalK gene (in place of the target gene) should grow. Replacement of GalK with a different gene sequence can be performed in a similar fashion, this time using GalK for negative selection. The addition of 2-deoxygalactose (DOG) to selection media will select clones in which GalK has been replaced since the product of GalK, galactokinase, metabolises DOG into a product that is highly toxic toE. Coli. Preferably, the host cell is BJ5183E. Colifor steps i) to iii) above and SW102 for further modifications. Preferably, an extra homology flank is included downstream of the adenovirus E1 region to enable simultaneous deletion of E1, as described in Example 1. Preferably, the method further includes deletion of the E3 region of the Ad-BAC vector genome. Deletion of the E3 region may be carried out by GalK recombineering, as described in Example 2. Preferably, the method further includes modifying the E4 region to optimise vector growth and yield. In one embodiment, the entire native E4 locus is replaced with the E4Orf6 gene from AdHu5. In a second embodiment, the native E4 locus is replaced with E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5, and the E4Orf1, E4Orf 2 and E4Orf 3 coding regions from AdY25, as described in Example 3. Preferably, the method further includes introducing phage lambda site specific recombination sites attR1 and attR2 at the Ad E1 locus as part of an Invitrogen Gateway@ destination cassette. Such a modification enables the efficient directional insertion of vaccine transgenes. Transgenes could also be inserted by recombineering, In-Fusion®, conventional ligation or gap repair. An eighth aspect of the present invention provides a Bacterial Artificial Chromosome (BAC) clone comprising a polynucleotide sequence encoding the viral vector according to the second aspect of the present invention. Preferably, the BAC clone comprises:a. a BAC backbone;b. the polynucleotide sequence according to the fifth aspect of the present invention. As described above, the viral vector according to the second aspect of the present invention may be replicated in a transformed cell line or helper virus (gutless vector system) which, if necessary, comprises the complement of any genes deleted from the virus. Such genes may be deleted from the virus in order to hinder replication in host cells, but are of course required in order to replicate the viral vector to produce immunogenic compositions according to the second aspect of the present invention. One can make use of any cell line permissive of wild type adenovirus replication that has been modified to express the functionally deleted genes, or a cell line which is not permissive of wild-type virus replication which has additionally or alternatively been modified to express CAR or integrins in addition to the functionally deleted genes. The present invention provides host cells comprising a Bacterial Artificial Chromosome (BAC) in accordance with the eighth aspect of the present invention, and suitable for propagation thereof. Preferably such host cells are bacteria, most preferablyE. coli. Suitable examples includeE. colistrains DH10B and SW1029. A ninth aspect of the present invention therefore provides a packaging cell or cell line producing or capable of producing the viral vector according to the second aspect of the present invention. The packaging cell or cell line comprises one or more nucleotide sequences which encode the viral vector of the second aspect of the present invention. Expression of these sequences results in the production of the viral vector. Some of the required genes may be provided by infection of the cell or cell line with a viral vector according to the second aspect. Preferably, the cell comprises the complement of any genes deleted or functionally deleted from the viral vector. Preferably, the cell comprises the complement of the AdY25 E1 gene. Preferably, the cell is an HEK293 cell or a PER.C6® cell. As described above, modification of the E4 locus of the adenoviral vector to include the E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5 increased the rate of hexon production, increasing the sensitivity of anti-hexon titre to allow quantification of the infectious titre of the viral vector, in particular those viral vectors developed for clinical use which do not contain a fluorescent marker gene. In addition, this modification was surprisingly found to increase the yield and rate of growth of the vector. One of skill in the art would appreciate that such a modification is expected to have a beneficial effect on a wide variety of adenoviruses, and not simply those derived from AdY25. A tenth aspect of the present invention therefore provides an adenoviral vector other than AdHu5 comprising a nucleic acid molecule, wherein said nucleic acid molecule comprises heterologous E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5. In one embodiment, the native E4 locus is deleted and replaced with heterologous E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5. Alternatively, nucleic acid molecule may comprise the native coding regions in addition to heterologous E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5. Preferably, the native coding regions are E4Orf1, E4Orf2 and E4Orf3. Preferred adenoviral vectors are selected from the group consisting of AdY25 and AdY68. Preferably, the adenoviral vector according to the tenth aspect lacks and E1 and an E3 locus. Merely for the convenience of those of skill in the art, a sample ofE. colistrain SW1029 (a derivative of DH10B) containing bacterial artificial chromosomes (BACs) containing the cloned genome of AdChOX1 (pBACe3.6 AdChOx1 (E4 modified) TIPeGFP, cell line name “AdChOx1 (E4 modified) TIPeGFP”) was deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. As described herein, the vector AdChOx1 is derived from chimpanzee adenovirus Y25, with deletion of E1 region, E3 region, modification of E4 region and insertion of TIPeGFP model antigen into E1 locus. TheE. colicontaining the BAC is a class I genetically modified organism. The BAC propagates within the bacteria during replication and can be maintained by selection with chloramphenicol. TheE. colistrain SW102 containing the bacterial artificial chromosomes into which the genomes are cloned can be propagated in Luria-Bertani broth or agar containing 12.5 μg/mL chloramphenicol at 32° C. The genome may be modified by genetic engineering inE. coliaccording to standard methods, as described in the specification, e.g. to insert an alternative recombinant antigen in place of TIPeGFP. Converting the BAC clones of the viral genomes into viruses (“rescue”) can be carried out by the following steps. TheE. colihost is propagated and the BAC DNA is purified from the bacteria according to standard methods. The DNA is linearised with the restriction endonuclease PmeI and transfected into HEK293 cells (or a similar E1 complementing cell line). The resulting adenovirus can then be propagated and purified for use as a vaccine, for example. All of these reagents and cells are publicly available. If the deposition were rescued, the resulting virus would be a class I genetically modified organism. In respect of all designated states to which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited material be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. Rule 32(1) EPC, Rule 13(1) and Schedule 1 of the UK Patent Rules 2007, Regulation 3.25(3) of the Australian Patent Regulations and generally similar provisions mutatis mutandis for any other designated state. A specific embodiment of the fifth aspect of the present invention provides a polynucleotide sequence encoding an adenoviral vector according to the second aspect of the present invention, wherein said polynucleotide sequence comprises or consists of the polynucleotide sequence of the viral vector AdChOX1, deposited in a BAC contained inE. colistrain SW1029 by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. The deposited BAC additionally comprises a transgene encoding the antigen TIPeGFP. In this aspect of the present invention, the polynucleotide sequence for AdChOX1 preferably does not include the sequence encoding the TIPeGFP antigen. A further embodiment of the present invention provides a host cell transduced with the viral vector according to the second aspect of the present invention, wherein said host cell is preferably a bacterium, more preferablyE. colistrain SW1029 containing a bacterial artificial chromosome (BAC) containing the cloned genome of AdChOX1 deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. The deposited BAC additionally comprises a transgene encoding the antigen TIPeGFP. In this aspect of the present invention, the polynucleotide sequence for AdChOX1 preferably does not include the sequence encoding the TIPeGFP antigen. Such a host cell may be used for BAC propagation. A specific embodiment of the seventh aspect of the present invention provides a method of producing the viral vector according to the second aspect of the present invention by generating a molecular clone of AdY25 in a Bacterial Artificial Chromosome (BAC), wherein said BAC is the BAC containing the cloned genome of AdChOX1, deposited inE. colistrain SW1029 by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. The deposited BAC additionally comprises a transgene encoding the antigen TIPeGFP. In this aspect of the present invention, the polynucleotide sequence for AdChOX1 preferably does not include the sequence encoding the TIPeGFP antigen. A specific embodiment of the eighth aspect of the present invention provides a Bacterial Artificial Chromosome (BAC) clone comprising the polynucleotide sequence according to the fifth aspect of the present invention, wherein said BAC is the BAC containing the cloned genome of AdChOX1, deposited inE. colistrain SW1029 by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. The deposited BAC additionally comprises a transgene encoding the antigen TIPeGFP. In this aspect of the present invention, the polynucleotide sequence for AdChOX1 preferably does not include the sequence encoding the TIPeGFP antigen. For the avoidance of doubt, it is hereby expressly stated that features described herein as ‘preferred’, ‘preferable’, “alternative” or the like may be present in the invention in isolation or in any combination with any one or more other features so described (unless the context dictates otherwise) and this constitutes and explicit disclosure of such combinations of features. All the features of each embodiment described above apply mutatis mutandis to all other embodiments of the present invention. EXAMPLES Example 1: Generation of a Molecular Clone of AdY25 in a Bacterial Artificial Chromosome Wild type chimpanzee adenovirus AdY25 was obtained from Goran Wadell of Umea University, Sweden. The virus was propagated to high titer in HEK293 cells and the viral DNA phenol extracted and sequenced. The nucleotide sequence of the wild type AdY25 virus is found in SEQ ID NO. 1. Based on the sequencing data, a BAC ‘rescue vector’ was constructed containing regions of homology to the left and right flanks of the viral genome (homology flanks were PCR amplified from viral DNA). Homologous recombination was then performed in BJ5183E. Colicells between viral DNA and the linearised rescue vector to incorporate the viral genome into the BAC vector. An extra homology flank downstream of the adenovirus E1 region was included to enable simultaneous deletion of E1 in order to render the new vector immediately replication incompetent. Phage lambda site specific recombination sites attR1 and attR2 were introduced at the Ad E1 locus as part of an Invitrogen Gateway® destination cassette to enable the efficient directional insertion of vaccine transgenes. A modified destination cassette was ligated into the AsiSI restriction site introduced at the E1 locus during isolation of the genomic clone. The resulting ΔE1 Ad-BAC vector was screened by both PCR and restriction digest before replication incompetent clones were transfected into E1 complementing HEK293 cells, where the new vector demonstrated the ability to produce infectious virions capable of replication and cytopathic effect in HEK293 cells. Example 2: Deletion of the Adenoviral E3 Region The ΔE1 Ad-BAC vector genome produced in accordance with Example 1 was further modified using GalK recombineering to delete the adenoviral E3 region and thus increase the insert capacity of the new vector by approximately 5 kb. The E3 region was deleted by recombination between the vector genome and a PCR amplified GalK cassette, flanked by 50 bp regions of homology either side of the E3 gene. Recombination was performed in SW102E. colicells, which have been specifically engineered to lack the GalK gene which is required for the utilisation of galactose as the sole carbon source. Recombinant cells were selected using mimimal media containing only galactose, in which only recombinants containing the GalK gene in place of the E3 locus were able to grow6. Example 3: Modification of the E4 Region and Effects Thereof i). Modification of E4 Region The E4 locus of the ΔE1 ΔE3 Ad-BAC vector genome produced in accordance with Example 2 was then modified. The E4 region was deleted by recombination in SW102E. Colicells between the vector genome and a PCR-amplified GalK cassette, flanked by 50 bp regions of homology either side of the E4 gene. Recombinant cells were selected using mimimal media containing only galactose. The GalK gene was then replaced with the required E4 open reading frames from AdHu5 and AdY25 in a similar manner to provide the 5 constructs listed inFIG.3C. Recombinant cells comprising the gene in place of the GalK gene were then selected using media comprising 2-deoxygalactose (DOG)6. ii). Effect of E4 Modification on Viral Yield HEK293 cells were infected with the following viral vectors at a multiplicity of infention of 9 and incubated at 37° C. for 48 hours before harvesting:i. AdHu5 (“Ad5”)ii. AdY25 E4 wildtype (“Y25E4 wt”)iii. AdY25 E4 AdHu5 E4Orf 6 (“Y25Ad5E4Orf”)iv. AdY25 E4 AdHu5 E4Orf 4, 6, 6/7 (“AdChOX1”) Infectious titre of the harvested material was measured by quantifying GFP positive foci 48 hours post infection. As can be seen inFIG.3A, the infectious titre of the AdY25-based viral vector comprising the wildtype E4 locus was significantly lower than that of AdHu5. Modification of the viral vector to replace the wildtype E4 locus with the E4Orf6 gene from AdHu5 signficantly increased the infectious titre. Replacement of the wildtype E4 locus with the E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5, and the E4Orf1, E4Orf2 and E4Orf3 coding regions from AdY25 (to create ChAdOX1) surprisingly further increased the infectious titre. iii). GFP Vs. Anti-Hexon Titre In order to assess vaccine vector immunogenicity and efficacy it is essential to develop a reliable method of quantifying the infectious titer of the virus. Traditionally, plaque assays in HEK293 cells have been the method of choice, but these require a long incubation period and titers are often inconsistent. Furthermore the plaque assay is inherently insensitive, not all infectious virions will induce plaque formation. One method is the single cell infectivity assay which simply involves quantifying the number of virally infected cells. The first recombinant AdY25-derived viral vectors expressed green fluorescent protein (GFP), enabling viruses that had initiated recombinant transgene expression within a cell to be visualised directly by fluorescence microscopy. However, an alternative method of assessing cell infectivity must be used where the vaccine antigen constructs do not contain a fluorescent marker gene, for example where the vaccine antigen constructs are for clinical use. An anti-hexon immunostaining assay has now been developed that enables visualisation of infected cells in which the viral hexon protein is being expressed. This assay uses a polyclonal anti-hexon antibody so can be used to titer virtually any adenovirus vaccine vector and we have found the assay to be reliable and consistent for both AdHu5 and AdCh63 based vectors. It does of course rely on the assumption that the rate of hexon production relative to transgene expression is consistent between vectors. The titers of GFP-expressing AdY25-derived viral vectors were compared by GFP and anti-hexon based assays. Titers were assessed at 48 hours post infection for AdHu5, AdC63, AdY25 E4 wildtype, and constructs A-E as described inFIG.3C, all expressing the TIPeGFP antigen. TIP is essentially an epitope string consisting of a number of strong murine T cell epitopes including Pb9 (a dominant CD8+ epitope from malarial antigen PbCSP) and P15 (a strong CD4+ epitope fromM. tuberculosisantigen 85A). The TIP epitope string is fused to the 5′ end of eGFP which enables transgene expression to be visualised directly and simplifies vaccine titration. FIG.3Billustrates the ratio of GFP foci to anti-hexon titer. For Ad5- and AdC63-based vectors, GFP titers were approximately twice as sensitive as anti-hexon titers. However, for AdY25-based vectors, the sensitivity of the anti-hexon assay varied considerably with E4 modification. For the AdY25 E4 wildtype vector, anti-hexon titers were over 40 fold less sensitive than GFP titers after 48 hrs, suggesting that the rate of hexon production is considerably slower than for AdHu5 and AdCh63 vectors. This was to be expected, given the poor yield of AdY25 E4 wildtype vector. Surprisingly, however, the construct A (“Y25Ad5E4Orf6”) was still 30 fold less sensitive by anti-hexon than by GFP. The best results were obtained with construct E (“ChAdOX1”), in which the wildtype E4 locus was replaced with the E4Orf4, E4Orf6 and E4Orf6/7 coding regions from AdHu5, and the E4Orf1, E4Orf2 and E4Orf3 coding regions from AdY25. iv). Hexon Expression The ratio of marker gene to hexon titre for ChAdOX1 viral vectors expressing TIPeGFP was measured using GFP and mCherry fluorescent transgenes in order to control for the sensitivity of the fluorescent detection. The results are provided inFIG.3D. In both cases, the marker generhexon titre ratio was approximately twofold, and thus the particular marker gene used did not affect the resulting marker gene:hexon titre ratio. The marker gene:hexon titre ratio for the ChAdOX1 vector is the same as that for HAdV-5, indicating that the E4 modification to the ChAdOX1 vector has been optimised. Example 4: Immunogenicity of AdY25-Based Vectors Immunogenicity was assessed using the model antigen TIPeGFP in order to determine whether comparable immunogenicity to AdC63 and AdC68 could be obtained in mice using an AdY25-based vector. Balb/c mice (4/group) were immunised intramuscularly with 109 infectious units (ifu) of each of the following viral vectors, all expressing the TIPeGFP antigen:v. AdCh63;vi. ΔE1 ΔE3 AdCh68; andvii. ChAdOX1. After 14 days post-prime, spleen immunogenicity against a strong CD8+ epitope (Pb9) was assessed by IFN-γ ELISpot The IFN-γ spleen ELISpot responses are shown inFIG.4. Responses elicited by ChAdOX1 were robust and comparable to those seen using AdCh63 and the AdCh68-based vector. These data support the continued development of AdY25-based vectors for clinical application. Example 5: Effect of E4 Modification on Immunogenicity of AdY25-Based Vectors The impact of two different E4 modifications on the immunogenicity of AdY25-based vectors was assessed using the following constructs:(i) AdY25 E4 wildtype (“E4 wt”)(ii) AdY25 E4AdHu5Orf6 (“E4Orf6”); and(iii) AdY25 E4AdHu5Orf4/6/7(“E4Orf4/6/7”). Balb/c mice (4/group) were immunised intramuscularly with either 106 ifu or 108 ifu of each vector. Responses to Pb9 and PI 5 epitopes were assayed two weeks post immunisation. Titers calculated once again on GFP to remove the effect of hexon production rates on vaccine titer. The effect of E4 modification on IFN-γ spleen ELISpot responses is shown inFIGS.5A and5B. The data indicate that E4 modification has no effect on vector immunogenicity. Therefore, such modifications can be used to enhance the rate of production of the viral vectors, without having a negative impact on the immunogenicity of the vectors. Example 6: Prevalence of Vector-Neutralising Antibodies The prevalence of vector neutralising antibodies in human sera from the UK and The Gambia against AdY25-based vectors and AdCh63-based vectors was assessed. HEK293 cells were infected with Y25Ad5E4Orf6-SEAP or AdCh63-SEAP (SEAP=Secreted Placental Alkaline Phosphatase). Recombinant adenoviruses were incubated with five serial dilutions of serum in FBS-DMEM before infection. The final serum dilutions were 1:18, 1:72, 1:288, 1:1 152, 1:4608; each serum sample was tested in duplicate. Supernatants were collected and assayed for SEAP concentration using CSPD (Tropix) according to the manufacturer's instructions. Luminescence intensity was measured using a Varioskan flash luminometer (Thermo Scientific). Neutralization titers were defined as the serum dilution required to reduce SEAP concentration by 50% compared to wells infected with virus alone. Neutralization titer was calculated by linear interpolation of adjacent values. As shown inFIG.6, the seroprevalence of neutralising antibodies against Y25Ad5E4Orf6 was surprisingly found to be much lower than that for AdCh63 in both the UK and The Gambia. Example 7: Humoral Immunogenicity of AdY25-Based Vectors Balb/c mice (6/group) were immunised with 108 infectious units of either of the following vectors, both expressing TIPeGFP:viii. ΔE1 ΔE3 AdCh68; orix. ChAdOX1. After 56 days post prime, mice were boosted with 106 pfu MVA-TIPeGFP. Serum was collected 50 days post-prime and 10 days post-boost to compare pre- and post-boost anti-GFP antibody responses. Responses were measured by endpoint ELISA. Statistical analyses were performed by one way ANOVA. As shown inFIG.7, humoral immunogenicity of the AdY25-based vector ChAdOX1 is superior to current chimpanzee adenovirus vector AdCh68, indicating an enhanced antibody response elicited by the AdY25-based vector in comparison to the AdCh68-based vector. Example 8: Induction of Immune Response AgainstMycobacterium tuberculosis A transgene encoding theMycobacterium tuberculosisprotein Ag85A was inserted into the E1 locus of ChAdOX1 under control of the human cytomegalovirus immediate early promoter, using the BAC technology as described in Example 1. The nucleotide sequence of the transgene (SEQ ID NO. 42) encodes residues 1 to 323 of the antigen, encoded by a sequence optimised to human codon usage (nucleotides 103 to 1071), fused at the N-terminus to tPA (the signal peptide from human tissue plasminogen activator)(nucleotides 1 to 102) and at the C-terminus to a PK tag (nucleotides 1072 to 1 104). The amino acid sequence of the Ag85A antigen is provided in SEQ ID NO.43. The BAC clone was transfected into HEK293 cells and the virus vector was amplified, purified and titred using the anti-hexon immunostaining assay described in Example 3. The immunogenicity of the vector was assessed in Balb/c mice immunized with varying doses, expressed in infectious units, of the vaccine, administered intramuscularly. After 14 days cellular immune responses to Ag85A were determined by IFN-γ ELIspot assay using splenocytes stimulated with synthetic peptides corresponding to the known immunodominant CD8+(pi 1; WYDQSGLSV (SEQ ID NO. 44)) and CD4+ T cell (pi 5; TFLTSELPGWLQANRHVKPT (SEQ ID NO. 45)) H-2d restricted epitopes in Ag85A. The results are shown inFIGS.8A and8B. These results indicate that the ChAdOX1 vector is capable of inducing immune responses againstMycobacterium tuberculosis. The magnitude of these responses is similar to that induced by vectors based on other adenoviruses. Example 9: Induction of Immune Response Against Influenza A A transgene encoding the nucleoprotein (NP) and matrix protein 1 (Ml) of influenza A virus was inserted into the E1 locus of ChAdOX1 under control of the human cytomegalovirus major immediate early promoter, using the BAC technology as described in Example 1. The nucleotide sequence of the transgene (SEQ ID NO. 46) encodes the influenza A nucleoprotein (nucleotides 1 to 1494) fused to the matrix protein 1 (nucleotides 1516 to 2274) and separated by a linker (nucleotides 1495 to 1515). The amino acid sequence of the NPM1 fusion protein is provided in SEQ ID NO. 47. The BAC clone was transfected into HEK293 cells and the virus vector was amplified, purified and titred using the anti-hexon immunostaining assay described in Example 3. A similar vector based on human adenovirus type 5 (HAdV-5) was similarly generated and titred for comparative purposes. The immunogenicity of the vector was assessed in Balb/c mice immunized with varying doses, expressed in infectious units, of the vaccine, administered intramuscularly. After 14 days cellular immune responses to NP were determined by IFN-γ ELIspot assay using splenocytes stimulated with synthetic peptides corresponding to the known immunodominant CD8+ T cell H-2d restricted epitope in NP ((TYQRTRALV) (SEQ ID NO. 48)). The results are shown inFIG.9. These results indicate that the ChAdOX1 vector is capable of inducing immune responses against influenza A virus and that, at the doses tested, these are similar to those induced by a HAdV-5 vector. The ChAdOX1-NPM1 vaccine has recently been produced for human clinical trials according to current good manufacturing practice at the University of Oxford Clinical Biomanufacturing Facility. This indicates the suitability of the vector for deployment as a medical product. REFERENCES 1. Buchbinder et al, Lancet, Vol 372, November 20082. Farina et al, J. Virol, December 2001, p11603-116133. Dudareva et al, Vaccine 27, 2009, 3501-35044. R. Wigand et al, Intervirology, Vo130; 1 19895. Roy et al, Hum. Gen. Ther., 2004, 15:519-5306. Warming et al. Nuc. Acid. Res, 2005, Vo133; 47. http://www.invitrogen.com/gateway8. Havenga et al, J.G.V., 2006, 87, 2135-2149. Wanning, S. et al. Nucleic Acids Res, 2005, February 24; 33(4): e36 | 103,672 |
11857641 | DETAILED DESCRIPTION Disclosed herein are methods and compositions for treating and/or preventing Hurler/Hurler-Scheie/Scheie (MPS I) syndrome in a human subject comprising insertion of a suitable transgene sequence in a target cell. The treatment employs engineered zinc finger nucleases (ZFNs) to site-specifically integrate a corrective copy of the enzyme iduronidase (hIDUA) transgene into the albumin locus of the subject's own hepatocytes in vivo. Once expressed from the integrated transgene, the hIDUA is active and able to degrade mucopolysaccharides glycosaminoglycans (GAG). The invention describes methods of prevention or treatment for MPS I subjects. Normally, IDUA enzyme is produced inside the cell and a small amount of it may leak out into the circulation due to cells' imperfect internal transport system. A steady state is established as extracellular enzyme is taken back up by receptors on the cells' surface. As a result, most of the enzyme normally produced in the body is found in the tissues, and there are generally very small concentrations of enzyme found in circulation. In contrast, ERT is an infusion directly into the bloodstream of a large bolus of enzyme designed to create high concentrations in the circulation to allow uptake into IDUA-deficient tissues. However, ERT only produces transient high levels of IDUA enzyme, followed by rapid clearance from the circulation within a matter of minutes to hours due to the short half-life of the enzyme, and because large amounts are taken up by the liver. This limits the effectiveness of ERT because it only provides a short window of exposure of enzyme to the tissues, and we know that enzyme uptake by the cells is a slow receptor-mediated process. Instead, an ideal therapy for MPS I would allow prolonged and sustained exposure of the IDUA enzyme to the tissues by producing and maintaining continuous, stable levels of enzyme in the circulation. Even low amounts of IDUA secreted continuously into the circulation could be adequate to reduce tissue GAGs and potentially provide efficacy for the compositions disclosed herein. ERT has been shown to increase the amount of IDUA activity in patient's leukocytes following treatment, presumably because the cells take up the enzyme from the plasma (leukocytes are lysosome-rich cells). For example, in a study of patients receiving recombinant IDUA, it was reported (see Kakkis et al. (2001)NEJM344(3):182-8) that the mean activity of IDUA in leukocytes was 0.04 U per mg prior to treatment, and following treatment, it was measured at 4.98 U per mg seven days after infusion (i.e. immediately prior to the next treatment). Thus measurement of IDUA in the circulating leukocytes can be useful for determining the presence of the enzyme in the blood. Lysosomal storage diseases (LSDs) are a group of rare metabolic monogenic diseases characterized by the lack of functional individual lysosomal proteins normally involved in the breakdown of waste lipids, mucopolysaccharides (i.e. glycosoaminoglycans (GAG)). These diseases are characterized by a buildup of these compounds in the cell since it is unable to process them for recycling due to the mis-functioning of a specific enzyme in the breakdown pathway. The pathophysiology of LSD was initially thought to be tied to the simple deposition of GAG, but current research has led to an appreciation of the complexities of these diseases. GAG storage appears to lead to the perturbation of cellular, tissue and organ homeostasis, and has also been linked to increased secretion of cytokine and inflammatory modulators leading to an activation of the inflammatory response (Muenzer (2014)Mol Gen Metabol111:63-72). Mucopolysaccharidosis type I (MPS I), also referred to as Hurler/Hurler-Scheie/Scheie syndrome, is a recessive lysosomal storage disorder. According to the National Institute of Neurological Disorders and Stroke (NINDS) factsheet for MPS I, the estimated incidence is 1 in about 100,000 births for severe MPS I, 1 in about 500,000 births for attenuated MPS I, and 1 in about 115,000 births for disease that falls between severe and attenuated. MPS I is associated with mutations in the gene encoding the iduronidase (IDUA) enzyme, which degrades and/or helps recycle glycosaminoglycans (sulfated carbohydrate polymers; GAGs). Mutations in the IDUA gene diminish or eliminate IDUA enzyme activity, which results in the accumulation of toxic GAGs in urine, plasma, and body tissues which leads to widespread tissue and organ damage. Depending upon the specific type of IDUA mutation (more than 100 different mutations have been described) and the levels of the resulting residual IDUA enzyme, patients will develop either Hurler syndrome (MPS I H) or the attenuated variants (MPS I H/S and MPS I S). It has been estimated that 50%-80% of all MPS I patients present with the severe form, which could be partly attributed to the relative ease of diagnosis (Muenzer et al., ibid). MPS I H patients show symptoms of developmental delay before the end of their first year as well as halted growth and progressive mental decline between ages 2-4 yrs. Other symptoms include organomegaly, corneal clouding, joint stiffness and skeletal deformities (including abnormal spinal bones), coarse facial features with enlarged tongue, hearing loss and hernias. The life expectancy of these MPS I H patients is less than 10 years. Patients with the attenuated form share most of these clinical manifestations but with less severe symptoms. In addition, there is no CNS involvement and therefore they do not suffer from mental retardation. Many of these patients can survive into adulthood but with significant morbidity. Current standard of care for MPS I include hematopoietic stem cell transplant (HSCT) for severe patients, and enzyme replacement therapy (ERT) given through frequent intravenous infusions. If patients suffering from the severe MPS I form (MPS I-H) can be diagnosed early (<2.5 yr), therapeutic intervention by HSCT (bone marrow or umbilical cord stems cells) can prevent or reverse most clinical features including neurocognition. Currently, almost all patients with MPS I H undergo HSCT. For MPS I the mortality rate after HSCT is 15% and survival rate with successful engraftment is 56% ERT with a polymorphic recombinant protein produced in Chinese Hamster Ovary cells, Aldurazyme®, has been in use since 2003. This enzyme has been shown to improve pulmonary function, hepatosplenomegaly, and exercise capacity and leads to improved health related quality of life. ERT should be instituted as early as possible. Limitations to enzyme replacement therapy includes the need for life-long treatment, development of neutralizing antibodies, inability to cross the blood brain barrier, continued cardiac, orthopedic, ocular complications and the inconvenience of weekly intravenous infusions. Together, these limitations underscore the urgent need to develop a broader array of curative therapies for MPS I. The objective and rationale for the methods and compositions disclosed herein is to abrogate or decrease the need for enzyme replacement therapy by in vivo genome editing. The proposed treatment employs engineered zinc finger nucleases (ZFNs) to site-specifically integrate a corrective copy of the iduronidase enzyme (hIDUA) transgene into the genome of the subject's own hepatocytes in vivo. Integration of the hIDUA transgene is targeted to intron 1 of the albumin locus, resulting in stable, high level, liver-specific expression and secretion of iduronidase into the blood. Placement of the huIDUA transgene under the control of the highly expressed endogenous albumin locus is expected to provide permanent, liver-specific expression of iduronidase for the lifetime of an MPS I patient. Patients with mild MPS I receiving weekly ERT were enrolled in the study. One patient has been dosed with 1e13 vg/kg of the compositions disclosed herein and two patients have been dosed with 5e13 vg/kg. None of the three patients enrolled in the study have received bone marrow transplant. Interim data results show dose-dependent increases in leukocyte IDUA enzyme activity in all three subjects treated with the methods and compositions disclosed herein. Leukocytes are an easily accessible target tissue for IDUA and therefore provide one estimate of tissue enzyme activity for patients with MPS I. In patients with MPS I who have received a bone marrow transplant, increased leukocyte IDUA activity is associated with successful engraftment and improved clinical outcomes. Administration of the composition described herein was generally well-tolerated. No treatment related serious adverse events (SAEs) have been reported. Of the 6 total adverse events (AEs) reported, all were mild or moderate and consistent with ongoing MPS I disease, and none were considered related to treatment with the compositions described herein. A dose-dependent increase in leukocyte IDUA activity was observed in all three patients treated with the compositions described herein, with activity levels rising above baseline and in the normal range (normal range is 6.0-71.4 nmol/hr/mg). Plasma IDUA activity was unchanged from baseline in all three patients. Baseline urine GAG measurements for the three patients were in a range considered to be at or slightly above normal. In the limited duration preliminary data set urine GAG measurements show no clear trend with no meaningful change at this time. Additional follow up is needed to observe whether any meaningful change in urine GAGs emerges. Second-generation, potentially more potent ZFN constructs (for example, SB-71557 and SB-71728) were designed to increase editing efficiency, among other improvements. The preclinical data showed three potential ZFN 2.0 advantages: (1) a 5- to 30-fold improvement in efficiency and potency due to structural changes; (2) the ability to function equally well in the patients who have a single nucleotide polymorphism (SNP) in the target locus in the albumin gene (approximately 20% of the population); and, (3) improved specificity (see U.S. Provisional Patent Application No. 62/758,786). These ZFN compositions will also be tested. General Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) Humana Press, Totowa, 1999. Definitions The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T. The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids. “Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10−6or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd. A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity. A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. The term “zinc finger nuclease” includes one ZFN as well as a pair of ZFNs (the members of the pair are referred to as “left and right” or “first and second” or “pair”) that dimerize to cleave the target gene. A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205. The term “TALEN” includes one TALEN as well as a pair of TALENs (the members of the pair are referred to as “left and right” or “first and second” or “pair”) that dimerize to cleave the target gene. Zinc finger and TALE binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 8,568,526; 6,140,081; 6,453,242; and 6,534,261; see also International Patent Publication Nos. WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496. A “selected” zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; and International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO 02/099084. “Recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing” in which the donor is used to re-synthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide. In the methods of the disclosure, one or more targeted nucleases as described herein create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site, and a “donor” polynucleotide, having homology to the nucleotide sequence in the region of the break, can be introduced into the cell. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide. Thus, the use of the terms “replace” or “replacement” can be understood to represent replacement of one nucleotide sequence by another, (i.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another. In any of the methods described herein, additional pairs of zinc-finger or TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell. In certain embodiments of methods for targeted recombination and/or replacement and/or alteration of a sequence in a region of interest in cellular chromatin, a chromosomal sequence is altered by homologous recombination with an exogenous “donor” nucleotide sequence. Such homologous recombination is stimulated by the presence of a double-stranded break in cellular chromatin, if sequences homologous to the region of the break are present. In any of the methods described herein, the first nucleotide sequence (the “donor sequence”) can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced. In other embodiments, the homology between the donor and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between donor and genomic sequences of over 100 contiguous base pairs. In certain cases, a non-homologous portion of the donor sequence can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1,000, that are homologous or identical to sequences in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence, and is inserted into the genome by non-homologous recombination mechanisms. Any of the methods described herein can be used for partial or complete inactivation of one or more target sequences in a cell by targeted integration of donor sequence that disrupts expression of the gene(s) of interest. Cell lines with partially or completely inactivated genes are also provided. Furthermore, the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.). “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage. A “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity). The terms “first and second cleavage half-domains;” “+ and − cleavage half-domains” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of cleavage half-domains that dimerize. An “engineered cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half-domain (e.g., another engineered cleavage half-domain). See, U.S. Pat. Nos. 7,888,121; 7,914,796; 8,034,598; and 8,823,618, incorporated herein by reference in their entireties. The term “sequence” refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length. A “disease associated gene” is one that is defective in some manner in a monogenic disease. Non-limiting examples of monogenic diseases include severe combined immunodeficiency, cystic fibrosis, lysosomal storage diseases (e.g. Gaucher's, Hurler's Hunter's, Fabry's, Neimann-Pick, Tay-Sach's etc), sickle cell anemia, and thalassemia. The “blood brain barrier” is a highly selective permeability barrier that separates the circulating blood from the brain in the central nervous system. The blood brain barrier is formed by brain endothelial cells which are connected by tight junctions in the CNS vessels that restrict the passage of blood solutes. The blood brain barrier has long been thought to prevent the uptake of large molecule therapeutics and prevent the uptake of most small molecule therapeutics (Pardridge (2005)NeuroRx2(1):3-14). “Chromatin” is the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores. A molecule of histone H1 is generally associated with the linker DNA. For the purposes of the present disclosure, the term “chromatin” is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin. A “chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes. An “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmIDUA and certain viral genomes. A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. An “exogenous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule. An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases. An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster. By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes. A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of fusion molecules include, but are not limited to, fusion proteins (for example, a fusion between a protein DNA-binding domain and a cleavage domain), fusions between a polynucleotide DNA-binding domain (e.g., sgRNA) operatively associated with a cleavage domain, and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein). Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure. A “gene” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. “Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP or TALEN as described herein. Thus, gene inactivation may be partial or complete. A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs. “Eukaryotic” cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells). “Red Blood Cells” (RBCs) or erythrocytes are terminally differentiated cells derived from hematopoietic stem cells. They lack a nuclease and most cellular organelles. RBCs contain hemoglobin to carry oxygen from the lungs to the peripheral tissues. In fact, 33% of an individual RBC is hemoglobin. They also carry CO2 produced by cells during metabolism out of the tissues and back to the lungs for release during exhale. RBCs are produced in the bone marrow in response to blood hypoxia which is mediated by release of erythropoietin (EPO) by the kidney. EPO causes an increase in the number of proerythroblasts and shortens the time required for full RBC maturation. After approximately 120 days, since the RBC do not contain a nucleus or any other regenerative capabilities, the cells are removed from circulation by either the phagocytic activities of macrophages in the liver, spleen and lymph nodes (˜90%) or by hemolysis in the plasma (˜10%). Following macrophage engulfment, chemical components of the RBC are broken down within vacuoles of the macrophages due to the action of lysosomal enzymes. “Secretory tissues” are those tissues in an animal that secrete products out of the individual cell into a lumen of some type which are typically derived from epithelium. Examples of secretory tissues that are localized to the gastrointestinal tract include the cells that line the gut, the pancreas, and the gallbladder. Other secretory tissues include the liver, tissues associated with the eye and mucous membranes such as salivary glands, mammary glands, the prostate gland, the pituitary gland and other members of the endocrine system. Additionally, secretory tissues include individual cells of a tissue type which are capable of secretion. The terms “operative linkage” and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous. With respect to fusion polypeptides, the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked. For example, with respect to a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to an activation domain, the ZFP or TALE DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to up-regulate gene expression. When a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to a cleavage domain, the ZFP or TALE DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site. A “functional” protein, polypeptide or nucleic acid includes any protein, polypeptide or nucleic acid that provides the same function as the wild-type protein, polypeptide or nucleic acid. A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989)Nature340:245-246; U.S. Pat. No. 5,585,245 and International Patent Publication No. WO 98/44350. A “vector” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors. A “reporter gene” or “reporter sequence” refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence. “Expression tags” include sequences that encode reporters that may be operably linked to a desired gene sequence in order to monitor expression of the gene of interest. The extracellular matrix that surrounds and binds certain types of cells is composed of numerous components, including fibrous structural proteins, such as various collagens, adhesive proteins like laminin and fibronectin, and proteoglycans that form the gel into which the fibrous structural proteins are embedded. Proteoglycans are very large macromolecules consisting of a core protein to which many long polysaccharide chains called glycosaminoglycans are covalently bound. Due to the high negative charge of the glycosaminoglycans, the proteoglycans are very highly hydrated, a property that allows the proteoglycans to form a gel-like matrix that can expand and contract. The proteoglycans are also effective lubricants. “Glycosoaminoglycans” or “GAGs” are long, linear polymers of unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit (except for keratan) consists of an amino hexose sugar (N-acetylglucosamine or N-acetylgalactosamine) along with an acidic uronic sugar (glucuronic acid or iduronic acid) or galactose. The exception to this general structure is keratan sulfate, which has galactose in place of the acidic hexose. Glycosaminoglycans are highly polar and attract water. All of the GAGs except hyaluronan are covalently linked to one of approximately 30 different core proteins to form proteoglycans. The core protein is synthesized on the rough endoplasmic reticulum and transferred to the Golgi where nucleoside diphosphate-activated acidic and amino sugars are alternately added to the nonreducing end of the growing polysaccharide by glycosyltransferases, resulting in the characteristic repeating disaccharide structure common to the GAGs. Heparin/heparan sulfate (HS GAGs) and chondroitin sulfate/dermatan sulfate (CS GAGs) are synthesized in the Golgi apparatus, where protein cores made in the rough endoplasmic reticulum are posttranslationally modified with O-linked glycosylations by glycosyltransferases forming proteoglycans. Keratan sulfate may modify core proteins through N-linked glycosylation or O-linked glycosylation of the proteoglycan. The fourth class of GAG, hyaluronic acid, is not synthesized by the Golgi, but rather by integral membrane synthases which immediately secrete the dynamically elongated disaccharide chain. Degradation of proteoglycans during normal turnover of the extracellular matrix begins with proteolytic cleavage of the core protein by proteases in the extracellular matrix, which then enters the cell via endocytosis. The endosomes deliver their content to the lysosomes, where the proteolytic enzymes complete the degradation of the core proteins and an array of glycosidases and sulfatases hydrolyze the GAGs to monosaccharides. The lysosomes contain both endoglycosidases, which hydrolyze the long polymers into shorter oligosaccharides, and exoglycosidases that cleave individual acidic- or aminosugars from the GAG fragments. Lysosomal catabolism of GAGs proceeds in a stepwise manner from the non-reducing end (seeFIG.1). If the terminal sugar is sulfated, then the sulfate bond must be hydrolyzed by a specific sulfatase before the sugar can be removed. When the sulfate has been removed, a specific exoglycosidase then hydrolyzes the terminal sugar from the nonreducing end of the oligosaccharide, thus leaving it one sugar shorter. Degradation continues in this stepwise fashion, alternating between removal of sulfates by sulfatases and cleavage of the terminal sugars by exoglycosidases. If removal of a sulfate leaves a terminal glucosamine residue, then it must first be acetylated to N-acetylglucosamine because the lysosome lacks the enzyme required to remove glucosamine. This is accomplished by an acetyltransferase that uses acetyl-CoA as the acetyl group donor. When the glucosamine residue has been N-acetylated it can be hydrolyzed by α-N-acetylglucosaminidase, allowing the continuation of the stepwise degradation of the GAG. The terminal sugar of heparan sulfate and dermatan sulfate are sulfated, which is removed by the IDS enzyme (iduronate sulfatase). The next step is the removal of the terminal sugar, which is catalyzed by the IDUA enzyme. In subjects with MPS I, the defective IDUA enzyme is not able to remove that terminal sugar, leading to a build-up of heparan and dermatan. The terms “subject” or “patient” are used interchangeably and refer to mammals such as human subjects and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the terms “subject” or “patient” as used herein means any mammalian subject to which the altered cells of the invention and/or proteins produced by the altered cells of the invention can be administered. Subjects of the present invention include those having MPS I disorder. Generally, the subject is eligible for treatment for MPS I. For the purposes herein, such eligible subject is one who is experiencing, has experienced, or is likely to experience, one or more signs, symptoms or other indicators of MPS I; has been diagnosed with MPS I, whether, for example, newly diagnosed, and/or is at risk for developing MPS I. One suffering from or at risk for suffering from MPS I may optionally be identified as one who has been screened for elevated levels of GAG in tissues and/or urine. As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life. As used herein, “delaying” or “slowing” the progression of MPS I means to prevent, defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As used herein, “at the time of starting treatment” refers to the time period at or prior to the first exposure to an MPS I therapeutic composition such as the compositions of the invention. In some embodiments, “at the time of starting treatment” is about any of one year, nine months, six months, three months, second months, or one month prior to a MPS I drug. In some embodiments, “at the time of starting treatment” is immediately prior to coincidental with the first exposure to an MPS I therapeutic composition. The term “wheelchair dependent” means a subject that is unable to walk through injury or illness and must rely on a wheelchair to move around. The term “mechanical ventilator” describes a device that improves the exchange of air between a subject's lungs and the atmosphere. As used herein, “based upon” includes (1) assessing, determining, or measuring the subject characteristics as described herein (and preferably selecting a subject suitable for receiving treatment; and (2) administering the treatment(s) as described herein. A “symptom” of MPS I is any phenomenon or departure from the normal in structure, function, or sensation, experienced by the subject and indicative of MPS I. “Severe MPS I” in subjects is characterized by delayed speech and developmental delay between 18 months to 3 years of age. The disease is characterized in severe MPS I subjects by organomegaly, hyperactivity and aggressiveness, neurologic deterioration, joint stiffness and skeletal deformities (including abnormal spinal bones), coarse facial features with enlarged tongue, heart valve thickening, hearing loss and hernias. “Attenuated form MPS I” in subjects are typically diagnosed later than the severe subjects. The somatic clinical features are similar to the severe subjects, but overall disease severity in milder with, in general, slower disease progression with no or only mild cognitive impairment. Death in the untreated attenuated form is often between the ages of 20-30 years from cardiac and respiratory disease. The term “supportive surgery” refers to surgical procedures that may be performed on a subject to alleviate symptoms that may be associated with a disease. For subjects with MPS I, such supportive surgeries may include heart valve replacement surgery, tonsillectomy and adenoidectomy, placement of ventilating tubes, repair of abdominal hernias, cervical decompression, treatment of carpal tunnel syndrome, surgical decompression of the median nerve, instrumented fusion (to stabilize and strengthen the spine), arthroscopy, hip or knee replacement, and correction of the lower limb axis, and tracheostomy (see Wraith et al. (2008)Eur J Pediatr.167(3):267-277; and Scarpa et al. (2011)Orphanet Journal of Rare Diseases,6:72). The term “immunosuppressive agent” as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDUA); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor-alpha antibodies (infliximab or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-tumor necrosis factor-beta antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (International Patent Publication No. WO 90/08187 published Jul. 26, 1990); streptokinase; TGF-beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxysperguahn; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al. (1991)Science251:430-432; International Patent Publication No. WO 90/11294; Janeway (1989)Nature341:482; and International Patent Publication No. WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9. “Corticosteroid” refers to any one of several synthetic or naturally occurring substances with the general chemical structure of steroids that mimic or augment the effects of the naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, glucocorticoid and betamethasone. A “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc. A “label” is used herein to refer to information customarily included with commercial packages of pharmaceutical formulations including containers such as vials and package inserts, as well as other types of packaging. Labels may also be of different colors. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. Nucleases The methods described herein can make use of one or more nucleases for targeted introduction of the IDUA transgene. Non-limiting examples of nucleases include ZFNs, TALENs, homing endonucleases, CRISPR/Cas and/or Ttago guide RNAs, that are useful for in vivo cleavage of a donor molecule carrying a transgene and nucleases for cleavage of the genome of a cell such that the transgene is integrated into the genome in a targeted manner. In certain embodiments, one or more of the nucleases are naturally occurring. In other embodiments, one or more of the nucleases are non-naturally occurring, i.e., engineered in the DNA-binding molecule (also referred to as a DNA-binding domain) and/or cleavage domain. For example, the DNA-binding domain of a naturally-occurring nuclease may be altered to bind to a selected target site (e.g., a ZFP, TALE and/or sgRNA of CRISPR/Cas that is engineered to bind to a selected target site). In other embodiments, the nuclease comprises heterologous DNA-binding and cleavage domains (e.g., zinc finger nucleases; TAL-effector domain DNA binding proteins; meganuclease DNA-binding domains with heterologous cleavage domains). In other embodiments, the nuclease comprises a system such as the CRISPR/Cas of Ttago system. A. DNA-Binding Domains In certain embodiments, the composition and methods described herein employ a meganuclease (homing endonuclease) DNA-binding domain for binding to the donor molecule and/or binding to the region of interest in the genome of the cell. Naturally-occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family (“LAGLIDADG” for example (LAGLIDADG” disclosed as SEQ ID NO: 39), see the DNA sequence for I-CreI below (Accession X01977, version x01977.1): 1gatccttgat caggaccctt gacagtttca ggtgggcagtttatttgggg cgaatgcctc61ctaaaaggta acggaggcgt gcaaaggttc cctcagtctggacggaaatc agacattgag121tgtaaaggca aaagggagct tgactgcaag acctacaagtcgagcagggg cgaaagaggc181cttagtgatc cgacggtgcc gcgtggaagg gccgtcgctcaacggataaa agttactccc241gggataacag gctgatcttc cccaagagtt cacatcgacgggaaggtttg gcacctcgat301gtcggctcat cacatcctcg gtctgtagta ggtccgaagggttgggctgt tcgcccatta361aagtggtacg tgagctgggt tcaaaacgta aataacactgcgtgtgcttg cagtaatgta421agcaaagtat cggcttatat cggtgaaacc ttcctattgttttaagtaca aactgtcgca481taaaccacat tcgtgggcaa tagatggcaa cgccgagggaagaccatttc tttttggttt541aataattcaa taaattaaat aaaacatctt atgaatacaaaatataataa agagttctta601ctctacttag cagggtttgt agacggtgac ggtagcataatcgctcaaat taagcctaat661cagtcttata aatttaagca tcagctatca ctcgcgttccaagtcacgca aaagacacag721agacgttggt ttttagacaa attagtggat gaaattggggttggttatgt aagagatagg781ggtagcgttt cggattatat tctaagcgaa atcaagcctttgcataattt tttaacacaa841ctacaacctt ttctaaaact aaaacaaaaa caagcaaatttagttttaaa aattatttgg901cggcttccgt cagcaaaaga atccccggac aaattcttagaagtttgtac atgggtggat961caaattgcag ctctgaatga ttcgaagacg cgtaaaacaacttctgaaac cgttcgtgct1021gtgctagaca gtttaagtga aaaaaagaaa tcgtccccgtagagacttta taaatttagc1081caatctctaa aagaatgttt acatacaatt tatttattgttgctcgattt ataggatatt1141ttctcgagag tgggaaagta taatacgccg actcctgccattaacagtag caggatgaag1201acatagtcca tgcctttacg aaagtaaagg ggttagttttaaagaccgca agttttattc1261ggctttaaaa tttcatgcgt gagacagttt ggtccatatccggtgtaggc gttagagcat1321tgagagtagc ctttcatagt acgagaggac ctgaaaggacatgccaattg tgtaccagtt1381ctcattccaa tgggaaacgc tgggtagcta cgcatggatagataactgct gaaagcatct1441aagtaggaag ctaaactcaa gatgagtgct ctctaaggccgcggctagac aagccgttat1501ataggtatca ggtgtacagt cagcaatggc tttagccgagatatactaaa ggccgtttga1561ttttgacctt tataatataa ttacataacc ccttgcgggtaactatcgtt tatgagctaa1621gct disclosed as SEQ ID NO:29), the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997)Nucleic Acids Res.25:3379-3388; Dujon et al. (1989)Gene82:115-118; Perler et al. (1994)Nucleic Acids Res.22:1125-1127; Jasin (1996)Trends Genet.12:224-228; Gimble et al. (1996)J Mol. Biol.263:163-180; Argast et al. (1998)J Mol. Biol.280:345-353 and the New England Biolabs catalogue. In certain embodiments, the methods and compositions described herein make use of a nuclease that comprises an engineered (non-naturally occurring) homing endonuclease (meganuclease). The recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997)Nucleic Acids Res.25:3379-3388; Dujon et al. (1989)Gene82:115-118; Perler et al. (1994)Nucleic Acids Res.22:1125-1127; Jasin (1996)Trends Genet.12:224-228; Gimble et al. (1996)J Mol. Biol.263:163-180; Argast et al. (1998)J Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell10:895-905; Epinat et al. (2003)Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006)Nature441:656-659; Paques et al. (2007)Current Gene Therapy7:49-66; U.S. Patent Publication No. 2007/0117128. The DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous cleavage domain. In other embodiments, the DNA-binding domain of one or more of the nucleases used in the methods and compositions described herein comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Pat. No. 8,586,526, incorporated by reference in its entirety herein. The plant pathogenic bacteria of the genusXanthomonasare known to cause many diseases in important crop plants. Pathogenicity ofXanthomonasdepends on a conserved type III secretion (T3 S) system which injects more than 25 different effector proteins into the plant cell. Among these injected proteins are transcription activator-like (TAL) effectors which mimic plant transcriptional activators and manipulate the plant transcriptome (see Kay et al. (2007)Science318:648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TAL-effectors is AvrBs3 fromXanthomonas campestgrispv.Vesicatoria(see Bonas et al. (1989)Mol Gen Genet218:127-136 and International Patent Publication No. WO 2010/079430). TAL-effectors contain a centralized domain of tandem repeats, each repeat containing approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for a review see Schornack S, et al. (2006)J Plant Physiol163(3):256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearumtwo genes, designated brg11 and hpx17 have been found that are homologous to the AvrBs3 family ofXanthomonasin theR. solanacearumbiovar 1 strain GMI1000 and in the biovar 4 strain RS1000 (See Heuer et al. (2007)Appl and Envir Micro73(13):4379-4384). These genes are 98.9% identical in nucleotide sequence to each other but differ by a deletion of 1,575 bp in the repeat domain of hpx17. However, both gene products have less than 40% sequence identity with AvrBs3 family proteins ofXanthomonas. See, e.g., U.S. Pat. No. 8,586,526, incorporated by reference in its entirety herein. Specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeated sequence comprises approximately 102 bp and the repeats are typically 91-100% homologous with each other (Bonas et al., ibid). Polymorphism of the repeats is usually located at positions 12 and 13 and there appears to be a one-to-one correspondence between the identity of the hypervariable diresidues (RVDs) at positions 12 and 13 with the identity of the contiguous nucleotides in the TAL-effector's target sequence (see Moscou and Bogdanove, (2009)Science326:1501 and Boch et al. (2009)Science326:1509-1512). Experimentally, the natural code for DNA recognition of these TAL-effectors has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds to T. These DNA binding repeats have been assembled into proteins with new combinations and numbers of repeats, to make artificial transcription factors that are able to interact with new sequences and activate the expression of a non-endogenous reporter gene in plant cells (Boch et al., ibid). Engineered TAL proteins have been linked to a FokI cleavage half domain to yield a TAL effector domain nuclease fusion (TALEN) exhibiting activity in a yeast reporter assay (plasmid based target). See, e.g., U.S. Pat. No. 8,586,526; Christian et al. (2010)Geneticsepub 10.1534/genetics. 110.120717). In certain embodiments, the DNA binding domain of one or more of the nucleases used for in vivo cleavage and/or targeted cleavage of the genome of a cell comprises a zinc finger protein. Preferably, the zinc finger protein is non-naturally occurring in that it is engineered to bind to a target site of choice. See, for example, See, for example, Beerli et al. (2002)Nature Biotechnol.20:135-141; Pabo et al. (2001)Ann. Rev. Biochem.70:313-340; Isalan et al. (2001)Nature Biotechnol.19:656-660; Segal et al. (2001)Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000)Curr. Opin. Struct. Biol.10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; and 2005/0267061, all incorporated herein by reference in their entireties. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227. In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 8,772,453; 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. Selection of target sites; ZFPs and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; and International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496. In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In certain embodiments, the DNA-binding domain of the nuclease is part of a CRISPR/Cas nuclease system, including, for example a single guide RNA (sgRNA). See, e.g., U.S. Pat. No. 8,697,359 and U.S. Patent Publication No. 2015/0056705. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the Cas (CRISPR-associated) locus, which encodes proteins (Jansen et al. (2002)Mol. Microbiol.43:1565-1575; Makarova et al. (2002)Nucleic Acids Res.30:482-496; Makarova et al. (2006)Biol. Direct1:7; Haft et al. (2005)PLoS Comput. Biol.1:e60) make up the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Thus, in the bacterial cell, several of the so-called ‘Cas’ proteins are involved with the natural function of the CRISPR/Cas system and serve roles in functions such as insertion of the alien DNA etc. In some embodiments, the CRISPR-Cpf1 system is used. The CRISPR-Cpf1 system, identified inFrancisellaspp, is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including in their guide RNAs and substrate specificity (see Fagerlund et al. (2015)Genom Bio16:251). A major difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize tracrRNA, and thus requires only a crRNA. The FnCpf1 crRNAs are 42-44 nucleotides long (19-nucleotide repeat and 23-25-nucleotide spacer) and contain a single stem-loop, which tolerates sequence changes that retain secondary structure. In addition, the Cpf1 crRNAs are significantly shorter than the ˜100-nucleotide engineered sgRNAs required by Cas9, and the PAM requirements for FnCpf1 are 5′-TTN-3′ and 5′-CTA-3′ on the displaced strand. Although both Cas9 and Cpf1 make double strand breaks in the target DNA, Cas9 uses its RuvC- and HNH-like domains to make blunt-ended cuts within the seed sequence of the guide RNA, whereas Cpf1 uses a RuvC-like domain to produce staggered cuts outside of the seed. Because Cpf1 makes staggered cuts away from the critical seed region, NHEJ will not disrupt the target site, therefore ensuring that Cpf1 can continue to cut the same site until the desired HDR recombination event has taken place. Thus, in the methods and compositions described herein, it is understood that the term “Cas” includes both Cas9 and Cfp1 proteins. Thus, as used herein, a “CRISPR/Cas system” refers both CRISPR/Cas and/or CRISPR/Cfp1 systems, including both nuclease, nickase and/or transcription factor systems. In some embodiments, other Cas proteins may be used. Some exemplary Cas proteins include Cas9, Cpf1 (also known as Cas12a), C2c1, C2c2 (also known as Cas13a), C2c3, Cas1, Cas2, Cas4, CasX and CasY; and include engineered and natural variants thereof (Burstein et al. (2017)Nature542:237-241) for example HF1/spCas9 (Kleinstiver et al. (2016)Nature529:490-495; Cebrian-Serrano and Davies (2017)Mamm Genome28(7):247-261); split Cas9 systems (Zetsche et al. (2015)Nat Biotechnol33(2):139-142), trans-spliced Cas9 based on an intein-extein system (Troung et al. (2015)Nucl Acid Res43(13):6450-8); mini-SaCas9 (Ma et al. (2018)ACS Synth Biol7(4):978-985). Thus, in the methods and compositions described herein, it is understood that the term “Cas” includes all Cas variant proteins, both natural and engineered. In certain embodiments, Cas protein may be a “functional derivative” of a naturally occurring Cas protein. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. In some cases, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein. Additional non-limiting examples of RNA guided nucleases that may be used in addition to and/or instead of Cas proteins include Class 2 CRISPR proteins such as Cpf1. See, e.g., Zetsche et al. (2015)Cell163:1-13. In some embodiments, the DNA binding domain is part of a TtAgo system (see Swarts et al. (2014)Nature507(7491):258-261; Swarts et al. (2012)PLoS One7(4):e35888; Sheng et al. (2014)Proc. Natl. Acad. Sci. U.S.A.111(2):652-657). In eukaryotes, gene silencing is mediated by the Argonaute (Ago) family of proteins. In this paradigm, Ago is bound to small (19-31 nt) RNAs. This protein-RNA silencing complex recognizes target RNAs via Watson-Crick base pairing between the small RNA and the target and endonucleolytically cleaves the target RNA (Vogel (2014)Science344:972-973). In contrast, prokaryotic Ago proteins bind to small single-stranded DNA fragments and likely function to detect and remove foreign (often viral) DNA (Yuan et al. (2005)Mol. Cell19:405; Olovnikov et al. (2013)Mol. Cell51:594; Swarts et al., ibid). Exemplary prokaryotic Ago proteins include those fromAquifex aeolicus, Rhodobacter sphaeroides, andThermus thermophilus. One of the most well-characterized prokaryotic Ago protein is the one fromT. thermophilus(TtAgo; Swarts et al., ibid). TtAgo associates with either 15 nt or 13-25 nt single-stranded DNA fragments with 5′ phosphate groups. This “guide DNA” bound by TtAgo serves to direct the protein-DNA complex to bind a Watson-Crick complementary DNA sequence in a third-party molecule of DNA. Once the sequence information in these guide DNAs has allowed identification of the target DNA, the TtAgo-guide DNA complex cleaves the target DNA. Such a mechanism is also supported by the structure of the TtAgo-guide DNA complex while bound to its target DNA (G. Sheng et al., ibid). Ago fromRhodobacter sphaeroides(RsAgo) has similar properties (Olovnikov et al., ibid). Exogenous guide DNAs of arbitrary DNA sequence can be loaded onto the TtAgo protein (Swarts et al., ibid.). Since the specificity of TtAgo cleavage is directed by the guide DNA, a TtAgo-DNA complex formed with an exogenous, investigator-specified guide DNA will therefore direct TtAgo target DNA cleavage to a complementary investigator-specified target DNA. In this way, one may create a targeted double-strand break in DNA. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNA systems from other organisms) allows for targeted cleavage of genomic DNA within cells. Such cleavage can be either single- or double-stranded. For cleavage of mammalian genomic DNA, it would be preferable to use of a version of TtAgo codon optimized for expression in mammalian cells. Further, it might be preferable to treat cells with a TtAgo-DNA complex formed in vitro where the TtAgo protein is fused to a cell-penetrating peptide. Further, it might be preferable to use a version of the TtAgo protein that has been altered via mutagenesis to have improved activity at 37 degrees Celsius. TtAgo-RNA-mediated DNA cleavage could be used to effect a panopoly of outcomes including gene knock-out, targeted gene addition, gene correction, targeted gene deletion using techniques standard in the art for exploitation of DNA breaks. Thus, the nuclease comprises a DNA-binding domain in that specifically binds to a target site in any gene into which it is desired to insert a donor (transgene). In certain embodiments the DNA-binding domains bind to albumin, e.g., DNA-binding domains of the ZFPs designated SBS-47171 and SBS-47898. See, e.g., U.S. Patent Publication No. 2015/0159172. B. Cleavage Domains Any suitable cleavage domain can be associated with (e.g., operatively linked) to a DNA-binding domain to form a nuclease. For example, ZFP DNA-binding domains have been fused to nuclease domains to create ZFNs—a functional entity that is able to recognize its intended nucleic acid target through its engineered (ZFP) DNA binding domain and cause the DNA to be cut near the ZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Natl Acad Sci USA93(3):1156-1160. More recently, ZFNs have been used for genome modification in a variety of organisms. See, for example, U.S. Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0188987; 2006/0063231; and International Publication WO 07/014275. Likewise, TALE DNA-binding domains have been fused to nuclease domains to create TALENs. See, e.g., U.S. Pat. No. 8,586,526. CRISPR/Cas nuclease systems comprising single guide RNAs (sgRNAs) that bind to DNA and associate with cleavage domains (e.g., Cas domains) to induce targeted cleavage have also been described. See, e.g., U.S. Pat. Nos. 8,697,359 and 8,932,814 and U.S. Patent Publication No. 2015/0056705. As noted above, the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease or a TALEN DNA-binding domain and a cleavage domain from a nuclease; a sgRNA DNA-binding domain and a cleavage domain from a nuclease (CRISPR/Cas); and/or meganuclease DNA-binding domain and cleavage domain from a different nuclease. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al. (1997)Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., Si Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.)Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains. Similarly, a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity. In general, two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains. Alternatively, a single protein comprising two cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing. Thus, in certain embodiments, the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides. However, any integral number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotide pairs or more). In general, the site of cleavage lies between the target sites. Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150; and 5,487,994; as well as Li et al. (1992)Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993)Proc. Natl. Acad. Sci. USA90:2764-2768; Kim et al. (1994a)Proc. Natl. Acad. Sci. USA91:883-887; Kim et al. (1994b)J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998)Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using zinc finger-FokI fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-FokI fusions are provided elsewhere in this disclosure. A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain. Exemplary Type IIS restriction enzymes are described in U.S. Pat. No. 7,888,121, incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003)Nucleic Acids Res.31:418-420. In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Pat. Nos. 8,772,453; 8,623,618; 8,409,861; 8,034,598; 7,914,796; and 7,888,121, the disclosures of all of which are incorporated by reference in their entireties herein. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets for influencing dimerization of the FokI cleavage half-domains. Exemplary engineered cleavage half-domains of FokI that form obligate heterodimers include a pair in which a first cleavage half-domain includes mutations at amino acid residues at positions 490 and 538 of FokI and a second cleavage half-domain includes mutations at amino acid residues 486 and 499. Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at 486 replaced Gln (Q) with Glu (E); and the mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage half-domains described herein were prepared by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to produce an engineered cleavage half-domain designated “E490K:I538K” and by mutating positions 486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce an engineered cleavage half-domain designated “Q486E:I499L”. The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. U.S. Pat. Nos. 7,914,796 and 8,034,598, the disclosures of which are incorporated by reference in their entireties. In certain embodiments, the engineered cleavage half-domain comprises mutations at positions 486, 499 and 496 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Gln (Q) residue at position 486 with a Glu(E) residue, the wild type Iso (I) residue at position 499 with a Leu (L) residue and the wild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E) residue (also referred to as a “ELD” and “ELE” domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490, 538 and 537 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue, the wild type Iso (I) residue at position 538 with a Lys (K) residue, and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as “KKK” and “KKR” domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type FokI), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as “KIK” and “KIR” domains, respectively). See, e.g., U.S. Pat. No. 8,772,453. In other embodiments, the engineered cleavage half domain comprises the “Sharkey” and/or “Sharkey mutations” (see Guo et al. (2010)J Mol. Biol.400(1):96-107). Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains (FokI) as described in U.S. Pat. Nos. 7,888,121; 7,914,796; 8,034,598; and 8,623,618. Alternatively, nucleases may be assembled in vivo at the nucleic acid target site using so-called “split-enzyme” technology (see, e.g. U.S. Patent Publication No. 2009/0068164). Components of such split enzymes may be expressed either on separate expression constructs, or can be linked in one open reading frame where the individual components are separated, for example, by a self-cleaving 2A peptide or IRES sequence. Components may be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain. Nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in U.S. Pat. No. 8,563,314. Expression of the nuclease may be under the control of a constitutive promoter or an inducible promoter, for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose. The Cas9 related CRISPR/Cas system comprises two RNA non-coding components: tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). To use a CRISPR/Cas system to accomplish genome engineering, both functions of these RNAs must be present (see Cong et al. (2013)Sciencexpress1/10.1126/science 1231143). In some embodiments, the tracrRNA and pre-crRNAs are supplied via separate expression constructs or as separate RNAs. In other embodiments, a chimeric RNA is constructed where an engineered mature crRNA (conferring target specificity) is fused to a tracrRNA (supplying interaction with the Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see Jinek et al. (2013)Elife2:e00471. doi: 10.7554/eLife.00471; Jinek et al. (2012)Science337:816-821 and Cong, ibid). The nuclease(s) as described herein may make one or more double-stranded and/or single-stranded cuts in the target site. In certain embodiments, the nuclease comprises a catalytically inactive cleavage domain (e.g., FokI and/or Cas protein). See, e.g., U.S. Pat. Nos. 9,200,266; 8,703,489 and Guillinger et al. (2014)Nature Biotech.32(6):577-582. The catalytically inactive cleavage domain may, in combination with a catalytically active domain act as a nickase to make a single-stranded cut. Therefore, two nickases can be used in combination to make a double-stranded cut in a specific region. Additional nickases are also known in the art, for example, McCaffery et al. (2016)Nucleic Acids Res.44(2):e11. doi: 10.1093/nar/gkv878. Epub 2015 Oct. 19. Thus, any nuclease comprising a DNA-binding domain and cleavage domain can be used. In certain embodiments, the nuclease comprises a ZFN made up of left and right ZFNs, for example a ZFN comprising a first ZFN comprising a ZFP designated SBS-47171 and a cleavage domain and a second ZFN comprising a ZFP designated SBS-47898 and a cleavage domain. In certain embodiments, the left and right (first and second) ZFNs of the ZFN are carried on the same vector and in other embodiments, the paired components of the ZFN are carried on different vectors, for example two AAV vectors, one designated SB-47171 AAV as shown in Table 1, SEQ ID NO:9 (an AAV2/6 vector carrying ZFN comprising the ZFP designated SBS-47171) and the other designated SB-47898 AAV as shown in Table 2, SEQ ID NO:12 (an AAV2/6 vector carrying ZFN comprising the ZFP designated SBS-47898). Target Sites As described in detail above, DNA domains can be engineered to bind to any sequence of choice in a locus, for example an albumin or other safe-harbor gene. An engineered DNA-binding domain can have a novel binding specificity, compared to a naturally-occurring DNA-binding domain. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual (e.g., zinc finger) amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of DNA binding domain which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Rational design of TAL-effector domains can also be performed. See, e.g., U.S. Patent Publication No. 2011/0301073. Exemplary selection methods applicable to DNA-binding domains, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237. Selection of target sites; nucleases and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Patent Publication Nos. 2005/0064474 and 2006/0188987, incorporated by reference in their entireties herein. In addition, as disclosed in these and other references, DNA-binding domains (e.g., multi-fingered zinc finger proteins) may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual DNA-binding domains of the protein. See, also, U.S. Pat. No. 8,586,526. In certain embodiments, the target site(s) for the DNA-binding domain(s) (is) are within an albumin gene. See, e.g., U.S. Patent Publication No. 2015/0159172. Donors As noted above, insertion of an exogenous sequence (also called a “donor sequence” or “donor”), for example for correction of a mutant gene or for increased expression of a gene encoding a protein lacking or deficient in MPS I disease (e.g., IDUA) is provided. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest. Described herein are methods of targeted insertion of a transgene encoding an IDUA protein for insertion into a chosen location. Polynucleotides for insertion can also be referred to as “exogenous” polynucleotides, “donor” polynucleotides or molecules or “transgenes.” The donor polynucleotide can be DNA or RNA, single-stranded and/or double-stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Pat. Nos. 8,703,489 and 9,005,973. The donor sequence(s) can also be contained within a DNA MC, which may be introduced into the cell in circular or linear form. See, e.g., U.S. Patent Publication No. 2014/0335063. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987)Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996)Science272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)). The donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted (e.g., highly expressed, albumin, AAVS1, HPRT, etc.). However, it will be apparent that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter. In some embodiments, the donor is maintained in the cell in an expression plasmid such that the gene is expressed extra-chromosomally. The donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into an albumin or other locus such that some (N-terminal and/or C-terminal to the transgene encoding the lysosomal enzyme) or none of the endogenous albumin sequences are expressed, for example as a fusion with the transgene encoding the IDUA protein(s). In other embodiments, the transgene (e.g., with or without additional coding sequences such as for albumin) is integrated into any endogenous locus, for example a safe-harbor locus. When endogenous sequences (endogenous or part of the transgene) are expressed with the transgene, the endogenous sequences (e.g., albumin, etc.) may be full-length sequences (wild-type or mutant) or partial sequences. Preferably the endogenous sequences are functional. Non-limiting examples of the function of these full length or partial sequences (e.g., albumin) include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier. Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. In certain embodiments, the exogenous sequence (donor) comprises a fusion of a protein of interest and, as its fusion partner, an extracellular domain of a membrane protein, causing the fusion protein to be located on the surface of the cell. This allows the protein encoded by the transgene to potentially act in the serum. In the case of treatment for MPS I disease, IDUA enzyme encoded by the transgene fusion acts on the metabolic products that are accumulating in the serum from its location on the surface of the cell (e.g., RBC). In addition, if the RBC is engulfed by a splenic macrophage as is the normal course of degradation, the lysosome formed when the macrophage engulfs the cell would expose the membrane bound fusion protein to the high concentrations of metabolic products in the lysosome at the pH more naturally favorable to that enzyme. In some cases, the donor may be an endogenous gene (IDUA) that has been modified. For instance, codon optimization may be performed on the endogenous gene to produce a donor. Furthermore, although antibody response to enzyme replacement therapy varies with respect to the specific therapeutic enzyme in question and with the individual subject, a significant immune response has been seen in many MPS I disease subjects being treated with enzyme replacement with wild-type IDUA. In addition, the relevance of these antibodies to the efficacy of treatment is also variable (see Katherine Ponder (2008)J Clin Invest118(8):2686). Thus, the methods and compositions of the current invention can comprise the generation of donor with modified sequences as compared to wild-type IDUA, including, but not limited to, modifications that produce functionally silent amino acid changes at sites known to be priming epitopes for endogenous immune responses, and/or truncations such that the polypeptide produced by such a donor is less immunogenic. MPS I disease subjects often have neurological sequelae due the lack of the missing IDUA enzyme in the brain. Unfortunately, it is often difficult to deliver therapeutics to the brain via the blood due to the impermeability of the blood brain barrier. Thus, the methods and compositions of the invention may be used in conjunction with methods to increase the delivery of the therapeutic into the brain, including but not limited to methods that cause a transient opening of the tight junctions between cells of the brain capillaries such as transient osmotic disruption through the use of an intracarotid administration of a hypertonic mannitol solution, the use of focused ultrasound and the administration of a bradykinin analogue. Alternatively, therapeutics can be designed to utilize receptors or transport mechanisms for specific transport into the brain. Examples of specific receptors that may be used include the transferrin receptor, the insulin receptor or the low-density lipoprotein receptor related proteins 1 and 2 (LRP-1 and LRP-2). LRP is known to interact with a range of secreted proteins such as apoE, tPA, PAI-1 etc, and so fusing a recognition sequence from one of these proteins for LRP may facilitate transport of the enzyme into the brain, following expression in the liver of the therapeutic protein and secretion into the blood stream (see Gabathuler (2010)Neurobiol Dis.37(1):48-57). In certain embodiments, the donor vectors is a vector as shown in SB-IDUA AAV (Table 5, SEQ ID NO:28). Compositions/Systems of the Invention The invention described herein utilizes three AAV vectors for practicing the method. Two vectors are used to deliver the right ZFN and the left ZFN and a third vector is used to provide the IDUA donor sequence (see Examples). In certain embodiments, the composition/systems comprising the 3 vectors which includes SB-47171 or SB-71557, SB-47898 or SB-71728 and SB-IDUA AAV. Cells Also provided herein are genetically modified cells, for example, liver cells or stem cells comprising a transgene encoding an IDUA protein, including cells produced by the methods described herein. The IDUA transgene may be expressed extra-chromosomally or can integrated in a targeted manner into the cell's genome using one or more nucleases. Unlike random integration, nuclease-mediated targeted integration ensures that the transgene is integrated into a specified gene. The transgene may be integrated anywhere in the target gene. In certain embodiments, the transgene is integrated at or near the nuclease binding and/or cleavage site, for example, within 1-300 (or any number of base pairs therebetween) base pairs upstream or downstream of the site of cleavage and/or binding site, more preferably within 1-100 base pairs (or any number of base pairs therebetween) of either side of the cleavage and/or binding site, even more preferably within 1 to 50 base pairs (or any number of base pairs therebetween) of either side of the cleavage and/or binding site. In certain embodiments, the integrated sequence does not include any vector sequences (e.g., viral vector sequences). Any cell type can be genetically modified as described herein to comprise a transgene, including but not limited to cells or cell lines. Other non-limiting examples of genetically modified cells as described herein include T-cells (e.g., CD4+, CD3+, CD8+, etc.); dendritic cells; B-cells; autologous (e.g., subject-derived). In certain embodiments, the cells are liver cells and are modified in vivo. In certain embodiments, the cells are stem cells, including heterologous pluripotent, totipotent or multipotent stem cells (e.g., CD34+ cells, induced pluripotent stem cells (iPSCs), embryonic stem cells or the like). In certain embodiments, the cells as described herein are stem cells derived from subject. The cells as described herein are useful in treating and/or preventing MPS I disease in a subject with the disorder, for example, by in vivo therapies. Ex vivo therapies are also provided, for example when the nuclease-modified cells can be expanded and then reintroduced into the subject using standard techniques. See, e.g., Tebas et al. (2014)New Eng J Med370(10):901. In the case of stem cells, after infusion into the subject, in vivo differentiation of these precursors into cells expressing the functional protein (from the inserted donor) also occurs. Pharmaceutical compositions (also referred to as “a formulation” or “article of manufacture” or “drug product” or “set of drug products”) comprising one or more of the compositions (nucleases, IDUA donors, cells, etc.) as described herein are also provided. The pharmaceutical compositions may include the same or different types of component compositions in any concentrations. For example, provided herein is an article of manufacture comprising a set of drug products, which include three separate pharmaceutical compositions as follows: a first pharmaceutical composition comprising a purified AAV vector carrying one member of a ZFN pair (e.g., a left ZFN); a second pharmaceutical composition comprising a purified AAV vector carrying the other member of a ZFN pair (e.g., a right ZFN); and a third pharmaceutical composition comprising a purified AAV vector carrying an IDUA donor. The left ZFNs may comprise the ZFN designated 47171 (e.g., drug product designated SB-A6P-ZLEF) or the ZFN designated 71557 (e.g., drug product designated SB-A6P-ZL2) and the right ZFN may comprise the ZFN designated 47898 (e.g., drug product designated SB-A6P-ZRIGHT) or the ZFN designated 71728 (e.g., drug product designated SB-A6P-ZL2). One, two or three of the three pharmaceutical compositions may be individually formulated in phosphate buffered saline (PBS) containing CaCl2, MgCl2, NaCl, sucrose and a Poloxamer (e.g., Poloxamer P188) or in a Normal Saline (NS) formulation. Any concentration can be used, including but not limited to the concentrations shown in Table 6. Further, the article of manufacture may include any ratio of the three pharmaceutical compositions can be used, for example 1:1:8 (left ZFN:right ZFN:IDUA donor). The pharmaceutical compositions (article of manufacture/set of drug products) are administered (e.g., intravenously) to a subject in need thereof such that IDUA is expressed in the subject, including at therapeutic levels (e.g., in plasma and/or blood leukocytes) for treatment of MPS I. The compositions may be administered separately or, preferably, the article of manufacture comprising a set of three drug products (ZFN1, ZFN2, and IDUA donor) are combined prior to administration, for example in an intravenous infusion bag. In addition, these formulations may be cryopreserved prior to administration to a subject. Delivery The nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered in vivo or ex vivo by any suitable means. Methods of delivering nucleases as described herein are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of the zinc finger, TALEN and/or Cas protein(s). Any vector systems may be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporated by reference herein in their entireties. Furthermore, it will be apparent that any of these vectors may comprise one or more of the sequences needed for treatment. Thus, when one or more nucleases and a donor construct are introduced into the cell, the nucleases and/or donor polynucleotide may be carried on the same vector or on different vectors. When multiple vectors are used, each vector may comprise a sequence encoding one or multiple nucleases and/or donor constructs. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor constructs in cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson (1992)Science256:808-813; Nabel & Felgner (1993)TIBTECH11:211-217; Mitani & Caskey (1993)TIBTECH11:162-166; Dillon (1993)TIBTECH11:167-175; Miller (1992)Nature357:455-460; Van Brunt (1988)Biotechnology6(10):1149-1154; Vigne (1995)Restorative Neurology and Neuroscience8:35-36; Kremer & Perricaudet (1995)British Medical Bulletin51(1):31-44; Haddada et al., inCurrent Topics in Microbiology and ImmunologyDoerfler and Bohm (eds.) (1995); and Yu et al. (1994)Gene Therapy1:13-26 (1994). Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91/17424 and WO 91/16024. The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal (1995)Science270:404-410; Blaese et al. (1995)Cancer Gene Ther.2:291-297; Behr et al. (1994)Bioconjugate Chem.5:382-389; Remy et al. (1994)Bioconjugate Chem.5:647-654; Gao et al. (1995)Gene Therapy2:710-722; Ahmad et al. (1992)Cancer Res.52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787). Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiarmid et al. (2009)Nature Biotechnology27(7):643). The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to subjects (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to subjects (ex vivo). Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been measured in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992)J. Virol.66:2731-2739; Johann et al. (1992)J. Virol.66:1635-1640; Sommerfelt et al. (1990)Virol.176:58-59; Wilson et al. (1989)J. Virol.63:2374-2378; Miller et al. (1991)J. Virol.65:2220-2224; International Patent Publication No. WO 94/26877). In applications in which transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al. (1987)Virology160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No. WO 93/24641; Kotin (1994)Human Gene Therapy5:793-801; Muzyczka (1994)J. Clin. Invest.94:1351. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al. (1985)Mol. Cell. Biol.5:3251-3260; Tratschin et al. (1984)Mol. Cell. Biol.4:2072-2081; Hermonat & Muzyczka (1984)PNAS81:6466-6470; and Samulski et al. (1989)J. Virol.63:03822-3828. At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al. (1995)Blood85:3048-305; Kohn et al. (1995)Nat. Med.1:1017-102; Malech et al. (1997)PNAS94(22):12133-12138). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al. (1995)Science270:475-480). Transduction efficiencies of 50% or greater have been measured for MFG-S packaged vectors. (Ellem et al. (1997)Immunol Immunother.44(1):10-20; Dranoff et al. (1997)Hum. Gene Ther.1:111-2. Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery system based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al. (1998)Lancet351(9117):1702-3; Kearns et al. (1996)Gene Ther.9:748-55). Other AAV serotypes, including by non-limiting example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9 and AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with the present invention. In some embodiments, AAV serotypes that are capable of crossing the blood brain barrier are used. Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for anti-tumor immunization with intramuscular injection (Sterman et al. (1998)Hum. Gene Ther.7:1083-9). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al. (1996)Infection24(1):5-10; Sterman et al. (1998)Hum. Gene Ther.9(7):1083-1089; Welsh et al. (1995)Hum. Gene Ther.2:205-18; Alvarez et al. (1997)Hum. Gene Ther.5:597-613; Topf et al. (1998)Gene Ther.5:507-513; Sterman et al. (1998)Hum. Gene Ther.7:1083-1089. Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al. (1995)Proc. Natl. Acad. Sci. USA92:9747-9751, reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells. Gene therapy vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual subject (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a subject, usually after selection for cells which have incorporated the vector. Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing nucleases and/or donor constructs can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Vectors suitable for introduction of polynucleotides described herein include non-integrating lentivirus vectors (IDLV). See, for example, Ory et al. (1996)Proc. Natl. Acad. Sci. USA93:11382-11388; Dull et al. (1998)J. Virol.72:8463-8471; Zuffery et al. (1998)J. Virol.72:9873-9880; Follenzi et al. (2000)Nature Genetics25:217-222; U.S. Patent Publication No 2009/054985. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g.,Remington's Pharmaceutical Sciences,17th ed., 1989). It will be apparent that the nuclease-encoding sequences and donor constructs can be delivered using the same or different systems. For example, a donor polynucleotide can be carried by a plasmid, while the one or more nucleases can be carried by an AAV vector. Furthermore, the different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration and/or intramuscular injection. The vectors can be delivered simultaneously or in any sequential order. Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition. Applications The methods of this invention contemplate the treatment and/or prevention of MPS I disease (e.g. a lysosomal storage disease). Treatment can comprise insertion of one or more corrective disease-associated genes (e.g., IDUA, etc.) into a safe harbor locus (e.g. albumin) in a cell for expression of the needed enzyme(s) and release into the blood stream. Once in the bloodstream, the secreted enzyme may be taken up by cells in the tissues, wherein the enzyme is then taken up by the lysosomes such that the GAGs are broken down. The transgene may encode a protein comprising a codon optimized transgene (e.g., IDUA); and/or a transgene in which epitopes may be removed without functionally altering the protein. In some cases, the methods comprise insertion of an episome expressing the corrective enzyme-encoding transgene into a cell for expression of the needed enzyme and release into the blood stream. Insertion into a secretory cell, such as a liver cell for release of the product into the blood stream, is particularly useful. The methods and compositions of the invention also can be used in any circumstance wherein it is desired to supply an IDUA transgene encoding one or more therapeutics in a hematopoietic stem cell such that mature cells (e.g., RBCs) derived from these cells contain the therapeutic. These stem cells can be differentiated in vitro or in vivo and may be derived from a universal donor type of cell which can be used for all subjects. Additionally, the cells may contain a transmembrane protein to traffic the cells in the body. Treatment can also comprise use of subject cells containing the therapeutic transgene where the cells are developed ex vivo and then introduced back into the subject. For example, HSC containing a suitable IDUA encoding transgene may be inserted into a subject via an autologous bone marrow transplant. Alternatively, stem cells such as muscle stem cells or iPSC which have been edited using with the IDUA encoding transgene may be also injected into muscle tissue. Thus, this technology may be of use in a condition where a subject is deficient in some protein due to problems (e.g., problems in expression level or problems with the protein expressed as sub- or non-functioning). Particularly useful with this invention is the expression of transgenes to correct or restore functionality in subjects with MPS I disease. By way of non-limiting examples, production of the defective or missing proteins accomplished and used to treat MPS I disease. Nucleic acid donors encoding the proteins may be inserted into a safe harbor locus (e.g. albumin or HPRT) and expressed either using an exogenous promoter or using the promoter present at the safe harbor. Alternatively, donors can be used to correct the defective gene in situ. The desired IDUA encoding transgene may be inserted into a CD34+ stem cell and returned to a subject during a bone marrow transplant. Finally, the nucleic acid donor may be be inserted into a CD34+ stem cell at a beta globin locus such that the mature red blood cell derived from this cell has a high concentration of the biologic encoded by the nucleic acid donor. The biologic-containing RBC can then be targeted to the correct tissue via transmembrane proteins (e.g. receptor or antibody). Additionally, the RBCs may be sensitized ex vivo via electrosensitization to make them more susceptible to disruption following exposure to an energy source (see International Patent Publication No. WO 2002/007752). In some applications, an endogenous gene may be knocked out by use of the methods and compositions of the invention. Examples of this aspect include knocking out an aberrant gene regulator or an aberrant disease associated gene. In some applications, an aberrant endogenous gene may be replaced, either functionally or in situ, with a wild type version of the gene. The inserted gene may also be altered to improve the expression and/or functionality of the therapeutic IDUA protein or to reduce its immunogenicity. In some applications, the inserted IDUA encoding transgene is a fusion protein to increase its transport into a selected tissue such as the brain. In some applications, provided herein is a method of improving or maintaining (slowing the decline) of functional ability in a human subject having MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In other applications, provided herein is a method of decreasing the need (dose level or frequency) for ERT in a subject with MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In yet another aspect, provided herein is a method of delaying the need for ERT initiation in a subject with MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In one aspect, provided herein is a method to delay, reduce or eliminate the need for supportive surgery in a subject with MPS I, comprising treating the subject with the compositions of the invention, as compared to a subject that has not received the compositions. In another aspect, provided herein is a method of delaying, reducing or preventing the need for a bone marrow transplant in a subject with MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In yet another aspect, provided herein is a method of improving the functional (delaying decline, maintenance) ability in a subject with MPS I by treating the subject with a standard dosing regimen of ERT in combination with treatment with the compositions as described herein as compared with a subject that has not been treated with the methods and compositions of the invention. In another aspect, provided herein is a method of suppressing disability progression in a human subject having MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In yet another aspect, provided herein is a method of delaying, reducing or preventing the need for the use of a medical ventilator device in a subject with MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. In another aspect, provided herein is a method of delaying onset of confirmed disability progression or reducing the risk of confirmed disability progression in a human subject having MPS I as compared to a subject that that has not been treated with the methods and compositions of the invention. In one aspect of the invention, provided herein is a method of reducing, stabilizing or maintaining urine GAGs in a subject with MPS I, comprising treating the subject with the composition of the invention. In yet another aspect, provided herein is a method of extending life expectancy in a subject with MPS I as compared with a subject that has not been treated with the methods and compositions of the invention. The following Examples relate to exemplary embodiments of the present disclosure in which the nuclease comprises a zinc finger nuclease (ZFN) or TALEN. It will be appreciated that this is for purposes of exemplification only and that other nucleases or nuclease systems can be used, for instance homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring of engineered homing endonucleases (meganucleases) DNA-binding domains and heterologous cleavage domains and/or a CRISPR/Cas system comprising an engineered single guide RNA. EXAMPLES Example 1 The preparation of polynucleotides and AAV vector comprising the polynucleotides is as follows: The AAV2/6 vector encoding the SB-47171 ZFN (left ZFN) comprises several structural features: the 5′ and 3′ ITRs of the AAV vector, the ApoE/hAAT hepatic control region and α1-anti-trypsin promoter, the human β-globin-IgG chimeric intron, the nuclear localization sequence, the ZFP 47171 ZFN binding domain, the FokI ELD nuclease domain, and a polyadenylation signal. The locations of the various elements are shown below in Table 1. TABLE 1Elements of SB-47171 AAV (SEQ ID NO: 9)SEQIDFeatureDescriptionPosition-annotationNOITR5′ inverted terminal repeat1-130- [plain text in1brackets]ApoE/ApoE Hepatic Control Region141-863-underlined2hAAT& α1-antitrypsin promoterChimericHuman β globin- IgG867-999-italics3Intronchimeric intronNLSNLS1016-1036-double4underline47171ZFP 47171 DNA-binding1055-1486-Bold5domainFokl-ELDFokl-ELD nuclease domain1493-2092- lower case6poly APolyadenylation signal2148-2370-7ITR3′ inverted terminal repeat2422-2529-8 The complete nucleotide sequence for the SB-47171 AAV2/6 vector is shown below. The specific annotations shown in Table 1 are indicated in the sequence text as shown in Table 1: (SEQ ID NO: 9)[CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG50GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG100GAGTGGCCAA CTCCATCACT AGGGGTTCCT]GCGGCCTAGTAGGCTCAGAG150GCACACAGGA GTTTCTGGGC TCACCCTGCC CCCTTCCAAC CCCTCAGTTC200CCATCCTCCA GCAGCTGTTT GTGTGCTGCC TCTGAAGTCC ACACTGAACA250AACTTCAGCC TACTCATGTC CCTAAAATGG GCAAACATTG CAAGCAGCAA300ACAGCAAACA CACAGCCCTC CCTGCCTGCT GACCTTGGAG CTGGGGCAGA350GGTCAGAGAC CTCTCTGGGC CCATGCCACC TCCAACATCC ACTCGACCCC400TTGGAATTTC GGTGGAGAGG AGCAGAGGTT GTCCTGGCGT GGTTTAGGTA450GTGTGAGAGG GGTACCCGGG GATCTTGCTA CCAGTGGAAC AGCCACTAAG500GATTCTGCAG TGAGAGCAGA GGGCCAGCTA AGTGGTACTC TCCCAGAGAC550TGTCTGACTC ACGCCACCCC CTCCACCTTG GACACAGGAC GCTGTGGTTT600CTGAGCCAGG TACAATGACT CCTTTCGGTA AGTGCAGTGG AAGCTGTACA650CTGCCCAGGC AAAGCGTCCG GGCAGCGTAG GCGGGCGACT CAGATCCCAG700CCAGTGGACT TAGCCCCTGT TTGCTCCTCC GATAACTGGG GTGACCTTGG750TTAATATTCA CCAGCAGCCT CCCCCGTTGC CCCTCTGGAT CCACTGCTTA800AATACGGACG AGGACAGGGC CCTGTCTCCT CAGCTTCAGG CACCACCACT850GACCTGGGAC AGTCAGGTAAGTATCAAGGT TACAAGACAG GTTTAAGGAG900ACCAATAGAA ACTGGGCTTG TCGAGACAGA GAAGACTCTT GCGTTTCTGA950TAGGCACCTA TTGGTCTTAC TGACATCCAC TTTGCCTTTC TCTCCACAGG1000CAATTCGCCA TGGCCCCCAA GAAGAAGAGG AAGGTGGGCA TCCACGGGGT1050ACCGGCCGCAATGGCAGAAC GGCCCTTCCA GTGCCGCATC TGCATGCGCA1100ACTTCAGCCA GTCGGGCAAC CTGTCCCGCC ACATCCGGAC TCATACCGGC1150GAAAAACCAT TCGCTTGTGA CATCTGCGGA AGAAAGTTTG CGCTGAAGCA1200GAACCTCTGC ATGCATACCA AGATTCACAC CGGAGAGAAG CCGTTTCAGT1250GTCGCATTTG CATGAGAAAG TTCGCCTGGG CCGATAACCT TCAGAATCAC1300ACCAAGATCC ACACCGGGGA AAAGCCGTTC CAGTGCCGGA TCTGCATGAG1350GAACTTCTCA ACGTCCGGAA ACCTGACCAG GCATATCCGG ACCCACACTG1400GGGAGAAGCC TTTCGCCTGC GACATTTGCG GTCGGAAGTT CGCCCGGCAA1450TCCCACTTGT GTCTCCACAC TAAGATCCAC CTGAGAGGAT CCcagctggt1500gaagagcgag ctggaggaga agaagtccga gctgcggcac aagctgaagt1550acgtgcccca cgagtacatc gagctgatcg agatcgccag gaacagcacc1600caggaccgca tcctggagat gaaggtgatg gagttcttca tgaaggtgta1650cggctacagg ggaaagcacc tgggcggaag cagaaagcct gacggcgcca1700tctatacagt gggcagcccc atcgattacg gcgtgatcgt ggacacaaag1750gcctacagcg gcggctacaa tctgcctatc ggccaggccg acgagatgga1800gagatacgtg gaggagaacc agacccggga taagcacctc aaccccaacg1850agtggtggaa ggtgtaccct agcagcgtga ccgagttcaa gttcctgttc1900gtgagcggcc acttcaaggg caactacaag gcccagctga ccaggctgaa1950ccacatcacc aactgcaatg gcgccgtgct gagcgtggag gagctgctga2000tcggcggcga gatgatcaaa gccggcaccc tgacactgga ggaggtgcgg2050cgcaagttca acaacggcga gatcaacttc agatcttgat aaCTCGAGTC2100215022002250230023502400245025002529 The AAV2/6 vector comprising SB-47898 similarly comprises several features, and these are shown below in Table 2. TABLE 2Elements of SB-47898 AAV (SEQ ID NO: 12)SEQIDFeatureDescriptionPosition- annotationNO:ITR5′ inverted terminal repeat1-130- [plain text1in brackets]ApoE/ApoE Hepatic Control Region141-863underlined2hAAT& α1-antitrypsin promoterChimericHuman β globin- IgG867-999italics3Intronchimeric intronNLSNLS1016-1036double4underline47898ZFP 47898 DNA-binding1055-1570Bold10domainFokl-KKRFokl-KKR nuclease domain1577-2170 lower case11poly APolyadenylation signal2226-24487ITR3′ inverted terminal repeat2500-26078 The complete nucleotide sequence for the SB-47898 AAV2/6 vector is shown below. The specific annotations shown in Table 2 are indicated in the sequence text as shown in Table 2. (SEQ ID NO: 12)[CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG50GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG100GAGTGGCCAA CTCCATCACT AGGGGTTCCT]GCGGCCTAGTAGGCTCAGAG150GCACACAGGA GTTTCTGGGC TCACCCTGCC CCCTTCCAAC CCCTCAGTTC200CCATCCTCCA GCAGCTGTTT GTGTGCTGCC TCTGAAGTCC ACACTGAACA250AACTTCAGCC TACTCATGTC CCTAAAATGG GCAAACATTG CAAGCAGCAA300ACAGCAAACA CACAGCCCTC CCTGCCTGCT GACCTTGGAG CTGGGGCAGA350GGTCAGAGAC CTCTCTGGGC CCATGCCACC TCCAACATCC ACTCGACCCC400TTGGAATTTC GGTGGAGAGG AGCAGAGGTT GTCCTGGCGT GGTTTAGGTA450GTGTGAGAGG GGTACCCGGG GATCTTGCTA CCAGTGGAAC AGCCACTAAG500GATTCTGCAG TGAGAGCAGA GGGCCAGCTA AGTGGTACTC TCCCAGAGAC550TGTCTGACTC ACGCCACCCC CTCCACCTTG GACACAGGAC GCTGTGGTTT600CTGAGCCAGG TACAATGACT CCTTTCGGTA AGTGCAGTGG AAGCTGTACA650CTGCCCAGGC AAAGCGTCCG GGCAGCGTAG GCGGGCGACT CAGATCCCAG700CCAGTGGACT TAGCCCCTGT TTGCTCCTCC GATAACTGGG GTGACCTTGG750TTAATATTCA CCAGCAGCCT CCCCCGTTGC CCCTCTGGAT CCACTGCTTA800AATACGGACG AGGACAGGGC CCTGTCTCCT CAGCTTCAGG CACCACCACT850GACCTGGGAC AGTCAGGTAA GTATCAAGGT TACAAGACAG GTTTAAGGAG900ACCAATAGAA ACTGGGCTTG TCGAGACAGA GAAGACTCTT GCGTTTCTGA950TAGGCACCTA TTGGTCTTAC TGACATCCAC TTTGCCTTTC TCTCCACAGG1000CAATTCGCCA TGGCCCCCAA GAAGAAGAGG AAGGTGGGCA TCCACGGGGT1050ACCGGCCGCA ATGGCAGAGA GGCCCTTTCA GTGCCGGATC TGCATGCGGA1100ACTTCTCCAC CCCACAACTT CTGGACCGAC ATATCCGCAC CCATACCGGG1150GAAAAGCCTT TCGCGTGCGA CATTTGCGGA CGGAAATTCG CGTTGAAGCA1200CAATCTCCTG ACCCACACTA AGATTCATAC TGGCGAAAAG CCGTTCCAGT1250GCCGCATCTG TATGAGGAAC TTCAGCGATC AGTCGAACCT GAACGCCCAC1300ATTCGGACTC ATACCGGAGA AAAGCCCTTT GCCTGCGATA TCTGCGGTCG1350CAAGTTCGCT AGGAACTTCT CACTGACCAT GCACACCAAA ATCCACACTG1400GAGAGCGGGG ATTCCAGTGT AGAATCTGTA TGCGCAACTT CTCCCTGCGG1450CACGACCTGG ACCGCCACAT CAGAACCCAC ACCGGGGAGA AGCCGTTCGC1500CTGCGACATC TGCGGCCGGA AGTTCGCCCA CCGGTCCAAC CTGAACAAGC1550ACACGAAGAT TCACCTCCGCGGATCCcagc tggtgaagag cgagctggag1600gagaagaagt ccgagctgcg gcacaagctg aagtacgtgc cccacgagta1650catcgagctg atcgagatcg ccaggaacag cacccaggac cgcatcctgg1700agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggaaag1750cacctgggcg gaagcagaaa gcctgacggc gccatctata cagtgggcag1800ccccatcgat tacggcgtga tcgtggacac aaaggcctac agcggcggct1850acaatctgcc tatcggccag gccgacgaga tgcagagata cgtgaaggag1900aaccagaccc ggaataagca catcaacccc aacgagtggt ggaaggtgta1950ccctagcagc gtgaccgagt tcaagttcct gttcgtgagc ggccacttca2000agggcaacta caaggcccag ctgaccaggc tgaaccgcaa aaccaactgc2050aatggcgccg tgctgagcgt ggaggagctg ctgatcggcg gcgagatgat2100caaagccggc accctgacac tggaggaggt gcggcgcaag ttcaacaacg2150gcgagatcaa cttctgataa CTCGAGTCTA GAGGATCTCG AGCCGAATTC2200225023002350240024502500255026002607 The AAV2/6 vector comprising SB-71557 similarly comprises several features, and these are shown below in Table 3. TABLE 3Elements of SB-71557 AAV (SEQ ID NO: 23)NucleotideSEQPosition-Feature/IDannotationDescriptionNO:Sequence1-1305′ ITR1CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG[plain textCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGin brackets]CGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT156-476ApoE15AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTT(Enhancer)CCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCunderlinedTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGG485-877hAAT17GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGA(Promoter)GCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCitalicsACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGT886-9335′ UTR18CTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGCABoldGAT943-1075Human β3GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAAglobin/IgGCTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCAchimericCCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGintron1089-115419GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG1161-1181Nuclear4CCCAAGAAGAAGAGGAAGGTClocalizationsignal1200-1631ZFP 7155720GCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCAGDNA-AACTTCAGTCAGTCCGGCAACCTGGCCCGCCACATCCGCACCCACbindingACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTdomainGCCCTGAAGCAGAACCTGTGTATGCATACCAAGATACACACGGGClower caseGAGAAGCCCTTCCAGTGTCGAATCTGCATGCAGAAGTTTGCCTGGCAGTCCAACCTGCAGAACCATACCAAGATACACACGGGCGAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTACCTCCGGCAACCTGACCCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCGCCGCTCCCACCTGACCTCCCATACCAAGATACACCTGCGG1638-2237FokI-ELD21CAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGnucleaseCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGdomainATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGN542DATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGC CCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGGAGAGATACGTGGAGGAGAACCAGACCCGGGATAAGCACCTCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCGACGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCTTGATAA2250-2841WPREmut622AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATT3′UTRCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTC TCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAACTGGATCCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCTCTCAATCCAGCGGACCTCCCTTCCCGAGGCCTTCTGCCGGTTCTGCGGCCTCTCCCGCGTCTTCGCTTTCGGCCTCCGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG2848-30707CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT3088-31953′ ITR8AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC[Bold textGCTCGCTCACTGAGGCCGCCCGGGCTTTGCCCGGGCGGCCTCAGTin brackets]GAGCGAGCGAGCGCGCAGSequence of 71557 AAV:(SEQ ID NO: 23)[CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG50GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG100GAGTGGCCAA CTCCATCACT AGGGGTTCCT] GCGGCCTAAG CTTGAGCTCT150TCGAAAGGCTCAGAGGCACA CAGGAGTTTC TGGGCTCACC CTGCCCCCTT200CCAACCCCTC AGTTCCCATC CTCCAGCAGC TGTTTGTGTG CTGCCTCTGA250AGTCCACACT GAACAAACTT CAGCCTACTC ATGTCCCTAA AATGGGCAAA300CATTGCAAGC AGCAAACAGC AAACACACAG CCCTCCCTGC CTGCTGACCT350TGGAGCTGGG GCAGAGGTCA GAGACCTCTC TGGGCCCATG CCACCTCCAA400CATCCACTCG ACCCCTTGGA ATTTCGGTGG AGAGGAGCAG AGGTTGTCCT450GGCGTGGTTT AGGTAGTGTG AGAGGGGTCC CGGGGATCTT GCTACCAGTG500GAACAGCCAC TAAGGATTCT GCAGTGAGAG CAGAGGGCCA GCTAAGTGGT550ACTCTCCCAG AGACTGTCTG ACTCACGCCA CCCCCTCCAC CTTGGACACA600GGACGCTGTG GTTTCTGAGC CAGGTACAAT GACTCCTTTC GGTAAGTGCA650GTGGAAGCTG TACACTGCCC AGGCAAAGCG TCCGGGCAGC GTAGGCGGGC700GACTCAGATC CCAGCCAGTG GACTTAGCCC CTGTTTGCTC CTCCGATAAC750TGGGGTGACC TTGGTTAATA TTCACCAGCA GCCTCCCCCG TTGCCCCTCT800GGATCCACTG CTTAAATACG GACGAGGACA GGGCCCTGTC TCCTCAGCTT850CAGGCACCAC CACTGACCTG GGACAGTCCT AGGTGCTTGT TCTTTTTGCA900GAAGCTCAGA ATAAACGCTC AACTTTGGCA GATACTAGTC AGGTAAGTAT950CAAGGTTACA AGACAGGTTT AAGGAGACCA ATAGAAACTG GGCTTGTCGA1000GACAGAGAAG ACTCTTGCGT TTCTGATAGG CACCTATTGG TCTTACTGAC1050110011501200ccgctatggc tgagaggccc ttccagtgtc gaatctgcat gcagaacttc1250agtcagtccg gcaacctggc ccgccacatc cgcacccaca ccggcgagaa1300gccttttgcc tgtgacattt gtgggaggaa atttgccctg aagcagaacc1350tgtgtatgca taccaagata cacacgggcg agaagccctt ccagtgtcga1400atctgcatgc agaagtttgc ctggcagtcc aacctgcaga accataccaa1450gatacacacg ggcgagaagc ccttccagtg tcgaatctgc atgcgtaact1500tcagtacctc cggcaacctg acccgccaca tccgcaccca caccggcgag1550aagccttttg cctgtgacat ttgtgggagg aaatttgccc gccgctccca16001650170017501800185019001950200020502100215022002250230023502400245025002550260026502700275028002850TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC290029503000TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT30503100GATGGAGTTG GCCACTCCCT CTCTGCGCGC TCGCTCGCTC ACTGAGGCCG3150CCCGGGCTTT GCCCGGGCGG CCTCAGTGAGCGAGCGAGCG CGCAG3195 The AAV2/6 vector comprising SB-71728 similarly comprises several features, and these are shown below in Table 4. TABLE 4Elements of SB-71728 AAV (SEQ ID NO: 26)NucleotideSEQPosition-Feature/IDannotationDescriptionNO:Sequence1-1305′ ITR1CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG[plain textCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGin brackets]CGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT156-476ApoE15AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTT(Enhancer)CCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCunderlinedTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGG485-877hAAT17GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGA(Promoter)GCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCitalicsACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGT886-9335′ UTR18CTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGCABoldGAT943-1075Human β3GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAAglobin/IgGCTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCAchimericCCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGintron1089-115419GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGAT TACAAGGATGACGATGACAAG1161-1181Nuclear4CCCAAGAAGAAGAGGAAGGTClocalizationsignal1200-1715ZFP 7172824GCCGCTATGGCTGAGAGGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTDNA-CAGTCAGTCCTCCGACCTGTCCCGCCACATCCGCACCCACACCGGCGAGAbindingAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCTGAAGCACAACdomainCTGCTGACCCATACCAAGATACACACGGGCGAGAAGCCCTTCCAGTGTCGlower caseAATCTGCATGCAGAACTTCAGTGACCAGTCCAACCTGCGCGCCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCGCAACTTCTCCCTGACCATGCATACCAAGATACACACCGGAGAGCGCGGCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCTGCGCCACGACCTGGAGCGCCACATCCGCACCCACACCGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAATTTGCCCACCGCTCCAACCTGAACAAGCATACCAAGATACACCTGCGG1722-2315FokI-KKR25CAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAnucleaseGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAdomainACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGA CGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGG ACACAAAGGCCTACAGCGGCGGCTACAATCTGAGCATCGGCCAGGCCGACGAGATGCAGAGATACGTGAAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCGCAAAACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCTGATAA2328-2919WPREmut622AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATT3′UTRCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTC TCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAACTGGATCCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCTCTCAATCCAGCGGACCTCCCTTCCCGAGGCCTTCTGCCGGTTCTGCGGCCTCTCCCGCGTCTTCGCTTTCGGCCTCCGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG2926-31487CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT3166-32733′ ITR8AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC[Bold textGCTCGCTCACTGAGGCCGCCCGGGCTTTGCCCGGGCGGCCTCAGTin brackets]GAGCGAGCGAGCGCGCAGComplete Sequence of 71728 AAV:(SEQ ID NO: 26)[CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG50GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG100GAGTGGCCAA CTCCATCACT AGGGGTTCCT] GCGGCCTAAG CTTGAGCTCT150TCGAAAGGCTCAGAGGCACA CAGGAGTTTC TGGGCTCACC CTGCCCCCTT200CCAACCCCTC AGTTCCCATC CTCCAGCAGC TGTTTGTGTG CTGCCTCTGA250AGTCCACACT GAACAAACTT CAGCCTACTC ATGTCCCTAA AATGGGCAAA300CATTGCAAGC AGCAAACAGC AAACACACAG CCCTCCCTGC CTGCTGACCT350TGGAGCTGGG GCAGAGGTCA GAGACCTCTC TGGGCCCATG CCACCTCCAA400CATCCACTCG ACCCCTTGGA ATTTCGGTGG AGAGGAGCAG AGGTTGTCCT450GGCGTGGTTT AGGTAGTGTGAGAGGGGTCC CGGGGATCTT GCTACCAGTG500GAACAGCCAC TAAGGATTCT GCAGTGAGAG CAGAGGGCCA GCTAAGTGGT550ACTCTCCCAG AGACTGTCTG ACTCACGCCA CCCCCTCCAC CTTGGACACA600GGACGCTGTG GTTTCTGAGC CAGGTACAAT GACTCCTTTC GGTAAGTGCA650GTGGAAGCTG TACACTGCCC AGGCAAAGCG TCCGGGCAGC GTAGGCGGGC700GACTCAGATC CCAGCCAGTG GACTTAGCCC CTGTTTGCTC CTCCGATAAC750TGGGGTGACC TTGGTTAATA TTCACCAGCA GCCTCCCCCG TTGCCCCTCT800GGATCCACTG CTTAAATACG GACGAGGACA GGGCCCTGTC TCCTCAGCTT850CAGGCACCAC CACTGACCTG GGACAGTCCT AGGTGCTTGT TCTTTTTGCA900GAAGCTCAGA ATAAACGCTC AACTTTGGCA GATACTAGTC AGGTAAGTAT950CAAGGTTACA AGACAGGTTT AAGGAGACCA ATAGAAACTG GGCTTGTCGA1000GACAGAGAAG ACTCTTGCGT TTCTGATAGG CACCTATTGG TCTTACTGAC1050110011501200ccgctatggc tgagaggccc ttccagtgtc gaatctgcat gcgtaacttc1250agtcagtcct ccgacctgtc ccgccacatc cgcacccaca ccggcgagaa1300gccttttgcc tgtgacattt gtgggaggaa atttgccctg aagcacaacc1350tgctgaccca taccaagata cacacgggcg agaagccctt ccagtgtcga1400atctgcatgc agaacttcag tgaccagtcc aacctgcgcg cccacatccg1450cacccacacc ggcgagaagc cttttgcctg tgacatttgt gggaggaaat1500ttgcccgcaa cttctccctg accatgcata ccaagataca caccggagag1550cgcggcttcc agtgtcgaat ctgcatgcgt aacttcagtc tgcgccacga1600cctggagcgc cacatccgca cccacaccgg cgagaagcct tttgcctgtg1650acatttgtgg gaggaaattt gcccaccgct ccaacctgaa caagcatacc1700175018001850190019502000205021002150220022502300235024002450250025502600265027002750280028502900295030003050AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA3100GGCCGCGTCG AGCGC[AGGAA CCCCTAGTGA TGGAGTTGGC CACTCCCTCT3200CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCTTTGC CCGGGCGGCC3250TCAGTGAGCG AGCGAGCGCG CAG] The AAV2/6 vector encoding the SB-IDUA transgene donor comprises several structural features: the 5′ and 3′ ITRs of the AAV vector, left and right homology arms (LA and RA) that have homology to the regions flanking the targeted cleavage site in the albumin gene, a splice acceptor derived from the human Factor IX exon 2 splice acceptor to ensure efficient joining of the transgene sequence to the albumin promoter, a codon optimized hIDUA cDNA sequence, and a polyadenylation signal sequence. The locations of the various elements are shown below in Table 5. TABLE 5Elements of IDUA AAV (SEQ ID NO: 28)FeatureSEQPositionDescriptionID NOSequence1-1305′ ITR1CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG[plain text inGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGbrackets]GAGTGGCCAACTCCATCACTAGGGGTTCCT271-550LA: Left13TTTATTCTATTTTCCCAGTAAAATAAAGTTTTAGTAAACTCTGCATCTTThomologyAAAGAATTATTTTGGCATTTATTTCTAAAATGGCATAGTATTTTGTATTTarmGTGAAGTCTTACAAGGTTATCTTATTAATAAAATTCAAACATCCTAGGTAitalicsAAAAAAAAAAAAGGTCAGAATTGTTTAGTGACTGTAATTTTCTTTTGCGCACTAAGGAAAGTGCAAAGTAACTTAGAGTGACTGAAACTTCACAGAATAGGGTTGAAGATTGAATTCATAACTATCCCAA557-584SA: Splice14ACTAAAGAATTATTCTTTTACATTTCAGacceptorBold587-2458hIDUA,27CACTTGGTCCACGTCGACGCTGCCAGAGCCCTGTGGCCGCTTCGAAGATTcodonTTGGAGGTCAACGGGTTTCTGTCCTCCCCTTCCCCACTCGCAAGCAGATCoptimizedAGTATGTACTGTCATGGGATCAACAGCTTAACCTCGCCTATGTCGGAGCAunderlinedGTGCCTCACCGCGGGATCAAGCAAGTAAGGACACATTGGCTCCTTGAACTCGTCACCACGAGAGGATCGACGGGAAGGGGGCTTTCGTACAACTTCACTCATCTCGATGGCTATTTGGATCTCCTCCGCGAGAATCAGTTGTTGCCAGGCTTCGAATTGATGGGATCGGCGAGCGGGCACTTTACAGACTTCGAGGACAAGCAGCAAGTGTTTGAGTGGAAGGACCTCGTGTCGTCGCTCGCGAGGAGATACATTGGTCGCTACGGTTTGGCGCATGTGTCAAAGTGGAACTTCGAAACGTGGAACGAGCCCGATCATCACGATTTTGACAACGTGTCAATGACCATGCAGGGTTTCCTTAACTATTACGACGCCTGTTCCGAGGGATTGAGGGCAGCATCACCGGCGCTTCGGCTGGGAGGGCCTGGTGATAGCTTTCATACACCACCTCGATCGCCACTTTCGTGGGGGCTGCTGCGCCATTGTCACGATGGTACGAACTTCTTCACCGGGGAAGCGGGGGTACGGCTTGATTACATCAGCCTCCACCGAAAGGGAGCGCGGTCAAGCATCTCGATTCTGGAGCAGGAGAAGGTAGTCGCTCAGCAGATCCGGCAACTCTTTCCCAAGTTCGCAGACACACCTATCTACAATGATGAGGCAGACCCACTTGTGGGATGGTCCCTTCCGCAGCCATGGCGCGCAGATGTGACTTATGCCGCGATGGTAGTGAAAGTCATCGCCCAGCACCAGAATCTGCTTCTTGCGAATACGACCAGCGCGTTTCCTTACGCGCTTTTGTCGAACGATAATGCCTTCCTGTCATATCACCCCCATCCGTTTGCGCAGAGGACTCTTACGGCGCGATTCCAAGTGAATAACACCAGACCGCCGCACGTGCAGCTGTTGCGAAAACCCGTGTTGACTGCGATGGGGCTTCTGGCGTTGCTTGATGAGGAACAACTCTGGGCTGAAGTGTCCCAGGCGGGGACAGTACTTGATAGCAATCATACAGTAGGCGTGTTGGCGTCGGCGCACCGACCGCAGGGACCCGCGGATGCTTGGAGGGCAGCGGTCCTGATCTACGCCTCGGACGATACTAGGGCACATCCCAACAGATCGGTCGCTGTCACCCTTCGCCTCAGAGGGGTCCCGCCTGGTCCCGGCTTGGTATACGTCACTAGATATCTCGACAATGGACTGTGCAGCCCCGACGGAGAGTGGCGGAGGCTGGGACGGCCGGTGTTTCCGACAGCCGAGCAGTTTAGACGGATGAGGGCCGCTGAGGACCCCGTGGCAGCGGCACCGAGGCCCCTCCCGGCAGGAGGTCGCCTCACTCTTCGACCGGCACTGCGGCTGCCGTCCCTTCTGCTCGTACACGTCTGCGCGCGACCCGAAAAGCCGCCTGGACAGGTAACCAGGCTCAGGGCGCTCCCCTTGACGCAGGGGCAGTTGGTACTTGTCTGGTCGGACGAACACGTGGGGTCCAAATGCTTGTGGACGTATGAAATTCAGTTTTCCCAAGACGGGAAAGCGTACACTCCGGTGTCGCGCAAACCCTCCACGTTCAACCTCTTCGTCTTTTCCCCAGACACGGGAGCCGTATCAGGGTCGTACCGAGTCAGAGCCCTCGATTATTGGGCGAGGCCTGGGCCGTTCTCGGACCCTGTACCATACTTGGAAGTGCCGGTGCCCAGGGGACCGCCCTCGCCTGGTAATCCT2471-2695poly A7CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTlowercaseTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG2702-2801RA: Right16CTATCCATTGCACTATGCTTTATTTAAAAACCACAAAACCTGTGCTGTTGhomologyATCTCATAAATAGAACTTGTATTTATATTTATTTTCATTTTAGTCTGTCTarmDoubleunderlined2948-30553′ ITR8AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG[BoldCTCACTGAGGCCGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAbracketed]GCGCGCAGComplete Sequence of IDUA AAV:(SEQ ID NO: 28)[CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG50GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG100GAGTGGCCAA CTCCATCACT AGGGGTTCCT] GCGGCCTAAG CTTGAGCGGA150GTTCCAATTG TACTGTACAG AACCATGGTC ACATGTTTAA CGCTAGCGTG200CCGACCTGGT AAACTGATCA GTGGGTGCAC TTAGGACTGC GTCTTACGCT250AATCACATGC GTGCGGCCGCTTTATTCTAT TTTCCCAGTA AAATAAAGTT300TTAGTAAACT CTGCATCTTT AAAGAATTAT TTTGGCATTT ATTTCTAAAA350TGGCATAGTA TTTTGTATTT GTGAAGTCTT ACAAGGTTAT CTTATTAATA400AAATTCAAAC ATCCTAGGTA AAAAAAAAAA AAGGTCAGAA TTGTTTAGTG450ACTGTAATTT TCTTTTGCGC ACTAAGGAAA GTGCAAAGTA ACTTAGAGTG500ACTGAAACTT CACAGAATAG GGTTGAAGAT TGAATTCATA ACTATCCCAA550GGTACCACTA AAGAATTATT CTTTTACATT TCAGCGCACT TGGTCCACGT600CGACGCTGCC AGAGCCCTGT GGCCGCTTCG AAGATTTTGG AGGTCAACGG650GTTTCTGTCC TCCCCTTCCC CACTCGCAAG CAGATCAGTA TGTACTGTCA700TGGGATCAAC AGCTTAACCT CGCCTATGTC GGAGCAGTGC CTCACCGCGG750GATCAAGCAA GTAAGGACAC ATTGGCTCCT TGAACTCGTC ACCACGAGAG800GATCGACGGG AAGGGGGCTT TCGTACAACT TCACTCATCT CGATGGCTAT850TTGGATCTCC TCCGCGAGAA TCAGTTGTTG CCAGGCTTCG AATTGATGGG900ATCGGCGAGC GGGCACTTTA CAGACTTCGA GGACAAGCAG CAAGTGTTTG950AGTGGAAGGA CCTCGTGTCG TCGCTCGCGA GGAGATACAT TGGTCGCTAC1000GGTTTGGCGC ATGTGTCAAA GTGGAACTTC GAAACGTGGA ACGAGCCCGA1050TCATCACGAT TTTGACAACG TGTCAATGAC CATGCAGGGT TTCCTTAACT1100ATTACGACGC CTGTTCCGAG GGATTGAGGG CAGCATCACC GGCGCTTCGG1150CTGGGAGGGC CTGGTGATAG CTTTCATACA CCACCTCGAT CGCCACTTTC1200GTGGGGGCTG CTGCGCCATT GTCACGATGG TACGAACTTC TTCACCGGGG1250AAGCGGGGGT ACGGCTTGAT TACATCAGCC TCCACCGAAA GGGAGCGCGG1300TCAAGCATCT CGATTCTGGA GCAGGAGAAG GTAGTCGCTC AGCAGATCCG1350GCAACTCTTT CCCAAGTTCG CAGACACACC TATCTACAAT GATGAGGCAG1400ACCCACTTGT GGGATGGTCC CTTCCGCAGC CATGGCGCGC AGATGTGACT1450TATGCCGCGA TGGTAGTGAA AGTCATCGCC CAGCACCAGA ATCTGCTTCT1500TGCGAATACG ACCAGCGCGT TTCCTTACGC GCTTTTGTCG AACGATAATG1550CCTTCCTGTC ATATCACCCC CATCCGTTTG CGCAGAGGAC TCTTACGGCG1600CGATTCCAAG TGAATAACAC CAGACCGCCG CACGTGCAGC TGTTGCGAAA1650ACCCGTGTTG ACTGCGATGG GGCTTCTGGC GTTGCTTGAT GAGGAACAAC1700TCTGGGCTGA AGTGTCCCAG GCGGGGACAG TACTTGATAG CAATCATACA1750GTAGGCGTGT TGGCGTCGGC GCACCGACCG CAGGGACCCG CGGATGCTTG1800GAGGGCAGCG GTCCTGATCT ACGCCTCGGA CGATACTAGG GCACATCCCA1850ACAGATCGGT CGCTGTCACC CTTCGCCTCA GAGGGGTCCC GCCTGGTCCC1900GGCTTGGTAT ACGTCACTAG ATATCTCGAC AATGGACTGT GCAGCCCCGA1950CGGAGAGTGG CGGAGGCTGG GACGGCCGGT GTTTCCGACA GCCGAGCAGT2000TTAGACGGAT GAGGGCCGCT GAGGACCCCG TGGCAGCGGC ACCGAGGCCC2050CTCCCGGCAG GAGGTCGCCT CACTCTTCGA CCGGCACTGC GGCTGCCGTC2100CCTTCTGCTC GTACACGTCT GCGCGCGACC CGAAAAGCCG CCTGGACAGG2150TAACCAGGCT CAGGGCGCTC CCCTTGACGC AGGGGCAGTT GGTACTTGTC2200TGGTCGGACG AACACGTGGG GTCCAAATGC TTGTGGACGT ATGAAATTCA2250GTTTTCCCAA GACGGGAAAG CGTACACTCC GGTGTCGCGC AAACCCTCCA2300CGTTCAACCT CTTCGTCTTT TCCCCAGACA CGGGAGCCGT ATCAGGGTCG2350TACCGAGTCA GAGCCCTCGA TTATTGGGCG AGGCCTGGGC CGTTCTCGGA2400CCCTGTACCA TACTTGGAAG TGCCGGTGCC CAGGGGACCG CCCTCGCCTG2450GTAATCCTTG ATAAAGATCT ctgtgccttc tagttgccag ccatctgttg2500tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact2550gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg2600tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt2650gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggACCGG27002750GATCTCATAA ATAGAACTTG TATTTATATT TATTTTCATT TTAGTCTGTC28002850GCTTAGCAAA CGCGTCTCCA ACGTTTCGCC GTTAACACCC CACATAGTGA2900GTGGTCTTAG TAGTCCGGGT GTTTAAACTG AAAGATAACT CGAGCGC[AGG2950AACCCCTAGT GATGGAGTTG GCCACTCCCT CTCTGCGCGC TCGCTCGCTC3000ACTGAGGCCG CCCGGGCTTT GCCCGGGCGG CCTCAGTGAG CGAGCGAGCG3050CGCAG]3055 Example 2 Compositions comprising the polynucleotides and AAVs as described in Example 1 were prepared as follows: The components were supplied in three capped vials: one for ZFN1 (SB-47171, white capped and labeled SB-A6P-ZLEFT or SB-71557, labeled as SB-A6P-ZL2); ZFN2 (SB47898, blue capped and labeled SB-A6P-ZRIGHT or SB-71728, labeled as SB-A6P-ZR2); and hIDUA Donor (hIDUA, orange capped and labeled SB-A6P-HRL). The product components were all purified AAV individually formulated in phosphate buffered saline (PBS) containing CaCl2, MgCl2, NaCl, sucrose and Kolliphor® (Poloxamer) P188 or in a Normal Saline (NS) formulation. Dose calculations were performed using the subject's weight and rounded to two decimal points. The calculations were done by multiplying the cohort dose by the subject weight at baseline, and then dividing by the vg/mL concentration. The three product component volumes were added together and the total volume determined. In addition, the volume of human serum albumin (HSA) intravenous solution for addition was calculated to achieve a final concentration of 0.25% HSA and finally the PBS or NS was added the required amount to achieve the correct component concentration. The product components were then added to an IV infusion bag containing 0.25% HSA in NS or PBS. Each product component was added separately and then the bag was mixed gently and transferred to the person responsible for infusion. The product was then infused into subjects at a rate of 100 mL/hour using an infusion pump (Sigma Spectrum). Example 3 Study Eligibility and Exclusion Criteria Key eligibility criteria for subjects in the study included: male or female ≥18 years of age; clinical diagnosis of attenuated MPS I (MPS HIS, MPS IS, MPS IH post-HSCT); IDUA deficiency confirmed by gene sequencing; Magnetic resonance imaging (MRI) negative for liver mass. Key exclusion criteria for subjects in the study included: known unresponsiveness to enzyme replacement therapy; neutralizing antibodies in the serum to AAV2/6; serious intercurrent illness or clinically significant organic disease (unless secondary to MPS I) such as cardiovascular, hepatic, pulmonary, neurologic, or renal disease. Receiving anti-retroviral therapy for hepatitis B or C, or active hepatitis B or hepatitis C or human immunodeficiency virus (HIV) 1/2; lack of tolerance to laronidase treatment with significant infusion-associated reactions (IARs) or occurrence of anaphylaxis; polymorphisms in the ZFN targeted region in the albumin locus; liver fibrosis score of 3 or 4 on a 0 to 4 point scale (Desmet et al. (1994)Hepatology19(6):1513-20) if subject has had a liver biopsy within 2 years of screening, markers of hepatic dysfunction; creatinine≥1.5 mg/dL; pregnant or breastfeeding female; contraindication to the use of corticosteroids; current treatment with systemic (iv or oral) immunomodulatory agent or steroid use; history of active malignancy in past 5 years; participation in prior investigational drug or medical device study within the previous 3 months; prior treatment with a gene therapy product; and elevated or abnormal α-fetoprotein. Study Design The study was performed on subjects with MPS I disease. The doses used in the cohorts are shown below in Table 6. Cohort 1 is considered the low dose, cohort 2 is the mid dose, and cohort 3 is the high dose. For all cohorts, total AAV dose includes 2 ZFN vectors and 1 donor vector in a fixed ratio of 1:1:8. TABLE 6Evaluation dosesZFN 1ZFN 2(SB-41717(SB-47898hIDUA donoror 71557)or 71728)(SB-IDUA)Total rAAVDoseCohortSubjectsvg/kgvg/kgvg/kgvg/kgDescription121.00e+121.00e+128.00e+121.00e+13Startingdose225.00e+125.00e+124.00e+135.00e+135x startingdose35To be determinedMaximallytolerateddose Subjects who received ERT prior to enrollment continued to receive ERT during the study and remain on their current schedule per standard of care; however, ERT was omitted during the week of infusion to facilitate accurate baseline testing (e.g., of urine GAG levels, and leukocyte and plasma IDUA activity) at ERT trough levels and to allow a week free of ERT after the infusion. To minimize the potential immune response to the AAV capsid protein, the engineered ZFNs, or the endogenous hIDUA, and to preserve hepatic function, prednisone or equivalent corticosteroid was administered prophylactically starting 2 days prior to infusion, and was tapered over a period of approximately 20 weeks. Clinical Endpoints Primary endpoint: The primary endpoint of this study were the safety and tolerability of the composition as assessed by incidence of adverse events and significant adverse events. Additional safety evaluations included: routine hematology, chemistry, and liver function laboratory tests, vital signs, physical exam, ECG, ECHO, and concomitant medications; cranial nerve exam and muscle strength testing; serial α-fetoprotein testing and MM of liver to evaluate for liver mass. Safety assessment was performed on all subjects. All reported adverse events were coded to a standard set of terms using the Medical Dictionary for Regulatory Activities (MedDRA) AE dictionary. The frequency of each event was summarized by severity and by relatedness to the study drug material. Key secondary endpoints included: change from baseline in: IDUA activity measured in plasma and leukocytes, total GAG, DS GAG, and HS GAG levels (expressed as a ratio to creatinine) measured in urine; AAV2/6 clearance measured by vector genomes in plasma, saliva, urine, stool, and semen by PCR. Urine GAG levels are a key biomarker of MPS I disease pathophysiology. Key exploratory endpoints included a change from baseline in: percentage and durability of gene modification at the albumin locus in liver tissue obtained at biopsy; imaging, functional and neurocognitive testing related to MPS I; liver and cerebrospinal fluid (CSF) GAG levels and any immune response to AAV2/6 and/or ZFNs. From consenting subjects, additional samples may be collected for future research objectives. Such future research objectives may include analysis of plasma markers of severity of disease, response to therapy (e.g., cytokines, soluble cell surface proteins, soluble receptors), and functional improvements (e.g., neurological function, musculoskeletal function), as well as determination of AAV virus inhibition, function, immunogenicity, or pharmacodynamics (e.g., antibodies, soluble receptors, AAV viral receptor inhibitors, cytokines, co-existing alternate serotype antibodies). Statistical Analysis and Data Analysis This was an exploratory Phase I study and thus there will be limited statistical power to evaluate efficacy and related biological endpoints. Therefore, analyses were primarily descriptive and exploratory in nature. This study will enroll 9 subjects (2 subjects in each of 2 cohorts, with potential enrollment of 5 additional subjects at the maximal tolerated dose). The selection of 2 subjects per cohort was not based on statistical calculations since this is a Phase I safety study to evaluate safety and tolerability. All tables, listings, and data summaries were performed in SAS version 9.2 or later. Patients The patient demographics are shown below in Table 7. Table 8 lists the exposure to treatment that each subject had at 32 weeks post trial initiation. TABLE 7Patient DemographicsSubject CharacteristicsOverall (N = 3)Age (Years)number of patients3Min-Max23.00,37.00Mean (SD)29.00(7.21)Median27.00Sex, n (%)Male1(33.3)Female2(66.7)Race, n (%)Asian2(66.7)White1(33.3) TABLE 8Treatment exposure (approximate)SubjectDose CohortFollow-Up (Weeks)1122229325 Observed Adverse Events All subjects reported treatment emergent adverse events (TEAEs), consistent with ongoing MPS I disease. Most were mild (grade1) and resolved without treatment. In general, the study drug was administered to three subjects with attenuated MPS I at a dose of up to 5e13 vg/kg and was generally well-tolerated. Study drug-related Adverse Events (AEs) were mild (Grade 1), and all were consistent with the ongoing MPS I disease. No SAEs were reported, and no AEs to the study drug were reported. No increase in liver function tests were reported. The AEs are shown below in Table 9. TABLE 9Study Drug-related Adverse EventsCohort 1Cohort 2Overall(N = 1)(N = 2)(N = 3)Preferred Termn [T]n [T]n[T]Any TEAE1 [2]2 [4]3 [6]1- Mild2- ModerateHeadache1[1]None1[1]Acne2[2]2[2]Upper respiratory1[1]None1[1]tract infectionMusculoskeletalNone1[1]1[1]stiffnessOropharyngeal painNone1[1]1[1] In Table 9, ‘N’ indicates the total number of subjects in each treatment group; ‘n’ indicates the number of subjects with an adverse event for each preferred term; and ‘[T]’ indicates the total number of adverse events. All subjects were tapered on prophylactic prednisone without the need for increased dosing. All subjects had normal AST and ALT readings throughout the period following treatment. Preliminary Plasma IDUA Measurements Plasma IDUA activity was measured at trough, which was defined as in the period immediately prior to ERT dosing when possible, and no less than 96 hours after the subject's last ERT infusion. The activity of α-L-iduronidase was determined by methods known in the art (see Example 4). In this study at this initial time point, plasma IDUA activity was not significantly changed from pre-treatment values. Leukocyte IDUA Results, Cohorts 1 and 2 IDUA levels in the subject's leukocytes were analyzed using methods known in the art (see Example 4). Because the subjects were enrolled in the study at different points in time, there were different time periods of post-dosing results reported as shown in Table 8. The results demonstrated that the treated subjects had IDUA levels in their leukocytes above the normal range lower limit. Increases in leukocyte IDUA activity into the normal range were observed in all three threated subjects at both the 1e13 and 5e13 vg/kg doses. Comparison of the IDUA levels found prior to dosing (seeFIG.2, Study Day post dosing “0” indicates the day compositions disclosed herein were administered to each subject) demonstrates that each subject had an increase in leukocyte IDUA activity. Urine Glycosaminoglycan Levels Results Determinations were made of total urine GAG levels as well as levels of dermatan sulfate and heparan sulfate. Methods used were those known in the art (see Example 4). The results of the urine GAG analysis are shown inFIG.4. These results are a preliminary read at this early timepoint. As the subjects progress in the clinical study, further data points will be analyzed for loss of urine GAGs. Summary of Results for Subjects 1-3 Also known as Hurler syndrome, MPS I is a rare inherited metabolic disease caused by a deficiency of IDUA, an enzyme needed to break down GAGs in the lysosomes. Without IDUA, the toxic buildup of GAGs in the cells can result in tissue and organ damage, musculoskeletal problems and other symptoms. The current standard-of-care treatment for MPS I is enzyme replacement therapy (ERT), given as weekly intravenous infusions. For severe MPS I patients, bone marrow transplant is also a common treatment. The study described herein contained two dose cohorts. One patient was treated in the first cohort at a dose of 1e13 vg/kg, and 2 patients were treated in the second cohort at 5e13. Safety data collected from all three patients showed that the administration of the study drug was generally well-tolerated with a favorable safety profile. Eight total adverse events were reported, all were mild or moderate, consistent with ongoing MPS I disease and resolved without treatment. None of the reported adverse events were determined to be related to study drug treatment. No serious adverse events or SAEs were reported and no persistent transaminitis was observed. In MPS I, leukocyte IDUA activity is commonly used to estimate levels of IDUA enzyme in the tissues of bone marrow transplant patients, as increased IDUA activity in leukocytes is associated with improved clinical outcomes in a bone marrow transplant setting. The results indicate a dose-dependent increase in leukocyte IDUA activity, with activity levels rising above baseline and in the normal range (normal range is 6.0-71.4 nmol/hr/mg). Plasma IDUA activity was unchanged from baseline in all three patients. Plasma IDUA activity was unchanged from baseline in all three patients. This may be due to the contrary PK/PD properties of the study drug and genome editing therapy and MPS I disease biology. Baseline urine GAG measurements for the three patients were in a range considered to be at or slightly above normal. In this limited duration data set, urine GAG measurements showed no clear trend or meaningful change. Additional follow-up is needed to determine whether any meaningful change in urine GAGs emerges. However, the early observations of increased leukocyte IDUA activity, a target tissue, observed in treated subjects treated with compositions as described herein was encouraging. Additional studies are performed using the composition disclosed herein comprising AAV SB-71557 and AAV SB-71728 (in place of 47171 and 47898) and an AAV hIDUA Donor. In pre-clinical studies, AAV SB-71557 and AAV SB 71728 demonstrated improved cutting efficiency (5- to 30-fold) and improved expression (5- to 20-fold increase) of IDUA (see U.S. Provisional application 62/728,226), the enzyme deficient in patients with MPS I. Example 4 IDUA Enzyme Assay Exemplary laboratory procedures that may be utilized are conducted as follows. To detect IDUA enzyme activity, there are many assays that can be used. One exemplary assay is as follows: The activity of α-L-iduronidase was determined by a fluorometric assay using 4-methylumbelliferyl α-L-iduronide (Glycosynth) as the substrate according to the established assay condition (Whitley et al. (1987)Am J Med Genet28:233-243; Whitley (1986)Birth Defects Orig Artic Ser.22(1):7-24. The 4MU-iduronide substrate was diluted with sodium formate buffer, 0.4 M, pH 3.5 in the narrow, well-established optimal range of pH (Hopwood et al. (1979)Clin ChimActa. 92:257-265, Whitley (1986), ibid), and at selected substrate concentrations. Then, 25 μL aliquots of substrate were mixed with 25 μL of biological sample (e.g. plasma, leukocytes, tissue homogenates). The mixture was incubated at 37° C. for 30 min, and 200 μL glycine carbonate buffer (pH 10.4) was added to quench the reaction. α-L-iduronidase catalyzed the cleavage of the non-fluorescent substrate (4MU-iduronide) into a fluorescent product (4-MU). 4-Methylumbelliferone (4-MU, Sigma) was used to make the standard curve. The resulting fluorescence was measured using a Bio-Tek plate reader with excitation at 355 nm and emission at 460 nm. α-L-iduronidase enzyme activity was expressed in units (nmol converted to product per hour) per mg protein as determined with a Pierce protein assay kit (Fisher). All reactions were run in triplicate. Another exemplary fluorometric assay, using 4-methylumbelliferyl α-1-iduronide (4-MU, Glycosynth, Cheshire, UK or Sigma Aldrich, St. Louis MO) as the substrate for measuring IDUA activity in leukocytes (Isman et al. (2005)Clin Chem51(3)) is as follows: Blood is obtained from healthy adult donors with informed consent. Leukocytes are fractionated with Ficoll-Paque as follows: Blood (10 mL) is drawn into evacuated tubes (Vacutainer; Becton Dickinson) containing sodium heparin, transferred to a 40-mL plastic centrifuge tube, diluted with 20 mL of Hanks Balanced Salt Solution (HBSS), and gently mixed. The diluted blood is gently layered on 15 mL of Ficoll-Paque in a 20×150 mm centrifuge tube and centrifuged at 360 g for 50 min at room temperature; the supernatant is carefully aspirated and discarded. The mononuclear cells at the interface with the plasma are pipetted into a plastic centrifuge tube, washed with HBSS, and centrifuged twice at 170 g for 10 min. The mononuclear pellets are then rinsed with saline solution (9 g/L NaCl) to remove residual HBSS and used for the experiments (hereafter referred to as the mononuclear fraction). The mononuclear fraction contains 90-93% lymphocytes and 3-5% monocytes when evaluated by Wright staining. The granulocyte/erythrocyte fraction that is present at the bottom of the initial Ficoll-Paque separation is washed twice with isotonic saline, and the erythrocytes are subsequently removed by hypotonic lysis, giving a granulocyte fraction (hereafter referred to as granulocytes) consisting of 94-98% granulocytes. Cell pellets are stored at −20° C., and all enzyme assays are carried out within 1-5 days after isolation of the cells. α-Iduronidase activity (EC 3.2.1.76) is determined by the method of Rome et al. (1979)Proc Natl Acad Sci USA76:2331-2334). Fluorescence is measured for this and all other assays with 4-methylumbelliferone-based substrates with an excitation wavelength of 365 nm and an emission wavelength of 450 nm; the results are compared with a calibration curve prepared with 4-methylumbelliferone. Results are reported as the mean ratio (with 95% confidence intervals) of enzyme activity in matched samples. A second assay known in the art (Aronovich et al. (1986)Am. J. Hum. Genet.58:75-85) is as follows: Leukocytes were prepared by differential sedimentation on dextran followed by two cycles of hypotonic hemolysis (Lichtman 1990). For some individuals, a lymphoblastoid cell line (LCL) was prepared by transformation with Epstein-Barr virus. The activity of IDUA was measured using fluorogenic substrate 4-methylumbelliferone (MU) CC-L-iduronide (Calbiochem) and expressed as nmol MU/mg protein/h, or nmol MU/ml plasma/h, as described elsewhere (Whitley et al. (1987)Am J Med Genet28:233-243). It is notable that the assay was originally developed to optimize human leukocyte IDUA activity at 37° C. with respect to reaction pH (3.3) and substrate concentration (2.85 mM, >10-fold above the Km). Under these conditions, the reaction was found to be linear with respect to reaction time for −3 h, although reactions were either 30 min or 2 h in the current study. Protein concentration was measured with Coomasie blue (Bio-Rad). Plasma IDUA enzyme activity was according to a previously published method (Wasteson and Neufeld (1982)Meth Enzymol83:573-578; Clarke et al. (1990)Clin Genet37:355-362). One unit of enzyme activity was defined as the percent of3H substrate converted to product. Specific enzymatic activity was reported as U/mg protein/h. 4-MU iduronide is diluted with sodium formate buffer (0.4 M, pH 3.5). Then, 25 μL aliquots of substrate (360 μM) are mixed with 25 μL aliquots of tissue homogenates. The mixture is incubated at 37° C. for 30 min, and 200 μL glycine carbonate buffer (pH 10.4) is added to quench the reaction. IDUA catalyzes the cleavage of the non-fluorescent substrate (4-MU iduronide) into a fluorescent product (4-MU). 4-methylumbelliferone (Sigma-Aldrich, St. Louis, MO) is used to make the standard curve. The resulting fluorescence is measured using a microplate reader (BioTek, Winooski, VT) with excitation at 355 nm and emission at 460 nm. IDUA enzyme activity is expressed in units (nmol converted to product per hour) per mg protein as determined with a Pierce protein assay kit (Thermo Fisher Scientific, Waltham, MA). All reactions are run in triplicate (Ou et al. (2018)Mol Genet Metab123(2):105-111). Another exemplary assay to measure IDUA activity from tissues is as follows: After sacrifice using a ketamine/xylazine cocktail (10 μL/g), mice were perfused transcardially with 1×PBS. Samples from the brain, heart, kidney, liver, spleen and lungs were immediately harvested and flash-frozen for IDUA and GAG analysis. Harvested mouse tissues were placed in 1 mL PBS in an Eppendorf tube on ice and homogenized using a motorized pestle. Then 11 μL of 10% Triton X-100 in PBS was added and the homogenate kept on ice for 10 min. Protein concentration in the clarified supernatant was estimated by the Bradford colorimetric method. IDUA activity was assayed as follow: Briefly, 25 μl of a solution of 50 μM 4-methylumbelliferyl alphα-L-iduronide made in 0.4 M sodium formate buffer, pH 3.5, containing 0.2% Triton X-100 was added to 25 μl of tissue homogenate and incubated for 1 h at 37° C. in the dark. The reaction was quenched by adding 200 μl of 0.5 M NaOH/glycine buffer, pH 10.3. Tubes were centrifuged for 1 min at 13,000 rpm at 4° C., the supernatant transferred to a 96 wells plate, and fluorescence read at 365 nm excitation wavelength and 450 nm emission wavelength using a Spectra Max Gemini XS fluorometric plate reader (Molecular Devices, Sunnyvale, CA). Note: Sodium formate, formic acid, 4-methylumbelliferone, glycine, NaOH, Triton X-100 and sodium azide were obtained from Sigma (St. Louis, MO) and 4-methylumbelliferyl alphα-L-iduronide from Glycosynth (Warrington, Cheshire, UK). IDUA activity in the tissue samples was calculated as: Activity in ng/h=(flourometric reading of the tissue sample×A)−B, where A and B were the values obtained from the curve fit equation of the standard curve generated using pure end product (4-methylumbelliferone). Specific activity of IDUA was expressed as nmol/h/mg protein in each sample (Garcia-Rivera et al. (2007)Brain Res Bull.74(6): 429-438). Total Urine Glycosaminoglycans (GAGs) Assay and Quantitative Urine Heparan Sulfate, Dermatan Sulfate and Chondroitin Sulfate Assay by MS/MS. A variety of assays exist to measure the level of GAGs in the urine. One exemplary assay is described as follows: Urine samples are collected during the study are analyzed for glycosaminoglycan levels using a Dimethyl Methylene Blue (DMB) Assay. Briefly, urine samples are stained for heparan sulfate by treating the sample with 1,9-dimethylmethylene blue dye resuspended in formic acid at a pH of 3.3, and measured for absorbance at a wave length of 520 nm. The concentration of heparan sulfate was normalized using the total concentration of creatinine protein identified in the urine sample. (see e.g. de Jong et al. (1989)Clin Chem35(7):1472-1479). Another exemplary assay for measuring total GAG present in a biological sample is as follows: The method involves (a) combining a serine protease (e.g., of the clotting cascade), a labeled substrate for the serine protease, an inhibitor of the serine protease, and a sample suspected of comprising one or more glycosaminoglycans under conditions and for a time suitable for cleavage of the labeled substrate by the serine protease to produce a detectable signal, (b) detecting the detectable signal, and (c) comparing the amount of detectable signal with a standard to determine the concentration of said one or more glycosaminoglycans in said sample, wherein said inhibitor of said serine protease is selected from the group consisting of heparin cofactor II and antithrombin III, and wherein said one or more glycosaminoglycans are selected from the group consisting of dermatan sulfate (DS) and heparin sulfate (HS). (See e.g. U.S. Patent Publication No. 2013/0189718). Another exemplary assay measures the types of GAGs present and is termed a multiplex assay (Langereis et al. (2015)PLoS One10(9):e0138622). This assay is based on enzymatic digestion the of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) found in the urine, followed by quantification by LC-MS/MS. This assay is a very sensitive assay and can be used to measure the exact types of GAGs in the urine. Another exemplary assay that can be used to determine the concentration of specific types of GAGs utilizes a RapidFire (RF, Agilent) high-throughput mass spectrometry system. Samples are absorbed to a matrix to concentrate and desalt, and then eluted directly into the MS/MS without chromatographic separation. Each sample is processed in less than ten seconds, yielding much faster throughput than conventional LC-MS/MS based methods (see Tomatsu et al. (2014)J Anal Bioanal Tech. March 1; 2014(Suppl 2):006.) AAV2/6 Clearance in Plasma, Saliva, Urine, Stool and Semen Detection of AAV in biological samples can be done by several methods known in the art. An exemplary shedding assay is for analysis of AAV2/6-donor and AAV2/6-ZFN vectors in human plasma, semen, saliva, urine, and feces samples, and to evaluate the recovery rate of DNA from the five matrices. Human plasma, semen, saliva, urine, and feces samples from human donors provided the source of matrix DNA for qPCR analysis. DNA isolation from human Plasma: An aliquot (200 μL) of human plasma sample was thawed, treated with proteinase K in the presence of 2 μg of salmon sperm DNA, prior to DNA isolation using QIAamp DNA Mini kit. The purified plasma DNA was dissolved in 100 μL of elution buffer AE. DNA isolation from human semen: An aliquot (up to 100 μL) of human semen sample was thawed, treated with proteinase K, and then processed for DNA isolation using QIAamp DNA Mini kit. The purified semen DNA was dissolved in 100 μL of elution buffer AE and the DNA concentration was determined by UV absorption at 260 nm with Nanodrop ND-8000 instrument. DNA isolation from human saliva: An aliquot (up to 200 μL) of human saliva sample was thawed, treated with proteinase K, and then processed for DNA isolation using QIAamp DNA Mini kit. The purified saliva DNA was dissolved in 100 μL of elution buffer AE and the DNA concentration was determined by UV absorption at 260 nm with Nanodrop ND-8000 instrument. DNA isolation from human urine: An aliquot (up to 200 μL) of human saliva sample was thawed, treated with proteinase K, and then processed for DNA isolation using QIAamp DNA Mini kit. The purified saliva DNA was dissolved in 100 μL of elution buffer AE and the DNA concentration was determined by UV absorption at 260 nm with Nanodrop ND-8000 instrument. DNA isolation from human feces: An aliquot (90-110 mg) of human feces sample was partially thawed, homogenized, and treated with proteinase K prior to DNA isolation using QIAamp Fast DNA Stool Mini Kit. The purified feces DNA was dissolved in 200 μL of Buffer ATE and the DNA concentration was determined by UV absorption at 260 nm with Nanodrop ND-8000 instrument. Each qPCR was performed on a standard 96-well plate in a 7900HT Fast Real Time PCR system. The plate with reaction mix was sealed with optical caps and all droplets spun down by centrifugation at 1500 rpm for 15 min before qPCR. The reaction for the donor AAV (SB-IDUA, SB-A6P-HNT) amplified and detected a 91 nucleotide amplicon. The reaction for detection of the ZFN DNA (SB-47171: SB-A6P-ZLEFT or SB-71557 and SB-47898: SB-A6P-ZRIGHT or SB-71728) amplified and detected a 96 nucleotide amplicon. Assay conditions used: Held at 50° C. for 2 minutes. Held at 95° C. for 10 minutes. 40 cycles at 95° C. for 15 seconds, and at 60° C. for 1 minute. Results were compared with a previously prepared standard curve using linearized MPS I or ZFN plasmid DNA. Gene Modification at the Albumin Locus in Liver Tissue Detection of gene modification through sequencing or other means is well known in the art. An exemplary assay is to determine the levels of insertions and deletions (indels) at the albumin gene in subject samples using the MiSeq next generation sequencing (NGS) platform. gDNA was isolated from liver tissue using standard procedures and diluted to 20 ng/mL. Samples were subjected to an adaptor PCR followed by a barcode PCR and loaded onto MiSeq cartridge for sequencing. Following conditions are used for PCR reactions: PCR reaction (Adaptor): 95° C. 3 minutes, [98° C. 20 seconds, 55° C. 15 seconds, 72° C. 15 seconds], repeat bracketed steps 29 times. Final extension at 72° C. for 1 minute. PCR reaction (Barcode): 95° C. 3 minutes, [98° C. 20 seconds, 60° C. 15 seconds, 72° C. 15 seconds], repeat bracketed steps 9 times. Final extension at 72° C. for 1 minute. All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety. Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting. | 182,213 |
11857642 | DETAILED DESCRIPTION OF THE INVENTION The present inventors have combined epigenetics, bioinformatics and neuroscience to find promoters which, when in the eye, drive gene expression only in rod photoreceptors. The nucleic acid sequence of the sequence of the invention is: (SEQ ID NO: 1)ATTCCGGTCACACGGCCAAGATTATTCCACCTGCGCTTTGAGCAATAGGGAGAGGGCTCTGGTGCCTCTTCCTGGAATTTGATTAATTCGCTTGAGTCAGTCACAGAATTTGAGGAAGCATTGATATTTGAAGATGTGTTCTTCTAAAGGATACAAATGAATATATGCATAGTGAGAGTTTAGGAGATAGG. The present invention hence provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence of at least 150 bp having at least 70% identity to said nucleic acid sequence of SEQ ID NO:1, wherein said isolated nucleic acid molecule specifically leads to the expression in rod photoreceptors of a gene operatively linked to said nucleic acid sequence coding for said gene. In some embodiments, the nucleic acid sequence is at least 150 bp, has at least 80% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 85% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 90% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 95% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 96% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 97% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 98% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has at least 99% identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 150 bp, and has 100% identity to said nucleic acid sequence of SEQ ID NO:1. The isolated nucleic acid molecule of the invention can additionally comprise a minimal promoter, for instance a SV40 minimal promoter, e.g. the SV40 minimal promoter or the one used in the examples. Also provided is an isolated nucleic acid molecule comprising a sequence that hybridizes under stringent conditions to an isolated nucleic acid molecule of the invention as described above. The present invention also provides an expression cassette comprising an isolated nucleic acid of the invention as described above, wherein said promoter is operatively linked to at least a nucleic acid sequence encoding for a gene to be expressed specifically in rod photoreceptors. The present invention further provides a vector comprising the expression cassette of the invention. In some embodiments, said vector is a viral vector. The present invention also encompasses the use of a nucleic acid of the invention, of an expression cassette of the invention or of a vector of the invention for the expression of a gene in rod photoreceptors. The present invention further provides a method of expressing gene in rod photoreceptors comprising the steps of transfecting an isolated cell, a cell line or a cell population (e.g. a tissue) with an expression cassette of the invention, wherein the gene to be expressed will be expressed by the isolated cell, the cell line or the cell population if said cell is, or said cells comprise, rod photoreceptors. In some embodiments, the isolated cell, cell line or cell population or tissue is human. The present invention also provides an isolated cell comprising the expression cassette of the invention. In some embodiments, the expression cassette or vector is stably integrated into the genome of said cell. A typical gene which can be operatively linked to the promoter of the invention is a gene encoding for a halorhodopsin or a channelrhodosin. In addition, the present invention also provides a kit for expressing gene in rod photoreceptors, which kit comprises an isolated nucleic acid molecule of the invention. As used herein, the term “promoter” refers to any cis-regulatory elements, including enhancers, silencers, insulators and promoters. A promoter is a region of DNA that is generally located upstream (towards the 5′ region) of the gene that is needed to be transcribed. The promoter permits the proper activation or repression of the gene which it controls. In the context of the present invention, the promoters lead to the specific expression of genes operably linked to them in the rod photoreceptors. “Specific expression”, also referred to as “expression only in a certain type of cell” means that at least more than 75% of the cells expressing the gene of interest are of the type specified, i.e. rod photoreceptors in the present case. Expression cassettes are typically introduced into a vector that facilitates entry of the expression cassette into a host cell and maintenance of the expression cassette in the host cell. Such vectors are commonly used and are well known to those of skill in the art. Numerous such vectors are commercially available, e.g., from Invitrogen, Stratagene, Clontech, etc., and are described in numerous guides, such as Ausubel, Guthrie, Strathem, or Berger, all supra. Such vectors typically include promoters, polyadenylation signals, etc. in conjunction with multiple cloning sites, as well as additional elements such as origins of replication, selectable marker genes (e.g., LEU2, URA3, TRP 1, HIS3, GFP), centromeric sequences, etc. Viral vectors, for instance an AAV, a PRV or a lentivirus, are suitable to target and deliver genes to rod photoreceptors using a promoter of the invention. The output of retinal cells can be measured using an electrical method, such as a multi-electrode array or a patch-clamp, or using a visual method, such as the detection of fluorescence. The methods using nucleic acid sequence of the invention can be used for identifying therapeutic agents for the treatment of a neurological disorder or of a disorder of the retina involving rod photoreceptors, said method comprising the steps of contacting a test compound with rod photoreceptors expressing one or more transgene under a promoter of the invention, and comparing at least one output of rod photoreceptors obtained in the presence of said test compound with the same output obtained in the absence of said test compound. Moreover, the methods using promoters of the invention can also be used for in vitro testing of vision restoration, said method comprising the steps of contacting rod photoreceptors expressing one or more transgene under the control of a promoter of the invention with an agent, and comparing at least one output obtained after the contact with said agent with the same output obtained before said contact with said agent. Channelrhodopsins are a subfamily of opsin proteins that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis, i.e. movement in response to light. Expressed in cells of other organisms, they enable the use of light to control intracellular acidity, calcium influx, electrical excitability, and other cellular processes. At least three “natural” channelrhodopsins are currently known: Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2), and Volvox Channelrhodopsin (VChR1). Moreover, some modified/improved versions of these proteins also exist. All known Channelrhodopsins are unspecific cation channels, conducting H+, Na+, K+, and Ca2+ ions. Halorhodopsin is a light-driven ion pump, specific for chloride ions, and found in phylogenetically ancient “bacteria” (archaea), known as halobacteria. It is a seven-transmembrane protein of the retinylidene protein family, homologous to the light-driven proton pump bacteriorhodopsin, and similar in tertiary structure (but not primary sequence structure) to vertebrate rhodopsins, the pigments that sense light in the retina. Halorhodopsin also shares sequence similarity to channelrhodopsin, a light-driven ion channel. Halorhodopsin contains the essential light-isomerizable vitamin A derivative all-trans-retinal. Halorhodopsin is one of the few membrane proteins whose crystal structure is known. Halorhodopsin isoforms can be found in multiple species of halobacteria, includingH. salinarum, andN. pharaonis. Much ongoing research is exploring these differences, and using them to parse apart the photocycle and pump properties. After bacteriorhodopsin, halorhodopsin may be the best type I (microbial) opsin studied. Peak absorbance of the halorhodopsin retinal complex is about 570 nm. Recently, halorhodopsin has become a tool in optogenetics. Just as the blue-light activated ion channel channelrhodopsin-2 opens up the ability to activate excitable cells (such as neurons, muscle cells, pancreatic cells, and immune cells) with brief pulses of blue light, halorhodopsin opens up the ability to silence excitable cells with brief pulses of yellow light. Thus halorhodopsin and channelrhodopsin together enable multiple-color optical activation, silencing, and desynchronization of neural activity, creating a powerful neuroengineering toolbox. In some embodiments, the promoter is part of a vector targeted a retina, said vector expressing at least one reporter gene which is detectable in living rod photoreceptors. Suitable viral vectors for the invention are well-known in the art. For instance an AAV, a PRV or a lentivirus, are suitable to target and deliver genes to rod photoreceptors. When working with isolated retina, optimal viral delivery for retinal cells can be achieved by mounting the ganglion cell side downwards, so that the photoreceptor side of the retina is exposed and can thus be better transfected. Another technique is slicing, e.g. with a razor blade, the inner limiting membrane of the retina, such that the delivering viruses can penetrate the inner membranes. A further way is to embed the retina in agar, slicing said retina and applying the delivery viruses from the side of the slice. The output of transfected cells can be measured using well-known methods, for instance using an electrical method, such as a multi-electrode array or a patch-clamp, or using a visual method, such as the detection of fluorescence. In some cases, the inner limiting membrane is removed by micro-surgery the inner limiting membrane. In other cases, recording is achieved through slices performed to the inner limiting membrane. Any source of retinal cells can be used for the present invention. In some embodiments of the invention, the retinal cells come from, or are in, a human retina. In other embodiments, the retina is from an animal, e.g. of bovine or of rodent origin. Human retina can be easily obtained from cornea banks where said retinas are normally discarded after the dissection of the cornea. Adult human retina has a large surface (about 1100 mm2) and can therefore be easily separated to a number of experimentally subregions. Moreover, retinas can also be used as an exquisite model for synaptic communication since the retina has synapses that are identical to the rest of the brain. As used herein, the term “animal” is used herein to include all animals. In some embodiments of the invention, the non-human animal is a vertebrate. Examples of animals are human, mice, rats, cows, pigs, horses, chickens, ducks, geese, cats, dogs, etc. The term “animal” also includes an individual animal in all stages of development, including embryonic and fetal stages. A “genetically-modified animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a sub-cellular level, such as by targeted recombination, microinjection or infection with recombinant virus. The term “genetically-modified animal” is not intended to encompass classical crossbreeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by, or receive, a recombinant DNA molecule. This recombinant DNA molecule may be specifically targeted to a defined genetic locus, may be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term “germ-line genetically-modified animal” refers to a genetically-modified animal in which the genetic alteration or genetic information was introduced into germline cells, thereby conferring the ability to transfer the genetic information to its offspring. If such offspring in fact possess some or all of that alteration or genetic information, they are genetically-modified animals as well. The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene, or not expressed at all. The genes used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof. A type of target cells for transgene introduction is the ES cells. ES cells may be obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981), Nature 292:154-156; Bradley et al. (1984), Nature 309:255-258; Gossler et al. (1986), Proc. Natl. Acad. Sci. USA 83:9065-9069; Robertson et al. (1986), Nature 322:445-448; Wood et al. (1993), Proc. Natl. Acad. Sci. USA 90:4582-4584). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection using electroporation or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with morulas by aggregation or injected into blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germline of the resulting chimeric animal (Jaenisch (1988), Science 240:1468-1474). The use of gene-targeted ES cells in the generation of gene-targeted genetically-modified mice was described 1987 (Thomas et al. (1987), Cell 51:503-512) and is reviewed elsewhere (Frohman et al. (1989), Cell 56:145-147; Capecchi (1989), Trends in Genet. 5:70-76; Baribault et al. (1989), Mol. Biol. Med. 6:481-492; Wagner (1990), EMBO J. 9:3025-3032; Bradley et al. (1992), Bio/Technology 10:534-539). Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. As used herein, a “targeted gene” is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles. In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, a nucleic acid contained in a clone that is a member of a library (e.g., a genomic or cDNA library) that has not been isolated from other members of the library (e.g., in the form of a homogeneous solution containing the clone and other members of the library) or a chromosome removed from a cell or a cell lysate (e.g., a “chromosome spread”, as in a karyotype), or a preparation of randomly sheared genomic DNA or a preparation of genomic DNA cut with one or more restriction enzymes is not “isolated” for the purposes of this invention. As discussed further herein, isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically. “Polynucleotides” can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms. The expression “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 50 degree C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. The terms “fragment,” “derivative” and “analog” when referring to polypeptides means polypeptides which either retain substantially the same biological function or activity as such polypeptides. An analog includes a pro-protein which can be activated by cleavage of the pro-protein portion to produce an active mature polypeptide. The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons). Polypeptides can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include, but are not limited to, acetylation, acylation, biotinylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, denivatization by known protecting/blocking groups, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, linkage to an antibody molecule or other cellular ligand, methylation, myristoylation, oxidation, pegylation, proteolytic processing (e.g., cleavage), phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).) A polypeptide fragment “having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of the original polypeptide, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the original polypeptide (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, in some embodiments, not more than about tenfold less activity, or not more than about three-fold less activity relative to the original polypeptide.) Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue. “Variant” refers to a polynucleotide or polypeptide differing from the original polynucleotide or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the original polynucleotide or polypeptide. As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. BIosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty—1, Joining Penalty—30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty—5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 impaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for instance, the amino acid sequences shown in a sequence or to the amino acid sequence encoded by deposited DNA clone can be determined conventionally using known computer programs. A preferred method for determining, the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty—I, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty—5, Gap Size Penalty—0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. Only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention. Naturally occurring protein variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes 11, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. “Label” refers to agents that are capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Labels that are directly detectable and may find use in the invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes and the like. A “fluorescent label” refers to any label with the ability to emit light of a certain wavelength when activated by light of another wavelength. “Fluorescence” refers to any detectable characteristic of a fluorescent signal, including intensity, spectrum, wavelength, intracellular distribution, etc. “Detecting” fluorescence refers to assessing the fluorescence of a cell using qualitative or quantitative methods. In some of the embodiments of the present invention, fluorescence will be detected in a qualitative manner. In other words, either the fluorescent marker is present, indicating that the recombinant fusion protein is expressed, or not. For other instances, the fluorescence can be determined using quantitative means, e.g., measuring the fluorescence intensity, spectrum, or intracellular distribution, allowing the statistical comparison of values obtained under different conditions. The level can also be determined using qualitative methods, such as the visual analysis and comparison by a human of multiple samples, e.g., samples detected using a fluorescent microscope or other optical detector (e.g., image analysis system, etc.). An “alteration” or “modulation” in fluorescence refers to any detectable difference in the intensity, intracellular distribution, spectrum, wavelength, or other aspect of fluorescence under a particular condition as compared to another condition. For example, an “alteration” or “modulation” is detected quantitatively, and the difference is a statistically significant difference. Any “alterations” or “modulations” in fluorescence can be detected using standard instrumentation, such as a fluorescent microscope, CCD, or any other fluorescent detector, and can be detected using an automated system, such as the integrated systems, or can reflect a subjective detection of an alteration by a human observer. The “green fluorescent protein” (GFP) is a protein, composed of 238 amino acids (26.9 kDa), originally isolated from the jellyfishAequorea victoria/Aequorea aequorea/Aequorea forskaleathat fluoresces green when exposed to blue light. The GFP fromA. victoriahas a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered. The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostablility and a shift of the major excitation peak to 488 nm with the peak emission kept at 509 nm. The addition of the 37° C. folding efficiency (F64L) point mutant to this scaffold yielded enhanced GFP (EGFP). EGFP has an extinction coefficient (denoted ε), also known as its optical cross section of 9.13×10-21 m2/molecule, also quoted as 55,000 L/(mol·cm). Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006. The “yellow fluorescent protein” (YFP) is a genetic mutant of green fluorescent protein, derived fromAequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm. As used herein, the singular forms “a”, “an,” and “the” include plural reference unless the context clearly dictates otherwise. A “virus” is a sub-microscopic infectious agent that is unable to grow or reproduce outside a host cell. Each viral particle, or virion, consists of genetic material, DNA or RNA, within a protective protein coat called a capsid. The capsid shape varies from simple helical and icosahedral (polyhedral or near-spherical) forms, to more complex structures with tails or an envelope. Viruses infect cellular life forms and are grouped into animal, plant and bacterial types, according to the type of host infected. The term “transsynaptic virus” as used herein refers to viruses able to migrate from one neurone to another connecting neurone through a synapse. Examples of such transsynaptic virus are rhabodiviruses, e.g. rabies virus, and alphaherpesviruses, e.g. pseudorabies or herpes simplex virus. The term “transsynaptic virus” as used herein also encompasses viral sub-units having by themselves the capacity to migrate from one neurone to another connecting neurone through a synapse and biological vectors, such as modified viruses, incorporating such a sub-unit and demonstrating a capability of migrating from one neurone to another connecting neurone through a synapse. Transsynaptic migration can be either anterograde or retrograde. During a retrograde migration, a virus will travel from a postsynaptic neuron to a presynaptic one. Accordingly, during anterograde migration, a virus will travel from a presynaptic neuron to a postsynaptic one. Homologs refer to proteins that share a common ancestor. Analogs do not share a common ancestor, but have some functional (rather than structural) similarity that causes them to be included in a class (e.g. trypsin like serine proteinases and subtilisin's are clearly not related—their structures outside the active site are completely different, but they have virtually geometrically identical active sites and thus are considered an example of convergent evolution to analogs). There are two subclasses of homologs—orthologs and paralogs. Orthologs are the same gene (e.g. cytochome ‘c’), in different species. Two genes in the same organism cannot be orthologs. Paralogs are the results of gene duplication (e.g. hemoglobin beta and delta). If two genes/proteins are homologous and in the same organism, they are paralogs. As used herein, the term “disorder” refers to an ailment, disease, illness, clinical condition, or pathological condition. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert, and is not toxic to the patient to whom it is administered. As used herein, the term “pharmaceutically acceptable derivative” refers to any homolog, analog, or fragment of an agent, e.g. identified using a method of screening of the invention, that is relatively non-toxic to the subject. The term “therapeutic agent” refers to any molecule, compound, or treatment, that assists in the prevention or treatment of disorders, or complications of disorders. Compositions comprising such an agent formulated in a compatible pharmaceutical carrier may be prepared, packaged, and labeled for treatment. If the complex is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol. Thus, the compounds and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor. For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated as a topical application, such as a cream or lotion. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, intraocular, subcutaneous or intramuscular) or by intraocular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically or prophylactically effective amounts of the compositions in pharmaceutically acceptable form. The composition in a vial of a kit may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes. In another embodiment, a kit further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of compositions by a clinician or by the patient. Rod photoreceptors, rod cells, or rods, are photoreceptor cells in the retina of the eye that can function in less intense light than the other type of visual photoreceptor, cone cells. Rods are concentrated at the outer edges of the retina and are used in peripheral vision. On average, there are approximately 90 million rod cells in the human retina. More sensitive than cone cells, rod cells are almost entirely responsible for night vision. However, because they have only one type of light-sensitive pigment, rather than the three types that human cone cells have, rods have little, if any, role in color vision. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Examples Gene Construct Whole genome high resolution DNA methylation maps were generated in the cell type of interest (rod) to identify candidate regulatory regions. Candidate enhancers were selected based on the presence of cell type specific DNA hypomethylation. The so-selected elements were screened for expression using an in vivo high throughput reporter assay in cones and rods. Rod specific sequence elements were then synthetized and cloned in front of a minimal promotor sequence ATCCTCACATGGTCCTGCTGGAGTTAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTA TCACATCCACTGTGTTGTTGTGAACTGGAATCCACTATAGGCCA (SEQ ID NO:2). ChR2-eGFP coding sequence was inserted immediately after this promoter and the optimized Kozak sequence (GCCACC), and followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 polyadenylation site. Retinal neurons were targeted using AAVs serotype 2/8 with a titer in the range of 3.43E+11 to 1.75E+12 GC/mL. Viral Transfection and Tissue Preparation For AAV administration, the eyes of anesthetized animals were punctured in the sclera close to the lens by a sharp 30 gauge needle. 2 microL of AAV particle suspension were injected subretinally by a Hamilton syringe. After 3 weeks, the isolated retinas were fixed for 30 min in 4% PFA in PBS, followed by a washing step in PBS at 4° C. Whole retinas were treated with 10% normal donkey serum (NDS), 1% BSA, 0.5% Triton X-100 in PBS for 1 h at room temperature. Treatment with monoclonal rat anti-GFP antibody (Molecular Probes Inc.; 1:500) and polyclonal rabbit anti-mouse Cone Arrestin antibody (Millipore: 1:200) in 3% NDS, 1% BSA, 0.5% Triton X-100 in PBS was carried out for 5 days at room temperature. Treatment with secondary donkey anti-rat Alexa Fluor-488 Ab (Molecular Probes Inc.; 1:200), anti-rabbit Alexa Fluor-633 and Hoechst, was done for 2 hr. Sections were washed, mounted with ProLong Gold antifade reagent (Molecular Probes Inc.) on glass slides, and photographed using a Zeiss LSM 700 Axio Imager Z2 laser scanning confocal microscope (Carl Zeiss Inc.). | 46,942 |
11857643 | DETAILED DESCRIPTION Fluorescent labels are widely used for imaging because they provide direct, quantitative, specific and sensitive detection of biomolecules including proteins and nucleic acids. Modified fluorescent labels that are sulfonated and/or PEG modified offer increased sensitivity compared to the basic unmodified dyes. However, even these modified fluorescent labels exhibit fluorescence quenching at certain dye to protein (D/P) ratios. Increase in fluorescence is seen with increasing molar excesses of the labeling dye, however, molar excesses which result in exceeding optimal dye to protein (D/P) ratios, typically result in quenching and/or precipitation of the biomolecule especially in fluorescent imaging application where spatial conformation of antigen/antibody or DNA/RNA interactions may cause static quenching. In some embodiments, the invention comprises methods of reducing quenching and/or increasing fluorescence signal with highly labeled conjugates (e.g., biomolecules with high dye to protein ratios (D/P)) that in standard conjugation results in decreased fluorescence. Modifications can be made on proteins, nucleic acids and other biomolecules (e.g., oligosaccharides). In some embodiments, the invention comprises compositions comprising fluorescently labeled biomolecules that exhibit increased fluorescent signal and/or reduced quenching, wherein the composition comprises a biomolecule, a spacer, and a fluorescent label, wherein the spacer and fluorescent label are not directly conjugated to each other. The invention also relates to compositions which exhibit enhanced fluorescence on a per fluorescent label basis, as well as methods for producing and using such compositions. By way of illustration, assume that a single fluorescent label attached to a biomolecule sets a baseline of 100% fluorescent emission. Further assume, when two fluorescent labels are attached to the same biomolecule, that each of the two fluorescent labels exhibits an average of 80% of the baseline fluorescent emission. The invention is directed, in part, to compositions and methods for increasing the average fluorescent emission above the 80% of the baseline. In some instances, compositions of the invention, as well as compositions used in methods of the invention, may be defined by one or more functional property. Examples of such properties are the numbers of fluorescent labels associated with a labeled molecule (e.g., a biomolecule), the average distance (measured in any of a number of different ways) between fluorescent labels on the labeled molecule, and/or the quantum yield of fluorescent labels on the labeled molecule. One measure of measuring fluorescent intensity is by measurement of Quantum Yield. Quantum Yield ((D) for fluorescent systems is effectively the emission efficiency of a given fluorophore and may be determined by the equation: Φ=NumberofPhotonsEmittedNumberofPhotonsAbsorbed Quantum Yield may also be used to measure quenching effects, as set out below in Example 8. Further, instruments, such as the Hamamatsu Absolute PL Quantum Yield Spectrometer (Hamamatsu Corp., Bridgewater N.J. 08807, C11347-11Quantaurus-QY Absolute PL Quantum Yield Spectrometer), that may be used to measure quantum yield are commercially available. As set out in Example 8 and Table 25, quantum yield of a fluorescently labeled molecule can be compared to that of the free fluorescent label. If the quantum yield of a single unit of the free fluorescent label under conditions where effectively no quenching occurs is set as one, then this can be used as a benchmark for comparison of the fluorescence generated by each fluorescent label attached to the labeled molecule. In many instances, compositions of the invention include fluorescently labeled molecules that are labeled with multiple fluorescent labels where the average amount of fluorescent emission on a per fluorescent label basis is at least 70% (0.7 Fluorescent Ratio) (e.g., from about 70% to about 99%, from about 70% to about 90%, from about 80% to about 99%, from about 85% to about 99%, from about 87% to about 99%, from about 90% to about 99%, from about 80% to about 95%, from about 85% to about 96%, etc.) of the fluorescent emission of the free fluorescent label. As set out in Example 8 and Table 25, fluorescent intensity may be determined is by the measurement of total fluorescence of a fluorescently labeled molecule compared to the fluorescence of the free label. If the fluorescence of a single unit of the free fluorescent label under conditions where effectively no quenching occurs is set as one, then this can be used as a benchmark for comparison of the fluorescence generated by each fluorescent label attached to the labeled molecule. In many instances, compositions of the invention include fluorescently labeled molecules that are labeled with multiple fluorescent labels where the average amount of fluorescent emission on a per fluorescent label basis is at least 70% (0.7 Fluorescent Ratio) (e.g., from about 70% to about 99%, from about 70% to about 90%, from about 80% to about 99%, from about 85% to about 99%, from about 87% to about 99%, from about 90% to about 99%, from about 80% to about 95%, from about 85% to about 96%, etc.) of the fluorescent emission of the free fluorescent label. As set out in Table 25, the brightness of a fluorescently labeled molecule compared to the free fluorescent label can be determined. Brightness is proportional to the product of quantum yield ((D), extinction coefficient (E) and number of dyes per molecule (N) as given in the equation: B=Φ×ε×N Thus the ratio of the brightness of the free fluorescent label to that of the labeled molecule can be used to describe the total fluorescence enhancement. As an example, the data in Table 25 sets as a benchmark ALEXA FLUOR™ 647 in deionized water. Further, this sets a benchmark of 100% quantum yield of free dye and a brightness ratio of 1.0. Amongst samples, the molecule AF647-20K8 had 73% of the quantum yield of the free dye but showed a fluorescent enhancement of 5.8× over the free dye. It is also shown that the sample AF647-10K4 had the highest percent quantum yield of the free dye (89%) but had a fluorescent enhancement of only 3.6× versus the free dye. These data show that the degree of fluorescent enhancement seen for these molecules can be directly correlated with the length of the arms. The data also show that when arm length is held constant and more fluorescently labeled arms are added to a polymer, then the fluorescent enhancement tends to increase. The invention thus includes compositions and methods for linking multiple fluorescent labels to individual molecules (e.g., biomolecules) such that the fluorescent labels are spaced in a manner that enhances fluorescent signal. This may be done by the reduction of quenching. One method for enhancing fluorescent signal is to spatially separate fluorescent labels present in a sample. This is especially useful when multiple fluorescent labels are attached to the same molecule (e.g., biomolecule) that is to be detected. In some aspects, the invention comprises methods of producing an antibody conjugated to a spacer and a fluorescent label, wherein a spacer agent is used to conjugate a spacer to an antibody, and wherein the spacer is not conjugated to the fluorescent label. Also encompassed are compositions comprising a spacer, an antibody, and a fluorescent label, wherein the spacer is not conjugated to the fluorescent label. In some embodiments, the spacer may be capable of reducing quenching of a plurality of fluorescent labels conjugated to an antibody. In some embodiments, a method of producing a nucleic acid conjugated to a spacer agent is encompassed, wherein the spacer agent is not directly conjugated to the fluorescent label. Such spacer agents may be capable of reducing quenching of a fluorescent label conjugated to a nucleic acid. In some embodiments, the invention includes compositions and methods related to the spatial separation fluorescent labels from the point on a molecule (e.g., biomolecule) to which they are conjugated to. In many instances, this will be done by connection for one or more fluorescent labels to a spacer and connection of the spacer to the molecule (e.g., biomolecule). Example of such compositions and methods are shown inFIG.18. Definitions This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. As used herein, a “biomolecule” refers to any molecule that may be included in a biological system, including but not limited to, a synthetic or naturally occurring protein or fragment thereof, glycoprotein, lipoprotein, amino acid, nucleoside, nucleotide, nucleic acid, oligonucleotide, DNA, RNA, carbohydrate, sugar, lipid, fatty acid, hapten, antibody, and the like. The terms “protein” and “polypeptide” are used herein in a generic sense to include polymers of amino acid residues of any length. The term “peptide” as used herein refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used. Additionally, unnatural amino acids, for example, β-alanine, phenylglycine and homoarginine are also included. Commonly encountered amino acids that are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L-isomer. The L-isomers are generally used. In addition, other peptidomimetics are also useful in the present invention. For a general review, see, Spatola, A. F., inChemistry and Biochemistry of Amino Acids. Peptides and Proteins. B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983). The term “antibody” as used herein refers to a protein of the immunoglobulin (Ig) superfamily that binds noncovalently to certain substances (e.g., antigens and immunogens) to form an antibody-antigen complex, including but not limited to antibodies produced by hybridoma cell lines, by immunization to elicit a polyclonal antibody response, by chemical synthesis, and by recombinant host cells that have been transformed with an expression vector that encodes the antibody. In humans, the immunoglobulin antibodies are classified as IgA, IgD, IgE, IgG, and IgM and members of each class are said to have the same isotype. Human IgA and IgG isotypes are further subdivided into subtypes IgA1, and IgA2, and IgG1, IgG2, IgG3, and IgG4. Mice have generally the same isotypes as humans, but the IgG isotype is subdivided into IgG1, IgG2aIgG2b, and IgG3subtypes. Thus, it will be understood that the term “antibody” as used herein includes within its scope (a) any of the various classes or sub-classes of immunoglobulin (e.g., IgA, IgD, IgG, IgM, and IgE derived from any animal that produced antibodies) and (b) polyclonal and monoclonal antibodies, such as murine, chimeric, or humanized antibodies. Antibody molecules have regions of amino acid sequences that can act as an antigenic determinant (e.g. the Fc region, the kappa light chain, the lambda light chain, the hinge region, etc.). An antibody that is generated against a selected region is designated anti-[region] (e.g., anti-Fc, anti-kappa light chain, anti-lambda light chain, etc.). An antibody is typically generated against an antigen by immunizing an organism with a macromolecule to initiate lymphocyte activation to express the immunoglobulin protein. The term antibody, as used herein, also covers any polypeptide or protein having a binding domain that is, or is homologous to, an antibody binding domain, including, without limitation, single-chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al.,Science242:423 (1988) and Huston et al.,Proc. Natl. Acad. Sci. USA85:5879 (1988)). These can be derived from natural sources, or they may be partly or wholly synthetically produced. Further, VHH antibodies may be used either as obtained from antigen stimulated cells or as engineered antigen binding proteins. The term “antibody fragments” as used herein refers to fragments of antibodies that retain the principal selective binding characteristics of the whole antibody. Particular fragments are well-known in the art, for example, Fab, Fab′, and F(ab′)2, which are obtained by digestion with various proteases and which lack the Fc fragment of an intact antibody or the so-called “half-molecule” fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody. Such fragments also include isolated fragments consisting of the light-chain-variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker. Other examples of binding fragments include (i) the Fd fragment, consisting of the VH and CHI domains; (ii) the dAb fragment (Ward et al.,Nature341:544 (1989)), which consists of a VH domain; (iii) isolated CDR regions; and (iv) single-chain Fv molecules (scFv) described above. In addition, arbitrary fragments can be made using recombinant technology that retains antigen-recognition characteristics. Exemplary VHH antibodies that may be used are single-domain antibody that are antibody fragments composed of a single monomeric variable antibody domain. Such antibody fragments typically have a molecular weight of only 12-25 kDa and are thus smaller than many other antibodies (150-160 kDa), which are composed of two heavy protein chains and two light chains. As used herein, an “antigen” refers to a molecule that induces, or is capable of inducing, the formation of an antibody or to which an antibody binds selectively, including but not limited to a biological material. Antigen also refers to “immunogen”. An antibody binds selectively to an antigen when there is a relative lack of cross-reactivity with or interference by other substances present. The term “reactive group” as used herein refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e., is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance. Reactive groups generally include nucleophiles, electrophiles and photoactivatable groups. Exemplary reactive groups include, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, alkynes and azides. As used herein, a “spacer,” “spacer molecule,” or “spacer agent” refers to a compound (e.g., an organic compound) that when conjugated to a biomolecule, directly or indirectly, is capable of enhancing fluorescence emitted from the biomolecule. This is believed to result from reduction of fluorescent quenching of a fluorescent label. Any number of compounds can act as spacers, exemplary compounds include NHS-acetate and various forms of polyethylene glycol (PEG). As used herein, the term “polyethylene glycol” or “PEG” refers to an oligomer or polymer of ethylene oxide. PEG polymer chain lengths may vary greatly but tended to have a molecular mass as high as 10,000,000 g/mol. PEGs are also available with different geometries. For example, branched PEGs typically have three to ten PEG chains emanating from a central core group. Star PEGs have 3 to 100 PEG chains emanating from a central core group. Comb PEGs have multiple PEG chains normally grafted onto a polymer backbone. Most PEGs include molecules with a distribution of molecular weights (i.e., they are polydisperse). The size distribution can be characterized statistically by its weight average molecular weight and its number average molecular weight, the ratio of which is called the polydispersity index. Exemplary PEG compounds that may be used in the practice of the invention include MS(PEG)4, MS(PEG)8, and MS(PEG)12(Thermo Fisher Scientific, Waltham, Mass., cat. nos. 22341, 22509B, and 22686, respectively), as well as branched chain PEG compounds, such as (Methyl-PEG12)3-PEG4-NHS Ester (Thermo Fisher Scientific, Waltham, Mass., cat. no. 22421). As used herein, the term “direct spacer” refers to a molecule that has at least one fluorescent labeled attached thereto and binds directly to a biomolecule. Direct spacers may be (1) a single polymer or (2) multiple polymers attached to a core. Examples of direct spacers include single-armed polymers and multi-armed polymers. As used herein, a “polymer” is a molecule composed of repeating subunits (typically at least 4 repeating subunits). Polymers may be synthetic or naturally occurring. The repeating units of a polymer need not be identical. For example, proteins are polymers that are composed of different amino acid subunits. Further, polymers need not be fully linear molecules and, thus, may be branched like dextrans. As used herein, the term “single-armed polymer” refers to an unbranched molecule that to which at least one fluorescent label is attached and having an unbranched structure (seeFIG.19). Examples of single-armed polymers that may be used in the practice of the invention are “linear” polysaccharides (e.g., amylose), polyethylene glycols, long-chain carbon molecules (e.g., Ahx), and polypeptides. In some instances, unbranched/linear polysaccharides are composed of monomers connected to each other by α1,4 linkages. As used herein, the term “multi-armed polymer” refers to a branched molecule that to which at least one fluorescent label is attached and having an unbranched structure (seeFIG.19). Examples of multi-armed polymers that may be used in the practice of the invention are branched polysaccharides (e.g., dextrans, glycogen), polyethylene glycols, branched long-chain carbon molecules (e.g., Ahx), and branched polypeptides. As used herein, the term “conjugation molecule” or “conjugation arm” refers linkers through which dyes are connected (e.g., covalently connected) to molecules (e.g., biomolecules). Conjugation molecule may be bound to a single dye molecule or multiple dye molecules (the same dye or different dyes). As used herein, the term “fluorescence” refers to an optical phenomenon in which a molecule absorbs a high-energy photon and re-emits it as a lower-energy (longer-wavelength) photon, with the energy difference between the absorbed and emitted photons ending up as molecular vibrations or heat. The term “fluorescent label,” “fluorescent dye,” “fluorophore” or “fluorescent moiety”, as used herein, refers to a compound, chemical group, or composition that is inherently fluorescent. Fluorophores may contain substituents that alter the solubility, spectral properties or physical properties of the fluorophore. Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a furan, a benzazole, a borapolyazaindacene and xanthenes including fluorescein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002). Reactive chemistries such as N-hydroxysuccinimide (NHS), maleimide and hydrazides as well as click chemistry (e.g. SITECLICK™) are currently being used for conjugation of florescent labels to biomolecules. As used herein, the term “conjugated” refers to a molecule being attached to another molecule, either directly or indirectly, either by covalent or noncovalent linkage. The term “dye conjugate” refers to a dye molecule bound covalently or non-covalently to another carrier molecule, such as an antibody and, in many instances, the dyes are bound covalently. The dye conjugate can be directly bound through a single covalent bond, cross-linked or bound through a linker, such as a series of stable covalent bonds incorporating 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, S and P that covalently attach the fluorescent dye to the antibody or another moiety such as a chemically reactive group or a biological and non-biological component. The conjugation or linker may involve a receptor binding motif, such as biotin/avidin. The term “near IR dye” or “near IR reporter molecule” or “NIR dye” or “NIR reporter molecule” as used herein indicates a dye or reporter molecule with an excitation wavelength of about 580 nm to about 800 nm. In many instances, the NIR dyes emit in the range of about 590 nm to about 860 nm. In many instances, NIR dyes are excited from about 680 to about 790 nm. In many instances, dyes include ALEXA FLUOR™ 660 Dye, ALEXA FLUOR™ 680 dye, ALEXA FLUOR™ 700 dye, ALEXA FLUOR™ 750 dye, and ALEXA FLUOR™ 790 dye. The NIR dyes are particularly advantageous for in vivo imaging because they can be selectively visualized without exciting endogenous materials present in living body. Some of the NIR dyes have a large stokes shift, such that the excitation and emission wavelengths are separated by at least 20, 30, 40, 50, 60, 70 or 80 nm. “Solid support” means a substrate material having a rigid or semi-rigid surface. Typically, at least one surface of the substrate will be substantially flat, although it may be desirable to physically separate certain regions with, for example, wells, raised regions, etched trenches, or other such topology. Solid support materials also include spheres (including microspheres), rods (such as optical fibers) and fabricated and irregularly shaped items. Solid support materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: poly(vinylidene difluoride) (PVDF), polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, polytetrafluoroethylene, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, Kieselguhr-polyacrylamide non-covalent composite, polystyrene-polyacrylamide covalent composite, polystyrene-PEG [poly(ethylene glycol)] composite, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The solid support may be particulate or may be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials. “Kit” means a packaged set of related components, typically one or more compounds or compositions. Detectable Biomolecules Disclosed herein, in some embodiments, are biomolecules that are detectably labeled with a plurality of fluorescent labels that also comprise a spacer agent. a. Spacers A spacer may be any molecule that is capable of enhancing fluorescent emissions when conjugated to a biomolecule that results from excitation of a plurality of fluorescent labels independently conjugated to the biomolecule. It is believed that this is due to the reduction of fluorescence quenching of the fluorescent labels. In some embodiments, the spacer comprises an acetyl (—C(O)CH3) group. In some embodiments, the spacer is an acetate molecule. In some embodiments, the acetate molecule is sulfo-NHS-acetate. In some embodiments, the spacer agent comprises polyethylene glycol (PEG). In some embodiments, the spacer agent comprises (PEG)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the spacer agent comprises MS-(PEG)n. In some embodiments, the spacer is selected from an alkanoyl, alkenoyl, and alkynoyl (C(O)CnHmin which n is 1 to 20 atoms, m>n, the carbon atoms can be connected to each other by single, double, and/or triple bonds, and the alkyl, alkenyl, and/or alkynyl groups can be further substituted. In some embodiments, specific substitutions include poly(ethylene)glycol moieties, such as (OCH2CH2Ox—(CH2)y—OR in which x is 1 to 20, y is 1 to 6, and R is H or C1-6alkyl. In some embodiments, specific substitutions include ammonium (—NH3+), quaternary ammonium ((—NR3+) groups in which R is C1-6alkyl, or phosphonium groups (—PQ3+) in which Q is aryl, substituted aryl, or C14 alkyl. In some embodiments, the spacer is selected from alkyl, alkenyl, or alkynyl groups (CnHmin which n is 1 to 20 atoms, m>n, the carbon atoms can be connected to each other by single, double, and/or triple bonds, and the alkyl, alkenyl, and/or alkynyl groups can be further substituted. In some embodiments, specific substitutions include negatively charged sulfonate groups (—OSO3—), carboxylate groups (—CO2—), phosphate groups (—OPO3—), and/or phosphonate groups (—PO3—). In some embodiments, other substitutions include poly(ethylene)glycol moieties, such as (OCH2CH2O), (CH2)yOR in which x is 1 to 20, y is 1-6, and R is H or C1-6alkyl. In some embodiments, other specific substitutions include ammonium (—NH3+), quaternary ammonium ((—NR3+) groups in which R is C1-6alkyl, or phosphonium groups (—PQ3+) in which Q is aryl, substituted aryl, or C1-6alkyl. In some embodiments, the spacer is positively charged. In some embodiments, the spacer agent comprises betaine (i.e., trimethylglycine). In some embodiments, the spacer agent is negatively charged. b. Fluorescent Label The fluorescent dyes described herein function as reporter molecules to confer a detectable signal, directly or indirectly, to the sample as a result of conjugation to a functional group on the protein, including, but not limited to, amine groups or thiol groups. This results in the ability to detect the total protein in a sample generally in combination with detection of a subset of the total protein of the sample. In such instances the total protein labels are detectable distinguished from the dye that labels a subset of the total protein in the sample. Where the detectable response is a fluorescence response, it is typically a change in fluorescence, such as a change in the intensity, excitation or emission wavelength, distribution of fluorescence, fluorescence lifetime, fluorescence polarization, or a combination thereof. The fluorescent dyes can be any fluorophore known to one skilled in the art. Typically the dye contains one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on chromophores or fluorophores known in the art. A wide variety of fluorophores that may be suitable for total protein labeling as described herein are already known in the art (RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (2002)). A fluorescent dye used in the methods and compositions described herein is any chemical moiety that exhibits an absorption maximum beyond 280 nm. Such chemical moieties include, but are not limited to, a pyrene, sulfonated pyrenes, sulfonated coumarins, sulfonated carbocyanines, sulfonated xanthenes, an anthracene, a naphthalene, an acridine, a stilbene, an indole an isoindole, an indolizine, a benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1, 3-diazole (NBD), a carbocyanine, a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, an isoquinoline, a chromene, a borapolyazaindacene, a xanthene, a fluorescein, a rosamine, a rhodamine, a rhodamine, benzo- or dibenzofluorescein, seminaphthofluorescein, a naphthofluorescein, a bimane, an oxazine or a benzoxazine, a carbazine, a phenalenone, a coumarin, a benzofuran, a benzphenalenone) and derivatives thereof. As used herein, oxazines include resorufins, aminooxazinones, diaminooxazines, and their benzo-substituted analogs. In one aspect the fluorescent dyes contain one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on chromophores or fluorophores known in the art. In one aspect the fluorophore is a xanthene that comprises one or more julolidine rings. In an exemplary embodiment, the dyes are independently substituted by substituents selected from the group consisting of hydrogen, halogen, amino, substituted amino, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive group, solid support and carrier molecule. In another embodiment, the xanthene dyes of this invention comprise both compounds substituted and unsubstituted on the carbon atom of the central ring of the xanthene by substituents typically found in the xanthene-based dyes such as phenyl and substituted-phenyl moieties. In many instances, dyes are rhodamine, fluorescein, borapolyazaindacene, indole and derivatives thereof. Choice of the reactive group used to attach the total protein labels or expression tag labels to the protein to be conjugated typically depends on the reactive or functional group on the substance to be conjugated and the type or length of covalent linkage desired. The types of functional groups typically present on the organic or inorganic substances (biomolecule or non-biomolecule) include, but are not limited to, amines, amides, thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles, hydrazines, hydroxylamines, disubstituted amines, halides, epoxides, silyl halides, carboxylate esters, sulfonate esters, purines, pyrimidines, carboxylic acids, olefmic bonds, or a combination of these groups. In proteins a variety of sites may occur including, but not limited to, amines, thiols, alcohols and phenols. Amine reactive fluorescent dyes that can be used in the protein labeling methods described herein include, but are not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 488, ALEXA FLUOR™ 500, ALEXA FLUOR™ 514, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 555, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 610-X, ALEXA FLUOR™ 633, ALEXA FLUOR™ 647, ALEXA FLUOR™ 660, ALEXA FLUOR™ 680, ALEXA FLUOR™ 700, ALEXA FLUOR™ 750, ALEXA FLUOR™ 790, AMCA-X™ BODIPY™ 630/650, BODIPY™ 650/665, BODIPY™ FL, BODIPY™ TMR, BODIPY™ TR, BODIPY™ TR-X, CASCADE BLUE™, Dinitrophenyl, Fluorescein, HEX™, JOE™, MARINA BLUE™, OREGON GREEN™ 488, OREGON GREEN™ 514, PACIFIC BLUE™, PACIFIC ORANGE™, RHODAMINE GREEN™ QSY™ 7 QSY™ 9, QSY™ 21, QSY™ 35, ROX™ RHODAMINE RED™, TET™, TAMRA™, tetramethyl rhodamine, FAM™, TEXAS RED™ and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE™). In some embodiments, the fluorogenic reagents/dyes that bind to tags attached to proteins used in the protein labeling methods described herein are biarsenical fluorophore, including, a biarsenical derivative of fluorescein, such as, by way of example only, FlAsH-EDT2 (4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2) (LUMIO™ Green, Life Technologies Corp., Carlsbad, Calif.), or a biarsenical derivative of resorufin such as, by way of example only, ReAsh-EDT2 (LUMIO™ Red, Life Technologies Corp., Carlsbad, Calif.), or may instead be an oxidized derivative, such as ChoXAsH-EDT2 or HoXAsH-EDT2. In addition, the biarsenical fluorophore can be a biarsenical derivative of other known fluorophores, including, but not limited to, the ALEXA FLUOR™ series described herein, such as, by way of example only, ALEXA FLUOR™ 350, ALEXA FLUOR™ 430, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 663 and ALEXA FLUOR™ 660, available commercially from Molecular Probes (Eugene, Oreg.). In some embodiments, the biarsenical fluorophore can be present at a concentration of at least about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM or more, and at a concentration of no more than about 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 4 μM, 3 μM, 2 μM or 1 μM. In some embodiments, the tag attached to a protein to which such fluorogenic dyes binds is a tetracysteine peptide motif, cys-cys-Xn-cys-cys (SEQ ID NO: 1), wherein each X is any natural amino acid, non-natural amino acid or combination thereof, and n is an integer from 2-100. In certain embodiments, n is an integer from 2-90, while in other embodiments n is an integer from 2-80. In certain embodiments, n is an integer from 2-70, while in other embodiments n is an integer from 2-60. In certain embodiments, n is an integer from 2-50, while in other embodiments n is an integer from 2-40. In certain embodiments, n is an integer from 2-30, while in other embodiments n is an integer from 2-20. In certain embodiments, n is an integer from 2-10, while in other embodiments n is an integer from 2-5. The natural amino acids if such motifs include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the tetracysteine tag has the sequence CCPGCC (SEQ ID NO: 2). In other embodiments a 12 amino acid peptide containing the tetracysteine motif is used including, but not limited to, the amino acid sequence, AGGCCPGCCGGG (SEQ ID NO: 3). In addition, the protein can be labeled with a single tetracysteine tag or the protein can be labeled with a plurality of tetracysteine tags including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tetracysteine tags. Such tags may be separated from one another within the primary amino acid sequence of the protein or directly multimerized in tandem as concatemers. In certain embodiments, the tetracysteine peptide has the sequence cys-cys-Xn-cys-X-cys-X (SEQ ID NO: 1), wherein each X is any natural amino acid, non-natural amino acid or combination thereof, and n is an integer from 2-100. In certain embodiments, n is an integer from 2-90, while in other embodiments n is an integer from 2-80. In certain embodiments, n is an integer from 2-70, while in other embodiments n is an integer from 2-60. In certain embodiments, n is an integer from 2-50, while in other embodiments n is an integer from 2-40. In certain embodiments, n is an integer from 2-30, while in other embodiments n is an integer from 2-20. In certain embodiments, n is an integer from 2-10, while in other embodiments n is an integer from 2-5. The natural amino acids if such motifs include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the tetracysteine tag has the sequence CCGGKGNGGCGC (SEQ ID NO: 4). The tetracysteine peptide tag or tags can be recombinantly fused to the protein desired to be labeled, either at the N-terminus, C-terminus, or in frame within the protein sequence; expression vectors for creating tetracysteine-fused recombinant proteins may readily be constructed using techniques known to one of skill in the art. In certain embodiments the tetracysteine-tagged protein is expressed recombinantly in host cells including, but not limited to, in bacterial host cells, in fungal host cells, in insect cells, in plant cells, or in mammalian cells. Such bacterial host cells include, but are not limited to, gram negative and gram positive bacteria of any genus, including, by way of example only,Escherichiasp. (e.g.,E. coli),Klebsiellasp.,Streptomycessp.,Streptococcussp.,Shigellasp.,Staphylococcussp.,Erwiniasp.,Klebsiellasp.,Bacillussp. (e.g.,B. cereus. B. subtilisandB. megaterium),Serratiasp.,Pseudomonassp. (e.g.,P. aeruginosaandP. syringae) andSalmonellasp. (e.g.,S. typhiandS. typhimurium). Suitable bacterial strains and serotypes suitable for the invention can includeE. coliserotypes K, B, C, and W. A typical bacterial host isE. colistrain K-12. The fungal host cells include, by way of example only,Saccharomyces cerevisiaecells, while the mammalian cells include, by way of example only, including human cells. In such embodiments, the protein sample containing the protein of interest is a lysate of the host cells, that can be unpurified, partially purified, or substantially purified prior to labeling and analysis using the methods described herein. In other embodiments, the tetracysteine-tagged protein is expressed in vitro, wherein the protein sample containing the protein of interest is the cell-free extract in which translation (and, optionally, transcription) is performed, or a partially purified or purified fraction thereof. In embodiments in which the extract permits coupled transcription and translation in a single cell-free extract, such as theE. coli-based EXPRESSWAY™ or EXPRESSWAY™ Plus systems (Life Technologies Corp., Carlsbad, Calif.), the sample is the cell-free extract in which transcription and translation commonly occur, or a fraction thereof. Alternatively, GATEWAY™ Technology (Life technologies Corp., Carlsbad, Calif.) is a universal cloning technology that can be used to express a gene of interest inE. coli. The protein to be labeled using the biarsenical dyes described herein can be any protein having a tetracysteine motif. The protein to which the tetracysteine tag or tags is fused or conjugated can be any protein desired to be labeled, either naturally-occurring or nonnaturally occurring. Naturally-occurring proteins may have known biological function or not, and may be known to be expressed or only predicted from genomic sequence. The protein, if naturally-occurring, can be a complete protein or only a fragment thereof. The tetracysteine-tagged protein can thus be an animal protein, such as a human protein or non-human mammalian protein, a fungal protein, a bacterial protein, including eubacterial and archaebacterial protein, a plant protein, an insect protein or a viral protein. In addition to the tetracysteine tag, other protein sequences can usefully be recombinantly appended to the proteins desired to be labeled. Among such additional protein sequences are linkers and/or short tags, usefully epitope tags, such as a FLAG tag, or a myc tag, or other sequences useful for purification, such as a polyhistidine (e.g., 6× his) tag. Alternatively, the tetracysteine tag or tags can be chemically conjugated to proteins to be labeled using art-routine conjugation chemistries. In some embodiments, the fluorescent label is positively charged. In some embodiments, the fluorescent label is negatively charged. In some embodiments, the excitation wavelength of the fluorescent label is between 350 and 850 nm. In some embodiments, the excitation wavelength of the fluorescent label is far red. In some embodiments, the excitation wavelength of the fluorescent label is near infrared. In some embodiments, the excitation wavelength of the fluorescent label is ultraviolet (UV). In some embodiments, the fluorescent label comprises a DYLIGHT™ fluor. In some embodiments, the DYLIGHT™ fluor is selected form DYLIGHT™ 350, DYLIGHT™ 405, DYLIGHT™ 488, DYLIGHT™ 550, DYLIGHT™ 594, DYLIGHT™ 633, DYLIGHT™ 650, DYLIGHT™ 680, DYLIGHT™ 755, and DYLIGHT™ 800. In some embodiments, the DYLIGHT™ fluor is conjugated to a PEG molecule (e.g., 2×PEG, 4×PEG, 8×PEG, or 12×PEG). In some embodiments, the fluorescent label comprises an ALEXA FLUOR™. In some embodiments, the ALEXA FLUOR™ is selected from ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 555, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 610, ALEXA FLUOR™ 633, ALEXA FLUOR™ 635, ALEXA FLUOR™ 647, ALEXA FLUOR™ 660, ALEXA FLUOR™ 680, ALEXA FLUOR™ 700, ALEXA FLUOR™ 750 and ALEXA FLUOR™ 790. In some embodiments, the fluorescent label comprises a moiety selected from xanthene; coumarin; cyanine; pyrene; oxazine; borapolyazaindacene; benzopyrylium; and carbopyronine. In some embodiments, the fluorescent label comprises fluorescein (e.g., Cy™2 or FITC). In some embodiments, the fluorescent label comprises rhodamine (e.g., TRITC or Cy™3). In some embodiments, the fluorescent label comprises MCA, coumarin, RHODAMINE RED™ TEXAS RED™, CASCADE BLUE™ Cy™S, Cy™5.5, IR™680, IR™800 and Cy™7. In some embodiments, the fluorescent label is a modified fluorescent label (e.g., the fluorescent label has been sulfonated or conjugated with PEG). In some embodiments, the fluorescent label is a fluorescent protein. In some embodiments, the fluorescent protein is a phycobiliprotein. Examples of phycobiliproteins useful in the present invention are allophycocyanin, phycocyanin, phycoerythrin, allophycocyanin B, B-phycoerythrin, phycoerythrocyanin, and b-phycoerythrin. The structures of phycobiliproteins have been studied and their fluorescent spectral properties are known. See A. N. Glazer, “Photosynthetic Accessory Proteins with Bilin Prosthetic Groups,” Biochemistry of Plants, Volume 8, M. D. Hatch and N. K. Boardman, EDS., Academic Press, pp. 51-96 (1981), and A. N. Glazer, “Structure and Evolution of Photosynthetic Accessory Pigment Systems with Special Reference to Phycobiliproteins,” The Evolution of Protein Structure and Function, B. S. Sigman and M. A. Brazier, EDS., Academic Press, pp. 221-244 (1980). In some embodiments, the fluorescent protein has absorption maxima of at least about 450 nm, often at least about 500 nm, having Stokes shifts of at least 15 nm, often at least about 25 nm, and has fluorescence emission maxima of at least about 500 nm, often at least about 550 nm. In some embodiments, the fluorescent label is a dipyrromethene boron difluoride dye, as disclosed in U.S. Patent Publication No. 2014/0349,893, incorporated by reference herein, in its entirety. The amine reactive fluorogenic reagents used in the protein labeling methods described herein include, but are not limited to, aroyl-2-quinoline-carboxaldehyde type reagents. Such reagents have been described in U.S. Pat. Nos. 5,459,272 and 5,631,374, each of which is herein incorporated by reference in their entirety. In some embodiments, the aroyl-2-quinoline-carboxaldehyde reagent used is 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or 3-(2-furoyl)quinoline-2-carboxaldehyde). In certain embodiments, the amine reactive fluorogenic reagent is 3-(2-furoyl)quinoline-2-carboxaldehyde), while in other embodiments, the amine reactive fluorogenic reagent is 3-(4 carboxybenzoyl)-quinoline-2-carboxaldehyde. c. Biomolecules A biomolecule that can be used in the compositions and methods disclosed herein include any biomolecule that is useful in molecular biology applications. In some embodiments, the biomolecule is an antibody (e.g., a primary antibody or a secondary antibody). In some embodiments, the biomolecule is an antibody fragment. Antibodies used in the practice of the invention may be antibody is polyclonal, monoclonal or engineered and may be from any source (e.g., shark, chicken or a mammal, such as a llama, human mouse, rabbit, goat, rat, etc.). Further, humanized antibodies may be used. In some embodiments, the antibody is chimeric. In some embodiments, the biomolecule is a protein or polypeptide. In some embodiments, the biomolecule is a recombinant polypeptide. In some embodiments, the biomolecule is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is an oligonucleotide (e.g., between 15 and 50 nucleotides in length). In some embodiments, the nucleic acid molecule is greater than 50 nucleotides in length, greater than 100 nucleotides in length, greater than 500 nucleotides in length, greater than 1 kb in length, greater than 2 kb in length, or greater than 5 kb in length. d. Conjugation of Fluorescent Dyes to Biomolecules After selection of an appropriate dye with the desired spectral characteristics, typically where the excitation wavelength is at least 580 nm, the dyes may be conjugated to a targeted carrier molecule, using methods well known in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra, (2002)). In many instances, conjugation to form a covalent bond consists of simply mixing the reactive compounds of the present invention in a suitable solvent in which both the reactive compound and the spacer molecule to be conjugated are soluble. The reaction, in many instances, proceeds spontaneously without added reagents at room temperature or below. For those reactive compounds that are photoactivated, conjugation is facilitated by illumination of the reaction mixture to activate the reactive compound. Chemical modification of water-insoluble substances, so that a desired compound-conjugate may be prepared, is, in many instances, performed in an aprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble materials is readily accomplished through the use of the instant reactive compounds to make them more readily soluble in organic solvents. Preparation of biomolecule (e.g., proteins) conjugates typically comprises first dissolving the biomolecule to be conjugated in aqueous buffer at about. 1-10 mg/mL at room temperature or below. For example, bicarbonate buffers (pH about 8.3), carbonate and borate buffers (pH about 9) are especially suitable for reaction with succinimidyl esters, phosphate buffers (pHs of about 7.2-8) for reaction with thiol-reactive functional groups and carbonate or borate buffers (pH about 9) for reaction with isothiocyanates and dichlorotriazines. The appropriate reactive compound is then dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in an amount sufficient to give a suitable degree of conjugation when added to a solution of the biomolecule to be conjugated. The appropriate amount of compound for any biomolecule (e.g., protein) or other component is conveniently predetermined by experimentation in which variable amounts of the compound are added to the biomolecule, the conjugate is chromatographically purified to separate unconjugated compound and the compound-biomolecule conjugate is tested in its desired application. Any number of buffers may be used for conjugation reactions, as well as for other set out herein. Using Examples 1 and 5 for purposes of illustration, phosphate buffered saline and borate buffer can be and are used in the conjugation reactions. It is also believed that carbonate buffer at pH 9.5 can be used. Along these lines, it is believed that higher pH conjugations require a lower molar excess of dye and spacer molecules. The invention thus includes compositions and methods for performing conjugation reactions where the pH is from about 4.0 to about 10.0 (e.g., from about 4.0 to about 10.0, from about 5.0 to about 10.0, from about 6.0 to about 10.0, from about 7.0 to about 10.0, from about 7.5 to about 10.0, from about 8.0 to about 10.0, etc.). Conjugation reactions may be performed in Borate Buffer 50 mM, pH 8.5 with a fluorescent dye, or a mixture of each fluorescent dye and a spacer selected from NHS-Acetate, NHS-MS(PEG)4, NHS-MS(PEG)8or NHS-MS(PEG)12at various molar excesses. The labeling reactions may be incubated for approximately 1 hour at room temperature (RT). The NHS activated dye and the NHS activated spacer agents may be combined before addition to the antibody so that both reactions are concurrent, allowing for a random spacing of the dye substitution and of the spacers. Buffers that may be used in the practice of the invention include 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (TRIS), borate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and carbonate buffers as examples. The concentration of the fluorogenic reagents used in the biomolecule (e.g., protein) labeling methods described herein are in the range from 50 nM to 100 mM (e.g., from about 50 nM to about 25 mM, from about 50 nM to about 10 mM, from about 50 nM to about 5 mM, from about 100 nM to about 10 mM, from about 100 nM to about 5 mM, from about 200 nM to about 5 mM, from about 50 nM to about 100 μM, from about 1 μM to about 100 μM, etc.). In certain embodiments such concentrations are obtained by dilution of a stock solution of the fluorogenic reagents having a concentration in the range from 100 nM to 200 mM (e.g., from about 10 μM to about 500 μM, from about 1 μM to about 100 μM, from about 100 μM to about 200 μM, etc.). In certain embodiments the concentration of the stock solution is 100 mM. In certain embodiments the concentration of the stock solution is 50 mM. In certain embodiments the concentration of the stock solution is 20 mM. In certain embodiments the concentration of the stock solution is 10 mM. In certain embodiments the concentration of the stock solution is 1 mM. In certain embodiments the concentration of the stock solution is 500 μM. In certain embodiments the concentration of the stock solution is 10 μM. In some embodiments, the concentration of the amine reactive fluorescent dyes used in the biomolecule (e.g., protein) labeling methods described herein are in the range from 50 nM to about 100 mM (e.g., from about 50 nM to about 50 mM, from about 50 nM to about 25 mM, from about 50 nM to about 10 mM, from about 50 nM to about 5 mM, from about 50 nM to about 5 μM, from about 50 nM to 100 μM, from about 100 nM to about 5 mM, etc.). In certain embodiments such concentrations are obtained by dilution of a stock solution of the fluorogenic reagents having a concentration in the range from 100 nM to 200 mM. In certain embodiments the concentration of the stock solution is 100 mM. In certain embodiments the concentration of the stock solution is 50 mM. In certain embodiments the concentration of the stock solution is 20 mM. In certain embodiments the concentration of the stock solution is 10 mM. In certain embodiments the concentration of the stock solution is 1 mM. In certain embodiments the concentration of the stock solution is 500 μM. In certain embodiments the concentration of the stock solution is from 10 μM to 500 μM. In certain embodiments the concentration of the stock solution is from 1 μM to 100 μM. In certain embodiments the concentration of the stock solution is 10 μM. In certain embodiments the concentration of the stock solution is from 100 μM to 200 μM. In some embodiments, the concentration of the proteins or protein fragments (e.g., antibody fragments) labeled using the methods described herein is in the range from 0.01 mg/mL to 200 mg/mL (e.g., from about 0.1 mg/mL to 100 mg/mL, from about 0.1 mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 10 mg/mL, from about 0.2 mg/mL to about 100 mg/mL, from about 0.2 mg/mL to about 50 mg/mL, from about 0.2 mg/mL to about 10 mg/mL, from about 0.3 mg/mL to 10 mg/mL, from about 0.4 mg/mL to about 10 mg/mL, from about 0.5 mg/mL to about 10 mg/mL etc.). In some instances, more than one dye molecule may be attached at each location on a biomolecule. One way of attaching more than one dye molecule at a single location on a biomolecule is through the use of conjugation molecules that bind to more than one dye molecule. These conjugation molecules are then connected to the biomolecule and carry with them multiple dye molecules. As an example, polymeric dendrimers, such as those set out in U.S. Patent Publication No. 2012/0256102, may be used multiple fluorescent dye molecules conjugated to a single polymeric backbone or core (referred to as “dendrimers” therein) for the attachment of these dye molecules to a biomolecule. These dendrimers may have regular or irregular branched polymeric network structures that allow for the chemical attachment of multiple dye molecules, multiple color dyes, and/or multiple functional groups, in a combinatorial fashion. Additional examples of conjugation molecules that may be used include many of the same molecules used as spacers. Thus, in some instances, the spacer molecule and the conjugation molecule will have the same structure with the exception that the conjugation molecule has dye molecules bound to it. Along these lines, various forms of PEG molecules may be used as conjugation molecules. Thus, the spacer molecule may contain the dye molecule (e.g., the fluorescent label) (seeFIG.18). The invention thus contemplates the use of conjugation molecules that each have, on average, from about two to about fifty (e.g., from about two to about forty-five, from about two to about forty, from about two to about thirty-five, from about two to about thirty, from about two to about twenty, from about five to about forty-five, from about ten to about forty-five, etc.) associated dye molecules. In many instances, the standard deviation in the average number of dye molecules bound to conjugation molecules will be less than 10%, 15%, and/or 20%. The degree of labeling may be measure for labeled biomolecules. Degree of labeling may be calculated as follows. First, the molarity of the labeled biomolecule is calculated using, for example, the formula: Proteinconcentration(M)=A280-(Amax×CF)ɛ×dilutionfactorε=protein molar extinction coefficient (e.g., the molar extinction coefficient of IgG is˜210,000 μM−1cm−1)Amax=Absorbance (A) of a dye solution measured at the wavelength maximum (λmax) for the dye moleculeCF=Correction factor; adjusts for the amount of absorbance at 280 nm caused by the dye (see Table 8)Dilution factor=the extent (if any) to which the protein:dye sample was diluted for absorbance measurement Molesdyepermoleprotein=Amaxofthelabeledproteinɛ′×proteinconcentration(M)×dilutionfactorε=molar extinction coefficient of the fluorescent dye TABLE 8Characteristics of Exemplary DyeWavelengthExtinctionCorrectionMaximumCoefficientFactorFluorophore(λmax)(ε−)(CF)DyLight ™ 350353 nm15,000M−1cm−10.1440DyLight ™ 405405 nm30,000M−1cm−10.5640DyLight ™ 488493 nm70,000M−1cm−10.1470DyLight ™ 550562 nm150,000M−1cm−10.0806DyLight ™ 594595 nm80,000M−1cm−10.5850DyLight ™ 633627 nm170,000M−1cm−10.1100DyLight ™ 650652 nm250,000M−1cm−10.0371DyLight ™ 680684 nm140,000M−1cm−10.1280DyLight ™ 755754 nm220,000M−1cm−10.0300DyLight ™ 800777 nm270,000M−1cm−10.0452Fluorescein494 nm68,000M−1cm−10.3000Isothiocyanate (FITC),NHS-Fluorescein,5-IAFTetramethyl-555 nm65,000M−1cm−10.3400rhodamine-5-(and6)-isothiocyanate(TRITC)NHS-Rhodamine570 nm60,000M−1cm−10.3400Texas Red ™ Sulfonyl595 nm80,000M−1cm−10.1800ChlorideR-Phycoerythrin566 nm1,863,000M−1cm−10.1700AMCA-NHS, AMCA-346 nm19,000M−1cm−10.1900Sulfo-NHS orAMCA-Hydrazide In some embodiments of the invention, enhanced fluorescence is observed for biomolecules comprising spacers with lower degree of labeling (DOL) than biomolecules without spacers. As an example, assume there is antibody that has been labeled with a fluorescent dye separately with and without a spacer. On an equivalent DOL basis, the antibody labeled with the dye that also has spacers bound to it may exhibit an enhancement in fluorescence of between 1.5 and 3.5 times, where 1 would present the same level of fluorescence for both antibodies. The point being that the amount of fluorescent signal on a per dye molecule basis increases for biomolecules bound to both dye and spacer. Following addition of the reactive compound to the component solution, the mixture is incubated for a suitable period (typically about 1 hour at room temperature to several hours on ice), the excess compound is removed by gel filtration, dialysis, HPLC, adsorption on an ion exchange or hydrophobic polymer or other suitable means. The compound-conjugate is used in solution or lyophilized. In this way, suitable conjugates can be prepared from antibodies, antibody fragments, and other targeting carrier molecules. The incubation temperatures used in the methods described herein can be room temperature, ambient temperature, or temperatures above room temperature, such as, by way of example only, at least about 26° C., 27° C., 28° C., 29° C., 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., even as high as 90° C., 95° C., 96 C, 97°, 98° C., 99° C. or 100° C. The first incubation temperature and the second incubation temperature used in the methods described herein can be the same or different. In some embodiments, the first incubation temperature is between 20° C. and 80° C., between 25° C. and 30° C., and/or at ambient or room temperature. In some embodiments, the second incubation temperature is between 20° C. and 80° C., between 65° C. and 75° C., and/or at approximately 70° C. In other embodiments, the second incubation temperature is at ambient or room temperature. The incubation times used in the methods described herein include, but are not limited to, for at least 30 seconds, at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, at least 1 hour, or any range herein. The first incubation time and the second incubation time used in the methods described herein can be the same or different. In one embodiment, the first incubation time is 0 to 60 minutes, 5 to 10 minutes, and/or at 5 to 10 minutes at room temperature. In some embodiments, the second incubation time is 0 to 20 minutes and/or approximately 10 minutes. In specific instances, the second incubation time is approximately 10 minutes at approximately 70° C. In other embodiments, the first incubation time is 1 to 3 hours at 25° C., the second incubation time is overnight at 25° C. and the third incubation time is 2 to 3 hours at 37° C. Conjugates of polymers, including biopolymers and other higher molecular weight polymers are typically prepared by means well recognized in the art (for example, Brinkley et al.,Bioconjugate Chem.,3:2 (1992)). In these embodiments, a single type of reactive site may be available, as is typical for polysaccharides) or multiple types of reactive sites (e.g., amines, thiols, alcohols, phenols) may be available, as is typical for proteins. Selectivity of labeling is best obtained by selection of an appropriate reactive dye. For example, modification of thiols with a thiol-selective reagent such as a haloacetamide or maleimide, or modification of amines with an amine-reactive reagent such as an activated ester, acyl azide, isothiocyanate or 3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained by careful control of the reaction conditions. When modifying polymers with the compounds, an excess of compound is typically used, relative to the expected degree of compound substitution. Any residual, unreacted compound or a compound hydrolysis product is typically removed by dialysis, chromatography or precipitation. Presence of residual, unconjugated dye can be detected by thin layer chromatography using a solvent that elutes the dye away from its conjugate. In all cases it is usually the case that the reagents be kept as concentrated as practical so as to obtain adequate rates of conjugation. In certain embodiments of the methods described herein a control protein or proteins may be labeled to monitor the effectiveness of labeling, either in a parallel reaction or, if readily resolvable from the protein desired to be labeled, by inclusion in the same reaction. In certain embodiments of the methods described herein, the proteins of the labeled sample can usefully be resolved in parallel with a series of fluorescent molecular weight standards. Usefully, the standards are spectrally matched to at least one fluorophore used to label the proteins. Such spectral matching can be accomplished, for example, by using tetracysteine-tagged protein standards that are labeled in parallel with the same biarsenical fluorophore used to label the protein sample, or by using standards having a fluorescent moiety that is spectrally matched to the biarsenical fluorophore or other fluorophore used to label the sample proteins. Examples of standards useful in the practice of the present invention include the BENCHMARK™ family of protein standards (Life Technologies Corp., Carlsbad, Calif.) and MARKL2™ Unstained Standard (Life Technologies Corp., Carlsbad, Calif.). The methods and compositions described herein can also be used to quantitate the amount of fluorescently labeled protein present in a sample. In certain embodiments, the methods described herein further comprise quantitating the amount of fluorescence from the biarsenical fluorophore. In certain embodiments, the methods described herein further comprise quantitating the amount of fluorescence from the amine reactive fluorescent dye. In certain embodiments, the methods described herein further comprise quantitating the amount of fluorescence from the fluorescent moiety from the amine reactive fluorogenic reagent. The quantitation can be done without resolution of the proteins present in the protein sample or after the proteins have been partially or fully resolved, as by electrophoresis, such as PAGE, 2D-PAGE, or IEF or chromatography or combinations thereof. e. Conjugation of Spacer Molecules to Biomolecule In some embodiments, the spacer molecule is conjugated to the biomolecule using NHS-ester chemistries are described in the examples herein, other chemistries such as maleimide, pyridyl disulfide and hyrazides, as well as the SITECLICK™ technology involving azide/alkyne may also be used for this conjugation strategy. In some embodiments, the spacer molecule is conjugated to a biomolecule (e.g., an antibody) at primary lysine side chains present on a protein, such as an antibody. In some embodiments, the concentration of a protein, protein fragment or other biomolecule labeled using the methods described herein is in the range from about 0.01 mg/mL to about 200 mg/mL (e.g., from about 0.1 mg/mL to about 100 mg/mL, from about 0.1 mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 10 mg/mL, from about 0.2 mg/mL to about 100 mg/mL, from about 0.2 mg/mL to about 50 mg/mL, from about 0.2 mg/mL to about 10 mg/mL, from about 0.3 mg/mL to about 10 mg/mL, from about 0.4 mg/mL to about 10 mg/mL, from about 0.5 mg/mL to about 10 mg/mL, etc.). In some embodiments, the dye to protein ratio of the fluorescent label to the antibody is between 1 and 50. In some embodiments, the dye to protein ratio of the fluorescent label to the antibody is between 5 and 30. In some embodiments, the dye to protein ratio of the fluorescent label to the antibody is between 1 and 20. In some embodiments, the spacer to protein ratio is between 1 and 50. In some embodiments, the spacer agent to protein ratio is between 5 and 30. In some embodiments, the spacer agent to protein ratio is between 1 and 20. In some embodiments, the spacer agent is added in molar excess to the plurality of fluorescent labels in an amount between 0.1 to 25, between 1 to 15, or between 2.5 to 10 fold. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 2.5 fold. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 5 fold. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 7.5 fold. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 10 fold. In some embodiments, the spacer agent is conjugated to a nucleic acid molecule. Protocols for conjugating moieties (e.g., fluorescent labels) to nucleic acids are described in the art (see. e.g., Rombouts et al.,Bioconjugate Chem.,27:280-207 (2016)). One method for labeling nucleic acid molecules is through the use of the ARES™ ALEXA FLUOR™ 488 DNA Labeling Kit (Thermo Fisher, cat. no. A21665). Amine-modified nucleotides may also be used to enable labeling of nucleic acid molecules. For example, 5-aminohexylacrylamido-dUTP (aha-dUTP) and 5-aminohexylacrylamido-dCTP (aha-dCTP) can be used to produce amine-modified DNA by conventional enzymatic incorporation methods such as reverse transcription, nick translation, random primed labeling or PCR. The amine-modified DNA can then be labeled with any amine-reactive dye or hapten. This two-step technique consistently produces a uniform and high degree of DNA labeling that is difficult to obtain by other methods. One method for achieving a high DOL, with spatially separated fluorescent labels, is set out inFIG.18.FIG.18shows the preparation of fluorescently labeled branched PEG molecules and the attachment of the resulting PEG molecules (direct spacers) to an antibody, where the PEG molecules are covalently linked to the antibody by CLICK-IT™ reactions (see Example 7). The PEG molecules shown inFIG.18are each covalently linked to seven fluorescent labels. Further, the fluorescent labels may be attached to the individual PEG molecules in a manner such that the labels have a specified “brush” length with respect to the labeled molecule. “Brush length” refers to the extended length of chemical groups that attach a fluorescent labels to a labeled molecule. As an example, assuming that the average monomer length in a PEG molecule is about 3.5 angstroms, when n=1 inFIG.18, then the brush length of the fluorescent labels would range from about 5 to about 10 angstroms. In many instances, n inFIG.18will not be 1. Further, PEG molecules typically vary in size and are described based upon an average molecular weight. Thus, using the PEG molecules set out inFIG.18for purposes of illustration, a population of PEG molecules with an average weight of 10,000 would have an n of around 30. Also, a population of PEG molecules with an average weight of 40,000 would have an n of around 120. In view of the fact that both the arm of a branched PEG molecule that links this molecule to the labeled molecule and the other arm all have a repeating region, the brush lengths may range from about 15 angstroms to about 800 angstroms (e.g., from about 25 to about 800, from about 150 to about 800, from about 450 to about 800, from about 600 to about 800, from about 65 to about 800, from about 25 to about 700, from about 40 to about 700, from about 28 to about 600, from about 28 to about 500, from about 70 to about 700, etc.). Thus, the invention includes compositions and methods for producing and using molecules that are covalently linked to at least one fluorescent label, wherein the fluorescent label(s) has a brush length of from about 22 angstroms to about 800 angstroms. As an alternative, compositions of the invention may be described by the number of covalent bonds between the molecules and the fluorescent label to which they are linked. Again using the branched PEG molecule set out inFIG.18for purposes of illustration, the number of intervening covalent bonds would be between about 24 and about 32, depending on where the fluorescent label is attached to the branched PEG molecule. The invention includes compositions and methods for producing and using molecules that are covalently linked to at least one fluorescent label, wherein the fluorescent label(s) are connected to the molecule by from about 16 to about 800 (e.g., from about 16 to about 700, from about 32 to about 800, from about 60 to about 800, from about 100 to about 800, from about 150 to about 800, from about 200 to about 800, from about 250 to about 800, from about 250 to about 700, from about 250 to about 650, from about 250 to about 600, from about 350 to about 800, etc.) intervening covalent bonds. The invention also relates, in part, to spacing out of multiple fluorescent labels attached to the same location on individual labeled molecules. Similar to what is set out above with respect to the separation of fluorescent labels from labeled molecules, multiple fluorescent labels attached to the same location on individual labeled molecules may be separated from each other by distances of from about 15 angstroms to about 800 angstroms (including the ranges set out above) and/or by from about 16 to about 800 (including the ranges set out above) intervening covalent bonds. As noted herein, aspects of the invention relate to the spacing of fluorescent labels. Table 9 shows estimated characteristics of a series of branched PEG molecules. It should be understood that these PEG molecules, as well as other molecules, exist in multiple formats at different times. For example, arms of a branch PEG molecule may be fully extended (brush) or fully coiled (mushroom), as well as effectively all conformations in between. Further, each arm may be in a different state of extension or coiling independent of other arms at any one time. Thus, in Table 9, the term “brush” refers to fully extended PEG arms and the term “mushroom” refers to fully coiled PEG arms. The mushroom state is modeled upon a very large Flory radius (distance between adjacent PEG arms) of 80 angstroms to get the smallest number. F-F distances in Table 9 are distances between two fluorophores on the termini of different arms, assuming all of the fluorophores are equidistant from each other. The maximum distance is double the arm length. Further, the nearest neighbor value assumes tetrahedral arrangement for 4 arms and spherical/cubic for 8 arms. Of course, the actual value of F-F distances at any one time point will typically be somewhere in between the brush distances and the mushroom distance. TABLE 9Branched PEG Arm Length (Estimated Fluorophore to Fluorophore (F—F) Distance)F—FF—FArmArmMaxNearestMaxNearestLengthLengthF—FNeighborF—FNeighborArmsEG/(Å)(Å)Dist.(Å)Dist. (Å),Dist. (Å),Dist. (Å),MWArmsMWArm*(BAL)**(MAL)**BAL (3)Brush (3)MAL (4)MAL (4)10000425006020826417340524210000812503010413208120261520000825006020826417241523040000850001194175283348110460*EG/Arm = Arm MW/EG MW ~ Arm MW/42 (MW = Molecule Weight)**Brush Arm Length (BAL) = EG/Arm * monomer length ~ EG/Arm * 3.5**Mushroom Arm Length (MAL), as determined by the equation below (L = . . .)(3) Fully extended, end of arms equidistant from each other (Dist. = Distance)(4) Fully coiled, end of arms equidistant from each otherThese calculations are based upon the JenKem following products: 4ARM-NH2HCl and 8ARM-NH2HClL=Na5/3D2/3a = 0.35 nm (3.5 A)N = 30, 60, 120D = 80 A Dextrans are another suitable material for connecting fluorescent labels to molecules. Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. Dextrans tend to be biologically inert due to their uncommon poly-(α-D-1,6-glucose) linkages. These linkages render them resistant to cleavage by most endogenous cellular glycosidases. They also usually have low immunogenicity and are generally branched molecules. Dextrans are commercially available with nominal molecular weights (MW) varying between 3000 daltons and 2,000,000 daltons. Dextrans suitable for use in the practice of the invention may be of any number of different molecule weights, including 3000, 10,000, 40,000, 70,000, 500,000, and 2,000,000 daltons (e.g., from about 4,000 to about 150,000, from about 6,000 to about 150,000, from about 8,000 to about 150,000, from about 15,000 to about 150,000, from about 10,000 to about 80,000, from about 12,000 to about 70,000, etc.). Dextrans usually have a degree of substitution (e.g., a DOL) of 0.2 to 2 of dye molecules per dextran molecule for dextrans in the 10,000 MW range. Further, dextrans typically contain 0.2-0.7 dyes per dextran in the 3000 MW range, 0.4-2 dyes per dextran in the 10,000 MW range, 1-4 dyes in the 40,000 MW range and 2-6 dyes in the 70,000 MW range. Thus, dextrans, as well as other labeled polymers, used in the practice of the invention may have a DOL ranging from about 0.03 to 0.3 fluorescent labels per 1,000 MW (e.g., from about 0.03 to about 0.25, from about 0.08 to about 0.3, from about 0.09 to about 0.3, from about 0.1 to about 0.3, from about 0.05 to about 0.2, from about 0.07 to about 0.25, etc. per 1,000 MW). Dextrans, as well as other polymers, may be labeled in a number of ways. For example, fluorescently labeled dextrans may be prepared by the reaction of a water-soluble amino dextran with a fluorescent label having a succinimidyl ester group. Fluorescently labelled dextran may also be prepared by the reaction of a native dextran with an isothiocyanate derivative of a fluorescent label such as FITC. Where appropriate, once the fluorescent label has been added, unreacted amines on the dextran may be capped to yield a neutral or charged dextran (i.e., negatively or positively charged). Further, even where capping is not performed, charged fluorescent labels may be used which can render dextrans anionic or cationic. Another type of polysaccharide that can be useful in the practice of the invention is amylose. Amylose is a linear polysaccharide composed of α-D-glucose units linked by α-1,4-glycosidic bonds. Due to hydrogen bonding, amylose tends to form spiral structures containing six glucose units per turn. Molecules of this type provide structure regularity that can be used space out fluorescent labels in consciously designed manner. The invention thus includes compositions, as well as methods for making and using such compositions, with fairly static structural features that may be employed for maintaining a uniform distance between fluorescent labels. In many instances, the distance between two fluorescent labels associated with such a molecule in a manner and under conditions that would result in two fluorescent labels would not vary in distance from each other by more than 30% (e.g., from about 5% to about 30%, from about 10% to about 30%, from about 15% to about 30%, from about 20% to about 30%, from about 10% to about 20%, etc.). Polypeptides may also be used in the practice of the invention. One exemplary such molecule is the branched polylysine molecule shown inFIG.21. This molecule may be made according to methods set out in U.S. Patent Publication No. 2010/0278750. A number of “R groups” are shown inFIG.21, representing locations where fluorescent labels may be attached. In such a molecule the R groups may be the same or different (e.g., R1and R2). Further, when a number of R groups are the same, these groups may act as fluorescent label attachments points where the R groups are not labeled to completion. By way of example,FIG.21shows a polymer with 33 R groups, 32 R1groups and 1 R2group. Assume that all of the R groups are of the same type. These R groups may be partially fluorescently labeled in a semi-random manner. By this is meant that conditions may be provided such that only a percentage of the R group receive a fluorescent label. Further, the labeling would be semi-random because some R group, due to their position in the polymer, would be more prone to receive a fluorescent label. Thus, the invention includes the design of fluorescently labeled polymers where the positioning and DOL are adjusted for a desired fluorescent effect In some instances, the average number of available attachment site on a per polymer basis that receive labels is in the range of 10% to 90% (e.g., from about 10% to about 85%, from about 15% to about 85%, from about 20% to about 85%, from about 20% to about 75%, from about 30% to about 75%, from about 30% to about 80%, from about 40% to about 80%, etc.). As noted above,FIG.21shows a polymer with R1and R2groups. If “directional” linkage of the polymer to a biomolecule (e.g., an antibody) is desired, the R2group may be different from the R1groups. If the R groups are the same, then attached would be “non-directional” in the sense that any R group in a suitable location on the polymer could serve as a conjugation point to the biomolecule. Of course, conditions would be adjusted to achieve a high level of fluorescence, while maintaining a high level of biological activity for the biomolecule (e.g., antigen binding). Maximization of biomolecule fluorescence may be partially independent of the number of fluorescent labels on the biomolecule of interest. Assume for example that there are seven fluorescent labels on a particular antibody, when labeled under a first set of conditions, and ten fluorescent labels on the same antibody, when labeled under the second set of conditions. Further assume that the amount of total fluorescence of the antibody labeled under the first set of conditions is greater than the amount of total fluorescence of the antibody labeled under the second set of conditions. In this instance, fewer fluorescent labels resulted in more fluorescence. Thus, other factors being equal (e.g., functional activity of the biomolecule), the first set of conditions would be preferred over the second set of conditions. Any number of linking groups may be used to attach fluorescent labels to biomolecules. These attachments may be non-covalent or covalent. Further, non-covalent or covalent can refer to one or all of (1) the linking of the fluorescent label to a polymer, (2) the linking of the polymer to a core (when present), and/or (3) the linking of the polymer or the core, when present, to the biomolecule. In many instances, polymers (with or without cores), as well as other types of spacers set out herein, may serve the function of increasing the fluorescent intensity of fluorescent labels attached to a biomolecule. One category of molecules that are useful in the practice of the invention are referred to as star polymers (see Ren et al.,Star Polymers. Chemical Reviews.116:6743-6836 (2016)). Star polymers are multi-arm molecules that contain a core and a series of linear polymers (referred to as “arms”). These arms typically contain terminal functionality to facilitate attachment of other molecules. Star polymers may be classified as homo-arm (containing arms of only one composition) or mikto-arm (containing arms of more than one composition, molecular weight or terminal functionality). Synthesis of star polymers is typically carried out using either core first, arm first or grafting onto approaches. The core effectively serves as a branching point for the arms. Any number of molecules can serve as cores for compositions of the invention. Examples of suitable cores include oligoglycerols (e.g. hexaglycerol), oligoerythritols (e.g., pentaerythritol, dipentaerythritol, tripentaerythritol), sorbitol, trimethylolpropane, silanes (e.g. 1,2-bis(methylsilyl)ethane), adamantanes, PAMAM (1-, 2-, and 3-generation (G-1-3) poly(amidamine)) dendrimers, polyethylene imine (PEI) branched polymers, peptides (e.g., polylysine, polyaspartic acid, etc.). The core may already have functional groups for initiation of polymerization or linking onto of arms or, as shown inFIG.20, may require chemical modification in order to facilitate the attachment of arms. Using hexaglycerol as an example, this compound is available commercially and may be used in the core first synthesis of star-shaped PEG polymers. StarPEGs with hexaglycerol cores may be used for the controlled release of drugs and for wound sealing. StarPEGs with hexaglycerol cores may be produced by the controlled polymerization of ethylene oxide from the hexaglycerol core. In this instance, the core is hexaglycerol and the arms are polyethylene glycol. The arms may be composed any number of linear polymers and will typically function as attachment points for fluorescent labels and for spacing these labels out from each other, as well as spacing the labels out from other fluorescent labels attached to the same labeled molecule (e.g., a biomolecule). Examples of suitable arms include polyethylene glycol, poly(vinyl pyrrolidone), polyglycerol, and polyvinyl alcohol, zwitterionic polymers (e.g. polysuflo betaines) as well as water soluble polymers. In many instances, polymers suitable for use in the invention will be non-charged. One area of antibodies that is particularly useful for the attachment of fluorescent labels, when present, is the Fc (fragment crystallizable) region. The Fc region is at the far end of antibodies with respect to the antigen bind site(s). This region of the antibody interacts with cell surface receptors called Fc receptors and proteins of the complement system. Typically, attachment of chemical entities at or near the Fc region has little impact on antigen binding by the antibody. However, interference with antigen binding events tends to increase with the size of chemical entities attached to the antibody. Further, large attached chemical entities tend to interfere with each other, due to steric hindrance, with respect to the ability to attach to a biomolecule. Thus, a series of factors may need to be balanced in order to produce a biomolecule (e.g., an antibody) with both a high level of fluorescence and a high level of functional activity (e.g., antigen binding ability). Some of these factors are as follows: (1) Size of the biomolecule, (2) location of attachment points on the biomolecule for fluorescent labeling, and (3) the size(s) and three dimensional structures of the fluorescent labeled molecules being attached to the biomolecules. With respect to antibodies, the invention includes antibodies that contain one or more of the following features:Fluorescently labeled polymers bound (e.g., covalently bound) at from an average 2 to 10 different locations on the antibody molecules.An average of from 3 to 80 fluorescent labels attached to each of the antibody molecules.Antigen binding affinity (KD) of the fluorescently labeled antibody molecule is decreased by no more than two (e.g., from about 0.5 to about 2.0, from about 1.0 to about 2.0, from about 0.5 to about 1.5, from about 0.75 to about 1.5, etc.) orders of magnitude as compared to the unlabeled form of the antibody.The average amount of fluorescent emission on a per fluorescent label basis bound to the antibody molecules is at least 60% (e.g., from about 60% to about 98%, from about 70% to about 98%, from about 80% to about 98%, from about 85% to about 98%, from about 80% to about 93%, etc.) that of the free fluorescent label. f. Biomolecule/Fluorescent Label/Spacer Combinations The invention is based, in part, on combinations of three components: Biomolecules, fluorescent labels, and spacers. With respect to proteins (e.g., antibodies), groups that may be used as attachments sites for fluorescent labels and spacers may not always be accessible for attachment, especially when the protein has not be denatured. Further, in many instances, it will be desirable to maintain proteins in undenatured form. Enhancement in fluorescent emission have been found to relate to various ratios of fluorescent labels and spacers used to label biomolecules (e.g., antibodies). Further, each biomolecule has the potential for requiring different ratios of components in the conjugation process to yield specified enhanced fluorescence levels. This may be due to different structure (e.g., primary, secondary, tertiary and quaternary structure) of biomolecules, such as antibodies, as well as characteristics of the particular fluorescent label and the particular spacer. In some instances, ratios may be based upon the respective weights of components. In other instances, ratios may be based upon molar ratios. A number of the figures and example of this application relate to molar ratios. In some instances (e.g., where the biomolecule is large and with a large number of conjugation sites), the use of component weights may be more suitable. For antibodies, as well as other biomolecules, the following ratios of biomolecule to fluorescent label to spacer used in the conjugation process may vary greatly but in most instances the amount of biomolecule will be lower than the amount of both fluorescent label and spacer. Further, the density and/or spacing of conjugation sites on a biomolecule is one factor that will often determine the optimum ratio of fluorescent label to spacer. This is so because, assuming enhanced fluorescence is due to reduced quenching, the lower the total or regional density of conjugations sites, the lower the amount of quenching would be expected to be. In any event, ratios of biomolecule to fluorescent label to spacer that may be used can be described as follows: B1:FL2-30:S2-20, where B is biomolecule, FL is fluorescent label and S is spacer. Specific ranges of ratios that may be used in the practice of the invention include ratios that fall within 1:2-25:2-20 (e.g., from about 1:2:2 to about 1:25:20, from about 1:5:2 to about 1:25:5, from about 1:10:2 to about 1:25:5, from about 1:5:2 to about 1:15:10, from about 1:10:5 to about 1:25:20, from about 1:10:5 to about 1:15:20, from about 1:10:5 to about 1:20:20, etc.), wherein the first number is the amount (e.g., moles) of biomolecule, the second number is the amount of fluorescent label, and the third number is the amount of spacer. In some instances, fluorescent label and spacer concentrations used for conjugation will be such that available attachments sites on biomolecules will be effectively saturated (e.g., at least 95% of available attachments sites will have bound thereto either a fluorescent label or a spacer). In such instances, the fluorescent label and spacer ratio may be a determining factor in the level for fluorescent enhancement. In many instances the ratio of fluorescent label to spacer will be between 10:1 to 10:50 (e.g., from about 10:1 to about 10:25, from about 10:1 to about 10:10, from about 10:1 to about 10:5, from about 10:5 to about 10:50, from about 10:5 to about 10:20, from about 10:3 to about 10:30, from about 10:5 to about 10:30, from about 10:10 to about 10:25, etc.). Spacers and dyes may be conjugated to a biomolecule at the same time or sequentially, with either the spacer or the dye being conjugated to the biomolecule first. In many instances, when the spacer and dye connect to the biomolecule at the same loci, they will be conjugated to the biomolecule at the same time. However, sequential conjugation could be used when the first conjugation reaction (e.g., of the spacer) is done under conditions where the binding sites are the biomolecule are not saturated, thus leaving binding sites available for a second conjugation reaction (e.g., of the dye). g. Buffers In some embodiments, the composition of the invention comprises one or more spacer, one or more fluorescent label in a buffer. Any of the fluorescent and spacer molecules disclosed herein may be used together with any buffer known in the art. In some embodiments, the compositions disclosed herein comprise any suitable buffer for a molecular biology application. In some embodiments, the buffer is a suitable storage buffer (e.g., borate buffer, phosphate buffer or carbonate buffer). In some embodiments, the buffer is suitable for buffering a detectable biomolecule, as disclosed herein, during use in a detection assay. Methods The instant invention has useful applications in basic research, high-throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, diagnostics, and medical therapeutics. The invention can be used in a variety of assay formats for diagnostic applications in the disciplines of microbiology, immunology, hematology and blood transfusion, tissue pathology, forensic pathology, and veterinary pathology. In some embodiments, the compositions described herein can be used in any molecular biology application wherein fluorescently labeled molecules are detected. For example, the detectable biomolecules, as disclosed herein, can be used in Western blotting, ELISA, flow cytometry, flow cytometry and applications involving FRET. The detectable biomolecules, as disclosed herein, can also be used in fluorescent immunohistochemistry (IHC), fluorescent immunocytochemistry (ICC), and in vivo imaging applications. In some embodiments, a method for determining the presence of a desired target in a biological sample is encompassed, the method comprising: a) contacting the biological sample with a composition comprising one or more fluorescent label, and one or more spacer molecules, wherein the spacer and fluorescent label are conjugated to the biomolecule but not to each other, b) detecting fluorescence emitted by the plurality of fluorescent labels; and c) determining the presence of the desired target in the biological sample when fluorescence emitted by the plurality of fluorescent labels is detected. In some embodiments, the biological sample comprises cell lysate. In some embodiments, the biological sample comprises intact cells. In some embodiments, the biological sample comprises a tissues sample. Further such tissue samples may be fixed. Further, compositions of the invention may be used for applications such as immunohistochemistry. In some embodiments, the biological sample comprises isolated protein. In some embodiments, the biological sample comprises recombinant protein. In some embodiments, the biological sample is immobilized on a solid support. In some embodiments, the biological sample comprises intact cells in fluid. In some embodiments, the biological sample is a live animal. In some embodiments, the live animal is a mammal. In some instances, the sample comprises tissues such as liver, lung, muscle and skin. In some embodiments, disclosed herein is a method for imaging a target antigen in a living body, wherein the method comprises; a) providing an antibody conjugated to a plurality of fluorescent labels and a spacer agent, as disclosed herein, that binds to the target antigen; b) introducing the antibody into the body to form a contacted body; c) illuminating the contacted body with an appropriate wavelength to form an illuminated body; and d) observing the illuminated body wherein the target antigen is imaged. In some embodiments, these antibodies or other targeted proteins or peptides specific for a target or antigen in a living body that has been conjugated with a fluorescent dye(s) having an excitation wavelength compatible with in vivo imaging, typically about 580 nm to about 800 nm. The target specific dye conjugates travel relatively freely within the circulating blood until their preferential sequestration occurs at a target pathological or non-pathological tissue sites such as a diseased or injury tissue sites. h. Kits The compositions of the invention may be incorporated into kits that facilitate the practice of various assays. The kits may be packaged with the composition in a dry form or with the composition in solution. The kits may optionally further include one or more buffering agents, typically present as an aqueous solution, sample preparation reagents, additional detection reagents, organic solvent, other fluorescent detection probes, standards, microspheres, specific cell lines, antibodies and/or instructions for carrying out an assay. Additional optional agents include components for testing of other cell functions in conjunction with the compound. In some embodiments, the kits comprise a biomolecule, spacer agent, and a fluorescent label, as disclosed herein. In some embodiments, the kit further comprises a buffer. In some embodiments, the biomolecule is already conjugated to the spacer agent and the fluorescent label. In some embodiments, the kit may comprise a polymer conjugated to fluorescent labels and a reactive group as a bioconjugation kit. Kits of the invention may further contain reagents used to prepare fluorescently labeled biomolecules (e.g., antibodies). Exemplary reagents include one or more of the following: fluorescent dyes, spacers that are either prelabeled or labeled according to instructions provided in the kits, and compounds that may be used to conjugate (1) spacers to biomolecules and/or (2) fluorescent labels to biomolecules. This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. EXAMPLES The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. Example 1: Fluorescent Western Blotting Methods a.) Antibody Labeling Using NHS Activated Fluorescent Dyes and Sulfo NHS-Acetate/NHS-Acetate and NHS-MS(PEG)4, NHS-MS(PEG)8and NHS-MS(PEG)12 NHS activated fluorescent dyes, such as DYLIGHT™ 650-4×PEG, were reconstituted in dimethylformamide (DMF) at 10 mg/ml. NHS-Acetate was prepared fresh in dimethyl formamide (DMF) at 1 mg/ml. NHS-MS(PEG)4(Cat. no. 22341 (Thermo Fisher Scientific), NHS-MS(PEG)8(cat. no. 22509 (Thermo Fisher Scientific), NHS-MS(PEG)12(cat. no. 22686 (Thermo Fisher Scientific), were reconstituted in DMF at 100 mg/ml. The PEG Reagents were further diluted to 1 mg/ml in DMF just prior to use. 1 mg of Goat Anti-Mouse (GAM) and Goat Anti-Rabbit (GAR) antibody at 7-10 mg/ml in Borate Buffer 50 mM (cat. no. 28384 (Thermo Fisher Scientific), pH 8.5 were labeled with each fluorescent dye, or a mixture of each fluorescent dye and a spacer selected from NHS-Acetate, NHS-MS(PEG)4, NHS-MS(PEG)8or NHS-MS(PEG)12at various molar excesses. The labeling reactions were incubated for approximately 1 hour at room temperature (RT). The NHS activated dye and the NHS activated spacer agents were combined before addition to the antibody so that both reactions were concurrent, allowing for a random spacing of the dye substitution and of the spacers. 100 mM MES Buffer at pH 4.7 was added to each sample to lower the pH from 8.5 to approximately 7.2. At this point the concentration of the conjugates was adjusted down to approximately 6 mg/ml to accommodate the final dilution in the storage buffer. The free dye was removed using the Dye Removal Resin (Thermo Fisher Scientific, cat. no. 22858) and 5 m Harvard columns (Harvard Apparatus, cat. no. 74-3820). 0.2 ml of the 50% purification resin slurry was used per mg of protein. The conjugates were diluted 1:50 with 0.1M Sodium Phosphate Buffer, pH 7.2 (PBS) and scanned using the UV Cary Spectrophotometer. OD scans (252 nm to 900 nm) were used to determine concentrations of conjugates and calculate mole dye/mole protein ratios (D/P). Finally, the conjugates were diluted to 1 mg/ml in STABILZYME™ NOBLE Storage Buffer (Surmodics, cat. no. SZ04) for long term storage. Serially diluted cell lysates (500 ng to 2 ng) were combined with SDS-PAGE sample buffer. The samples were heated for five minutes at 95° C., and loaded onto Thermo Fisher Scientific Tris Glycine SDS-PAGE gels (Novex Gels, 4-20%, 10-well, cat. no. WT4202BX10). Gels were electrophoresed according to manufacturer's instructions and then transferred to nitrocellulose membranes using a semi-dry transfer method. The membranes were blocked for thirty minutes with SEA BLOCK™ Blocking Buffer (Thermo Fisher Scientific, cat. no. 0037527). The primary antibodies were prepared to a final concentration of 0.1 to 2.5 μg/ml in SEA BLOCK™ Blocking Buffer. The blots were incubated with the antibody prepared in the SEA BLOCK™ Blocking Buffer for one hour with shaking at room temperature (RT). The antibody solution was decanted and the membranes were washed two times for ten minutes in 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.2 (TBST). The fluorescently labeled secondary antibody conjugates were diluted to a final concentration of 20 to 1000 ng/mL in SEA BLOCK™ Blocking Buffer. The washed membrane was incubated with the relevant secondary antibody conjugate with agitation for 30 to 60 minutes. The buffer was decanted and the membranes were washed six times for five minutes with TBST. The membranes were imaged using a compatible fluorescent imager. Results are shown inFIG.8for an experiment testing the effect of addition of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(3.75×) spacer to a GAM secondary antibody conjugated to DYLIGHT™ 488 at 5×-20× molar excess in a western blotting assay. A431 cell lysate was diluted 3-fold from 1 μg/well. The primary antibody rabbit used was anti-Hsp90 diluted 1/5000 from 1 mg/ml. All DYLIGHT™ secondary antibodies were diluted 1/5000 from 1 mg/ml stock. In a Western blot application, there is noticeable increase of fluorescent intensity over the base conjugate (made without the spacer) at each dye molar excess from 7.5× to 20× for DYLIGHT™ 488-GAR conjugated with the addition of NHS Acetate or MS(PEG)4. Results demonstrating the effect of addition of NHS Acetate (5×) or MS(PEG)4(5×) spacer to a GAM secondary antibody conjugated to DYLIGHT™ 650-4×PEG (at 7.5× Dye) in a Western blot assay are shown inFIG.9. HeLa cell lysate was diluted 4-fold from 0.5 μg/well. Primary antibody mouse anti-PDI was diluted to 1/5000 of 1 mg/ml. All DYLIGHT™ secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. NHS acetate added at 5× molar excess to GAM-DYLIGHT™ 650-4×PEG-7.5× conjugation improved intensity by 1.5-fold. NHS acetate added at 3.75× molar excess to GAM-DYLIGHT™ 650-4×PEG-7.5× conjugation improved intensity by 1.4-fold. Results demonstrating the effect of addition of NHS Acetate (2.5×, 5×) and MS(PEG)4spacer to a GAR-DYLIGHT™ 800-4×PEG secondary antibody in a Western blot assay are shown inFIG.10and Table 10. A431 cell lysate was serially diluted 1:1. Primary antibodies rabbit anti-Hsp90 and anti-Cyclophilin B were diluted 1/5000. All DYLIGHT™ secondary antibodies were diluted to 1/20,000 of 1 mg/ml stock. The addition of MS(PEG)4(3.75× and 5×) and NHS Acetate (2.5-5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 800-4×PEG conjugate by 20-100% at different molar excesses of the dye in this Western blotting application. TABLE 10Effect of ±NHS Acetate (2.5X, 5X) or MS(PEG)4(5X) on GAR-DYLIGHT ™ 800-4xPEG in Western Blot Assays+2.5X NHS+5X NHS+5XFold Improvement (WB)NAAcetateAcetateMS(PEG)4GAR-DYLIGHT ™ 800-1.01.41.21.84xPEG_5xGAR-DYLIGHT ™ 800-1.01.51.21.74xPEG_7.5xGAR-DYLIGHT ™ 800-1.01.71.51.94xPEG_10xGAR-DYLIGHT ™ 800-1.01.91.52.04xPEG_15xNA = No Addition Results demonstrating the effect of addition of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(5×) and MS(PEG)8(5×) spacer to a GAM-DYLIGHT™ 550-2×PEG conjugate (at 12.5× molar excess of dye) in a western blotting assay are shown inFIG.11. HeLa cell lysate was diluted 4-fold from 0.5 μg/well and was stained with anti-PDI primary antibody diluted to 1/5000 of 1 mg/ml. All DYLIGHT™ secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. This experiment showed that the addition of MS(PEG)4(5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 550-2×PEG conjugates by at least by 2-fold in a Western Blot assay. Conjugates prepared with longer chain MS(PEG)8did not show significant improvement over the base conjugate in this experiment. Results demonstrating the effect of addition of NHS Acetate (2.5×, 5×) and MS(PEG)4(5×) spacer to GAM-DYLIGHT™ 680-4×PEG-GAR (at 10× molar excess of dye). are shown inFIG.12. HeLa cell lysate was diluted 4-fold from 0.5 μg/well and anti-PDI primary antibody was diluted to 1/5000 of 1 mg/ml. All DYLIGHT™ 680-4×PEG-GAR secondary antibodies were diluted to 1/20000 of 1 mg/ml stock. This experiment shows that that the addition of MS(PEG)4(5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 680-4×PEG conjugate by 3 to 4-fold in a Western blotting assay. Example 2: Dot Blot Assays Serial dilutions (1:1) Mouse or Rabbit IgG were made from chosen stock concentration. The dilutions were placed in a 96-well plate from highest to lowest using a 20 μL 12-channel multi pipette. 1 or 2 μL of the 11 serial dilutions was carefully spotted onto Nitrocellulose membranes. The membranes were allowed to dry overnight and then blocked with 2% BSA blocking buffer in TBST. The membranes were incubated with agitation for one hour at room temperature. The blocking solution was decanted from the container. The secondary antibody conjugates were diluted in TBS or blocking buffer. Secondary antibody conjugate dilutions varied depending on the conjugated label: 1:5,000 (DYLIGHT™ 488 and 550-2×PEG conjugates); 1:10,000 (DYLIGHT™ 650-4×PEG conjugates); and 1:20,000 (DYLIGHT™ 680-4×PEG and DYLIGHT™ 800-4×PEG conjugates). The membranes were incubated with the relevant secondary antibody conjugates with agitation for 30 to 60 minutes. The membranes were washed five times for five minutes with TBST buffer. The membranes were imaged with a suitable imager, for example, ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm). Results depicted inFIG.3and Table 11 show the effect of NHS acetate (2.5×, 5× and10x) and MS(PEG)4(3.75×) spacer to GAM-DYLIGHT™ 488 (at 5×-20× molar excess of dye) in a dot blot assay. In dot blot applications, the DYLIGHT™ 488-GAM conjugates made with the addition of NHS Acetate and MS(PEG)4resulted in definite improvement in fluorescent intensity ranging from 1.2 to 1.8-fold over the base conjugate (made without the spacer) at the various dye molar excesses from 7.5× to 20×. TABLE 11Effect of NHS Acetate (2.5X, 5X and 10X) or MS(PEG)4(3.75x to 10X) addition inthe conjugation of GAM-DYLIGHT ™ 488 at 5X-20X molar excess in a Dot Blot+2.5X+5XFOLD OFNHSNHSNHS+10X+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4DYLIGHT ™1.01.51.10.31.00.6488_5xDYLIGHT ™1.01.21.61.81.01.3488_7.5xDYLIGHT ™1.01.61.31.31.30.8488_10xDYLIGHT ™1.01.41.00.81.61.21.1488_15xDYLIGHT ™1.01.00.7061.51.31.0488_20xNA = No Addition Results shown inFIG.4and Table 12 demonstrate the effect of addition of NHS acetate (2.5× and 5×) and MS(PEG)4(3.75×) to GAM-DYLIGHT™ 488 (at 7.5×-20× molar excess of dye) in a dot blot assay. In this experiment, a different secondary antibody source was used. NHS acetate and MS(PEG)4added to the conjugation mixture provided a significant improvement of signal intensity as compared to the base conjugates at 5×, 15× and 20× dye molar excesses; ranging from 1.2 to 2.6-fold. TABLE 12Effect of NHS Acetate (2.5X, 5X and 10X) orMS(PEG)4(3.75X) addition in the conjugation of GAM-DYLIGHT ™ 488 at 5X-20X molar excess in a Dot Blot AssayFOLD OFNo+2.5X NHS+5X NHS+3.75XIMPROVEMENTadditionAcetateAcetateMS(PEG)4DYLIGHT ™ 488_7.5X1.01.51.10.3DYLIGHT ™ 488_10x1.01.61.61.3DYLIGHT ™ 488_15x1.01.41.01.6DYLIGHT ™ 488_20x1.01.00.71.5 Results shown inFIG.5and Table 13 demonstrate the effect of addition of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(3.75×) to GAM-DYLIGHT™ 550-2×PEG-GAR (at 10×-20× molar excess of dye) in a dot blot assay. Mouse IgG was serially diluted 1:1 from 1000 ng/dot. All DYLIGHT™ 550-2×PEG-GAR secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. NHS acetate and MS(PEG)4added to the conjugation mix provided an improvement of signal intensity as compared to the base conjugates at each respective dye molar excesses. Improvement ranged from 1.2 to 1.6-fold. TABLE 13Effect of the addition of NHS Acetate (2.5X, 5X and 10X)or MS(PEG)4 (3.75X) in the conjugation of GAM-DYLIGHT ™ 550-2xPEG-GAR at 10X-20X molar excess in a Dot Blot AssayFOLD OFNo+2.5X NHS+5X NHS+3.75XIMPROVEMENTadditionAcetateAcetateMS(PEG)4GAR-DYLIGHT ™ 550-1.01.231.351.322xPEG_10xGAR-DYLIGHT ™ 550-1.00.770.980.932xPEG_12.5xGAR-DYLIGHT ™ 550-1.01.121.11.522xPEG_15xGAR-DYLIGHT ™ 550-1.00.91.511.632xPEG_20x Results shown inFIG.6and Table 14 demonstrate the effect of the addition NHS Acetate (2.5×, 5× and10x) and MS(PEG)4(3.75×) to GAM-DYLIGHT™ 650-4×PEG-GAM (at 10×-20× molar excess) in a dot blot assay. Mouse IgG was serially diluted 1:1 from 1000 ng/dot. All DYLIGHT™ 650-4×PEG-GAR secondary antibodies were diluted to 1/10000 of 1 mg/ml stock. Both NHS Acetate and (MS)PEG4brought significant improvement in sensitivity and signal/background over the initial base conjugates. NHS acetate added at 2.5× molar excess to GAM-DYLIGHT™ 650-4×PEG-15× improved intensity by 1.7-fold. The improvement provided by NHS Acetate showed 1.3-fold better performance than with the conjugate prepared with the highest molar excess of dye (20×). All MS(PEG)4added to GAM-DYLIGHT™ 650-4×PEG-15× conjugation improved fluorescence intensity by 1.8 to 2.2-fold and performed better than the corresponding highest base conjugate GAM-DYLIGHT™ 650-4×PEG-20×. TABLE 14Effect of NHS Acetate (2.5X, 5X and 10X) or MS(PEG)4(3.75X)addition in the conjugation of GAM-DYLIGHT ™650-4xPEG-GAM at (10X-20X) in a Dot Blot Assay+2.5X+5X+10XFOLD OFNHSNHSNHS+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4GAM-DYLIGHT ™1.01.01.21.11.91.1650-4xPEG_5xGAM-DYLIGHT ™1.01.71.51.81.21.6650-4xPEG_7.5xGAM-DYLIGHT ™1.01.22.91.81.31.5650-4xPEG_10xGAM-DYLIGHT ™1.01.61.51.02.21.02.2650-4xPEG_15xGAM-DYLIGHT ™1.01.00.80.81.60.91.3650-4xPEG_20xNA = No Addition Results shown inFIG.7and Table 15 demonstrate the effect of the addition of NHS Acetate (2.5×, 5× and10x) and MS(PEG)4(3.75×, 5×,10x) spacers to GAM-DYLIGHT™ 800-4×PEG-in a dot blot assay. Mouse IgG was serially diluted 1:2 from 1000 ng/dot. All DYLIGHT™ 800-4×PEG-GAR secondary antibodies were diluted to 1/20000 of 1 mg/ml stock. This experiments shows that the addition of MS(PEG)4(3.75× and 5×) and NHS Acetate (5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 800-4×PEG conjugate by 1.5 to 6-fold in a dot blot application. TABLE 15Effect of the addition of NHS Acetate (2.5X, 5X and 10X) or MS(PEG)4(3.75X,5X, 10X) GAM-DYLIGHT ™ 800-4xPEG- in a Dot Blot Assay+2.5X+5X+10XFOLD OFNHSNHSNHS+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4GAM-DYLIGHT ™1.01.70.23.15.51.8800-4xPEG_5xGAM-DYLIGHT ™1.00.61.41.82.91.3800-4xPEG_7.5xGAM-DYLIGHT ™1.01.71.32.10.71.0800-4xPEG_10xGAM-DYLIGHT ™1.01.31.61.23.11.31.9800-4xPEG_15xGAM-DYLIGHT ™1.01.31.81.53.21.31.6800-4xPEG_20xNA = No Addition Results shown inFIG.11demonstrate the effect of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(5×) and MS(PEG)8(5×) spacers addition to GAM-DYLIGHT™ 550-2×PEG-GAR (at 12.5× molar excess of dye) in a dot blot assay. Mouse IgG was diluted 3 fold from 0.5 pig/well. All DYLIGHT™ secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. These dot blot assays showed that the addition of MS(PEG)4(5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 550-2×PEG conjugates by at least by 2-fold. Conjugates prepared with longer chain MS(PEG)8did not show significant improvement over the base conjugate. Results shown inFIG.12demonstrate the effect of addition of NHS Acetate (2.5×, 5×) and MS(PEG)4(5×) spacers to GAM-DYLIGHT™ 680-4×PEG-GAR (at 10× molar excess of dye) in a dot blot assay. Mouse IgG was serially diluted 1:2 from 1000 ng/dot. All DYLIGHT™ 680-4×PEG-GAR secondary antibodies were diluted to 1/20000 of 1 mg/ml stock. These dot blot assays show that the addition of MS(PEG)4(5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of base DYLIGHT™ 680-4×PEG conjugate. Example 3: Plate Assay Methods To prepare plates, eleven (1:1) serial dilutions of Mouse or Rabbit IgG starting from 10 g/ml were prepared. 100 μL of each dilution was placed in 96-well plates in the corresponding well from 1-11 from highest to lowest using a 300 μL 12-channel multi pipette; PBS was added to the last column (#12; negative control). This was repeated from row A to H. The plates were incubated overnight and then blocked and incubated with SUPERBLOCK™ Blocking Buffer (Thermo Fisher, cat. not. 37515) as follows: two times two hundred L for 5 minutes followed by one times 200 μL for ten minutes. The plates were allowed to dry and then stored desiccated at 4° C. Mouse IgG- or Rabbit IgG-coated plates were washed two times 200 μL with PBST 20 then one time with PBS. The secondary antibody conjugates were diluted in TBS or PBS. Secondary antibody conjugates were diluted 1:100 (DYLIGHT™ 488 and 550-2×PEG, DYLIGHT™ 650-4×PEG DYLIGHT™ 680-4×PEG and DYLIGHT™ 800-4×PEG conjugates). 100 μL of the relevant conjugates GAM in Mouse IgG-coated plate, GAR in Rabbit IgG coated plate were added to the plate well. Each dilution was added to different rows for each conjugate to be tested. All comparisons were made on the same plate. The plates were incubated for sixty minutes. Plates were washed three time 200 μL with TBST or PBST buffer. 100 μL of PBS was added to each row in each well. The fluorescence intensity was measured using the VariosKan instrument or Image the fluorescent signal with a suitable imager, such as ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm). Example 4: Immunofluorescence (IFC) Methods (i.e., Cellular Imaging Methods) Method #1: Frozen U2OS cell plates stored at −80° C. were thawed for thirty minutes at 50° C. Storage buffer (PBS) was removed and the cells were permeabilized for fifteen minutes (100 μl/well) with 0.1% Triton-X100 in 1×PBS buffer. Plates were blocked for thirty minutes in 2% BSA/PBS-0.1% Triton-X100 blockers. Primary antibody Mouse anti-PDI or Rabbit anti-HDAC2 (10 μg/ml) (cat. no. PA1-861, Life Technologies Corp., Carlsbad, Calif.), diluted in 2% BSA/PBS-0.1% Triton-X100 was added to the plate and incubated for one hour at RT. Negative controls contain only 2% BSA/PBS-0.1% Triton-X100 blocker. After incubation, the primary antibody solution was removed from the plate and the plate was washed three times 100 l/well PBST and one time 100 l/well PBS. Next, GAM or GAR secondary antibodies labeled with various molar excesses of dyes were diluted to 4 μg/ml in PBS and incubated for one hour at room temperature. The plates were washed three times 100 μl/well PBST and 1×100 l/well PBS and Hoechst 33342 (cat. no. 62249, Thermo Scientific, Waltham, Mass.), (diluted to 0.1 μg/ml in PBS) was added to each well (100 l/well). The plates were scanned on ARRAYSCAN™ Plate Reader VTI3, 20× objective. Method #2: Certain experiments were done in A549 cells in 384-well plates. Primary antibody was used at the same concentration (1 μg/ml) with varying dilutions of secondary. pH2AX measurements in etoposide (50 μM for 3 hours, Tocris Bioscience, cat no. 12-261-00) treated cells were used to measure B Signal/Noise (S/N, also referred to as Signal to Background), and brightness was used to compare different antibodies. Secondary antibody conjugates were tested at 4 different dilutions (0.5, 1, 2 and 4 mg/ml). Standard procedures were used for antibody staining: 4% formaldehyde fixation for fifteen minutes. Permeabilization was performed in 0.5% Triton x-100 for ten minutes. Blocking was performed with 3% BSA for thirty minutes. Primary antibody incubation was carried out at RT for one hour. This was followed by three washes with PBS. Secondary antibody conjugates were incubated at RT for one hour followed by three washes with PBS. The cells were analyzed on ARRAYSCAN™ VTI (Thermo Fisher). Results shown inFIG.13and Tables 16 and 17 demonstrate the effect of NHS acetate (2.5× and 5×) and MS(PEG)4(3.75×) spacer addition to GAM-DYLIGHT™ 488 (13A) and GAR-DYLIGHT™ 488 ((13B) at 7.5× to 20× molar excess of dye) in a cellular imaging application. DYLIGHT™ 488-GAM and DYLIGHT™ 488-GAR. A549 cells were stained with a pH2Ax primary antibody diluted to 1/1000 of the 1 mg/ml stock. All DYLIGHT™ 488 secondary antibodies were diluted to 1/250 of the 1 mg/ml stock. NHS acetate added to the conjugation mix provided an improvement of signal/background as compared to the base conjugates at 15× dye molar excesses; ranging from 1.4 to 1.5-fold (GAM) and 1.1 to 1.6-fold (GAR). For GAM conjugates the most significant improvement was observed with NHS Acetate at 5× and with MS(PEG)4at 3.75×, and for GAR conjugates the more noticeable improvement was observed with NHS Acetate at 2.5× and with MS(PEG)4at 3.75×. TABLE 16Effect of NHS Acetate (2.5X, 5X and 10X) orMS(PEG)4(3.75x) addition in the conjugation ofGAM-DYLIGHT ™ 488 at 5X-20X molar excessin a Cellular Imaging application - DYLIGHT ™ 488-GAMFOLD OF+2.5X NHS+5X NHS+3.75XIMPROVEMENTNAAcetateAcetateMS(PEG)4DYLIGHT ™ 488_7.5x1.001.071.141.23DYLIGHT ™ 488_10x1.001.041.131.17DYLIGHT ™ 488_15x1.001.111.481.53DYLIGHT ™ 488_20x1.000.721.091.07NA = No Addition TABLE 17Effect of NHS Acetate (2.5X, 5X and 10X) orMS(PEG)4(3.75x) addition in the conjugation ofGAR-DYLIGHT ™ 488 at 5X-20X molar excessin a Cellular Imaging application - DYLIGHT ™ 488-GARFOLD OF+2.5X NHS+5X NHS+3.75XIMPROVEMENTNAAcetateAcetateMS(PEG)4DYLIGHT ™ 488_7.5x1.001.131.071.13DYLIGHT ™ 488_10x1.001.201.211.02DYLIGHT ™ 488_15x1.001.601.311.67DYLIGHT ™ 488_20x1.001.231.141.11NA—No Addition Results shown inFIG.14and Table 18 demonstrate the effect of NHS Acetate (2.5×, 5× and10x) and MS(PEG)4(3.75×, 5× and10x) on GAM-DYLIGHT™ 550-2×PEG-GAM (at 7.5× to 20× molar excess of dye) in a cellular fluorescence imaging application. U2OS cells were stained with an anti-PDI primary antibody diluted to 1/100 of the 1 mg/ml stock. All DYLIGHT™ 550-2×PEG-GAM secondary antibodies were diluted to 1/250 of the 1 mg/ml stock. In this cellular imaging application, the addition of 5×NHS Acetate generated about 50% improvement as compared to the base conjugate (made without the additives) for DYLIGHT™ 550-2×PEG GAM conjugate at 12.5× dye molar excess, and addition of 3.75×MS(PEG)4resulted in about 50% improvement over the base conjugates at 20× dye molar excess. TABLE 18Effect of 2.5X to 10X NHS Acetate and MS(PEG)43.75X to 10X on fluorescenceintensity of GAM-DYLIGHT ™ 550-2xPEG in a Cellular Imaging application+2.5X+5X+10XFOLD OFNHSNHSNHS+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4GAM-DYLIGHT ™1.00.50.70.50.90.7550-2xPEG_7.5xGAM-DYLIGHT ™1.01.71.70.91.41.5550-2xPEG_10xGAM-DYLIGHT ™1.01.82.00.91.11.2550-2xPEG_12.5xGAM-DYLIGHT ™1.02.22.21.91.31.91.1550-2xPEG_15xGAM-DYLIGHT ™1.01.71.31.01.21.21.0550-2xPEG_20xNA = No Addition Results are shown inFIG.15and Table 19 for an experiment testing the effect of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(3.75×, 5×, 10×) spacer to GAM-DYLIGHT™ 650-4×PEG) in a cellular imaging application. U2OS cells were stained with anti-PDI primary antibody diluted to 1/100 of 1 mg/ml stock. All DYLIGHT™ 650-4×PEG-GAM secondary antibodies were diluted to 1/250 of 1 mg/ml stock. In this cellular imaging application, the addition of NHS Acetate-5× generated about 70% improvement as compared to the base conjugate (made without the additives) for DYLIGHT™ 650-4×PEG-GAM conjugate at 20× molar excess, and MS(PEG)4-3.75× showed about 90% improvement over the base conjugates at 20× molar excess. TABLE 19Effect of the addition of NHS Acetate (2.5X, 5X and 10X) or MS(PEG)4(3.75X,5X, 10X) GAM-DYLIGHT ™ 650-4xPEG-Cellular Imaging application+2.5X+5XFOLD OFNHSNHSNHS+10X+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4GAM-DYLIGHT ™1.01.01.00.91.21.1650-4xPEG_5xGAM-DYLIGHT ™1.00.50.50.50.41.6650-4xPEG_7.5xGAM-DYLIGHT ™1.01.31.70.91.01.5650-4xPEG_10xGAM-DYLIGHT ™1.00.80.80.80.80.72.2650-4xPEG_15xGAM-DYLIGHT ™1.01.00.91.11.91.11.3650-4xPEG_20xNA = No Addition Results are shown inFIG.16and Table 20 for an experiment testing the effect of the addition of NHS Acetate (2.5×, 5× and 10×) and MS(PEG)4(3.3.75×, 5×, 10×) spacer to the detectable fluorescence level of GAM-DYLIGHT™ 680-4×PEG) in a cellular imaging application. U2OS cells were stained with mouse anti-PDI primary antibody diluted to 1/100 of 1 mg/ml stock. All DYLIGHT™ 680-4×PEG-GAM secondary antibodies were diluted to 1/250 of 1 mg/ml stock: In this cellular imaging application, the addition of NHS Acetate-5× generated about 70% improvement for dyes conjugates at both 7.5× and 10× as compared to the base conjugate (made without the additives) for DYLIGHT™ 680-4×PEG. GAM conjugate at molar excesses of 15× molar excess and MS(PEG)4-3.75× showed about 80% improvement over the base conjugates at 15× molar excesses. TABLE 20Effect of the addition of NHS Acetate (2.5X, 5X and 10X) or MS(PEG)4(3.75X,5X, 10X) GAM-DYLIGHT ™ 680-4xPEG-Cellular Imaging application+2.5X+5XFOLD OFNHSNHS+10X+3.75X+5X+10XIMPROVEMENTNAAcetateAcetateAcetateMS(PEG)4MS(PEG)4MS(PEG)4GAM-DYLIGHT ™1.00.80.70.71.00.8680-4xPEG_5xGAM-DYLIGHT ™1.01.21.71.11.11.1680-4xPEG_7.5xGAM-DYLIGHT ™1.01.71.71.11.51.5680-4xPEG_10xGAM-DYLIGHT ™1.01.41.21.11.81.31.4680-4xPEG_15xGAM-DYLIGHT ™1.00.80.91.11.01.11.1680-4xPEG_20xNA = No Addition Results The use of spacer agents such as NHS-Acetate, NHS-MS(PEG) and NHS-Betaine increased fluorescent signal sensitivity and intensity above optimal D/P levels that typically result in quenching. This was demonstrated by labeling goat anti-mouse (GAM) and goat anti-rabbit (GAR) secondary antibodies with NHS-DYLIGHT™ 488, NHS-DYLIGHT™ 550 2×PEG, NHS-DYLIGHT™ 650 4×PEG, NHS-DYLIGHT™ 680 4×PEG, NHS-DYLIGHT™ 800 4×PEG in combination with spacer agents which include, but are not limited to, NHS-Acetate and NHS-MS(PEG)4, NHS-MS(PEG)8and NHS-MS(PEG)12. Our calculations of D/P value following dye conjugation and purification demonstrated that the addition of the spacer agents did not make significant differences to the D/P ratios indicating that these reagents and the fluorophores labeled different primary amines on the antibody (i.e., they don't compete for the same primary amines). Conjugates made with different dye and spacer agents were tested in a variety of applications including IFC, Western blotting, dot blotting or IgG Binding plate-based assays. In each instance, with certain spacer agents at certain molar excess values of the spacer agent to the dye, an increase in fluorescence intensity was observed when spacer agents were used as compared to controls lacking spacer agents. In addition to the above described experiments, antibodies labeled with NHS-Rhodamine and conjugated with a variety of NHS-Betaine concentrations (Betaine 2.5, Betaine 5, Betaine 10 molar ratios) displayed an increase in total fluorescence when the antibodies were conjugated with Betaine as the spacer modification reagent. SeeFIG.17and Tables 21 and 22 below. Amongst the different Betaine chain lengths, Betaine 10 had a positive effect on TAMRA™ at all molar excesses of the dyes and on ALEXA FLUOR™ 555 above a D/P ratio of 12 (data not shown). TABLE 21Effect of 2.5X to 10X Betaine on fluorescenceof TAMRA ™-GAM conjugatesNHS-NHS-TAMRA ™/RelativeTotalBetaineTAMRA ™IgGQuantumFluor-MRMRDOLYieldescence2.55NANANA2.5106.30.42.522.5208.60.32.58555.90.563.35107.10.342.415206.40.181.151055.40.563.0210108.40.54.2102015.70.345.34054.20.542.270106.70.422.8102011.40.384.33MR = Molar RatioDOL = Degree of Labeling Example 5: Reaction of Goat Anti-Mouse IgG (GAM) and 5-(and-6)-Carboxytetramethylrhodamine, Succinimidyl Ester (5(6)-TAMRA™-SE) with and without N,N,N-Trimethylglycine-N-Hydroxysuccinimide Ester Bromide (Betaine-SE) TAMRA™-SE was weighed out and made up as a stock solution at 10 mg/mL in anhydrous DMSO and Betaine-SE was weighed out and made up at a stock of 4 mg/mL in anhydrous DMSO. The DMSO solutions were then transferred into reaction vials with the TAMRA™-SE+/−Betaine-SE measured into the vials based on a 5, 10, or 20-fold molar ratio of dye to IgG and the equivalent of molar ratio 0 or 10 Betaine-SE to IgG also added to the vials. Separately, 0.417 mL (3.5 mg) of a 8.4 mg/mL solution of GAM in 10 mM potassium phosphate, 150 mM sodium chloride buffer (PBS) was measured into a plastic tube and the pH raised to >8.0 with 42 μL of 1M sodium bicarbonate, pH 9.0. 0.5 mg of the GAM solution was added to the reaction vials containing SE and reacted for 1 hour at RT. The dye-protein conjugates were separated from free dye and Betaine by size exclusion chromatography using 5-0.75×20 cm columns packed with BIORAD™ BIO-GEL™ P-30 fine in PBS and eluted with same. The initial protein-containing band from each column was collected. Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV/Vis spectrometer, and the degree of substitution (DOS) or moles dye/mole GAM was determined for each sample. Fluorescence emission spectra were obtained using a Perkin Elmer LS 55 Fluorescence Spectrometer, using samples with matched optical density at 545 nm and excited at 545 nm. Emission data collected from 550-750 nm. Relative quantum yield (RQY) was measured as the area of the sample spectrum/area of the dye standard spectrum. Total fluorescence was then calculated as the product of the RQY*DOS. TABLE 22Total Fluorescent Output of DifferingMolar Ratios of TAMRA ™/GAMTAMRA ™/GAM (Molar Ratios)51020TAMRA ™-GAM and Betaine-SE added at MR = 103.024.25.34TAMRA ™-GAM (No Betaine-SE)2.272.814.33 Example 6: Reaction of Goat Anti-Mouse IgG (GAM) and ALEXA FLUOR™ 488 Carboxylic Acid, Succinimidyl Ester, Dilithium Salt (AF488-SE) with and without 1,3-Propane Sultone (3-Hydroxy-1-Propanesulfonic Acid γ-Sultone) AF488-SE was weighed out and made up as a stock solution at 10 mg/mL in anhydrous DMSO and Propane sultone was weighed out and made up at a stock of 1 mg/mL in E-Pure H2O. 0.357 mL (4.0 mg) of a 11.2 mg/mL solution of GAM in 10 mM potassium phosphate, 150 mM sodium chloride buffer (PBS) was measured into a plastic tube and the pH raised to >8.0 with 36 μL of 1M sodium bicarbonate, pH 9.0. 0.5 mg of the GAM solution was transferred to reaction vials and reacted with a 0, 2, 5, or 10-fold molar excess of propane sultone for 2 minutes and then AF488 stock was added to the mixtures in an 8 or 15-fold molar excess over the GAM and let react for 1 hour at room temperature. The dye-protein conjugates were separated from free dye and Propane sultone by size exclusion chromatography using 5-0.75×20 cm columns packed with BIORAD™ BIO-GEL™ P-30 fine in PBS and eluted with same. The initial protein-containing band from each column was collected. Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV/Vis spectrometer, and the degree of substitution (DOS) or moles dye/mole GAM was determined for each sample. Fluorescence emission spectra were obtained using a Perkin Elmer LS 55 Fluorescence Spectrometer, using samples with matched optical density at 475 nm and excited at 475 nm. Emission data collected from 480-800 nm. Relative quantum yield (RQY) was measured as the area of the sample spectrum/area of the dye standard spectrum. Total fluorescence was then calculated as the product of the RQY*DOS. TABLE 23Total Fluorescent Output of DifferingMolar Ratios of AF 488/GAMAF488/GAM (Molar Ratios)815AF488-GAM + propanesultone/GAM MR = 22.523.67AF488-GAM + propanesultone/GAM MR = 52.623.73AF488-GAM + propanesultone/GAM MR = 102.693.86AF488-GAM control (no propanesultone)3.56 Example 7: Labeling of SK3 Mouse Anti-Human CD4 Azide with 20 kDa 8-Arm PEG Amine (20K8 PEG) Modified with ALEXA FLUOR™ 647 NHS Ester, Tris(Triethylammonium Salt) (AF647-SE) ALEXA FLUOR™ 647 NHS/Succinimidyl Ester (Thermo Fisher Scientific, cat. no. A37573), abbreviated as “AF647-SE”, was weighed out and made up as a stock solution at 32 mM in anhydrous DMSO (Thermo Fisher Scientific, D12345). CLICK-IT™ SDP Ester sDIBO Alkyne (sDIBO) (Thermo Fisher Scientific, cat. no. C20025) was made up as a stock solution at 9 mg/mL in anhydrous DMSO. 8arm PEG Amine (hexaglycerol), HCl salt (JenKem, Piano, Tex. 75024, cat. no. 8ARM-NH2HCl), abbreviated as “20K8 PEG”, having the following structure: was weighed out and prepared as a stock solution at 40 mg/mL in anhydrous DMSO. To a plastic tube, 300 μL of 20K8 PEG stock solution, 176 μL of sDIBO stock solution (2.4-fold molar excess/20K8 PEG) and 6 μL of neat Triethylamine (TEA) was added and allowed to react for 3 hours at 25° C. After reaction, 300 μL of the AF647-SE stock solution (2-fold molar excess/PEG-amine) was added to the tube and the reaction proceeded overnight at 25° C. The AF647-20K8 PEG-sDIBO constructs were purified from free dye and sDIBO by size exclusion chromatography using a 1×30 cm column packed with BIORAD™ BIO-GEL™ P-10F in PBS and eluted in the same. The initial dye-containing fractions were collected and concentrated using EMD Millipore AMICON™ Ultra-4 10 kDa centrifugal filters. Azido (PEO)4propionic acid, succinimidyl ester (Thermo Fisher Scientific, cat. no. A10280), abbreviated as “Azide-SE”, was weighed out and made up as a stock solution at 10 mM in anhydrous DMSO (Thermo Fisher Scientific, D12345). 266 μL SK3 mouse anti-human CD4 antibody (2.5 mg) and 134 μL PBS were added to a plastic tube and the pH was raised to >8.0 with 50 μL 1M sodium bicarbonate, pH 9.0. 8.3 μL of Azide-SE stock solution (5 fold molar excess/antibody) and 42 μL of DMSO were added to the antibody solution. The reaction was allowed to proceed for 2 hours at 25° C. The azido-SK3 antibody was purified using 2 mL BIORAD™ BIO-GEL™ P-30M spin columns. Azido-SK3 antibody was prepared with 5-fold to 20-fold excess of Azide-SE to antibody. 24.2 μL of a 2 mM solution of AF647-20K8 PEG-sDIBO (concentration of DIBO) and 116 μL of a 4.3 mg/mL azido-SK3 were combined in a plastic tube. 360 μL of PBS were added to bring the final concentration of the solution to 100 μM AF647-20K8 PEG-sDIBO (concentration of DIBO) and 1 mg/mL azido-SK3 antibody. The click reaction was allowed to proceed for 2 hours at 37° C. followed by quenching with 5 mM NaN3for 1 hour at room temperature. AF647-20K8 PEG-SK3 conjugates were diluted to 0.5 mg/mL in PBS. Conjugation reactions were carried out at final DIBO concentrations between 100 and 600 μM, with azido-SK3 antibody at 1-3 mg/mL, at reaction temperatures between 25° C. and 37° C., for 2 hours to 20 hours. Specific conditions per experimental run are set out in Table 24. 24.2 μL of a 2 mM solution of AF647-20K8 PEG-sDIBO (concentration of DIBO) and 116 μL of a 4.3 mg/mL azido-SK3 were combined in a plastic tube. 360 μL of PBS were added to bring the final concentration of the solution to 100 μM AF647-20K8 PEG-sDIBO (concentration of DIBO) and 1 mg/mL azido-SK3 antibody. The click reaction was allowed to proceed for 2 hours at 37° C. followed by quenching with 5 mM NaN3for 1 hour at room temperature. Conjugates were purified and concentrated using EMD Millipore AMICON™ Ultra-4 100 kDa centrifugal filters. AF647-20K8 PEG-SK3 conjugates were diluted to 0.5 mg/mL in PBS. Conjugation reactions were carried out at final DIBO concentrations between 100 and 600 AM, with azido-SK3 antibody at 1-3 mg/mL, at reaction temperatures between 25° C. and 37° C., for 2 hours to 20 hours. Specific conditions per experimental run are set out in Table 24. TABLE 24Conjugation ConditionsClickIncubation conditionSK3starPEGSK3-N3mg/DIBOtime/ExampleMR1DOS2starPEG3mL4μM5temp6sa153HG 20 kD11002 h/37° C.sa2106.1HG 20 kD11002 h/37° C.sa32012HG 20 kD11002 h/37° C.sa453HG 20 kD12002 h/37° C.sa5106.1HG 20 kD12002 h/37° C.sa62012HG 20 kD12002 h/37° C.sa2*106.1HG 20 kD110020 h/25° C.sa753TP 20 kD11002 h/37° C.sa8106.1TP 20 kD11002 h/37° C.sa92012TP 20 kD11002 h/37° C.sa1053TP 20 kD12002 h/37° C.sa11106.1TP 20 kD12002 h/37° C.sa122012TP 20 kD12002 h/37° C.sa8*106.1TP 20 kD110020 h/25° C.sa13106.1HG 20 kD36003 h/37° C.sa142012HG 20 kD36003 h/37° C.1MR: Molar Ratio; x-fold excess used to tag SK3 antibody with Azide-SE:antibody (mole/mole).2DOS: degree of substitution, number of azide groups incorporated per antibody molecule.3starPEG: indicates MW of starPEG (20 kD) and core. HG: hexaglycerol, TP: tripentaerythritol.4In the click incubation SK3 was present at indicated concentrations in mg/mL (specified for each example).5The starPEG DIBO during the click conjugation was present at the indicated concentrations in μM.6The click reactions were carried out for the indicated times at the indicated temperatures. Example 8: Quantum Yield Characterization of Branched PEG AF647 Constructs To prepare samples for quantum yield measurement, solutions of AF647 Branched PEG constructs (AF647-2K4, AF647-10K4, AF647-10K8 and AF647-20K8) were diluted to a final dye concentration of 0.16 μM in deionized water. Quantum yield (0) was measured using a Hamamatsu Absolute PL Quantum Yield Spectrometer. Quantum yields for the Branched PEG constructs were compared to that of the free dye to determine the degree of quenching in the final constructs. Additionally, the brightness was determined to determine the fluorescent enhancement achieved using the branched PEG spacers. The smallest construct, AF647-2K4 (2,000 molecular weight branched PEG with four arms) showed the greatest quenching (20% QY of the free dye, 0.2 Fluorescent Ratio) and had the lowest overall improvement in the total fluorescence. The greatest fluorescence enhancement was seen for higher molecular weight constructs, with either 4 or 8 arms (AF647-10K4 and AF647-20K8) where up to 89% of the fluorescence quantum yield was retained of the free dye (Fluorescent Ratio of 0.9), and up to 5.8 fold improvement in brightness was noted. TABLE 25Effect of Branched PEG spacers on the percent quantum yield(QY) and brightness (B) of ALEXA FLUOR ™ 647Dyes/% QY ofBright-Bright-Mole-QuantumFree Dyeness, BnessculesYield(Fluores-(QY × ε ×Ratio toSample(N)(QY)cent Ratio)N) AUFree DyeAF64710.404100% (1.0)1.09E+051.0AF647-2K440.08722% (0.2)9.40E+040.9AF647-10K440.35989% (0.9)3.88E+053.6AF647-10K880.14536% (0.4)3.13E+052.9AF647-20K880.29573% (0.7)6.37E+055.8An assumption is made that effectively one fluorescent label is attached to the terminus of each arm. In other words, an assumption is made that the PEG molecules have been labeled to saturation, thus N = number of arms/polymer.ε = 270,000 cm−1M−1for ALEXA FLUOR ™ 647 Example 9: Flow Cytometry Evaluation of AF647-20K8 PEG-SK3 Constructs Freshly collected anti-coagulated whole blood (human) was lysed using ACK lysis buffer for 20 minutes at room temperature. White blood cells were isolated by centrifugation (400×g, 5 mins.) and washes in 1% bovine serum albumin/PBS twice (1% BSA/PBS). After isolation, the total number of white blood cells was determined using the COUNTESS™ Automated Cell Counter and then diluted to 10 million cells per mL. One million cells/well in a 96 well plate were stained with the AF647-20K8 PEG-SK3 conjugates using a 7 point titration of 1 μg to 0.015 μg of antibody. Stained cells were washed twice with 1% BSA/PBS. Analysis of the stained cells was carried out using the ATTUNE™ N×T Flow Cytometer and compared to APC (Thermo Fisher Scientific, cat. no. MHCD0405), ALEXA FLUOR™ 488 (Invitrogen, cat. no. MHCD0420), FITC (Thermo Fisher Scientific, cat. no. MA1-81103) and BRILLIANT VIOLET™ 605 (BioLegend, San Diego, Calif., cat. no. 300555) CD4 conjugates. FIG.22shows a histogram plot of CD4 positive lymphocyte cells as a function of fluorescence intensity in the RL1 channel of the ATTUNE™ N×T Flow Cytometer. ALEXA FLUOR™ 647 conjugated to CD4 is shown as a dashed line, and the AF647-20K8 PEG-SK3 conjugates are shown as dotted or solid lines. Compared to the AF647 conjugate alone, the StarPEG conjugates show a greater that 0.5 log increase in brightness.FIG.23shows plotted signal to noise (S/N) and percent positive (% Positive) as a function of conjugate concentration in the flow cytometry experiment. It is shown that the StarPEG constructs (here B1 and B2) have up to 2.5 fold increase in S/N versus the APC CD4 benchmark conjugate, and up to 2 fold increase in S/N versus the AF647 CD4 benchmark conjugate while retaining the ability to accurately assess the number of CD4 positive cells in the sample. Example 10: Conjugation of ALEXA FLUOR™ 488 to an Amino Dextran Scaffold Preparation of 70 kD amino dextran AF488 scaffold: 10 mg of amino dextran (70,000 μMW, 20 amino groups; Thermo Fisher Scientific, Cat. No. D1862) was dissolved in 1.2 ml of dry DMSO containing 1.0 μl of DIEA. 0.9 mg of ALEXA FLUOR™ 488 succinimidyl ester lithium salt (643 MF; Thermo Fisher Scientific, Cat. No. A20000) was added to solution and the mixture was stirred for 3.5 hours at ambient temperature. The solution was diluted with 12 mL of ethyl acetate and the resulting suspension was centrifuged. The supernatant was discarded and the solid material was shaken with 10 mL of fresh ethyl acetate and centrifuged. This washing was repeated 3 more times with 10 mL of fresh ethyl acetate and the resulting precipitate was dried in vacuum. The solid was re-dissolved in 0.5 ml of water and solution put in 10 cm Spectra/Por Dialysis membrane (Spectrum Labs, MWCO 12-14,000 flat width 10 mm) clipped from both side. The dialysis membrane was slowly stirred in 1 L of water for 1 week. The water was replaced twice per day. The dialysis membrane was open from one end and solution was lyophilized to give amino dextran ALEXA FLUOR™ scaffold. The measured DOL is 9.7 and relative QY is 0.6 (referenced to QY of ALEXA FLUOR™ 488). Attaching thiol linker to 70 kD amino dextran AF488 scaffold: Amino dextran AF488 scaffold (4.5 mg) was dissolved in 0.5 mL of DMSO containing 0.055 μL of N,N-Diisopropylethylamine (DIEA). Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (20 μg) was added to solution and the mixture was kept at ambient temperature overnight and then capped with acetic acid succimidyl ester (1.0 mg, 3 hrs). The solution was diluted with 10 mL of ethyl acetate. The resulting suspension was centrifuged and supernatant discarded. The solid was shaken with 10 mL of fresh ethyl acetate and centrifuged. The washing was repeated 5 more times. The resulting solid was dried in vacuum. The measured DOL is 0.74. This material was re-dissolved in 2 mL of water and 16 mg of DT was added to solution. The mixture was stirred for 5 min and loaded on G15 SEPHADEX™) column, the product was eluted with DE water as green fluorescent solution which was used for conjugation to SMCC modified streptavidin. The determined concentration was 48 μM (by dye adsorption). Conjugation of amino dextran AF488 scaffold modified with thiol linker to SMCC modified streptavidin: SMCC modified streptavidin (35 μL solution in water) was treated with 1, 2, 3 and 4 equivalents of thiol modified amino dextran AF488 scaffold (48 μM solution in water). The reaction was carried out at ambient temperature for 3 hours and after that reaction mixture was kept overnight at 4° C. overnight. The conjugates are purified on P100 size exclusion column with 10 nM PBS buffer. TABLE 26Streptavidin Labeled with 70 kD Amino dextran AF488 Scaffold(average of 9.7 molecules of dye per scaffold)DOL by ScaffoldDOL by Dye(Avg.)(Avg.)QYBrightness0.99.20.524.81.110.20.505.11.8180.539.52.625.50.5413.7 TABLE 27Streptavidin labeled with AF488 dye per scaffoldDOL (Avg.)QYBrightness1.50.701.03.00.601.84.00.552.14.50.401.85.00.341.7 Results: As shown in Tables 26 and 27, conjugates made from the scaffold are brighter as compared to conjugates made from single AF488 dye. Also, QY of AF488 fluorophore drops from 0.70 to 0.34 for single dye conjugation in contrast to almost constant QY for labeling with the amino dextran scaffold. | 138,359 |
11857644 | DETAILED DESCRIPTION OF EMBODIMENTS It should be noted that “prepared by . . . ” is synonymous with “include”. In addition, the term “include”, “have”, “contain” or any other variant thereof is intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus/device need not be limited to those listed elements, but may include other elements that are not explicitly listed or inherent to the process, method, article or device. The term “consist of” excludes any unspecified elements or components. This term has a closed meaning when used in claims, indicating the exclusion of other unspecified materials except for conventional impurities associated therewith. When the phrase “consist of” appears in a clause in the body of a claim rather than immediately following the subject matter, it is only to limit the element described in such clause; other elements are not excluded from the claim as a whole. When amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood as specifically disclosing all ranges formed by any pairing of any upper limit or preferred value with any lower limit or preferred value, regardless of whether such range is separately disclosed. For example, a range “1-5” should be interpreted to include ranges of “1-4”, “1-3”, “1-2”, “1-2 and 4-5”, and “1-3 and 5”. Unless otherwise indicated, the numerical range described herein is intended to include its endpoints and all integers and fractions within the range. In this application, the parts and percentages are expressed by weight unless otherwise indicated. The phrase “part by weight” refers to a basic measurement unit indicating the mass ratio relationship of multiple components. For example, 1 part can represent any unit of mass, such as 1 g and 2.689 g. If it is specified that there are a parts by weight of component A and b parts by weight of component B, it means a mass ratio of A to B is a:b, or indicates that the mass of component A is aK and the mass of component B is bK (K is an arbitrary value, representing a multiplication factor). It should be noted that the sum of the parts by weight of all the components is not limited to 100 parts. The term “and/or” indicates that one or both of the specified conditions may occur, for example, “A and/or B” includes “A”, “B”, and “a combination of A and B”. Provided is a multi-signal fluorescent probe for early diagnosis of a tumor, where structural formula of the multi-signal fluorescent probe is shown as follows: A method for preparing the multi-signal fluorescent probe is also provided, including: adding 2-methoxyphenothiazine and ethyl iodide into a mixture of DCM and acetonitrile followed by a first reaction to obtain a reaction mixture; and subjecting the reaction mixture to a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine; adding boron tribromide into the 10-ethyl-2-methoxy-10H-phenothiazine under an inert gas, followed by a second reaction under an ice bath and a second post-treatment to obtain 10-ethyl-10H-phenothiazin-2-ol; and mixing the 10-ethyl-10H-phenothiazin-2-ol, malonic acid, zinc chloride and phosphorus oxychloride followed by a third reaction and a third post-treatment to obtain the multi-signal fluorescent probe. In an embodiment, the first reaction is performed at 65-75° C. for 8-12 h. In an embodiment, the first reaction is performed at 65° C., 70° C., 75° C. or any temperature between 65-75° C. for 8 h, 10 h, 12 h or any duration between 8-12 h. In an embodiment, the first post-treatment includes: subjecting the reaction mixture to column chromatography to obtain the 10-ethyl-2-methoxy-10H-phenothiazine. In an embodiment, the second reaction is performed under stirring in a dark environment for 4-6 h. In an embodiment, the second post-treatment includes: dropwise adding a red solution resulted from the second reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and concentrating the organic layer to obtain the 10-ethyl-10H-phenothiazin-2-ol. In an embodiment, the third reaction is performed under reflux and stirring at 80-90° C. for 24-36 h. In an embodiment, the third reaction is performed at 80° C., 85° C., 90° C. or any temperature within 80-90° C. for 24 h, 30 h, 36 h or any duration within 24-36 h. In an embodiment, the third post-treatment includes: dropwise adding a brown viscous solution resulted from the third reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and subjecting the organic layer to concentration and column chromatography to obtain the multi-signal fluorescent probe. In an embodiment, the pH adjustment is performed with a 20 wt. % sodium hydroxide aqueous solution. The present disclosure also provides a use of the multi-signal fluorescent probe in the detection of ONOO−and Na2S2in cells. The present disclosure will be described in detail below with reference to the embodiments. Obviously, described below are merely some embodiments of this disclosure, and are not intended to limit the disclosure. Unless otherwise specified, the materials and reagents used in the following embodiments are available commercially, and the experiments are carried out using conventional methods. A synthesis route of the multi-signal fluorescent probe is illustrated as follows: EXAMPLE (S1) Synthesis of 10-ethyl-2-methoxy-10H-phenothiazine (1a) 2.0 g (8.72 mmol) of 2-methoxyphenothiazine and 3.26 g (20.93 mmol) of ethyl iodide were added into 20 mL of DCM-acetonitrile mixture. The reaction mixture was reacted at 65° C. for 8-12 h. (1b) After cooled to room temperature, the reaction mixture was subjected to column chromatography to obtain 616 mg of white solid as 10-ethyl-2-methoxy-10H-phenothiazine (yield: 27.5%). (S2) Synthesis of 10-ethyl-10H-phenothiazin-2-ol (2a) 5.84 g (23.31 mmol) of boron tribromide (BBr3) was slowly added into a solution of the 10-ethyl-2-methoxy-10H-phenothiazine (1.0 g, 3.89 mmol) in DCM under the protection of nitrogen gas. The reaction mixture was stirred on an ice bath in the dark for 4-6 h to obtain a red solution. (2b) The red solution was slowly dropwise added into ice water, and the resultant mixture was adjusted to pH 6-7 with a 20 wt. % sodium hydroxide aqueous solution. (2c) The reaction mixture was subjected to multiple extractions with DCM, and the resultant organic layers were collected, combined and subjected to rotary evaporation to obtain 600 mg of a light green solid as 10-ethyl-10H-phenothiazin-2-ol (yield: 63.46%). (S3) Synthesis of 4-chloro-11-ethylpyrano[2,3-b]phenothiazin-2(11H)-one (the multi-signal fluorescent probe) (3a) The 10-ethyl-10H-phenothiazin-2-ol, malonic acid and zinc chloride were mixed in a phosphorus oxychloride solution, and reacted under stirring and air reflux at 80° C. for 24 h to obtain a brown viscous solution. (3b) The brown viscous solution was slowly dropwise added into ice water, and the resultant mixture was adjusted to pH 6-7 with a 20 wt. % sodium hydroxide aqueous solution. (3c) The reaction mixture was subjected to multiple extractions with DCM, and the resultant organic layers were collected, combined, and subjected to rotary evaporation and column chromatography to obtain a yellow solid as 4-chloro-11-ethylpyrano[2,3-b]phenothiazin-2(11H)-one (yield: 30.3%) The1H-NMR spectrum of the multi-signal fluorescent probe was shown inFIG.1. The multi-signal fluorescent probe synthesized herein was capable of distinguishing and detecting ONOO−and Na2S2produced in organisms. Unless otherwise specified, the experimental procedures were similar to those of other probes. Spectral properties of the multi-signal fluorescent probe were investigated as follows. The multi-signal fluorescent probe was dissolved with dimethyl sulfoxide (DMSO) to obtain a 1 mM probe solution. A 1 mM ONOO−aqueous solution and a 1 mM Na2S2aqueous solution were prepared. 20 μL of the 1 mM probe solution, 980 μL of analytically pure CH3CN, the required amount of the 1 mM ONOO−or Na2S2aqueous solution and the required amount of phosphate buffered saline (PBS) were added into a 2 mL sample tube. A volume ratio of an organic phase to an aqueous phase was kept at 5:5 for all tests (a total volume of each test sample was 2 mL). For example, when it was required to explore the fluorescence intensity of the multi-signal fluorescent probe after reacted with 20 mM ONOO−, the experiment was formulated as follows. L of the 1 mM probe solution, 980 μL of analytically pure CH3CN, 20 μL of the 1 mM ONOO−aqueous solution and 980 μL of PBS were added into a 2 mL sample tube. The reaction mixture was mixed evenly under shaking at room temperature for 15 min. Then, the fluorescence emission intensity of the reaction mixture could be measured under the 350 nm excitation wavelength. Other steps were performed as above. The limit of detection (LOD) for ONOO−was 42.12 nM, and the LOD for Na2S2was 38.45 nM. Fluorescence spectra of the multi-signal fluorescent probe in the detection of peroxynitrite and sodium disulfide were shown inFIG.2. Accordingly, the highly-sensitive discrimination and detection for ONOO−and Na2S2were realized by using the multi-signal fluorescent probe provided herein. The multi-signal fluorescent probe provided herein is efficient and simple for the detection of oxidative and reductive substances, and can be used to discriminate and detect ONOO−and Na2S2simultaneously. The multi-signal fluorescent probe can undergo different reactions respectively with ONOO−and Na2S2under the same condition to generate different fluorescent matters which will emit green fluorescence and yellow fluorescence under specific wavelength. Therefore, ONOO−and Na2S2can be simultaneously distinguished and detected, and the simultaneous dual-channel fluorescence imaging of endogenous ONOO−and Na2S2in cells can be realized, which will facilitate the development of multi-signal bio-fluorescent probes. The multi-signal fluorescent probe provided herein utilizes a dual-channel fluorescent signal to discriminate and detect ONOO−and Na2S2simultaneously, developing the fields of analysis and detection and medical early diagnosis. Regarding a method for detecting ONOO−and Na2S2in cells, unless otherwise specified, a probe molecule is dissolved at room temperature for analytical detection, where a volume ratio of an organic phase to an aqueous phase is 5:5. The organic phase is acetonitrile (CH3CN), and the aqueous phase is formed by PBS (pH=7.4) and an aqueous solution of the analyte. The multi-signal fluorescent probe for reactive oxygen was dissolved in a solution, in which a volume ratio of DMSO to the aqueous phase was 5:5. The multi-signal fluorescent probe emitted 492 nm green light under 380 nm excitation wavelength after reacted with ONOO−, and emitted 548 nm yellow light under 410 nm excitation wavelength after reacted with Na2S2. Accordingly, a specific analyte can be detected by the specific excitation and fluorescence emission signal. The ONOO−and Na2S2can be distinguished by using different excitation wavelengths and fluorescence emission signals. The multi-signal fluorescent probe can detect the ONOO−and Na2S2simultaneously, and has no significant response to amino acids, sulfur-containing derivatives and amine derivatives, such as OH, t-BuO,1O2, NO, O2−, GSH, Cys, Hcy, SO2, NAC, H2S, F-, Cl-, Br−, I−, NAC, Gly, Ala, His, Met, Thr, Lys, Asp, Glu, Pro, Ser, NaHS, NaHSO3, EtSH, PhSH, n-Butylamine, and aniline. The LOD for ONOO−is 42.12 nM, and the LOD for Na2S2is 38.45 nM. In summary, the multi-signal fluorescent probe can realize the high-sensitivity discrimination and detection of ONOO−and Na2S2. It should be noted that described above are merely illustrative of the disclosure, and are not intended to limit the disclosure. Although the disclosure has been illustrated and described in detail above, it should be understood that those skilled in the art could still make modifications and replacements to the embodiments of the disclosure. Those modifications and replacements made by those skilled in the art based on the content disclosed herein without departing from the scope of the disclosure shall fall within the scope of the present disclosure defined by the appended claims. In addition, the features of various embodiments may be combined in the absence of contradiction. The contents in the background are merely for better understanding of the general background of the application, and should not be considered as admitting or in any way implying that the content belongs to the prior art known to those skilled in the art. | 12,878 |
11857645 | DETAILED DESCRIPTION OF THE INVENTION I. General The present invention provides novel, compositions and methods for visualizing tissue under illumination with near-infrared radiation using fusion compounds comprising near IR, closed chain, sulfo-cyanine dyes and prostate specific membrane antigen (PSMA) ligands. Surprisingly, the fusion compounds of present invention demonstrated significantly increased fluorescence compared other combinations of dyes to PSMA ligands. II. Definitions The abbreviations used herein have their conventional meaning within the chemical and biological arts. The term “metal ion” as used herein refers to elements of the periodic table that are metallic and that are positively charged as a result of having fewer electrons in the valence shell than is present for the neutral metallic element. Metals that are useful in the present invention include metals capable of forming pharmaceutically acceptable compositions. Useful metals include Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, and Ba. One of skill in the art will appreciate that the metals described above can each adopt several different oxidation states. In some instances, the most stable oxidation state is formed, but other oxidation states are useful in the present invention. The term “alkyl” as used herein refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, C1-C6alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together. The term “cycloalkyl” as used herein refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene and adamantane. For example, C3-8cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane. The term “haloalkyl” as used herein refers to alkyl as defined above where some or all of the hydrogen atoms are substituted with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro” defines a compound or radical which has at least two available hydrogens substituted with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy. As used herein, the term “halogen” refers to fluorine, chlorine, bromine and iodine. The term “aryl” as used herein refers to a monocyclic or fused bicyclic, tricyclic or greater, aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl may be phenyl, benzyl or naphthyl, preferably phenyl. “Arylene” means a divalent radical derived from an aryl group. Aryl groups can be mono-, di- or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; all of which are optionally further substituted, for instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to two adjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. An example for oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl. The term “heteroaryl” as used herein refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted, especially mono- or di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl represents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl represents preferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, and most preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl. Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted. Similarly, substituents for the aryl and heteroaryl groups are varied and are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO2, —CO2R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)2R′, —NR′—C(O)NR″R′″, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —N3, —CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″ and R′″ are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl. As used herein, the term “visualization” refers to methods of obtaining graphic images of tissue by any means, including illumination with near-infrared radiation. The term “near-infrared radiation” or “near IR radiation” refers to optical radiation with a wavelength in the range of about 700 nm to 1400 nm. References herein to the optionally plural term “wavelength(s)” indicates that the radiation may be a single wavelength or a spectrum of radiation having differing wavelengths. As used herein, the term “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate; most preferably said patient is a human. The term “tissue” as used herein includes, but is not limited to, allogenic or xenogenic bone, neural tissue, fibrous connective tissue including tendons and ligaments, cartilage, dura, fascia, pericardia, muscle, heart valves, veins and arteries and other vessels, dermis, adipose tissue, glandular tissue, prostate tissue, kidney tissue, brain tissue, renal tissue, bladder tissue, lung tissue, breast tissue, pancreatic tissue, vascular tissue, tumor tissue, cancerous tissue, or prostate tumor tissue. As used herein, the term “sterile” refers to a system or components of a system free of infectious agents including bacteria, viruses, and bioactive RNA or DNA. As used herein, the term “non-toxic” refers to the non-occurrence of detrimental effects when administered to a vertebrate as a result of using a pharmaceutical composition at levels effective for visualization of tissue under illumination with near-infrared radiation (therapeutic levels). The term “unit dosage form” as used herein encompasses any measured amount that can suitably be used for administering a pharmaceutical composition to a patient. Preferably, the unit dosage form is between 0.01 and 8 mg/kg, or 0.01 and 5 mg/kg, or 0.01 and 1 mg/kg. Suitable dosage ranges also include 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg. As recognized by those skilled in the art, when another form (e.g., another salt the pharmaceutical composition) is used in the formulation, the weight can be adjusted to provide an equivalent amount of the pharmaceutical composition. The term “oral dosage form” as used herein refers to its normal meaning in the art (i.e., a pharmaceutical composition in the form of a tablet, capsule, caplet, gelcap, geltab, pill and the like). The term “injectable dosage form” as used herein refers to its normal meaning in the art (i.e., refer to a pharmaceutical composition in the form of solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.) “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a patient and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, and the like. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention. Pharmaceutically acceptable carriers include but not limited to any adjuvants, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizing agents, isotonic agents, solvents or emulsors. As used herein, the term “machine detectable identifier” includes identifiers visible or detectable by machines including medical devices. In some instances, the medical device is a tele-surgical system. Machine detectable identifiers may facilitate the access or utilization of information that is directly encoded in the machine detectable identifier, or stored elsewhere. Examples of machine detectible identifiers include microchips, radio frequency identification (RFID) tags, barcodes (e.g., 1-dimensional or 2-dimensional barcode), data matrices, quick-response (QR) codes, and holograms. One of skill in the art will recognize that other machine detectible identifiers are useful in the present invention. III. Compositions A. Compounds The compounds of the present invention comprise fusion compounds of formulas I, II, and III which absorb light at wavelengths in the near-infrared regions of the electromagnetic spectrum. In some embodiments, the present invention provides a composition comprising a compound having the formula: wherein,R1, R2, R3, R4, R5, R6, and R7are each independently hydrogen, C1-4alkyl, substituted C1-4alkyl, or —CO2T;X is a single bond, —O—, or —S—;subscripts a, b, c, d, e, f, g, and h are each independently an integer from 1 to 6, andT is each independently a metal ion, H, or a negative charge; Z is each independently H, C1-6substituted or unsubstituted alkyl, C6-12substituted or unsubstituted aryl, or a C6-12substituted or unsubstituted heteroaryl; andwherein, the composition is adapted for visualization of tissue under illumination with near-infrared radiation. In some other embodiments, the composition has the formula: In some other embodiments, the composition has the formula: The compounds of the present invention may exist as salts. The present invention includes such salts. Examples of applicable salt forms include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates, (−)-tartrates or mixtures thereof including racemic mixtures, succinates, benzoates and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in art. Also included are base addition salts such as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like. Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other salts include acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of pharmaceutically acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference. Pharmaceutically acceptable salts include salts of the active compounds which are prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively non-toxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science,1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents. Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. In some embodiments, the present invention provides a pharmaceutical composition including a pharmaceutically acceptable excipient. B. Synthesis The compounds described in the above embodiments may be made using procedures known in the art. In general, fusion compounds of the present invention can be synthesized by attaching near IR, closed chain, sulfo-cyanine dyes to prostate specific membrane antigen ligands via a linkage. The materials used can be determined by the desired structure, and the type of linkage used. For example, the prostate specific membrane antigen ligands used in the compositions of the present invention can be synthesized as described in PCT application WO 2010/108125 and is incorporated herein in its entirety. Compounds can assembled by reactions between different components, to form linkages such as ureas (—NRC(O)NR—), thioureas (—NRC(S)NR—), amides (—C(O)NR— or —NRC(O)—), or esters (—C(O)O— or —OC(O)—). Urea linkages can be readily prepared by reaction between an amine and an isocyanate, or between an amine and an activated carbonamide (—NRC(O)—). Thioureas can be readily prepared from reaction of an amine with an isothiocyanate. Amides (—C(O)NR— or —NRC(O)—) can be readily prepared by reactions between amines and activated carboxylic acids or esters, such as an acyl halide or Nhydroxysuccinimide ester. Carboxylic acids may also be activated in situ, for example, with a coupling reagent, such as a carbodiimide, or carbonyldiimidazole (CDI). Esters may be formed by reaction between alcohols and activated carboxylic acids. Triazoles are readily prepared by reaction between an azide and an alkyne, optionally in the presence of a copper (Cu) catalyst. Prostate specific membrane antigen ligands can also be prepared by sequentially adding components to a preformed urea, such as the lysine-urea-glutamate compounds described in Banerjee et al. (J. Med. Chem. vol.51, pp. 4504-4517, 2008). Other urea-based compounds may also be used as building blocks. Exemplary syntheses of the near IR, closed chain, sulfo-cyanine dyes used in the compositions of the present invention are described in U.S. Pat. Nos. 6,887,854 and 6,159,657 and are incorporated herein in their entirety (FIG.1). Additionally, some IR, closed chain, sulfo-cyanine dyes of the present invention are commercially available, including DyLight 800 (ThermoFisher). As mentioned above, fusion compositions of the present invention can be synthesized via attachment of near IR, closed chain, sulfo-cyanine dyes to prostate specific membrane antigen ligands by reacting a reactive amine on the ligand with a near IR dye (FIG.2). A wide variety of near IR dyes are known in the art, with activated functional groups for reacting with amines. C. Formulation The compositions of the present invention can be prepared in a wide variety of oral, parenteral and topical dosage forms. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. The compositions of the present invention can also be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compositions described herein can be administered by inhalation, for example, intranasally. Additionally, the compositions of the present invention can be administered transdermally. The compositions of this invention can also be administered by intraocular, insufflation, powders, and aerosol formulations (for examples of steroid inhalants, see Rohatagi,J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann.Allergy Asthma Immunol.75:107-111, 1995). Accordingly, the present invention also provides pharmaceutical compositions including a pharmaceutically acceptable carrier or excipient. For preparing pharmaceutical compositions from the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa. (“Remington's”). In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5% or 10% to 70% of the compounds of the present invention. Suitable solid excipients include, but are not limited to, magnesium carbonate; magnesium stearate; talc; pectin; dextrin; starch; tragacanth; a low melting wax; cocoa butter; carbohydrates; sugars including, but not limited to, lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins including, but not limited to, gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound (i.e., dosage). Pharmaceutical compositions of the invention can also be used orally using, for example, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the compositions of the present invention mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the compositions of the present invention may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. Aqueous solutions suitable for oral use can be prepared by dissolving the compositions of the present invention in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity. Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. Oil suspensions can be formulated by suspending the compositions of the present invention in a vegetable oil, such asarachisoil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto,J. Pharmacol. Exp. Ther.281:93-102, 1997. The pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be formulated for administration via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao,J. Biomater Sci. Polym. Ed.7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g.,Gao Pharm. Res.12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles,J. Pharm. Pharmacol.49:669-674, 1997). Both transdermal and intradermal routes afford constant delivery for weeks or months. In another embodiment, the compositions of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a body cavity or lumen of an organ. The formulations for administration will commonly comprise a solution of the compositions of the present invention dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well-known techniques including radiation, chemical, heat/pressure, and filtration sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the compositions of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed,J. Microencapsul.13:293-306, 1996; Chonn,Curr. Opin. Biotechnol.6:698-708, 1995; Ostro,Am. J. Hosp. Pharm.46:1576-1587, 1989). Lipid-based drug delivery systems include lipid solutions, lipid emulsions, lipid dispersions, self-emulsifying drug delivery systems (SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). In particular, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants and co-surfactants that can disperse spontaneously in aqueous media and form fine emulsions (SEDDS) or microemulsions (SMEDDS). Lipids useful in the formulations of the present invention include any natural or synthetic lipids including, but not limited to, sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®. In some embodiments the compositions of the present invention are sterile and generally free of undesirable matter. The compounds and compositions may be sterilized by conventional, well known techniques including heat/pressure, gas plasma, steam, radiation, chemical, and filtration sterilization techniques. For example, terminal heat sterilization can be used to destroy all viable microorganisms within the final formulation. An autoclave is commonly used to accomplish terminal heat-sterilization of drug products in their final packaging. Typical autoclave cycles in the pharmaceutical industry to achieve terminal sterilization of the final product are 121° C. for 15 minutes. The compositions of the present invention can be autoclaved at a temperature ranging from 115 to 130° C. for a period of time ranging from 5 to 40 minutes with acceptable stability. Autoclaving is preferably carried out in the temperature range of 119 to 122° C. for a period of time ranging from 10 to 36 minutes. The compositions can also be sterilized by dry heat as described by Karlsson, et al., in U.S. Pat. No. 6,392,036, which discloses a method for the dry heat sterilization that can be used for drug formulations. The compositions can also be sterilized as described in WO 02/41925 to Breath Limited, which discloses a rapid method, similar to pasteurization, for the sterilization of compositions. This method entails pumping the composition to be sterilized through stainless steel pipes and rapidly raising the temperature of the composition to about 130-145° C. for about 2-20 seconds, subsequently followed by rapid cooling in seconds to ambient conditions. The compositions can also be sterilized by irradiation as described by Illum and Moeller inArch. Pharm. Chemi. Sci., Ed.2, 1974, pp. 167-174). The compositions can also be sterilized by UV, x-rays, gamma rays, e beam radiation, flaming, baking, and chemical sterilization. Alternatively, sterile pharmaceutical compositions according to the present invention may be prepared using aseptic processing techniques. Aseptic filling is ordinarily used to prepare drug products that will not withstand heat sterilization, but in which all of the ingredients are sterile. Sterility is maintained by using sterile materials and a controlled working environment. All containers and apparatus are sterilized, preferably by heat sterilization, prior to filling. The container (e.g., vial, ampoule, infusion bag, bottle, or syringe) are then filled under aseptic conditions. In some embodiments, the compounds and compositions of the present invention are non-toxic and generally free of detrimental effects when administered to a vertebrate at levels effective for visualization of tissue under illumination with near-infrared radiation. Toxicity of the compounds and compositions of the present invention can be assessed by measuring their effects on a target (organism, organ, tissue or cell). Because individual targets typically have different levels of response to the same dose of a compound, a population-level measure of toxicity is often used which relates the probabilities of an outcome for a given individual in a population. Toxicology of compounds can be determined by conventional, well-known techniques including in vitro (outside of a living organism) and in vivo (inside of a living organism) studies. For example, determination of metabolic stability is commonly examined when assessing the toxicity of a compound as it is one of several major determinates in defining the oral bioavailability and systemic clearance of a compound. After a compound is administered orally, it first encounters metabolic enzymes in the gastrointestinal lumen as well as in the intestinal epithelium. After it is absorbed into the bloodstream through the intestinal epithelium, it is first delivered to the liver via the portal vein. A compound can be effectively cleared by intestinal or hepatic metabolism before it reaches systemic circulation, a process known as first pass metabolism. The stability of a compound towards metabolism within the liver as well as extrahepatic tissues will ultimately determine the concentration of the compound found in the systemic circulation and affect its half-life and residence time within the body. Cytochrome P450 (CYP) enzymes are found primarily in the liver but also in the intestinal wall, lungs, kidneys and other extrahepatic organs and are the major enzymes involved in compound metabolism. Many compounds undergo deactivation by CYPs, either directly or by facilitated excretion from the body. Also, many compounds are bioactivated by CYPs to form their active compounds. Thus, determining the reactivity of a compound to CYP enzymes is commonly used to assess metabolic stability of a compound. The Ames reverse mutation Assay is another common toxicology assay for assessing the toxicity of a compound. The Ames Assay, utilizes several different tester strains, each with a distinct mutation in one of the genes comprising the histidine (his) biosynthetic operon (Ames, B. N., et al., (1975)Mutation Res.31:347-363). The detection of revertant (i.e., mutant) bacteria in test samples that are capable of growth in the absence of histidine indicates that the compound under evaluation is characterized by genotoxic (i.e. mutagenic) activity. The Ames Assay is capable of detecting several different types of mutations (genetic damage) that may occur in one or more of the tester strains. The practice of using an in vitro bacterial assay to evaluate the genotoxic activity of drug candidates is based on the prediction that a substance that is mutagenic in a bacterium is likely to be carcinogenic in laboratory animals, and by extension may be carcinogenic or mutagenic to humans. In addition, the human ether-a-go-go related gene (hERG) assay can be used to evaluate the potential cardiotoxicity of a compound. Cardiotoxicity can arise when the QT interval is prolonged leading to an elevated risk of life-threatening arrhythmias. The QT interval is a measure of the time between the start of the Q wave and the end of the T wave in the heart's electrical cycle. The QT interval represents electrical depolarization and repolarization of the ventricles. A lengthened QT interval has most commonly been associated with loss of current through hERG potassium ion channels due to direct block of the ion channel by drugs or by inhibition of the plasma membrane expression of the channel protein (Su et al.J. Biomol Screen2011, 16, 101-111). Thus, an in vitro hERG screening assay can be used to detect disruption or inhibition of the hERG membrane trafficking function and assess potential cardiotoxicity of a compound. Other methods of assessing the toxicity of compounds include in vivo studies which administer relatively large doses of a test compound to a group of animals to determine the level which is lethal to a percentage of the population (mean lethal dose LD50or mean lethal concentration LC50). Toxicity of a compound can also be assessed in vivo by examining whether a compound produces statistically significant negative effects on cardiac, blood pressure, central nervous system (CNS), body weight, food intake, gross or microscopic pathology, clinical pathology, or respiratory measures in an animal. For example, in a set of in vitro studies evaluating the metabolic stability of a composition of formula III, it was shown that the compound appears to be stable in rat, dog and human plasma, does not appear to be broken down into metabolites, and does not show any significant reactivity to 9 major CYP liver enzymes. Additionally, the composition of formula III does not show any mutagenicity at any of the tested doses in the Ames reverse mutation assay, a widely used in vitro method that determines the ability of a chemical to cause mutations in DNA. Further safety pharmacology was assessed in vitro using the human ether-a-go-go related gene (hERG) assay for determining possible cardio-toxic effects. Studies utilizing this assay determined that the composition of formula III shows only a small (11%) inhibition of hERG function at the highest tested concentration (30 times higher than levels effective for visualization), indicating even at this concentration the composition of formula III is unlikely to translate into any clinically threatening physiological cardiac changes. Furthermore, toxicology of the composition of formula III was also investigated in non-clinical in vivo studies in both rats and dogs. A study evaluating the effects of the composition of formula III on cardiac and respiratory safety in radio-telemetry monitored dogs showed no statistically significant negative effects on cardiac, blood pressure or respiratory measures at doses up to 80 times higher than those required for visualization. Additional safety pharmacology performed over 28 days in rats indicated doses as high as 160 times visualization levels per day showed no biologically meaningful effects on the central nervous system (CNS). Finally, a series of toxicology studies in dogs showed doses as high as 100 times visualization levels showed no significant effects on body weight, food intake, gross or microscopic pathology as well as clinical pathology (clinical serum chemistry, hematology, coagulation and urinalysis). No mortality was observed at any tested dose of the composition of formula III in rat, dog or pig animal models. In some embodiments, the compositions of the present invention can be lyophilized in a sterile container for convenient dry storage and transport. A ready-to-use preparation can subsequently be made by reconstituting the lyophilized compositions with sterile water. The terms “lyophilization,” “lyophilized,” and “freeze-dried” refer to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to enhance stability of the lyophilized product upon storage. In some embodiments, the composition can be contained within a sterile container, where the container has a machine detectable identifier which is readable by a medical device. Examples of machine detectible identifiers include microchips, radio frequency identification (RFID) tags, barcodes (e.g., 1-dimensional or 2-dimensional barcode), data matrices, quick-response (QR) codes, and holograms. One of skill in the art will recognize that other machine detectible identifiers are useful in the present invention. In some cases, the machine detectable identifier can include a microchip, an integrated circuit (IC) chip, or an electronic signal from a microchip that is detectable and/or readable by a computer system that is in communication with the medical device. In some cases, the machine detectable identifier includes a radio frequency identification (RFID) tag. RFID tags are sometimes called as transponders. RFID tags generally are devices formed of an IC chip, an antenna, an adhesive material, and are used for transmitting or receiving predetermined data with an external reader or interrogator. RFID tags can transmit or receive data with a reader by using a contactless method. According to the amplitude of a used frequency, inductive coupling, backscattering, and surface acoustic wave (SAW) may be used. Using electromagnetic waves, data may be transmitted or received to or from a reader by using a full duplex method, a half duplex (HDX) method, or a sequential (SEQ) method. In some cases, the machine detectable identifier can include a barcode. Barcodes include any machine-readable format, including one-dimensional and two-dimensional formats. One-dimensional formats include, for example, Universal Product Code (UPC) and Reduced Space Symbology (RSS). Two-dimensional formats, or machine-readable matrices, include for example, Quick Response (QR) Code and Data Matrix. In some cases, the medical device can be configured to detect the machine detectable identifier. In one example, the medical device is a tele-surgical system that includes a special imaging mode (e.g., a fluorescence imaging mode) for use with dyes such as those described in this disclosure. One example of a tele-surgical system that includes a fluorescence imaging mode is described in U.S. Pat. No. 8,169,468, entitled “Augmented Stereoscopic Visualization for a Surgical Robot,” which is hereby incorporated in its entirety herein. In some cases, medical devices can incorporate an imaging device that can scan, read, view, or otherwise detect a machine detectable identifier that is displayed to the imaging device. In one aspect, the medical device will permit a user to access the fluorescence imaging mode of the medical device only if the medical device detects the presence of a known machine detectable identifier that corresponds to a dye identified as being compatible for use with the medical device. In contrast, if the medical device does not detect a known machine detectable identifier, the medical device will not permit a user to access the fluorescence imaging mode and associated functionality. Imaging devices can include optical scanners, barcode readers, cameras, and imaging devices contained within a tele-surgical system such as an endoscope. Information associated with the machine detectable identifier can then be retrieved by the medical device using an imaging device. Upon detection of the identifier, an automatic process may be launched to cause a predetermined action to occur, or certain data to be retrieved or accessed. The information encoded onto the machine detectable identifier may include instructions for triggering an action, such as administering a composition of the present invention to a patient. In some embodiments, the machine detectable identifier includes unencrypted e-pedigree information in the desired format. The e-pedigree information can include, for example, lot, potency, expiration, national drug code, electronic product code, manufacturer, distributor, wholesaler, pharmacy and/or a unique identifier of the salable unit. In some embodiments, the sterile container having a machine detectable identifier includes a fluid outlet configured to mate with the medical device. In some cases, the fluid outlet of the machine detectable identifier is mechanically affixed to the medical device. D. Administration The compounds and compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods. The compounds and compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods. Transdermal administration methods, by a topical route, can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the compounds and compositions of the present invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including the compounds and compositions of the present invention and any other agent. Alternatively, the various components can be formulated separately. The compositions of the present invention, and any other agents, can be present in any suitable amount, and can depend on various factors including, but not limited to, weight and age of the patient, state of the disease, etc. Suitable dosage ranges include from about 0.01 and 8 mg/kg, or about 0.01 and 5 mg/kg, or about 0.01 and 1 mg/kg. Suitable dosage ranges also include 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg. The composition can also contain other compatible compositions. The compositions described herein can be used in combination with one another, with other active compositions known to be useful for visualization of tissue under illumination with near-infrared radiation, or with compositions that may not be effective alone, but may contribute to the efficacy of the active composition. The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described, or portions thereof, it being recognized that various modifications are possible within the scope of the invention claimed. Moreover, any one or more features of any embodiment of the invention may be combined with any one or more other features of any other embodiment of the invention, without departing from the scope of the invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. IV. Methods for Visualization of Tissue The present invention generally provides novel, compositions and methods for visualizing tissue under illumination with fusion compounds comprising near-infrared radiation using near IR, closed chain, sulfo-cyanine dyes and prostate specific membrane antigen ligands. In some embodiments, the present invention provides a method for visualization of tissue expressing PSMA, the method comprising, administering to a patient a composition of a compound having the formula: wherein,R1, R2, R3, R4, R5, R6, and R7are each independently hydrogen, C1-4alkyl, substituted C1-4alkyl, or —CO2T;X is a single bond, —O—, or —S—;subscripts a, b, c, d, e, f, g, and h are each independently an integer from 1 to 6;T is each independently a metal ion, H, or a negative charge; Z is each independently H, C1-6substituted or unsubstituted alkyl, C6-12substituted or unsubstituted aryl, or a C6-12substituted or unsubstituted heteroaryl; and where the compound is administered in an amount sufficient for imaging tissue under illumination with near-infrared radiation; imaging the tissue under illumination with near-infrared radiation; and obtaining at least one image of tissue from the patient using the composition. In some embodiments, the method administers to a patient a pharmaceutical composition comprising a unit dosage form of a compound of formula I wherein, the composition is sterile, non-toxic, and adapted for administration to a patient; and wherein, the unit dosage form of the compound delivers to the patient an amount between 0.01 and 8 mg/kg. In some cases, the method further comprises obtaining the image during administration, after administration, or both during and after administration of the composition. In some cases, the method further comprises intravenously injecting a composition of formula I into a patient. In some cases, the composition is injected into a circulatory system. In some embodiments, the present invention provides a use of the composition adapted for administration to a patient to obtain visualization of tissue expressing PSMA under illumination with near-infrared radiation wherein the unit dosage form delivers to the patient an amount is between 0.01 and 8 mg/kg. In some cases, the use is adapted for administration to a human patient to obtain visualization of human tissue under illumination with near-infrared radiation wherein the unit dosage form delivers to the patient an amount between 0.01 and 8 mg/kg. In some embodiments, the method further comprises visualizing a patient area on which surgery is or will be performed, or for viewing a patient area otherwise being examined by a medical professional. In some cases, the method further comprises performing a surgical procedure on the patient areas based on the visualization of the surgical area. In some cases, the method further comprises viewing a patient area on which an ophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, or neurological procedure is or will be performed. In some cases, the method further comprises obtaining ex vivo images of at least a portion of the patient. In some embodiments, improvement in visualization of tissue can be achieved through the use of dyes capable of targeted visualization of tissue, including by fusion compositions of near IR, closed chain, sulfo-cyanine dyes and prostate specific membrane antigen (PSMA) ligands. The PSMA, while expressed in prostate tumor epithelium, is also expressed in the neovasculature of many solid tumors (Chang et al.,Cancer Res., vol. 59, pp. 3192-3198, 1999; Chang et al.,Clin. Cancer Res., vol. 5, pp. 2674-2681, 1999; Gong et al.,Cancer Metastasis Rev., vol. 18, pp. 483-490, 1999; Chang et al.,Mol. Urol., vol. 3, pp. 313-320, 1999; Baccala et al.,Urology, vol. 70, pp. 385-390, 2007; Chang et al.,Urology, vol. 57, pp. 801-805, 2001 Milowsky et al.,J Clin. Oncol., vol. 25, pp. 540-547, 2007). As a result, PSMA ligands have been used to identify renal, bladder, lung, breast, colorectal, and pancreatic tumors (Milowsky et al.,J Clin. Oncol., vol. 25, pp. 540-547, 2007). PSMA ligands have proved to be an effective vascular targeting agent in human subjects. Other reports have further studied PSMA expression in certain tumor types. For example, Baccala et al. noted that clear cell renal cell carcinoma expresses significantly more PSMA in its neovasculature than does the papillary variety (Baccala et al.,Urology, vol. 70, pp. 385-390, 2007). Furthermore, angiomyolipoma, a benign renal lesion, has been found not to express PSMA. As an enzyme with an extracellular active site, PSMA represents an excellent target for imaging and therapy directed toward solid tumor neovasculature in addition to prostate cancer itself. PSMA-based agents can report on the presence of this marker, which is increasingly recognized as an important prognostic determinate in PCa (Murphy et al.,Urology, vol. 51, pp. 89-97, 1998). It is also the target for a variety of new PCa therapies (Gal sky et al.,J Clin Oneal, vol. 26, pp. 214 7-2154, 2008). Accordingly, compositions of the present invention comprising fusion compounds of near IR, closed chain, sulfo-cyanine dyes and PSMA ligands can be used to visualize a variety of tumor tissues. In some cases, the tumor tissue being visualized is prostate tissue, kidney tissue, brain tissue, renal tissue, bladder tissue, lung tissue, breast tissue, pancreatic tissue, vascular tissue, tumor tissue, cancerous tissue, or prostate tumor tissue. In some cases, the tissue being visualized is cancerous tissue. In some cases, the tissue being visualized is prostate tissue. In some cases, the tissue being visualized is prostate tumor tissue. In some cases, the method further comprises the use of compositions of the present invention in combination with the da Vinci Surgical System's Firefly fluorescence, in providing an augmented view that enhances the difficult-to-visualize tissue to increase surgical efficacy, reduce the injury rate, and possibly speed up surgery by providing the surgeon with confidence and security that he/she is removing all intended tissue. For example, positive margin rates during prostate cancer surgery can be as high as 30%, meaning that cancerous tissue is left within a man despite attempts to remove it all. Such patients frequently require subsequent therapy, including but not limited to external beam radiation therapy, brachytherapy, ablative and hormonal therapy. In addition, rates of erectile dysfunction and incontinence, resulting from nerve injury during prostate surgery can approach 50% and 20% respectively. Compositions of the present invention comprising fusion compounds of near IR, closed chain, sulfo-cyanine dyes and PSMA ligands can also be used to increase contrast between prostatic and nerve tissue, helping to avoid resulting injury to nerves and sphincter tissue. Accordingly, the use of compositions of the present invention in combination with the da Vinci Surgical System's Firefly fluorescence, provide an augmented view that enhances the difficult-to-visualize tissue to reduce positive surgical margin rates, reduce adverse side effects, and possibly speed up surgery. V. Examples Example 1. Visualization of Tumor Tissue Under Illumination Four similar tumor tissues were treated with four different fusion compounds comprising different dyes and targeting ligands. The four fusion compounds include DyLight800 and PSMA-targeting ligand, IRDye800CW and a PSMA-targeting ligand, ZW800 and PSMA-targeting ligand, and ICG and a PSMA-targeting ligand. The treated tumor tissue in each of the four images was then exposed to narrow band, near IR excitation light in order to produceFIGS.1A-1D. The narrow band, near IR excitation light included light wavelengths expected to correspond to the excitation maxima wavelength for each of the fluorophores associated with each of the different compounds. As shown inFIG.2, the fusion compound comprising DyLight800 and a PSMA-targeting ligand exhibited significantly more fluorescence. The significant differences in fluorescence may be due to at least one of the following reasons: (1) Attaching ZW800, IRDye800CW, or ICG to PSMA-targeting ligand adversely affected the binding affinity of the PSMA-targeting ligand to PSMA binding sites on tumor tissue; and (2) Attaching ZW800, IRDye800CW, or ICG to PSMA-targeting ligand adversely affected the fluorescent properties of the ZW800, IRDye800CW, or ICG fluorescent moiety. In contrast, conjugating DyLight800 to PSMA-targeting ligand had neither of these adverse effects. Example 2. Visualization of Prostatic Tissue Under Illumination 20 mg of a compound of formula III in 10 ml of sterile water can be administered intravenously to patients. Laparoscopic ports can then be placed and the da Vinci Surgical System connected to the ports. The endoscope of the system can then be directed at the prostate of the patient, and laser excitation at approximately 800 nm can be used to excite the composition of formula III within the prostate. A small amount of blue and green light can also be emitted in order to allow visualization of the background anatomy. Approximately 2-24 hours after administration, visualization of the prostate and prostate tumor tissue can be achieved as the composition of formula III has bound to PSMA. | 58,093 |
11857646 | DETAILED DESCRIPTION Provided herein are improved methods for preparing phospholipid-based ultrasound contrast agents (UCA). These improvements are based in part on the surprising discovery that certain phospholipid-based formulations, intended for use in preparing ultrasound contrast agents, are susceptible to the presence and amount of certain divalent metal cations. Specifically, it was unexpectedly found that divalent metal cations, such as calcium, at certain concentrations, when introduced into a phospholipid-based formulation used to generate the leading ultrasound contrast agent, DEFINITY®, caused phospholipid and potentially other components of the formulation to precipitate out of solution, thereby rendering the formulation unusable. Such formulations are typically made in large scale batches and thus the inadvertent addition of calcium, for example, would render an entire batch unusable. This can lead to reduced manufacturing capability. It has also been found, surprisingly, that certain phospholipids are more susceptible to precipitation induced by the presence of divalent metal cations such as calcium. Specifically, DPPA in non-aqueous solvent such as propylene glycol is more likely to precipitate in the presence of certain concentrations of divalent metal cations such as calcium. This same sensitivity was not observed, or not observed to the same degree, with other phospholipids such as DPPC and DPPE. This differential precipitation profile can easily result in a phospholipid formulation, and ultimately a UCA, having a different phospholipid composition than planned or desired. Thus, not only can the presence of divalent metal cations reduce total yield of a UCA (e.g., due to the non-filterability of a precipitate-containing phospholipid formulation such as the phospholipid suspensions described herein), it can also interfere with the phospholipid distribution of the UCA. This is problematic because it may result in UCA formulations of wholly unknown phospholipid content. As is well known in the pharmaceutical arts, the composition of such UCA formulations must remain constant and robustly reproducible, and batch-to-batch variability must be avoided or minimized to the greatest extent possible. This disclosure therefore provides improved methods for preparing phospholipid formulations such as phospholipid solutions and phospholipid suspensions, as described herein. These methods improve the yields of such formulations by reducing the likelihood of phospholipid precipitation. They also produce, in a more robust and reproducible manner, phospholipid formulations having their intended phospholipid profiles and distributions. These methods take advantage of the novel and surprising findings described herein and provide phospholipid formulations of the desired phospholipid content and proportion without resorting to detecting precipitate. Phospholipid Formulations, Generally Provided herein are methods for preparing improved phospholipid solutions, phospholipid suspensions and ultimately UCA formulations. As will be described in greater detail, in some instances, the UCA formulations may be formed from non-aqueous phospholipid solutions that are combined with a gas such as perflutren. In other instances, the UCA formulation may be formed from combining the non-aqueous phospholipid solution with an aqueous solvent to form a phospholipid suspension that is combined with a gas such as perflutren. These and other phospholipid-containing compositions are collectively referred to herein as phospholipid formulations. Each of the specific formulations will be described in greater detail below. The phospholipid formulations of this disclosure may comprise the three phospholipids that are used in the manufacture of the FDA-approved DEFINITY® microspheres. These three phospholipids are(1) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (referred to herein as DPPC),(2) 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (referred to herein as DPPA), and(3)N-(methoxy polyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (referred to herein as MPEG5000-DPPE). The phospholipid formulations of this disclosure may comprise DPPC and MPEG-5000-DPPE. In some instances, modified forms of one or more of these phospholipids may be used. For example, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) may be conjugated to polyethylene glycol (PEG). The PEG conjugated to DPPE, or to another phospholipid, may have a molecular weight (MW, or length) selected from 1000-10,000, in some non-limiting instances. More typically, the PEG may have a MW of about 5000, in which case it is referred to as PEG5000, and when conjugated to DPPE is referred to as PEG5000-DPPE. The PEG is typically conjugated to a phospholipid such as DPPE at the phospholipid head group rather than at the aliphatic chain end. The PEG may have a hydroxy or a methoxy terminus, and may be referred to as HO-PEG5000 or as MPEG5000, respectively. When conjugated to a DPPE, as an example, the conjugate may be referred to as HO-PEG5000-DPPE or as MPEG5000-DPPE. The full chemical name of the latter conjugate is N-(methoxy polyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, mono sodium salt (referred to herein as MPEG5000-DPPE). DPPA, DPPC and MPEG5000-DPPE may be used in molar percentages of about 77-90 mole % DPPC, about 5-15 mole % DPPA, and about 5-15 mole % DPPE, including MPEG5000-DPPE. Preferred ratios of each phospholipid include weight % ratios of 6.0 to 53.5 to 40.5 (DPPA:DPPC:MPEG5000-DPPE) or a mole % ratio of 10 to 82 to 8 (10:82:8) (DPPA:DPPC:MPEG5000-DPPE). The remainder of this disclosure will refer specifically to DPPA, DPPC and MPEG5000-DPPE for convenience and brevity, but it is to be understood that the teachings provided herein are intended to encompass methods that utilize and/or compositions that comprise these or other phospholipids singly or in combination such as but not limited to a combination of DPPC and MPEG5000-DPPE. Various methods provided herein involve measuring the divalent metal concentration of the components used to make the phospholipid formulations described herein. Of particular importance are the components used to make the phospholipid solutions, particularly since precipitation appears to be a phenomenon first observed at the phospholipid solution step rather than at the phospholipid suspension step. Methods that may be used to measure divalent metal cation concentration, such as calcium and magnesium concentration, are described in greater detail herein including in the Examples. Some methods may involve measuring the divalent metal cation concentration of only one component, such as for example MPEG5000-DPPE. Other methods may involve measuring the divalent metal cation concentration of two or more of the components such as for example two or three of the phospholipids. In some embodiments, the components may be combined together before the measurement is made. Still other methods involve measuring the divalent metal cation concentration of all the components, including the non-aqueous solvent, used to make the phospholipid formulation such as the phospholipid solution. Such measurement may be made before or after the components are combined. For example, measurement may be made of individual components used to make a phospholipid solution or it may be made of the phospholipid solution itself. Various other methods provided herein involve selecting components used to make the phospholipid formulations such as the phospholipid solutions, based on their divalent metal cation concentration. More specifically, the methods involve selecting one or more components that have been characterized or identified as having no or low divalent metal cation concentration, including no or low calcium concentration or no or low magnesium concentration. Some methods may involve selecting one component, such as MPEG5000-DPPE, characterized or identified as having no or low divalent metal cation concentration, including no or low calcium concentration or no or low magnesium concentration. Some methods may involve selecting two or more or all components based on their combined divalent metal cation concentration. Thus, it is contemplated that DPPA, DPPC and MPEG5000-DPPE, as well as other components such as but not limited to non-aqueous solvent and/or its individual components, may be individually characterized as having no or low divalent metal cation concentration but that when used together their combined divalent metal cation concentration will no longer satisfy the requirement of no or low divalent metal cation concentration and will cause precipitation. Thus, in these and other instances, two, three or all of the components such as two or three of the phospholipids may be selected such that their combined divalent metal cation concentration is characterized as no or low divalent metal cation concentration. Phospholipid Solution As used herein, a phospholipid solution refers to a composition comprising one or more phospholipids in a non-aqueous solvent. The phospholipid solution may minimally comprise DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent. The phospholipid solution may minimally comprise DPPC and MPEG5000-DPPE in a non-aqueous solvent. A non-aqueous solvent, as used herein, is a solvent that causes phospholipids to dissolve thereby forming solution (i.e., a phospholipid solution). Preferably, the non-aqueous solvent present in the phospholipid solution is pharmaceutically acceptable, particularly since it is carried through to the final UCA formulation that is administered to a subject including a human subject. In certain embodiments, the non-aqueous solvent used to make the phospholipid solution is not or does not comprise methanol or toluene or methyl t-butyl ether (MTBE). The non-aqueous solvent of the phospholipid solution may be a single solvent or it may be combination of solvents. Non-aqueous solvents include but are not limited to propylene glycol (which may be referred to herein as PG) and glycerol (which may be referred to herein as G). Both are provided as liquid stocks. In some instances, the non-aqueous solvent of the phospholipid solution may be PG alone or it may be a mixture of PG and G (which may be referred to as PG/G). A non-aqueous solvent that comprises at least PG may be referred to herein as a PG-comprising non-aqueous solvent. The PG/G mixtures include ratios ranging from 5:1 to 1:5 (weight by weight). In some embodiments, a PG:G w/w ratio of 1:1 is used (and is referred to herein as a 1:1 mixture). The phospholipid solution may further comprise one or more buffers. Such buffers are those capable of buffering a non-aqueous solvent such as those recited above. Examples include, without limitation, an acetate buffer (e.g., a combination of sodium acetate and acetic acid), a benzoate buffer (e.g., a combination of sodium benzoate and benzoic acid), and a salicylate buffer (e.g., a combination of sodium salicylate and salicylic acid). Other buffers that may be used include a diethanolamine buffer, a triethanolamine buffer, a borate buffer, a carbonate buffer, a glutamate buffer, a succinate buffer, a malate buffer, a tartrate buffer, a glutarate buffer, an aconite buffer, a citrate buffer, a lactate buffer, a glycerate buffer, a gluconate buffer, and a tris buffer. In some embodiments, an acetate buffer is used. The buffer used in the non-aqueous solvent may be a non-phosphate buffer intending that it is not a phosphate buffer. The buffer concentration will vary depending on the type of buffer used, as will be understood and within the skill of the ordinary artisan to determine. The buffer concentration in the non-aqueous solvent may range from about 1 mM to about 100 mM, including about 1 mM to about 50 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM, including about 5 mM. Accordingly, the phospholipid solution may comprise one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueous solvent that is or that comprises PG, and optionally a buffer such as acetate buffer. The phospholipid solution may be made in a number of ways, several of which are described in greater detail below. In general, the non-aqueous solvent may be warmed prior to contact with the phospholipids, and if used the buffer may first be present in the solvent prior to contact with the phospholipids. The solvent and then solution may be stirred to facilitate dissolution of the phospholipids. Significantly, it has been found that phospholipid precipitation associated with divalent metal cations occurs in the non-aqueous solvent and thus in the process of making the phospholipid solution. Thus, as described herein various methods include steps of measuring divalent metal cation concentration of the various components used to make the phospholipid solution, including the phospholipids whether individually or collectively, the non-aqueous solvent such as the PG and G, the buffer such as the acetate buffer, if used, and the like. In some embodiments, the divalent metal cation concentration of the phospholipid suspension may be measured, instead of or in addition to measuring the divalent metal cation concentration of the phospholipid solution. A visual observation of the phospholipid solution may be made to detect precipitate, although this is not required.FIG.1is a photograph showing various phospholipid solutions having differing degrees of precipitate. In some embodiments, the phospholipid solution is then used to prepare the phospholipid suspension described in greater detail below. In some embodiments, the phospholipid solution is directly contacted with gas such as a perfluorocarbon gas to make phospholipid encapsulated gas microspheres, without first contacting the phospholipid solution with an aqueous solvent. That is, in some instances, the phospholipid-encapsulated gas microspheres are made through contact and vigorous shaking (referred to as activation) of the (non-aqueous) phospholipid solution and the gas. Such microspheres may then be contacted with an aqueous solvent to form a UCA. Phospholipid suspension As used herein, a phospholipid suspension refers to an aqueous phospholipid formulation comprising phospholipid solution and an aqueous solvent. The phospholipid suspension may comprise one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE. A phospholipid suspension will minimally comprise one or more phospholipids such as one or more phospholipids, a non-aqueous solvent such as PG, and an aqueous solvent. An aqueous solvent, as used herein, is or comprises water as its major component (by weight). An aqueous solvent may further comprise one or more salts, and thus may be referred to as an aqueous saline solvent. It may additionally or alternatively comprise a buffer, and thus may be referred to as an aqueous buffered saline solvent or an aqueous buffered solvent. Preferably, the aqueous solvent, regardless of whether it includes salt(s) or buffer(s) is pharmaceutically acceptable, since like the phospholipid solution it is carried through to the final UCA formulation that is administered to a subject including a human subject. Salts that may be included in the aqueous solvent include but are not limited to sodium chloride. Buffers that may be included in the aqueous solvent include but are not limited to phosphate buffer, acetate buffer, benzoate buffer, salicylate buffer, diethanolamine buffer, triethanolamine buffer, borate buffer, carbonate buffer, glutamate buffer, succinate buffer, malate buffer, tartrate buffer, glutarate buffer, aconite buffer, citrate buffer, lactate buffer, glycerate buffer, gluconate buffer, and a tris (tris(hydroxymethyl)methylamine) buffer. Typically, either the non-aqueous solvent or the aqueous solvent comprises a buffer, but not both. The buffer concentration will vary depending on the type of buffer used, as will be understood and within the skill of the ordinary artisan to determine. The buffer concentration in the aqueous solvent may range from about 1 mM to about 100 mM, including about 1 mM to about 50 mM, or about 10 mM to about 30 mM, or about 20 mM to about 30 mM, or about 20 mM to about 25 mM, including about 25 mM. Accordingly, the phospholipid suspension may comprise one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueous solvent that is or that comprises PG, an aqueous solvent that may comprise one or more salts such as sodium chloride, and optionally a buffer such as acetate buffer or a phosphate buffer. Phospholipid suspensions may be physically characterized as phospholipids suspended, rather than dissolved, in an aqueous solvent. The phospholipid suspension is generally made by contacting the phospholipid solution, which is non-aqueous, with the aqueous solvent. The aqueous solvent may already comprise any salts and/or any buffers or alternatively those may be added after contact with the phospholipid solution. The aqueous solvent may be stirred in order to ensure mixing of the phospholipid solution with the aqueous solvent. The aqueous solvent may also be warmed prior to contacting with phospholipid solution which in some instances may also be warmed. Surprisingly, the divalent metal cation concentration of the aqueous solvent is not as important as that of the non-aqueous phospholipid solution (and its combined components). For example, it has been found unexpectedly that once a precipitate-free phospholipid solution is prepared, it can be combined with an aqueous solvent that has a high divalent metal cation concentration without inducing any discernable phospholipid precipitate. Thus, it has been found surprisingly that the phospholipid sensitivity to high divalent metal cation content exists only in the phospholipid solution or in the presence of the non-aqueous solvent, but not beyond that point. Similarly, it has been found that once the precipitate is formed in the phospholipid solution, contact with the aqueous solvent, even if warmed, does not lead to its dissolution. This differential sensitivity of phospholipids, and in particular, DPPA to high divalent metal cation levels, such as calcium levels, was not heretofore appreciated and was considered a surprising finding. While the divalent metal cation concentration of the aqueous solvent does not appear to induce precipitation of one or more phospholipids, it does surprisingly induce precipitation of other components, including most notably phosphate such as may be present if a phosphate buffer is used in the aqueous solvent. Thus, in some instances, the methods provided herein may further include measuring divalent metal cation concentration of components used to make the aqueous phospholipid suspensions that comprise phosphate. Alternatively, the methods may include selecting individual components or combined components that are characterized, individually or in combination, as having no or low divalent metal cation concentration. The phospholipid suspension may then be used to prepare the phospholipid-encapsulated gas microspheres. Phospholipid-Encapsulated Gas Microspheres, and UCA Formulations Comprising them As will be apparent, the phospholipid-based ultrasound contrast agents of this disclosure are phospholipid-encapsulated gas microspheres. These microspheres may be made in a number of ways. For example, a phospholipid solution may be contacted with an aqueous solvent to form a phospholipid suspension, and the phospholipid suspension may be contacted with a gas such as a perfluorocarbon gas to form the phospholipid-encapsulated gas microspheres. As another example, the non-aqueous phospholipid solution may be contacted with a gas such as a perfluorocarbon gas to form the phospholipid-encapsulated gas microspheres. In either instance, the phospholipid formulation, be it a non-aqueous phospholipid solution or an aqueous phospholipid suspension, is combined with the gas in a manner sufficient to create the phospholipid-encapsulated gas microspheres. This usually involves vigorous shaking or other agitation. Sufficient shaking or agitation is typically achieved using a device, such as a VIALMIX®, and is not typically achieved manually. The phospholipid solution or the phospholipid suspension are provided in a container, such as a vial, having a gas headspace. A perfluorocarbon gas, such as perflutren, is introduced into the headspace of such containers, usually through a process of gas exchange. It is this vial that is then vigorously shaken in order to form the phospholipid-encapsulated gas microspheres. This process, known as activation, is carried out by the end user or medical personnel just prior to administration into a subject. The microspheres comprise gas, such as a perfluorocarbon gas including but not limited to perflutren gas, in their internal cavity. The phospholipid shell that encapsulates the gas may be arranged as a unilayer or a bilayer, including unilamellar or multilamellar bilayers. The microspheres may have a mean diameter of less than 10 microns, or less than 6 microns, or less than 3 microns, or more preferably less than 2 microns. These mean diameters intend that, when a population of microspheres is analyzed, the mean diameter of the population is less than microns, or less than 6 microns, or less than 3 microns, or more preferably less than 2 microns. The microspheres may have a mean diameter in the range of 0.5 to 3 microns, or 1 to 2 microns, or 1.4 to 1.8 microns, or 1.4 to 1.6 microns. The mean diameter may be about 1.4 microns. The process of generating phospholipid-encapsulated gas microspheres is known as activation. Formulations that comprise a sufficient concentration of phospholipid-encapsulated gas microspheres may be referred to herein as activated formulations. It will be appreciated that the concentration of the gas microspheres that is “sufficient” will depend on whether the gas microspheres are made using the phospholipid solution (without intervening use of an aqueous solvent) or are made using the phospholipid suspension. Typically, the UCA formulation being administered to a subject will comprise on the order of about at least 1×107microspheres per ml of administered formulation, or at least 5×107microspheres per ml, or at least 7.5×107microspheres per ml, or at least 1×108microspheres per ml, or at least 1×109microspheres per ml, or about 5×109microspheres per ml. The range of microsphere concentration may be, in some instances, 1×107to 1×1010microspheres per ml of administered formulation, and more typically 5×107to 5×109microspheres per ml. Depending on how they are made, the gas microspheres may be present in a non-aqueous solvent or in an aqueous solvent. Regardless, prior to administration to a subject, they are typically diluted in an aqueous solution that may be a saline solution, or a buffered aqueous solution, or a buffered saline solution. The UCA formulation to be administered, typically intravenously, to a subject including a human subject may have a pH in the range of 4-8 or in a range of 4.5-7.5. In some instances, the pH may be in a range of about 6 to about 7.5, or in a range of 6.2 to about 6.8. In still other instances, the pH may be about 6.5 (e.g., 6.5+/−0.5 or +/−0.3). In some instances, the pH may be in a range of 5 to 6.5 or in a range of 5.2 to 6.3 or in a range of 5.5 to 6.1 or in a range of 5.6 to 6 or in a range of 5.65 to 5.95. In still another instance, the pH may be in a range of about 5.7 to about 5.9 (e.g., +/−0.1 or +/−0.2 or +/−0.3 either or both ends of the range). In another instance, the pH may be about 5.8 (e.g., 5.8+/−0.15 or 5.8+/−0.1). The gas is preferably substantially insoluble in the phospholipid formulations provided herein, including the phospholipid solution and the phospholipid suspension. The gas may be a non-soluble fluorinated gas such as sulfur hexafluoride or a perfluorocarbon gas. Examples of perfluorocarbon gases include perfluoropropane, perfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane, perfluorohexane. Examples of gases that may be used are described in U.S. Pat. No. 5,656,211 and are incorporated by reference herein. In an important embodiment, the gas is perfluoropropane. Divalent Metal Cations, and Methods of Measuring Same Divalent metal cations are divalent metal ions with a valence of 2. These include: barium(2+), beryllium(2+), cadmium(2+), calcium(2+), chromium(2+), cobalt(2+), copper(2+), europium(2+), gadolinium(2+), germanium(2+), iron(2+), lanthanum(2+), lead(2+), magnesium(2+), manganese(2+), mercury(2+), nickel(2+), osmium(2+), platinum(2+), ruthenium(2+), strontium(2+), tin(2+), uranium(2+), vanadium(2+), yttrium(2+), and zinc(2+). In some embodiments, the divalent metal cations of interest are calcium, magnesium and manganese. In some embodiments, the divalent metal cations of interest are calcium and magnesium and therefore only calcium and magnesium are measured or components are selected based only on their calcium and magnesium content. In some embodiments, the divalent metal cation of interest is calcium, and therefore only calcium is measured or components are selected based on their calcium concentration. Effect of Divalent Metal Cations As described herein, divalent metal cations may be present in one or more of the components used to make the UCA formulations. Their presence may not be appreciated until such components are combined with the non-aqueous solvent to form the phospholipid solution, at which point phospholipid precipitation may be induced for example, or until such components are combined with the aqueous solvent to form the phospholipid suspension, at which point phosphate precipitation may be induced for example. Surprisingly, it was discovered in accordance with this disclosure that the MPEG5000-DPPE phospholipid stock contained calcium and magnesium at sufficiently high concentrations to cause precipitation of at least the DPPA phospholipid once combined. Thus, the divalent metal cations may have different effects on different phospholipids, and it may not be readily apparent to the user whether a phospholipid (or other component) contains such cations at concentrations sufficient to induce precipitation. The inventors discovered in the process of preparing various UCA formulations that a non-aqueous solvent became cloudy when combined with a phospholipid blend comprising DPPA, DPPC and MPEG5000-DPPE phospholipids. It was further determined that the cloudy appearance was likely due to the precipitation of the DPPA phospholipid. Unbeknownst to the inventors, however, was the fact that the MPEG5000-DPPE contained high concentrations of calcium and magnesium ions and that those calcium and magnesium concentrations were likely the cause of the DPPA precipitation. Interestingly, such cations did not appear to affect the ability of MPEG5000-DPPE to remain in solution and thus a user would not appreciate that fact until such phospholipid was combined with the others in the non-aqueous mixture. Further studies, described in greater detail herein, found that precipitation occurred when a MPEG5000-DPPE stock later characterized as having a high calcium concentration was combined with DPPA, regardless of the order of addition or the presence of other components such as other phospholipids such as DPPC and DPPA. The sensitivity of DPPA to precipitate in a non-aqueous solvent comprising PG in the presence of sufficiently high divalent metal cation concentration such as calcium and magnesium concentration yet not in an aqueous solvent having similarly high concentrations of either or both calcium and magnesium was even more surprising. In other words, the concentrations of calcium that caused DPPA precipitation in non-aqueous solvent comprising PG did not cause DPPA precipitation in the aqueous solvent, and this too was surprising. No or Low Divalent Metal Cation Concentration As used herein, components are selected that are characterized or identified as having no or low divalent metal cation concentration, which includes no or low calcium concentration. Such divalent metal cation concentration is expressed as a weight by weight measure (i.e., weight of the divalent metal cation per unit weight of the underlying matrix or solvent in which the component of interest is present). A microgram per gram concentration may be alternatively referred to as parts per million or ppm. A no or low calcium concentration of such component will further depend upon how much of that component is used or in other words how much such component is diluted to form the phospholipid solution or the phospholipid suspension. In the simplest case, only one component is of interest, and only its calcium concentration is measured or only that component is selected based on its calcium concentration. Based on this disclosure, one of ordinary skill in the art will understand and be able to determine how much calcium will be tolerated in that component in order to avoid precipitation in the phospholipid solution or the phospholipid suspension. As an example, calcium concentration in a phospholipid stock (which is typically provided as a solid such as a powder) is expressed in weight of calcium per gram of phospholipid. An example is a calcium weight per gram of MPEG5000-DPPE or calcium weight per gram DPPC. When two components such as two phospholipids are combined, the measure may be weight of calcium per gram of MPEG5000-DPPE and DPPC combined. No divalent metal cation, such as no calcium, refers to a concentration of such cation that is undetectable using the methods known in the art and/or provided herein. No or low divalent metal cation concentration in a phospholipid stock will depend on the particular component. No or low divalent metal cation concentration in an MPEG5000-DPPE phospholipid stock is less than 510 micrograms/gram (i.e., micrograms of divalent metal cation per gram of MPEG5000-DPPE) (also referred to as less than 510 ppm), including less than 345 ppm, less than 230 ppm, less than 115 ppm, less than 57.5 ppm, and less than 11.5. No or low divalent metal cation concentration in a DPPC phospholipid stock is less than 390 micrograms/gram (i.e., micrograms of divalent metal cation per gram of DPPC) (also referred to as less than 390 ppm), including less than 270 ppm, less than 180 ppm, less than 90 ppm, less than 45 ppm, and less than 9 ppm. No or low divalent metal cation concentration in a DPPA phospholipid stock is less than 3440 micrograms/gram (i.e., micrograms of divalent metal cation per gram of DPPA) (also referred to as less than 3440 ppm), including less than 2340 ppm, less than 1560 ppm, less than 780 ppm, less than 390 ppm, and less than 78 ppm. No or low divalent metal cation concentration in a phospholipid blend is less than 210 micrograms/gram (i.e., micrograms of divalent metal cation per gram of phospholipid blend or the combined weight of MPEG5000-DPPE and DPPC and DPPA) (also referred to as less than 210 ppm), including less than 150 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm, and less than 5 ppm. No or low divalent metal cation concentration in propylene glycol is less than 3.1 micrograms/gram (i.e., micrograms of divalent metal cation per gram of propylene glycol) (also referred to as less than 3.1 ppm), including less than 2.1 ppm, less than 1.4 ppm. less than 0.7 ppm, less than 0.35 ppm, and less than 0.07 ppm. No or low divalent metal cation concentration in a 1:1 (weight to weight) propylene glycol and glycerol mixture is less than 10.4 micrograms/gram (i.e., micrograms of divalent metal cation per gram of propylene glycol and glycerol combined) (also referred to as less than 10.4 ppm), including less than 7.8 ppm, less than 5.2 ppm, less than 2.6 ppm, less than 1.3 ppm, and less than 0.26 ppm. No or low divalent metal cation concentration in glycerol is less than 20.4 micrograms/gram (i.e., micrograms of divalent metal cation per gram of glycerol) (also referred to as less than 20.4 ppm), including less than 15.3 ppm, less than 10.2 ppm, less than 5.10 ppm, less than 2.6 ppm, less than 0.51 ppm. No or low divalent metal cation concentration in a phospholipid solution that comprises only propylene glycol as the non-aqueous solvent is less than 3.1 micrograms/gram (i.e., micrograms of divalent metal cation per gram of all components of the phospholipid solution) (also referred to as less than 3.1 ppm), including less than 2.1 ppm, less than 1.4 ppm. less than 0.7 ppm, less than 0.35 ppm, and less than 0.07 ppm. As will be appreciated based on the composition of the phospholipid solution, the major component by weight is the non-aqueous solvent, in this particular case, propylene glycol. It is to be understood that the same concentration limits apply to calcium concentration and magnesium concentration, as well as combined calcium and magnesium concentrations. This disclosure contemplates measurement of divalent metal cation concentration in one or more components of the phospholipid solution, including for example one, two or all three of the phospholipid stocks, optionally together with measurement of divalent metal cation concentration of the non-aqueous solvent such as propylene glycol and/or glycerol, depending on the nature of the phospholipid solution. If any of these components contain a divalent metal cation concentration in excess of those levels recited above, then it is expected that once a phospholipid solution is made with such component, such phospholipid solution will be prone to precipitation. Some embodiments contemplate that a single component will be analyzed for its divalent metal cation concentration and that if such concentration is a “no or low divalent metal cation concentration” then the component may be combined with the remaining components even if such components were not analyzed for their divalent metal cation concentration. Components of a phospholipid solution include the phospholipid stocks, whether provided individually or as a blend, non-aqueous solvents such as propylene glycol and glycerol, and optionally buffers. Some embodiments contemplate that more than one but less than all components of the phospholipid solution will be measured for their divalent metal cation concentration. In some instances, if one of the components have a divalent metal cation concentration that is in excess of the “no or less divalent metal cation concentration” limit listed above, then it may not be used to prepare the phospholipid solution (or phospholipid blend). In some instances, no one component may be characterized or identified as having a divalent metal cation concentration that is more than the “no or low divalent metal cation concentration”. However, when such components are used in combination, the combined divalent metal cation concentration may be determined based on the amount each contributes to the phospholipid solution. It is contemplated that the combined divalent metal cation concentration may or may not exceed the “no or low divalent metal cation concentration” as defined above for the phospholipid solution. As discussed throughout, the various levels set forth herein while referring to divalent metal cations, apply equally to calcium. The Examples demonstrate that the lowest calcium concentration at which precipitation of phospholipid is apparent is about 0.7 microgram calcium per gram of non-aqueous solvent (see Examples 1 and 2), otherwise referred to as about 0.7 ppm. Thus, a no or low divalent metal cation concentration such as a no or low calcium concentration in a non-aqueous phospholipid solution is less than 0.7 ppm, less than 0.35 ppm, or less than 0.07 ppm. It is to be understood that a divalent metal cation concentration in the phospholipid solution of less than 0.7 ppm will also be regarded as a no or low divalent metal ion concentration. Divalent metal cation concentration in an aqueous phospholipid suspension may be provided as a weight of divalent metal cation per gram of aqueous solvent. Typically, the phospholipid suspension is formed by diluting the non-aqueous phospholipid solution about 20-fold into the aqueous solvent. Thus, a no or low divalent metal ion concentration in a phospholipid suspension is less than 0.035 micrograms per gram of phospholipid suspension. Similarly, the calcium concentration in a phospholipid suspension originating from the phospholipid solution is less than 0.035 micrograms per gram of phospholipid suspension. The concentration at which the divalent metal cations cause phospholipids to precipitate may be temperature dependent. At higher temperatures, higher concentrations of cations may be tolerated before precipitation is observed. At lower temperatures, lower concentrations of cations may cause the precipitation to occur. As an example, at temperatures of about 55° C. (e.g., 50-60° C.), no or low divalent metal cation concentration is a divalent metal cation concentration of less than 0.7 micrograms calcium per gram of phospholipid solution or of non-aqueous solvent. This level may be slightly lower if the phospholipid solution is prepared at a lower temperature. Alternatively, this level may be slightly higher if the phospholipid solution is prepared at a higher temperature. Calcium Sources As demonstrated in the Examples, the phospholipid solution is unexpectedly and uniquely sensitive to particular levels of calcium. This unique sensitivity was not heretofore recognized. Given the effect of calcium on the preparation of the phospholipid solution, and thus ultimately on the UCA, it is important to measure and thus control the calcium concentration of the phospholipid solution. Calcium may be present in each of the components of the phospholipid solution, including the phospholipid stocks and the non-aqueous solvents, as described below. Calcium and magnesium are divalent alkaline earth metals in group 2 of the periodic table. Calcium is the fifth-most-abundant element by mass in the Earth's crust and the cation Ca2+ is also the fifth-most-abundant dissolved ion in seawater. It is found at various levels in tap water depending on the on the “hardness”. The total water hardness is the sum of the molar concentrations of Ca2+ and Mg2+, ranging from soft at 0-60 ppm to very hard ≥181. Calcium and magnesium are also found in varying concentration in the crude glycerol extract from biodiesels. Level of calcium and magnesium were reported to range from 12 to 163 ppm and 4 to 127 ppm respectively depending on the seed oil for biodiesel production (J. C. Thompson 2006 Applied Engineering in Agriculture Vol. 22(2): 261-265). A major supply of glycerol comes from this biodiesel byproduct. The crude glycerol extract can be purified by treatment with activated carbon to remove organic impurities, alkali to remove unreacted glycerol esters, and ion exchange to remove salts. High purity glycerol (>99.5%) is obtained by multi-step distillation; vacuum is helpful due to the high boiling point of glycerol (290° C.). Industrially, propylene glycol is produced from propylene oxide. Different manufacturers use either non-catalytic high-temperature process at 200° C. (392° F.) to 220° C. (428° F.), or a catalytic method, which proceeds at 150° C. (302° F.) to 180° C. (356° F.) in the presence of ion exchange resin or a small amount of sulfuric acid or alkali. Final products contain 20% propylene glycol, 1.5% of dipropylene glycol and small amounts of other polypropylene glycols. Further purification produces finished industrial grade or USP/JP/EP/BP grade propylene glycol that is typically 99.5% or greater. Propylene glycol can also be converted from glycerol, a biodiesel byproduct. The calcium and magnesium contents of Pharmacopeia grade propylene glycol and glycerol are not quantified as a certificate of analysis requirement of US Pharmacopeia, European Pharmacopeia, British Pharmacopeia or Japanese Pharmacopeia. Phospholipid DPPA contains a phosphate which can be ionized at appropriate pH. The pKa for the two hydroxyl groups of the phosphate are 6.2 and 1.8 (Tatulian Ionization and Binding, 511-552 Phospholipid Handbook, Ed. G Ceve 1993). DPPA is commercially available as different salt forms. Usually, the Na salt is used but the Ca salt is also available. DPPC is a zwitterion and therefore does not require a counter ion. MPEG5000-DPPE is a modified DPPE which has a pKa of 1.9 and 9.3 for the hydroxyl of the phosphate and the amine of the ethanolamine (Tatulian Ionization and Binding, 511-552 Phospholipid Handbook, Ed. G Ceve 1993). MPEG500-DPPE is available as the Na salt form. Methods of Measuring Divalent Metal Cation Concentration Quantitation of divalent metal cations concentration can be performed using one of several known techniques. These include atomic spectroscopy methods such as atomic absorption spectroscopy (AAS), flame photometry or flame atomic emission spectrometry (FAES), inductively coupled plasma-atomic emission spectrometry (ICP-AES), and other methods such as inductively coupled plasma-mass spectroscopy or complexometric titration. The spectroscopic approaches utilize absorption or emission characteristics of the metal ions of alkali metals (Group 1) and alkaline earth metals (Group II) metals when dissociated due to thermal energy provided by a flame source. ICP-MS is a type of mass spectrometry which is capable of detecting metals at very low concentrations. This is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ions. Some of these methods are used in the Examples. Complexometric titration is another method for detecting divalent metal cation concentration. This method uses EDTA (ethylenediaminetetraacetic acid) complexation with calcium and magnesium ions to compete with a color indicator. This allows rapid colorimetric quantitation. EDTA forms a complex with calcium and magnesium ions. A blue dye called Eriochrome Black T (ErioT) is used as the indicator. This blue dye also forms a complex with the calcium and magnesium ions, changing color from blue to pink in the process. The dye-metal ion complex is less stable than the EDTA-metal ion complex. For the titration, the sample solution containing the calcium and magnesium ions is reacted with an excess of EDTA. The indicator is added and remains blue as all the Ca2+ and Mg2+ ions present are complexed with the EDTA. A back titration is carried out using a solution of magnesium chloride. This forms a complex with the excess EDTA molecules until the end-point, when all the excess EDTA has been complexed. The remaining magnesium ions of the magnesium chloride solution then start to complex with ErioT indicator, immediately changing its color from blue to pink Methods of Synthesis The disclosure provides methods for preparing phospholipid solutions and phospholipid suspensions intended for use with a perfluorocarbon gas to form a UCA formulation comprising phospholipid-encapsulated gas microspheres. In preferred embodiments, the phospholipids are DPPA, DPPC and DPPE such as MPEG5000-DPPE. The phospholipid solution may be made in a number of ways, as described below. These methods are characterized broadly as blend and non-blend methods. The starting phospholipid stocks may be in solid (e.g., powder) form or in liquid form. Blend Methods Blend methods refer to methods in which the phospholipids are intimately blended with each other in order to render a solid phospholipid mixture that is more uniform (and thus more homogenous) with respect to its phospholipid content and phospholipid distribution and in some instances has higher purity, as compared to simple mixtures of phospholipids. This method creates a homogenous dispersion of the three phospholipids by dissolving or suspending them in an appropriate blend solvent system, and then separates the evenly distributed phospholipids from the solvent. The separation of the blend solvent from the phospholipids can involve drying, lyophilization, distillation, and the like, or it can include precipitation using an additional blend solvent. Blend solvents for neutral lipids are relatively non-polar solvents such as diethyl ether or chloroform. More polar blend solvents such as alcohol (e.g., methanol and ethanol) are required for membrane-associated lipids which are themselves more polar. Chloroform may also be used, particularly for lipids of intermediate polarity. When mixed with methanol, chloroform becomes a general solvent. Dichloromethane (or methylene dichloride) is a similar extractant but less oxidizable. Hexane may be used for lipids of low polarity. It can be used to extract neutral lipids from water/alcohol mixtures. Petroleum ether is a mixture of various hydrocarbons with 5-8 carbon atoms and may be used in place of hexanes in some instances. Other blend solvents include without limitation cyclohexane and toluene. As will be described in greater detail herein, certain blends are formed by contacting one or more desired phospholipids (e.g., DPPA, DPPC and MPEG5000-DPPE) in a blend solvent system to first dissolve such phospholipids, optionally then concentrating such solution, then either removing the blend solvent or precipitating the phospholipid blend from such solvent. The blend solvent is not to be confused with the non-aqueous solvent that is later used to dissolve the phospholipids, thereby forming a phospholipid solution. It should also be clear that this precipitation is a desired event and is not to be confused with the undesirable phospholipid precipitation that can occur during the later step of forming the phospholipid solution by the presence of calcium, another divalent cation, or a combination of divalent cations. Some methods of preparing phospholipid solutions involve contacting a phospholipid blend with the non-aqueous solvent. There are various ways of making phospholipid blends, including but not limited to organic solvent (or blend solvent, as used herein) dissolution-precipitation methods and aqueous suspension-lyophilization methods. The organic solvent dissolution-precipitation method is described in detail in U.S. Pat. No. 8,084,056 and in published International Application No. WO99/36104, the entire contents of both of which are incorporated herein by reference. One embodiment of this method involves the following steps: (a) Contacting the desired phospholipids (e.g., DPPA, DPPC and MPEG5000-DPPE or DPPC and MPEG5000-DPPE) with a first blend solvent system. This system is typically a combination of solvents, for example CHCl3/MeOH, CH2Cl2/MeOH, and toluene/MeOH. It may be desirable to warm the resultant solution to a temperature sufficient to achieve complete dissolution. Such a temperature is preferably about 25 to 75° C., more preferably about 35 to 65° C. After dissolution, undissolved foreign matter may be removed by hot-filtration or cooling to room temperature and then filtering. Known methods of filtration may be used (e.g., gravity filtration, vacuum filtration, or pressure filtration). (b) The solution is then concentrated to a thick gel/semisolid. Concentration is preferably done by vacuum distillation. Other methods of concentrating the solution, such as rotary evaporation, may also be used. The temperature of this step is preferably about 20 to 60° C., more preferably 30 to 50° C. (c) The thick gel/semisolid is then dispersed in a second blend solvent. The mixture is slurried, preferably near ambient temperature (e.g., 15-30° C.). Useful second blend solvents are those that cause the phospholipids to precipitate. The second blend solvent is preferably methyl t-butyl ether (MTBE). Other ethers and alcohols may be used. (d) The solids produced upon addition of the second blend solvent are then collected. Preferably the collected solids are washed with another portion of the second blend solvent (e.g., MTBE). Collection may be performed via vacuum filtration or centrifugation, preferably at ambient temperature. After collection, it is preferred that the solids are dried in vacuo at a temperature of about 20-60° C. The resultant solid is referred to herein as a phospholipid blend. Certain of the methods described herein use phospholipids in the form of a phospholipid blend, including a phospholipid blend made according to any of the blend methods set forth above. Some methods use phospholipid blends, excluding the phospholipid blend made according to the methanol/toluene/MTBE method set forth above wherein a methanol and toluene mixture is used as the first blend solvent and MTBE is used as the second blend solvent. For clarity, the method set forth above is referred to herein as the methanol/toluene/MTBE phospholipid blend method. As used herein, a phospholipid blend is distinguished from other phospholipid mixtures, including those mixtures that are made from simply combining phospholipids in their solid (including powder) forms, as described herein. In the aqueous suspension-lyophilization methods, phospholipids are suspended in water at an elevated temperature and then concentrated by lyophilization. The organic solvent dissolution-precipitation process is preferred over the aqueous suspension/lyophilization process for a number of reasons as outlined in U.S. Pat. No. 8,084,056 and published PCT application WO 99/36104, including the uniformly distributed phospholipid solid that results using the organic dissolution method. Some blend methods that are not the methanol/toluene/MTBE phospholipid blend method recited above use a blend solvent system other than methanol and toluene. In these methods, the phospholipids are combined in a methanol-free and toluene-free condition (also referred to as a methanol and toluene-free condition) to form a phospholipid blend. Thus, a methanol and toluene-free condition refers to a condition that does not include both of these solvents. Some blend methods combine the phospholipids in an blend solvent to form the phospholipid blend and then evaporate the blend solvent completely, for example either by drying or distillation, to form the dried phospholipid blend. It is this dried phospholipid blend that is then contacted with the non-aqueous solvent such as PG in the methods provided herein. Some blend methods combine the phospholipids in an aqueous solvent and then lyophilize the mixture to form a lyophilized phospholipid composition. Other blend methods combine the phospholipids with other solvent systems such as but not limited to (1) an ethanol and cyclohexane (e.g., 1:1, v:v) mixture, and (2) tertiary butanol (t-butanol or 1,1 dimethyl ethanol), in place of water. Following dissolution in these various solvents, the compositions are lyophilize. Lyophilization can be performed by freezing over an isopropanol/CO2 bath or an acetone/CO2 bath and drying on a Virtis Lyophilizer until the product appears dry and flocculent in appearance. Various blend methods that are not the methanol/toluene/MTBE phospholipid blend method recited above are described in published EP 0923383 (WO1997040858), the entire contents of which are incorporated by reference herein. Some blend methods that are not the methanol/toluene/MTBE phospholipid blend method recited above combine the phospholipids in a blend solvent, which may be a mixture of toluene and methanol, to form a phospholipid blend, and then precipitate such phospholipid blend in the absence of MTBE. In these methods, the precipitation occurs in an MTBE-free condition. In other blend methods, DPPA, DPPC and MPEG5000-DPPE (or DPPC and MPEG5000-DPPE) may be combined in their dry, solid forms and such combination then may be actively and intimately mixed in dry form (e.g., manually stirring the powders or using a mixing device such as a tumbler silo mixer, an orbiting screw mixer, a ribbon mixer, an extruder, a cyclomix, a henschel mixer, a lodige type mixer, an Eirich type mixer, or other type of device designed for pharmaceutical powder mixing, with the aim of preparing a uniform blend of phospholipids (e.g., uniform phospholipid dispersion throughout the mixture). Reference can be made to Deveswaran et al. Research J. Pharm. and Tech.2 (2): April-June. 2009) for additional methodologies for generating a uniform dry product. Non-Blend Methods In contrast to blend methods, certain non-blend methods involve simple mixing of solid phospholipids and this tends to result in a less uniform (and thus less homogenously dispersed or more heterogeneously dispersed) mixture. These latter mixtures are referred to herein as phospholipid mixtures (or non-blend phospholipid mixtures) in order to distinguish them from phospholipid blends. Some ways of preparing phospholipid solutions involve simply contacting phospholipids in their solid forms with the non-aqueous solvent. The phospholipids may be contacted with the non-aqueous solvent simultaneously or sequentially. If sequentially, any order of addition may be used. The phospholipids may be added individually to the non-aqueous solvent or they may be first combined together, in any combination, and then added to the non-aqueous solvent. Thus, it is contemplated that the phospholipids may be added to the non-aqueous solvent individually and simultaneously, individually and sequentially, combined and simultaneously, and partially combined and sequentially. An example of the latter is an instance where two of the phospholipids are combined together in solid form and then contacted with the non-aqueous solvent before or after the remaining phospholipid is contacted with the non-aqueous solvent. Thus, as an example, DPPA, DPPC and MPEG5000-DPPE (or DPPC and MPEG5000-DPPE) phospholipids may be added individually to a non-aqueous solvent. Such individual addition may be sequential or simultaneous addition. If sequential, the order of addition can be any order although in some instances DPPA may be added second or last since it is the least abundant and least soluble and its dissolution can be facilitated by the presence of one of the other phospholipids. In some instances, regardless of whether the phospholipids are provided as individually, or as a simple mixture, or as a phospholipid blend, they are then dissolved in a non-aqueous solvent comprising PG or a PG/G mixture, as described above to form the phospholipid solution. The phospholipid solution may be combined with gas or it may combined with an aqueous solvent to form a phospholipid suspension which in turn is contacted with gas. In other instances, a phospholipid blend may be prepared by combining the phospholipids in, for example, water, or in an ethanol and cyclohexane (e.g., 1:1, v:v) mixture, or in tertiary butanol (t-butanol or 1,1 dimethyl ethanol), and such mixture is then lyophilized, and the dried product is resuspended in an aqueous solvent. In these instances, the final resuspended product may be combined with a gas such as perflutren. This disclosure contemplates that any or all components used in this preparation may be analyzed for their divalent metal cation concentration, and such individual or combined divalent metal cation concentration may be quantified and used to select and/or prepare a UCA. Methods of Preparing Ultrasound Contrast Agent, Including Activation Phospholipid encapsulated gas microspheres are formed by combining and vigorously shaking a phospholipid solution or a phospholipid suspensions with a gas such as a perfluorocarbon gas. This process is referred to herein as activation. The UCA formulation so formed minimally comprises phospholipids, non-aqueous solvent such as PG, and gas, and thus activation minimally results in gas-filled phospholipid microspheres. The phospholipids may be present in an aqueous solution such as is the case with DEFINITY®, or they may be present in a non-aqueous solution such as is the case with novel UCA formulations including for example DEFINITY-II, described in greater detail herein. Thus, in some instances, activation comprises shaking an aqueous phospholipid suspension in the presence of a gas, such as a perfluorocarbon gas (e.g., perflutren). In other instances, activation comprises shaking a phospholipid solution in the presence of a gas, a perfluorocarbon gas (e.g., perflutren). It is to be understood that perflutren, perflutren gas and octafluoropropane are used interchangeably herein. Shaking, as used herein, refers to a motion that agitates a solution, whether aqueous or non-aqueous, such that gas is introduced from the local ambient environment within the container (e.g., vial) into the solution. Any type of motion that agitates the solution and results in the introduction of gas may be used for the shaking. The shaking must be of sufficient force or rate to allow the formation of foam after a period of time. Preferably, the shaking is of sufficient force or rate such that foam is formed within a short period of time, as prescribed by the particular UCA formulation. Thus in some instances such shaking occurs for about 30 seconds, or for about 45 seconds, or for about 60 seconds, or for about 75 seconds, or for about 90 seconds, or for about 120 seconds, including for example for 30 seconds, or for 45 seconds, or for 60 seconds, or for 75 seconds, or for 90 seconds, or for 120 seconds. In some instances, the activation may occur for a period of time in the range of 60-120 seconds, or in the range of 90-120 seconds. The disclosure contemplates that, in some instances, the shaking time (or duration) will vary depending on the type of UCA formulation being activated. For example, in some instances, an aqueous UCA formulation may be shaken for shorter periods of time than a non-aqueous UCA formulation. The disclosure contemplates that, in such instances, the shaking rate (or shaking speed, as those terms are used interchangeably herein) may be constant. Thus an activation or shaking means such as an activation or shaking device may be set to shake at one rate (defined in terms of number of shaking motions per minute, for example) for two or more different pre-determined periods of time. The disclosure further contemplates that, in some instances, the shaking rate will vary depending on the type of UCA formulation being activated. For example, in some instances, an (aqueous) phospholipid suspension may be shaken at a slower shaking rate than a (non-aqueous) phospholipid solution. The disclosure contemplates that, in such instances, the shaking time (or duration, as those terms are used interchangeably herein) may be constant. DEFINITY® may be activated with a VIALMIX®, as described below. DEFINITY® activation, which involves vigorous shaking of an (aqueous) phospholipid suspension in the presence of perflutren, lasts for about 45 seconds with a VIALMIX®. Unless indicated otherwise, the term “about” with respect to activation time intends a time that is +/−20% of the noted time (i.e., 45+/−9 seconds). DEFINITY-II may be activated with a VIALMIX® as well. DEFINITY-II activation, which involves vigorous shaking of a (non-aqueous) phospholipid solution in the presence of perflutren, lasts for about 60 to 120 seconds. In some instances, DEFINITY-II is activated for about 75 seconds (i.e., 75+/−15 seconds). DEFINITY-II may be activated for longer periods of time including 90-120 seconds The shaking may be by swirling (such as by vortexing), side-to-side, or up and down motion. Further, different types of motion may be combined. The shaking may occur by shaking the container (e.g., the vial) holding the aqueous or non-aqueous phospholipid solution, or by shaking the aqueous or non-aqueous solution within the container (e.g., the vial) without shaking the container (e.g., the vial) itself. Shaking is carried out by machine in order to standardize the process. Mechanical shakers are known in the art and their shaking mechanisms or means may be used in the devices of the present disclosure. Examples include amalgamators such as those used for dental applications. Vigorous shaking encompasses at least 1000, at least 2000, at least 3000, at least 4000, at least 4500, at least 5000 or more shaking motions per minute. In some instances, vigorous shaking includes shaking in the range of 4000-4800 shaking motions per minute. VIALMIX® for example targets shaking for 4530 “figure of eight” revolutions per minute, and tolerates shaking rates in the range of 4077-4756 revolutions per minute. Vortexing encompasses at least 250, at least 500, at least 750, at least 1000 or more revolutions per minute. Vortexing at a rate of at least 1000 revolutions per minute is an example of vigorous shaking, and is more preferred in some instances. Vortexing at 1800 revolutions per minute is most preferred. The shaking rate can influence the shaking duration needed. A faster shaking rate will tend to shorten the duration of shaking time needed to achieve optimal microbubble formation. For example, shaking at 4530 rpm for a 45 second duration will achieve 3398 total revolutions on a VIALMIX©. Shaking at 3000 rpm would require 68 seconds to achieve the same number of revolutions. The duration and shake speed required will also be influenced by the shape of the travel path and amplitude of shaking. The velocity the liquid in the container reaches and the forces exerted upon change of direction will influence gas incorporation. These aspects will be impacted upon based on the shaker arm length and path, the container shape and size, the fill volume and the formulation viscosity. Water has a viscosity of approximately 1.14 cps at 15° C. (Khattab, I. S. et al., Density, viscosity, surface tension, and molar volume of propylene glycol+ water mixtures from 293 to 323 K and correlations by the Jouyban-Acree model Arabian Journal of Chemistry (2012). In contrast, propylene glycol has a viscosity of 42 cps at 25° C. (Khattab, I. S. et al., Density, viscosity, surface tension, and molar volume of propylene glycol+ water mixtures from 293 to 323 K and correlations by the Jouyban-Acree model Arabian Journal of Chemistry (2012) and glycerol has a viscosity of 2200 cps at 15° C. (Secut J B, Oberstak H E Viscosity of Glycerol and Its Aqueous Solutions. Industrial and Engineering Chemistry 43. 9 2117-2120 1951). DEFINITY-II has a high viscosity of 1150 cps at 15° C. Since DEFINITY® is predominantly water it has a much lower viscosity than DEFINITY-II. The formation of gas-filled microspheres upon activation can be detected by the presence of a foam on the top of the aqueous or non-aqueous solution and the solution becoming white. Activation is carried out at a temperature below the gel state to liquid crystalline state phase transition temperature of the phospholipid employed. By “gel state to liquid crystalline state phase transition temperature”, it is meant the temperature at which a phospholipid layer (such as a lipid monolayer or bilayer) will convert from a gel state to a liquid crystalline state. This transition is described for example in Chapman et al., J. Biol. Chem. 1974 249, 2512-2521. The gel state to liquid crystalline state phase transition temperatures of various phospholipids will be readily apparent to those skilled in the art and are described, for example, in Gregoriadis, ed., Liposome Technology, Vol. I, 1-18 (CRC Press, 1984) and Derek Marsh, CRC Handbook of Lipid Bilayers (CRC Press, Boca Raton, Fla. 1990), at p. 139. Vigorous shaking can cause heating of the formulation based on the shake speed, duration, shaker arm length and path, the container shape and size, the fill volume and the formulation viscosity. It will be understood by one skilled in the art, in view of the present disclosure, that the phospholipids or phospholipid microspheres may be manipulated prior to or subsequent to being subjected to the methods provided herein. For example, after the shaking is completed, the gas-filled microspheres may be extracted from their container (e.g., vial). Extraction may be accomplished by inserting a needle of a syringe or a needle-free spike (e.g., PINSYNC®) into the container, including into the foam if appropriate, and drawing a pre-determined amount of liquid into the barrel of the syringe by withdrawing the plunger or by adding an aqueous liquid, mixing and drawing a pre-determined amount of liquid into the barrel of the syringe by withdrawing the plunger. As another example, the gas-filled microspheres may be filtered to obtain microspheres of a substantially uniform size. The filtration assembly may contain more than one filter which may or may not be immediately adjacent to each other. Methods of Using Ultrasound Contrast Agent to Image a Subject Also provided herein are methods of use of phospholipid-encapsulated gas microspheres and formulations thereof. The gas microspheres and formulations thereof may be used in vivo in human or non-human subjects, or they may be used in vitro. They may be used for diagnostic or therapeutic purposes or for combined diagnostic and therapeutic purposes. When used in human subjects, phospholipid-encapsulated gas microspheres and formulations thereof may be used directly (neat) or may be diluted further in a solution, including a pharmaceutically acceptable solution, and administered in one or more bolus injections or by a continuous infusion. Administration is typically intravenous injection. Imaging is then performed shortly thereafter. The imaging application can be directed to the heart or it may involve another region of the body that is susceptible to ultrasound imaging. Imaging may be imaging of one or more organs or regions of the body including without limitation the heart, blood vessels, the cardiovasculature, the liver, the kidneys and the head. Subjects of the invention include but are not limited to humans and animals. Humans are preferred in some instances. Animals include companion animals such as dogs and cats, and agricultural or prize animals such as but not limited to bulls and horses. UCAs are administered in effective amounts. An effective amount will be that amount that facilitates or brings about the intended in vivo response and/or application. In the context of an imaging application, such as an ultrasound application, the effective amount may be an amount of phospholipid-encapsulated gas microspheres that allow imaging of a subject or a region of a subject. EXAMPLES 1 Examples Methods 1.1 Phospholipids and Phospholipid Blends and Reagents Phospholipids were used as either individual powders, combined together as powders and used as a mixture or blended together by dissolving and drying (details described below). The measured content of the individual phospholipids was used to estimate the final calcium or magnesium concentrations in the non-aqueous concentrate or the aqueous preparation unless direct measurements in the blend was made. Solvents with low calcium were used for all studies. 1.1.1 Phospholipid Blend One phospholipid blend (LB) was prepared by dissolving DPPC, DPPA, MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) in toluene/methanol, concentrated with vacuum and warming and then slurried by the addition of Methyl t-butyl ether (MTBE). The solid material was collected, washed with MTBE and dried (consistent with U.S. Pat. No. 8,084,056). Alternatively, DPPC, DPPA, and MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) were solubilized at 55° C. in methanol. The methanol was then evaporated and the solids recovered as phospholipid blend. Similarly, DPPC, DPPA, and MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) were combined together as solid powders and the powders were mixed together with a spatula. 1.1.1.1 Residual Solvent Method for Phospholipid Blend Residual solvent in phospholipid blend was determined by FID using GC headspace. Sample was weighed, transferred into a separate 20 cc headspace vials and dissolved in N-methylpyrrolidone. A set of residual solvent standards was prepared in N-methylpyrrolidone. Standards and samples were analyzed by FID using GC headspace. The concentration of each residual solvent was calculated from the calibration curve for that solvent. 1.1.2 Calcium Measurements Calcium levels were quantified in individual lipid, lipid blend, glycerol and propylene glycol using either ICP-MS or AA. Magnesium and other metal ions were also measured with these methods in some samples 1.1.1.2 ICP-MS (Inductively Coupled Plasma—Mass Spectrometry) Method Samples were prepared by weighing into a pre-cleaned quartz digestion vessel. Matrix spikes were added and then mixed with nitric acid and hydrochloric acid. The samples were digested in a closed-vessel microwave digestion system. After cooling internal standard solution were added and diluted and analyzed by ICP-MS using He collision mode. 1.1.1.3 AA (Atomic Absorption Spectroscopy) Method Samples were prepared by weighing into a dry “trace metals cleaned” digestion vessel and dissolved with nitric acid and hydrochloric acid and reflux with H2O2. The sample solution was washed with water and filtered. A set of standards were used to calibrate the AA and then the absorbance of the samples read from the calibration curve. Results for individual lipid, phospholipid blend, and formulations solvents are provided in Table 1. TABLE 1Ca+2and Mg+2 Level in Individual Lipid, Lipid Blend and SolventaMaterialsCa+2(ppm)bMg+2(ppm)bPhospholipid Blend, Lot 1cNot detectedNot detectedPhospholipid Blend Lot 2c,d37054MPEG5000-DPPE (high Ca+2) Lot 1980150MPEG5000-DPPE (high Ca+2) Lot 2520110MPEG5000-DPPE (low Ca+2)4Not determinedDPPC Lot 1Not detectedNot detectedDPPC Lot 27Not determinedDPPA19Not determinedPropylene glycolNot detectedNot determinedGlycerol0.7Not determinedaDetermined by ICP-MSbppm = parts per million and is equivalent to μg/gcPhospholipid blend consists of DPPC, DPPA and MPEG5000-DPPE (0.401:0.045:0.304[wt:wt:wt])dLipid blend, Lot 2 prepared using MPEG5000-DPPE (high Ca+2) Lot 1 1.2 Aqueous Formulation Preparation 1.2.1 Non-Aqueous Phospholipid Concentrate Phospholipid concentrates were prepared by adding the individual lipids (DPPC, DPPA, and MPEG5000-DPPE low Ca+2, or MPEG5000-DPPE high Ca+2, or a combination) in any order, adding phospholipid blend (LB), or adding LB containing high levels of Ca+2to 25-115 mL of propylene glycol (PG), or 1:1 v/v propylene glycol/glycerol (PG/G), or glycerol vehicle with constant stirring at 55° C. to 70° C. In some cases, lipid concentrate was prepared without DPPA or with calcium acetate added prior to lipid addition. 1.2.2 Aqueous Formulation Aqueous formulations were prepared by adding: dibasic sodium phosphate, heptahydrate; monobasic sodium phosphate, monohydrate; sodium chloride; propylene glycol; glycerol and finally non-aqueous phospholipid concentrate to 400 to 500 mL of water with constant stirring at 55° C. to 70° C. In some cases, calcium acetate was added prior to, or after addition of the non-aqueous phospholipid concentrate to the bulk compounding solution. In other cases the phosphate buffer was not included. 1.3 Non-Aqueous Formulation Preparation 1.3.1 Non-Aqueous Formulation Individual lipids (DPPC, DPPA, and MPEG5000-DPPE low Ca+2or MPEG5000-DPPE high Ca+2, or a combination of both) in any order, LB, or LB containing high levels of Ca+2were added to 25 to 100 mL of propylene glycol (PG) containing 0.005 M acetate buffer (90/10, sodium acetate/glacial acetic acid) vehicle with constant stirring at 60° C.±5° C. Following dissolution, glycerol was then added to produce the non-aqueous formulation. 1.4 Calcium and/or Magnesium Additions Using Stock Solutions 1.4.1 Initial Studies Stock solutions of calcium acetate, magnesium acetate alone and a mixture of both were prepared in propylene glycol (25.4 μg Ca+2/g, 28.0 μg Mg+2/g and 14.0 μg Ca+2/g with 12.7 μg Mg+2/g respectively). The individual stock solutions were added in aliquots up to a total of 1 mL in 33 mL of propylene glycol containing lipid blend (15 mg/mL). The solutions were compared to propylene glycol alone, and the solution first showing cloudiness recorded. 1.4.2 Follow-Up Studies with Reference Scale Stock solutions of calcium acetate, monohydrate were prepared in propylene glycol (299 Ca+2μg/g), propylene glycol and glycerol (299 Ca+2μg/g), or water (6085 Ca+2μg/g) and vehicle matched when added to the propylene glycol, the non-aqueous phospholipid concentrate or the aqueous formulation (before or after addition of the non-aqueous phospholipid concentrate). The maximum added calcium stock was always <12% of the total volume. Some non-aqueous phospholipid concentrates were titrated with calcium acetate. The appearance was evaluated on a 0, +, ++, +++ scale by visual inspection.FIG.1provides the scale used for the determinations, and was generated using low Ca+2lipids (DPPC, DPPA and MPEG5000-DPPE; 0.401:0.045:0.304 [wt:wt:wt]) formulated in propylene glycol at 15 mg total lipid/mL. 1.5 Filtration 1.5.1 Aqueous Formulation Prepared samples of phospholipid aqueous suspensions were held at 55° C. prior to filtration. Samples were placed in a 55° C. temperature controlled 60 mL syringe with a 13 mm Hydrophilic Polyvinylidene Fluoride (PVDF) 0.22 μm membrane syringe filter attached. A 5 psi nitrogen head pressure was applied to the syringe. Flow rate was determined by weighing the filtered solution over time with readings every 30 seconds. Flow rates per time point were calculated and the average flow between 9 to 10 minutes was compared to the initial flow (0 to 1 minute) and expressed as a percentage. A pre-filtration sample was collected along with samples throughout the filtration for phospholipid concentration analysis. 1.5.2 Non-Aqueous Formulation Prepared samples of phospholipid non-aqueous solutions were held at 60° C. prior to filtration. Samples were placed in a 60° C. temperature controlled 60 mL syringe with 25 mm Hydrophilic Polyethersulfone (PES) 0.2 μm membrane syringe filter attached. A 10 psi nitrogen head pressure was applied to the syringe. Flow rate was determined by weighing the filtered solution over time with readings every 30 seconds. Flow rates per time point were calculated and the average flow between 8 to 9 minutes was compared to the initial flow (0 to 1 minute) and expressed as a percentage. Flow rates in clear solutions were seen to increase with time as the filter warmed. Samples were collected as outlined above. 1.6 Phospholipid Assay In some cases samples were assayed for phospholipid content. The sample was transferred to a HPLC vial and analyzed by reverse phase HPLC separation and Corona Charged aerosol detection (CAD; HPLC With Charged Aerosol Detection for the Measurement of Different Lipid Classes, I. N. Acworth, P. H. Gamache, R. McCarthy and D. Asa, ESA Biosciences Inc., Chelmsford, MA, USA; J. Waraska and I. N. Acworth, American Biotechnology Laboratory, January 2008) and quantified versus reference standards. 1.7 Product Preparation and Testing 1.7.1 Aqueous Formulation Filtered aqueous formulation (see section 1.5.1) was aliquoted (1.76 mL) into 2 cc Wheaton vials the headspace air replaced with perfluoropropane (PFP) gas, the vial sealed with a West grey butyl stopper, and crimped with an aluminum seal. 1.7.2 Non-Aqueous Formulation Filtered aqueous formulation (see section 1.5.2) was aliquoted (0.35 mL) into 2 cc Wheaton vials the headspace air replaced with perfluoropropane (PFP) gas, the vial sealed with a West grey butyl stopper, and crimped with an aluminum seal. 1.7.3 Sysmex Microsphere Sizing Samples were analyzed for number and size distribution using a particle sizer (Malvern FPIA-3000 Sysmex). Aqueous or non-aqueous samples were optimally activated using a VIALMIX®, a portion of the activated product diluted with saline and then transferred to the sample vessel of the Sysmex. The Sysmex uses an appropriate sheath solution and analyzes the sample using both low and high power fields to generate sizing data for the specified size range (1 to 80 μm in the current studies). 1.7.4 Ultrasound Contrast of Activated Product Acoustic attenuation was measured for selected samples using a Philips Sonos 5500 clinical ultrasound imaging system. Following optimal activation with a VIALMIX® 10 microliter samples were pipetted into a 250 mL beaker containing 200 ml of 0.9% saline at room temperature. A round, vaned, 38 mm diameter stirring bar maintained solution uniformity and served as an acoustic reflector. The s3 clinical transducer of the ultrasound system was positioned at the top of the beaker, just into the solution and 4.8 cm above the upper margin of the stirring bar. Five seconds of 120 Hz images were then acquired digitally and written to disk beginning 10 seconds after introduction of the sample. The US system was used in IBS mode, TGC was fixed at the minimal value for all depths, and LGC was disabled. The mechanical index (MI) was 0.2 with power set 18 dB below maximum. The receive gain was fixed at 90 and the compression at 0. For each sample tested, US data acquisition was acquired prior to (blank) and after sample iniection. Image analysis was performed using Philips QLab version 2.0, which read files produced by the US system and calculated values in dB for IBS mode. Regions of interest were drawn on the stirring bar and the dB values exported to Excel. These were then averaged over the full 5 second (approximately 360 video frame) acquisition. Attenuation measurements were obtained by subtracting the averaged sample ROI value from the averaged blank ROI value (both in dB). This was divided by twice the distance between the US transducer and the upper margin of the stirring bar to yield attenuation in dB/cm. Values were then divided by the calculated microbubble concentration in the beaker and expressed in terms of dB attenuation per centimeter per million microbubbles/mL. Example 1: Effect of Calcium Addition to Non-Aqueous Phospholipid Solution This example demonstrates the effect of calcium and magnesium ions on phospholipid precipitation. Example 1.1: Initial Studies on the Effect of Calcium and Magnesium Addition to Non-Aqueous Solution In initial studies, lipid blend (LB, Lot 1) characterized as having low divalent metal ion concentration (Table 1 in example methods), was added to propylene glycol at 55°±5° C. and stirred. It was verified by visual observation that the phospholipids had fully dissolved and the resulting solution was clear. This LB solution was titrated with calcium (25.4 μg Ca+2 magnesium (28.0 μg Mg+2/g) or a combination (1:1 to make a solution containing 14.0 μg Ca+2/g and 12.7 μg Mg+2/g) and showed cloudiness at 3.60 μg Ca+2/g, 4.23 μg Mg+2/g and 2.35 μg/g combined metal ion/g non-aqueous phospholipid solution, respectively. Example 1.2: Follow-Up Studies on the Effect of Calcium Addition to Non-Aqueous Solution The experiment was conducted as follows: DPPC, DPPA and MPEG5000-DPPE powder, characterized as having low divalent metal ion concentration (Table 1 in example methods), were added either individually (in the sequence shown in Table 2) or as a mixture or as a blend added to heated (55° C.±5° C.) and stirred propylene glycol (PG) or a 1:1 mixture of propylene glycol and glycerol (PG/G). It was verified by visual observation that the phospholipids had fully dissolved and the resulting solution was clear (=0: see example methods Section 1.4.2,FIG.1for rating scale).FIG.2illustrates the appearance of a lipid concentrate in propylene glycol upon the successive additions of DPPC, MPEG5000-DPPE, DPPA and calcium acetate stock (1 mL of a 299 μg Ca+2per mL of stock was added to produce a lipid concentrate with an 11.1 μg Ca+2per g of solution). The lipid concentrate did not turn cloudy until the calcium was added. Phospholipid solutions in PG or 1:1 mixture PG/G were titrated by a series of small additions of calcium. After each addition, the solution was assessed for clarity (see example methods Section 1.4.2,FIG.1for rating scale) and the lowest calcium concentration producing a +, ++, and +++ score is shown in Table 2.FIG.3shows representative solutions for the Study 4 titration. TABLE 2Effect of Calcium addition to Non-aqueous phospholipid solutionObserved cloudinessthresholds(μg Ca+2/g) forOrder of lipid additionNon-titrationMPEG5000-aqueouswith calciuma,bStudyDPPCDPPEDPPASolvent++++++1c132PG1.52.6>5.72c231PG1.52.9>11.13c312PG2.34.611.14c123PG1.82.9>5.75dphospholipid BlendPG1.85.711.16ephospholipid Mixture (dry)PG1.85.711.17f132PG & GLipids not dissolved8g132PG & G2.619.235.8aDefined in methods Section 1.4bTitrated with calcium acetate stock solutions (299 μg Ca+2/g of stock solution)cDPPA (0.9 mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE (6.08 mg/mL) final concentration was achieved by individual phospholipid addition to propylene glycol (25 mL)dPhospholipid blend (15 mg/mL), made using methanol to dissolve phospholipids at 55° C. followed by drying, dissolved in 25 mL propylene glycolePhospholipid mixture: DPPA, DPPC, and MPEG5000-DPPE (0.045:0.401:0.304), powders were stirred together and used for compounding in 25 mL propylene glycol. The final concentration is 15 mg/mLfPhospholipid solution made by adding individual phospholipids [DPPA (0.9 mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE (6.08 mg/mL)] to 25 mL, 1:1 (v/v) propylene glycol:glycerolgPhospholipid solution made by adding individual phospholipids [DPPA (0.225 mg/mL), DPPC (2.00 mg/mL) and MPEG5000-DPPE (1.70 mg/mL)] to 100 mL, 1:1 (v/v) propylene glycol:glycerol Calcium titration produced a clear concentration dependent precipitation in the phospholipid solution irrespective of the how the phospholipids were added (individually, as a mixture or as a blend) to the propylene glycol (see Table 2). The lipids were not soluble in either glycerol alone or in 25 mL of 1:1 PG/G but did achieve a clear solution when added to 100 mL of 1:1 PG/G (Study 8). Calcium produced a concentration dependent precipitation in this lipid solution consistent with initial findings (see Table 2). Overall, these titration studies indicated the lowest calcium, magnesium and combined concentrations that produced precipitation was 1.5 μg Ca+2/g, 4.23 μg Mg+2/g, and 2.35 μg combined metal ion/g non-aqueous phospholipid solution. Example 2: Effect of Phospholipid Solution Components Containing Calcium when Mixed Example 2.1: Calcium in PG Study 9 was conducted as follows: DPPC, MPEG5000-DPPE and DPPA powder, characterized as having low calcium concentration (see Table 1 in example methods), were added individually (in the sequence shown in Table 3) to heated (55° C.±5° C.) and stirred PG containing 11 μg/g calcium. Clarity was assessed (see Section 1.4) and the solution was clear after DPPC dissolved, turned and stayed cloudy after addition of DPPA, and remained cloudy after addition of MPEG5000-DPPE. The cloudiness observed was scored as +++(FIG.1, Section 1.4). This contrasted with the clear solution produced when these phospholipids (including DPPA) were added to PG containing low calcium (starting solution for Study 1). This was further emphasized by Study 12, where only phospholipids DPPC and MPEG5000-DPPE containing high Ca+2levels, were dissolved and this solution stayed clear even with the presence of calcium. Example 2.2: Calcium in Lipid Blend from MPEG5000-DPPE Initial experiments were performed on phospholipid blend (made using toluene and methanol to dissolve and adding MTBE to precipitate out the lipid blend) containing DPPC, DPPA and either low (not detected Ca+2and 1 μg Mg+2/g, MPEG5000-DPPE) or high (980 μg Ca+2/g and 150 μg Mg+2/g, MPEG5000-DPPE Lot 1) calcium and magnesium containing MPEG5000-DPPE, respectively, were added to heated (55° C.±5° C.) and stirred propylene glycol. The two lipid blends were mixed to provide samples having approximately 0, 1.75, 4.11 and 12.9 μg combined Ca+2& Mg+2/g of non-aqueous phospholipid solution. The 1.75 μg combined Ca+2& Mg+2/g of non-aqueous phospholipid solution showed cloudiness. Follow-up study 10 and 11 were conducted as follows: phospholipid blend (made using toluene and methanol to dissolve and adding MTBE to precipitate out the phospholipid blend) containing DPPC, DPPA and either high (980 ppm Ca+2, 150 ppm Mg+2, Lot 1) or low (4 ppm Ca+2) calcium containing MPEG5000-DPPE were added to heated (55° C.±5° C.) and stirred PG. Clarity was assessed (see Section 1.4) and slight cloudiness was observed (+; see example methods in Section 1.4) with the phospholipid blend containing high calcium (measured as 370 ppm Ca+2and 54 ppm Mg+2). This contrasted with the clear solution produced by dissolving low calcium (non-detectable levels of Ca+2and Mg+2) containing phospholipid blend (see Table 3). TABLE 3Effect of Phospholipid solution components containingcalcium when mixedOrder of phospholipidadditionNon-Ca+2(Mg+2)ObservedMPEG5000-aqueousμg/gCloudinessStudyDPPCDPPEDPPASolvent[source]Levela9b123PG11.2 (0.0)+++c[added to PGbeforelipid addition]12d12—PG5.8 (0.9)0e[High Ca+2MPEG5000-DPPE]10Phospholipid blendPG0.0 (0.0)0containing low Ca+2 f[LB Lot 1]11Phospholipid blendPG5.36 (0.8)+++containing high Ca+2 f[LB Lot 2]aDefined in methods Section 1.4bDPPA (0.9 mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE (6.08 mg/mL) final concentration was achieved by individual phospholipid addition to propylene glycol (25 mL)cSolution was clear when DPPC solubilized, remained clear after addition of MPEG5000-DPPE turned cloudy after addition of DPPAdDPPC (8.02 mg/mL) and MPEG5000-DPPE containing Ca+2(6.08 mg/mL; 980 ppm Ca+2and 150 ppm Mg+2); final concentration was achieved by individual phospholipid addition to propylene glycol (25 mL), no DPPA addedeSolution was clear upon addition of DPPC and MPEG5000-DPPEfPhospholipid blend (15 mg/mL) made using toluene and methanol to dissolve phospholipids and adding MTBE to precipitate out the phospholipid blend, dissolved in 25 mL propylene glycol Example 2.3: Calcium from MPEG5000-DPPE Added Individually Studies 13 through 17 were conducted as follows: DPPA and DPPC, characterized as having low calcium concentration (see Table 1 in example methods), were added individually (in the sequence shown in Table 4) to heated (55° C.±5° C.) and stirred PG. MPEG5000-DPPE containing different proportions of “low” and “high” calcium and magnesium material was added. Clarity was assessed (see example methods in Section 1.4,FIG.1) and a calcium and magnesium concentration dependent precipitation was observed (see Table 4 andFIG.4). TABLE 4Calcium and Magnesium from MPEG5000-DPPE added as an individualcomponentOrderPercentagecMetal ionof lipidMPEG5000-MPEG5000-Non-ConcentrationObservedadditionDPPEDPPEaqueous(μg/g)CloudinessStudyDPPCDPPA(Low Ca+2)(high Ca+2)SolventCa+2Mg+2TotalLevela13b121000PG0.10.00.1014b127525PG0.70.10.8+15b125050PG1.3031.6++16b122575PG1.90.42.3++17b120100PG3.10.63.7+++1812n/pdn/pdPG & GeNot addedCloudy,DPPA notdissolvedaDefined in methods Section 1.4bDPPA (0.9 mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE (6.08 mg/mL) final concentration was achieved by individual phospholipid addition to propylene glycol (25 mL)cPercentages of MPEG5000-DPPE; low Ca+2[4 ppm] and high Ca+2[520 ppm Ca+2, 110 ppm Mg+2] relative to totaldn/p = not performed; Phospholipids [DPPC (8.02 mg/mL) and DPPA (0.9 mg/mL)] not solubilized in propylene & glycol solvent systemePropylene glycol and glycerol 50:50 (v/v) Summary of Example 2 Overall these studies have demonstrated the addition of calcium, either in the non-aqueous solvent or via the phospholipid blend or when added as MPEG5000-DPPE as an individual compound, all caused precipitation. The concentration where effects were seen were similar for Example 2 compared to those in Example 1. The lowest calcium concentration that produced cloudiness (+) was at 0.7 μg/g Ca+2(0.8 μg/g total Ca+2and Mg+2). This is a similar concentration to the 1.5 to 2.6 μg/g see in Example 1. Example 3: Addition of Non-Aqueous Phospholipid Solution to Aqueous Solvent Example 3.1: Effect of Calcium in Non-Aqueous Phospholipid Solution on Addition to Aqueous Solvent A series of studies were performed to examine the impact of calcium in the non-aqueous phospholipid solution prior to transferring into the aqueous formulation. These involved the steps of: 1) preparing a non-aqueous phospholipid solution, 2) preparing an aqueous solution and 3) combining solutions from 1 and 2. Example 3.1.1: Preparing Non-Aqueous Solution: Calcium Added to Non-Aqueous Solution after Phospholipids Dissolved Consistent with Example 2, the first step in studies 19, 20 and 22 were as follows: DPPC, DPPA, and MPEG5000-DPPE powder, characterized as having low calcium concentration (see Table 1 in example methods), were added individually (in the sequence shown in Table 5) to heated (55° C.±5° C., with the exception of study 22, which was heated to 70° C.) and stirred propylene glycol. It was verified by visual observation that the phospholipids had fully dissolved and the resulting solution was clear (see example methods in Section 1.4,FIG.1). A solution of calcium acetate [Ca(OAc)2] in propylene glycol was added as indicated in Table 5, the solution was stirred and observed for changes in appearance, as compared to a solvent blank and the assessment of clarity was recorded. Upon addition of calcium acetate, the solutions turned cloudy. These propylene glycol concentrates were transferred to the aqueous phase as described below. Example 3.1.2 Preparing Non-Aqueous Solution: Calcium in MPEG5000-DPPE The first step studies 21 and 25 were as follows: DPPC, DPPA (not included in study 25), and calcium containing MPEG5000-DPPE powder (980 ppm, MPEG5000-DPPE Lot 1; see Table 1 in example methods), were added individually (in the sequence shown in Table 5) to heated (55° C.±5° C.) and stirred in PG. Clarity was assessed and significant cloudiness observed in study 21 (+++; see example methods in Section 1.4,FIG.1) after addition of DPPA, and remained cloudy after addition of MPEG5000-DPPE, whereas no cloudiness was observed in study 25 which did not contain DPPA. These non-aqueous phospholipid solutions were transferred to the aqueous phase as described below. Example 3.1.3: Preparing Non-Aqueous Solution: Calcium in Lipid Blend from MPEG5000-DPPE Consistent with Example 2, the first step in studies 23 and 24 were conducted as follows: phospholipid blend (made using toluene and methanol to dissolve and adding MTBE to precipitate out the lipid blend) containing DPPC, DPPA and either low (4 ppm, lot 2 or high (980 ppm, MPEG5000-DPPE Lot 1) calcium containing MPEG5000-DPPE, respectively, were added to heated (55° C.±5° C.) and stirred propylene glycol. Clarity was assessed and significant cloudiness observed (+++; see example methods in Section 1.4,FIG.1) with the phospholipid blend containing high calcium. This contrasted with the clear solution produced by dissolving low calcium containing phospholipid blend (see Table 5). These non-aqueous phospholipid solutions were transferred to the aqueous phase as described below. Example 3.1.4: Preparing Non-Aqueous Solution: Calcium in PG Prior to Adding Phospholipids Consistent with Example 2, the first step in studies 28 and 30 were conducted as follows: DPPC, MPEG5000-DPPE and DPPA powder, characterized as having low calcium concentration (see Table 1 in example methods), were added individually (in the sequence shown in Table 5) to heated (55° C.±5° C.) and stirred PG either containing 11 μg/g calcium or calcium added after phospholipid addition, respectively. Clarity was assessed (see example methods in Section 1.4,FIG.1) and in study 28 the solution was clear after DPPC and MPEG5000-DPPE dissolved but turned and stayed cloudy after addition of DPPA. In study 30 the solution was clear after DPPC, DPPA and MPEG5000-DPPE were dissolved, and turned cloudy after addition of Ca+2. The cloudiness for both studies was scored as +++(see Table 5). These non-aqueous phospholipid solutions were transferred to the aqueous phase as described below. Example 3.2: Preparing Aqueous Solution For all studies the aqueous solution was prepared as follows: In a separate vessel Sodium Chloride (NaCl), Sodium Phosphate Dibasic Heptahydrate (Na2HPO4·7H2O), and Sodium Phosphate Monobasic (NaH2PO4·H2O) were added to water in a stirred vessel, and mixed until dissolved. Propylene glycol and glycerol were also added, as needed, so the final addition of phospholipid concentrate will reconstitute to an 8:1:1 water: glycerol: propylene glycol composition. This stirred solution was maintained at 55° C.±5° C. (with the exception of study 22 where the aqueous solution was maintained at 70° C.). Example 3.3: Combining Non-Aqueous and Aqueous Solutions For all studies, the addition of the non-aqueous phospholipid concentrate to the aqueous solution was done as follows: The warm phospholipids in propylene glycol were added and stirred at 100 to 150 rpm. Visual observations were recorded and the time for full dispersion or dissolution was (either clear or cloudy) noted. These aqueous suspensions were then collected and filtered through a 0.2 um filter at 55° C. under 5 psi head pressure. Flow rate was measured and samples collected for phospholipid measurement (see example methods for procedure). Pre- and post-filtration samples were assayed to determine the level of phospholipid loss associated with filtration. TABLE 5Effect of divalent metal ion in non-aqueous phospholipid solution onaddition to aqueous solventAqueous suspensionbPercent ofNon-aqueous phospholipid concentrateaInitialPhospho-Ca+2(Mg+2)Filtration% phospholipidlipidContainsAppearance afterconcentration inRate at 9post filtationdadditionCa+2(Mg+2) μg/g PGObservedphosphateaddition toaqueousto 10MPEG5000-Studyto PG[Ca+2source]cloudinesscbufferaqueous[μg/g water]minutesDPPCDPPADPPE19C, E, A0.0 (0.0)0YesClear0 (0)64.61011009920C, E, A13.7 (0)e+++YesCloudy0.81.3;957694[Calcium acetate(0)blockedadded after lipids]filter21C, A, E3.1 (0.7)f+++YesCloudy0.29.0;967895[in MPEG5000-(0.03)blockedDPPE]filter22gC, A, E21.4 (0)e[Calcium+++Yes;Cloudy1.28.5;987597acetate added afterat(0)blockedlipids]70° C.filter25C, E5.8 (0,9)h0YesClear0.382.1100nd99[in MPEG5000-(0.04)DPPE]23LBi0 (0)0YesClear0 (0)92.0991009924LBi5.36 (0.8)+++YesSlightly0.35.2 blocked986596[Lipid Blend]cloudy(0.04)filter28C, E, A11.2 (0.0)k+++YesCloudy0.642.710124100[Calcium acetate in(0)PG, thenphospholipid added]30C, A, E21.4 (0.0)g+++NoCloudy1.29.0 blocked894286[Calcium acetate(0)filteradded after lipids]aPhospholipid concentrate was prepared at 15 mg/mL by dissolving DPPC (C), MPEG5000-DPPE (E) and DPPA (A) in the ratio of 0.401:0.304:0.045 in propylene glycol in the order listed at 55° C.bPhospholipid concentrates were added to a compounding vessel containing: water (800 mg); dibasic sodium phosphate, heptahydrate (2.16 mg); monobasic sodium phosphate, monohydrate (2.34 mg); sodium chloride (4.84 mg), glycerol (126 mg), and propylene glycol (51.75 mg) per mL of compounding solution. Materials were combined at 55° C., in the order listed.cDefined in methods Section 1.4.2dHPLC with CAD detection described in Section 1.6e1 mL, 2 mL and 2 mL (Study 20, 22, and 30, respectively) of a stock 299 μg Ca+2per g PG, after lipid addition prior to transfer to the aqueous compounding solutionfMPEG5000-DPPE containing Ca+2(6.08 mg/mL; 520 and 110 ppm Ca+2and Mg+2) used for experimentgAll compounding performed at 70° C.hMPEG5000-DPPE containing Ca+2(6.08 mg/mL; 980 and 150 ppm Ca+2and Mg+2) used for experimentiMade using toluene and methanol to dissolve lipids and adding MTBE to precipitate out the lipid blend. Resulting lipid blend added to 25 mL propylene glycol (15 mg/mL). Study 23 used low Ca+2lipid blend and Study 24 used lipid blend containing Ca+2and Mg+2(370 and 54 μg/g, respectively).kAdded 1 mL of a stock 299 μg Ca+2per g PG, prior to addition of lipids Consistent with the previous examples, these studies showed precipitation occurred in the non-aqueous phospholipid solution when high calcium or calcium and magnesium were present. This occurred regardless of if the calcium was present in the propylene glycol prior to the phospholipid addition, added after the phospholipid addition or added with one of the components of the phospholipids (either with MPG5000 DPPE or in a phospholipid blend). Once the precipitate was formed it did not disperse when mixed with aqueous solvent. This resulted in a cloudy aqueous preparation that had a reduced rate of filtration initially and often blocked the 0.2 μm filter (Table 5;FIG.5). The filtrate of cloudy aqueous preparations was clear but phospholipid measurement indicated consistently reduced levels of DPPA. This effect was apparent for both individually added phospholipids and phospholipids added as a blend. Example 3.4: Effect of Non-Aqueous Phospholipid Solution Addition to Aqueous Solvent Containing Calcium A series of studies were performed to examine the impact of calcium in the aqueous solution on phospholipid suspension preparation. These involved the steps of: 1) preparing a non-aqueous phospholipid solution, 2) preparing an aqueous solution and 3) combining solutions from 1 and 2. Example 3.4.1: Preparing Non-Aqueous Solution Consistent with Example 1, the first step in Studies 26, 27 and 29 were conducted as follows: DPPC, DPPA and MPEG5000-DPPE powder, characterized as having low calcium concentration (see Table 1 in example methods), were added individually (in the sequence shown in Table 6) to heated (55° C.±5° C.) and stirred propylene glycol. It was verified by visual observation that the phospholipid had fully dissolved and the resulting solution was clear. These propylene glycol concentrates were transferred to the aqueous phase as described below. Example 3.4.2: Preparing Aqueous Solution In a separate vessel Sodium Chloride (NaCl), Sodium Phosphate Dibasic Heptahydrate (Na2HPO4·7H2O), and Sodium Phosphate Monobasic (NaH2PO4·H2O) were added to water in a stirred vessel, and mixed until dissolved (for study 29 the Phosphate salts were excluded from the formulation). Propylene glycol and glycerol were also added as needed, so the final addition of non-aqueous phospholipid solution will reconstitute an 8:1:1 water: glycerol: propylene glycol composition. In some studies, a solution of calcium acetate [Ca(OAc)2] in water was added as indicated in Table 6. This aqueous solution was stirred, maintained at 55° C.±5° C. It was identified that the addition of 48.4 μg/g calcium caused a marked flocculation in the aqueous solution in the absence of any phospholipids (see Table 6, study A). At 12.2 μg/g calcium no precipitation was produced in the aqueous solution (see Table 6, study B). Example 3.4.3: Combining Non-Aqueous and Aqueous Solutions For all studies, the addition of the non-aqueous phospholipid concentrate to the aqueous solution was done as follows: the warm phospholipid dissolved in propylene glycol was added and stirred at 100 to 150 rpm. Visual observations were recorded and the time for full dispersion or dissolution is stable (either clear or cloudy) noted. For study 27, the aqueous formulation was initially clear. Calcium was titrated and at concentrations ≥30.4 μg/g a cloudy precipitate was formed (see Table 6). It was noted, however, that the aqueous solution without phospholipid had a marked precipitated at 48.4 μg/g (Study A: Table 6). In study 27, at calcium levels where the aqueous solution alone was not effected (12.2 μg/g based on study B, Table 6), no effect was seen on aqueous Phospholipid formulation clarity. This was confirmed in study 26, where calcium was added to the aqueous solution (12.2 μg/g) prior to combining with the non-aqueous phospholipid concentrate. This was further extended in study 29, where the phosphate buffer was excluded from the aqueous solution. Initially, calcium was added to the aqueous solution (12.2 μg/g) prior to combining with the non-aqueous phospholipid concentrate and the formulation was clear. Additional calcium was added after the phospholipid addition to the formulation up to 96 μg/g and no precipitation was observed. The aqueous formulations from study 26 and 29 were then collected and filtered through a 0.2 μm filter at 55° C. under 5 psi head pressure. Flow rate at 10 minutes was not reduced compared to initial flow; all the sample was filtered and overall filtration was similar to preparations not containing calcium (see studies 19, 23 and 25). Pre- and post-filtration samples were collected and compared to determine loss of phospholipids associated with filtration. No meaningful loss of Phospholipid was apparent (see Table 6). TABLE 6Effect of non-aqueous phospholipid solution addition to aqueous solvent containing divalent metal ionsAqueous suspensionbAppearanceNon-aqueousCalciumafter lipid% phospholipidlipid concentrateconcentrationconcentrateContainsPercent of Initialpost filtrationdPhospholipidObservedin aqueousaddition toPO4Filtration Rate at 9-MPEG5000-Studyaddition to PGbcloudinessc(μg Ca+2/g)aqueousbuffer10 minutesDPPCDPPADPPE27C, E, A0Titration 0 toClear to 12.2,Yesn/a48.7 μgslightly cloudyCa+2/gat 30.4 andcloudy withprecipitate at36.5 μg Ca+2/gwater26C, E, A012.2eClearYes114.3991019829C, A, E012.2eClearNo100.8999998An/an/a48.4ePrecipitateYesn/an/an/an/aBn/an/a12.2eClearYesn/an/an/an/aaAll compounding performed at 55° C. Non-aqueous phospholipid solutions were added to aqueous compounding vessel containing: water (800 mg); sodium phosphate heptahydrate (2.16 mg/mL), sodium phosphate monohydrate (2.34 mg/mL), sodium chloride (4.84 mg/mL), glycerol (126 mg), and propylene glycol (51.75 mg) unless otherwise indicated in footnotes.b“A” is DPPA (0.9 mg/mL), “C” is DPPC (8.02 mg/mL) and “E” is MPEG5000-DPPE (6.08 mg/mL) which were added in the order listed, to 25 mL propylene glycolcDefined in methods Section 1.4dHPLC with CAD detection described in Section 1.6ePrior to addition of lipid concentrate to aqueous compounding vessel, 1 mL, 1 mL, 4 mL and 1 mL of calcium acetate concentrate (6.085 mg Ca+2per g of water) was added for studies 26, 29, A and B, respectively. These studies demonstrate that calcium is not causing phospholipid precipitation in the aqueous formulation even up to 96 μg/g. However, at calcium levels higher than 12.2 μg/g the phosphate salts start to precipitate. Example 4: Effect of Divalent Metal Ions on Phospholipid Dissolving in Buffered Propylene Glycol and with the Addition of Glycerol Example 4.1: Calcium titration in buffered non-aqueous phospholipid concentrate Studies 31 and 32 were conducted as follows: DPPC, DPPA and MPEG5000-DPPE powder, characterized as having low calcium concentration (see Table 1 in example methods), were added either individually (in the sequence shown in Table 7) or as a phospholipid blend (made using toluene and methanol to dissolve and adding MTBE to precipitate out the lipid blend) to heated (55° C.±5° C.) and stirred acetate buffered propylene glycol. It was verified by visual observation that the lipid had fully dissolved and the resulting solution was clear (see example methods Section 1.4). A solution of calcium acetate [Ca(OAc)2] in propylene glycol was used to titrate the phospholipid solution by a series of small additions. The solution was stirred and observed for changes in appearance during the titration, as compared to a solvent blank after each addition and the assessment of clarity was recorded. A cloudiness score (see Section 1.4,FIG.1, for method) based on this assessment was made and the lowest calcium concentration producing a +, ++, and +++ score is shown in Table 7. TABLE 7Effect of calcium on phospholipid dissolving in buffered propylene glycolOrder of lipid additionaCalciumObserved cloudinessMPEG5000-concentrationthresholds (μg/mL Ca+2)bStudyDPPCDPPADPPELipid blend(μg/mL Ca+2)++++++31n/an/an/a1cTitrationr5.811.222.332132n/aTitrations11.317.0>33.5aIndividual lipids [DPPC (4.01 mg), DPPA (0.45 mg), and MPEG5000-DPPE (3.04 mg), in the order listed] or lipid blend (7.5 mg) were added to each mL of propylene glycol containing sodium acetate (0.74 mg) and acetic acid (0.06 mg), at 60° C. with stirring.bDefined in methods Section 1.4cMade using toluene and methanol to dissolve lipids and adding MTBE to precipitate out the lipid blend. Example 4.2: Calcium Titration in Buffered Non-Aqueous Phospholipid Concentrate from MPEG5000-DPPE Studies 33 through 36 were conducted as follows: DPPC, DPPA and either high (980 ppm, Lot 1 or low Ca+2(4 ppm) containing MPEG5000-DPPE, were added individually (in the sequence shown in Table 8) or as a phospholipid blend (made using toluene and methanol to dissolve and adding MTBE to precipitate out the lipid blend) to heated (55° C.±5° C.) and stirred acetate buffered propylene glycol. Clarity was assessed (see example methods Section 1.4) and cloudiness was observed (+ or ++; see example methods,FIG.1) with the phospholipid blend containing high calcium. This contrasted with the clear solution produced by dissolving low calcium containing phospholipid blend (see Table 8). Example 4.3: Glycerol Addition To these buffered non-aqueous phospholipid solutions, glycerol was transferred with stirring at 300 rpm. Many gas bubbles were trapped in the mixing solution but cleared once the stirrer was stopped. Visual observations were recorded and the clarity level (either clear or cloudy) noted. These PG/G solutions were then collected and filtered through a 0.2 μm filter at 60° C. under 10 psi head pressure. Flow rate was measured and samples collected for phospholipid measurement. Pre- and post-filtration samples were compared to determine loss of phospholipids associated with filtration. TABLE 8Effect of Calcium phospholipid dissolving in buffered propylene glycol and glycerol addedPropylene glycol with added glycerolbPercent ofAcetate/Propylene glycolCa+2(Mg+2)Initial% phospholipid postconcentrateaAppearanceconcentrationFiltrationfiltrationdLipid addition toCa+2(Mg+2)Observedafter additionproductRate at 8-9MPEG 5000StudyPG[μg/g]cloudinesscof glycerol[μg/g]minuteDPPCDPPADPPE33LBe0 (0)0clear0174.297909634LBe2.7+cloudy1.6 (0.2)96.7;1018698(0.4)blockedfilter35C, A, E0 (0)0clear0245.2971009836C, A, Ef2.9++cloudy1.7 (0.2)19.8:9980100(0.4)blockedfilteraIndividual lipids [DPPC (4.01 mg), DPPA (0.45 mg), and MPEG5000-DPPE (3.04 mg), in the order listed] or lipid blend (7.5 mg) were added to each mL of propylene glycol containing sodium acetate (0.74 mg) and acetic acid (0.06 mg), at 60° C. with stirring.bPropylene glycol containing acetate buffer and phospholipids is diluted 1:1 (v/v) with glycerol.cDefined in methods Section 1.4dHPLC with CAD detection described in Section 1.6eLipid blend made using toluene and methanol to dissolve lipids and adding MTBE to precipitate out the lipid blend, low Ca+2 (Lot 1) for study 33, and high Ca+2 (370 Ca+2and 54 ppm Mg+2; Lot 2) for Study 34.fMPEG5000-DPPE containing Ca+2(3.04 mg/mL; 980 and 150 ppm Ca+2and Mg+2; Lot 1) used in this experiment These studies showed precipitation occurred in the buffered non-aqueous phospholipid solution in a calcium concentration dependent manner. This occurred regardless of whether the buffered non-aqueous phospholipid solution was made with individual phospholipids or a lipid blend and at concentrations that were not meaningfully different. The concentration to cause initial precipitation for the buffered solution was higher (5.8 to 11.3 μg/g Ca+2) than for the non-buffered solutions (1.5-2.3 μg Ca+2/g: see Table 2, studies 1 through 4) indicating an influence of the buffer. Calcium from the lipid blend caused precipitation in the buffered non-aqueous phospholipid solution as was seen in the non-buffered solution. Once the precipitate was formed it did not disperse when mixed with glycerol. This results in a cloudy non-aqueous formulation that had a reduced rate of filtration initially and often blocked the 0.2 μm filter (Table 8,FIG.6). The filtrate of cloudy preparations was clear but phospholipid measurement indicated slightly reduced levels of DPPA. Example 5: Microsphere Formation and Acoustic Detection of Manufactured Product Example 5.1: Aqueous Phospholipid Suspension Studies 37 and 38 were conducted as follows: filtered materials from study 19 and 23 were prepared in vials (see examples method Section 1.7.1). Following VIALMIX®, activation samples were analyzed for microsphere size and number (see methods Section 1.7.3) and clinical ultrasound acoustic attributes (see methods Section 1.7.4), see Table 9. TABLE 9Aqueous phospholipid suspension Microsphere number and Size and acousticactivity.MeanMicrosphereAcousticMeanMicrosphere(SD)Diameterper mLAttenuationc(microns)a(×109)b(dB/cm/106StudyProduction basisN = 2N = 2bubbles/mL)37Individual phospholipids with low1.38, 1.363.73, 2,928.9 (0.3)Ca+2measured in MPEG-5000 DPPEand other components38Phospholipid blend with low1.34, 1.353.4, 2.59.0 (1.3)Ca+2measured in MPEG-5000 DPPEand other componentsaMean microsphere diameter for microspheres ranging from 1 to 80 microns.bMean microsphere concentration for microspheres ranging from 1 to 80 microns.csee example methods section for details These studies demonstrate an aqueous phospholipid suspension can be produced using individual phospholipids or a phospholipid blend when the components have a low calcium concentration. Both products have microsphere diameter within the specification of DEFINITY® (see DEFINITY® package insert) and have strong ultrasound acoustic attenuation on a clinical ultrasound machine. Aspects and Embodiments Various aspects and embodiments provided by this disclosure are listed below. Clause 1. A method for preparing a phospholipid suspension, comprisingproviding DPPA, DPPC and MPEG5000-DPPE stocks,measuring calcium concentration of one or more of the DPPC, DPPA and MPEG5000-DPPE stocks,combining the DPPA, DPPC and/or MPEG5000-DPPE stocks with a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 2. The method of clause 1, further comprising measuring calcium concentration of the non-aqueous solvent. Clause 3. The method of clause 1, wherein the combined measured calcium concentration of the DPPA, DPPC and/or MPEG-DPPE stocks is low. Clause 4. The method of clause 1 or 3, wherein the combined measured calcium concentration of the DPPA, DPPC and/or MPEG-DPPE stocks and the non-aqueous solvent is low. Clause 5. The method of clause 1, wherein the calcium concentrations of the DPPC, DPPA and MPEG5000-DPPE stocks are measured. Clause 6. The method of clause 2, wherein the calcium concentrations of the DPPC, DPPA and MPEG5000-DPPE stocks are measured and the combined measured calcium concentration of the DPPA, DPPC, MPEG-DPPE stocks and the non-aqueous solvent is low. Clause 7. A method for preparing a phospholipid suspension, comprisingproviding DPPA, DPPC and MPEG5000-DPPE stocks,measuring calcium concentration of one or more of the DPPC, DPPA and MPEG5000-DPPE stocks,combining DPPA, DPPC and/or MPEG5000-DPPE stocks having a combined measured low calcium concentration with a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 8. The method of clause 7, wherein the calcium concentration of the non-aqueous solvent is measured and the DPPA, DPPC, MPEG500-DPPE stocks and the non-aqueous solvent have a combined measured low calcium concentration. Clause 9. A method for preparing a phospholipid suspension, comprisingcombining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueous solvent, each with a characterized calcium concentration to form a phospholipid solution, wherein the combined characterized calcium concentration of the MPEG5000-DPPE stock, the DPPA stock, the DPPC stock and the non-aqueous solvent is a low calcium concentration, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 10. A method for preparing a phospholipid suspension, comprisingselecting a MPEG5000-DPPE stock, a DPPA stock and a DPPC stock, one, two or all three of which have a characterized calcium concentration, wherein the combined characterized calcium concentration is a low calcium concentration,combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 11. A method for preparing a phospholipid suspension, comprisingselecting a MPEG5000-DPPE stock, a DPPA stock and a DPPC stock, each with characterized calcium concentration, wherein the combined characterized calcium concentration is a low calcium concentration,combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 12. The method of clause 11 wherein the non-aqueous solvent has a characterized calcium concentration, and the combined characterized calcium concentration of the MPEG5000-DPPE, DPPA and DPPC stocks and the non-aqueous solvent is low. Clause 13. A method for preparing a phospholipid suspension, comprisingmeasuring calcium concentration of a MPEG5000-DPPE stock,combining a MPEG5000-DPPE stock having a measured low calcium concentration with a DPPA stock, a DPPC stock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 14. The method of clause 11, wherein the non-aqueous solvent comprises (i) propylene glycol or (ii) propylene glycol and glycerol. Clause 15. The method of clause 13 or 14, wherein the non-aqueous solvent comprises a buffer. Clause 16. The method of clause 13 or 14, wherein the non-aqueous solvent comprises an acetate buffer. Clause 17. The method of clause 13 or 14, wherein the aqueous solvent comprises a buffer. Clause 18. The method of clause 13 or 14, wherein the aqueous solvent comprises a phosphate buffer. Clause 19. The method of any one of clauses 13-18, wherein the DPPC, DPPA and MPEG5000-DPPE stocks are individually combined with the non-aqueous solvent to form the phospholipid solution. Clause 20. The method of any one of clauses 13-18, wherein the DPPC, DPPA and MPEG5000-DPPE stocks are sequentially combined with the non-aqueous solvent, in an order-independent manner, to form the phospholipid solution. Clause 21. The method of any one of clauses 13-18, wherein the DPPC, DPPA and MPEG5000-DPPE stocks are combined with each other to form a phospholipid mixture and the phospholipid mixture is then combined with the non-aqueous solvent to form the phospholipid solution. Clause 22. The method of any one of clauses 13-18, wherein the DPPC, DPPA and MPEG5000-DPPE stocks are combined with each other to form a phospholipid blend, and the phospholipid blend is combined with the non-aqueous solvent to form the phospholipid solution. Clause 23. The method of clause 22, wherein the phospholipid blend is formed using an organic solvent dissolution-precipitation process comprising dissolving the DPPC, DPPA and MPEG5000-DPPE stocks into a mixture of methanol and toluene, optionally concentrating the phospholipid/methanol/toluene mixture, and then contacting the concentrated phospholipid/methanol/toluene mixture with methyl t-butyl ether (MTBE) to precipitate the phospholipids to form the phospholipid blend. Clause 24. The method of any one of clauses 13-23, wherein the low calcium concentration is less than 115 ppm. Clause 25. The method of any one of clauses 13-24, further comprising placing the phospholipid suspension in a vial and introducing a perfluorocarbon gas into the headspace of the vial. Clause 26. The method of clause 25, further comprising activating the phospholipid suspension with the perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres. Clause 27. The method of clause 26, further comprising administering the ultrasound contrast agent to a subject and obtaining one or more contrast-enhanced ultrasound images of the subject. Clause 28. The method of any one of clauses 13-27, further comprising measuring calcium concentration of the DPPA stock and/or DPPC stock and/or phospholipid mixture and/or phospholipid blend. Clause 29. A method for preparing a phospholipid suspension, comprisingmeasuring calcium concentration of a DPPC stock,combining a DPPC stock having a measured low calcium concentration with a DPPA stock, a MPEG5000-DPPE stock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 30. The method of clause 29, wherein the low calcium concentration is less than 90 ppm. Clause 31. A method for preparing a phospholipid suspension, comprisingmeasuring calcium concentration of a DPPA stock,combining a DPPA stock having a measured low calcium concentration with a DPPC stock, a MPEG5000-DPPE stock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 32. The method of clause 31, wherein the low calcium concentration is less than 780 ppm. Clause 33. A method for preparing a phospholipid suspension, comprisingmeasuring calcium concentration of a non-aqueous solvent,combining a non-aqueous solvent having a measured low calcium concentration with a DPPA stock, a DPPC stock, and a MPEG5000-DPPE stock, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 34. The method of clause 33, wherein the low calcium concentration is less than 0.7 ppm. Clause 35. A method for preparing a phospholipid suspension, comprisingselecting a MPEG5000-DPPE stock characterized as having no or low calcium concentration,combining said MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 36. The method of clause 35, wherein the MPEG5000-DPPE stock is further characterized as having no or low divalent metal cation content. Clause 37. A method for preparing a phospholipid suspension, comprisingcombining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueous solvent to form a phospholipid solution characterized as having no or low calcium concentration, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 38. A method for imaging a subject comprisingcombining a phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres,administering the ultrasound contrast agent to a subject, andobtaining one or more contrast-enhanced ultrasound contrast images of the subject, wherein the phospholipid suspension is prepared by the method of any one of clauses 1-37. Clause 39. A method for imaging a subject comprisingcombining a phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres,administering the ultrasound contrast agent to a subject, andobtaining one or more contrast-enhanced ultrasound contrast images of the subject, wherein the phospholipid suspension is prepared by a method comprisingmeasuring calcium concentration of MPEG5000-DPPE stock,combining a MPEG5000-DPPE stock having a measured low calcium concentration with a DPPA stock, a DPPC stock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form the phospholipid suspension. Clause 40. A method for imaging a subject comprisingcombining a phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres,administering the ultrasound contrast agent to a subject, andobtaining one or more contrast-enhanced ultrasound contrast images of the subject, wherein the phospholipid suspension is prepared by a method comprisingselecting a MPEG5000-DPPE stock characterized as having no or low calcium concentration,combining said MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 41. A method for imaging a subject comprisingcombining a phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres,administering the ultrasound contrast agent to a subject, andobtaining one or more contrast-enhanced ultrasound contrast images of the subject, wherein the phospholipid suspension is prepared by a method comprisingcombining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueous solvent to form a phospholipid solution characterized as having no or low calcium concentration, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 42. A method for preparing a phospholipid suspension, comprising individually combining DPPA, DPPC and MPEG5000-DPPE stocks with a propylene glycol (PG)-comprising non-aqueous solvent, in a low or no calcium condition, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 43. A method for preparing a phospholipid suspension, comprising sequentially combining DPPA, DPPC and MPEG5000-DPPE stocks with a PG-comprising non-aqueous solvent, in a low or no calcium condition, in an order-independent manner, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 44. A method for preparing a phospholipid suspension, comprisingcombining, in a methanol and toluene-free condition, DPPA, DPPC and MPEG5000-DPPE stocks to form a phospholipid blend,combining the phospholipid blend with a PG-comprising non-aqueous solvent, in a low or no calcium condition, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 45. A method for preparing a phospholipid suspension, comprisingcombining DPPA, DPPC and MPEG5000-DPPE stocks with a blend solvent to form a phospholipid blend,evaporating the blend solvent to form a dried phospholipid blend,combining the dried phospholipid blend with a PG-comprising non-aqueous solvent, in a low or no calcium condition, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 46. A method for preparing a phospholipid suspension, comprisingcombining DPPA, DPPC and MPEG5000-DPPE stocks with a blend solvent to form a phospholipid blend,precipitating, in a MTBE-free condition, the phospholipid blend using a second blend solvent,combining the precipitated phospholipid blend with a non-aqueous solvent, in a low or no calcium condition, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Clause 47. The method of any one of clauses 42-46, wherein the no or low calcium concentration is less than 0.7 ppm. Clause 48. The method of any one of clauses 42-47, further comprising combining the phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres. Clause 49. The method of clause 48, further comprising administering the ultrasound contrast agent to a subject and obtaining one or more contrast-enhanced ultrasound images of the subject. Clause 50. A method for imaging a subject comprisingcombining a phospholipid suspension with a perfluorocarbon gas to form an ultrasound contrast agent comprising phospholipid-encapsulated gas microspheres,administering the ultrasound contrast agent to a subject, andobtaining one or more contrast-enhanced ultrasound contrast images of the subject, wherein the phospholipid suspension is prepared by the method of any one of clauses 42-47. Clause 51. A composition comprising a phospholipid solution comprising DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent and having a low calcium concentration. Clause 52. A composition comprising a phospholipid solution comprising DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent, wherein the DPPA, DPPC and MPEG5000-DPPE and the non-aqueous solvent have a combined characterized calcium ion content that is low. Clause 53. The composition of clause 51 or 52, wherein the non-aqueous solvent comprises propylene glycol. Clause 54. The composition of clause 51 or 52, wherein the non-aqueous solvent comprises propylene glycol and glycerol. Clause 55. The composition of any one of clause 51-54, wherein the non-aqueous solvent comprises a buffer. Clause 56. The composition of clause 55, wherein the buffer is acetate buffer. Clause 57. The composition of any one of clauses 51-56, further comprising a perfluorocarbon gas. Clause 58. The composition of clause 57, wherein the perfluorocarbon gas is perflutren. Clause 59. A method of ultrasound contrast imaging a subject comprising(a) activating a phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres, wherein the phospholipid suspension comprises a phospholipid solution having one or more phospholipids and a non-aqueous solvent, one or more of which has a characterized low calcium concentration,(b) administering the lipid-encapsulated gas microspheres to a subject, and(c) obtaining an ultrasound image of the subject. Clause 60. The method of clause 59, wherein the one or more phospholipids comprise DPPC and MPEG-5000-DPPE. Clause 61. The method of clause 59, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE. Clause 62. The method of clause 61, wherein DPPA, DPPC and MPEG5000-DPPE are present in a mole % ratio of 10 to 82 to 8 (10:82:8). Clause 63. The method of any one of clauses 60-62, wherein the characterized low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm. Clause 64. The method of any one of clauses 59-63, wherein the non-aqueous solvent comprises (a) propylene glycol, or (b) propylene glycol and glycerol. Clause 65. The method of any one of clauses 59-64, wherein the characterized low calcium concentration for the non-aqueous solvent is less than 0.7 ppm. Clause 66. The method of any one of clauses 59-65, wherein the phospholipid solution has no detectable phospholipid precipitate. Clause 67. A method of ultrasound contrast imaging a subject comprising(a) activating a phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres, wherein the phospholipid suspension comprises a phospholipid solution having one or more phospholipids and a non-aqueous solvent and made under a methanol and toluene free condition and a methyl t-butyl ether free condition, wherein one or more of the phospholipids and non-aqueous solvent has a low calcium concentration,(b) administering the lipid-encapsulated gas microspheres to a subject, and(c) obtaining an ultrasound image of the subject. Clause 68. The method of clause 67, wherein the one or more phospholipids comprise DPPC and MPEG-5000-DPPE. Clause 69. The method of clause 67, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE. Clause 70. The method of clause 69, wherein DPPA, DPPC and MPEG-5000-DPPE are present in a mole % ratio of 10 to 82 to 8 (10:82:8). Clause 71. The method of any one of clauses 67-70, wherein the low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm. Clause 72. The method of any one of clauses 67-71, wherein the non-aqueous solvent comprises (a) propylene glycol, or (b) propylene glycol and glycerol. Clause 73. The method of any one of clauses 67-72, wherein the low calcium concentration for the non-aqueous solvent is less than 0.7 ppm. Clause 74. The method of any one of clauses 67-73, wherein the phospholipid solution has no detectable phospholipid precipitate. Clause 75. A method for preparing lipid-encapsulated gas microspheres comprisingcombining one or more phospholipids and a non-aqueous solvent to form a phospholipid solution, wherein one or more of the phospholipids and/or the non-aqueous solvent has a characterized low calcium concentration,combining the phospholipid solution with an aqueous solution to form a phospholipid suspension, andactivating the phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres. Clause 76. The method of clause 75, wherein the one or more phospholipids comprise DPPC and MPEG-5000-DPPE. Clause 77. The method of clause 75, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE. Clause 78. The method of clause 77, wherein DPPA, DPPC and MPEG-5000-DPPE are present in a mole % ratio of 10 to 82 to 8 (10:82:8). Clause 79. The method of any one of clauses 75-78, wherein the characterized low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm. Clause 80. The method of any one of clauses 75-79, wherein the non-aqueous solvent comprises (a) propylene glycol or (b) propylene glycol and glycerol. Clause 81. The method of any one of clauses 75-80, wherein the characterized low calcium concentration for the non-aqueous solvent is less than 0.7 ppm. Clause 82. The method of any one of clauses 75-81, wherein the phospholipid solution has no detectable phospholipid precipitate. Clause 83. A method for preparing lipid-encapsulated gas microspheres comprisingcombining one or more phospholipids and a non-aqueous solvent, in a methanol and toluene free and methyl t-butyl ether free condition, to form a phospholipid solution, wherein one or more of the phospholipids and/or the non-aqueous solvent has a low calcium concentration.combining the phospholipid solution with an aqueous solution to form a phospholipid suspension, andactivating the phospholipid suspension with a perfluorocarbon gas, to form lipid-encapsulated gas microspheres. Clause 84. The method of clause 83, wherein the one or more lipids comprise (a) DPPC and MPEG-5000-DPPE, or (b) DPPA, DPPC and MPEG-5000-DPPE and/or (c) DPPA, DPPC and MPEG-5000-DPPE in a mole % ratio of 10 to 82 to 8 (10:82:8). Clause 85. The method of clause 84, wherein the low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm. Clause 86. The method of any one of clauses 83-85, wherein the non-aqueous solvent comprises (a) propylene glycol, or (b) propylene glycol and glycerol. Clause 87. The method of any one of clauses 83-86, wherein the low calcium concentration for the non-aqueous solvent is less than 0.7 ppm. Clause 88. The method of any one of clauses 83-87, wherein the phospholipid solution has no detectable phospholipid precipitate. Clause 89. The method of any one of clauses 67-74, wherein one or more of the phospholipids or the non-aqueous solvent has a characterized low calcium concentration. Clause 90. The method of clause 89, wherein the characterized low calcium concentration is determined using atomic absorption spectroscopy. Clause 91. The method of any one of clauses 67-74, wherein the low calcium concentration is determined using atomic absorption spectroscopy. EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. | 139,085 |
11857647 | DETAILED DESCRIPTION OF THE INVENTION As used herein, the term “Tetrofosmin” includes not only tetrofosmin per se but also its pharmaceutically acceptable salts such as tetrofosmin disulfosalicylate, tetrofosmin sulfosalicylate, tetrofosmin hydrobromide and tetrofosmin hydrochloride. As used herein, the term “composition”, as in pharmaceutical composition, is intended to encompass a diagnostic product comprising tetrofosmin or its pharmaceutically acceptable salts, and the other inert ingredient(s) (pharmaceutically acceptable excipients). Such pharmaceutical compositions are synonymous with “formulation” and “dosage form”. As used herein, the term “lyophilized kit”, is intended to encompass a kit which comprises a freeze-dried composition comprising tetrofosmin or its pharmaceutically acceptable salts and one or more pharmaceutically acceptable excipients. As used herein, the term “non-radioactive composition”, as in pharmaceutical composition, is intended to encompass a freeze-dried composition comprising tetrofosmin or its pharmaceutically acceptable salts, and one or more pharmaceutically acceptable excipients. As used herein, the term “radiopharmaceutical composition”, as in pharmaceutical composition, is intended to encompass a composition comprising99mTc-pertechenetate solution, tetrofosmin or its pharmaceutically acceptable salts, and one or more pharmaceutically acceptable excipients. As used herein, the term “excipient” means an inactive component i.e., which do not have diagnostic function such as a biocompatible reductant, transchelator, pH adjusting agent, filler, radioprotectant, and the like. The excipients that are useful in preparing a pharmaceutical composition are generally safe, non-toxic and are acceptable for human pharmaceutical use. Reference to an excipient includes both one and more than one such excipient. Combination of excipients performing the same function may also be used to achieve desired composition characteristics. As used herein, the term “reductant or reducing agent” means a compound which is capable of reducing the technetium from a high oxidation state (such as Tc(VII)) to lower oxidation states of technetium. Suitable reductants as per the present invention include, but are not limited to, sodium dithionite, sodium bisulphite, formamidine sulphinic acid, tin, iron(II) or copper(I), sodium borohydride. Biocompatible reductant preferably comprises stannous ions, metallic tin or an alloy thereof in the form of Tin (II), and salts of Tin (II) such as stannous chloride dihydrate, stannous tartrate, stannous phosphate, stannous citrate etc. As used herein, the term “transchelator” also referred to as transfer ligand or intermediate ligand, a compound which reacts rapidly with technetium to form a weak complex and is then displaced from this complex by the ligand and accordingly ensures reduced risk of formation of reduced hydrolyzed technetium (RHT) due to rapid reduction of pertechnetate competing with technetium complexation. Suitable transchelators are salts of organic acids with a biocompatible cation, particularly “weak organic acids” with a pKa in the range 3 to 7. Suitable such weak organic acids include, but are not limited to, gluconic acid, acetic acid, citric acid, tartaric acid, glucoheptonic acid, benzoic acid, phenols or phosphonic acids. Suitable salts are acetates, citrates, tartrates, gluconates, glucoheptonates, benzoates, phenolates or phosphonates. Two or more transchelators may be used in combination to achieve the desired results. As used herein, the term “pH-adjusting agents” refers to a compound or mixture of compounds capable of maintaining the pH of the radiopharmaceutical composition within limits acceptable for human administration. Preferably pH of the radiopharmaceutical composition is about 4.0 to about 10. pH adjusting agents suitable for use in the present invention include, but are not limited to, pharmaceutically acceptable buffers such as tricine, phosphate or TRIS [tris(hydroxymethyl)aminomethane]; pharmaceutically acceptable bases such as sodium bicarbonate, sodium carbonate, or mixtures thereof. As used herein, the term “filler” refers to bulking agent which eases handling of material during production of lyophilized kit or radiopharmaceutical composition. As used herein, the term “radioprotectant” means a compound which prevents degradation reactions, such as redox reactions, by trapping highly reactive free radical species such as oxygen containing free radicals generated from the radiolysis of water. Radioprotectants of the present invention include, but are not limited to para-aminobenzoic acid, gentisic acid, maleic acid, anthranilic acid or their pharmaceutically acceptable salts thereof and combinations thereof. As used herein, the term “about” means±approximately 20% of the indicated value, such that “about 10 percent” indicates approximately 8 to 12 percent. “Biocompatible carrier” as used herein refers to a liquid in which the radiopharmaceutical is suspended or dissolved, such that the composition is physiologically tolerable. The biocompatible carrier can be an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which is isotonic); sugars (e.g. sucrose or glucose), sugar alcohols (e.g. mannitol or sorbitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols etc.). The biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Preferably, the biocompatible carrier is pyrogen-free water for injection or isotonic saline. As used herein, the term “radiochemical purity” refers to proportion of the total radioactivity in the sample which is present as the desired radiolabelled product. The radiochemical purity can be measured in curie, millicurie, becqurels. As used herein, the term “HPLC” refers high performance Liquid chromatography used to measure content of various components in a sample using US Pharmacopeial methods. As used herein, the term “GC” refers gas chromatography used to measure content of various components in a sample using US Pharmacopeial methods. As used herein, the term “TGA refers Thermo Gravimetric Analysis used to measure content of various components in a sample using US Pharmacopeial methods. In one aspect, the present invention provides a stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salts thereof with one or more pharmaceutically acceptable excipients, wherein the composition does not contain ascorbate or ascorbic acid as a radioprotectant. In yet another aspect of the present invention provides a stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salts and one or more pharmaceutically acceptable excipients, with the proviso that the radioprotectant is not ascorbic acid. The radioprotectant according to the present invention is selected from the group consisting of gentisic acid, para-amino benzoic acid, maleic acid, anthranilic acid, or their pharmaceutically acceptable salts thereof and combinations thereof, wherein the composition is free of antimicrobial preservatives. In yet another aspect of the present invention provides a lyophilized, non-radioactive kit which upon reconstitution with99mTc-pertechnetate solution gives a stable99mTc-tetrofosmin radiopharmaceutical composition comprising (i) tetrofosmin or its pharmaceutically acceptable salts and one or more pharmaceutically acceptable excipients and (ii) a vial containing99mTc-pertechnetate. According to one embodiment of this aspect, the kit is unit dose or multi dose. In one aspect, the non-radioactive kit comprises (i) first container containing tetrofosmin or pharmaceutically acceptable salts, reductants, transchelator, radioprotectant selected from gentisic acid, para amino benzoic acid, benzoic acid, maleic acid, anthranilic acid optionally preservative other than parabens (ii) second container comprising buffer, pH adjusting agents, transchelator, diluents, fillers, solvents or other pharmaceutically acceptable excipients. According to one embodiment of the above aspects, wherein the pharmaceutically acceptable excipients are selected from the group comprising biocompatible reductant, transchelator, pH adjusting agent, filler and radio protectant selected from the group consisting of gentisic acid, para-amino benzoic acid, maleic acid, anthranilic acid or their pharmaceutically acceptable salts thereof or combinations thereof. According to one aspect of the present invention, the non-radioactive composition comprises tetrofosmin or its pharmaceutically acceptable salts; a radio protectant selected from group consisting of gentisic acid, maleic acid, para amino benzoic acid, anthranilic acid; a transchelator selected from gluconic acid, acetic acid, citric acid, tartaric acid, glucoheptonic acid, benzoic acid, phenol or phosphonic acid or their pharmaceutically acceptable salts thereof and combinations thereof; wherein the composition is free of ascorbic acid and antimicrobial preservatives. According to another aspect of the present invention, the radiopharmaceutical composition comprises a (a)99mTc complex of tetrofosmin; (b) tetrofosmin or its pharmaceutically acceptable salts thereof selected from sulfosalicylate, disulfosalcylate; (c) at least one or more radio protectant selected from group consisting of gentisic acid, maleic acid, para amino benzoic acid, anthranilic acid or their pharmaceutically acceptable salts or combinations thereof; (d) at least one or more transchelator selected from gluconic acid, acetic acid, citric acid, tartaric acid, glucoheptonic acid, benzoic acid, phenol or phosphonic acid or their pharmaceutically acceptable salts thereof and combinations thereof; (e) a reducing agent such as stannous chloride dihydrate, stannous tartrate, stannous phosphate, stannous citrate; wherein the composition is free of ascorbic acid and antimicrobial preservative. In another aspect of the present invention, a process for producing a lyophilized composition, useful for diagnostic imaging, comprising the steps of: (a) preparing an aqueous solution of tetrofosmin or pharmaceutically acceptable salts; (b) addition of at least one or more radioprotectant selected from the group consisting of, gentisic acid, maleic acid, para amino benzoic acid, anthranilic acid or their pharmaceutically acceptable salts; (c) addition of at least one or more reducing agent such as stannous chloride dihydrate, stannous tartrate, stannous phosphate, stannous citrate or their pharmaceutically acceptable salts thereof; (d) addition of at least one or more transchelator selected from gluconic acid, acetic acid, citric acid, tartaric acid, glucoheptonic acid, benzoic acid, phenol or phosphonic acid or their pharmaceutically acceptable salts thereof; (e) lyophilizing said solution. In one aspect of the present invention, the radiopharmaceutical composition comprises99mTc complex of tetrofosmin; tetrofosmin or its pharmaceutically acceptable salts thereof selected from sulfosalicylate, disulfosalcylate, hydrobromide, hydrochloride; a radio protectant selected from group consisting of gentisic acid, maleic acid, para amino benzoic acid, anthranilic acid or their pharmaceutically acceptable salts thereof and combinations thereof; wherein the composition is free of ascorbate or ascorbic acid; a transchelator selected from gluconic acid, acetic acid, citric acid, tartaric acid, glucoheptonic acid, benzoic acid, phenol or phosphonic acid or their pharmaceutically acceptable salts thereof; wherein the composition has a radiochemical purity of at least 90% after 12 hours of storage at temperature 2-30° C. In yet another aspect of the present invention provides a process for the preparation of multiple unit patient doses of radiopharmaceutical composition of tetrofosmin comprising: reconstituting the lyophilized composition with either a sterile solution of99mTc-pertechnetate or first a biocompatible carrier followed by a sterile solution of99mTc-pertechnetate to obtain desired99mTc-tetrofosmin radiopharmaceutical to be withdrawn into a clinical grade container to obtain desired doses when required. In an another aspect of the present invention, provides use of a stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salts and one or more pharmaceutically acceptable excipients in the scintigraphic delineation of regions of reversible myocardial ischemia in the presence or absence of infarcted myocardium and for the evaluation of ventricular function. In an embodiment a stable non-radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salts thereof and gentisic acid as radioprotectant characterized in that said composition retains at least 95% w/w of total tetrofosmin after storage at temperature 2-30° C. for at least three months, preferably five months, more preferably six months. In an embodiment a radiopharmaceutical composition comprises (i) a99mTc complex of tetrofosmin; (ii) tetrofosmin disulfosalicylate (iii) gentisic acid as radioprotectant; wherein the molar ratio of tetrofosmin to gentisic acid is in the range 0.01:1.0 to 1.0:1.0. In an embodiment a stable non-radiopharmaceutical composition comprising tetrofosmin, gentisic acid, wherein the molar ratio of tetrofosmin to gentisic acid is in the range 0.01:1.0 to 1.0:1.0 characterized in that said composition retains at least 95% w/w of total tetrofosmin after storage at temperature 2-30° C. for at least six months. In an another aspect, the present invention provides a tetrofosmin composition after reconstitution with99mTc-pertechnetate which has a radiochemical purity of at least 90% after 12 hours of storage at temperature 2-30° C. In an another aspect, the present invention provides a tetrofosmin composition after reconstitution with99mTc-pertechnetate which has a radiochemical purity of at least 90% after 12 hours after storage of composition at temperature 2-30° C. for six months. In an embodiment a stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salts thereof, gentisic acid, wherein the composition is stable at room temperature for at least six months. In an embodiment a stable lyophilized tetrofosmin composition comprises tetrofosmin or its pharmaceutically acceptable salts thereof having moisture content of less than 10%. In accordance with still another embodiment of the present invention, there is provided a suitable clinical grade container or vial or pre-filled syringes or ampoules that are suitable for safe administration to patients. In another embodiment of the invention, the stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salt thereof, wherein the composition comprises one or more reducing agent. Reducing agents like stannous are susceptible to oxidation or hydrolysis hence, the composition comprising such agents also become susceptible to oxidation. In one embodiment of the present invention the compositions are prepared in controlled environment using inert gas. The final lyophilized compositions are prepared in such a controlled atmosphere that lyophilized vial comprises less than 5 percent of oxygen content in vial head space. Maintaining an appropriate level of stannous in the composition is important for reducing technetium to lower oxidation state which is required for complexation with tetrofosmin. In another embodiment, the present invention provides a stable pharmaceutical composition comprising less than 5% of the oxygen content in the vial head space. The headspace gas above the composition in the vial is suitably an inert gas. By the term “inert gas” is meant a gas which would be used to provide an “inert atmosphere’ to the composition. Such a gas does not undergo chemical reactions with organic compounds and is hence compatible with a wide range of synthetic compounds even on prolonged storage over many hours or even weeks in contact with the gas. Suitable gases include but not limited to helium, neon, argon, nitrogen, carbon dioxide or combinations thereof. Each vial of composition includes tetrofosmin or its pharmaceutically acceptable salts from about 0.1 mg to about 50 mg. In yet another embodiment, the present invention relates to stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salt, wherein the amount of reducing agent in the radiopharmaceutical composition is in a molar excess to a99mTc-compound. Preferably, the reductant used in the present invention is between about 0.01% to about 15% (w/w). Another embodiment of the present invention encompasses a stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salt and a pharmaceutically acceptable transchelator. Combination of transchelators performing the same function may also be used to achieve desired formulation characteristics. In another embodiment, the present invention includes a stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salt, wherein the composition comprises one or more pharmaceutically acceptable pH-adjusting agents such as tricine, phosphate or TRIS [tris(hydroxymethyl)aminomethane]; pharmaceutically acceptable bases such as sodium bicarbonate, sodium carbonate, or mixtures thereof. In another embodiment, the present invention includes a stable non-radioactive composition comprising tetrofosmin or its pharmaceutically acceptable salt, wherein the pH ranges from about 4.0 to 10.0. The stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salt, is provided in either a suitable clinical grade container like vial or pre-filled syringes that are suitable for safe administration in patients. The pre-filled syringe may optionally be provided with a syringe shield to protect the operator from radioactive dose. The stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salt, is compatible with clinical grade container closure system. The present invention provides non-radioactive kits for the preparation of the stable99mTc radiopharmaceutical composition. Such kits comprise conventional non-radioactive freeze-dried vials containing the necessary reactants and are intended to be reconstituted with99mTc-pertechnetate (TcO4−) from a supply of99mTc to give the desired sterile99mTc radiopharmaceutical composition. The compositions according to the present invention comprises a radioactivity of 1 mCi to 2400 mCi. In a separate embodiment, the present invention relates to non-radioactive kits for the preparation of the stable99mTc radiopharmaceutical composition, wherein the kits comprise: (i) non-radioactive freeze-dried vial containing tetrofosmin, reductant and transchelator; buffering agent, radio protectant; (ii) vial containing99mTc-pertechnetate (TcO4—) from a supply of99mTc. In yet another independent embodiment, the present invention relates to the use of stable radiopharmaceutical composition comprising tetrofosmin or its pharmaceutically acceptable salt in the scintigraphic delineation of regions of reversible myocardial ischemia in the presence or absence of infarcted myocardium and for the evaluation of ventricular function. The composition as per present invention can be used for diagnosis of other organs also. Having described the invention with reference to certain preferred embodiments, other embodiments will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following examples describing in detail method for the preparation of radiopharmaceutical compositions comprising tetrofosmin. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention. Following examples are set out to illustrate the invention and do not limit the scope of the invention. EXAMPLES The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature Example 1: Lyophilized Composition Per Vial Comprising Tetrofosmin is Given in Table 1 TABLE 1Amount (% w/w)ExampleExampleExampleExampleIngredientsABCDTetrofosmin13.00-15.0013.14-14.5314.7214.19DisulfosalicylateStannous0.37-0.50.37-0.460.440.43ChlorideDihydrateSodium D-12.00-16.0012.65-15.4714.9714.43GluconateSodium50.00-62.0050.59-61.8454.8952.91BicarbonateGentisic Acid*15.00-17.0013.54-15.0014.9718.03*Para-amino benzoic acid, Maleic acid, Anthranilic acid can also be used Procedure: Approximately 90% of the total volume of Water for Injection (WFI) was added to a preparation vessel. The WFI was deoxygenated. Tetrofosmin, reducing agent selected from stannous chloride dihydrate, transchelator selected from Sodium D-gluconate, radioprotectant selected from Gentisic acid, para-amino benzoic acid, maleic acid, anthranilic acid and combinations thereof and sodium hydrogen carbonate were dissolved. The solution obtained was deoxygenated and final volume and the bulk solution was deoxygenated. The solution was sterile filtered. The vials were partially stoppered and then lyophilized. Example 2: Radiolabelling Procedure Test vial was placed in a suitable lead pot and stored under a mild atmosphere of nitrogen. A sterile venting needle was inserted through the rubber septum. Up to 2.4 Ci of technetium99mTc generator eluate diluted with about 15.0 mL sodium chloride injection was injected into the vial. Before removing the syringe from the vial, 2 mL of gas was withdrawn from above the solution and then the venting needle was removed. The contents of the vial were mixed gently to ensure complete solubilization of the powder and incubated at room temperature for 15 minutes. RCP was checked at 30 minutes and 12 hrs. The reconstituted product was stored at temperature 2-8° C. for 12 hrs. Example 3: Radiochemical Purity Analysis by Ascending Paper Chromatography RCP of the reconstituted test formulations and the marketed formulation (Myoview® 30 mL) were measured using two chromatographic systems System 1: Stationary phase: ITLC-SA (1×10 cm strip); Mobile phase: acetone and dichloromethane (65:35))] System 2: Stationary phase: ITLC-SG (2×20 cm strip); Mobile phase: acetone and dichloromethane (65:35)) Method: To determine the amount of free technetium, a 10 to 20 μL volume of injection was applied about 1.0 cm from the bottom of a non-heated 1×10-cm glass fiber paper impregnated with silicic acid strip (ITLC-SA/ITLC-SG). The chromatogram was immediately developed by ascending chromatography using a solvent system consisting of a mixture of acetone and dichloromethane (65:35). The chromatogram was allowed to air-dry. The radioactivity distribution of the chromatogram was determined by scanning with Bioscan. The relative front (RF) value, which is defined as the ratio of component position to the total distance travelled by the solvent front, of the technetium99mTc tetrofosmin spot was found approximately at 0.5. The RCP based on the % ROI (region of interest) was recorded on the Bioscan report. The results of test formulation Example A and B are illustrated in Table 2: TABLE 2Radiochemical Purity of99mTcTestTetrofosmin (%)FormulationActivity added30 minutes12 hourExample A1.0 Ci97.4594.63257 mCi98.4998.43Example B2.21 Ci10095.42.01 Ci99.6495.35Myoview ®1.86 Ci97.1292.96(30 mL) Radiochemical purity of test composition of Example C was compared with commercially available Myoview (30 mL) stored at temperature 2-8° C. for six months. The results are illustrated in Table 3. TABLE 3Stored at 2-8° C.Time postRadio chemicalCompositionlabellingpurity (%)Myoview1 hour90.312 hour87.2Example C1 hour99.0(6 Month)12 hour95.5 It is apparent from the above results that compositions according to the present invention provide superior radiochemical purity in comparison to the commercially available Myoview® (30 mL) compositions. Example 5: Animal Bio-Distribution Data Bio-distribution was determined after technetium-99m labeling of composition of Example A. Aliquots of solution were intravenously injected into tail vein of rats. % Injected dose (ID)/organ were determined at 10 minutes, 1 hour, 2 hours and 4 hours post injection. Table 4 shows the comparison of Bio-distribution (10 minutes post injection) in animals between test formulation (Example A) and commercial formulation (Myoview®). Table 5 shows the comparison of Bio-distribution (1 hour post injection) in animals between test formulation (Example A) and commercial formulation. Table 6 shows the comparison of Bio-distribution (2 hour post injection) in animals between test formulation (Example A) and commercial formulation. Table 7 shows the comparison of Bio-distribution (4 hour post injection) in animals between test formulation (Example A) and commercial formulation. TABLE 4Test Formulation and Myoview ® Bio-distribution(10 minutes post injection)% ID/organOrganExample AMyoview ®Blood0.780.76Liver7.865.42Kidneys7.635.83Stomach1.641.39Intestines18.8519.83Muscle0.330.28Spleen0.990.95Lung1.091.27Heart1.841.54 TABLE 5Test Formulation and Myoview ® Bio-distribution(1 hour post injection)% ID/organOrganExample AMyoview ®Blood0.340.32Liver2.691.95Kidneys3.532.45Stomach1.140.94Intestines33.6126.3Muscle0.450.36Spleen0.530.37Lung0.810.91Heart1.941.49 TABLE 6Test Formulation and Myoview ® Bio-distribution(2 hours post injection)% ID/organOrganExample AMyoview ®Blood0.20.21Liver1.441.21Kidneys2.351.65Stomach0.920.79Intestines35.2927.01Muscle0.330.31Spleen0.280.17Lung0.50.75Heart1.531.06 TABLE 7Test Formulation and Myoview ® Bio-distribution(4 hours post injection)% ID/organOrganExample AMyoview ®Blood0.150.16Liver0.710.97Kidneys1.061.17Stomach0.530.63Intestines33.739.81Muscle0.320.23Spleen0.110.1Lung0.340.64Heart1.291.41 It is apparent from the above results that test formulations according to the present invention provide comparable bio-distribution with respect to the commercially available Myoview® (30 mL) compositions. Example 6 Lyophilized test formulation prepared in Example C was subjected to stability testing at temperature of 5°±3° C. for 6 months and content of tetrofosmin and gentisic acid was analyzed by High Performance Liquid Chromatography (HPLC) method, whereas content of stannous chloride dihydrate was measured by voltammeter, headspace oxygen content by Gas Chromatography and water content was analyzed by Thermo Gravimetric Analysis (TGA). The prepared dosage form was found to be stable and exhibited following values (refer Table 8): TABLE 8Stored at 5° C. ± 3° C.TestAcceptableInitial1 Month3 Months6 MonthsParameterslimits(%)(%)(%)(%)Assay of90%-110%100.199.1103.5101.9tetrofosminContent ofNLT 32%92.191.489.489StannousChlorideDihydrateHeadspaceNMT 2%0.21.00.60.6Oxygen (%) Example 7 Lyophilized formulation prepared in Example D was subjected to stability testing at temperature of 5°±3° C. for 3 months (refer Table 9) and 25° C./60% RH for 1 month (refer Table 10). The content of tetrofosmin and gentisic acid was analyzed by High Performance Liquid Chromatography (HPLC) method, whereas content of stannous chloride dihydrate was measured by voltammeter, headspace oxygen content by GC and water content was analyzed by TGA. The prepared dosage form was found to be stable and exhibited following values. The prepared dosage form was found to be stable and exhibited following values (refer Table 9 and 10): TABLE 9TestStored at 5° C. ± 3° C.ParametersInitial (%)1 Month (%)3 Months (%)Assay of105.4100.9102.2tetrofosminContent of84.384.983.7StannousChlorideDihydrateContent of100.8100.599.9Gentisic AcidHeadspace0.540.620.66Oxygen (%)Water content6.24.55.9 TABLE 10Stored at 25 ° C./60% RHTest ParametersInitial (%)1 Month (%)Assay of tetrofosmin105.4101.4Content of Stannous84.383.0Chloride DihydrateContent of Gentisic100.8100.4AcidHeadspace Oxygen (%)0.540.59Water content6.24.9 Example 8 Lyophilized formulation prepared in Example B was subjected to stability testing at 25° C./60% RH for 6 months and content of tetrofosmin was analyzed by High Performance Liquid Chromatography (HPLC) method, whereas content of stannous chloride dihydrate was measured by voltammeter. The prepared dosage form was found to be stable and exhibited following assay values (refer Table 11): TABLE 11Stored at 25 ° C./60% RHTestAcceptableParameterslimitsInitial6 MonthsAssay of90%-110%101.7%100.3%tetrofosminContent ofNLT 32%83.6%81.6%StannousChlorideDihydrate While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those skilled in the art without departing from the scope, and spirit of this invention. | 29,358 |
11857648 | 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compounds, kits, and methods for targeted molecular imaging and/or therapy, wherein targeting molecules interact with corresponding biomarkers on a biological subject. In certain non-limiting embodiments, the biological subject is a normal or diseased or degenerated cell, tissue, or other structure of interest. In certain non-limiting embodiments, the biological subject is a tumor or cancer cell. The present invention further relates to compounds/compositions/molecules/chelators and kits for targeted molecular imaging and/or therapy. The present invention also relates to in vitro high-throughput screening methods for identifying the appropriate spacer length for multivalent targeted molecular imaging and/or therapy compounds. In certain non-limiting embodiments, the disclosed targeted molecular imaging and/or targeted drug delivery methods allow for prolonged retention of the fast-clearing detectable label and/or active agent, which consequently increases cellular uptake significantly. In certain non-limiting embodiments, the disclosed methods provide increased sensitivity and/or increased specificity. For example, the disclosed methods can convert low-affinity monovalent targeting molecule (˜μM) into one with high avidity (˜nM). In certain non-limiting embodiments, the disclosed targeted molecular imaging and/or targeted drug delivery methods can be broadly applied to various dual/multi-biomarker combinations and targets (e.g., tumors or cancer). In certain non-limiting embodiments, the present invention provides a targeted molecular imaging and/or targeted drug delivery compound that binds more tightly to a cell of interest (e.g., a tumor or cancer cell) which can result in less non-specific binding and less false positive results. A tighter binding targeted molecular imaging and/or targeted drug delivery compound can result in smaller amounts of compound dissociating from the biological subject of interest. In certain non-limiting embodiments, the tighter binding compound increases cellular update and/or decreases uptake by non-targeted cells. By way of example, but not limitation, in the case of tumor cells, achieving high avidity can significantly enhance binding affinity on a tumor that overexpresses two targeted biomarkers simultaneously, but not increase the binding affinity on non-tumor tissues that express only one (or none) of the two targeted biomarkers, thus tumor/non-tumor ratio will increase significantly. In certain non-limiting embodiments, by increasing the local concentration of the targeting molecules due to increased total binding sites and by increasing the circulation time of the targeting molecules due to improved pharmacokinetic (such as clearance properties and excretion rates), the methods of the present invention provide a higher potential for clinical translation as the targeting ligand/molecule incorporated radioactive/drug molecule can accumulate at the site of interest, thereby increasing the uptake. For example, the density of targeted receptors can be increased by targeting an appropriate combination of complementary cell-surface receptors. In certain non-limiting embodiments, due to the change in size and lipophilicity, the multimers can also have improved pharmacokinetic performance. In certain non-limiting embodiments, the invention provides a chelator for combining a targeting molecule to at least one of the same targeting molecule, at least one different targeting molecule, and/or a dye molecule. In certain non-limiting embodiments, the chelator is able to couple at least two targeting molecules. In certain non-limiting embodiments, the chelator is able to take part in solid phase peptide synthesis. In certain non-limiting embodiments, the chelator can simplify the process of developing targeted monomer, homodimers, heterodimers, and multimodalities as diagnostic tracers and/or radiotherapy agents. In certain non-limiting embodiments, the invention provides an in vitro high-throughput screening platform for optimizing the length of spacers between the targeting molecules of the multimer. In certain non-limiting embodiments, the in vitro high-throughput screening platform is a sensitive assay which can utilize targeting molecules in the nM range for each test. In certain non-limiting embodiments, the method combines click chemistry and radio chemistry to optimize the spacer length. In certain non-limiting embodiments, cells can be used as a screening platform via on-site (i.e., in vitro) formation of multimers (e.g., heterodimers). In certain non-limiting embodiments, the targeting molecules of the multimer can be functionalized separately with a reactive group (e.g. clickable group) and a photolabile group (e.g., clickable groups). The term “biomarker”, as used herein, refers to a marker (e.g., including but not limited to proteins (including monomeric and multimeric proteins, glycoproteins, lipoproteins, etc.), carbohydrates, lipids, nucleic acids and combinations thereof) that allows detection of a disease or disorder in an individual, including detection of disease or disorder in its early stages. Diseases or disorders include but are not limited to disorders of proliferation, including but not limited to cancer autoimmune conditions, degenerative conditions, vascular disorders, neurological disorders, and infectious diseases; biomarkers associated with numerous diseases and disorders in human and nonhuman animals are known in the art. In certain non-limiting embodiments, the presence or absence of a biomarker is determined by imaging. In certain non-limiting embodiments, the presence or absence of a biomarker in a biological sample of a subject is compared to a reference control. The term “active agent” refers to an agent that is capable of having a physiological effect when administered to a subject. In certain non-limiting embodiments, the term “active agent” refers to a protein, peptide, small molecule, or radiopharmaceutical. In certain non-limiting embodiments, the active agent is a chemotherapeutic agent. In certain non-limiting embodiments, the active agent is an immunotherapeutic agent. The term “therapeutically effective amount”, as used herein, refers to that amount of active agent sufficient to treat, prevent, or manage a disease. Further, a therapeutically effective amount with respect to the second targeting probe of the disclosure can mean the amount of active agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease, which can include a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The term can encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapeutic agent. The term “biological subject”, as used herein, refers to, but is not limited to, a protein, virus, cell, tissue, organ or organism. In certain non-limiting embodiments, the biological subject can be a normal or diseased or degenerated or infected cell, tissue, or organ. In certain non-limiting embodiments, the cell can be a tumor or cancer cell. The term “functionalized”, as used herein, refers to a modification of an existing molecular segment to introduce a new functional group that is capable of undergoing a reaction with another functional group (e.g., an azide). Ranges disclosed herein, for example “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y. For clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections:(i) Targeted molecular imaging and/or drug delivery compounds and methods of use;(ii) Chelators and methods for making the targeted molecular imaging and/or drug delivery compounds;(iii) High-throughput screening platform for optimizing the length of spacers between the targeting molecules of the multimer;(iv) Biomarkers; and(v) Kits. 5.1. Targeted Molecular Imaging and/or Drug Delivery Compounds and Methods of Use The present invention provides targeted molecular imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the invention provides two components or targeting molecules that each interacts with at least one biomarker (e.g., on a cell). 5.1.1. Targeting Molecules The present invention provides for a targeted molecular imaging and/or targeted drug delivery compound having at least one first targeting molecule. In certain non-limiting embodiments, the invention provides for a targeted molecular imaging and/or targeted drug delivery compound having at least one first targeting molecule and at least one second targeting molecule. In certain non-limiting embodiment, the targeted molecular imaging and/or targeted drug delivery compound can have at least one, at least two, at least three, at least four, or at least five different targeting molecules directed to the same or different biomarkers. In certain non-limiting embodiment, the targeted molecular imaging and/or targeted drug delivery compound can have at one, two, three, four, five, or more targeting molecules. In certain non-limiting embodiment, the targeted molecular imaging and/or targeted drug delivery compound can have more than one of each targeting molecule. In certain non-limiting embodiments, the targeting molecule can be an antibody, protein, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide that binds to the biomarker. In certain non-limiting embodiments, the targeting molecule is the active agent. In certain non-limiting embodiments, the targeting molecule can be internalizable or non-internalizable. In certain non-limiting embodiments, the targeting molecule can be a protein. In certain non-limiting embodiments, the first targeting probe is an antibody. The term “antibody” as used herein, includes, but is not limited to antibodies, antibody derivatives, organic compounds derived there from, monoclonal antibodies, antibody fragments, modified antibodies, single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or di-, oligo- or multimers thereof. In certain non-limiting embodiments, modified antibodies includes synthetic antibodies, chimeric or humanized antibodies, or mixtures thereof, or antibody fragments which partially or completely lack the constant region, e.g., Fv, Fab, Fab′ or F(ab)′2 etc. In certain non-limiting embodiments, the antibody is a monoclonal antibody. In certain non-limiting embodiments, the targeting molecule is commercially available. In certain non-limiting embodiments, the targeting molecule can be made against a specific biomarker by any technique understood by those of skill in the art. In certain non-limiting embodiments, the targeted molecular imaging compound comprises one or two detectable labels. In certain non-limiting embodiments, the detectable label is an imaging label, and/or therapeutic probe. In certain non-limiting embodiments, the imaging label can be, but is not limited to,110In,1111n,177Lu,52Fe,62Cu, 64Cu,32P, 11C,13N, 150,67Cu,67Ga,68Ga,86Y,90Y,18F,89Zr,94mTc,94Tc,99mTc,120I,123I,124I,125I,131I,154-158Gd,186Re,188Re,51Mn,52mMn,55Co,72As,75Br,76Br,82mRb,83Sr, and other gamma-, beta- or positron-emitters. In certain non-limiting embodiments, the therapeutic probe is therapeutic radioisotope, such as but not limited to67Cu,177Lu,90Y,131I,212Bi,211At or225Ac. In certain non-limiting embodiments, the therapeutic probe is an anticancer drug, such as, doxorubicin, paclitaxel, fluorouracil, etc. In certain non-limiting embodiments, the targeted drug delivery compound comprises one or two active agents. In certain non-limiting embodiments, the active agent can be, but is not limited to, a protein, peptide, small molecule, peptide nucleic acid (PNA), or radiopharmaceutical. In certain non-limiting embodiments, the detectable label is a dye molecule. In certain non-limiting embodiments, the compound can have more than one dye molecule. In certain non-limiting embodiments, the dye molecule is attached to one type of targeting molecule (one or more of the one type). In certain non-limiting embodiments, the dye molecule is attached to at least two types of targeting molecules (at least one of each). In certain non-limiting embodiments, the dye molecule can be, but is not limited to, cyanine dyes (Cy3, Cy3.5, Cy5, Cy7, Cy5.5, Cy7.5), GFP, Calcein, FITC, FluorX, Alexa dyes, Rhodamine dyes, 5-FAM, Oregon Green, Texas Red. In certain non-limiting embodiments, the active agent can be, but is not limited to, trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib, dacomitinib, KD-019 erlotinib, cisplatin, carboplatin, gemcitabine, pemetrexed, irinotecan, 5-fluoruracil, paclitaxel, docetaxel, or capecitabine. In certain non-limiting embodiments, the radiopharmaceutical can be111In-ibritumomab tiuxetan,90Y-ibritumomab tiuxetan,131I-tositumomab,131I-labetuzumab,131I-rituximab,212Pb-trastuzumab,131-trastuzumab,111In-trastuzumab,188Re-trastuzumab. Table 1 below provides non-limiting examples of targeting molecules (i.e., the first targeting molecule and/or the second targeting molecule) that bind specific biomarkers. TABLE 1Examples of targeting moleculesTargeting Moleculepeptide (or smallBiomarkersantibodymolecule) ligandCD13N/ANGR (peptide)Integrin α4β1N/ALLP2A (peptide)uPARN/AAE105 (peptide)AE105mut (peptide)gastrin-releasingN/ABBN(7-14) (peptide)peptide (GRP)SSTR2N/ATyr(3)-octreotate(peptide)CCR5N/ADAPTA (peptide)Integrin αvβ3EtaracizumabRGD (peptide)RAD (peptide)EGFRCetuximabErlotinib(small molecule)VEGFBevacizumabN/ACA19-91116NS19-9, Human 5B1N/ACD40CP-870,893N/APD-L1AtezolizumabN/A The structure of LLP2A (CAS number 874148-50-2) is In certain non-limiting embodiments, the targeted molecular imaging and/or targeted drug delivery compounds comprise at least one first targeting molecule, at least one second targeting molecule, and a detectable label and/or active agent. In certain non-limiting embodiments, the targeted molecular imaging and/or targeted drug delivery compounds comprise a first targeting molecule, a second targeting molecule, and a detectable label and/or active agent. In certain non-limiting embodiments, the targeted molecular imaging compounds comprise a first targeting molecule, a second targeting molecule, a detectable label, and optionally an active agent. In certain non-limiting embodiments, first targeting molecule and second targeting molecule can be a protein. In certain non-limiting embodiments, the detectable label can be an imaging label. In certain non-limiting embodiments, the imaging label can be64Cu,68Ga, or18F. In certain non-limiting embodiments, the first targeting molecule can be, but is not limited to, uPAR targeting molecules. In certain non-limiting embodiments, the uPAR targeting molecule can be, but are not limited to uPA, ATF (amino terminal fragment of urokinase), AE105, or AE105mut. In certain non-limiting embodiments, the first targeting molecule can be, but is not limited to, a CD13 targeting molecule. In certain non-limiting embodiments, the CD13 targeting molecule can be, but is not limited to peptides containing the Asn-Gly-Arg (NGR) motif. In certain non-limiting embodiments, the CD13 targeting molecule can be a peptide such as, but is not limited to, cyclo(cNGRc), cyclo(cPNGRc), cyclo(NRGyK), linear cNGRc, or linear cPNGRc. In certain non-limiting embodiments, the second targeting molecule can be, but is not limited to, integrin αvβ3 targeting molecules. In certain non-limiting embodiments, the integrin αvβ3 targeting molecule can be, but is not limited to a protein with an exposed arginine-glycine-aspartic acid (RGD) tripeptide sequence or arginine-alanine-aspartic acid (RAD) sequence. In certain non-limiting embodiments, the integrin αvβ3 targeting molecule can be the peptide such as, but not limited to, cyclco(RGDyK) (RGD) or cyclo(RADyK) (RAD). In certain non-limiting embodiments, the biomarker can be, but is not limited to, CD13 and/or integrin αvβ3 (See e.g.,FIG.1). In certain non-limiting embodiments, the biomarker can be, but is not limited to, uPAR and/or integrin αvβ3. In certain embodiments, the invention provides for the use of the above-described compounds for imaging a cell, tissue, or structure of interest in a subject in need of such treatment, for example a subject having a disease or disorder, at risk of having a disease or disorder, or being screened/tested for a disease of disorder. According to such methods, a subject is administered an effective amount of at least one first targeting molecule and a detectable label. In related embodiments, said subject may be further administered a second targeting molecule, and a chelator compound, as described above. Said method may be used, for example, to diagnose a tumor, an infection, a degenerative condition, etc. in a subject. In certain embodiments, said method may be used to determine the spread of disease, for example, the presence or absence of tumor metastasis or invasion in an organ or structure (e.g., bone). 5.1.2. Active Agent Delivery In certain non-limiting embodiments, a subject is provided a therapeutically effective amount of a targeted drug delivery compound of the invention. In certain embodiments, the invention provides methods of treating a disease such as, but not limited to, cancer, congestive heart failure, diabetes, asthma, emphysema, infarction, ischemia, arteriosclerosis, toxicity, mental disease, depression or arrhythmia. One of skill in the art can select the proper biomarker(s) to target the active agent to the diseased cell. Accordingly, in certain embodiments, the invention provides for the use of the above-described compounds for treating a disease or disorder of a subject or a cell, tissue or structure of interest in the subject comprising administering to the subject, an effective amount of at least one first targeting molecule and a detectable label. In related embodiments, said subject may be further administered a second targeting molecule, and a chelator compound, as described above. In certain non-limiting embodiments, the subject includes any human or nonhuman animal. In certain non-limiting embodiments, the subject is a pediatric patient. In certain non-limiting embodiments, the subject is an adult patient. In certain non-limiting embodiments, nonhuman animal includes, but is not limited to, all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, rodents, rabbits, horses, cows, chickens, amphibians, reptiles, etc. In certain non-limiting embodiments, the targeted molecular imaging compound can be administered by, but not limited to, injection (e.g, intravenous, subcutaneous, intraperitoneally), infusion, inhalation, orally, topically, parenterally, transdermally, rectally or via an implanted reservoir. 5.1.3. Molecular Imaging In certain non-limiting embodiments, after the administration of targeted molecular imaging compound, the subject is imaged. In certain non-limiting embodiments, imaging can be conducted by Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Planar gamma camera, X-ray CT, planar X-ray, Magnetic Resonance Imaging (MRI), optical imager, or other diagnostic imaging technique. In certain non-limiting embodiments, the subject includes any human or nonhuman animal. In certain non-limiting embodiments, the subject is a pediatric patient. In certain non-limiting embodiments, the subject is an adult patient. In certain non-limiting embodiments, nonhuman animal includes, but is not limited to, all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, rodents, rabbits, horses, cows, chickens, amphibians, reptiles, etc. The targeted molecular imaging compound can be administered by the same routes as disclosed for the targeted drug delivery compound. 5.2 Chelators and Methods for Making the Targeted Molecular Imaging and/or Drug Delivery Compounds The present invention provides targeted molecular imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the invention provides two components or targeting molecules that each interacts with at least one biomarker (e.g., on a cell). In certain non-limiting embodiments, a chelator can be used to attach various moieties of the targeted molecular imaging and/or targeted drug delivery compounds. For example, the chelator can attach various targeting molecules together (seeFIG.2by way of example). In certain non-limiting embodiments, the chelator can also attach the detectable label, dye molecule, and/or active agent to at least one targeting molecule. In certain non-limiting embodiments, the chelator can also attach the detectable label, dye molecule, and/or active agent to at least two targeting molecules. In certain non-limiting embodiments, the chelator can be bound to one targeting molecule. In certain non-limiting embodiments, the chelator can be bound to two targeting molecules. In certain non-limiting embodiments, the chelator can be bound to more than one of the same targeting molecules. In certain non-limiting embodiments, the chelator can be bound to more than one type of targeting molecule. In certain non-limiting embodiments, the chelator can be bound to two types of targeting molecule. In certain non-limiting embodiments, the chelator can be bound to one targeting molecule (e.g., either the first or second targeting molecule) for monomers. In certain non-limiting embodiments, the chelator can be bound to two of the same targeting molecules (e.g., either two of the first or two of the second targeting molecule) for homodimers. In certain non-limiting embodiments, the chelator can be bound to two different targeting molecules (e.g., the first and second targeting molecules) for heterodimers. In certain non-limiting embodiments, the chelator can be bound to one targeting molecule (e.g., either the first or second targeting molecule) and one dye molecule for multimodalities. In certain non-limiting embodiments, the chelator can be attached to one or more of the targeting molecules via a spacer. In certain non-limiting examples, the spacer can be a polymer or a biomolecule. In certain non-limiting embodiments, the polymer can be synthetic or natural. In certain non-limiting examples, the polymer can be polyethylene glycol (PEG). For example, the polymer can have a molecular weight of between about 5 and 40 Da, about 40 Da, up to about 100 Da, up to about 200 Da, up to about 300 Da, up to about 400 Da, up to about 1,000 Da, up to about 10,000 Da, up to about 25,000 Da, up to about 30,000 Da, up to about 35,000 Da, or up to about Da 40,000, or for further example, from about 40 Da to about 100,000 Da, from about 40 Da to about 5,000 Da, from about 40 Da to about 10,000 Da, from about 40 Da to about 25,000 Da, from about 1,000 Da to about 25,000 Da, from about 200 Da to about 100,000 Da, from about 10,000 Da to about 100,000 Da, from about 25,000 to about 100,000 Da, or from about 25,000 Da to about 50,000 Da. For further example, the polymer can be polyacrylic acid; hydroxyethyl starch (HES); poly lactide-co-glycolide; poly-D, L-p-dioxanonepoly lacticacid-ethylene glycol block copolymer (PLA-DX-PEG); poly (ortho) esters; poly-glutamate; polyaspartates; a polymer of α-β-unsaturated monomers, such as (meth) acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid or anhydride, etc.; a comonomer comprising vinyl ethers, vinyl esters, vinylamine amides, olefins, diallyl dialkyl ammonium halides, preferably vinyl ether; poly (diethylenglycoladipat); polyethyleneimine; polyglycolide; polyurea; Polylimonen (or Polylimo); poly (2-methyl-1, 3-propylene adipate); a graft polymer; graft (block) polymer with other polymers. In certain non-limiting embodiments, the polymer is linear, branched, or dendrimic. In certain non-limiting embodiments, the polymer is PEG. In certain non-limiting embodiments, the PEG spacer can have a molecular weight of about 44 Da to 20 kDa. In certain non-limiting embodiments, the PEG spacer can comprise non-PEG portions and/or non-PEG monomers. In certain non-limiting embodiments, the spacer can comprise about 2 to about 30 monomers. In certain non-limiting embodiments, the spacer can comprise about 2 to about 20, about 2 to about 10, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, or about 4 to about 5 monomers. In certain non-limiting embodiments, the spacer can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 55, at least 26, at least 27, at least 28, at least 29, or at least 30 monomers. In certain non-limiting embodiments, the spacer can comprise about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 monomers. In certain non-limiting embodiments, the chelator comprises a mutlifunctional chelator having general Formula I: where the chelating core groups is selected from NOTA, NETA, CB-TE2A, CB-TE1A1P, TETA, Pycu2A, DiAmSar, DOTA, DTPA, PCTA, DFO, etc. In certain embodiments, the chelating core is a group that can coordinate certain metal ions and form a stable chelate. The chelating core is the key group for complexing radiometal. In certain non-limiting embodiments, the chelator combines a carboxylic acid or active ester group for amide bond connection, and an azide group suitable for azide-alkyne based click chemistry, in addition to a chelating core that can coordinate with a radioistope, such as64Cu,68Ga, Al18F, etc. In certain non-limiting embodiments, the chelator comprises a 1, 4, 7-triazacyclonenonane (TACN)-based chelator. In certain non-limiting embodiments, the chelator comprises a 1,4,7,10-tetraazacyclododecane(cyclen)-based chelator. In certain non-limiting embodiments, the chelator comprises a 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-based chelator. In certain non-limiting embodiments, the chelator comprises NOTA, DOTA, L-NETA, N3—NOtB2or N3-DOtB3. Examples 1 and 2 provide sample synthetic schemes in accordance with the present invention. In addition to the metal chelating core, the disclosed chelator can contain two bioorthogonal functional groups: carboxylic acid group and azide group for attaching the first targeting molecule via assisted amide formation and the second targeting molecule (or, detectable label, or active agent) via click chemistry, respectively. Compared to other molecule chelators containing one functional group only, these newly developed bifunctional chelators (BFCs) demonstrated several advantages. First, the synthetic strategy can be more straightforward due to the use of a BFC that serves as both a chelator and a spacer; therefore, extensive protection and/or deprotection and/or multifunctional spacer preparation is not required. Secondly, condition optimization is not needed because the reactions (SPPS and click reaction) can easily be completed in nearly quantitative yield, which facilitates the ease of preparations. Maximum utilization of SPPS and click reaction allows for the use of only one chromatography purification step to obtain a pure imaging probe. Finally, this is a universal and robust platform that can be applied to prepare the multivalent and multimodal imaging probes containing any interested ligand(s), dye(s) and other functional moieties, not limited to the ones exemplified here. In certain non-limiting embodiments, the chelator can be modified to be suitable for use for solid phase peptide synthesis (SPPS) by reducing the azide to amide group using commonly-used reduction agents such as PPh3. SPPS is based on the amide forming reaction between carboxylic acid and an amino group. The reduction of an azide provides an amino group for the following amino acid conjugation. Thus, with an amino group, the chelator is compatible to the SPPS system. In certain non-limiting embodiments, automatic peptide synthesis can be used to simplify the synthesis process after on-resin reduction of azide group to amide group. The automatic peptide synthesis works on the same principle as SPPS, which can save time and effort. In certain non-limiting embodiments, the chelator can be synthesized by conjugating an active pendant arm, bearing both an azide group and a acarboxylic acid or ester group, to a chelating core by a nucleophilic substitution reaction. In certain non-limiting embodiments, the detectable labels can be attached by incubating the chelator with radionuclides. In certain non-limiting embodiments, the second detectable labels can be attached by reacting the chelator with dye through click chemistry, esterification reaction, amidation reaction, or another conjugating reaction. In certain non-limiting embodiments, the active agent can be added by reacting the chelator with the active agent through click chemistry, esterification reaction, amidation reaction, or another conjugating reaction. 5.2. High-Throughput Screening Platform for Optimizing the Length of Spacers Between the Targeting Molecules of the Multimer In certain non-limiting embodiments, the invention provides an in vitro high-throughput screening platform for optimizing the length of spacers between the targeting molecules of the imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the in vitro high-throughput screening platform is a sensitive assay that only utilizes targeting molecules in the nM range for each test. Using fewer targeting molecules can reduce the cost of the screening assay. In certain non-limiting embodiments, the invention provides reactions involving only one to two steps.FIG.3is a non-limiting example of an in vitro high-throughput screening assay of the invention. In certain non-limiting embodiments, the method combines click chemistry and radio chemistry to optimize the spacer length. In certain non-limiting embodiments, cells can be used as a screening platform via on-site formation of targeted molecular imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the targeting molecules of the targeted molecular imaging and/or targeted drug delivery compounds can be functionalized separately with a nonactivated photolabile functional group (i.e., photo-triggerable functional group) or a reactive functional group that binds to the photoliable functional group once activated by a photon generating source. In certain non-limiting embodiments, the high-throughput screening platform comprises exposing cells to a first functionalized targeting molecule and a second functionalized targeting molecule, wherein either the first functionalized targeting molecule and/or second functionalized targeting molecule comprises spacers of different lengths between the targeting molecule and the reactive functional group. In certain non-limiting embodiments, either the first functionalized targeting molecule or second functionalized targeting molecule comprises spacers of a set length between the targeting molecule and the reactive functional group. In certain non-limiting embodiments, the cells are exposed to photon energy to activate a nonactivated photolabile functional group, which allows the two targeting molecules to be linked via their respective spacers. In certain non-limiting embodiments, the assay can be quenched with excess radio-metal labeled chelators that are able to bind to the unbound activated photolabile functional group. In certain non-limiting embodiments, the amount of bound radio-metal labeled chelators can be measured. In certain non-limiting embodiments, the decrease in measured radioactivity indicates that the spacer length is appropriate or optimized. In certain non-limiting embodiments, the first functionalized targeting molecule comprises a photolabile functional group. In certain non-limiting embodiments, the photolabile functional group can be, but is not limited to, Photo-OIDBO or Photo-tertrazole. In certain non-limiting embodiments, and the second functionalized targeting molecule comprises a reactive functional group that only binds to the photolabile functional group once the photolabile functional group has been exposed to photon energy. In certain non-limiting embodiments, the reactive functional group of the second functionalized targeting molecule can be, but is not limited to, an azide or an alkene. 5.2.1. Preparation of Multivalent Compounds In certain non-limiting embodiments, the first functionalized targeting molecule is a Nonactivated Photolabile Functional Group-(Monomer) n-Targeting Molecule (exemplified as p-ODIBO inFIG.3) that comprises spacers (e.g., PEG) of various monomer lengths. In certain non-limiting embodiments, the spacer can comprise about 2 to about 30 monomers (as discussed above). For example, but not by way of limitation, n can equal 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 30 monomers. In certain non-limiting embodiments, the Nonactivated Photolabile Functional Group-(Monomer) n-Targeting Molecules can be prepared by first forming NH2-(Monomer) n-Targeting Molecules by adding Boc-(Monomer) n-NHS to the targeting molecule of interest followed by Boc deprotection. For example, the Boc-(Monomer) n-NHS can be combined with the targeting molecule in a suitable buffer (e.g., phosphate buffered saline (PBS)) followed by deprotection with trifluoroacetic acid (TFA) (e.g., 95%). The prepared NH2-(Monomer) n-Targeting Molecule can then be mixed with Nonactivated Photolabile Functional Group-NHS to produce Nonactivated Photolabile Functional Group-(Monomer) n-Targeting Molecules. In certain non-limiting embodiments, the second functionalized targeting molecule is a Reactive Functional Group-Spacer-Targeting Molecule (exemplified as N3inFIG.3), which comprises a spacer with a set monomer length. In certain non-limiting embodiments, the spacer can comprise about 2 to about 30 monomers (as discussed above). For example, but not by way of limitation, the spacer can be 4 or 8 monomers. In certain non-limiting embodiments, the reactive functionalized targeting molecule can be prepared by mixing the targeting molecule and Reactive Functional Group-Spacer-NHS in a suitable buffer (e.g., PBS). In certain non-limiting embodiments, the reactive functionalized targeting molecule comprises spacers of different lengths rather than the photolabile functionalized targeting molecule. As one non-limiting example, it can be more convenient to test the spacer length using a reactive functionalized targeting molecule instead of a photolabile functionalized targeting molecule, as the former can be easier to prepare. By way of example, and not limitation, the photolabile functionalized targeting molecule can be a photo-ODIBO-PEGn-RGD peptide (e.g., n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20). Also by way of example, and not limitation, the reactive functionalized targeting molecule can be N3-PEGn-AE105 and/or N3-PEGn-NGR peptides. 5.2.2. In Vitro High-Throughput Assay In certain non-limiting embodiments, Reactive Functional Group-Spacer-Targeting Molecules can be mixed with one set of Nonactivated Photolabile Functional Group-(Monomer) n-Targeting Molecules with a spacer having a particular monomer length. For example, in such embodiments, there can be one mixture of each spacer length combination. In certain non-limiting embodiments, Nonactivated Photolabile Functional Group-Spacer-Targeting Molecules can be mixed with one set of Reactive Functional Group-(Monomer) n-Targeting Molecules with a spacer having a particular monomer length. In certain non-limiting embodiments, the reactive functionalized targeting molecules and nonactivated photolabile functionalized targeting molecules can be mixed in about a 1:1 molar ratio to prepare mixed-targeting molecule stock solutions. This ratio can be adjusted depending on the densities of the two targeted receptors. In certain non-limiting embodiments, each reaction mixture comprises functionalized targeting molecules each with one specific spacer length. In certain non-limiting embodiments, one of the mixed-targeting molecule stock solutions can be added to cells comprising the biomarkers of interest. It is desirable to have a large excess of targeting molecules present. In certain non-limiting embodiments, after the targeting molecules bind to the targeted biomarker, the unbound targeting molecules will be washed off (e.g., using a suitable buffer). In certain non-limiting embodiments, the cells are exposed to photon energy (including but not limited to laser and/or other light sources) for between, for example, about 1 min and 1 hour (inclusive), i.e., for a period of time effective to convert the nonactivated photolabile functional group on the functionalized targeting molecule to the activated photolabile functional group. In certain non-limiting embodiments, radiolabeled reactive functional groups that bind to the activated photolabile functional group are added to the cells. In certain non-limiting embodiments, the cells are incubated 2-4 hours before adding the radiolabeled reactive functional group. In certain embodiments, the radiolabeled functional group can be a N3-Radioactive Element-Chelator (e.g., N3-(64Cu)NOTA), and can, in non-limiting embodiments, be added 2-4 hours after the photo irradiation to allow sufficient time for the click reaction between two different targeting molecules. The purpose of adding this N3-Radioactive Element-Chelator is to detect the amount of non-reacted photolabile functional group for measuring the extent of the click reaction between the two different targeting molecules. In certain non-limiting embodiments, the radiolabeled reactive functional groups bind to the “excess” activated photolabile group that is bound to the biomarker but did not bind to the reactive group of a functionalized targeting molecule. In certain non-limiting embodiments, the cells are washed with an appropriate buffer to remove excess radiolabeled reactive groups before detecting the level of radioactivity by methods known to those of skill in the art. In certain non-limiting embodiments, the combination of functionalized targeting molecules with the lowest radio-counts, containing the lowest “excess” radiolabeled reactive groups, indicates that the corresponding spacers are of an appropriate or optimal length. By way of example, and not limitation, the Reactive Functional Group-Spacer-Targeting Molecule (e.g., N3-PEG4-AE105) can be mixed with one or more (e.g., about ten) Nonactivated Photolabile Functional Group-(Monomer) n-Targeting Molecules (e.g., photo-ODIBO-PEGn-RGD peptides; n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20) in a 1:1 molar ratio to prepare one or more (e.g., about ten) mixed-targeting molecules stock solutions. Each of the mixed-targeting molecules stock solutions can be added to separate cell culture wells pre-seeded with cells and the cells can be incubated with the mixed-targeting molecules (e.g., until binding equilibrium is achieved). In certain embodiments, the cells are pre-seeded in 24, 48, 96, 384, or 1536 well plates. Following incubation with the mixed targeting molecules, the cells can be washed with a suitable buffer (e.g., PBS) to remove unbound targeting molecules. The cells can then be exposed to photon energy (e.g., a UV lamp (365 nm)) to activate the photolabile functional group (e.g., to generate azide-active “ODIBO”), subsequently triggering ligation between the reactive group and the activated photolabile group (e.g., N3-PEGn-AE105 and ODIBO-PEGn-RGD) bound to the biomarkers on the cells. Following incubation to allow the two targeting molecules to bind (e.g., 2-4 hours), radiolabelled reactive groups (e.g., N3-(64Cu)NOTA) can be added to the cells, which will bind to the activated photolabile groups not bound to the reactive groups of the functionalized targeting molecules. The unbound radiolabelled reactive group can be washed away, and the plate of cells can be processed to be read with a plate reader (e.g., a high-throughput MicroBeta2 Plate Counter) to measure the radiolabelled reactive groups. 5.2.3. Cell Lines In certain embodiments, this method can be applied using various cell cultures, including but not limited to, primary cell cultures, tissue explants, or transformed cell cultures known in the art. Non-limiting examples of such cell cultures include: Primary-hBM SC; Primary-hSkin FB; Primary-cow CC; Primary-rat BMSC; Primary-h CC; MC3T3-E1; Primary-hUVEC; Primary-rabbit CC; NIH 3T3; Primary-CC; Primary-rat Liver Hep; Primary-hSkin Keratinocyte; MG63; HEP-G2; L929; Primary-BM SC; Primary-rabbit BM SC; Primary-pig CC; Primary-hBone OB; MCF-7; Primary-rat Heart CM; Primary-h Foreskin FB; Primary-hAdipose SC; Primary-hFB; Primary-hAdipose SC; Primary-FB; Primary-ratAortaSMC; Primary-Bone; Primary-dog CC; 3T3 (nonspecific); C2C12; MDA-MB-231; SaOS-2; Primary-mouse BM SC; Primary-rat CC; Primary-h Mesoderm Mes Pre C; Primary-rat Brain Neuronal; PC12; Primary-Cancerous; Primary-h Skin EC; Primary-rat BM OB; Primary-mouse Embryo SC; MCF-10A; Primary-h Bone OB-like; Primary-goat BMSC; Primary-h Aorta SMC; MDCK (Madin-Darby Canine Kidney); Primary-hi DAnnulus C; Primary-ratBone OB; Primary-h Adipose Preadipocyte; Primary-SC; Primary-rat Skeletal Muscle Myoblast; Primary-Heart CM; Primary-cow AortaEC; Primary-dog BM SC; Primary-sheep BM SC; Primary-sheep CC; Primary-pig BMSC; Primary-cow BMSC; Primary-h BladderSMC; Primary-pig Aorta EC; Primary-h Cornea Epi C; Primary-h Aorta EC; Primary-h Cornea FB; Primary-pig Aorta SMC; Primary-mouse Liver Hep; A549; Primary-Bone OB; Primary-h Bladder Uro; Primary-h UV SMC; Swiss 3T3; Primary-Liver Hep; Primary-h Lig FB; Primary-h Coronary Artery SMC; Primary-OB-like; Primary-h Teeth Mes Pre C; HT1080; Primary-rat Heart FB; Primary-pig HV Intersticial C; C3A; Primary-h Breast Cancerous; Primary-h Foreskin Keratinocyte; Primary-h Oral Mucosa Keratinocyte; Primary-mouse Ovary Oocytes; Primary-h Vase SMC; 3T3-L1; Primary-h Lung FB; Primary-chicken Ganglia Neuronal; Primary-h U CStC; Primary-cow Aorta SMC; Primary-mouse Embryo FB; Primary-h Bronchi EpiC; CHO-K1; Primary-h Liver Hep; Primary-hSaphVEC; Primary-hTeethPDL; Primary-rat Skin FB; Primary-pig Liver Hep; PC-3; Primary-SMC; Primary-hMVEC; Primary-mouseFB; Primary-h Nasal Chondrocyte; Primary-hCorneaKeratinocyte; Primary-hOvaryCancerous; Primary-h U CBSC; Primary-rat Heart EC; Primary-Vasc; Primary-mouse Skin FB; Primary-h Tendon TC; Primary-rat Brain Astrocyte; Primary-rat Nerve SC; Ha CaT; Primary-h Gingiva FB; Primary-Neural; Primary-cow Bone OB; Primary-rat Adipose SC; Primary-mouse Bone OB; Primary-h Teeth PC; Primary-h Blood Mononuclear; Primary-rat Hippocampus Neuronal; D3; HeLa; HEK293; C17.2; Primary-h Skin Melanocyte; Primary-h Blood EC-like; HOSTE85; Primary-h UC SC-like; Primary-h Cornea SC; Primary-rat Aorta EC; Primary-h Saph VSMC; Primary-h UCBEC; Primary-mouse Heart CM; D10RL UVA; Primary-h Coronary Artery EC; Primary-h Aorta Myo FB; HT-29; Primary-h Tendon FB; RAW 264; Primary-rat Dental Pulp SC; 3T3-J2; H1; Primary-pig Teeth; Primary-rat Sciatic Schwann; Primary-rabbit Bone OB-like; Primary-sheep Aorta EC; Primary-rabbit Cornea Epi C; Primary-h Ovary Epi C; Primary-rabbit Ear Chondrocyte; SH-SY5Y; Primary-h Teeth FB; Primary-h Oral Mucosa FB; Primary-rabbit FB; C6; Primary-rat Testes Stertoli; Primary-cow Arterial EC; Primary-pigHVEC; Primary-cow Nucleus Pulposus Cells; Primary-rat Ganglia Neuronal; Primary-dog Bladder SMC; Primary-Vasc SMC; 129/SV; Primary-pig Ear Chondrocyte; ED27; Primary-rabbit Bone B; Primary-h Brain Glioblast; Primary-rat Adipose Preadipocyte; Primary-h Cartilage Synov; Primary-rat Pancreas Insulin; Primary-hEC; Primary-sheep Aorta SMC; Primary-h Endometrium EpiC; U251; Primary-h Endometrium StC; Primary-pig Bladder SMC; Primary-h HVIintersticial C; Primary-pig Esoph SMC; Primary-h NP Neuronal; Primary-rabbit Aorta SMC; Primary-h NSC; Primary-rabbit CorneaFB; Primary-h ral Cancerous; Primary-rabbit Lig FB; Primary-h SC; Primary-rat BMOB-like; Primary-h Skeletal Muscle Myoblast; COS-7; C-28/12; HK-2; Primary-h Uterus Cancerous; Primary-rat Ventricle CM; Primary-h Vase EC; Primary-sheep Carotid Artery SMC; HCT-116; ROS 17/2.8; Primary-h Vocal FB; UMR-106; Primary-mouse Aorta SMC; H9; R1; Primary-rat Fetal Neuronal; Primary-chicken Ear EpiC; Huh7; Primary-rat Vasc SMC; Primary-h NP SC; ES-D3; IMR-90; Primary-rat Bladder SMC; 293T; Primary-h Foreskin VascularEC; Primary-h Placenta EC; Primary-h Lung EpiC; Primary-h Prostate EpiC; U-87 MG; Primary-dog Carotid Artery SMC; Primary-rabbit Cornea StC; Primary-dog ID Annulus Fibrosus; Primary-chicken Embryo Chondrocyte; Primary-EC; HFF; Vero; HFL-1; Primary-h Adipose FB; Primary-cow FB; Primary-h UTSMC; Primary-rat Ventricle FB; AH 927; Primary-sheep Vasc FB; DU-145; ST2; B16.F10; Primary-h Nasal EpiC; Primary-ID Annulus C; Primary-h Dental Pulp SC; 3H10T1/2; Primary-Heart Valve; Primary-h Bone Alveolar; Primary-rabbit Tendon FB; Primary-mouse Kidney Insulin; HEPM; Primary-baboon Aorta SMC; HTK; Primary-mouse MDSC; Primary-rat Esoph EpiC; Primary-mouse Nerve SC; Primary-h Fetus OB-like; Primary-mouse Skeletal Muscle SC; hFOB 1.19; Primary-Nerve Schwann; Primary-h Ganglia Neuronal; Caco-2; Primary-h Kidney Renal; Primary-h Breast EpiC; Primary-h Liver SC; Primary-pig Bladder Uro; Primary-h Lung EC; Primary-h Breast FB; Primary-sheep Jugular Vein EC; Primary-pig Esoph EpiC; Primary-h Lymph EC; Primary-chicken CC; Primary-h Lymph TCell; Primary-h Colon Adenocarcinoma; Primary-h Mammary EC; Primary-pig Vocal FB; Primary-h Mammary EpiC; Primary-rabbit Adipose SC; Primary-h Cornea EC; H9c2; Primary-h UT StC; Primary-cat Heart CM; Primary-mouse Pancreas EpiC; HS-5; Primary-sheep Skeletal Muscle Fetus Myoblast; Primary-cow ID; Primary-mouse BM OCpre; Primary-cow Knee Meniscus C; Hep-3B; Primary-cow Lig FB; HL-1; HuS-E/2; RWPE1; Primary-cow Retina EpiC; Primary-hVascMyoFB; IEC-6; Primary-mouse Fetal Hep; HS68; OVCAR-3; Primary-dog Knee MeniscusC; Primary-rabbit Mesoderm Mes PreC; Primary-dog Lig FB; Primary-rat Lung Alveolar; Primary-dog Skin Keratinocyte; CRL-11372; Primary-dog Vase SMC; HMEC-1; Primary-Embryo SC; T-47D1; Pimary-goatCC; Primary-h UVSC-like; Primary-guineapig Ear EpiC; Primary-Ligament; Primary-guineapig Skin FB; Primary-mouse Cortical Neuronal; Primary-hAdipose Adipocyte; Primary-mouse Liver SC; Primary-h Adipose FB-like; CAL72; J774; P19; Primary-h Amniotic fluid; Primary-rabbit Cornea EC; Primary-h Amniotic FSC; Primary-rat BMFB-like; ARPE-19; Primary-rat Kidney Mesangial; K-562; Primary-rat Nasal Ensheathing; Primary-h Bladder StC; Primary-chicken Embryo Proepicardium; ATDC5; Primary-sheep FB; Kasumi-1; Primary-Skeletal Muscle; Primary-h Bone Mes PreC; HMT-3522; Primary-h Bone Periosteal; A431; Primary-h Brain EC; Primary-h UTFB; KLE; 143b OST; BALB/3T3; Primary-h Vasc FB; LLC-PKI; Primary-h Vasc Pericyte; BHK21-C13; Primary-Mammary EpiC; M.DUNNI; C4-2B; ZR-75; HEC-1B; Primary-h Gingiva Keratinocyte; U178; Primary-h HN Cancerous; Primary-mouse Mammary EpiC; Primary-h Keratinocyte; Primary-mouse Sciatic N Schwann; OVCA429; Primary-h Kidney EpiC; Primary-pig Esoph FB; MBA-15; Primary-pig Mandible FB-like; Primary-h Liver Cancerous; Primary-rabbit Bladder Uro; GD25beta1A; Primary-rabbit ID AnnulusC; HSC-T6; Primary-rabbit NP Neuronal; DOV13; HEY; Primary-h Mammary FB; HTB-94; BZR-T33; Primary-chicken CorneaFB; MiaPaCa2; Primary-rat Mucosa Ensheathing; Primary-hOvaryFB; Primary-rat Salivary Acinar; Primary-h Ovary Oocyte; Primary-rat Testes Germ; Primary-h Pancreas Cancerous; Primary-chicken Embryo StC; Primary-h Pancreas Stellate Cells; Primary-sheep Carotid Artery FB; MLO-Y4; Primary-chicken Retina SC-like; Primary-h Prostate Cancerous; Primary-chicken Ten TC; Primary-h Saph V Myo FB; Primary-Synoviocyte; MTLn3; Primary-Vase EC; Primary-h Skeletal Muscle Pre; RT4-D6P2T; C2; SCA-9; HOC-7; T31; Primary-h UC EpiC; TR146; HCS-2/8; EA.hy926; Primary-rat Ebryo; SW480; Primary-sheep Fetus CC; Primary-dog Pancreas Insulin; KS-IMM; BPH-1; Primary-rat Pancreas SC; M2139; RIN-5F; Primary-hGallbladderCancerous; E14/TG2a; M4E; HES3; G8; Primary-hConjunctivaFB; Primary-dogSaphVEC; LN CaP; Primary-dog Saph V SMC; M4T; Primary-h Fetus CC; BR-5; Primary-pig UT Uro; Primary-Hippocampus Neuronal; PE-0041; Primary-dog Skin FB; Primary-rabbit Skeletal Muscle MyoBlast; Primary-cow Denta ipulp; CGR8; Primary-dog Teeth PDL; Primary-rat Fetus Hep; Primary-dog Tendon FB; Primary-rat Mammary; Primary-h Knee C; Primary-rat SMC; BRC6; Primary-sheep Artery FB; Primary-dog Vasc EC; Primary-cow Mammary Alveolar; pZIP; 293 cell line; BMC9; Primary-h Lung Cancerous; SKOV-3; IOSE; TEC3; MCF-12A; Primary-rabbitBladderEpiC; Gli36DeltaEGFR; Primary-rabbit Conjunctiva EpiC; Primary-h Lung Neuronal; Primary-rabbit Endometrium EpiC; 1205Lu; Primary-rabbit MDSC; 3T3-A31; Primary-rabbit Tendon Tenocyte; MDA-MB-435; Primary-h Cancerous; Primary-cow EC; Primary-rat Cornea FB; Primary-EpiC; Primary-rat Fetal Cardiac; Primary-h Meninges Arachnoidal; COS-1; Primary-Eye; Primary-rat Liver Oval C; GLUTag-INS; Primary-rat Oral Mucosa Keratinocyte; GM3348; CRFK; 21NT; Primary-rat Testes EC; Primary-h Nasal FB; Primary-h Dura MaterSC; Primary-h Nasal OB; Primary-dog NP Neuronal; Primary-h Nasal Secretory; Primary-sheep Lung FB; AC-1M59; BHPrE1; MIN6; Primary-UT; MKN28; RAT-2; MLO-A5; RT112; CRL-2266; S91; GM5387; SK-ChA-1; Primary-horse CC; SPL201; Primary-horse Tendon FB; Primary-h Fetus Mes PreC; D283; Primary-pig Thyroid EpiC; H1299; Par-C10; AE-6; Primary-rabbit Blood Platelet; Primary-goat Carotid EC; Primary-rabbit Bone OC; Primary-goat Carotid FB; Primary-cow Comea FB-like; Primary-h Pancreas SC; Primary-rabbit CT Pericyte; Primary-goat Carotid SMC; Primary-rabbit Esophagus SMC; Primary-h Parotid Acinar; Primary-baboon Blood EC; A498; Primary-h Bronchi SMC; Primary-h Placenta SC; Primary-rabbit Sphincter SMC; Primary-cow Retina SC; 7F2; MM-Sv/HP; A10; Primary-h Prostate StC; Primary-buffalo Embryo SC-like; Primary-h Salivary Cancerous; CHO-4; Primary-h Salivary Salisphere; Primary-rat Cortical Neuronal; H13; Primary-rat Embryo Neuronal; Primary-guineapig Pancreas EpiC; Primary-rat Fetal OB; H144; CNE-2; MPC-11; 21PT; Primary-cow Synovium; Primary-rat Liver EC; Primary-cow Fetus CC; BEAS-2B; H2122; LM2-4; Detroit 551; C18-4; FLC4; Ishikawa; Primary-rat Skin Keratinocyte; H35; Primary-rat Tendon; Primary-h SMC; HTR8; Primary-h Synovial CC; E8.5; H460M; HL-60; MUM-2B; CRL-1213; MUM-2C; CRL-12424; W20-17; Lovo; Primary-dog Blood EC; Primary-sheep Nasal CC; HAK-2; Primary-sheep Skin FB; Primary-h Testes Sertoli; Primary-h Thyroid Cancerous; Primary-Trachea; Primary-h Trachea; LRM55; Primary-h UASC-like; Primary-Colon FB; Primary-hUASMC; r-CHO; HAT-7; RN22; HC-11; Primary-h Eye Vitreous; AEC2; S2-020; HCC1937; CRL-2020; AG1522; SCC-71; N18-RE-105; SK-N-AS; Primary-h Uterus SMC; SLMT-1; IMR-32; STO; NB4; Swan 71; Primary-h Alveolar Perosteum; Primary-dog Oral Mucosa EpiC; Primary-h Amnion EP; Primary-h Fetus Schwann; Primary-dog Bone OB; Primary-pig UTSMC; 184A1; Panc 1; NCTC 2544; 46C; Primary-cow Cornea EC; B6-RPE07; Primary-hamster EC; cBAL111; Primary-hamster Retina Neuronal; HEPA-1Clc7; NEB1; CCE; NHPrE1; Primary-rabbit Conjunctiva FB; 410; Hepa RG; Primary-Keratinocyte; PMC42-LA; Primary-dog Cartilage Synov; 21MT; NOR-PT; Primary-rabbit Endometrium StC; Primary-Lymphnode Lymphocyte; DLD-1; Primary-Lymphnode TCell; Primary-rabbit Lacrimal Gland Acinar; AB2.1; primary-rabbit Lung Pneumocyte; Primary-monkey Embryo; ES-2; Primary-monkey Kidney FB-like; Primary-rabbit Penis SMC; Primary-mouse Adipose StC; Primary-rabbit Skin FB; NR6; Primary-Blood SC; Primary-mouse BM Macrophage; 786-0; AT2; Primary-rat Adrenal Chromaffin; AT3; CCF-STTGI; Primary-mouse Bone Calvarial; Primary-rat Bladder Uro; HCT-8/E11; CE3; Primary-mouse Brain Neuronal; CFK2; Primary-mouse Breast Cancerous; L6; Primary-mouse Chondrocytes; HeyA8; Primary-mouse Colon EpiC; Primary-rat Cortical Astrocyte; Primary-dog CFB; Primary-buffalo Ovary EpiC; Primary-dog Cornea Chondrocyte; Primary-rat Embryo CM; Primary-mouse Embryo Neuronal; A2780; C5.18; Primary-dog MV EpiC; Primary-mouse Esophagus SC; Primary-rat Fetal Renal; HEK001; A357; EFO-27; Primary-chicken Bone OB; Primary-mouse Fetal Lung; Primary-rat Heart SC-like; Primary-mouse Germ; Primary-rat Kidney; EN Stem-A™; Primary-rat Lacrimal Acinar; U-251 MG; Primary-dog Myofibroblasts; A4-4; Primary-rat Liver SC-like; Primary-cow Brain EC; Primary-rat Lung FB; Primary-mouse Kidney Renal; BEL-7402; NT2; HIAE-101; Primary-h BM Mononuclear; Primary-rat Ovary; Primary-mouse Lymph FB-like; Primary-rat Pancreas Islets; Primary-dog Esophageal EpiC; Primary-rat Renal EpiC; Primary-mouse Mast; Primary-chicken Embryo Blastoderm; NTera2/c1.D1; G-415; Null; Primary-rat Small Intestine; Primary-mouse Ovary Cumulus C; Primary-rat Teeth SC-like; HEL-299; Primary-rat Tendon Tenocyte; KB; b-End-2; Primary-mouse Pancreas Insulin; Primary-rat Vase EC; Primary-mouse Salivary Salisphere; Primary-h Duodenum EpiC; Primary-h Bone Fetus OB; Primary-Respiratory EpiC; Primary-mouse Skeletal Muscle Myoblast; Primary-sheep Amniotic fluid; OC2; Primary-chicken Heart CM; Daudi; Primary-shee pArtery MyoFB; Primary-mouse SkinKeratinocyte; Primary-sheep Bone OB-like; Primary-mouse Small Intestine; Primary-chicken Heart ECM; Primary-mouse Spleen Tcell; LNZ308; Primary-mouse Teeth Odontoblast; Primary-sheep ID Annulus Fibrosus; Primary-mouse Testes SC; Primary-sheep Jugular Vein SMC; Primary-mouse Testes Sperm; Primary-sheep Lung SC; Primary-mouse UT Uro; Primary-sheep Saph VEC; Primary-mouse Uterus EpiC; Primary-sheep Skin EC; OCT-1; Primary-sheep Vasc EC; HELF; Primary-sheep Vasc SMC; CAC2; HL-7720; OPC1; Primary-Teeth PDL; Primary-dog Heart SC; Primary-UCB Mononuclear; Primary-pig Artery Carotid EC; Primary-h Endometriotic CystStC; Primary-pig Artery Carotid SMC; Primary-Colon Cancerous; Primary-pig Artery Coronary SMC; QCE-6; Primary-pig Bladder FB; R221A; OSCORT; LS180; B35; RIF-1; Calu-1; RL-65; Calu-3; Primary-cow Adrenal ChrC; B5/EGFP; RT-112; Primary-pigEC; RW.4; Primary-pig ESC; S2-013; OVCAR-5; S5Y5; Primary-h Bone OC-like; SA87; INT-407; SAV-I; Primary-pig Fetus Hep; SCC-68; P69; HNPSV-1; CaSki; SK-CO15; Primary-pig Iliac EC; SK-N-DZ; Hep2; SKOV3Ip.1; Primary-pig Mandible Ameloblast; SNB 19; Primary-cow Joint Synovial; Primary-h Fetus FB; Primary-pig Mandible Odontoblast; SW1353; Primary-pig NP Neuronal; SW948; Primary-pig Oral MucosaEpiC; CRL-2102; Primary-pig PancreasIslets; T4-2; Primary-pig PulmonarySMC; TE-85; Primary-pig Salivary Acinar; THP-1; Primary-pig SynoviumSC; BME-UV1; KG-1; D4T; HUES-9; Primary-mouse Hippocampus Neuronal; ECV304; NRK; Primary-mouse Kidney Mesangial; D407; I0T1/2 cell line; and Primary-h Foreskin Melanocyte. 5.3. Biomarkers In certain non-limiting embodiments, the first targeting molecule and the second targeting molecule target at least one biomarker of a biological subject of interest. In certain non-limiting embodiments, the first and second targeting molecules can target the same or different biomarker(s) of a biological subject of interest. In certain non-limiting embodiments, when the first and second targeting molecule are targeting two different biomarkers, the biomarkers are expressed on the same cell. In certain non-limiting embodiments, there is only one targeting molecule targeting one biomarker. In certain non-limiting embodiments, the biomarker can be expressed on the surface of the cell or internally. In certain non-limiting embodiments, the biomarker can be a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, bacterial surface protein, etc. In certain non-limiting embodiments, the biomarker is an integrin. In certain non-limiting embodiments, the biological subject is a protein, virus, cell, tissue, organ or organism. In certain non-limiting embodiments, the cell can be, but is not limited to, a tumor, cancer, or diseased cell. In certain non-limiting embodiments, the first and second targeting molecules bind to a cell (including a tumor or cancer) such as, but not limited to, pancreatic cancer, breast cancer, colorectal cancer, NSCLC, lung cancer, bone cancer, skin cancer, cancer of the head or neck, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin's Disease, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer, biliary cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, CNS neoplasm, spinal axis cancer, brain stem glioma, glioblastoma multiform, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma and pituitary adenoma tumors, or tumor metastasis. In certain non-limiting embodiments, the level of biomarkers are lower than would be detectable by other methods. In certain non-limiting embodiments, the current method is able to detect early stages of the disease (e.g., cancer). In certain non-limiting embodiments, the current method is able to detect low levels of biomarker presence. In certain non-limiting embodiments, the biomarker can be epidermal growth factor receptor (EGFR), integrin α1β1, integrin α2β1, integrin α3β1, integrin α4β1, integrin α5β1, integrin α6β1, integrin αvβ3, uPAR, gastrin-releasing peptide (GRP), SSTR2, SSTR3, SSTR4, SSTR5, Folate receptor, CCR5, CXCR4, plectin-1, VEGF, CA19-9, PD-I1, Her2/neu, 5-alpha reductase, α-fetoprotein, AM-1, APC, APRIL, BAGE, β-catenin, Bc12, ber-ab1 (b3a2), CA 125, CASP-8/FLICE, Cathepsins, CD13, CD19, CD20, CD21, CD23, CD22, CD38, CD33, CD35, CD40, CD44, CD45, CD46, CD5, CD52, CD55, CD59 (791Tgp72), CDC27, CDK4, CEA, c-myc, COX-2, Cytokeratin, DCC, DcR3, E6/E7, EGFR, EMBP, Ena78, Estrogen Receptor (ER), FGF8b and FGF8a, FLK 1/KDR, G250, GAGE-Family, gastrin 17, Gastrin-releasing hormone (bombesin), GD2/GD3/GM2, GnRH, GnTV, gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1, hCG, Heparanase, Her3, HMTV, Hsp70, hTERT (telomerase), IGFR1, IL 13R, iNOS, Ki 67, KIAA0205, K-ras, H-ras, N-ras, KSA (CO17-1A), LDLR-FUT, MAGE Family (MAGE1, MAGE3, etc.), Mammaglobin, MAP17, Melan-A/MART-1, mesothelin, MIC A/B, MT-MMP's, such as MMP2, MMP3, MMP7, MMP9, Mox1, Mucin, such as MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1, NY-ESO-1, Osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PDGF, Plasminogen (uPA), PRAME, Probasin, Progenipoietin, Progesterone Receptor (PR), PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX gene family, STAT3, STn (mucin assoc.), TAG-72, TGF-α, TGF-β, Thymosin β-15, IFN-γ, TPA, TPI, TRP-2, Tyrosinase, VEGF, ZAG, p161NK4, Myo D1, Glutathione, or S-transferase. In certain non-limiting embodiments, the biomarker can be epidermal growth factor receptor (EGFR), integrin αvβ3, uPAR, gastrin-releasing peptide (GRP), SSTR2, CCR5, integrin U401, VEGF, CA19-9, CD13, CD40, or PD-L1. In certain non-limiting embodiments, the biomarker is uPAR and/or integrin αvβ3. In certain non-limiting embodiments, the biomarker is CD13 and/or integrin αvβ3. Integrins are cell adhesion molecules that mediate cell-cell and cell-matrix interactions and contribute to migration, proliferation, angiogenesis, tumor invasion, and metastasis. Integrin αvβ3serves as a receptor for extracellular matrix proteins with exposed arginine-glycine-aspartic (RGD) tripeptide sequence. Importantly, integrin αvβ3usually expresses at very low (or undetectable) levels in most adult epithelia cells, but are highly upregulated in various tumor cells. Recent expression analysis demonstrated that the patients with high αvβ3expression showed significantly shorter survival times than those with low αvβ3-expression. Its restricted expression during tumor growth, invasion, and metastasis presents an interesting molecular target for diagnosis and treatment of the rapidly growing and metastatic tumors and, therefore, αvβ3is an example of one of the biomarkers of the invention. Aminopeptidase N (APN)/CD13, a transmembrane protease, is another important biomarker. Similar to integrin αvβ3, CD13 is also up-regulated in the angiogenic vessels in the tumor but only barely expressed in the normal blood vessels, and high expression of CD13 has been observed in a number of human solid tumors, including melanoma, prostate, lung and ovarian cancer and pancreatic cancer. NGR sequence containing peptides have shown high efficiency/selectivity in binding with CD13. Thus, CD13 provides another example biomarker in accordance with the invention. uPAR is another important biomarker for cancer imaging, as both clinical studies and laboratory research revealed that overexpression of uPA/uPAR is strongly correlated with poor prognosis in malignant tumors. Moreover, uPAR is overexpressed in various malignancies (normally expresses several thousand receptors per cell), but absent or very poorly expressed in normal and adjacent tissues. Thus, uPAR is an example of another biomarker of the invention. In certain non-limiting embodiments, multivalent compounds of the present invention directed to integrin αvβ3and CD13 (e.g., CNGRC-(68Ga)NOTA-RGDyK heterodimer (“CNGRC” disclosed as SEQ ID NO: 1)) can be used to detect early stages of the cancer. In certain non-limiting embodiments, multivalent compounds of the present invention directed to integrin αvβ3and CD13 can be used to detect low levels of integrin αvβ3and/or CD13. 5.4. Kits The present invention further provides kits that can be used to practice the invention. For example, and not by way of limitation, a kit of the present invention can comprise at least one imaging and/or drug delivery compound. In certain non-limiting embodiments, a kit of the present invention can optionally comprise instructions on how to use the kit for molecular imaging and/or targeted drug delivery. In certain non-limiting embodiments, a kit can further comprise an administration device such as a syringe and/or catheter and/or introducer sheath. In certain non-limiting embodiments, the imaging and/or drug delivery compound comprises a monomer with a detectable label and/or active agent. In certain non-limiting embodiments, the imaging and/or drug delivery compound comprises a homodimer with a detectable label and/or active agent. In certain non-limiting embodiments, the imaging and/or drug delivery compound comprises a heterodimer with a detectable label and/or active agent. In certain non-limiting embodiments, the imaging and/or drug delivery compound comprises a targeting molecule with a dye molecule with a detectable label and/or active agent. The present invention further provides kits for preparing the imaging and/or drug delivery compound. In certain non-limiting embodiments, the kit of the present invention contains the first targeting molecule (in dry or liquid form) and/or the second targeting molecule (in dry or liquid form) and/or the chelator for assembly into the imaging and/or drug delivery compound. When the molecule is provided in dry form, the kit can contain the appropriate buffer or solvent to create a solution or composition. The present invention further provides kits for determining the optimal length of spacers of the imaging and/or drug delivery compound. In certain non-limiting embodiments, the kit of the present invention contains the first targeting molecule (in dry or liquid form) and/or the second targeting molecule (in dry or liquid form) and/or spacers of different length and/or a radio-metal labeled chelator for a high-throughput screening platform. In certain non-limiting embodiments, the kit can contain the appropriate buffer or solvent to create perform the high-throughput assay. The following Examples are offered to more fully illustrate the disclosure, but is not to be construed as limiting the scope thereof. Methods and materials described in the examples are hereby incorporated by reference into the detailed description of the invention. 6. EXAMPLES Example 1: Synthesis of a Metal Chelator of the Invention The 1, 4, 7-triazacyclonenonane (TACN)-based chelator (N3-NOtB2) was prepared as shown in Scheme 1: 4-amino-2-hydroxybutanoate (2) MeCOCl (15 mL) was dropwise added to anhydrous methanol (100 mL) at 0° C. The resulting mixture was stirred at room temperature for 1 h. Then, starting material (1) (10 g, 84 mmol) was added and the mixture was stirred for 2 h at room temperature. Solvent was removed under reduced pressure, and the residue was treated with ether (50 mL) to obtain (2) (12.67 g, 88.5%) as a white solid after filtration. No further purification is needed suggested by TLC and NMR.1H NMR (400 MHz, D2O) 64.53-4.44 (m, 1H, —CH(COOH)—), 3.82 (s, 3H, —CH3), 3.27-3.11 (m, 2H, —CH2N3), 2.31-2.19 (m, 1H, —CH2CH2N3), 2.13-1.98 (m, 1H, —CH2CH2N3).13C NMR (101 MHz, D2O) δ 175.16, 68.50, 52.89, 36.66, 30.42. ESI-MS: observed, m z (M+H)+=133.92, calculated, (M+H)+=134.08. methyl 4-azido-2-hydroxybutanoate (3) Imidazole-1-sulfonyl azide hydrochloride (2.5 g, 12 mmol) was added to the slurry of (2) (1.7 g, 5 mmol), K2CO3(3.2 g, 23 mmol), and CuSO4·5H2O (30 mg, 100 μmol) in MeOH (30 mL) and the mixture was stirred overnight. The mixture was concentrated, diluted with H2O (100 mL), acidified with conc. HCl and extracted with EtOAc (50×3 mL). The combined organic layers were dried (MgSO4), filtered and concentrated to obtain crude (3) (1.22 g, 76.6%) as a colorless liquid. The crude was used in the next step without further purification. For NMR spectra, a little crude was purified by silica gel chromatography (DCM/MeOH, 10:1) to give pure (3) as a colorless liquid.1H NMR (400 MHz, CDCl3) δ 4.31 (dd, J=7.6, 4.0 Hz, 1H, —CH(COOH)—), 3.83 (s, 3H, —CH3), 3.59-3.43 (m, 2H, —CH2N3), 2.93 (brs, 1H, —OH), 2.15-2.04 (m, 1H, —CH2CH2N3), 1.99-1.86 (m, 1H, —CH2CH2N3).13C NMR (101 MHz, CDCl3) δ 175.06, 67.60, 52.81, 47.18, 33.16. methyl 4-azido-2-(tosyloxy)butanoate (4) TsCl (2.7 g, 14 mmol) was added to a solution of (3) (1.5 g, 9.4 mmol) and TEA (3 g, 30 mmol) in DCM (50 mL) and the mixture was stirred at room temperature overnight. The mixture was then washed by water (30×2 mL), dried over MgSO4, and filtered. Concentration of the filtrate and flash chromatography (EtOAc/Hexane, 1:4) gave (4) (2.37 g, 70.8%) as a white solid.1H NMR (400 MHz, CDCl3) δ 7.84 (d, J=8.3 Hz, 2H, Ar—H), 7.38 (d, J=8.1 Hz, 2H, Ar—H), 4.97 (dd, J=7.6, 5.0 Hz, 1H, —CH(COOH)—), 3.72-3.68 (m, 3H, —CH3), 3.47-3.39 (m, 1H, —CH2CH2N3), 3.37-3.27 (m, —CH2CH2N3), 2.47 (s, 3H, Ar—CH3), 2.13-1.98 (m, 2H, —CH2N3).13C NMR (101 MHz, CDCl3) δ 168.68, 145.46, 132.89, 129.88, 128.08, 74.36, 52.77, 46.36, 31.53, 21.69. di-tert-butyl 2,2′-(7-(4-azido-1-methoxy-1-oxobutan-2-yl)-1,4,7-triazonane-1,4-diyl)diacetate (5) To a slurry of NO2A(tBu) (0.8 g, 2.24 mmol) and Cs2CO3(1.1 g, 3.36 mmol) in MeCN (20 mL), (4) (0.95 g, 2.67 mmol) was added and the mixture was heated at 50° C. for 1 d. After cooling to room temperature, the mixture was filtered and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH, 10:1) to give (5) (0.73 g, 65.2%) as a yellowish liquid.1H NMR (400 MHz, CDCl3) δ 3.73-3.60 (m, 3H, —OCH3), 3.58-3.44 (m, 2H, —CH2N3), 3.39 (s, 1H—NCH—), 3.28 (s, 4H, 2*—CH2CO—), 3.02-2.58 (m, 12H, 6*—NCH2—), 2.01-1.79 (m, 2H, —CH2CH2N3), 1.43 (s, 18H, 6*—CH3).13C NMR (101 MHz, CDCl3) δ 173.51, 171.54, 80.70, 63.83, 59.40, 56.11, 55.77, 53.31, 51.23, 48.52, 29.52, 28.21. ESI-MS: observed, m z (M+H)+=499.266, calculated, (M+H)+=499.32. ESI-HRMS: observed, m z (M+H)+=499.3239, calculated, (M+H)+=499.3239. 4-azido-2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)butanoic acid (N3-NOtB2, 6) To a solution of (5) (100 mg, 0.2 mmol) in pyridine (2 mL), LiI (134 mg, 1 mmol) was added and the mixture was stirred for 4 h at room temperature. The mixture was treated with DCM (20 mL), and washed with saturated citric acid (10×2 mL) and water (20 mL). The organic layer was dried over MgSO4, filtered and concentrated to give crude (6). Pure (6) (50 mg, 51.5%) as a white solid was obtained after purification with silica gel chromatography (DCM/MeOH, 20:3).1H NMR (400 MHz, CDCl3) δ 3.72 (d, J=7.1 Hz, 1H, —NCH(COOH)—), 3.70-3.41 (m, 6H, 2*—CH2CO— &—CH2N3), 3.43-2.75 (m, 12H, 6*—NCH2—), 2.36-2.21 (m, 1H, —CH2CH2N3), 1.91 (dd, J=12.9, 5.2 Hz, 1H, —CH2CH2N3), 1.46 (s, 18H, 6*—CH3).13C NMR (101 MHz, CDCl3) δ 172.17 (—COOH), 169.51 (2*—CO2C(CH3)3), 82.28 (—CO2C(CH3)3), 63.12 (—NCH(COOH)—), 56.35 (2*—NCH2CO2C(CH3)3), 50.79 (—N—CH2—), 49.80 (—N—CH2—), 49.16 (—N—CH2—), 49.04 (—CH2N3), 28.62 (—CH2CH2N3), 28.12 (6*—CH3). ESI-MS: observed, m z (M+H)+=485.45, calculated, (M+H)+=485.31. ESI-HRMS: observed, m z (M+H)+=485.3064, calculated, (M+H)+=485.3082. N3—NOtB2 was synthesized with an overall yield of 15%. Example 2: Synthesis of a Metal Chelator of the Invention The TACN-based chelator (N3-DOtB3) was prepared as shown in Scheme 2: tri-tert-butyl 2,2′,2″-(10-(4-azido-1-methoxy-1-oxobutan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (S2) To a slurry of (S1) (DO3A(tBu)) (51.4 mg, 0.1 mmol) and Cs2CO3(49 mg, 0.15 mmol) in MeCN (1 mL), (4) (38 mg, 0.12 mmol) was added and the mixture was shaken at 50° C. for 1 d. After cooling to room temperature, the mixture was filtered and concentrated. The reaction was monitored by LC-MS. Pure (S2) (30 mg, 38.4%) was obtained after purification with silica gel chromatography (DCM/MeOH, 10:1)1H NMR (500 MHz, CDCl3) δ 3.70-3.18 (m, 10H, —CHCO— & —NCH2CO— & —OCH3), 3.04-2.07 (m, 16H—NCH2—), 1.97-1.81 (m, 2H, —CH2N), 1.71-1.63 (m, 2H, —CH2CH2N3), 1.48-1.45 (m, 27H, 9*—CH3). ESI-MS: observed, m z (M+H)+=656.455, calculated, (M+H)+=656.435. ESI-HRMS: observed, m z (M+H)+=656.4322, calculated, (M+H)+=656.4347. 4-azido-2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)butanoic acid (S3) To a solution of S2 (10 mg, 0.015 mmol) in pyridine (0.5 mL), LiI (10 mg, 0.075 mmol) was added and the mixture was shaken for 2 h at room temperature. The mixture was treated with DCM, and washed with saturated citric acid and water. The organic layer was dried over MgSO4, filtered and concentrated to give crude (S3). Silica gel chromatography (DCM/MeOH, 10:1) was applied to obtain pure S3 (3 mg, 31.2%).1H NMR (500 MHz, CDCl3) δ 3.87-3.74 (m, 2H, —CH2CO—), 3.63-3.53 (m, 1H, —NCH(COOH)—), 3.46-3.33 (m, 4H, —CH2CO—), 3.08-1.98 (m, 18H, —NCH2— & —CH2N), 1.67-1.56 (m, 2H, —CH2CH2N3), 1.51-1.46 (m, 27H, 9*—CH3). ESI-MS: observed, m z (M+H)+=642.439, calculated, (M+H)+=642.419. ESI-HRMS: observed, m z (M−H)−=640.4046, calculated, (M−H)−=640.4034. N3-DOtB3 was synthesized with an overall yield of 12%. Example 3: Synthesis of an AE105-Dimers of the Invention The TACN-based chelator was prepared as provided in Example 1. The TACN-based chelator was then attached to the AE105 peptide via solid-phase synthesis (SPS). In particular, N3—NOtB2was attached to the N-terminal of peptide AE105 with a high yield via SPS. Peptides were prepared on resin (Resin-AE105*, Resin-AE105-PEG8-NH2) using standard SPS protocol by a peptide synthesizer. Compound 6 (from Example 1) (3 eq.) was coupled to the resin by mixing them with HATU (5 eq.) and DIEA (10 eq.) in DMF for 2 h at room temperature. Moiety-A-NOTA-N3(7) was then obtained after cleavage from resin support using TFA/H2O/TIS/phenol (90:5:2.5:2.5) and HPLC purification. To obtain hetero-dimeric, homo-dimeric, and dual-modality compounds, the monomer (AE105-NOTA-N3) was cleaved from the Rink amide resin, and was then conjugated to a BCN-functionalized RGDyk (or AE105 or cynaine dyes Cy3 or Cy5) via a strain-promoted alkyne-azide cycloaddition (SPAAC) in high yield as outlined in Scheme 3: Rendered by the metal-free click reaction, >95% yield was achieved after incubating the monomer (AE105-NOTA-N3) with 1.0 equivalent of BCN-attached peptides or dye overnight at 37° C. The resulting compounds could be used directly. HPLC purification through gradient elution staring from 0% acetonitrile 100% water to 100% acetonitrile could further increase the puritiy of those synthesized probes. The above prepared probes were successfully labeled with64Cu,68Ga, and Al18F at 37° C., 70° C., and 90° C., respectively. Generally, nearly 100% labeling yield could be achieved for64Cu and68Ga labeling when a 1 nmol probe and 1 mCi radioactivity were used.FIG.5provides representative examples of the radio-HPLC results of the radiolabeling. All above compounds could be labeled with64Cu under mild conditions, with a specific activity of 1.0 mCi/nmol. The resulting radiotracers showed great stabilities in human serum after being incubated at 37° C. for 24 hours (with <2%64Cu dissociation). The resulting heterodimer (AE105-NOTA-RGD;FIG.6) and monomers (AE105-NODAGA and RGD-NODAGA;FIG.6) were radiolabeled with64Cu at 70° C. in NH4OAc buffer (pH˜6.8), and their serum stabilities were evaluated. The resulting radiotracers remained intact after being incubated at 37° C. for 24 hours, showing great serum stability.FIG.7provides radio-HPLC results demonstrating that there was no significant64Cu-disassociation from the probe after incubating in serum for 1 day. The good serum stability demonstrates that the probe is able to stay intact during the circulation in the blood stream in vivo. The larger molecular weight and size will also increase retention of the probe in the blood. Radiolabeling of compounds: For Cu-64 labeling, all compounds was conducted in a 0.1 M NH4OAC buffer (pH=6.8). Briefly,64CuCl2(usually in 0.1 N HCl) was first buffered in a 0.1 M NH4OAC buffer (pH=6.8), and then the prepared NOTA-bioconjugates were added. The resulting mixture was vortexed for 10 sec and incubated in a thermomixer at 37° C. for 0.5 h, after which the64Cu incorporation yield was determined by radio-HPLC. FIG.8depicts a dimer made with N3-DOtB3-AE105-PEG4-DOTA-PEG4-RGD. Cell stain study: Cells were seeded in an 8 well chamber slide (100,000 cells per well) 24 h prior to the experiment. Before the experiment, cells were washed twice with PBS twice and added culture media. Then block agent (10 μg AE105) was added to half of the wells as cold block to determine in vitro non-specific uptake and incubated for 1 h. Then, AE105-NOTA-NHCO-Cy3 (10 pmol per well) was added to each well and further incubated for 2 h. Media was then removed and the cells were washed twice with PBS. After fixing the cells using 1% Paraformaldehyde, the nucleus was stained by DAPI. The slide was sealed and observed under fluorescence microscopy (40×, oil). As shown inFIG.9, staining and blocking were observed on U87MG human glioblastoma cell line, which confirmed the strong affinity of peptide AE105 towards uPAR receptor and indicated that AE105-NOTA-NHCO-Cy3 could also be used as optical probe. Example 4: Cell-Uptake and Binding Studies Cell uptake and stain studies were measured in U87MG human cancer cells. In particular, a comparison was made between the AE105-RGD heterodimer (FIG.4) and the monomers AE105-NODAGA and RGD-NODAGA (FIG.6). Cell-uptake assay: U87MG human cancer cells were purchased from American Type Culture Collection (Manassas, VA). All cell handling was aseptically performed in a laminar flow hood. The U87MG cells were cultured in Dulbecco's Modified Eagle Medium, supplemented with 10% FBS, penicillin (100 unit/mL), streptomycin (100 μg/mL) L-glutamine (300 μg/mL) and sodium pyruvate (100 mg/mL), glucose (4.5 g/L) and maintained at 37° C., 5% CO2. Cells were seeded in 12-well plates (200,000 cells per well) 24 h prior to the experiment. Before the experiment, cells were washed with 1 mL HBSS twice and 1 mL media (DMEM with 0.1% BSA and 1 mM Mn2+) was added to each well. Cells were then incubated with the64Cu-labeled conjugates (10 pmol64Cu-GYK12,64Cu-RGD or64Cu-AE105 per well). At each time point (1, 2 and 4 h) radioactive media was aspirated. The cells were washed twice with HBSS (pH 7.2) and dissolved in 0.5% SDS. The radioactivity in each fraction was measured with a gamma counter. The protein content of each cell lysate sample was determined. The measured radioactivity associated with the cells was normalized to same amount of cell protein per well. The cell uptake was expressed as the percentage added dose after decay correction. The heterodimer showed significant improvements on the cell-uptake as compared to the monomers AE105-NODAGA and RGD-NODAGA at all examined time points (p<0.1) (FIG.10). Cell saturation binding assay: Cells were seeded in 24-well plates (100,000 cells per well) 24 h prior to the experiment. Before the experiment, cells were washed with 1 mL HBSS twice and 0.5 mL binding media (HBSS with 0.1% BSA and 1 mM Mn2+) was added to each well. Then block agents (10 μg AE105 and/or 10 μg RGD) were added to half of the wells as cold block to determine in vitro non-specific binding, followed by64Cu-AE105-RGD,64Cu-RGD and64Cu-AE105 in increasing concentrations (1-100 nM). The samples were incubated for 2 h on ice (4° C.). After incubation, the radioactive media was removed. Cell pellets were rinsed with ice cold binding buffer (1 mL) twice and dissolved in 0.5% SDS solution. The radioactivity in each fraction was measured in a gamma counter. The protein content of each cell lysate sample was determined (BCA Protein Assay Kit, Pierce). The measured radioactivity associated with the cells was normalized to the amount of cell protein present (fmol/mg). The results showed that64Cu-GYK12 exhibited significantly enhanced Bmax(488±73 fmol/mg) and binding affinity (9.9±4.2 nM), compared to those of two64Cu-monomers:64Cu-AE105 (Bmax269±43 fmol/mg, Kd65±49 nM), and64Cu-RGD (Bmax260±44 fmol/mg, Kd91±36 nM). Statistical analysis: All the experiments were performed in triplicate. Comparisons between different groups of experiments were made using the two-way ANOVA test (GraphPad Prism 6). When more than two data sets were compared, a two-way ANOVA analysis with Bonferroni post-tests were applied. P values <0.05 were considered statistically significant. Example 5: PET Imaging Using the AE105-RGD Heterodimer In vivo PET/CT imaging was conducted in NCr nude mice bearing U87MG tumor xenografts. Nude mice were injected with U87MG cells (5 million cells in 150 μL PBS) into the subcutaneous flank of the right shoulder. Either AE105-RGD heterodimer (FIG.4), AE105-NODAGA, or RGD-NODAGA were injected into bloodstream via tail vein injection. Blocking studies were conducted for the heterodimer studies by co-injecting 100 times of AE105 and RGD. Small animal PET/CT was performed at 1 h and 4 h post injection of tracers. Organ uptakes were determined by analysis of ROI. For AE105-RGD dimer, ex vivo biodistribution was also performed. The organs of mice were taken and counted using Gama-counter. All xenografted tumors were visible at both 1 h and 4 h p.i. (FIG.11). Organ uptake was determined from PET imaging quantitation. The results showed that higher tumor uptake (p<0.01) was observed with mice injected with64Cu-heterodimer (1 h 3.13±0.49% ID/g, 4 h 3.27±0.25% ID/g), compared to that of the mice injected with two monomeric PET probes:64Cu-AE105 (1 h 1.45±0.15% ID/g, 4 h 1.55±0.31% ID/g) and64Cu-RGD (1 h 1.50±0.42% ID/g, 4 h 1.73±0.51% ID/g). The tumor to muscle ratio of64Cu-heterodimer was 7.6±1.9 at 4 h, which is significantly higher than that of64Cu-AE105 (4.2±1.1). The tumor to liver ratio of64Cu-heterodimer was 1.5±0.4 at 4 h, which is also significantly higher than that of64Cu-AE105 (0.43±0.1). The high intestine uptake can be attributed to the fact that αvβ3integrin and uPAR are also highly expressed in intestine in young mice. In a blocking study performed by co-injecting unlabeled cyclo(RGDyK) and AE105 (100 μg for each one) (FIG.12), the tumor uptake of64Cu-heterodimer was reduced to 1.01±0.18% ID/g (4 h, p<0.01), which further confirmed the specificity of the heterodimer for targeting αvβ3integrin and uPAR in U87MG xenografts. Ex vivo biodistribution was also conducted for AE105-RGD to validate the PET imaging quantitation data. The results are shown inFIG.13. Statistical analysis: All the experiments were performed in triplicate. Comparisons between different groups of experiments were made using the two-way ANOVA test (GraphPad Prism 6). When more than two data sets were compared, a two-way ANOVA analysis with Bonferroni post-tests were applied. P values <0.05 were considered statistically significant. Example 6: Synthesis of Heterodimers for Dual Targeting This Example provides an alternative method of synthesizing heterodimers for dual targeting. This method can be modified to prepare heterodimers combining various peptides. A first peptide (Peptide A) can be prepared on resin (Resin-Peptide A-PEGn-NH2) using standard SPS protocol by a peptide synthesizer. N3—NOtB2can be coupled to the resin and peptide. The resulting Peptide A-PEGn-NOTA-N3can be cleaved from the resin support using TFA and HPLC purification. The heterodimer can be prepared by further combination with Peptide B (e.g., Peptide B-PEG4-BCN), as shown in Scheme 4. As shown in Scheme 4, the Peptides A and B can be selected from a number of suitable peptides. For example, they can be selected to target integrin and CD13. As such, Peptide A can be selected from AE105 and AE105mut and Peptide B can be selected from cyclo(RGDyK) and cyclo(RADyK). Additionally, the PEG spacer can be varied in length, e.g., from n=0 to n=12. For example, the heterodimers of AE105 and cyclo(RGDyK) are the same as described above in Example 4 (i.e., GYK4, GYK8, GYK12, and GYK16). However, this Example further demonstrates that heterodimers can be prepared with alternative targeting molecules, such as AE105mut and cyclo(RADyK). The prepared heterodimers can be radiolabeled for in vitro and/or in vivo evaluation respectively. Cell uptake and/or efflux assays can be used to identify one or more heterodimers with the greatest potential for cell uptake and retention, as will be described in greater detail in Example 13, below. Additionally, a cell saturation binding assay can be performed as described in Example 4 to evaluation the Bmax and binding affinity of the heterodimers as compared to the monomers. Example 7: Synthesis of an Integrin-CD13 Dual Targeting Compound The integrin-CD13 dual targeting compound was prepared as shown in Scheme 4 (Scheme 4 discloses “CNGRC” as SEQ ID NO: 1): Using the bifunctional chelator (BFC) N3—NOtB2, the peptidic ligands c(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1) and c(RGDyK), targeting CD13 and αvβ3 respectively, were linked covalently via a metalfree click reaction and an amide formation reaction.FIG.1depicts a dimer made with N3—NOtB2-c(CNGRC)-PEG4-NOTA-PEG4-RGD (“CNGRC” disclosed as SEQ ID NO: 1). The Fmoc-PEG4-OH and TACN-based chelators were attached to the cyclo(CNGRC) peptide (“CNGRC” disclosed as SEQ ID NO: 1) via solid-phase synthesis (SPS) sequentially. In particular, the N3—NOtB2was attached to the N-terminus of the peptide cyclo(CNGRC)-PEG4-NH2 (“CNGRC” disclosed as SEQ ID NO: 1) with a high yield via SPS. Peptide on resin (Resin-CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1) was prepared using standard SPS protocol by a peptide synthesizer, and then the side chain of cysteine was deprotected and then cyclized by treating with thallium(III) trifluoroacetate. Fmoc-PEG4-OH and N3—NOtB2were attached to the resin sequentially, by mixing them with HATU (5 eq.) and DIEA (10 eq.) in DMF for 2 h at room temperature. The c(CNGRC)-PEG4-NOTA-N3(“CNGRC” disclosed as SEQ ID NO: 1) was then obtained after cleavage from the resin support using TFA/H2O/TIS/phenol (90:5:2.5:2.5) and HPLC purification, and then ligated with cyclo(RGDyK)-PEG4-BCN (prepared by mixing BCN-PEG4-NHS with cyclo(RGDyK) in pH˜8.5 PBS buffer) via strain-promoted alkyne-azide cycloaddition (SPAAC) between N3and BCN moieties. Rendered by the triazole formation after metal-free click reaction, the purified heterodimers (NGR-NOTA-RGD) were successfully labeled with64Cu,68Ga and Al18F at 37° C., 70° C., and 90° C., respectively. Labeling results were monitored by radio HPLC. Labeling yields were above 90% for64Cu and68Ga, close to 50% for Al18F. Example 8: Synthesis of an Integrin-CD13 Dual Targeting Compound The integrin-CD13 dual targeting compound was prepared as shown in Scheme 5. The solid phase synthesis (Scheme 4) is more convenient from the aspect of synthesis as coupling agents used in the amide formation (the reaction between the amino group from the peptide and the carboxylic acid group from the BFC) can be easily removed. However, the solution phase synthesis (Scheme 5) consumed less amount of peptides and BFC; thus it can be suitable for small scale preparation when the amount of peptide and/or BFC available is limited. Using the bifunctional chelator (BFC), N3—NOtB2was conjugated to the fully protected c(RGDyK) via an amide formation reaction and then the protection group was removed in strong acid conditions. The resulting peptide was ligated to BCN-c(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1) via metal-free click reaction. FIG.1depicts a dimer made with N3—NOtB2-c(CNGRC)-PEG4-NOTA-PEG4-RGD (“CNGRC” disclosed as SEQ ID NO: 1). The protected linear RGDyK was prepared via solid-phase synthesis (SPS), and then was cleaved from resin using 2% TFA in DCM. Cyclization of protected RGDyK was performed by treating with Diphenyl phosphoryl azide (DPPA). After ivDde on the lysine was deprotected, Fmoc-PEG4-OH was attached to the primary amine on the side chain of lysine, and then the Fmoc was deprotected using 20% piperidine in DMF. After HPLC purification, the resulting protected cyclo(RGDyK)-PEG4-NH2was conjugated with the N3—NOtB2using EDCI and DMAP. The purified cyclo(RGDyK)-PEG4-NOTA-N3was ligated with cyclo(CNGRC)-PEG4-BCN (“CNGRC” disclosed as SEQ ID NO: 1) (prepared by mixing BCN-PEG4-NHS with cyclo(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1) in pH˜8.5 PBS buffer) via strain-promoted alkyne-azide cycloaddition (SPAAC) between N3and BCN moieties. Rendered by the triazole formation after metal-free click reaction, the purified heterodimers (NGR-NOTA-RGD) were successfully labeled with64Cu,68Ga, and Al18F at 37° C., 70° C., and 90° C., respectively. Labeling results were monitored by the radio HPLC. Labeling yields were above 90% for64Cu and68Ga, close to 50% for Al8F. Example 9: PET Imaging Using the c(cNGRc)-c(RGDyK) Heterodimer in the Subcutaneous Xenograft Mouse Model In vivo PET/CT imaging was conducted in NCr nude mice bearing bxpc3 (human pancreatic adenocarcinoma cell line) and 4T1 (a murine breast cancer cell line that overexpresses integrin αvβ3 and CD13) tumor xenografts. Mice were injected with bxpc3 cells (1 million cells in 150 μL PBS) into the subcutaneous flank of the right shoulder and 4T1 cells (1 million cells in 150 μL PBS) into the subcutaneous flank of the left shoulder. Either the CNGRC-(68Ga)NOTA-RGDyK heterodimer (“CNGRC” disclosed as SEQ ID NO: 1), (68Ga)NOTA(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1), or (68Ga)NOTA(RGDyK) were injected into the bloodstream via tail vein injection. Blocking studies were conducted for the heterodimer studies by co-injecting 100 times of cyclo(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1) and cyclo(RGDyK). Small animal PET/CT was performed at 1 hour post injection of tracers (FIG.14). The heterodimer CNGRC-(68Ga)NOTA-RGDyK (“CNGRC” disclosed as SEQ ID NO: 1) showed improved enhanced in in vivo performance (such as longer blood retention, better tumor/non-tumor ratios). Example 10: PET Imaging Using the c(cNGRc)-c(RGDyK) Heterodimer in the Orthotopic Xenograft Mouse Model In vivo PET/CT imaging was conducted in Balb/c mice. One week after the orthotropic implantation of 1×106luciferase-transfected KPCP cancer cells into the pancreas of Balb/c mice, the mice were used for PET imaging. Either the CNGRC-(68Ga)NOTA-RGDyK heterodimer (“CNGRC” disclosed as SEQ ID NO: 1), (68Ga)NOTA(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1), or (68Ga)NOTA(RGDyK)] were injected into the bloodstream via tail vein injection. Blocking studies were conducted for the heterodimer studies by co-injecting 100× of CNGRC (SEQ ID NO: 1) and RGDyK. Small animal PET/CT was performed at 1 hour post injection of tracers (FIGS.15A-15C). The heterodimer CNGRC-(68Ga)NOTA-RGDyK (“CNGRC” disclosed as SEQ ID NO: 1) showed improved in in vivo performance (such as longer blood retention, better tumor/non-tumor ratios) (FIG.15A). Uptakes of the RGD-NGR heterodimer in muscle, blood, liver, spleen, kidney, pancrease, and orthotopic tumor were 0.10% ID/g, 0.10% ID/g, 1.8% ID/g, 1.10% ID/g, 2.2% ID/g, 0.36% ID/g, and 1.4% ID/g, respectively. Example 11: PET Imaging Using the c(cNGRc)-c(RGDyK) Heterodimer in the Spontaneous Transgenic Mouse Model In vivo PET/CT imaging was conducted in the genetically engineered KCH (Pdx1-Cre;K-RasG12D/+; HMGB1−/−) mouse model. High mobility group box 1 (HMGB1) is a critical regulator of autophagy, a major pathway for degradation of effete proteins and damaged organelles, and the conditional genetic ablation of HMGB1 limited to the pancreas inhibits autophagy, promotes proliferation, activates normally quiescent pathways, and renders mice extraordinarily sensitive to K-RasG12D/+-driven pancreatic carcinogenesis. The progression of PanINs from low grade PanINI to high grade PanIN3 could be observed as early as seven days (normally three-nine months) after birth in KCH (Pdx1-Cre;K-RasG12D/+;HMGB1−/−). PET imaging normally was performed˜6 weeks old KCH mice. Either the CNGRC-(68Ga)NOTA-RGDyK (“CNGRC” disclosed as SEQ ID NO: 1) heterodimer, (68Ga)NOTA(CNGRC) (“CNGRC” disclosed as SEQ ID NO: 1), or (68Ga)NOTA(RGDyK)] were injected into the bloodstream via tail vein injection. Blocking studies were conducted for the heterodimer studies by co-injecting 100× of CNGRC (SEQ ID NO: 1) and RGDyK. Small animal PET/CT was performed at 1 hour post injection of tracers (FIGS.16A-16D). The heterodimer CNGRC-(68Ga)NOTA-RGDyK (“CNGRC” disclosed as SEQ ID NO: 1) showed improved in vivo performance (such as longer blood retention, better tumor/non-tumor ratios) (FIG.16A). Uptakes of the RGD-NGR heterodimer in muscle, blood, liver, spleen, kidney, and malignant pancreas were 0.38% ID/g, 0.23% ID/g, 4.7% ID/g, 3.1% ID/g, 5.0% ID/g, and 5.9% ID/g, respectively. In addition, as compared to the clinically widely-used18F-FDG, which did not show any specific tumor uptake in the spontaneous transgenic mouse model (FIG.16B), the heterodimer CNGRC-(68Ga)NOTA-RGDyK (“CNGRC” disclosed as SEQ ID NO: 1) showed specific tumor uptake and good tumor/non-tumor ratios (FIG.16A). Example 12: In Vitro High-Throughput Screening Platform In a proof of concept study, a platform for screening different lengths of PEG spacers (PEG4, 8, 12 and 16) between RGD and AE105 was developed for the heterodimer targeting to avow and uPAR. Results showed that PEG12 was the best spacer, which was validated by the in vivo PET imaging. Preparation of chemicals for the above optimization are outlined inFIG.3(RGD and AE105 are peptides targeting to biomarker αvβ3 and uPAR, respectively):1) Ten NH2—PEGn-RGD peptides containing spacers of various PEG lengths (n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20) can be prepared by adding the corresponding Boc-PEGn-NHS to RGD in a PBS buffer (pH=8.2), followed by Boc deprotection with 95% TFA. Then, the prepared NH2-PEGn-RGD is mixed with photo-ODIBO-NHS in a PBS buffer (pH=8.2) to produce photo-OIDBO-PEGn-RGD (abbreviated to p-ODIBO inFIG.3).2) N3-PEG4-AE105 can be prepared by mixing AE105 with N3-PEG4-NHS in a PBS buffer (pH=8.2). In Vitro Optimization Procedures:1) N3-PEG4-AE105 will be mixed with one of the ten photo-ODIBO-PEGn-RGD peptides (n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20) in a 1:1 molar ratio to prepare ten mixed-targeting molecule stock solutions;2) One of the ten mixed-targeting molecule stock solutions will be added into a well in 96-well plate that is pre-seeded with cells, (in total ten wells are needed for ten mixed-targeting molecule solution);3) After the targeting molecules bind to the targeted receptors, the unbound targeting molecules will be washed off using a PBS buffer;4) A UV lamp (365 nm) will be applied to deprotect the azide-inactive photo-ODIBO and generate azide-active “ODIBO”, subsequently triggering ligation between the N3-PEG4-AE105 and ODIBO-PEGn-RGD (both bind to biomarkers on the cells);5) After being incubated for an additional 2-4 h, N3-(64Cu)NOTA will be added to react with the “excess” ODIBO-PEGn-RGD (that binds to cancer cells, but does not react to N3-PEG4-AE105);6) The excess N3-(64Cu)NOTA will be washed off using a PBS buffer; and N3-(64Cu)NOTA can be retained on cells only after it ligates to the “excess” ODIBO-PEGn-RGD.7) The 96-well plate will be then be loaded into a high-throughput MicroBeta2 Plate Counter to measure the N3-(64Cu)NOTA ligated to “excess” ODIBO-PEGn-RGD on cells. The well with the lowest radio-counts, containing the lowest “excess” ODIBO, should get the highest amount of ligation product (between AE105-PEG4-N3 and ODIBO-PEGn-RGD), thus the corresponding spacers are of an appropriate length. Example 13: High-Throughput Screening Platform for Heterodimer Spacer Optimization A high throughput cell-based universal platform for rapid heterodimer spacer optimization has been developed to generate heterodimers with high avidity effects. By using the developed platform, the repetition of the traditional approach, which requires repeated synthesis and evaluation of a heterodimer library, is avoided. The platform can screen heterodimers with various spacers to identify a heterodimer with the best performance in in vitro and/or in vivo evaluations. Methodology Two ligands of interest, RGD (targeting to integrin αvβ3) and AE105 (targeting to urokinase-type plasminogen activator receptor (uPAR)), were functionalized with a photo ODIBO group and N3group, respectively, for the in-situ formation of a heterodimer. Herein, the photo-ODIBO group is a photo-triggered metal-free click chemistry moiety, which can be deprotected to ODIBO and react with azide via the strain-promoted alkyne-azide cycloadditions (SPAAC) upon UV 365 nm irradiation. To offer the capability of high-throughput screening and facilitate its application in research groups, the preparation of chemical tools was designed to avoid complex purifications (seeFIG.23). In particular, photo-ODIBO-PEG4-RGD was prepared via treating RGD dissolved in DMSO with 6 eqv. DIEA and 3 eqv. ODIBO-PEG4-NHS. After the pegylation was completed, 1×PBS was added to the reaction mixture so that the excess photo-ODIBO-PEG4-NHS could be hydrolyzed to non-cell reactive photo-ODIBO-PEG4-COOH. Parallel synthesis of four N3functionalized AE105 analogues with different spacers was conducted via in a similar way via incubating AE105 with N3-PEGn-NHS (one of the four selected PEG spacers for each analogue), followed by hydrolyzing excess NHS with 1×PBS. Without further purification, the resulting photo-ODIBO-PEG4-RGD and four N3-PEGPn-AE105 solutions could be directly applied in the following heterodimer spacer optimization experiments. Spacer optimization was performed as illustrated inFIG.17: the photo-ODIBO-PEG4-RGD and one of the N3-PEGn-AE105 prepared above were mixed and then added into a 96-well plate that pre-seeded with u87MG cells (a human brain cancer cell line which over expressed both integrin αvβ3 receptor and uPAR). Those cells were pre-fixed with 4% paraformaldehyde to minimize the internalization. After a 2 h incubation to allow sufficient binding of RGD and AE105 to integrin αvβ3 and uPAR respectively, the excess (unbound) ligands were washed off using PBS buffer. Then the plate was irradiated with a UV lamp (365 nm) for 2 minutes to deprotect the azide-inactive photo-ODIBO to the azide-active “ODIBO”, triggering the metal-free click reaction between the N3-PEGn-AE105 and the ODIBO-PEG4-RGD. After being incubated for an additional 2 h to allow the completion of the metal-free click reactions, (64Cu)NOTA-N3was added as a radio scavenger to click with the “excess” ODIBO-PEG4-RGD (that binds to cells, but did not click with N3-PEGn-AE105). Upon the removal of unbound (64Cu)NOTA-N3, the (64Cu)NOTA-N3clicked to ODIBO-PEGn-RGD was measured on MicroBeta2 Plate Counter. One group without UV irradiation was used as a background control to get counts resulting from the non-specific binding of (64Cu)NOTA-N3. After subtracting the background counts due to the non-specific binding, the well with the lowest radioactivity counts contained the least amount of (64Cu)NOTA-click-PEG4-RGDso as the highest amount of the in situ generated heterodimer (AE105-PEGn-click-PEG4-RGD), indicating the corresponding spacer length (PEGn+4) will be the most suitable for achieving high avidity. Compared with the traditional strategy, this platform avoided the abundant synthesis and evaluation of a heterodimer library consisting of heterodimers bearing varied spacers. In addition, owning to the high sensitivity of the beta counter, ligands were consumed at a nanomole scale for each test so that the cost of expensive starting materials was significantly reduced. Taking into account advantages of convenience, sensitivity, and capability on high throughput screening, this universal rapid spacer optimization platform can greatly facilitate the development of heterodimeric pharmaceuticals for research and/or clinical applications. Results and Discussion Firstly, the distance between one integrin αvβ3 receptor and one uPAR on a cell surface was estimated to select spacers of proper length for screening, and it was found that the possible distance between two receptors could be 5 nm or less. Given that the length of a single bond was around 1.5 Å (0.15 nm), the length of a PEG4unit consisting of 12 single bonds would be around 1.2 nm, when taking the bond angle into account. Therefore, to cover the length from 1 nm to 5 nm, 4 spacers consisting of PEG4, PEG8, PEG12, and PEG16were selected for the screening purpose (see Table 2). TABLE 2Spacers selected for in vitro screeningPEG unitsPEG unitsattached toattached toTotal PEGEstimatedentranceRGDAE105unitslength (nm)14041.224482.4348123.64412164.8 Then RGD-PEG4-photo-ODIBO and AE105-PEGn-N3(n=0, 4, 8, and 12) were prepared as shown inFIG.23. Due to the use of 3 eqv. R-PEG-NHS ester, conversion yields of peptidic ligands reached above 95% within 30 minutes, as monitored by HPLC.FIG.18shows an example of converting RGD into RGD-PEG4-photo-ODIBO, in which RGD, RGD-PEG4-photo-ODIBO, photo-ODIBO-PEG4-COOH, and photo-ODIBO-PEG4-NHS were eluted at 13, 19, 20, and 21 minutes respectively. InFIG.18, the HPLC conditions were as follows: 0-2 minutes, 100% H2O; 2-12 minutes, changing from 100% H2O to 80% H2O and 20% ACN; 12-22 minutes, changing from 80% H2O and 20% ACN to 10% H2O and 90% ACN; 22-26 minutes, 10% H2O and 90% ACN; 26-27 minutes changing from 10% H2O and 90% ACN to 100% H2O; 27-35 minutes, 100% H2O with a flow rate of 1.5 ml/min. Based on the quantitative results obtained from the HPLC spectra, after the reaction mixture was stirred for 0.5 h at room temperature, the RGD conversion yield was above 95%, and less than 5% photo-ODIBO-PEG4-NHS was hydrolyzed to photo-ODIBO-PEG4-COOH. After the addition of 1×PBS, the reaction mixture was allowed to stand overnight to maximize hydrolysis of photo-ODIBO-PEG4-NHS, and only RGD-PEG4-photo-ODIBO as well as photo-ODIBO-PEG4-COOH remained in the reaction mixture. Similar observations were obtained when the AE105-PEGn-N3(n=0, 4, 8, and 12) was prepared. Because neither photo-ODIBO-PEG4-COOH nor N3-PEGn-COOH would bind to cells due to the lack of a targeting ligand, they were washed away together with unbound RGD-PEG4-photo-ODIBO or AE105-PEGn-N3. Therefore, the resulting five reaction mixtures can be directly applied in the cell based screening without further purification. Subsequently, the RGD-PEG4-photo-ODIBO was parallelly mixed with either AE105-PEG0-N3or AE105-PEG4-N3or AE105-PEG8-N3or AE105-PEG12-N3, resulting in four groups of stock solutions each containing both RGD-PEG4-photo-ODIBO and one of the AE105-PEGn-N3(n=0, 4, 8, 12). As illustrated inFIG.17, the four groups of stock solutions were applied in the designed cell based screening assay using u87MG cells pre-fixed with 4% paraformaldehyde. In addition to the four experimental groups, a negative control group was prepared in which cells were treated with RGD-PEG4-photo-ODIBO and NH2—PEG0-AE105; thus, no heterodimer could be generated in this negative control group as there was no ligation between ODIBO and NH2. Additionally, there was a background control group, in which no UV irradiation was applied; thus, the amount of (64Cu)NOTA-N3detected was caused by its non-specific binding on cells. After subtracting the non-specific binding recorded in the background control group, the specific bindings of (64Cu)NOTA-N3in different groups caused by its ligation with the RGD-PEG4-ODIBO were compared. As shown inFIG.19, the groups treated with N3-PEG4-AE105 and N3—PEG8-AE105 exhibited less amount of the specific binding of (64Cu)NOTA-N3, suggesting that RGD-PEG8-AE105 and RGD-PEG12-AE105 were the two most abundant heterodimers formed on u87MG cells. Accordingly, the two corresponding spacers (entrance 2 & 3) were the most potent among the four tested spacers. Finally, the result obtained from the above screening assay was validated both in vitro and in vivo. The four RGD-AE105 heterodimers possessing various PEG spacers (PEG4, PEG8, PEG12and PEG16, respectively) were prepared as described in the previous examples. The prepared heterodimers were then radiolabeled with either Cu-64 or Ga-68 for in vitro and in vivo evaluation respectively. Based on the cell uptake result as shown inFIG.20A, the PEG16-containing heterodimer exhibited the highest cell uptake, followed by the PEG12-, PEG8-, and PEG4-contained heterodimers with 4 h uptake values of 0.46%, 0.38%, 0.24% and 0.16% respectively. In the cell efflux study (FIG.20B), the PEG8and PEG12-containing heterodimers showed the best cell retention, followed by the PEG4-, and PEG16-containing heterodimers with 2 h retention values of 44%, 43%, 35% and 26%, respectively. Taking into account both the cell uptake and efflux results among the four tested heterodimers, the PEG12-containing heterodimer demonstrated the highest potential in this in vitro evaluation, consistant with the result obtained from the designed cell based screening assay. Further in vivo validation was conducted by comparing PET imaging results obtained from mice bearing u87MG xenografts. As shown inFIG.21, tumors could be visualized by using all the four tested heterodimeric PET tracers while the PEG12-contained heterodimer exhibited the highest tumor to background contrast, followed by the PEG8-, PEG16-, and PEG4-containing heterodimers. Quantitative tumor uptake values were subsequently revealed by the region of interest (ROI) analysis (FIG.22). The tumor uptake value of the PEG12-containing heterodimer was 2.8%, while those of PEG8-, PEG16-, and PEG4-containing heterodimers were 2.4%, 2.1% and 1.7%, respectively, reaffirming the result obtained from the designed cell based screening assay. Collectively, results from both in vitro and in vivo evaluations successfully validated the accuracy and reliability of the rapid spacer-optimization platform. The selected AE105-PEG12-RGD was further compared with two corresponding monomer AE105 and RGD via PET imaging of u87MG xenografts on nude mice. Superior imaging results were obtained in mice administrated with the heterodimer, indicating its better in vivo performance than the two monomer counterparts due to the avidity effects. Thus, using a photo-triggered metal-free click reaction, a universal in vitro screening platform can be established for simplifying the spacer optimization process involved in developing high avidity heterodimers, which can be broadly applied to various dual-biomarker combinations and different diseases. The developed screening platform was successfully applied in the spacer optimization of the integrin αvo3-uPAR dual-targeted heterodimeric ligand. The accuracy and reliability of this platform was further validated via both in vitro and in vivo evaluations, in which heterodimers containing all the tested spacers were prepared and evaluated individually. In addition to the demonstrated capability of high throughput screening (as shown inFIG.17), this universal platform can significantly accelerate and/or enhance the application of the dual-receptor-targeting strategy in various biomedical fields, particularly when targeted receptors are expressed in low abundance and/or when high affinity (and/or specificity) monovalent ligands are not available. Example 14: Exploring Spacer Lengths Eight NH2—PEGn-RGD peptides containing spacers of various PEG lengths (n=2, 4, 6, 8, 10, 12, 14, 16) will be prepared by adding the corresponding Boc-PEGn-NHS to RGD in a PBS buffer (pH=8.2), followed by Boc deprotection. Photo-ODIBO-NHS, prepared using previously reported procedures, will then be mixed with the prepared NH2—PEGn-RGD in a PBS buffer (pH=8.2) to produce photo-OIDBO-PEGn-RGD. N3-PEG4-cetuximab will be prepared using previously reported procedures. N3—PEG4-cetuximab and the eight photo-ODIBO-PEGn-RGD peptides (n=2, 4, 6, 8, 10, 12, 14, 16) will be used for in vitro screening (at 4° C. to minimize the internalization of targeting probes). As shown inFIG.11: 1): eight mixed-ligands stock solutions will be prepared by mixing N3-PEG4-cetuximab with one of the eight photo-OIDBO-PEGn-RGD peptides; 2) U87MG cells will be cultured in a 96-well plate; 3) one of the above eight mixed-ligands stock solution will be added into each well (eight wells in total) pre-seeded with U87MG; 2) after the ligands bind to the targeted receptors, the excess (unbound) targeting ligands will be washed off using a PBS buffer (repeated 5 times to ensure complete removal); 3) a UV lamp (365 nm) will be applied to deprotect the azide-inactive photo-ODIBO and generate azide-active “ODIBO”, subsequently triggering ligation between the N3-PEG4-cetuximab and ODIBO-PEGn-RGD; 4) after being incubated for an additional 2 h,64Cu-labeled N3—NOTA will be added to click with the “excess” ODIBO-PEGn-RGD (that binds to cells, but does not click to N3-PEG4-cetuximab); and 5) the excess N3-(64Cu)NOTA will be removed, and the N3-(64Cu)NOTA clicked to “excess” ODIBO-PEGn-RGD will be measured on MicroBeta2 Plate Counter. One group without UV irradiation will be used as a negative control to get counts from the non-specific binding of N3-(64Cu)NOTA. After subtracting the non-specific binding, the specific binding of N3-(64Cu)NOTA obtained from the eight ODIBO-PEGn-RGD (n=2, 4, 6, 8, 10, 12, 14, 16) will be compared. The well with the lowest specific binding will contain the highest amount of clicking product (between cetuximab-PEG4-N3and ODIBO-PEGn-RGD), thus the corresponding spacer will be the most potent. The ODIBO-PEGn-RGD containing the most potent PEG spacer will click with Tz-NOTA-N3and then be radiolabeled with64Cu, and the resulting Tz-(64Cu)NOTA-PEGn-RGD will be used for the in vitro avidity studies on U87MG cells. Tz-(64Cu)NOTA-RGD (without a PEG spacer) will be used as a negative control because the distance between RGD and cetuximab in the resulting heterodimer is too short to achieve avidity effect (proved in preliminary study,FIG.5B). Briefly, Tz-(64Cu)NOTA-PEGn-RGD/TCO-PEG4-cetuximab ligation product (cetuximab-PEG4-(64Cu)NOTA-PEGn-RGD) will be used for cell uptake/efflux, binding affinity and Bmax measurements on U87MG cells. After high avidity effect is confirmed on the above ligation product, in vivo evaluation will be performed then. Mice bearing U87MG xenografts will be pre-injected with 100 μg of TCOPEG4-cetuximab, and 24 h later, ˜250-350 pCi of Tz-(64Cu)NOTA-PEGn-RGD (or Tz-(64Cu)NOTA-RGD in the negative control group) will be injected. Then 1 h dynamic PET scans will be performed at multiple time points (p.i., 4, 18, and/or 28 h). As cetuximab is cleared through the liver, kinetics on tumor and liver at mid and late time points can be evaluated. At mid/late time points (4, 18, 28 h) when most of the un-ligated Tz-(64Cu)NOTAPEGn-RGD has been washed off, observation of relatively slower tumor washing out and faster liver clearing (compared to that from Tz-(64Cu)NOTA-RGD) can indicate the much stronger binding with tumor cells, and thus an avidity effect of in vivo ligation product (cetuximab-PEG2-(64Cu)NOTA-PEGn-RGD) is being achieved. Various references are cited in this document, which are hereby incorporated by reference in their entireties herein. | 110,458 |
11857649 | DETAILED DESCRIPTION In a particular embodiment, the curable substance can be used as construction material in an additive manufacturing process using a digital data model, preferably in 3D printing and in particular for producing a product for use in the field of optics, preferably for the production of lenses and/or filters. The curable substance of the invention for producing a material can be produced by mixing the following starting materials:one or more polymerizable monomer(s),one or more strontium, zirconium, lead, barium, bismuth or rare earth compound(s) which is/are soluble in the monomer or in the monomer mixture,one or more curing initiator(s) and optionallyone or more auxiliaries. At least one of the polymerizable monomers is preferably selected from the group consisting of free-radically curable monomers, and at least one of the polymerizable monomers is particularly preferably selected from the group consisting of acrylic acid, acrylates, methacrylic acid or methacrylates or derivatives thereof. Examples of suitable free-radically curable monomers of acrylic acid or methacrylic acid are: methyl methacrylate, ethyl methacrylate, isopropyl methacrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl methacrylate, 2-hydroxy-1,3-dimethacryloxypropane, n-butyl methacrylate, isobutyl methacrylate, hydroxypropyl methacrylate, glycidyl methacrylate, 2-methoxyethyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate, 2,2-bis(methacryloxyphenyl)propane, 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane, 2,2-bis(4-methacryloxydiethoxyphen-yl)propane, 2,2-bis(4-methacryloxypolyethoxyphenyl)propane, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, butylene glycol dimethacrylate, neopentyl glycol dimethacrylate, 1,3-butanediol dimethacrylate, 1,4-butanediol dimethacrylate, 1,6-hexanediol dimethacrylate, trimethylolpropane trimethacrylate, trimethylolethane trimethacrylate, pentaerythritol trimethacrylate, trimethylolmethane trimethacrylate, pentaerythritol tetramethacrylate and also methacrylates having a urethane bond in their derivatized compound. Very particular preference is given to using the following curable substances or dental materials selected from the group consisting of methacrylic acid, butyl diglycol methacrylate, urethane dimethacrylate, isobornyl methacrylate, tetrahydrofurfuryl methacrylate, 1,4-butanediol dimethacrylate, 2-[[(butylamino)carbonyl]oxy]ethyl acrylate, bisphenol A dimethacrylate and/or methyl methacrylate. For polymerization, the abovementioned monomers are mixed as main constituent, e.g. of dental materials, with at least one curing initiator for free-radical polymerization and optionally with additional monomers and with one or more strontium, zirconium, lead, barium, bismuth or rare earth compound(s) described below and optionally with auxiliaries. The mixtures obtained in this way can be cured by free-radical polymerization. Both the curable compositions and also the cured products, materials, are provided by the present invention. The metals of the “rare earths” of the Periodic Table of the Elements include the chemical elements of the third transition group of the Periodic Table, with the exception of actinium, and the lanthanides, a total of 17 elements. According to the definitions of inorganic nomenclature, this group of chemically similar elements is also referred to as rare earth metals. The compounds derived therefrom are, inter alia, subject matter of the present invention as per claim1. The rare earth compounds used according to the invention are derived firstly from the lighter rare earth metals, for example scandium (Sc, 21), lanthanum (La, 57), cerium (Ce, 58), praseodymium (Pr, 59), neodymium (Nd, 60), promethium (Pm, 61), samarium (Sm, 62) and europium (Eu, 63), and secondly from the heavier rare earth metals, for example yttrium (Y, 39), gadolinium (Gd, 64), terbium (Tb, 65), dysprosium (Dy, 66), holmium (Ho, 67), erbium (Er, 68), thulium (Tm, 69), ytterbium (Yb, 70), lutetium (Lu, 71). In addition, the present invention encompasses compounds of the elements strontium (Sr, 38), zirconium (Zr, 40) lead (Pb, 82) barium (Ba, 56) and bismuth (Bi, 83) which are soluble in the monomer or the monomer mixture. Suitable initiators for the free-radical polymerization are the initiators which are well known from the prior art for hot curing, cold curing and photocuring. Suitable initiators are indicated, for example, in the Encyclopedia of Polymer Science and Engineering, vol. 13, Wiley-Interscience Publishers, New York 1988. Customary thermal initiators are, for example, azo compounds such as azobis(isobutyr-onitrile) (AIBN) or azobis(4-cyanovaleric acid) or peroxides such as dibenzoyl peroxide, dilauryl peroxide, tert-butyl peroctoate, tert-butyl perbenzoate or di(tert-butyl)peroxide. UV curing initiators which are preferred for the purposes of the present invention are embodied by compounds from the group of phosphine oxides, preferably diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) and/or 2,4,6-trimethylbenzoylphenylphosphinate (TPO-L) and/or bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (BAPO), and/or camphorquinone and/or a compound from the group of the thioxanthones. For the purposes of the present invention, auxiliaries are first and foremost organic acids, with the organic acid preferably being an organic carboxylic acid. The organic carboxylic acid is particularly preferably an aliphatic and/or aromatic carboxylic acid and/or an aralkylcarboxylic acid. The aliphatic carboxylic acid or aralkylcarboxylic acid can be a branched and/or unbranched carboxylic acid and/or a substituted or unsubstituted, saturated and/or unsaturated carboxylic acid and/or a carboxylic acid derivative which is functionalized on the carboxyl moiety. An aralkylcarboxylic acid derivative is very particularly preferably selected from the group consisting of phenyl acetic acid and 3-phenylpropionoic acid and trans-cinnamic acid. Further suitable organic acids are unsaturated polymerizable monocarboxylic, dicarboxylic or polycarboxylic acids or derivatives thereof, with such carboxylic acids or carboxylic acid derivatives being able to have from 3 to 25, preferably from 3 to 15 and particularly preferably from 3 to 9, carbon atoms and being able to be branched or unbranched, substituted, preferably by a phenyl substituent, or unsubstituted. Examples which may be mentioned are acrylic acid, fumaric acid, maleic acid, citraconic acid, cinnamic acid, itaconic acid, sorbic acid and mesconic acid, which can also be used in mixtures. Furthermore, the auxiliary can preferably be embodied by a complex former which is capable of forming a complex with an ion of the rare earth metals or of strontium, zirconium, lead, barium and/or bismuth. The complex former preferably has at least one carbonyl function and/or at least one carboxyl function which is/are capable of forming a coordinate bond with an ion of the rare earth metals and/or of strontium and/or of zirconium and/or of lead and/or of barium and/or of bismuth. The complex former particularly preferably has an acetylacetone or acetylacetonate moiety. The complex former very particularly preferably has a polymerizable moiety which is embodied by a free-radically polymerizable function and which can preferably be an ethylenically unsaturated group, for example optionally multiply unsaturated carboxylic acids which preferably comprise methacryl moieties. The complex former is even more preferably selected from the group consisting of 2-methacryloyloxyethyl acetoacetate (AAEMA), bis(2-methacryloyloxyethyl) pyromellitate, methacryloyloxyethyl phthalate, methacryloyloxyethyl maleate and methacryloyloxyethyl succinate. In practical terms, the strontium, zirconium, lead, barium, bismuth and/or rare earth compound(s) are provided in the form of compounds from which the corresponding monomer-soluble compounds are formed in-situ in concentrations which make radiopacity possible in the later polymer. In a preferred embodiment, the lead, barium, bismuth and/or rare earth compound(s) are provided in the form of their compounds or complexes which are soluble in the monomers or monomer mixture(s). Due to the presence of metal ions in solution, the problem of sedimentation at the low viscosity required for additive manufacture, as occurs, for example, when using fillers, does not occur. Consequently, the solutions or printing materials can be stored without problems, so that the risk of blocking of the printer nozzles by agglomerated particles in ink-jet-based systems can be avoided. Furthermore, the materials do not contain any particles which can interfere in photopolymerization by absorbing and scattering the incident light. The polymerized or cured materials obtained are clear and colorless and do not have any particles which can be regarded as defects and could thus impair the mechanical properties, as is evidenced for various elements (nickel and copper are not according to the present invention) inFIG.1. The macromolecular materials obtained after polymerization can also be polished very well since they do not contain any interfering filler particles. The X-ray contrast agents dissolved in the polymer are always homogeneously distributed in the resin mixture and the polymerized materials, which ensures excellent reproducibility, as is evidenced byFIG.2. FIG.2shows, in the first row, X-ray images (taken at a voltage of 55 kV applied to the X-ray tubes) of illustrative samples (denoted correspondingly). The first row of the image of the test specimens commences with the standard aluminum (Al) followed by a sample which does not have any metal content at all (0). This is followed by pictures of samples having a content of 5 and 10% by weight of praseodymium (correspondingly denoted by 5 Pr and 10 Pr). In the second row, X-ray images of test specimens containing 5 and 10% by weight of erbium (5 Er 5 and 10 Er) are shown, followed by pictures of samples containing in each case 5% by weight of ytterbium (5 Yb) or barium (5 Ba). The last row shows, inter alia, the X-ray image of a sample containing 5% of weight of lead (5 Pb). It is thus also possible to form complex radiopaque constructions which can be produced in classical manual work, e.g. using autopolymers. The additively manufactured constructions fit as intended since the polymerization shrinkage which occurs is taken into account or included in the calculation beforehand by the CAD/CAM software. The use of polymerizable strontium, zirconium, barium, lead, bismuth or rare earth compounds significantly reduces migration. Finally, the inventive systems or curable substances have an adjustable, low viscosity in the range from 500-3000 mPas, while the composites known from the prior art are in the range significantly above 40 000 mPas. EXAMPLES 1. Production of Radiopaque, Praseodymium-Containing Polymer for the Production of Drilling Templates on a DLP Printer with 385 nm.1.21 g of praseodymium carbonate5.03 g of methacrylic acid2.03 g of butyl diglycol methacrylate6.96 g of urethane dimethacrylate0.31 g of TPO-L. The mixture results in a clear, green solution, which, after the polymerization reaction, forms a clear, green platelet. The radiopacity is about 70% of Al at somewhat less than 5% of Pr.2. Production of Radiopaque, Europium-Containing Polymer1.22 g of europium carbonate4.06 g of 3-phenylpropionic acid2.18 g of methacrylic acid2.60 g of isobornyl methacrylate5.32 g of urethane dimethacrylate0.37 g of TPO-L The mixture results in a clear, slightly yellowish solution, which, after the polymerization reaction, forms a clear, yellowish platelet. The radiopacity is about 73% of Al at somewhat less than 5% of Eu.3. Production of radiopaque, erbium-containing polymer2.34 g of erbium carbonate2.51 g of 3-phenylpropionoic acid2.91 g of phenyl acetic acid3.01 g of methacrylic acid2.00 g of isobornyl methacrylate3.17 g of butyl glycol methacrylate0.27 g of TPO The mixture results in a clear, pink solution, which, after the polymerization reaction, forms a clear, pink platelet. The radiopacity is about 85% of Al at 10% of Er.4. Production of radiopaque lead-containing polymer for use as printing material, e.g. for the production of X-ray-absorbing windows3.28 g of lead oxide3.02 g of 3-phenylpropionoic acid3.24 g of methacrylic acid0.66 g of isobornyl methacrylate4.93 g of urethane dimethacrylate0.33 g of TPO-L The mixture results in a clear, slightly brownish solution, which, after the polymerization reaction, forms a clear, light-brown platelet. The radiopacity is 162% of Al at somewhat less than 20% of lead.5. Production of radiopaque, barium-containing polymer I1.26 g of barium hydroxide, anhydrous2.99 g of methacrylic acid0.99 g of trans-cinnamic acid4.03 g of butyl diglycol methacrylate2.08 g of methacrylic anhydride7.78 g of urethane dimethacrylate0.36 g of TPO The mixture results in a clear, slightly yellowish solution, which, after the polymerization reaction, forms a clear, slightly yellowish platelet. The radiopacity is about 70% of Al at somewhat less than 5% of Ba.6. Production of radiopaque, barium-containing polymer II2.49 g of barium hydroxide, anhydrous6.03 g of methacrylic acid0.54 g of phenylacetic acid6.67 g of urethane dimethacrylate0.36 g of TPO The mixture results in a clear, slightly yellowish solution, which, after the polymerization reaction, forms a clear, slightly yellowish platelet through which it is possible to read without problems. The radiopacity is about 100% of Al at somewhat less than 10% of Ba.7. Production of holmium-containing polymer suitable for applications in optics and for surface coating1.19 g of holmium carbonate4.04 g of 3-phenylpropionoic acid2.17 g of methacrylic acid0.66 g isobornyl methacrylate5.88 g of urethane dimethacrylate0.36 g of TPO-L The mixture results in a clear, yellowish solution, which, after the polymerization reaction, forms a clear, yellowish platelet. The radiopacity is about 67% of Al at somewhat less than 5% of Ho. Under illumination with artificial light, the monomer mixture and platelet have a distinct pink color, although they merely appear light-yellow in daylight (FIG.1, test specimen G).8. Production of a zirconium-containing polymer2.27 g of zirconium(IV) methacrylate3.29 g of tetrahydrofurfuryl methacrylate1.06 g of methacrylic acid2.05 g of triethylene glycol dimethacrylate7.41 g of phenylglycerol dimethacrylate0.36 g of TPO-L The mixture results in a clear, yellowish solution, which, after the polymerization reaction, forms a clear, yellowish platelet.9. Production of a strontium-containing polymer0.28 g of strontium hydroxide4.39 g of tetrahydrofurfuryl methacrylate2.04 g of methacrylic acid2.71 g of mono-2-methacryloyloxyethyl succinate5.95 g of bisphenol A glycidyl methacrylate0.32 g of TPO-L The mixture results in a clear, virtually colorless solution, which, after the polymerization reaction, forms a clear platelet. The present invention thus provides a curable substance for producing a material, which substance can be produced by mixing starting materials, wherein:one or more polymerization monomer(s),one or more strontium, zirconium, lead, barium, bismuth or rare earth compound(s) which is/are soluble in the monomer or in the monomer mixture,one or more curing initiator(s) and optionallyone or more auxiliaries are used as starting material to be mixed. The present invention preferably provides a curable substance for which at least one of the polymerizable monomers is selected from the group consisting of free-radically curable monomers. Furthermore, the present invention preferably provides a curable substance for which at least one of the polymerizable monomers is selected from the group consisting of acrylic acid, acrylates, methacrylic acid and methacrylates and derivatives thereof. Furthermore, the present invention preferably provides a curable substance for which the acrylic acid derivative and/or the methacrylic acid derivative is selected from the group consisting of acrylic esters, methacrylic esters, acrylamide and methacrylamide. Furthermore, the present invention preferably provides a curable substance for which the acrylic acid derivative and/or methacrylic acid derivative is selected from the group consisting of methacrylic acid, butyl diglycol methacrylate, urethane dimethacrylate, isobornyl methacrylate, tetrahydrofurfuryl methacrylate, 1,4-butanediol dimethacrylate, 2-[[(butylamino)carbonyl]oxy]ethyl acrylate, bisphenol A dimethacrylate and methyl methacrylate. Furthermore, the present invention preferably provides a curable substance in which the lead, barium, bismuth or rare earth compound is a polymerizable strontium, zirconium, lead, barium, bismuth or rare earth compound and/or an inorganic or organic rare earth salt or a complex. Furthermore, the present invention preferably provides a curable substance in which the lead, barium, bismuth and/or rare earth compound is present in a concentration which makes radiopacity possible. Furthermore, the present invention preferably provides a curable substance in which the curing initiator is a UV curing initiator. Furthermore, the present invention preferably provides a curable substance in which the curing initiator consists of a two-component redox system and which contains, as auxiliary, a pulverulent component which after mixing with the liquid component gives a self-curing substance. Furthermore, the present invention preferably provides a curable substance in which the UV initiator is from the group of phosphine oxides, preferably diphenyl(2,4,6-tri-methylbenzoyl)phosphine oxide (TPO) and/or 2,4,6-trimethylbenzoylphenyl phosphinate (TPO-L) and/or bis(2,4,6-trimethylbenzoyl)phenyl phosphine oxide (BAPO) and/or camphorquinone and/or a compound from the group of thioxanthones. Furthermore, the present invention preferably provides a curable substance in which the auxiliary is an organic acid. Furthermore, the present invention preferably provides a curable substance in which the organic acid is an organic carboxylic acid. Furthermore, the present invention preferably provides a curable substance in which the organic carboxylic acid is an aliphatic, aromatic and/or an aralkyl carboxylic acid. Furthermore, the present invention preferably provides a curable substance in which the carboxylic acid is a substituted or unsubstituted, saturated and/or unsaturated branched and/or unsaturated carboxylic acid and/or a carboxylic acid derivative correspondingly functionalized on the carboxyl moiety. Furthermore, the present invention preferably provides a curable substance in which the carboxylic acid derivative is phenylacetic acid and/or 3-phenylpropionic acid and/or trans-cinnamic acid. Furthermore, the present invention preferably provides a curable substance in which the auxiliary is a complex former which is capable of forming a complex with an ion of the rare earth metals. Furthermore, the present invention preferably provides a curable substance in which the complex former has at least one carbonyl function and/or at least one carboxyl function which is/are capable of forming a coordinate bond with an ion of the rare earth metals. Furthermore, the present invention preferably provides a curable substance in which the complex former has an acetylacetone or acetylacetonate moiety. Furthermore, the present invention preferably provides a curable substance in which the complex former has a polymerizable moiety. Furthermore, the present invention preferably provides a curable substance in which the polymerizable moiety is represented by at least one free-radically polymerizable group. Furthermore, the present invention preferably provides a curable substance in which the complex former is selected from the group consisting of 2-methacryloyloxyethyl acetoacetate (AAEMA), bis(2-methacryloyloxyethyl) pyromellitate, methacryloyloxyethyl phthalate, methacryloyloxyethyl maleate, methacryloyloxyethyl succinate and derivatives thereof. Furthermore, the present invention preferably provides a curable substance in which the complex former, but preferably the entire curable mixture, has, at room temperature, a vapor pressure of less than 1 mbar at 20° C., particularly preferably less than 0.3 mbar at 20° C. and very particularly preferably a vapor pressure of less than 0.1 mbar at 20° C. Furthermore, the present invention preferably provides a curable substance, where the curable substance after polymerization gives a material which is transparent to electromagnetic waves in the range of visible light and may be colored due to the metal ions present. Furthermore, the present invention preferably provides a curable substance for use in a procedure for the surgical or therapeutic treatment of the human or animal body and/or for use in a diagnostic procedure carried out on the human or animal body, preferably for specific usein a therapeutic procedure for temporary or permanent filling of a dental cavity and also in a therapeutic procedure astooth filling material,dental cement,dental underfilling material,as flowable composite material (flow material),as crown material,as inlay and/or onlay,as drilling templateand/or as stump buildup materialand/or in a diagnostic procedure asdrilling templateX-ray contrast agent. In addition, the present invention provides a method for producing a curable substance, which comprises the following steps:(i) production or provision of the starting materials as defined in any of claims1to21, orproduction or provision of intermediates derived from the starting materials as defined above,(ii) mixing of the starting materials produced or provided as per step (i) or the intermediates produced or provided as per (i) so as to result in each case in the curable substance. The present invention preferably further provides for the use of a curable substance, as defined above, in 3D printing. The present invention particularly preferably further provides for the use of a curable substance, as defined above, as construction material in an additive manufacturing process using a digital data model. The present invention very particularly preferably further provides for the use of the curable substance, as defined above, for producing a dental product, preferably for producing a dental product selected from the group consisting of artificial teeth, inlays, onlays, crowns, bridges, milling blanks, implants and finished tooth parts and also drilling templates. In addition, the present invention provides for the use of a curable substance for producing a product for use in the field of optics, preferably for producing lenses and/or filters. The present invention further provides a method for producing a dental product by means of an additive manufacturing process using a digital data model, which comprises the steps:(i) production or provision of a curable dental material as defined above, preferably production by the abovementioned method, and(ii) processing of the curable dental material produced or provided in the additive manufacturing process using a digital data model so as to result in the dental product or a precursor of the dental product, where the dental product is preferably selected from the group consisting of artificial teeth, inlays, onlays, crowns, bridges, milling blanks, implants, drilling templates and finished tooth parts. The present invention further provides a cured substance or a material obtainable by polymerization of polymerizable monomers in a curable substance as defined above. The present invention further provides a kit comprisingone or more than one syringe and(i) one, two or more than two curable substances as defined above and/or(ii) one, two or more than two base pastes and one, two or more catalyst pastes, with a curable substance, as defined above, being obtainable by mixing of a base paste and the appropriate catalyst paste. | 24,131 |
11857650 | COMPARATIVE EXAMPLE 1 US 20060241205 ‘Filler Materials For Dental Composites’ In example E and G to I of US 20060241205, pastes are disclosed containing a polymerizable resin mixture and silane-treated glass flakes in combination with a further filler. The silane-treated glass flakes have a thickness of 5 μm and either an average glass flake dimension of about 15 microns or of about 160 microns. In case of examples E and H the ratio of flakes to silane-treated glass filler is 40 to 60. Following those examples, pastes containing silanized flakes of 350 nm and 5 μm, respectively, and 0.6 μm Ba glass were prepared. The total filler content of 74.0% and a flake to 0.6 μm Ba glass ratio of 40 to 60 was used. The flakes used for paste preparation are listed in the following table 4: TABLE 4experimental pastes containing flakes of various nominal thicknessUntreated Glass FlakesMedianPasteNominalGrindingflake sizeFIG.No.NameThickness1)timed3,50no.3)1ECR GF350ca. 350nm1 h17.1 μmFIG. 2nmM2ECR GF350ca. 350nm2 h11.4 μmFIG. 3nmM3ECR GF0074-6μmno grinding30.8 μmFIG. 74ECR GF0074-6μm212.0 μmFIG. 8passages2)1)Nominal flake thickness acc. to manufactures technical data sheet2)Instead of circulating the suspension via the pearl mill, the flake suspension was pumped once via the pearl mill and collected at the mill outlet in a separated bucket (=1 passage). When necessary this procedure was repeated.3)SEM pictures were taken after silanization according to example 2 After grinding the flakes were silanized according to Example 2 and the pastes prepared according to example 3. The results of the flexural strength and E-modulus determinations of the four pastes are summarized in Table 5: TABLE 5experimental pastes containing flakes of various nominal thicknessFlexural strength1)E-modulusPaste No.MPaGPa116112.1216311.7314410.241419.31)determined according to ISO 4049: 2009 From Table 5 it can be seen that for an identical formulation the flexural strength and E-modulus is favorably higher in case of the thinner flakes (nominal thickness of 350 nm, paste no. 1 and 2) compared to the thicker flakes (nominal thickness of 5 μm, paste no. 3 and 4). | 2,167 |
11857651 | DETAILED DESCRIPTION A major limitation of adding titanium dioxide nanoparticles (TiO2NPs) to dental resins has been that the UV wavelengths necessary for activation of TiO2are or can be dangerous to human cells and tissues. In addition, polymer-based dental materials, when exposed to UV wavelengths, have been demonstrated to undergo significant polymer degradation. As a solution to these problems, the present disclosure describes various novel doped TiO2nanoparticles (doped TiO2NPs) for use in polymer-based dental materials (and other materials), such as but not limited to adhesive resins and bonding agents. Examples of such doped TiO2NPs include but are not limited to nitrogen-doped titanium dioxide (TiO2-xNxNPs), also referred to herein as N—TiO2NPs, nitrogen/silver co-doped titanium dioxide (N—Ag—TiO2NPs), nitrogen/fluorine co-doped titanium dioxide (N—F—TiO2NPs), and nitrogen/phosphorus or nitrogen/phosphate co-doped titanium dioxide (N—P—TiO2NPs). The doped TiO2NPs have use, for example, as antibacterial, bond-promoting, and bioactive materials in polymer-based dental biomaterials. Further, doping and co-doping of titanium dioxide with ions such as nitrogen, fluorine, phosphate, and silver shift the absorption behavior of titanium dioxide from the UV range into the visible spectrum. Before further describing various embodiments of the compositions and methods of the present disclosure in more detail by way of exemplary description, examples, and results, it is to be understood that the embodiments of the present disclosure are not limited in application to the specific details of methods and compositions as set forth in the following description. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. The inventive concepts of the present disclosure are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive, and it is not intended that the present disclosure be limited to these particular embodiments. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the embodiments of the present disclosure may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. It is intended that all alternatives, substitutions, modifications and equivalents apparent to those having ordinary skill in the art are included within the scope of the present disclosure. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure. Thus, while the compositions and methods of the present disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the formulations, compounds, or compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit and scope of the inventive concepts of the present disclosure. All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. Further, all patents, published patent applications, and non-patent publications referenced in any portion of this application (particularly U.S. Ser. No. 62/431,604) are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As utilized in accordance with the methods and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The use of the word “a” or “an” when used in conjunction with the tell “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y and Z. As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-80 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, as well as fractional values within the range, including 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, includes ranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000, for example. As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACS, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. As used herein the qualifiers “about” or “approximately” are intended to include not only the exact value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, stress exerted on various parts or components, observer error, wear and tear, and combinations thereof, for example. The term “about” or “approximately”, where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ±20% or +10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance (e.g., reaction) occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time, or to at least 90%, at least 95%, at least 98%, or at least 99% completion. As used herein any reference to “one embodiment” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment. The term “pharmaceutically acceptable” refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity, irritation and/or allergic response commensurate with a reasonable benefit/risk ratio. By “biologically active” or “bioactive” is meant the ability to modify or affect the physiological system of an organism without reference to how the active agent has its physiological effects. “Antibacterial” refers to the ability to inhibit the growth of and/or to kill bacteria. As used herein, “pure,” or “substantially pure” means an object species (e.g., an imaging agent) is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term “pure” or “substantially pure” also refers to preparations where the object species (e.g., an imaging agent) is at least 60% (w/w) pure, or at least 70% (w/w) pure, or at least 75% (w/w) pure, or at least 80% (w/w) pure, or at least 85% (w/w) pure, or at least 90% (w/w) pure, or at least 92% (w/w) pure, or at least 95% (w/w) pure, or at least 96% (w/w) pure, or at least 97% (w/w) pure, or at least 98% (w/w) pure, or at least 99% (w/w) pure, or 100% (w/w) pure. The terms “subject” and “patient” are used interchangeably herein and will be understood to refer to a warm blooded animal, particularly a mammal, and more particularly, humans. Animals which fall within the scope of the term “subject” as used herein include, but are not limited to, dogs, cats, rats, mice, guinea pigs, chinchillas, horses, goats, ruminants such as cattle, sheep, swine, poultry such as chickens, geese, ducks, and turkeys, zoo animals, Old and New World monkeys, and non-human primates. “Treatment” refers to therapeutic treatments. “Prevention” refers to prophylactic or preventative treatment measures. The term “treating” refers to administering the composition to a patient for therapeutic purposes. The terms “therapeutic composition,” and “pharmaceutical composition” refer to an active agent-containing composition (e.g., a resin composition comprising doped TiO2nanoparticles, such as N—TiO2NPs) that may be administered to or used in a subject by any method known in the art or otherwise contemplated herein, wherein administration or use of the composition brings about an effect or result as described elsewhere herein. In addition, the compositions of the present disclosure may be designed to provide delayed, controlled, extended, and/or sustained effects using formulation techniques which are well known in the art. The term “effective amount” refers to an amount of an active agent (doped TiO2nanoparticles) as defined herein (e.g., N—TiO2NPs) which is sufficient to exhibit a detectable antibacterial and/or bioactive effect or result without excessive adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the inventive concepts. The effective amount for a patient will depend upon the type of patient, the patient's size and health, the nature and severity of the condition to be treated or diagnosed, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein. As noted above, in certain embodiments, the present disclosure is directed to dental compositions, such as but not limited to, dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth) containing doped or co-doped titanium dioxide nanoparticles such as described herein. The dental compositions may be used in dentistry, for example, as restorative materials, adhesives, bonding agents, cements, sealants, coatings and in the fabrication of partial and complete dentures). These dental compositions have better optical and antibacterial properties when compared to the unaltered commercial dental compositions (e.g., resins) or those comprising undoped TiO2, and have bioactive properties that can improve the service lives of polymer-based dental biomaterials. Particular applications of the presently disclosed compositions containing doped or co-doped titanium dioxide nanoparticles include the development of dental products such as adhesive resins (e.g., bonding agents) with antibacterial functionalities. However, the resins can also be used in secondary applications after small changes in the functionalization of the nanoparticles. Secondary applications include the development of self-cleaning and antibacterial paints and coatings for health care facilities such as universities, hospitals, private practices, spas and saloons, ambulances (cars, helicopters and planes), and medical devices. These paints and coatings may also be used in public spaces where the control of cross-contamination is important, such as passenger trains, airplanes, cruise-ships, bus and train stations and ultimately in regular businesses and houses. The presently disclosed doped TiO2compositions can be used, for example, as or in resin cements (dental and orthopedic), composite resins, denture bases, denture teeth, dental implants, orthodontic brackets and wires, metallic bands and elastomers, catheters, and stents. The presently disclosed antibacterial resins can also be used as antibacterial coatings in hospitals, dental clinics, furniture, equipment, medical devices and hand-held metallic instruments, or for imparting antibacterial properties to indoor and outdoor paints. In certain non-limiting embodiments, the compositions of the present disclosure which contain the doped TiO2NPs comprise a resin-based matrix, containing least one monomeric component selected from the group: acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols, such as but not limited to ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). The composition may comprise a polymeric material selected from the groups: acrylate resins, methacrylate resins, and dimethacrylate esters resins, epoxy resins, polycarbonate, silicone, polyester, polyether, polyolefin, synthetic rubber, polyurethane, nylon, polystyrene, polyvinylaromatic, polyamide, polyimide, polyvinylhalide, polyphenylene oxide, polyketone, and copolymers and blends thereof. The composition may comprise a solvent selected from the group: water, ethanol, methanol, toluene, ethyl ether, cyclohexane, iso-propanol, chloroform, ethyl acetate, acetone, hexane, and heptanes. An inorganic filler such as silicon dioxide or glass ceramics. The composition may include a coupling agent such as a silane, a photoinitiator such as camphorquinone (CQ), phenylpropanedione (PPD), or lucirin (TPO) for initiating polymerization, and a catalyst to control the rate of the polymerization reaction. In non-limiting embodiments, the composition may comprise a volume to volume ratio of doped TiO2to curable resin material in a range of 1% to 80% (v/v), 5% to 50% (v/v), or 10% to 40% (v/v), for example. EXAMPLES The present disclosure will now be discussed in terms of several specific, non-limiting, examples and embodiments. The examples described below, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the present disclosure. Example 1 Methods Synthesis of TiO2Nanoparticles (TiO2NPs) and doped TiO2NPs TiO2NPs for use in the present disclosure can be synthesized using any appropriate method and are not limited to the methods disclosed herein. The NPs can be doped during production of the TiO2NPs (Process 1), or can be doped after production of the TiO2NPs (Process 2). For example, the doped TiO2NPs can be produced using a two-step method wherein TiO2NPs are produced in the first step (Process 1) and are then processed in a second step (Process 2) to produce the doped TiO2NPs. In non-limiting embodiments, Process 1 utilizes a solvothermal method, and Process 2 utilizes the nitrogen-doping of the TiO2. In a non-limiting version of Process 1, titanium (IV) butoxide (TB, 97%) (5-10 mmol) was added to a mixture of X mmol oleic acid (OA, 90%), Y mmol oleylamine (OM, 70%), and 100 mmol absolute ethanol (X+Y=50). X and Y can be varied while leaving the molar ratio of titanium n-butoxide (TB) and surfactants unchanged (i.e., TB/(OA+OM)=1:10) to gain different OA/OM ratios, which lead to the formation of different shapes of NPs. For example, to synthesize TiO2with truncated rhombic shape, 5 mmol of TB was added to a mixture of 25 mmol OA, 25 mmol OM, and 100 mmol absolute ethanol. The obtained mixture in a 40 mL Teflon cup was stirred for 10 min before being transferred into a 100 mL Teflon-lined stainless steel autoclave containing 20 mL of a mixture of ethanol and water (96% ethanol, v/v). The concentration of ethanol was used at the azeotropic point so that the amount of water vapor did not change much during the crystallization process. The system was then heated at 180° C. for 18 h. The obtained white precipitates were washed several times with ethanol and then dried at room temperature. The as-synthesized TiO2NP products were dispersed in nonpolar solvent, such as toluene. TiO2NPs synthesized by varying the OA/OM molar ratio, When the OA/OM mole ratio is 4:6, rhombic-shaped TiO2NPs with uniform size are obtained. By increasing the OA/OM molar ratio to 5:5, smaller TiO2NPs with truncated rhombic shape can be produced. A further increase of this ratio up to 6:4 leads to the formation of spherical particles with an average size of 13 nm. In a particular non-limiting example of Process 1, a solution comprised of 1.7 g of Ti(IV)-butoxide (Aldrich, 97%), 4.6 g ethanol (Decon Labs, 100%), 6.8 g oleylamine (Aldrich, 70%), and 7.1 g oleic acid (Aldrich, 90%) was prepared, then mixed with 20 mL of 4% H2O in ethanol (18-MΩ Milli-Q; Decon Labs). Each solution was clear before mixing, but the final mixture immediately clouded due to formation of micelles and likely some hydrolysis. This solution was then split into two portions (around 20 mL/portion), and each portion was placed into a high-pressure reaction vessel (Paar Series 5000 Multiple Reactor System) and reacted at 180° C. for 24-hours. The vessels were stirred via external magnetic field and Teflon-coated stir bars. The reaction vessels were Teflon-lined. Upon cooling, the solutions were decanted and rinsed 3 times with anhydrous ethanol to remove extraneous surfactants resulting in pure TiO2NPs which were readily dispersible into 20-30 mL ethanol, but did not form clear solutions. The TiO2NPs formed were stored in ethanol. In a particular non-limiting embodiment of Process 2 for making doped TiO2NPs, a portion of the TiO2NPs in ethanol (manufactured in Process 1) are then reacted with an equal volume of triethylamine (Sigma-Aldrich Co., LLC.), also using the high-pressure reaction vessel, at 140° C. for 12 hours. Upon cooling the now-nitrogen-doped TiO2NPs (N—TiO2NPs) are rinsed 3 times with anhydrous ethanol. The exact concentration of TiO2in ethanol/triethylamine varies, but in every instance there is an excess of triethylamine. The final N—TiO2NP ethanol solution yields a gravimetrically-determined concentration of particles typically in the range of 35 mg/mL. In at least certain embodiments, the N/Ti molar ratio of the N—TiO2NPs was in a range of 0.1% to 3.4%. Synthesis of Co-Doped-TiO2NPs Nitrogen and Silver co-doping. N—Ag—TiO2NPs can be formed following the reaction steps of Process 1 using Ti(IV)-butoxide. Ag is provided by adding silver acetlyacetonate and N is provided using tetramethyl ammonium hydroxide as the dopant sources in a wt:wt:wt N:Ag:Ti ratio of, for example, 1:1:18, which provides a 5%/5%/90% N/Ag/Ti composition. For example in one embodiment, components sufficient to provide 0.085 g N and 0.085 g Ag can be combined with a component comprising Ti (e.g., Ti(IV)-butoxide) are used. The TiO2NPs form as in Process 1 but with the N and Ag dopants in place. Nitrogen and Fluorine co-doping. N—F—TiO2NPs can be formed following the reaction steps of Process 1 using Ti(IV)-butoxide. F and N are provided by adding ammonium fluoride as the dopant sources in a wt:wt:wt N:Ag:Ti ratio of, for example, 1:1:18, which provides a 5%/5%/90% N/F/Ti composition. For example in one embodiment, components sufficient to provide 0.085 g N and 0.085 g F can be combined with a component comprising Ti (e.g., Ti(IV)-butoxide) are used. The TiO2NPs form as in Process 1 but with the N and F dopants in place. Phosphate coating of TiO2NPs. In this variation, undoped TiO2NPs, N—TiO2NPs, or co-doped TiO2NPs are treated to provide a coating of phosphate on the outer surface of the particles. The nanoparticles are dispersed into commercial phosphate buffered saline (PBS), which is a neutral pH solution of sodium phosphate. Upon reaction as usual, the particles become phosphate-derivatized and are amenable to further mineralization. The phosphate coated TiO2NPs are designated herein as P—TiO2NPs. The phosphate coated N—TiO2NPs are designated herein as N—P—TiO2NPs. The phosphate coated N—Ag—TiO2NPs are designated herein as N—Ag—P—TiO2NPs. The phosphate coated N—F—TiO2NPs are designated herein as N—F—P—TiO2NPs. Characterization of N—TiO2NPs UV-VIS Spectroscopy TiO2(P25, Evonik Degussa GmbH, Germany) and N—TiO2(Oak Ridge National Laboratory, TN) nanoparticles in ethanol suspension were individually characterized regarding its optical absorbance with a Cary®50 (Agilent Technologies, Santa Clara, CA) spectrophotometer using the transmittance method. Aliquots (20 μL) of each material (either P25 or N—TiO2, 40 mg/mL in ethanol) were individually placed in a quartz microcell. Each sample was then placed inside of the spectrophotometer's chamber between the light source and the photodetector and the intensity of light that reached the photodetector was measured from 190 nm-900 nm in 2 nm increments (FIG.1). Scanning Electron Microscopy (SEM) Aliquots (5 μL) of N—TiO2NPs suspended in ethanol in the as-synthesized concentration (200 proof, 40 mg/mL, Oak Ridge National Laboratory, USA) were placed onto a polished silicon wafer. N—TiO2NP samples were air-dried in room temperature until all the solvent had been evaporated. Individual specimens were then mounted onto standard aluminum SEM pin stubs (diameter ⅛″) and were sputter coated with a thin-layer (˜4 nm) of iridium using a sputter coater (K575D, Emitech Sample Preparation, UK) prior to the imaging process. Adhesive samples containing 50, 67 and 80% (v/v) were mounted directly onto standard aluminum SEM pin stubs using double-sided carbon tape and silver paste for electrical grounding to the stub. The adhesive samples were sputter coated using the same procedures described for the N—TiO2in suspension. Both, N—TiO2NPs in suspension and immobilized in dental adhesive resins were imaged using a Zeiss Neon 40 EsB SEM at 5 kV (FIG.2). Energy dispersive X-ray spectroscopy (EDS) and EDS compositional mapping was performed using an Oxford INCA 250 microanalysis system with an analytical drift detector at 15 kV (FIG.3). Transmission Electron Microscopy (TEM) N—TiO2NPs suspended in ethanol (100%, 0.032 mg/mL, Oak Ridge National Laboratory, USA) were dispersed by brief sonication in an ultrasound bath (Bransonic 220, Branson Ultrasonics, USA). A drop of suspended TiO2NPs was placed on holey carbon coated copper grids. The drop was allowed to adsorb for 1-2 min, then wicked with filter paper to remove excess fluid, and dried before viewing in a JEOL 2000FX transmission electron microscope. Images were made on Carestream® Kodak® electron image film SO-163 (Eastman Kodak Company, USA) and digitized with an Epson Perfection V750-M Pro scanner (Epson America, Inc. USA). X-ray spectra were collected using a Kevex thin window detector and EDS software (IXRF Systems Inc., USA) (FIG.4). Specimen Fabrication Disk shaped specimens (diameter=12.00 mm, thickness ≅15 μm) of OptiBond Solo Plus (OBSP) adhesive resin (Kerr Corp., USA) and experimental adhesive resins containing 50, 67 and 80% (v/v) of N—TiO2NPs, were manually fabricated by individually dispensing 10 μL of each material onto the surfaces of separated glass coverslips (No. 2, VWR International, LLC). Then both the unaltered and experimental adhesive resins were uniformly spread over glass coverslips using disposable flexible applicators (Kerr Corp., USA) and were polymerized using blue light irradiation (1000 mW/cm2, 1 min) emitted from a broadband LED light-curing unit (VALO, Ultradent Products, Inc., USA). Specimens of both unaltered and experimental adhesive resins were then UV-sterilized (254 nm, 800,000 μJ/cm2, UVP Crosslinker, model CL-1000, UVP, USA). Similarly, P—TiO2NPs, N—P—TiO2NPs, N—Ag—TiO2NPs, N—Ag—P—TiO2NPs, N—F—TiO2NPs, and N—F—P—TiO2NPs can be mixed with adhesive resins including but not limited to OBSP to form doped-TiO2dental resins. Bacterial Strain Streptococcus mutansstrain UA159 (JM10::pJM1-ldh, luc+, Spr, luc under the control of the ldh promoter) was utilized for this project. The selection of antibiotic-resistant colonies was performed on TH plates (Todd-Hewitt, BD Difco, USA) supplemented with 0.3% yeast extract (EMB, Germany) and 800 μg/mL of spectinomycin (MP Biomedicals, USA). The plates were incubated under anaerobic conditions at 37° C. for 48 h. Antibacterial Behavior of N—TiO2NPs in Suspension In order to assess the antibacterial efficacy of the N—TiO2in ethanol suspension (200 proof, 40 mg/mL, Oak Ridge National Laboratory, USA),S. mutansbiofilms were grown in sterile microcentrifuge tubes (Safe-Lock Tubes, Eppendorf North America, USA). Planktonic cultures ofS. mutans(UA159-ldh, JM10) were grown in THY culture medium at 37° C. for 16 hours. Planktonic cultures having optical density (OD600) levels≥0.900 were used as inoculum to grow the biofilms. A 1:500 dilution of the inoculum was added to 0.65×THY+0.1% (w/v) sucrose biofilm growth medium. Inoculum aliquots (1.00 mL) were added to separate sterile microcentrifuge tubes and biofilms were grown for 24-hours (static cultures, anaerobic conditions, 37° C.). After the growth period, biofilms were replenished with 1.00 mL of fresh 1×THY+1% (w/v) glucose culture medium and were incubated at 37° C. for 1 hour. Replenished biofilms (n=15/group/irradiation condition) were then exposed to the nanoparticles diluted in growth media [1×THY+1% (v/v)] in the concentrations of 19%, 25% and 30% (v/v) with or without blue light irradiation (1000 mW/cm2, 1 min) provided by a commercially available broadband LED light-curing unit (VALO, Ultradent Products, Inc., USA). Biofilms that were not exposed to the nanoparticles comprised our negative control (n=45). Positive control groups (n=15/concentration) were comprised by biofilms that were exposed to ethanol aqueous solutions (200 proof, AAPER Alcohol and Chemical Co., Shelbyville, KY) in concentrations of 19%, 25% and 30% (v/v) with or without light irradiation (1000 mW/cm2, 1 min). Following the treatment, the suspension containing either N—TiO2NPs in suspension or ethanol aqueous solution was carefully aspirated. Immediately after, biofilms were replenished with 1.00 mL of fresh 1×THY+1% (w/v) glucose sterile culture medium. The microcentrifuge tubes containing the replenished biofilms were then sonicated to facilitate the removal of the adherent biomass using a sonicator (Q700 sonicator, QSonica, USA) connected to a water bath (4° C.; 4 cycles of 1 minute, 15 seconds interval between cycles; power 230±10 W, total energy ≈ 78 kJ). Antibacterial behavior of N—TiO2NPs immobilized in dental adhesive resins In order to assess the antibacterial efficacy of experimental adhesive resins containing 50%, 67% and 80% (v/v) of N—TiO2NPs (Oak Ridge National Laboratory, USA),S. mutansbiofilms were grown against the surfaces of sterile specimens of both unaltered and experimental adhesive resins. Planktonic cultures ofS. mutans(UA159-ldh, JM10) were grown in THY culture medium at 37° C. for 16 hours. Planktonic cultures having optical density (OD600) levels≥0.900 were used as inoculum to grow the biofilms. A 1:500 dilution of the inoculum was added to 0.65×THY+0.1% (w/v) sucrose biofilm growth medium. Inoculum aliquots (2.5 mL) were dispensed into the wells of sterile 24-well microtiter plates (Falcon, Corning, USA) containing sterile specimens. Biofilms were grown for either 3- or 24-hours (static cultures, anaerobic conditions, 37° C.) with or without continuous light irradiation provided by a prototype LED device (410±10 nm, 3 h irradiation=38.75 J/cm2, 24 h irradiation=310.07 J/cm2). After the growth period, biofilms were replenished with 2.5 mL of fresh 1×THY+1% (w/v) glucose culture medium and were incubated at 37° C. for 1 hour. Replenished biofilms were transferred into individual sterile polypropylene tubes (3 mL, ConSert Vials, Thermo Fisher Scientific, USA) containing 1.0 mL of fresh 0.65×THY+0.1% (w/v) sucrose medium. Vials containing the specimens were sonicated to facilitate the removal of the adherent biomass using a sonicator (Q700 sonicator, QSonica, USA) connected to a water bath (4° C.; 4 cycles of 1 minute, 15-second interval between cycles; power 230±10 W, total energy ≈ 78 kJ). Colony-Forming Units (CFU/mL) Biofilms grown either in the microcentrifuge tubes or on the surfaces of both unaltered and experimental dental adhesive resins were sonicated to allow the antibacterial efficacy assessment using the colony-forming units method. Immediately after sonication procedures, inoculum aliquots (10 μL) from each specimen were diluted in 90 μL of 0.65× THY+0.1% (w/v) sucrose sterile culture medium. Serial dilutions were then carried out in 0.65×THY+0.1% (w/v) sucrose sterile culture medium for all samples using a multi-channel pipette (5-50 μL, VWR, USA). Aliquots (10 μL) of each dilution were then plated in triplicate (total: 30 μL/sample/dilution) using THY plates supplemented with 800 μg of spectinomycin. Staining and Confocal Laser Scanning Microscopy A separate set of specimens was fabricated as described above. Biofilms were then grown on the surfaces of unaltered and experimental adhesive resins, using the conditions above in preparation for staining and confocal microscopy. The biofilms on all specimens were stained using BacLight™ LIVE/DEAD fluorescent stains (1.67 μM each of Syto 9 to stain live cells and propidium iodide to stain dead/damaged cells, Molecular Probes, USA) and kept hydrated prior to confocal microscopy. The full thickness of biofilms on all specimens was imaged by confocal microscopy at three randomly selected locations per specimen, in order to gain a representative sample for each specimen, using a Leica TCS SP2 MP confocal laser scanning microscope (CLSM) with Ar (488 nm) and He/Ne (543 nm) lasers for excitation of the fluorescent stains. A 63× water immersion microscope objective lens was used and serial optical sections were recorded from the bottom of the specimen to the top of the biofilm at 0.6 μM intervals in the z-direction. Representative 3-D reconstruction images of live and dead/damaged cells in the 24-hour biofilms grown on adhesive resins were generated using Velocity software (Version 4.4.0, Velocity Software solutions Pvt. Ltd., India) to facilitate visualization of biofilm distribution in all groups investigated. Contact Angle Goniometry A separate set of specimens (n=4/group/concentration) was fabricated as described above in preparation for the contact angle goniometry at oral temperature (37° C.). Immediately after fabrication, specimens of each group were left undisturbed (10 min) inside of the environmental chamber of a contact angle goniometer (OCA15-Plus, Dataphysics Instruments, Germany) for thermal equilibration prior to testing. The wettability of water was tested at oral temperature by displacing a 2 μL drop of ultrapure pure water onto four random locations of each specimen (16 drops/group). The profiles of the axisymmetric drops were recorded using a high-speed and high-definition CCD camera (1 min, 25 frames/sec). The evolution of drop profiles over time was analyzed using the SCA20 software (Dataphysics Instruments, Germany) and the Laplace-Young equation was used to calculate the contact angles at time=0 s (θINITIAL) and time=59 s (θFINAL). Results UV-Vis Spectroscopy FIG.1represents the UV-vis spectroscopy results for both the undoped and nitrogen-doped titanium dioxide nanoparticles. It is possible to observe that doped samples displayed higher absorption levels throughout the range of wavelengths considered, which confirms that nitrogen was successfully incorporated into the crystal lattice of titanic. SEM, EDS and TEM Characterization of N—TiO2NPs Suspended in Ethanol FIG.2represents the SEM pictures of the N—TiO2NPs at different magnifications (500× to 50,000×). Even though it is possible to observe a strong agglomeration behavior of the nanoparticles in the as-synthesized concentration (40 mg/mL in 100% ethanol), these pictures indicate that nanoparticles fabricated by the solvothermal method described above have an approximately spherical shape, smooth surfaces and most of the nanoparticles exhibit some faceting. FIG.3represents the EDS pictures of the compositional analysis of the N—TiO2NPs in the as-synthesized concentration (40 mg/mL). The mapping of elements indicates large quantities of titanium (Ti), oxygen (O), carbon (C) and silicon (Si). The visible peaks present in the EDS compositional spectrum confirm the presence, and the relative amounts (in wt %) of the elements in the samples investigated. The results of the analysis of compositional characterization of the N—TiO2NPs by EDS revealed that Ti (40.9%), O (39.3%), C (13.3%) and Si (6.5%) were the major components found in N—TiO2NP samples. The presence of silicon is related with the wafer substrate in which the samples were imaged. However, under the conditions used herein the doping element (nitrogen) could not be mapped. It is believed that the combination of factors like the low atomic number of nitrogen (Z=7), and the complete overlap between the Ti Lλ (0.395 keV) and the N Kα (0.392 KeV) peaks made the mapping of nitrogen in the N—TiO2NP samples impossible. The characterization of light elements such as Be, B, N and F is difficult due to their low photon energies, low yield of x-rays and low energy to noise ratio.FIG.4represents the TEM pictures (500,000× magnification) and compositional analysis of N—TiO2NPs. The TEM images presented confirm the SEM findings regarding the morphologies of the N—TiO2NPs and demonstrated that synthesized nanoparticles had sizes varying around 10 nm. In addition, it is also possible to observe that even for a very diluted sample (1:1250 in 100% ethanol) the nanoparticles still display a strong agglomeration behavior. SEM and EDS Characterization of N—TiO2NPs Immobilized in Dental Adhesive Resins FIG.5represents the SEM pictures (500× and 5,000× magnifications) of both unaltered and experimental dental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2. It is possible to observe (FIG.5C-H) that adhesive resins containing higher N—TiO2NP concentrations resulted in materials with rougher surfaces due to the higher presence of particles at the surface level. In addition, it is possible to observe that materials containing 67% and 80% (v/v) presented particles (FIG.5E-H) that were not covered by the adhesive matrix when compared to the remaining groups. This finding can be observed by the presence of circular-shaped particulates of very intense brightness. FIG.6represents the results of the compositional analysis of both unaltered and experimental dental adhesive resins. It is possible to observe on image A that the elements composing the unaltered adhesive resins were mainly barium, silicon, oxygen and carbon, which is in agreement with the composition expected for an unaltered dental adhesive resin. Images B to D demonstrate increasing amounts of titanium and oxygen, which can be observed on the images by the presence of increasing amounts of pink (Ti) and yellow dots (O). Contact Angle Goniometry The results obtained from the assessment of the wettability of water at times 0 s (θINITIAL) and 59 s (θFINAL) on both, unaltered and experimental dental adhesive resins, are presented in a self-explanatory graph of mean and standard deviation values (FIG.7). The results demonstrate that, independent of the group considered, initial contact angles (t=0 s) had values that were consistently higher than the values of the final contact angles (t=59 s). The SNK post hoc test demonstrates that similar initial contact angle values were obtained in all groups tested. Although final contact angles were smaller in value than initial contact angles, a similar trend of wettability behavior could still be noticed, where no significant differences among the groups tested could be observed. Antibacterial Behavior of N—TiO2NPs in Suspension The results of the antibacterial activity of N—TiO2NPs againstS. mutansbiofilms grown in microcentrifuge tubes were determined by the colony-forming units (CFU/mL) method and are presented inFIG.8as mean and standard deviation values, and % survival vs. % treatment efficacy (Table 1). The results demonstrated that all N—TiO2NPs and ethanol concentrations tested (19%, 25% or 30% (v/v)] significantly decreased the viability ofS. mutansbiofilms when compared to the control group (intact biofilms). It is also possible to observe that the combination of ethanol and visible light produced viability results that were higher when compared to both experimental groups (N—TiO2NPs or ethanol only) and were comparable to the control group (intact biofilms), which suggest that blue light may act as a biomodulator in situations of low cytotoxic stress. TABLE 1Survival rate and Treatment efficacySurvivalTreatmentrate (%)efficacy (%)Control Group (n = 45)100.00%0.00%19% (v/v) N_TiO2(n = 15)5.58%94.42%25% (v/v) N_TiO2(n = 15)0.55%99.45%30% (v/v) N_TiO2(n = 15)0.24%99.76%19% (v/v) EtOH (n = 15)10.71%89.29%25% (v/v) EtOH (n = 15)0.92%99.08%30% (v/v) EtOH (n = 15)0.73%99.27%19% (v/v) EtOH + light (n = 15)93.79%6.21%25% (v/v) EtOH + light (n = 15)7.79%92.21%30% (v/v) EtOH + light (n = 15)9.65%90.35%Table 1:S. mutanssurvival rate and antibacterial efficacy of the groups were investigated. The survival rate (Sr) and Treatment efficacy (Te) were calculated using the following equations: Sr = (Nf/N0)100% and Ae = (N0− Nf/N0)100%, where N0is the initial population and Nfis the viable population after the treatments. Antibacterial Behavior of N—TiO2NPs Immobilized in Dental Adhesive Resins The results of the antibacterial efficacy of N—TiO2NPs immobilized in dental adhesive resins against 3- or 24-hourS. mutansbiofilms grown against the surfaces of specimens of both unaltered and experimental dental adhesive resins under dark or continuous light irradiation conditions were determined using the colony-forming units method (CFU/mL) and are presented as mean and standard deviation values (FIGS.9and10). The results presented indicate, that regardless of the experimental groups tested or periods of time considered (either 3- or 24-hour), biofilms grown under continuous-light irradiation conditions (410±10 nm, 3-hour irradiation=38.75 J/cm2, 24-hour irradiation=310.07 J/cm2) displayed lower viability levels when compared to biofilms pertaining to either the control group or to experimental groups where biofilms were grown without light irradiation. It is also possible to observe that biofilms grown under continuous-light irradiation, displayed similar viability levels independent of the material investigated. In addition, the results of the viability levels of biofilms grown in dark conditions, indicate that experimental adhesive resins have antibacterial properties that are not dependent on light irradiation. Confocal Laser Scanning Microscopy (CLSM) The CLSM analysis of 24-hourS. mutansbiofilms grown on the surfaces of both unaltered and experimental dental adhesive resins are presented inFIG.11. The 3D rendering images revealed that the morphology, biovolume and viability of the cells within the investigated biofilms were significantly altered based on the N—TiO2NP concentration (50%, 67% and 80% [v/v]) and light irradiation condition (with or without). The results clearly demonstrate that, independent of the experimental group considered, all biofilms grown under continuous-light irradiation conditions (FIGS.11B, D, F, H) expressed higher instances of red fluorescence, which denotes that these biofilms had lower viability levels than the biofilms grown in dark conditions, which predominantly fluoresced green (FIGS.11A, C, E, G). These findings demonstrate that the wavelength and dose of energy used (410±10 nm, 3-hour irradiation=38.75 J/cm2, 24-hour irradiation=310.07 J/cm2) during the biofilms growth significantly impacted the ability ofS. mutansto sustain viable biofilms. It is also noticeable on the CLSM results (FIGS.11Fand H) that the combination of continuous-light irradiation and experimental materials with higher nanoparticles concentration (67% and 80%) supported biofilms displaying the least amount of biovolume and viability, which can be noted on the images by the presence of extremely sparse micro colonies displaying intense red colors. The results obtained for biofilms pertaining to non-irradiated groups indicate that experimental materials containing 50%, 67% and 80% (v/v) of N—TiO2NPs in dark conditions displayed antibacterial properties that were not dependent on light irradiation and further confirm the CFU/mL results. This finding can be observed specially inFIGS.11Eand G by the presence of colonies displaying colors that are a mix of red, green and yellow. In addition, it is also possible to observe, that biofilms grown under dark conditions produced biofilms of similar biovolume and thickness, as noted by the large chained amorphous colonies (FIGS.11A, C, E, G) regardless of group parameters. This finding indicates that the amounts of dead colonies present on the images are directly proportional to increasing amounts of the N—TiO2NPs in the materials investigated. Color Stability The objective of the color analysis was to investigate the effect of the incorporation of 5%, 10%, 15% or 20% (v/v) of N—TiO2into the dental adhesive resin OPTIBOND SOLO PLUS (OPTB). Disk shaped specimens were fabricated and were subjected to 500, 1000, 2500 and 5000 thermal cycles between two water baths (5° C. and 55° C., dwell time 15 sec.). Digital color analysis was then performed immediately after the fabrication of the specimens and at the completion of each thermal cycle proposed. The color stability of specimens was assessed in terms of total color change (ΔE) using the CIELab color space. The color analysis performed immediately after the fabrication of specimens demonstrated that experimental materials containing varying concentrations of N—TiO2NPs displayed color changes that were comparable to the unaltered OPTB (FIG.12). After the completion of 500 thermal cycles it is possible to observe that specimens pertaining to experimental groups containing higher N—TiO2NPs had the least amount of total color change as compared to OPTB. After the completion of 5000 thermal cycles it became clear that experimental materials containing either 10%, 15% or 20% (v/v) N—TiO2NPs have displayed the least amount of color variation when compared to OPTB. It is possible to observe that specimens containing 5% (v/v) of N—TiO2have undergone to color changes that were similar in intensity to the color changes observed in specimens fabricated with the unaltered dental adhesive resin. These findings indicate that incorporation of N—TiO2NPs into OPTB rendered materials having improved color stability properties in comparison to the color stability properties of the unaltered and commercially available OPTB. The larger presence of metal oxide nanoparticles with cores resistant to degradation by water and temperature variation could explain the findings regarding the color stability reported in the present research. Bioactivity of N—TiO2NPs and N—P—TiO2NPs The in vitro testing of the bioactivity of experimental dental adhesive resins containing 20% (v/v) of either N—TiO2NPs or N—P—TiO2NPs was conducted to demonstrate the ability of experimental materials to spontaneously deposit a crystalline layer of amorphous calcium phosphate upon exposure to Dulbecco's phosphate buffer solution (DPBS). Specimens fabricated with OPTB-only served as the control group. SEM and EDS analyses were used to characterize the bioactivity of experimental dental adhesive resins. The SEM and EDS representative images presented inFIGS.13-15represent the results obtained with the in vitro bioactive testing of unaltered OPTB, as well as with N—P—TiO2NPs and N—TiO2NPs, respectively. It is possible to observe from the EDS compositional analysis (FIG.13E) that specimens fabricated with unaltered OPTB were able to promote the precipitation of very small amounts of calcium (Ca, 0.4%), and phosphorous (P, 0.7%).FIG.14Erepresents the EDS compositional analysis results of specimens fabricated with experimental dental adhesive resins containing 20% (v/v) of N—P—TiO2NPs. It is possible to observe that these materials promoted the highest precipitation of Ca (6.6%) and P (5.6%).FIG.15Eshows the EDS compositional analysis results of specimens fabricated with 20% (v/v) of N—TiO2. The results have demonstrated that these materials promoted an intermediate precipitation of Ca (4.2%) and P (3.0%). In addition, the EDS mapping of individual elements further confirmed the findings of the compositional analysis performed. Discussion In at least certain embodiments, doped TiO2NPs were obtained via a two-step fabrication process. In a first step, undoped TiO2NPs were synthesized. In a second step, nitrogen-doping, or co-doping of the TiO2NPs was carried out. After the doping process, the obtained single-doped NPs had their initial visual aspect altered from a bright white—into a yellow—pale suspension, which indicates that the doping process was carried out successfully. The results of the UV-vis spectroscopy of both undoped and N—TiO2NPs are presented inFIG.1, which shows that N—TiO2NPs had higher levels of light absorption when compared to the behavior observed for undoped TiO2NPs. The nanoparticles had significant absorption behavior in the visible region (between 400 nm and 600 nm). The SEM analysis of nanoparticles presented inFIG.2revealed important aspects related to morphologies and agglomeration levels of nanoparticles. The layered and amorphous structures visible in the images suggest that N—TiO2NPs have spherical shapes, smooth surfaces and display a strong agglomeration behavior in ethanol (40 mg/mL). The use of surfactants is one approach that could be used to improve the dispersability behavior of N—TiO2NPs. However, the use of surfactants decreases the possibility of oxidation reactions taking place on the N—TiO2NP surfaces due to the creation of a physical barrier, thereby diminishing their antibacterial behavior. Thus the methods of the present work were designed to maximize the photocatalytic behavior of N—TiO2NPs. The SEM images demonstrate a physical association of nanoparticles due to the drying process that is required for SEM imaging. The results of the compositional characterization of the nanoparticles using EDS are presented inFIG.3. The analysis revealed that Ti (40.9%), O (39.3%), C (13.3%) and Si (6.5%) were the major components found in N—TiO2NP samples. However, under the conditions of the present work, the doping element (nitrogen) could not be mapped. Apparently the combination of factors like the low atomic number of nitrogen (Z=7), and the complete overlap between the Ti Lλ, (0.395 keV) and the N Kα(0.392 KeV) peaks made the mapping of nitrogen in the N—TiO2NP samples excessively difficult. Characterization of the NPs by TEM is presented inFIG.4. The images demonstrated that N—TiO2NPs have mostly spherical shapes, smooth surfaces and a homogeneous distribution of sizes, with individual NP sizes ranging around 10 nm. It is also possible to observe that N—TiO2NPs still tend to have strong agglomeration behaviors even for very diluted samples (1:1250 or 0.032 mg/mL). Control over the nanoparticle agglomeration levels is a key factor in the optimization of photocatalytic reactions, because agglomeration can decrease the nanoparticle surface to volume ratio, decrease the amount of free surface area that is actually available for oxidative reactions to take place and, increase the amount of recombination centers present in the bulk of the photocatalyst, thereby adversely impacting the overall photocatalytic behavior of any light responsive material. SEM and EDS analyses were used to characterize the surface properties and compositions of specimens fabricated with both unaltered and experimental dental adhesive resins. The SEM results demonstrated the successful incorporation of nanoparticles into the polymer matrix, which can be observed by the presence of increasing amounts of particulates on the surfaces of specimens that were fabricated with higher nanoparticles concentrations. In addition, specimens fabricated with higher nanoparticles content displayed rougher surfaces when compared to specimens of OPTB resin due to the strong presence of exposed particulates. The compositional analysis performed using EDS further corroborates our SEM findings regarding the successful incorporation of nanoparticles into the OPTB. It is possible to observe on the EDS images, that specimens of OPTB displayed barium (Ba), Si, O and C as its major chemical components, which is an expected composition. The compositional mapping of specimens fabricated with experimental dental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2NPs clearly demonstrate higher concentrations of Ti and O, which can be noticed by observing increasing amounts of pink (Ti) and yellow (O) dots on the images. These results agree with the compositions expected for samples fabricated with experimental materials. The wettability analysis using the measurement of contact angles was made necessary in the present work to investigate the impact of the incorporation of nanoparticles on the wettability characteristics of OPTB. The measurement of contact angles at the solid-liquid-vapor interface is considered to be the most widely known technique used to investigate the wettability of solid surfaces. The hydrophobicity behavior of dental composites is an important factor in the longevity of resin-based materials because it affects the initial absorption of water, which regulates the attachment of oral bacteria. The wettability findings reported in the present work demonstrated that the incorporation of N—TiO2NPs into OPTB promoted the attainment of experimental materials with wettability properties that were not significantly different when compared to the control group. From the clinical perspective, the fact that there were no statistical differences between the groups is important because in order to promote the establishment of an adequate adhesive layer, dental adhesive resins must compete with water from the dentine substrate to wet the collagen fibrils. Adhesive materials must come into intimate contact with the dentine substrate to allow for the proper micromechanical surface attachment. When observing results of the assessment of the antibacterial efficacy of N—TiO2NPs in suspension (FIG.8and Table 1), it is possible to see thatS. mutansbiofilms displayed similar CFU/mL values regardless of treatment with either nanoparticles or ethanol. This indicates that the N—TiO2NPs suspended in 100% ethanol did not present a strong antibacterial effect againstS. mutansbiofilms in the conditions investigated. The analysis of the survival rates (Sr) and treatment efficacy (Te) for the same experimental groups discussed (Table 1) further corroborates this finding. Oxidative photocatalytic reactions are inhibited in the presence of ethanol because some reactive species of oxygen, such as hydroxyl radicals, are strongly quenched. In the same direction, Hydroxyl radicals and hydrogen peroxide appeared to be the major species associated with the antibacterial effects observed againstStaphylococcus epidermidis. Results from antibacterial assays performed herein with experimental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2NPs againstS. mutansbiofilms grown for either 3-hour or 24-h, with or without continuous-light irradiation, are presented inFIGS.9-10. These results have demonstrated that, independent of growth time (either 3-hour or 24-h), or light irradiation conditions (with or without light), experimental groups containing higher N—TiO2NP concentrations were more antibacterial in nature when compared to the control group, which indicates the establishment of a concentration-dependent antibacterial mechanism. The CLSM images presented inFIG.11illustrate and further corroborate the results of the antibacterial assays performed on dental adhesive resins. These results confirm a decrease in cells viability and biovolume when specimens were fabricated with higher concentrations of doped TiO2NPs while also being irradiated with continuous visible light irradiation, and therefore align the expected results with the representative CSLM images. It is interesting to note, that while it was expected that the N—TiO2NPs would affect the viability ofS. mutansbiofilms when exposed to visible light, it is apparent that there is a toxicity effect (“dark toxicity”) absent exposure of the adhesive to light. Although many photosensitizers are able to increase bactericidal effects, they usually require an irradiation light source in order to elicit reduced viability. However, the dark toxicity observed was statistically significant due to decrease in viability between the OPTB control and the 80% N—TiO2NP adhesive resin in dark conditions in both the 3-hour and 24-hour biofilms. It is also supported by the 24-hour CLSM images that visually show a change in viability, but not necessarily the structure of the biofilm. Both our CLSM images as well as our CFU/mL results show not only a decrease in biovolume, but also a highly toxic effect when biofilms are grown on an adhesive containing N—TiO2NPs in the presence of blue light. Since it was previously established that the N—TiO2NPs do have some degree of dark toxicity, the high degree of bactericidal effects may have been due to a two-fold mechanism; the restriction of EPS in the formation of the biofilm, as well as the toxicity of the nanoparticles themselves. In the present work, a titanium dioxide-based photocatalyst was successfully prepared by doping TiO2NPs with nitrogen using a simple solvothermal method. These NPs were demonstrated to have superior visible light absorption levels when compared to pure TiO2NPs due to the contribution of substitutional nitrogen in the crystal lattice of titania. The visible light-driven antibacterial efficacy of N—TiO2NPs was investigated for nanoparticles suspended in ethanol and incorporated in a commercially available dental adhesive resin (OPTB). It was demonstrated that nanoparticles in suspension have only a limited antibacterial behavior againstS. mutansbiofilms probably due to the use of ethanol as a solvent, which is a well-known potent hydroxyl scavenger. The present work has shown for the first time that specimens fabricated with experimental dental adhesive resins containing either 50%, 67% or 80% (v/v) of N—TiO2NPs were shown to have strong antibacterial behavior in both, dark and light irradiated conditions, when compared to the antibacterial behavior of unaltered dental adhesive resins (e.g., OPTB). This indicates that N—TiO2NPs comprise a feasible antibacterial agent against oral cariogenic biofilms. The present work has also demonstrated that experimental materials had similar wettability behaviors when compared to the unaltered adhesive resins, which is important from the clinical perspective. Example 2 Polymerization shrinkage, poor adhesive infiltration and incomplete enveloping of dentin matrix are important limitations of current dental adhesive resins. The approach of this example to solving these problems was the development of a bioactive and bond-promoting adhesive resin containing N—TiO2NPs. The spontaneous and light-stimulated deposition of hydroxyapatite on the surfaces of specimens fabricated with both experimental [20% (v/v) N—TiO2NPs] and unaltered dental adhesive resin (OPTB) was investigated. Experimental resins were synthesized by adding 20% (v/v) N—TiO2NPs (Oak Ridge National Laboratory) to OPTB. Thin-films (n=12/group; d=12 mm, t=15 μm) were fabricated and light-cured (40 sec, 457±15 nm) on acid-etched glass cover slips in preparation for bioactivity testing. Thin-films were then irradiated (405±15 nm) for 1, 3, and 8 hours either in air or water (2.5 mL) conditions. Specimens were then immersed in pre-heated DPBS (Dubelcco's Phosphate-Buffered Saline, with Calcium and Magnesium Chloride) aqueous solution (40 mL/specimen, 60° C.) and were stored in dark conditions (37° C.) for 7 days. Solutions of DPBS were subsequently replenished at 72-h and 120-h. Specimens were air dried in dark conditions (minimum of 24-h) and were sputter-coated with either iridium or gold in preparation for Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy analyses. Chemical compositional data provided by EDS was analyzed using GLM and SNK post hoc tests (α=0.05). Mean deposition values of calcium and phosphorous ranged from 0.60 and 1.13 wt % [Control, irradiated (1 h) in water] to 6.73 and 6.13 wt % [20% (v/v) N—TiO2NPs, irradiated (8 h) in air], respectively. Significant differences were found in the interaction “material*irradiation time*irradiation condition” (p<0.05). It was concluded that experimental dental adhesive resins containing 20% (v/v) of N—TiO2NPs that were irradiated for 8 h in air conditions, were demonstrated to have bioactive properties that were stimulated by visible light irradiation, as hypothesized. Example 3 Caries is the primary reason of dental restoration failure. The objective of this example was to assess the wettability, color stability and fracture toughness of adhesives containing N—TIO2NPs. Experimental adhesives were synthesized by adding 5%, 10%, 15% and 20% (v/v) of N—TiO2NPs to OPTB. Unaltered adhesive resin (OPTB) served as the control group. Dentin specimens (a=5 mm2, t=1 mm) were fabricated to test wettability. Disk-shaped (d=7.64 mm, t=1.75 mm) and SENB (17.6×2.0×4.0 mm) specimens were fabricated for wettability and fracture toughness testing. Wettability of adhesives was tested in an OCA15-Plus goniometer. Profiles of adhesive drops were analyzed (25 frames/s, 37° C.) to determine contact angle at time=0 s (θINITIAL) and time=30 s (θFINAL). Color stability (n=5/concentration) was tested using an image analysis software after 0, 500, 1,000, 2,500 and 5,000 thermal cycles (5° C.-55° C., 15 s dwell). Fracture toughness specimens were tested with an Instron system using ASTM Standard D5045-99. Data was analyzed using GLM and SNK post hoc tests (α=0.05). Mean θINITIALvalues ranged from 95.87° (Control) to 49.69° (20% N—TiO2NPs) and for θFINALfrom 30.98° (5% N—TiO2) to 25.00° (20% N—TiO2NPs). Mean L*, a* and b* values ranged from 79.93, −5.06 and 3.65 (5% N—TiO2) to 84.51, −4.50 and 4.33 (15% N—TiO2NPs), respectively. No significant differences (p>0.05) were observed for initial or final wettability. Significant differences among groups were found for color stability (p<0.0001). Mean K1cvalues ranged from 0.431 MPa (20% N—TiO2NPs) to 4.317 MPa (5% N—TiO2NPs), but results were rejected because they couldn't be validated using Standard D5045-99. It was concluded that adhesive resins containing N—TiO2NPs had comparable wettability and better color stability than unaltered adhesive resin (control), as hypothesized. Example 4 The antibacterial efficacy of unaltered and experimental (doped) dental adhesive resins against non-disrupted cariogenic (caries producing) biofilms was further assessed in terms of relative luminescence units (RLUs) using a real-time luciferase-based bioluminescence assay. Toward this end, experimental dental adhesive resins containing either N—TiO2NPs (5%-30%, v/v), N—F—TiO2NPs (30%, v/v) and N—Ag—TiO2NPs (30%, v/v) were synthesized by dispersing the nanoparticles in OBSP adhesive resin using a sonicator (4 cycles of 1 min, intervals of 15-sec between cycles; Q700, QSonica, USA). Two non-antibacterial (OBSP, and Scotchbond Multipurpose, 3M ESPE, USA) and one antibacterial (Clearfil SE Protect, Kuraray, Noritake Dental Inc., Japan) commercially available dental adhesive resins were also tested for antibacterial functionalities.Streptococcus mutansbiofilms were grown (UA 159-ldh, JM 10; 37° C., microaerophilic) on the surfaces of disk-shaped specimens (n=18/group, d=6.0 mm, t=0.5 mm) for either 24 or 48 hours with or without continuous visible light irradiation (405±15 nm). One set of specimens was fabricated with OBSP and was treated with Chlorhexidine 2% (2 min) that served as our control group. Results for the antibacterial efficacies of both unaltered and experimental dental adhesive resins containing either doped or co-doped TiO2NPs under continuous visible light irradiation for either 24 or 48 hours, demonstrated that all groups tested displayed similar antibacterial behaviors under continuous visible light irradiation. Such findings suggest that under the conditions investigated (wavelength and power intensity), visible light irradiation had a very strong antibacterial behavior that took place independently of the antibacterial activity of the substrate where biofilms were grown (either antibacterial or not). Such impact made impossible the determination of the materials' real antibacterial efficacies under such light irradiation conditions. Experiments were then conducted under dark conditions; bacteria were grown in dark conditions for either 24 and 48 hours. The results indicated that the TiO2-containing adhesive resins were more antibacterial than commercially available non-antibacterial dental adhesive resins (such as OptiBond Solo Plus and Scotchbond Multipurpose). The experimental dental adhesive resins containing 30% (v/v) of nanoparticles (N—TiO2NPs, N—F—TiO2NPs and N—Ag—TiO2NPs) displayed antibacterial efficacies in dark conditions that were similar to Clearfil SE Protect (Fluoride-releasing material, Kuraray, Noritake Dental Inc., Japan).S. mutansbiofilms grown on specimens treated with chlorhexidine 2% (2 min) displayed the lowest RLU values amongst all groups investigated, thereby confirming the strong antibacterial behavior of non-immobilized chlorhexidine. In addition, the antibacterial effect was demonstrated to be concentration-dependent, wherein experimental adhesive resins containing higher concentrations of antibacterial nanoparticles (either doped or co-doped) displayed stronger antibacterial effects against non-disruptedS. mutansbiofilms. Since long intra-oral irradiation periods (24-hour and 48-hour) are impractical and clinically not feasible, associated with the fact that these materials are intended to be used in the oral cavity's dark conditions, these results were considered of paramount importance and clinically relevant for the commercialization pathway of recently developed antibacterial and bioactive nano-filled dental adhesive resins. Optical and mechanical properties of both unaltered and experimental dental adhesive resins containing 5%-30% (v/v, 5% increments) of N—TiO2NPs were assessed in terms of color stability and biaxial flexure strength. Color stability (n=5) and biaxial flexure strength (n=8) specimens (d=6.0 mm, t=0.5 mm) were fabricated and tested using a color analysis software (ScanWhite, Darwin Syst., Brazil) and an Instron universal testing machine (cross-head rate=1.27 mm/min), respectively. Color stability measurements were performed immediately after specimen fabrication and after water storage (1, 2, 3, 4, 5, 6 months; 37° C.). The color stability results demonstrated that specimens fabricated using either unaltered or experimental dental adhesive resins containing N—TiO2NPs (5%-30%, v/v) were subjected to color changes induced by long-term water storage. The highest color variations were observed at two months of water storage (37° C.) for specimens pertaining to experimental groups containing either 5% or 10% of N—TiO2NPs. Specimens fabricated with unaltered OptiBond Solo Plus have demonstrated color variations that were similar to the color variations observed for the experimental group containing 20% N—TiO2NPs. Specimens fabricated with 30% N—TiO2NP-containing dental adhesive resins have shown the least amount of color variation throughout the investigation time (6-mo), and therefore, were considered as the most color stable amongst all materials investigated. From the esthetic standpoint, the human eye can only detect differences in color above a certain threshold (ΔE≥3). In at least one embodiment, dental composition specimens fabricated with at 30% N—TiO2NPs displayed color variations that were either lower than or close to the human eye detection capability, thereby corroborating the long-term use of these highly esthetic experimental dental adhesive resins. In at least certain embodiments, the dental compositions contain at least 5% to 80% (v/v) of doped-TiO2NPs as disclosed herein, such as at least 5% (v/v), at least 6% (v/v), at least 7% (v/v), at least 8% (v/v), at least 9% (v/v), at least 10% (v/v), at least 11% (v/v), at least 12% (v/v), at least 13% (v/v), at least 14% (v/v), at least 15% (v/v), at least 16% (v/v), at least 17% (v/v), at least 18% (v/v), at least 19% (v/v), at least 20% (v/v), at least 21% (v/v), at least 22% (v/v), at least 23% (v/v), at least 24% (v/v), at least 25% (v/v), at least 26% (v/v), at least 27% (v/v), at least 28% (v/v), at least 29% (v/v), at least 30% (v/v), at least 31% (v/v), at least 32% (v/v), at least 33% (v/v), at least 34% (v/v), at least 35% (v/v), at least 36% (v/v), at least 37% (v/v), at least 38% (v/v), at least 39% (v/v), at least 40% (v/v), at least 41% (v/v), at least 42% (v/v), at least 43% (v/v), at least 44% (v/v), at least 45% (v/v), at least 46% (v/v), at least 47% (v/v), at least 48% (v/v), at least 49% (v/v), at least 50% (v/v), at least 51% (v/v), at least 52% (v/v), at least 53% (v/v), at least 54% (v/v), at least 55% (v/v), at least 56% (v/v), at least 57% (v/v), at least 58% (v/v), at least 59% (v/v), at least 60% (v/v), at least 61% (v/v), at least 62% (v/v), at least 63% (v/v), at least 64% (v/v), at least 65% (v/v), at least 66% (v/v), at least 67% (v/v), at least 68% (v/v), at least 69% (v/v), at least 70% (v/v), at least 71% (v/v), at least 72% (v/v), at least 73% (v/v), at least 74% (v/v), at least 75% (v/v), at least 76% (v/v), at least 77% (v/v), at least 78% (v/v), at least 79% (v/v), or at least 80% (v/v), with the balance comprising the curable adhesive resin material, and optionally other components as described elsewhere herein. The present results demonstrate that experimental dental adhesive resins containing varying concentrations of N—TiO2NPs display biaxial flexure strengths that are either similar or better than the strength observed for specimens fabricated with the unaltered OBSP. No differences were observed among the flexure strengths of experimental groups, thereby indicating that the presently disclosed materials can behave very similar to commercially available materials when subjected to masticatory forces. Specimens (d=6.0 mm, t=0.5 mm) of the unaltered resins and experimental dental adhesive resins containing 30% N—TiO2NPs, 30% N—F—TiO2NPs and 30% N—Ag—TiO2NPs were fabricated and characterized using the state of the art scanning electron microscope. This dual focused ion-beam microscope (Dual-FIB SEM/EDS) is capable, through a destructive process, to characterize and map the chemical composition and distribution of elements in three dimensions. The 3-D characterization and localization of components clearly demonstrated that experimental materials containing co-doped nanoparticles (e.g., 30% v/v, N—F—TiO2NPs) displayed an optimized dispersion of filler particles (part of the original composition) when compared to the filler particle distribution observed on specimens fabricated with the unaltered dental adhesive resin. The 3-D images demonstrated that the experimental adhesive resins had more filler particles per unit volume with a more homogeneous size distribution than the filler fraction and size distribution observed on OptiBond Solo Plus samples. In addition, results showed that larger and more agglomerated filler particles tend to result in a polymer matrix containing more pores per unit volume. This finding was corroborated by the pore-size distribution calculated for the unaltered samples and experimental dental adhesive resin samples, where it is possible to observe that the quantity and sizes of pores formed in experimental materials were smaller when compared to the unaltered OptiBond Solo Plus samples. In at least one embodiment, the present disclosure includes a dental composition, comprising doped and/or coated TiO2NPs, and a curable resin material, wherein the curable resin material comprises a polymer precursor component. The TiO2NPs may comprise at least one dopant or coating selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO4(phosphate). As noted above, in non-limiting embodiments, the dental composition may comprise a volume to volume ratio of doped TiO2NPs to curable resin material in a range of 1% to 80% (v/v), 5% to 50% (v/v), or 10% to 40% (v/v), for example. The polymer precursor component may be photocurable. The polymer precursor may be selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols. The polymer precursor may be selected from the group consisting of ethylenedimethacrylate (“EDMA”), bisphenol A glycidyl methacrylate (“BisGMA”), triethyleneglycol dimethacrylate (“TEGDMA”), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). The dental composition may comprise at least one solvent. The at least one solvent may be selected from the group consisting of water, ethanol, methanol, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, acetone, hexane, and heptanes. The dental composition may comprise a polymerization initiator. The dental composition may comprise a filler. The dental composition may be selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth. The dental composition may comprise bioactive and/or antibacterial activity in the absence of visible or ultraviolet light. The dental composition may be used to form a hardened dental article after a photocuring step. In at least one embodiment, the disclosure includes an in vivo dental process, comprising applying the dental composition to at least one of a dental restorative and a dental substrate, and causing the dental restorative to be bonded to the dental substrate via the dental composition after a step of photocuring the dental composition. Accordingly, the present disclosure is directed to at least the following non-limiting embodiments: Clause 1. In at least one embodiment the present disclosure includes a dental composition, comprising doped TiO2nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component. Clause 2. The dental composition of clause 1, wherein the doped TiO2nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO4(phosphate). Clause 3. The dental composition of clause 1 or 2, wherein the doped TiO2nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO4. Clause 4. The dental composition of any one of clauses 1-3, comprising a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 1% to 80% (v/v). Clause 5. The dental composition of any one of clauses 1-4, comprising a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 5% to 50% (v/v). Clause 6. The dental composition of any one of clauses 1-5, comprising a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 10% to 40% (v/v). Clause 7. The dental composition of any one of clauses 1-6, wherein the polymer precursor component is photocurable. Clause 8. The dental composition of any one of clauses 1-7, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols. Clause 9. The dental composition of any one of clauses 1-8, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). Clause 10. The dental composition of any one of clauses 1-9, further comprising at least one solvent. Clause 11. The dental composition of any one of clauses 1-10, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes. Clause 12. The dental composition of any one of clauses 1-11, further comprising a polymerization initiator. Clause 13. The dental composition of any one of clauses 1-12, further comprising a filler. Clause 14. The dental composition of any one of clauses 1-13, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth. Clause 15. The dental composition of any one of clauses 1-14, comprising bioactive and/or antibacterial activity in the absence of visible or ultraviolet light. Clause 16. A kit for forming a dental composition, the kit comprising doped TiO2nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component. Clause 17. The kit of clause 16, wherein the doped TiO2nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO4(phosphate). Clause 18. The kit of clause 16 or 17, wherein the doped TiO2nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO4. Clause 19. The kit of any one of clauses 16-18, comprising sufficient doped TiO2nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 1% to 80% (v/v). Clause 20. The kit of any one of clauses 16-19, comprising sufficient doped TiO2nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 5% to 50% (v/v). Clause 21. The kit of any one of clauses 16-20, comprising sufficient doped TiO2nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 10% to 40% (v/v). Clause 22. The kit of any one of clauses 16-21, wherein the polymer precursor component is photocurable. Clause 23. The kit of any one of clauses 16-22, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols. Clause 24. The kit of any one of clauses 16-23, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). Clause 25. The kit of any one of clauses 16-24, further comprising at least one solvent. Clause 26. The kit of any one of clauses 16-25, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes. Clause 27. The kit of any one of clauses 16-26, further comprising a polymerization initiator for combining with the doped TiO2nanoparticles, and curable resin material. Clause 28. The kit of any one of clauses 16-27, further comprising a filler for combining with the doped TiO2nanoparticles, and curable resin material. Clause 29. The kit of any one of clauses 16-28, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth. Clause 30. The kit of any one of clauses 16-29, wherein the dental composition has bioactive and/or antibacterial activity in the absence of visible or ultraviolet light. Clause 31. A hardened dental article formed from the dental composition of any one of clauses 1-15, after the dental composition has been photocured. Clause 32. An in vivo dental process, comprising: applying a dental composition to a dental surface, the dental composition comprising doped TiO2nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component; and causing the dental composition to be bonded to the dental surface by photocuring the dental composition. Clause 33. The dental process of clause 32, wherein the dental surface is at least one of a dental restorative and a dental substrate. Clause 34. The dental process of clause 32 or 33, wherein the doped TiO2nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO4(phosphate). Clause 35. The dental process of any one of clauses 32-34, wherein the doped TiO2nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO4. Clause 36. The dental process of any one of clauses 32-35, wherein the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 1% to 80% (v/v). Clause 37. The dental process of any one of clauses 32-36, wherein the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 5% to 50% (v/v). Clause 38. The dental process of any one of clauses 32-37, wherein the dental composition comprises a volume to volume ratio of doped TiO2nanoparticles to curable resin material in a range of 10% to 40% (v/v). Clause 39. The dental process of any one of clauses 32-38, wherein the polymer precursor component is photocurable. Clause 40. The dental process of any one of clauses 32-39, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols. Clause 41. The dental process of any one of clauses 32-40, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). Clause 42. The dental process of any one of clauses 32-41, wherein the dental composition further comprises at least one solvent. Clause 43. The dental process of any one of clauses 32-42, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes. Clause 44. The dental process of any one of clauses 32-43, wherein the dental composition further comprises a polymerization initiator. Clause 45. The dental process of any one of clauses 32-44, wherein the dental composition further comprises a filler. Clause 46. The dental process of any one of clauses 32-45, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth. Clause 47. The dental process of any one of clauses 32-46, wherein after curing, the dental composition has bioactive and/or antibacterial activity in the absence of visible or ultraviolet light. Clause 48. The dental process of any one of clauses 32-47, wherein the dental surface has been acid-etched prior to the application of the dental composition thereon. While the present disclosure has been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the present disclosure be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the present disclosure as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the inventive concepts of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be the most useful and readily understood description of procedures as well as of the principles and conceptual aspects of the present disclosure. Changes may be made in the formulation of the various compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the present disclosure. Further, while various embodiments of the present disclosure have been described in claims herein below, it is not intended that the present disclosure be limited to these particular claims. | 86,978 |
11857652 | MODE FOR DISCLOSURE Hereinafter, in order to describe the present invention in more detail, it will be described with reference to the following embodiments. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided by way of example to aid in a specific understanding of the present invention. Unless otherwise specified, % described herein can be understood to mean % by weight. [Dental Gel-Wrap Drug Delivery System] As follows, a kit including gel and wrap was prepared. InFIG.1, the kit100includes wrap107and gel103. The wrap107includes an adhesive layer101and backing layer102. This kit includes an active ingredient for whitening teeth in the gel, and the wrap includes flavor, but does not include an active ingredient for whitening teeth. Table 1 below shows the composition of the gel, and Table 2 below shows the composition of the adhesive layer of the wrap. TABLE 1IngredientGelHydrogen peroxide6.0Glycerin20.0Carbomer1.8Sodium hydroxide0.08Sodium saccharin0.1Purified waterTo 100 TABLE 2IngredientWrap1Wrap2Wrap3Wrap4Glycerin26.634.030.233.1HCO-405.04.4SPAN803.3PVP59.242.547.744.2Pullulan0.05HPMC5.0EC8.5Sodium phosphate1.51.51.5tribasicSodium hydroxide0.3Saccharin0.60.60.50.5Flavor5.65.75.05Ethanol2.8Purified waterTo 100To 100To 100To 100 The manufacturing method is as follows. Gel—Raw materials excluding a neutralizing agent are added to a mixing tank, stirred homogeneously, and after confirming that there is no dissolution or lumped mass, carbomer is neutralized to increase the viscosity. The viscosity is adjusted in the range of 20,000 to 480,000 after rotation using Brookfield Viscometer (RV) Spindle No. 7 at 20 rpm for 15 seconds. Wrap—After putting raw materials into a mixing tank and stirring it homogeneously, the mixture was applied to a certain thickness using a comma coater, dried by blowing dry hot air (40-60° C.), and then the backing layer (Low density polyethylene (LDPE)) was laminated thereto. (Solvent casting) [Method of use] Gels and wraps having the compositions of Table 1 and Table 2 above were prepared, respectively, and used for tooth whitening. The gel was used in a fixed amount, and stored in a syringe pump with a shut-off valve to prevent leakage. A fixed amount of gel discharged from the syringe pump was applied to the tooth surface. When used about 0.2-0.6 g per use, the gel flows into the teeth as well as the tooth surface, so it was effective for whitening between teeth. The wrap was detached from the release liner and then attached to the tooth surface. [Tackiness Measurement] It was measured using a TA/TX analyzer. After attaching the backing layer of the wrap product to the bottom of the device using double-sided tape, the release liner was remove from the adhesive layer and the Ball tackiness was measured. (Dry/Wet) The experimental method is schematically shown inFIG.2. InFIG.2, the wrap107was attached to double-sided tape104. Once the wrap was attached, a ball105was placed on the adhesive layer of the wrap101. After a specific amount of time, the ball105was removed from the adhesive layer101. Wetting method: Purified water is sprayed once using a spray. Pressing time (5 sec), Pressing force (5 gf), Peeling speed (10 mm/sec) Adhesion according to the prescription is shown in Table 3. TABLE 3F average(gf)F Max(gf)F Min(gf)(S.D.)Wrap 1150.166.7110.7(35.039)Wrap 2143.980.0115.1(26.914)Wrap 343.121.729.5(10.041)Wrap 499.378.591.6(11.378)*F Max(gf): Maximum force until falling,F Min(gf): Minimum force until falling Adhesion according to wetting is shown in Table 4. TABLE 4F average(gf)NumberWettingF Max(gf)F Min(gf)(S.D.)Wrap 4Dry99.378.591.6(11.378)Wet178.3143.1161.0(17.638) The force when peeling off the Wrap from PET was measured, and the experimental method is shown inFIG.3. The TA/TX analyzer was used.As illustrated inFIG.3, after attaching the release liner of the Wrap product106to the bottom of the device using double-sided tape104, the backing layer and a cylindrical tip (diameter 5 mm)108were attached by pressing the backing layer with the tip with double-sided tape109attached. Then, the force when the Wrap fell from the release liner was measured, and the tip was attached to one end of the Wrap size (10 mm×25 mm).Pressing time (5 sec), Pressing force (1000 gf), Peeling speed (10 mm/sec) TABLE 5F average(gf)NumberF Max(gf)F Min(gf)(S.D.)Wrap 1733.8665.3710.7(39.338)Wrap 2351.0245.9308.0(55.055)Wrap 3325.2225.4274.6(53.096)Wrap 4362.5200.8306.7(91.744) [Comparison of Wrap Curling Degree] The experimental method is as follows, and is illustrated inFIG.4. 1. After uniformly cutting the Wrap into 70 mm in length and 20 mm in width, the release liner is attached and fixed to the bottom of the device using double-sided tape. 2. Using tape, Zwick universal tester and the backing layer of the Wrap are attached. 3. The Wrap was peeled off from the release liner by pulling at a certain speed. (13 mm/sec) 4. The length of the Wrap was measured without unfolding the Wrap again. The curling results using the wrap of Table 2 are shown in Table 6 below. TABLE 6NumberLength after peeling (mm)Wrap 144Wrap 263Wrap 362Wrap 468 Wrap1was easily curled up when peeling, so that length was shortened. A photograph of the experimental results of Wrap1and Wrap4is shown inFIG.5. | 5,520 |
11857653 | DETAILED DESCRIPTION OF THE INVENTION Applicants have discovered significant advantages in providing benefits to the oral cavity using poorly-soluble calcium compounds and fluoride. In particular, the claimed methods tend to exhibit both significant remineralization and pH buffering, as compared to other methods including methods using highly-soluble calcium compounds. As used herein, the term “poorly-soluble calcium compound” refers to any calcium-containing compound having a solubility in water at neutral pH of less than 0.5 g/L. In certain embodiments, the poorly-soluble calcium compound has a solubility of about 0.25 g/L or less, about 0.1 g/L or less, or about 0.05 g/L or less. Examples of suitable poorly-soluble calcium compounds include poorly-soluble calcium salts including, but not limited to, calcium phosphate, calcium carbonate, calcium oxalate, calcium silicate, combinations of two or more thereof, and the like. In certain preferred embodiments, the poorly-soluble calcium compound comprises calcium carbonate. The poorly soluble calcium-containing compounds may be delivered to the oral cavity in any suitable form, including but not limited to a solid form, for example, in a powder, tablet, confection, chewing gum, and the like, or in liquid form, for example, in a mouthwash, mouth rinse, toothpaste, gel, and the like. In certain embodiments, the poorly soluble calcium compound is introduced to the oral cavity in the form of a tablet. In certain embodiments, the poorly soluble calcium compound is introduced to the oral cavity via mouthwash or mouthrinse. The concentration of calcium in the delivery vehicle depends at least in part on the form of the selected delivery vehicle. Generally, it is desired to deliver a concentration of poorly soluble calcium in the oral tissue that is effective for reacting with the administered fluoride to form calcium-bound fluoride deposits in plaque, on teeth and in oral tissue. By way of example, to deliver these concentrations of poorly soluble calcium in the oral cavity, the calcium concentration in a pre-rinse that is used before a fluoride rinse or dentifrice preferably is between about 0.1 percent to about 20 percent, or from about 1 percent to about 15 percent, or from about 5 percent to about 10 percent by weight. When formulated as a tablet, the total calcium content is between about 0.1 percent to about 50 percent, or from about 1 percent to about 15 percent, or from about 1 percent to about 5 percent by weight. When formulated in a dentifrice, the calcium content is between about 0.2 percent to about 50 percent, or from about 1 percent to about 25 percent, or from about 5 percent to about 15 percent of poorly soluble calcium by weight. In some embodiments, poorly soluble calcium-containing compounds may be delivered in the form of tablet, such as the tablets which generate a fluid that can be moved throughout the oral cavity as described in U.S. Patent Publication No. 20180140521 to Geonnotti III, et al., and U.S. Patent Publication No. 20180140554A1 to Wittorff, et al., herein incorporated by reference in their entirety. In certain preferred embodiments, the tablets are chewable, dissolvable tablets, having a hardness that allows for biting and chewing the tablet by a user. They may be of any suitable size/weight for use in generating a fluid for use in the methods. Weights such as greater than about 1 gram or greater, or about 1.2 grams or greater, or about 1.5 grams or greater. In certain preferred embodiments, the tablets are from about 1.0 grams to about 3 grams, or from about 1.1 grams to about 2.5 grams, or from about 1.5 grams to about 2 grams. The tablets may be of any appropriate thickness, including a thickness of from about 5 to about 15 millimeters (mm), or from about 6 to about 12 mm, from about 7 to about 8 mm. The tablets may also have a diameter, diagonal, or longest edge length of any suitable size including from about 5 to about 20 mm, or from about 10 to about 18 mm, or from about 12 to about 16 mm. The tablets may be prepared via any of a variety of tableting methods known in the art. Conventional methods of tablet production include direct compression (“dry blending”), dry granulation followed by compression, wet granulation followed by drying and compression, application of energy to a blend of materials to be tableted, including applying heat, microwave, infrared, and other energies, combinations of two or more thereof, and the like. The tablets may comprise any of a variety of materials suitable for use therein. In certain embodiments, the tablets comprise at least one carbohydrate. Examples of carbohydrates include but are not limited to sugars such as dextrose, dextrose monohydrate, lactose, glucose, fructose, maltodextrin, sucrose, corn syrup solids and mannose; carbohydrate alcohols, such as sugar alcohols including sorbitol, lactitol, xylitol, erythritol, mannitol, maltitol, isomalt, and polyols; and combinations of two or more thereof. In certain preferred embodiments, the tablets comprise one or more sugar alcohols selected from the group consisting of xylitol, erythritol, maltitol, and isomalt, including, for example, xylitol, maltitol and combinations thereof, or xylitol, erythritol, isomalt and combinations thereof including combinations of xylitol, erythritol, and isomalt. In certain preferred embodiments, the tablets comprise erythritol alone or in combination with one or more additional sugar alcohols. In certain preferred embodiments, the tablets comprise xylitol alone or in combination with one or more additional sugar alcohols. In other preferred embodiments, the tablets comprise one or more sugar alcohols selected from the group consisting of sorbitol, lactitol, xylitol, mannitol, maltitol, isomalt, and combinations two or more thereof. In some embodiments, the carbohydrates in the tablet may be Zerose™ 16952F erythritol supplied by Cargill, or Zerose™ DC 16966 erythritol also supplied by Cargill. In certain embodiments, the tablets comprise both erythritols. In certain embodiments, the tablets comprise a total amount of all carbohydrates in amount of at least 40% by weight of the tablet, including from about 40 to about 99%, or from about 75 to about 95%, or from about 80 to about 90% by weight of the tablets. In certain preferred embodiments, the carbohydrates comprise one or more sugar alcohols, and the tablet comprises a total amount of sugar alcohols of at least 40% by weight of the tablet, including from about 60 to 99%, or from about 80 to about 90% by weight of the tablets. In certain embodiments, the tablets comprise at least 20% of each of two or more carbohydrates, including at least 20% of xylitol and at least 20% of one or more other sugar alcohols. In certain embodiments, the tablet comprises from about 20-50% of xylitol, including mixtures comprising from about 20-50% xylitol and from about 20-50% of erythritol, and from about 20-40% of isomalt. In certain embodiments, the tablets comprise from about 20-50% xylitol and at least 20% of one other sugar alcohol selected from the group consisting of sorbitol, lactitol, mannitol, maltitol, isomalt, and combinations two or more thereof. The tablets may be sugar-free. As will be readily understood by one of skill in the art, such tablets may contain sugar alcohols but are nevertheless substantially free of sugars such as glucose, dextrose, sucrose, or oligomers/polymers of sugar molecules. In addition to poorly soluble calcium-containing compounds, the tablets may comprise one or more active ingredients for use in providing an oral care benefit. In certain embodiments, the active ingredients include, but are not limited to, any of a variety of actives for providing benefits such as mouth cleaning, including debris removal, antimicrobial, including anti-plaque, anti-gingivitis, and reduction in malodor, biofilm disruption, prevention of bacterial attachment, modification of oral microbial community structure, modification of the metabolic profile of oral microbes, antiviral activity, anti-inflammatory, pH balance, tooth whitening, stain prevention, anti-sensitivity, anti-caries, enamel strengthening, breath freshening, oral hydration/dry mouth relief, erosion repair and prevention, active delivery and retention, sensory enhancement, mouth feel alteration, pain relief, wound healing, and the like. In addition, in certain embodiments, the tablets also comprise essential oils. Essential oils are volatile aromatic oils which may be synthetic or may be derived from plants by distillation, expression or extraction, and which usually carry the odor or flavor of the plant from which they are obtained. Useful essential oils may provide antiseptic activity. Some of these essential oils also act as flavoring agents. Useful essential oils include but are not limited to citra, thymol, menthol, methyl salicylate (wintergreen oil), eucalyptol, carvacrol, camphor, anethole, carvone, eugenol, isoeugenol, limonene, osimen, n-decyl alcohol, citronel, a-salpineol, methyl acetate, citronellyl acetate, methyl eugenol, cineol, linalool, ethyl linalaol, safrola vanillin, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil, laurel oil, cedar leaf oil, gerianol, verbenone, anise oil, bay oil, benzaldehyde, bergamot oil, bitter almond, chlorothymol, cinnamic aldehyde, citronella oil, clove oil, coal tar, eucalyptus oil, guaiacol, tropolone derivatives such as hinokitiol, avender oil, mustard oil, phenol, phenyl salicylate, pine oil, pine needle oil, sassafras oil, spike lavender oil, storax, thyme oil, tolu balsam, terpentine oil, clove oil, and combinations thereof. In certain preferred embodiments, the tablet comprises one or more bioactive essential oils selected from the group consisting of menthol, thymol, eucalyptol, and methyl salicylate. In certain preferred embodiments, the tablet comprises menthol and at least one other essential oil selected from thymol, eucalyptol, and methyl salicylate. In certain preferred embodiments, the tablet comprises menthol and eucalyptol, menthol, eucalyptol and thymol, or menthol, eucalyptol, thymol, and methyl salicylate. Thymol, [(CH3)2CHC6H3(CH3)OH, also known as isopropyl-m-cresol], is only slightly soluble in water but is soluble in alcohol, and its presence is one of the reasons alcohol was necessary in the well-established, high alcohol commercial mouth rinses. Methyl salicylate, [C6H4OHCOOCH3, also known as wintergreen oil], additionally provides flavoring to the together with its antimicrobial function. Eucalyptol (C10H18O, also known as cineol) is a terpene ether and provides a cooling, spicy taste. Eucalyptol may be used in place of thymol in certain formulations in the same amount if desired. Menthol (CH3C6H9(C3H7)OH), also known as hexahydrothymol) is also only slightly soluble in alcohol, and is fairly volatile. Menthol, in addition to any antiseptic properties, provides a cooling, tingling sensation. Other suitable antimicrobial agents include Halogenated Diphenyl Ethers, 2′,4,4′-trichloro-2-hydroxy-diphenyl ether (Triclosan), 2,2′-dihydroxy-5,5′-dibromo-diphenyl ether, Halogenated Salicylanilides, 4′5-dibromosalicylanilide, 3,4′,5-trichlorosalcylanilide, 3,4′,5-tribromosalicylanilide, 2,3,3′,5-tetrachlorosalicylanilide, 3,3′,5-tetrachlorosalicylanilide, 3,5, dibromo-3′-trifluoromethyl salicylanilide, 5-n-octanoyl-3′-trifluoromethyl salicylanilide, 3,5-dibromo-4′-trifluoromethyl salicylanilide, 3,5-dibromo-3′-trifluoro methyl salicylanilide (Flurophene), Benzoic Esters, Methyl-p-Hydroxybenzoic Ester, Ethyl-p-Hydroxybenzoic Ester, Propyl-p-Hydroxybenzoic Ester, Butyl-p-Hydroxybenzoic Ester, Halogenated Carbanilides, 3,4,4′-trichlorocarbanilide, 3-trifluoromethyl-4,4′-dichlorocarbanilide 3,3′,4-trichlorocarbanilide, Phenolic Compounds (including phenol and its homologs, mono- and poly-alkyl and aromatic halo (e.g. F, Cl, Br, I)-phenols, resorcinol and catechol and their derivatives and bisphenolic compounds), 2 Methyl-Phenol, 3 Methyl-Phenol, 4 Methyl-Phenol, 4 Ethyl-Phenol, 2,4-Dimethyl-Phenol, 2,5-Dimethyl-Phenol, 3,4-Dimethyl-Phenol, 2,6-Dimethyl-Phenol, 4-n-Propyl-Phenol, 4-n-Butyl-Phenol, 4-n-Amyl-Phenol, 4-tert-Amyl-Phenol, 4-n-Hexyl-Phenol, 4-n-Heptyl-Phenol, 2-Methoxy-4-(2-Propenyl)-Phenol (Eugenol), Mono- And Poly-Alkyl And Aralkyl Halophenols, Methyl-p-Chlorophenol, Ethyl-p-Chlorophenol, n-Propyl-p-Chlorophenol, n-Butyl-p-Chlorophenol, n-Amyl-p-Chlorophenol, sec-Amyl-p-Chlorophenol, n-Hexyl-p-Chlorophenol, Cyclohexyl-p-Chlorophenol, n-Heptyl-p-Chlorophenol, n-Octyl-p-Chlorophenol, O-Chlorophenol, Methyl-o-Chlorophenol, Ethyl-o-Chlorophenol, n-Propyl-o-Chlorophenol, n-Butyl-o-Chlorophenol, n-Amyl-o-Chlorophenol tert-Amyl-o-Chlorophenol, n-Hexyl-o-Chlorophenol, n-Heptyl-o-Chlorophenol, p-Chlorophenol, o-Benzyl-p-Chlorophenol, o-Benzyl-m-methyl-p-Chlorophenol o-Benzyl-m,m-dimethyl-p-Chlorophenol, o-Phenylethyl-p-Chlorophenol, o-Phenylethyl-m-methyl-p-Chlorophenol, 3-Methyl-p-Chlorophenol, 3,5-Dimethyl-p-Chlorophenol, 6-Ethyl-3-methyl-p-Chlorophenol, 6-n-Propyl-3-methyl-p-Chlorophenol, 6-iso-Propyl-3-methyl-p-Chlorophenol, 2-Ethyl-3,5-dimethyl-p-Chlorophenol, 6-sec Butyl-3-methyl-p-Chlorophenol, 2-iso-Propyl-3,5-dimethyl-p-Chlorophenol, 6-Diethylmethyl-3-methyl-p-Chlorophenol, 6-iso-Propyl-2-ethyl-3-methyl-p-Chlorophenol, 2-sec Amyl-3,5-dimethyl-p-Chlorophenol, 2-Diethylmethyl-3,5-dimethyl-p-Chlorophenol, 6-sec Octyl-3-methyl-p-Chlorophenol, p-Bromophenol, Methyl-p-Bromophenol, Ethyl-p-Bromophenol, n-Propyl-p-Bromophenol, n-Butyl-p-Bromophenol, n-Amyl-p-Bromophenol, sec-Amyl-p-Bromophenol, n-Hexyl-p-Bromophenol, cyclohexyl-p-Bromophenol, o-Bromophenol, tert-Amyl-o-Bromophenol, n-Hexyl-o-Bromophenol, n-Propyl-m,m-Dimethyl-o-Bromophenol, 2-Phenyl Phenol, 4-chloro, 2-methyl phenol, 4-chloro-3-methyl phenol, 4-chloro-3,5-dimethyl phenol, 2,4-dichloro-3,5-dimethylphenol, 3,4,5,6-terabromo-2-methylphenol, 5-methyl-2-pentylphenol, 4-isopropyl-3-methylphenol, 5-chloro-2-hydroxydiphenylemthane, Resorcinol And Its Derivatives, Resorcinol, Methyl-Resorcinol, Ethyl-Resorcinol, n-Propyl-Resorcinol, n-Butyl-Resorcinol, n-Amyl-Resorcinol, n-Hexyl-Resorcinol, n-Heptyl-Resorcinol, n-Octyl-Resorcinol, n-Nonyl-Resorcinol, Phenyl-Resorcinol, Benzyl-Resorcinol, Phenylethyl-Resorcinol, Phenylpropyl-Resorcinol, p-Chlorobenzyl-Resorcinol, 5-Chloro-2,4-Dihydroxydiphenyl Methane, 4′-Chloro-2,4-Dihydroxydiphenyl Methane, 5-Bromo-2,4-Dihydroxydiphenyl Methane, 4′-Bromo-2,4-Dihydroxydiphenyl Methane, Bisphenolic Compounds, Bisphenol A, 2,2′-methylene bis(4-chlorophenol), 2,2′-methylene bis(3,4,6-trichlorophenol) (hexachlorophene), 2,2′-methylene bis(4-chloro-6-bromophenol), bis(2-hydroxy-3,5-dichlorophenyl) sulfide, bis(2-hydroxy-5-chlorobenzyl) sulfide, menthoxy-1,2-propanediol, ortho-methoxy cinnamic aldehyde, menthyl-3-hydroxybutanoate, combinations of two or more thereof, and the like. Other antimicrobial agents include, but are not limited to: hexetidine; fatty acid compounds such as caproic acid, caprilic acid, capric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, linolelaidic acid, arachidonic acid vitamin E, vitamin E acetate, apigenin and mixtures thereof; long chain fatty alcohols such as described in US Patent publication US 20110123462 to Mordas et al., herein incorporated by reference in its entirety, (examples of which include, but are not limited to 1-decen-3-ol; cis-4-decen-1-ol, trans-2-decen-1-ol, cis-2-nonen-1-ol, cis-4-decenal, trans-2-decenal, cis-7-decenal, cis-5-octen-1-ol, trans-2-octen-1-ol, 1-octen-3-ol, cis-3-nonen-1-ol, trans-2-nonen-1-ol, cis-6-nonen-1-ol, 9-decen-1-ol, trans-2-undecen-1-ol, trans-2-dodecen-1-ol, trans-2-octenal, trans-2-nonenal, 6-nonenal, cis-2-decenal, trans-2-undecenal, trans-2-dodecenal, cis-3-octen-1-ol, 3-octen-2-ol, 10-undecen-1-ol, trans-2-tridecen-1-ol, stereoisomers thereof and mixtures thereof); cyclic sesquiterpene alcohols, such as farnesol; N′-alkyl-L-arginine alkyl ester (e.g., Lauroyl Arginine Ethyl Ester) and salts such as described in U.S. Pat. No. 5,874,068 to Engelman et al., herein incorporated by reference in its entirety; Amino acid derivative compounds as described in U.S. Patent Publication No. 20160145203 to Gambogi, et al., herein incorporated by reference in its entirety; antimicrobial peptides, such as retrocyclin (RC101), protegrin-1 (PG1) or KSL-W; and surfactants, including cationic surfactants such as cetylpyridinium chloride, chlorhexedine and mixtures thereof. Additionally, antimicrobial extracts of certain botanical or fruits may be included, including proanthocyanidins (PACs) found in cranberry such as, flavan-3-ols (and polymers of), procyanidins (and polymers of), terpenes (and polymers of), hydroxybenzole acids, hydroxycinnamic acids, anthocyanidins (and polymers of), flavonols (and polymers of), and other cyanidins and peonidins. Oils such as peppermint oil and sage oil are also useful herein. Other suitable actives include fluoride ion sources such as sodium fluoride, sodium monofluorophosphate, stannous fluoride, and amine fluorides (providing, for example, about 1-5000 ppm of fluoride ion, optionally about 200-1150 ppm of fluoride ion); anti-calculus agents, such as water-soluble pyrophosphate salts such as alkali metal pyrophosphates, polyacrylates and copolymers of maleic anhydride or acid and methyl vinyl ether, polyamino propane sulfonic acid (AMPS), zinc citrate trihydrate, polypeptides (such as polyaspartic and polyglutamic acids), and mixtures of two or more thereof, water-soluble pyrophosphate salts such as alkali metal pyrophosphates; chelating agents such as tartaric acid and pharmaceutically-acceptable salts thereof, citric acid and alkali metal citrates and mixtures thereof tooth desensitization agents which reduce tooth sensitivity including potassium salts such as potassium nitrate and potassium chloride and strontium salts such as strontium chloride and strontium acetate; tooth whitening agents and vitamins such as vitamin A; as well as pigments and colorants such as inorganic white pigments, inorganic colored pigments, pearling agents, filler powders and the like, as well as talc, mica, magnesium carbonate, magnesium silicate, aluminum magnesium silicate, silica, titanium dioxide, zinc oxide, red iron oxide, brown iron oxide, yellow iron oxide, black iron oxide, ferric ammonium ferrocyanide, manganese violet, ultramarine, nylon powder, polyethylene powder, methacrylate powder, polystyrene powder, silk powder, crystalline cellulose, starch, titanated mica, iron oxide titanated mica, bismuth oxychloride, and mixtures of two or more thereof. Enzymes are another type of active that may be used in the tables. Useful enzymes include those that belong to the category of proteases, lytic enzymes, plaque matrix inhibitors and oxidases: Proteases include papain, pepsin, trypsin, ficin, bromelin; cell wall lytic enzymes include lysozyme; plaque matrix inhibitors include dextranases, mutanases; and oxidases include glucose oxidase, lactate oxidase, galactose oxidase, uric acid oxidase, laccase, peroxidases including horse radish peroxidase, myeloperoxidase, lactoperoxidase, chloroperoxidase. The oxidases also have whitening/cleaning activity, in addition to antimicrobial properties. Ingredients which are metabolized by oral bacteria to cause a benefit effect in the oral cavity may also be included in these tablets, including arginine, arginine monohydrochloride, and inulin-type fructans, maltodextrin, fructooligosaccharides and galactooligosaccharides. Additionally, these tablets may be used to deliver probiotic strains of bacteria, including certain species of lactobacilli and bifidobacteria,Saccharomycesspp, streptococci, enterococci and commensalEscherichia coli. The tablet may also be used to deliver pharmaceutical actives to treat oral diseases or disease symptoms which occur in the oral cavity or the oropharynx, such as anesthetics, antibiotics, antifungals, antiviral, and anti-inflammatory compounds. In certain embodiments, the tablets for use in the present invention may comprise any of a variety of salivation agents (also known as salivary stimulants or salivary agents). Suitable salivation agents include food organic acids such as citric, lactic, malic, succinic, ascorbic, adipic, fumaric, tartaric acids, parasympathomimetic drugs, such as choline esters like pilocarpine hydrochloride, or cholinesterase inhibitors, and combinations of two or more thereof. In certain preferred embodiments, the salivation agents comprise citric acid, succinic acid, or a combination thereof alone or in combination with other salivation agents. In certain preferred embodiments, the salivation agent comprises jambu oleoresin extract. The salivation agent may be present in any suitable amount for use in the present invention including, from about 0.001 to about 5% by weight of the tablet, including from about 0.01 to about 3%, from about 0.01 to about 1% from about 0.01 to about 0.5%, from about 0.01 to about 0.25%, and from about 0.01 to about 0.1% by weight of the tablet. The tablets for use in the present invention may comprise any of a variety of additional ingredients suitable for use in the tablets including, for example, sweeteners, lubricants, fillers, adsorbents, disintegrants, glidants, superdisintegrants, flavor and aroma agents, antioxidants, preservatives, texture enhancers, coloring agents, and the like, and mixtures of two or more thereof. In certain embodiments, the tablets may comprise additional sweeteners including, but not limited to, synthetic or natural sugars; artificial sweeteners such as saccharin and its salts including sodium saccharin, aspartame, acesulfame and its salts including potassium acesulfame, thaumatin, glycyrrhizin, sucralose, dihydrochalcones, alitame, miraculin, monellin, stevioside, and combinations of two or more thereof. In certain preferred embodiments, the tablets comprise sucralose, potassium acesulfame, or a combination thereof. The tablets may comprise any suitable total amounts of additional sweeteners including from 0.001 to about 8% by weight, including from about 0.02 to about 8%, from about 0.1 to about 3%, from about 0.1 to about 1%, and from about 0.1 to about 0.5% by weight of the tablet. The tablets may also include lubricant materials in certain embodiments. Suitable lubricants include, but are not limited to, long chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glycerides waxes, and mixtures thereof. Such materials may be present in any suitable amount including from about 0.01 to about 5%, including from about 0.1 to about 5%, from about 0.5 to about 3%, including from about 0.5 to about 2% by weight of the tablet. Suitable fillers include, but are not limited to, water insoluble plastically deforming materials (e.g., microcrystalline cellulose or other cellulosic derivatives), and mixtures thereof. Suitable adsorbents include, but are not limited to, water-insoluble adsorbents such as dicalcium phosphate, tricalcium phosphate, silicified microcrystalline cellulose (e.g., such as distributed under the PROSOLV brand (PenWest Pharmaceuticals, Patterson, N.Y.)), magnesium aluminometasilicate (e.g., such as distributed under the NEUSILIN brand (Fuji Chemical Industries (USA) Inc., Robbinsville, N.J.)), clays, silicas, bentonite, zeolites, magnesium silicates, hydrotalcite, veegum, and mixtures thereof. Suitable disintegrants include, but are not limited to, sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starches, microcrystalline cellulose, and mixtures thereof. Examples of superdisintegrants include, but are not limited to, croscarmellose sodium, sodium starch glycolate and cross-linked povidone (crospovidone). In one embodiment, the tablet contains up to about 5% by weight of such superdisintegrant. Examples of flavors and aromatics include, but are not limited to, essential oils including distillations, solvent extractions, or cold expressions of chopped flowers, leaves, peel or pulped whole fruit containing mixtures of alcohols, esters, aldehydes and lactones; essences including either diluted solutions of essential oils, or mixtures of synthetic chemicals blended to match the natural flavor of the fruit (e.g., strawberry, raspberry and black currant); artificial and natural flavors of brews and liquors, e.g., cognac, whisky, rum, gin, sherry, port, and wine; tobacco, coffee, tea, cocoa, and mint; fruit juices including expelled juice from washed, scrubbed fruits such as lemon, orange, and lime; spear mint, pepper mint, wintergreen, cinnamon, cacoe/cocoa, vanilla, liquorice, menthol, eucalyptus, aniseeds nuts (e.g., peanuts, coconuts, hazelnuts, chestnuts, walnuts, colanuts), almonds, raisins; and powder, flour, or vegetable material parts including ginger. Examples of antioxidants include, but are not limited to, tocopherols, ascorbic acid, sodium pyrosulfite, butylhydroxytoluene, butylated hydroxyanisole, edetic acid, and edetate salts, and mixtures thereof. Examples of preservatives include, but are not limited to, citric acid, tartaric acid, lactic acid, malic acid, acetic acid, benzoic acid, and sorbic acid, and mixtures thereof. Examples of texture enhancers include, but are not limited to, pectin, polyethylene oxide, and carrageenan, and mixtures thereof. In one embodiment, texture enhancers are used at levels of from about 0.1% to about 10% percent by weight. In one embodiment, the tablets further contain one or more effervescent couples. In one embodiment, effervescent couple contains one member from the group consisting of sodium bicarbonate, potassium bicarbonate, calcium carbonate, magnesium carbonate, and sodium carbonate, and one member selected from the group consisting of citric acid, malic acid, fumaric acid, tartaric acid, phosphoric acid, and alginic acid. In one embodiment, the combined amount of the effervescent couple(s) in the powder blend/tablet is from about 2 to about 20 percent by weight, such as from about 2 to about 10 percent by weight of the total weight of the powder blend/tablet. In some embodiments, the tablets may be made by mixing its ingredients and heating them until they melt into a syrup, then exposing the mixture to pressurized carbon dioxide gas (about 600 pounds per square inch, or psi) and allowing it to cool. The process causes tiny high-pressure bubbles to be trapped inside the candy. When the tablet is placed in the mouth and comes into contact with saliva, the tablet breaks and dissolves, releasing the carbon dioxide from the bubbles, resulting in a popping and sizzling sound and leaving a slight tingling sensation. The tablets are substantially free of gum bases as used conventionally to manufacture gums, as opposed to chewable, dissolvable tablets. In preferred embodiments, the tablets of the present invention are not sufficiently heated to form hard candies nor lozenges as such terms and their manufacture is understood in the art and which product forms are intended to dissolve more slowly than chewable, dissolvable tablets. Any suitable fluoride-containing compounds/sources of fluoride or may be used in the methods of the present invention. Examples of suitable compounds used as fluoride sources include sodium fluoride, sodium monofluorophosphate, stannous fluoride, amine fluorides, titanium tetrafluoride, combinations of two or more thereof, and the like. The fluoride-containing compounds may be delivered to the oral cavity in any suitable form, including but not limited to a solid form, for example, in a powder, tablet, confection, chewing gum, and the like, or in liquid form, for example, in a mouthwash, mouth rinse, toothpaste, gel, and the like. In certain embodiments, the fluoride-containing compound is introduced to the oral cavity via a tablet. In certain embodiments, the fluoride-containing compound is introduced to the oral cavity via mouthwash. Any of a variety of commercially available fluoride containing products, e.g. rinses, toothpastes, and the like, may be used herein. The concentration of fluoride in the delivery vehicle/product depends at least in part on the form of the selected delivery vehicle. Generally, it is desired to deliver a concentration of fluoride in the oral tissue that is effective for reacting with the administered calcium to form calcium-bound fluoride deposits in plaque, on teeth and in oral tissue. The delivery vehicle should provide, for example, about 0.01-5000 ppm of fluoride ion, or about 100-1500 ppm of fluoride ion, or about 100-500 ppm of fluoride ion. By way of example, to deliver these concentrations of fluoride in the oral cavity, the fluoride concentration in a fluoride rinse is between about 50 ppm to about 1000 ppm. When formulated as a tablet, the total fluoride content preferably is between about 0.01 ppm to about 10 ppm fluoride. When formulated in a dentifrice, the fluoride content preferably is between about 500 ppm to about 5000 ppm, or about 500 to about 1500 ppm of fluoride. The embodiments of the present invention can be used with any commercial fluoride product (e.g., mouthwash, mouth rinse, dentifrice, gels/foams, varnishes, lozenges, tablets, chewing gum, medical supplements) specifically formulated to take advantage of the chemistry described herein, or in regions which have water fluoridation. In some embodiments, fluoride compounds may be delivered in the form of a mouthwash or dentifrice. Mouthwashes and rinses are usually antiseptic solutions intended to reduce the microbial load in the oral cavity, although other mouthwashes might be given for other reasons such as for their analgesic, anti-inflammatory or anti-fungal action. Additionally, some rinses act as saliva substitutes to neutralize acid and keep the mouth moist in xerostomia (dry mouth). Cosmetic mouth rinses temporarily control or reduce bad breath and leave the mouth with a pleasant taste. Fluoride mouthwashes and rinses typically use the sodium fluoride form, though stannous fluoride may also be used. They can be professionally-applied by a dental professional or used at home. The most common fluoride compound used in mouth rinse is sodium fluoride. Fluoride mouth rinses range from about 0.02% to about 0.2% (about 100 to about 1,000 ppm) in concentration, or about 0.02% to about 0.1% In some embodiments, daily fluoride rinses use a fluoride content of about 0.02%. After a fluoride mouth rinse treatment, the fluoride in the mouth rinse is retained in the saliva. Toothpaste is a paste or gel dentifrice used with a toothbrush to clean and maintain the aesthetics and health of teeth. Toothpaste is used to promote oral hygiene: it is an abrasive that aids in removing dental plaque and food from the teeth, assists in suppressing halitosis, and delivers active ingredients. Fluoride toothpastes may contain up to about 1.1% (5,000 ppm) fluoride in the form of sodium fluoride, or about 0.454% stannous fluoride. Typically, toothpaste has between about 0.22% (1,000 ppm) and about 0.312% (1,450 ppm) fluoride in the form of sodium fluoride or sodium monofluorophosphate. Most toothpastes with fluoride contain mild abrasives to remove heavier debris and light surface staining. These abrasives include calcium carbonate, silica gels, magnesium carbonates and phosphate salts. Fluoride is available in several forms during toothbrushing. It is available as a free ionic fluoride which can react with the tooth structure, interfere with the metabolism of bacteria in plaque, or absorb to the oral mucosa. It is also available as profluoride compounds which can precipitate in the mouth during toothbrushing and release ionic fluoride. Anti-sensitivity toothpastes with fluoride are also available for those who have sensitive teeth. Some anti-sensitivity toothpastes with fluoride on the market contain the ingredients called strontium chloride or potassium nitrate which help to alleviate tooth sensitivity. In some embodiments, fluoride containing foams/gels, varnishes, lozenges, of supplements may be used. Professionally-applied, or home-applied fluoride containing gels and foams may also be used. Typically, these fluoride gels include 2.0% neutral sodium fluoride and 1.23% acidulated phosphate fluoride. They are usually applied using a foam mouth tray which is held in the mouth by gently biting down. The application usually lasts for approximately four minutes. Some gels are made for home application with the use of a custom tray. The user holds a fluoride treatment against their teeth overnight or several minutes during the day. The concentration of fluoride in these gels is much lower than in professional products, typically containing between about 0.15% and 0.5% about fluoride. Fluoride varnish has practical advantages over gels in ease of application and use of smaller volume of fluoride than required for gel applications. The principle of fluoride varnish is to apply fluoride salt in a very high concentration (approximately 50,000 ppm) directly onto the surface of the teeth. Fluoride varnish is a resin-based application that is designed to stay on the surface of the teeth for several hours. As this varnish rests on the tooth's surface, saliva dissolves the fluoride salt, which in turn allows fluoride ions to be released. Later, the fluoride is re-released into the oral cavity from these reservoirs which acts as protection for the teeth against cavities. Fluoridated lozenges and medical fluoride supplements may also be used as the fluoride source in the present invention. Fluoridated lozenges contain about 1 mg fluoride each and are meant to be held in the mouth and sucked. The dissolved lozenge is swallowed slowly, so the use of lozenges is both a topical and a systemic therapy. Medical fluoride supplements are typically in the form of tablets, lozenges, or liquids (including fluoride-vitamin preparations). In some embodiments, the fluoride source in the present invention could be fluoridated water. Water fluoridation is the controlled addition of fluoride to a public water supply reduce tooth decay. Its use in the U.S. began in the 1940s and is now used for about two-thirds of the U.S. population on public water systems and for about six percent of people worldwide. It is the most cost-effective way to induce fluoride into the oral cavity. In use, the poorly soluble calcium compounds are orally administered in a delivery vehicle prior to the administration of the fluoride. In some embodiments, the poorly soluble calcium delivery vehicle is used just prior to the use of a fluoride-containing product. In these cases, the fluoride-containing compound is used as soon as practicable after use of the poorly soluble calcium compound delivery vehicle. In other embodiments, the administration of the fluoride-containing product is significantly after administration of the poorly soluble calcium compound. The administration of the fluoride-containing product may be one minute, or five minutes, or ten minutes, or thirty minutes, or one hour, or two hours, or four hours, or eight hours, or twelve hours or more after administration of the poorly soluble calcium compound or other times sufficient to allow most or all of the calcium to migrate from the oral cavity into the oral tissue. In one embodiment, the fluoride-containing compound is administered about one minute, or ten minutes, or twenty minutes, or one hour or more after the administration of the poorly soluble calcium-containing compound. In some embodiments, the vehicle with the poorly soluble calcium-containing compound is administered at numerous points in the day, with the fluoride-containing compound being administered at numerous points in the day, or only once a day. Healthy human saliva has a pH of 7.4. When acidic foods and drinks are consumed, the pH within the mouth decreases and can cause the pH in the dental plaque to fall rapidly below 5.0. This happens through the production of acids as the bacteria metabolize the ingested nutrients. Tooth decay can occur when the pH level in the mouth is 5.5 or below. When the mouth undergoes dramatic or long-lasting periods of low pH, it can cause cavity-causing bacteria to grow. Dental plaque that is kept at 7.0 or greater does not experience a shift to cavity-causing bacteria even when exposed to sugar. Calcium containing compounds, as they dissolve, may raise the pH in the oral cavity. The slow dissolution of poorly soluble calcium sources as discussed herein in the oral cavity can act as a buffer to prevent the rapid fall of pH to 5.5 or below. In some embodiments, a user can administer the poorly soluble calcium compound delivery vehicle immediately, or soon after ingesting food, such as a meal or snack. For example, within one minute, or two minutes, or five minutes, or ten minutes, or fifteen minutes, or thirty minutes after ingesting food. So, in some embodiments, the poorly soluble calcium compound delivery vehicle is administered two, or three, or four, or six, or eight or more times over the course of a day such as after meals or snacks, to help maintain poorly soluble calcium compound in plaque, on teeth, and in oral tissue. Fluoride compound may also be administered one, two, or three, or four or more times in conjunction with, the administration poorly soluble calcium compound. For example, a user may administer the poorly soluble calcium compound delivery vehicle after each meal or snack and administer the fluoride compound after one or more of the meals/snacks, or just at the end of the day. In other embodiments, the order of administration may be reversed (i.e. fluoride is administered prior to administration of calcium). Such a reverse system also is expected provide an increase in oral deposition of Ca—F. As with the administration of calcium followed by fluoride, it may be desirable to delay the administration of calcium subsequent to the administration of fluoride. In some embodiments, methods of the present invention comprise introducing into the oral cavity a tablet of the present invention. Any of a variety of know means may be used in the introducing step. For example, a tablet may be placed by hand into a user's mouth, the tablet may be introduced via an applicator, packaging, container, dosing apparatus, or other article or machine suitable for such purpose. In certain embodiments, chewing the tablet generates a fluid in the oral cavity while the tablet dissolves, generating a fluid comprising ingredients from the tablet. The tablet may be chewed for any time sufficient to generate fluid in accord with embodiments comprising a chewing step, including, for example, chewing for at least five seconds, including at least ten seconds, at least fifteen seconds, at least twenty seconds, or at least thirty seconds. In certain preferred embodiments, the tablet is chewed for about ten seconds or for at least ten seconds. The fluid generated may be forced around the oral cavity. The forcing step comprises applying any suitable amount of force within the oral cavity to move fluid in any one or more directions, e.g., from side to side, up, down, back, forth, forward, back, around, onto and/or through teeth, gums, cheek, and/or another surface in the oral cavity. In certain embodiments, the fluid is forced (a) from a lingual surface of the oral cavity toward or onto a buccal and/or labial surface of the oral cavity, (b) from a buccal and/or labial surface of the oral cavity toward or onto a lingual surface of the oral cavity, or both (a) and (b). In certain embodiments, the fluid is forced around the oral cavity using muscular movements of the cheeks and/or tongue. In certain embodiments, the fluid is forced around the oral cavity with the lips closed. In certain preferred embodiments, the forcing step comprises forcing at least a portion of the fluid generated around the mouth, with lips closed, using muscular movements of the cheeks and tongue. The fluid may be forced in any suitable manner in accord with the present invention, including, for example, by swishing, rinsing, washing, swirling, gargling, agitating, threshing, sloshing, irrigating, actuating, gushing, douching, swooshing, splooshing, squooshing, pushing, maneuvering, mixing, twisting, flowing, bathing, circulating, distributing, dispersing, wetting, moving, and the like, the fluid in any one or more directions, or otherwise using the fluid as a mouthwash, mouth rinse, or other liquid oral care product. The fluid may be forced/moved within the oral cavity for any suitable period of time including at for at least five seconds, including at least ten seconds, at least fifteen seconds, at least twenty seconds, or at least thirty seconds. In certain preferred embodiments, the fluid is agitated for about thirty seconds or for at least thirty seconds. In certain embodiments, the fluid generated in the present methods may be swallowed/ingested by a user or may be expelled/spit out after the moving step. In certain preferred embodiments, the methods comprise swallowing at least a portion of the fluid. However, upon swallowing/ingesting the fluid, some of the poorly soluble calcium-containing compound remains in the oral cavity, such as in or in the plaque, teeth or oral tissue. EXAMPLES The present invention will be further understood by reference to the following specific examples that are illustrative of the methods of the present invention. It is to be understood that many variations of the methods would be apparent to those skilled in the art. The following examples are only illustrative. Example 1: pH Cycling Study A pH cycling study was performed. The study was developed to simulate the spike in acid production after three meals during the course of a day. Bovine enamel specimens were polished and artificial lesions formed by immersion of the specimens in a lesion forming solution consisting of 0.1 M lactic acid and 0.2% Carbopol C907 which was 50% saturated with hydroxyapatite and adjusted to pH 5.0. The average specimen surface microhardness (SMH) was determined from four indentations on the surface of each specimen, using a Vickers hardness indenter at a load of 200 g for 15 seconds. The lesion surface hardness range was 25-45 VEIN and average lesion depth was approximately 70 microns. Specimens were divided into subgroups of 18 specimens balanced by post-demineralization surface microhardness (SMH) values. Remineralization efficacy was evaluated under two different cyclic treatment regimens. Both regimens consisted of three one-minute fluoride treatment periods (100 ppm, pH=6.5), and three one-hour acid challenges in the lesion forming solution, with calcium introduced during the acid challenges. For the remaining time (approximately 21 hours), the specimens were in artificial saliva consisting of 0.213 g/L calcium chloride dihydrate, 0.738 g/L potassium phosphate monobasic, 0.738 g/L potassium chloride, 0.381 g/L sodium chloride, and 2.2 g/L of porcine mucin. The difference between the two treatment regimens was the relative order of the fluoride treatments and the acid challenges. In Regimen I, shown in Table 1, the demineralization event (Demin: Lactic Acid Challenge) occurred first, followed by the remineralization event (Remin: Artificial Saliva Soak). In Regimen II, shown in Table 2, the order of events was reversed, and the remineralization event occurred first, followed by the demineralization event. TABLE 1pH cycling Regimen ITimeEvent9:00-9:20 a.m.Demin: Lactic Acid Challenge (15 mL)9:20-9:30 a.m.+Calcium Treatment9:30-9:40 a.m.+5 mL Lactic Acid9:40-9:50 a.m.+5 mL Lactic Acid9:50-10:00 a.m.+5 mL Lactic Acid10:00-10:30 a.m.Remin: Artificial Saliva Soak10:30-10:32 a.m.Fluoride Treatment10:32 a.m.-12:00 p.m.Remin: Artificial Saliva Soak12:00-12:20 p.m.Demin: Lactic Acid Challenge (15 mL)12:20-12:30 p.m.+Calcium Treatment12:30-12:40 p.m.+5 mL Lactic Acid14:40-12:50 p.m.+5 mL Lactic Acid12:50-1:00 p.m.+5 mL Lactic Acid1:00-1:30 p.m.Remin: Artificial Saliva1:30-1:32 p.m.Fluoride Treatment1:32-3:00 p.m.Remin: Artificial Saliva Soak3:00-3:20 p.m.Demin: Lactic Acid Challenge (15 mL)3:20-3:30 p.m.+Calcium Treatment3:30-3:40 p.m.+5 mL Lactic Acid3:40-3:50 p.m.+5 mL Lactic Acid3:50-4:00 p.m.+5 mL Lactic Acid4:00-4:30 p.m.Remin: Artificial Saliva Soak4:30-4:32 p.m.Fluoride Treatment4:32 p.m.-9:00 a.m.Remin: Overnight Artificial Saliva Soak TABLE 2pH cycling Regimen IITimeEvent9:00-9:02 a.m.Fluoride Treatment9:02-9:30 a.m.Remin: Artificial Saliva Soak9:30-9:50 a.m.Demin: Lactic Acid Challenge (15 mL)9:50-10:0 a.m.+Calcium Treatment10:00-10:10 a.m.+5 mL Lactic Acid10:10-10:20 a.m.+5 mL Lactic Acid10:20-10:30 a.m.+5 mL Lactic Acid10:30 a.m.-12:00 p.m.Remin: Artificial Saliva Soak12:00-12:02 p.m.Fluoride Treatment12:02-12:30Remin: Artificial Saliva Soak12:30-12:50 p.m.Demin: Lactic Acid Challenge (15 mL)12:50-1:00 p.m.+Calcium Treatment1:00-1:10 p.m.+5 mL Lactic Acid1:10-1:20 p.m.+5 mL Lactic Acid1:20-1:30 p.m.+5 mL Lactic Acid1:30-3:00 p.m.Remin: Artificial Saliva Soak3:00-3:02 p.m.Fluoride Treatment3:02-3:30 p.m.Remin: Artificial Saliva Soak3:30-3:50 p.m.Demin: Lactic Acid Challenge (15 mL)3:50-4:00 p.m.+Calcium Treatment4:00-4:10 p.m.+5 mL Lactic Acid4:10-4:20 p.m.+5 mL Lactic Acid4:20-4:30 p.m.+5 mL Lactic Acid4:30 p.m.-9:00 a.m.Remin: Overnight Artificial Saliva Soak During the acid challenge periods (Demin: Lactic Acid Challenge), a 15 ml aliquot of the lesion forming solution was transferred to a treatment beaker containing six enamel specimens. Calcium compounds at equimolar calcium concentration, were introduced to the solution 20 minutes after the start of the acid challenge. The masses of the calcium compounds are shown in Table 3. To simulate clearance of the calcium by saliva, 5 mL of fresh lesion forming solution was added every 10 minutes. TABLE 3Mass of powder to be introducedduring demineralization periodsCalcium compoundMass (mg)Alpha-Tricalcium Phosphate25Calcium Carbonate24Calcium Chloride Dihydrate35 The pH cycling regimens were repeated for five consecutive days. Two poorly soluble calcium compounds (Alpha-Tricalcium Phosphate and Calcium Carbonate) were compared to one highly soluble calcium compound (Calcium Chloride Dihydrate). As a negative control, the calcium sources, as well as the fluoride source, were replaced with water. Also, for comparison purposes, a study was performed with only the fluoride source. After five days of treatment and pH cycling, remineralization efficacy was measured from change in surface microhardness (SMH) and enamel fluoride uptake (EFU). The surface microhardness was measured as described above, where the indentations were measured next to the baseline indentations. The fluoride content was determined by micro-drilling to a depth of 100 micron into each tooth. The enamel powder from the drill hole was collected, dissolved (20 microliter of HClO4, 40 microliter Citrate/EDTA Buffer and 40 microliter deionized (DI) water) and analyzed for fluoride by comparison to a similarly prepared standard curve. Tables 4 and 5 summarize the results of pH cycling Regimen I and II, respectively. TABLE 4Results of 5-Day treatment and pH cycling by regimen ISMH (VHN)EFU (micro g/cm3)Water30.1(1.2)151(19)100 ppm F48.7(4.4)2705(124)100 ppm F + aTCP85.3(6.3)2726(135)100 ppm F + CaCO393(3.9)2942(67)100 ppm F + CaCl283.4(6.4)2900(14) TABLE 5Results of 5-Day treatment and pH cycling by regimen IISMH (VHN)EFU (micro g/cm3)Water12.6(1.3)80(5)100 ppm F20.2(1.9)1509(54)100 ppm F + aTCP27.1(3.3)1696(138)100 ppm F + CaCO322.4(1.2)1737(99)100 ppm F + CaCl257.7(9.3)2352(190) Table 4 shows that after five days of Regimen I treatment and pH cycling, the surface microhardness (SMH) of all calcium treated specimens were significantly higher than that of the negative control (water only) specimens, as well as that of the specimens only treated with 100 ppm F. The SMH of the specimens only treated with 100 ppm F was also significantly higher than that of the negative control (water only). The SMH of the specimens treated with the two poorly soluble calcium compounds (Alpha-Tricalcium Phosphate and Calcium Carbonate) were comparable to the SMH of the highly soluble calcium compound (Calcium Chloride Dihydrate) treated specimens. The table also shows that after five days of Regimen I treatment and pH cycling, the enamel fluoride uptake (EFU) of all calcium treated specimens were significantly higher than that of the negative control (water only) specimens, and equivalent to the EFU of the specimens only treated with 100 ppm F. The EFU of the specimens only treated with 100 ppm F was significantly higher than that of the negative control (water only) specimens. The EFU of the specimens treated with the two poorly soluble calcium compounds (Alpha-Tricalcium Phosphate and Calcium Carbonate) were comparable to the EFU of the highly soluble calcium compound (Calcium Chloride Dihydrate) treated specimens. Table 5 shows that after five days of Regimen II treatment and pH cycling, the surface microhardness (SMH) of all calcium treated specimens were significantly higher than that of the negative control (water only) specimens. The SMH of the specimens only treated with 100 ppm F was also significantly higher than that of the negative control (water only) specimens. The SMH of the specimens treated with the two poorly soluble calcium compounds (Alpha-Tricalcium Phosphate and Calcium Carbonate) were comparable to the SMH of the specimens only treated with 100 ppm F. The highly soluble calcium compound (Calcium Chloride Dihydrate) treated specimens had the highest values of SMH. The table also shows that after five days of Regimen II treatment and pH cycling, the enamel fluoride uptake (EFU) of all calcium treated specimens were significantly higher than that of the negative control (water only) specimens. The EFU of the specimens only treated with 100 ppm F was also significantly higher than that of the negative control (water only) specimens. The EFU of the specimens treated with the two poorly soluble calcium compounds (Alpha-Tricalcium Phosphate and Calcium Carbonate) were equivalent to the EFU of the specimens only treated with 100 ppm F, while the EFU of the highly soluble calcium compound (Calcium Chloride Dihydrate) treated specimens were the highest values of EFU. Example 2: pH Study The ability of highly soluble and poorly soluble forms of calcium to influence oral cavity pH was evaluated by the addition of calcium compounds at equimolar calcium concentration (8 mM Ca) to the lesion forming solution described in Example 1. Powders were added and allowed to stir for 30 minutes at 37 degrees C. before measuring the pH. The change in pH following addition of alpha-tricalcium phosphate, calcium carbonate, and calcium chloride are given in Table 6. The insoluble forms of calcium resulted in an increase in pH of the lesion forming solution and the soluble calcium resulted in a drop in pH. TABLE 6Change in pH of lesion forming solutionfollowing addition calcium compoundsCalcium compoundΔpHAlpha-Tricalcium Phosphate+0.33Calcium Carbonate+0.64Calcium Chloride Dihydrate−0.14 Example 3: Dissolution of Calcium Compounds in Plaque Fluid Dissolution of calcium compounds in plaque fluid was simulated using multicomponent thermodynamic speciation modelling, implemented using the software Geochemist's Workbench. The initial plaque fluid composition was modeled after starved plaque fluid from caries-free individuals. To simulate the low pH conditions following eating the initial pH was set to pH 5. Precipitation was suppressed for all minerals except for the mineral being modeled. FIG.1is a plot of pH versus the amount of mineral added for the highly soluble and poorly soluble forms of calcium generated from the software model. The plot shows that addition of alpha-tricalcium phosphate and calcium carbonate to simulated acidic plaque fluid results in an increase in pH from pH 5 to pH 6 or above when saturation is reached. Addition of soluble calcium results in a decrease in pH. FIG.2is a graph of HA saturation level versus the amount of mineral added for the highly soluble and poorly soluble forms of calcium. The plot shows that addition of alpha-tricalcium phosphate or calcium carbonate to simulated acidic plaque fluid results in an increase in hydroxyapatite supersaturation. At the solubility limit of alpha-tricalcium phosphate or calcium carbonate, hydroxyapatite supersaturation is at least five orders of magnitude greater than with addition of soluble calcium. Example 4: Saliva pH and Buffer Capacity The impact of calcium carbonate on saliva pH and buffer capacity was measured from saliva collected before and after consumption of a tablet containing 112 mg (7%) of calcium carbonate. Five subjects were asked to chew the tablet for 10 seconds, swish the generated liquid for 40 seconds and then swallow. Saliva was collected for five minutes prior to tablet use, and for five minutes immediately following. The collected saliva was homogenized by vortexing for 20 seconds and the pH measured using a pH electrode. The buffer capacity, defined as the mM of HCl required to drop the pH by one unit, was determined by acid titration. 0.1 M HCl was added 20-40 uL at a time to 0.5 mL of saliva, and the pH measured after each addition until the pH dropped below pH=5. The buffer capacity was calculated from the slope of the linear regression of mM of HCl added over the change in pH. TABLE 7Average saliva pH and buffer capacity before and afteruse of a tablet containing calcium carbonate (n = 5).Buffer CapacitypH(mM HCl)Before tablet6.9(0.2)7.4(0.5)use (baseline)After tablet use7.7(0.1)5.5(0.3) | 54,079 |
11857654 | DETAILED DESCRIPTION Definitions and General Techniques For simplicity and clarity of illustration, the figures illustrate the general manner of construction. Descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the present disclosure. Additionally, elements in the drawing are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of embodiments of the present disclosure. The same reference numerals in different figures denotes the same elements. The terms “first,” “second,” “third,” “fourth,” and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms “include,” and “have,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, device, or apparatus that comprises a list of elements is not necessarily limited to those elements but may include other elements not expressly listed or inherent to such a process, method, system, article, device, or apparatus. The terms “left,” “right,” “front,” “back,” “top,” “bottom,” “over,” “under,” and the like in the description and in the claims, if any, are used for descriptive purposes and not necessarily for describing permanent relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the apparatus, methods, and/or articles of manufacture described herein are, for example, capable of operation in other orientations than those illustrated or otherwise described herein. No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include items and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include items (e.g., related items, unrelated items, a combination of related items, and unrelated items, etc.), and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise. The present invention may be embodied in other specific forms without departing from its spirit or characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, health monitoring described herein are those well-known and commonly used in the art. No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. As defined herein, two or more elements are “integral” if they are comprised of the same piece of material. As defined herein, two or more elements are “non-integral” if each is comprised of a different piece of material. The present invention may be embodied in other specific forms without departing from its spirit or characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. As defined herein, “approximately” can, in some embodiments, mean within plus or minus ten percent of the stated value. In other embodiments, “approximately” can mean within plus or minus five percent of the stated value. In further embodiments, “approximately” can mean within plus or minus three percent of the stated value. In yet other embodiments, “approximately” can mean within plus or minus one percent of the stated value. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. The nomenclatures used in connection with, and the procedures and techniques of embodiments herein, and other related fields described herein are those well-known and commonly used in the art. All compositions and ingredients including percentage of ingredient according to this invention is to be construed by weight basis unless state otherwise. The following terms and phrases, unless otherwise indicated, shall be understood to have the following meanings.One Teaspoon (tsp) equal to 4.2 gram (g or gm) or 4.9 milliliters (ml).One Tablespoon (tbsp) equal to 14.7 gram (g or gm) or 14.78 milliliters (ml).One drop equal to 0.05 milliliters (ml). “Composition” herein refers to oral composition or dental composition. The term “oral composition” includes, but is not limited to, prevention and/or treatment of oral diseases, maintenance of oral health, reduction, or elimination of bad breath, whitening of teeth and prevention of gingival degradation and/or prevention of caries. It is intended to include various embodiments of compositions that are useful for all aspects of oral hygiene. “Hydroxyapatite”, also called hydroxyapatite (HA), is a naturally occurring mineral form of calcium apatite with the formula Ca5(PO4)3(OH), but it is usually written Ca10(PO4)6(OH)2to denote that the crystal unit cell comprises two entities. Hydroxyapatite is the hydroxyl end member of the complex apatite group. The OH−ion can be replaced by fluoride, chloride, or carbonate, producing fluorapatite or chlorapatite. It crystallizes in the hexagonal crystal system. Pure hydroxyapatite powder is white. Naturally occurring apatite can, however, also have brown, yellow, or green colorations, comparable to the discolorations of dental fluorosis. Up to 50% by volume and 70% by weight of human bone is a modified form of hydroxyapatite, known as bone mineral. Carbonated calcium-deficient hydroxyapatite is the main mineral of which dental enamel and dentin are composed. Hydroxyapatite crystals are also found in the small calcifications, within the pineal gland and other structures, known asCorpora arenaceaor ‘brain sand’. In an embodiment, remineralization of tooth enamel involves the reintroduction of mineral ions into demineralized enamel. Hydroxyapatite is the main mineral component of enamel in teeth. During demineralization, calcium and phosphorus ions are drawn out from the hydroxyapatite. The mineral ions introduced during remineralization restore the structure of the hydroxyapatite crystals. In an embodiment, hydroxyapatite is used within dentistry and oral and maxillofacial surgery, due to its chemical similarity to hard tissue. In an embodiment, synthetic hydroxyapatite (SHA) is proven to provide successful outcomes in alveolar socket preservation. Socket grafting using synthetic hydroxyapatite can result in successful bone regeneration. “Nanohydroxyapatite” or nano-hydroxyapatite or nano hydroxyapatite or like “NHAP” is a form of calcium crystal. It is nano sized form of hydroxyapatite. It works by remineralizing, it replaces missing sections of minerals that have dissolved out of enamel or bone by bonding directly to the bone or tooth surfaces. It can also stimulate new bone growth by acting on the cells that cause regrowth. Nanohydroxyapatite has properties such as high surface energy, high electrostatic field, strong polarization force, and high affinity for enamel and cementum, and it can enhance enamel surface remineralization, face microhardness, tooth whitening and reinforce the resistance to acid. Nanohydroxyapatite can also adsorb amino acids and polysaccharides like protein and glucose inhibiting the production of cariogenic speckle and managing dental caries and these two types of periodontal disease. Nanohydroxyapatite possesses a remineralizing effect on teeth and can be used to prevent damage from carious attacks. In the event of an acid attack by cariogenic bacteria, nanohydroxyapatite particles can infiltrate pores on the tooth surface to form a protective layer. Furthermore, nanohydroxyapatite may have the capacity to reverse damage from carious assaults by either directly replacing deteriorated surface minerals or acting as a binding agent for lost ions. In the future, there are possibilities for using nanohydroxyapatite for tissue engineering and repair. The main and most advantageous feature of nanohydroxyapatite is its biocompatibility. It is chemically like naturally occurring hydroxyapatite and can mimic the structure and biological function of the structures found in the resident extracellular matrix. Therefore, it can be used as a scaffold for engineering tissues such as bone and cementum. It may be used to restore cleft lips and palates and refine existing practices such as preservation of alveolar bone after extraction for better implant placement. NHAP in toothpaste may combat dental hypersensitivity. They aid in the repair and remineralization of the enamel, thus helping to prevent tooth sensitivity. Tooth enamel can become demineralized due to various factors, including acidic erosion and dental caries. If left untreated, this can lead to the exposure of dentin and subsequent exposure of the dental pulp. NHAP in toothpaste showed positive results in aiding the remineralization of dental enamel. In an embodiment, NHAP is in the form of a particle. In an embodiment, NHAP is equal or more effective than fluoride. In an embodiment, NHAP in composition is about 0.025% w/w, 0.05% w/w, 0.1% w/w, 0.5% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, 35% w/w, 40% w/w, 45% w/w, 50% w/w or more. In an another embodiment, a range of NHAP in the composition is selected from a minimum value being either 0.025% w/w, 0.05% w/w, 0.1% w/w, 0.2% w/w, 0.3% w/w, 0.4% w/w, 0.5% w/w, 1% w/w, 5% w/w, 12% w/w, 17% w/w, 20% w/w, 22% w/w, 25% w/w, 30% w/w, 32% w/w, 35% w/w, 37% w/w, 40% w/w and a maximum value being either 10% w/w, 13% w/w, 15% w/w, 20% w/w, 25% w/w, 35% w/w, 40% w/w, 45% w/w, 50% w/w, 55% w/w, 60% w/w, 70% w/w or more. “Particle” means a substance being a solid having a shape which can be geometrically determined. The shape can be regular or irregular. Particles can typically be analyzed with respect to e.g., grain size (e.g., mean grain or particle size) and grain or particle size distribution. In an embodiment, particle size of NHAP can vary from 0.1 nm to 100 nm. In an embodiment, a size range of NHAP particles is selected from 0.1 nm, 1 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm. Maximum size of nanoparticle particles is selected from 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm or more. These NHAP particles can be in the form of suspension. In an embodiment, particle size of NHAP can vary from 0.1 nm to 100 nm. In an embodiment, a size range of NHAP particles is selected from a minimum value being either 0.1 nm, 1 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm and a maximum value being either 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm or more. In an embodiment, a size of particle is less than 100 nm, less than 75 nm, less than 50 nm, less than 25 nm, less than 20 nm, less than 10 nm or lesser. “Powder” is a dry, bulk solid composed of many particles that may flow freely when shaken or tilted. Powders are a special subclass of granular materials, although the terms powder and granular are sometimes used to distinguish separate classes of material. In particular, powders refer to those granular materials that have the finer grain sizes, and that therefore have a greater tendency to form clumps when flowing. In an embodiment, particle size of NHAP powder can vary from 0.5 micron, 1 micron, 2 micron, 3 micron, 4 micron, 5 micron, 6 micron, 7 micron, 8 micron, 9 micron, 10 micron or more. In an embodiment, a paste or gel might become a powder after it has been thoroughly dried but is not considered a powder when it is wet because it does not flow freely. Substances like dried clay, although are dry solids composed of very fine particles, are not powders unless crushed because they have too much cohesion between the grains, and therefore do not flow freely like a powder. A liquid flow differently than a powder, because a liquid cannot resist any shear stress and therefore it cannot reside at a tilted angle without flowing (that is, it has zero angle of repose.) A powder on the other hand is a solid, not a liquid, because it may support shear stresses and therefore may display an angle of repose. In an embodiment, nanohydroxyapatite, has been shown to be biomimetic and biocompatible and more effective than fluoride at remineralizing teeth. “Biomimetic,” refers to the body recognizing, and “Biocompatibility” is related to the behavior of biomaterials. Biocompatibility refers to the ability to perform as a substrate that will support the appropriate cellular activity, including the facilitation of molecular and mechanical signaling systems, to optimize tissue regeneration, without eliciting any undesirable effects in those cells, or inducing any undesirable local or systemic responses in the eventual host. “Teeth”, as used herein, refers to natural teeth as well as artificial teeth or dental prosthesis. A tooth (plural teeth) is a hard, calcified structure found in the jaws (or mouths) of many vertebrates and used to break down food. Some animals, particularly carnivores and omnivores, also use teeth to help with capturing or wounding prey, tearing food, for defensive purposes, to intimidate other animals often including their own, or to carry prey or their young. The roots of teeth are covered by gums. Teeth are not made of bone, but rather of multiple tissues of varying density and hardness that originate from the embryonic germ layer, the ectoderm. The term, “baby teeth” or “primary teeth”, also informally known as, milk teeth, or temporary teeth, are the first set of teeth in the growth and development of humans and other diphyodonts, which include most mammals but not elephants, kangaroos, or manatees which are polyphyodonty. Deciduous teeth develop during the embryonic stage of development and erupt (break through the gums and become visible in the mouth) during infancy. They are usually lost and replaced by permanent teeth, but in the absence of their permanent replacements, they can remain functional for many years into adulthood. Dental caries, also known as tooth decay, is one of the most prevalent chronic diseases among children worldwide. This oral condition involves bacterial infection which demineralizes and destroys tooth tissues. In primary dentition, extensive tooth decay is the most common dental disease. An extensive carious lesion affects at least half of a tooth and possibly involves the pulp. The term “permanent teeth” or “permanent dentition” is comprised of 32 teeth. There are 16 teeth in the maxilla and 16 in the mandible. In each arch there are two central incisors, two lateral incisors, two canines, four premolars, and six molars. “Remineralization”, as used herein, means the in-situ generation of hydroxyapatite in teeth. It includes layers in teeth from 10 nm to 6 microns and, preferably, from about 75 nm to 5 microns and, most preferably, from 150 nm to 4 microns. Tooth whitening or tooth bleaching is the process of lightening the color of teeth. “Tooth sensitivity” is a dental pain, which is sharp in character and of short duration, arising from exposed dentin surfaces in response to stimuli, typically thermal, evaporative, tactile, osmotic, chemical, or electrical; and which cannot be ascribed to any other dental disease. Teeth sensitivity can be reduced by applying the composition of the present invention to the tooth surface according to the method of the present invention. The composition may be applied by any device or applicator, using traditional methods, as described in detail elsewhere in this specification, or typically associated with dental use. In one embodiment, a composition that reduces dental sensitivity using one or more human fingers is applied to one or more teeth. In an embodiment, the present invention decreases tooth sensitivity by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% after application. “Abrasive” is broadly defined as material, often a mineral, that is used to shape or finish a workpiece through rubbing which leads to part of the workpiece being worn away by friction. According to present invention, abrasive includes insoluble particles designed to help remove plaque from the teeth. The removal of plaque inhibits the accumulation of tartar (calculus) helping to minimize the risk of gum disease. In an embodiment, abrasive could be, but not limited to, particles of aluminum hydroxide (Al(OH)3), calcium carbonate (CaCO3), sodium bicarbonate, various calcium hydrogen phosphates, various silicas and zeolites. Abrasives which may be used in conventional amounts such as 20-80%, or more, of the formulations. Abrasives include, without limitation, particles of aluminum hydroxide (Al (OH)3), alumina trihydrate and/or dehydrate; calcium pyrophosphate; magnesium trisilicate; insoluble sodium metaphosphate, bicarbonates such as sodium bicarbonate, calcium carbonate (CaCO3), dibasic calcium phosphate, calcium hydrogen phosphates, silicas including dental silica thickener, zeolites, liponite, laponite, hydroxyapatite (Ca5(PO4)3OH), fluorapatite, and mica. In an embodiment, the amount of abrasive may vary from about 3-60 wt. % of total dental composition for example from about 3 wt. % to about 10 wt. %, from about 10 wt. % to about 20 wt. %, from about 20 wt. % to about 30 wt. %, from about 30 wt. % to about 40 wt. %, from about 40 wt. % to about 50 wt. % or from about 50 wt. % to about 60 wt. % of total dental composition. In an another embodiment, a range of abrasive in the composition is selected from a minimum value being either about 0.5% w/w, about 1% w/w, about 5% w/w, about 12% w/w, about 17% w/w, about 20% w/w, about 22% w/w, about 25% w/w, about 30% w/w, about 32% w/w, about 35% w/w, about 37% w/w, about 40% w/w and a maximum value being either about 10% w/w, about 13% w/w, about 15% w/w, about 20% w/w, about 25% w/w, about 35% w/w, about 40% w/w, about 45% w/w, about 50% w/w, about 55% w/w, about 60% w/w, about 70% w/w or more. “Sweetener” is a substance that can be used to sweeten another substance. In one embodiment, sweeteners can be natural or artificial, but are not sugars in the conventional sense. So, glucose, sucrose and fructose are generally not included, or if included only included in relatively small amounts. One category of useful “natural” sweeteners are sugar alcohols including, for example, xylitol, arabitol, ribitol, mannitol, isomalt, lactitol, maltitol, sorbitol, erythritol, and monk fruit. Not only can these provide sweetness, they also often adjust and enhance viscosity. “Artificial” sweeteners may be used in place of some or all of the other sweeteners. These include saccharine, aspartame,Stevia, sucralose, and derivatives (SPLENDA). Honey may also be used. In an embodiment, total amount of sweeteners may vary widely with the number and type used, their relative sweetness, the flavor to be used in the formulations, and the degree of taste masking that may be required. Amount of sweeteners may vary from about 0.25% wt./wt. as much as about 50% wt./wt. But generally, the total amount of sweeteners, particularly when using natural sweeteners such as sugar alcohols, ranges from between about 5 to about 40% wt./wt. In certain embodiments, the amount of sweetener may vary from about 5 to about 10% wt./wt., from about 10% to about 20% wt./wt., from about 20% to about 30% wt./wt., or from about 30% to about 40% wt./wt. In an embodiment, the amount of sweetener is typically from 0.005% to 5% by weight of composition for example, from about 0.005% to about 0.05%, optionally from about 0.05% to about 1%, optionally from about 1% to about 2%, or optionally from about 2% to about 3%, optionally from about 3% to about 4%, optionally from about 4% to about 5%, by weight of the composition of a sweetener. “Xylitol” is a chemical compound with the formula C5H12O5, or HO(CHOH)3OH; specifically, one particular stereoisomer with that structural formula. It is a colorless or white crystalline solid that is soluble in water. It can be classified as a polyalcohol and a sugar alcohol, specifically an alditol. “Erythritol” is a chemical compound, a sugar alcohol, used as a food additive and sugar substitute. It is naturally occurring and is made from corn using enzymes and fermentation. Its formula is C4H10O4, or HO(CHOH)2OH; specifically, one particular stereoisomer with that formula. “Stevia” is a natural sweetener and sugar substitute derived from the leaves of the plant speciesStevia rebaudiana, native to Brazil and Paraguay.Steviais probably the healthiest option, followed by xylitol, erythritol, and yacon syrup. Natural sugars like maple syrup, molasses, and honey are less harmful than regular sugar and even have health benefits.Steviahas no calories, and it is 200 times sweeter than sugar in the same concentration. In an embodiment, sweetener is low-calorie sweetener. “Low-calorie sweetener” refers to a sweetener with a calorie value less than 3. In an embodiment, calorie value of a low-calorie sweetener is less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2, less than 1.5, less than 1, less than 0.5 or less. In an embodiment, low-calorie sweetener is a sugar substitute that provides a sweet taste like that of sugar while containing significantly less food energy than sugar-based sweeteners thus making it a zero-calorie (non-nutritive) or low-calorie sweetener. In an embodiment, artificial sweeteners may be derived through manufacturing of plant extracts or processed by chemical synthesis. Sugar alcohols such as erythritol, xylitol, and sorbitol are derived from sugars. In 2017, sucralose was the most common sugar substitute used in the manufacture of foods and beverages. For example, but not limited to Aspartame, Cyclamate, Steviol glycosides (Stevia), Acesulfame potassium (Ace-K) etc. Ace-K is 200 times sweeter than sucrose (common sugar), as sweet as aspartame, about two-thirds as sweet as saccharin, and one-third as sweet as sucralose. Like saccharin, it has a slightly bitter aftertaste, especially at high concentrations. The term “Alkaline” refers to a part of pH scale. A substance is alkaline if it has a pH over 7. In an embodiment, alkaline pH ranges from above 7 and up to 14. A pH of 14 is completely alkaline. In an embodiment, a composition according to one or more embodiments of this invention has a pH of about 8 and above, about 9 or above, about 10 or above, about 11 or above, about 12 or above, or about 13 or above. “Natural oil” and the like refers to oil derived from plant or animal sources. As used herein, these phrases encompass natural oil derivatives as well, unless otherwise indicated. These are triglycerides in which the glycerin is esterified with three fatty acids. They are the main constituent of vegetable oil and animal fats. In an embodiment, natural oil is also called as natural oil polyols, also known as NOPs or bio polyols, are polyols derived from vegetable oils by several different techniques. The primary use for these materials is in the production of polyurethanes. There are a limited number of naturally occurring vegetable oils (triglycerides) which contain the unreacted hydroxyl groups that account for both the name and important reactivity of these polyols. Castor oil is the only commercially available natural oil polyol that is produced directly from a plant source: all other NOPs require chemical modification of the oils directly available from plants. Ninety percent of the fatty acids that make up castor oil is ricinolein acid which has a hydroxyl group on C-12 and a carbon-carbon double bond. Other vegetable oils—such as soybean oil, peanut oil, and canola oil-contain carbon-carbon double bonds, but no hydroxyl groups. There are several processes used to introduce hydroxyl groups onto the carbon chain of the fatty acids, and most of these involve oxidation of the C—C double bond. “Natural oil derivatives” refers to compounds and/or mixture of compounds derived from a natural oil using any one or combination of methods known in the art, including but not limited to saponification, transesterification, esterification, amidification, amination, hydrogenation (partial or full), isomerization, oxidation, reduction, and the like, and combinations thereof. Chain lengths of the fatty acids in naturally occurring triglycerides can be of varying lengths, but 16, 18 and 20 carbons are the most common. In an embodiment, coconut oil is a natural oil. In an embodiment, natural oils are selected without limitation from castor oil, coconut oil, corn oil, sesame oil, almond oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, soybean oil, rapeseed oil, mango butter, hemp seed oil, sweet almond oil, jojoba oil, apricot oil, or palm kernel oil, coconut oil, grape seed oil, sunflower oil, avocado oil, tea tree oil, or shea oil, cinnamon bark oil, cocoa oil, coffee oil, cognac oil, ravensara oil, tansy blue oil, vanilla oil or yarrow oil. In an embodiment, amount of natural oil or its derivatives in the composition is about 0.05% v/w, about 0.1% v/w, about 0.15% v/w, about 0.2% v/w, about 0.25% v/w, about 0.3% v/w, about 0.35% v/w, about 0.4% v/w, about 0.45% v/w, about 0.5% v/w, about 0.75% v/w, about 1% v/w, about 1.25% v/w, about 1.5% v/w, about 1.75% v/w, about 2% v/w, about 2.25% v/w, about 2.5% v/w, about 3% v/w, about 3.5% v/w, about 4% v/w, about 4.5% v/w, about 5% v/w, about 5.5% v/w, about 6% v/w, about 6.5% v/w, about 7% v/w, about 7.5% v/w, about 8% v/w, about 8.5% v/w, about 9% v/w, about 9.5% v/w, about 10% v/w or more. “Glycerin” is also called glycerol. It is a simple polyol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. It is also written as glycerin or glycerol or glycerin. Glycerin is a natural ingredient found in most oral care products to better preserve and sweeten them. A natural agent, glycerin in toothpaste also helps retain the moisture of the paste so it doesn't dry out in the tube. Glycerin is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known as glycerides. Due to having antimicrobial and antiviral properties, it is widely used in FDA approved wound and burn treatments. It is also widely used as a sweetener in the food industry and as a humectant in pharmaceutical formulations. Owing to the presence of three hydroxyl groups, glycerol is miscible with water and is hygroscopic in nature. A mouthwash or gargle historically has been a liquid product used to clean the oral cavity and freshen the breath. The ability to obtain glycerin commercially either as 96% material or 99+% is a feature of glycerin which endows it with a very important capability in oral care product manufacturing. Water and glycerin are mixed with the particles (including erythritol and rice flour) to form a pickering emulsion. Glycerin can also act as a humectant to prevent water loss, and any number of flavoring agents can be added from natural sources (such as jasmine extract, grapefruit oil, etc.). Glycerin may form a dispersed or discontinuous phase in the pickering emulsion. Glycerin may be added including but not limited to a concentration of about 3% or more, about 4% or more, about 5% or more, and any ranges between and including the weight percentages provided. In an exemplary embodiment, the dental care products contain glycerin at the following concentrations: at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% by weight, at least about 35% by weight, or any concentration between and including the provided percent concentrations of the dental care product. In an embodiment, teeth whitening composition comprises peroxide compounds. The term “teeth whitening” is also cleaning of teeth. In one embodiment, suitable peroxide compounds comprise hydrogen peroxide and organic peroxides including urea peroxide, glyceryl peroxide, or benzoyl peroxide. A preferred peroxide is hydrogen peroxide. Typically, the peroxide compound can be employed in the composition of the present invention in amounts so that at least about 1% by weight of the composition comprises a peroxide. Preferably, the peroxide compound comprises from about 2 to about 30% by weight of the composition for example from about 2% to about 5%, from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25% or from about 25% to about 30% by weight of the composition. More preferably, the peroxide comprises from about 3 to about 15% by weight of the composition. In an embodiment, teeth whitening composition comprises about 6 wt % hydrogen peroxide or less, in particular about 0.1 wt % to about 6 wt % hydrogen peroxide. In an embodiment, range of hydrogen peroxide in the composition vary from 0.1 wt %, 0.5 wt %, 1 wt %, 1.5 wt %, 2 wt %, 2.5 wt %, 3 wt %, 3.5 wt %, 4 wt %, 4.5 wt %, 5 wt %, 5.5 wt %, or 6 wt %. In an embodiment, the composition does not contain glycerin. In an embodiment, toothpaste does not contain glycerin. In an embodiment, calcium carbonate is a main ingredient to help with binding (instead of glycerin) in a composition. In an embodiment, the composition contains glycerin less than 5% w/W, 4% w/W, 3% w/W, 2% w/W, 1% w/W, 0.5% w/W or 0.1% w/W of the formulation. “Fluoride” is an inorganic, monatomic anion of fluorine, with the chemical formula F−, whose salts are typically white or colorless. Fluoride salts typically have distinctive bitter tastes and are odorless. Fluoride can be added to water or toothpaste to help prevent unhealthy teeth. Fluoride is the simplest fluorine anion. In terms of charge and size, the fluoride ion resembles the hydroxide ion. The fluoride in toothpaste has beneficial effects on the formation of dental enamel and bones. Sodium Fluoride (NaF) is the most common source of fluoride, but Stannous Fluoride (SnF2), and Sodium Monofluorophosphate (Na2PO3F) are also used. Fluoride-containing toothpaste can be acutely toxic if swallowed in large amounts, but instances are exceedingly rare and result from prolonged and excessive use of toothpaste (i.e., several tubes per week). Approximately 15 mg/kg body weight is the acute lethal dose, even though an amount as small as 5 mg/kg may be fatal to some children. The risk of using fluoride is low enough that the use of full-strength toothpaste (1350-1500 ppm fluoride) is advised for all ages. However, smaller volumes are used for young children, for example, a smear of toothpaste until three years old. In an embodiment, the composition does not contain fluoride. “Sodium bicarbonate”, commonly known as baking soda or bicarbonate of soda, is a chemical compound with the formula NaHCO3. It is a salt composed of sodium cation and Bicarbonate anion. Sodium Bicarbonate is a white solid that is crystalline, but often appears as a fine powder. It is a chemical compound with the formula NaHCO3. It is a salt composed of a sodium cation (Na+) and a bicarbonate anion (HCO3−). It has a slightly salty, alkaline taste resembling that of washing soda (Sodium Carbonate). The natural mineral form is nahcolite. It is a component of the mineral natron and is found dissolved in many mineral springs. It has weak disinfectant properties, and it may be an effective fungicide against some organisms. “Flavoring agents” are key food additives with hundreds of varieties like fruit, nut, seafood, spice blends, vegetables and wine which are natural flavoring agents. Besides natural flavors, there are chemical flavors that imitate natural flavors. Some examples of chemical flavoring agents are alcohols that have a bitter and medicinal taste, esters are fruity, ketones and pyrazines provide flavors to caramel, phenolics have a smokey flavor and terpenoids havecitrusor pine flavor. In an embodiment, toothpaste flavors are Spearmint, Peppermint, Wintergreen, Cinnamon, Bourbon, Rye, Anise, Clove, Caraway, Coriander,Eucalyptus, Nutmeg, Menthol, and Thyme. In an embodiment, there could be fun flavors, like Vanilla, Strawberry, Bubblegum, and Jasmine etc. Flavors include all commercially available flavors as well as custom formulations. In an embodiment, flavoring agents could be natural flavoring substances. These flavoring substances are obtained from plant or animal raw materials, by physical, microbiological, or enzymatic processes. They can be used in either their natural state or processed for human consumption but cannot contain any nature-identical or artificial flavoring substances. In an embodiment, flavoring agents could be nature-identical flavoring substances. These are obtained by synthesis or isolated through chemical processes, which are chemically and organoleptically identical to flavoring substances naturally present in products intended for human consumption. In an embodiment, artificial flavoring substances could also be used as a flavoring agent. Artificial flavoring substances are not identified in a natural product intended for human consumption. These are typically produced by fractional distillation and chemical manipulation of naturally sourced chemicals, crude oil, or coal tar. Despite their chemical differences, they have the same sensory characteristics as natural ones. The majority of artificial flavors are specific and often complex mixtures of singular naturally occurring flavor compounds that are combined to either imitate or enhance a natural flavor. These mixtures are formulated by flavorists to give a food product a unique flavor and to maintain flavor consistency between different product batches or after recipe changes. The list of known flavoring agents includes thousands of molecular compounds, and the flavor chemist (flavorist) can often mix these together to produce many common flavors. Many flavorants consist of esters, which are often described as being “sweet” or “fruity”. In an embodiment, flavoring agents are present in an amount from about 0.01% to about 5% by weight of the composition for example, from about 0.01% to about 0.05%, 0.05% to about 1%, 1% to about 1.5%, 1.5% to about 2%, 2% to about 2.5%, 2.5% to about 3%, 3% to about 3.5%, 3.5% to about 4%, 4% to about 4.5% by weight of the composition. “Surfactant” is a compound that lowers the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, or dispersants. Surfactants have specific physical properties resulting in decreases in the interfacial tension between different phases (i.e., oil and water) and corresponding micelle formation depending on the hydrophile-lipophile properties of the surfactants. Resultant dispersions of oil and water can be monophasic, biphasic or triphasic systems. “Suspension” is a heterogeneous mixture in which the solute particles do not dissolve, but get suspended throughout the bulk of the solvent, left floating around freely in the medium. The internal phase (solid) is dispersed throughout the external phase (fluid) through mechanical agitation, with the use of certain excipients or suspending agents. “Suspending agents” are hydrophilic colloids, such as but not limited to cellulose derivatives, acacia, and xanthan gum that are added to a suspension to increase viscosity, inhibit agglomeration, and decrease sedimentation. In an embodiment, a suspending agent includes at least one ethoxylated or ethoxylated-amidated plant oil. The suspending agent may have two different ethoxylated or ethoxylated-amidated plant oils. The suspending agent may have at least three different ethoxylated or ethoxylated-amidated plant oils. The ethoxylated or ethoxylated-amidated plant oil may be, for example, ethoxylated or ethoxylated-amidated plant oil forms of castor oil, coconut oil, corn oil, sesame oil, almond oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, soybean oil, or rapeseed oil. In some embodiments, the suspending agent includes one or more of ethoxylated coconut oil, ethoxylated castor oil or ethoxylated-amidified coconut oil. In some embodiments, the suspending agent has at least one ethoxylated or ethoxylated-amidated plant oil, and at least one nonionic alkyl glycoside cross polymer. In some embodiments, the only surfactants in the composition are alkyl glycoside cross polymers. In an embodiment, the suspending agent could be a surfactant. In some embodiments, the composition comprises from about 0.01% to about 5%, optionally from about 0.05% to about 3%, optionally from about 0.05% to about 1%, or optionally from about 0.05% to about 0.5%, by weight of the composition of a suspending agent. In an embodiment, the NHAP in a suspension is 10-50 nm in size. In an embodiment, a size range of NHAP particles is selected from a minimum value being either 1 nm, 5 nm, 10 nm, 20 nm or 25 nm and the maximum value being either 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm or more. In an embodiment, NHAP suspension could be produced synthetically by wet chemical precipitation according to WO2005077508. The process is relatively simple and can be described by the following 3 steps: 1. prepare inorganic solutions from calcium and phosphorous salts; 2. feed the solutions to reactor where they precipitate as a NHAP suspension; 3. concentrate this suspension to 15.5±0.5% wt. NHAP to obtain a paste product. The process is carried out in purified water and at room temperature. There are no complex compounds or chemicals. In an embodiment, the starting agents for preparing the NHAP suspension are inorganic calcium, phosphorous, and potassium salts, as well as water. In an embodiment, NHAP in toothpaste are <50 nanometers (i.e., 0.050 microns), with a nano-rod shape having a width between 5-20 nm (typically close to 10 nm) and length below 50 nm (typically between 30 to 40 nm). These small nano-sized particles are very effective in doing dentine tubule occlusion and enamel remineralization. “Plant product” signifies a product of plant origin in an unprocessed state or having undergone only simple preparation, such as milling, drying, or pressing, but excluding plants. In an embodiment, plant product comprises a natural oil. In an embodiment, a plant product can also be prepared by chemical synthesis (both semi synthesis and total synthesis). The term plant product has also been extended for commercial purposes to refer to cosmetics, dietary supplements, and foods produced from natural sources. “Dental product” herein signifies specially fabricated material, designed for use in dentistry. There are many different types of dental products, and their characteristics vary according to their intended purpose. Dental composition can be used in different forms and are not limited to toothpaste, tooth serum, floss, tooth wipes, gels, powder, tablets, lozenges, chewing gums, mouth strips, balms, dental filling material, desensitizing agents for teeth, whitening agents for teeth, tooth varnish, dental cements, and the like. Dental composition is alternately called an oral composition. In an embodiment, composition is meant for used as a dental product. “Floss” is a cord of thin filaments used in interdental cleaning to remove food and dental plaque from between teeth in particular, places difficult or impossible to reach with a toothbrush. Its regular use as part of oral cleaning is designed to maintain oral health. In an embodiment, the floss comprises nanohydroxyapatite (NHAP). In an embodiment, the floss comprises ecofriendly vegan material which further comprises fibers. The fibers include but are not limited to seaweed, cotton floss, bamboo charcoal, polyester, corn, hemp, cotton, or silk. “Wipes” are small, saturated, gauze-like pads formulated to help prevent tooth decay. These wipes are one way to administer antimicrobial agents to the mouth in order to reduce the amount of harmful oral bacteria, such as Streptococcus mutans. In an embodiment, the tooth wipes are organic and biodegradable and comprises 100% cotton with 2×2 gauze. Tooth wipes are used for cleaning and wiping baby teeth, adult teeth, or animal teeth after drinking tea, coffee, lemon water, sugary drinks, acidic drinks, or any non-water drinks, eating colored foods like blueberries or foods with sugar (like candies etc). In an embodiment, tooth wipes contain NHAP, which is effective in reducing sensitivity and remineralizing teeth. In an embodiment, tooth serum according to this embodiment, has about 10% NHAP. In another embodiment, tooth serum has NHAP about 15% NHAP, 20% NHAP, 25% NHAP, 30% NHAP, 40% NHAP, 50% NHAP or more in the formulation. The composition can be packaged in a plastic laminate, a metal tube, or a conventional single compartment dispenser in the form of toothpaste or gel. It can be applied to the surface of the teeth by any physical means, such as a toothbrush, fingertip, or through an applicator directly to the sensitive area. Types of solid dosage form include lozenges, chewing gums, tablets, mouth strips, balms, and the like. These can be packaged in conventional consumer-friendly packaging. In an embodiment, a tooth wipe is comprised of NHAP, coconut oil, and flavor. In an embodiment, because toothpastes are not available in plastic, the toothpaste/serum is kept in a glass jar, while the wipes are kept in aluminum containers. Bamboo or glass/stainless steel floss will be used. In an embodiment, toothpaste composition with ingredients in 1 oz. comprises Xylitol (9 tsp), Sodium Bicarbonate (2 tsp), NHAP suspension (1.05 tsp, to make 1% NHAP in the paste), Coconut Oil (5 tsp), Peppermint Oil (8 drops), Salt (0.02 tsp). In an embodiment, toothpaste is alkaline. The pH of the toothpaste is more than 7, more than 8, more than 9 or more than 10, more than 11 or more. Alkaline toothpaste does not contain glycerin and surfactants. This toothpaste contains less chemicals and preservatives than the majority of toothpastes in the market. Toothpaste contains NHAP (1%-65%) in solution with baking soda, coconut oil and peppermint oil. In an embodiment, the NHAP in solution could be about 1%-15%, 1%-20% or 1%-25%, 5%-10% 5%-20%, 5%-25%, 5%-30%, 1%-30% or 1%-40% in solution of toothpaste. In an embodiment, the toothpaste could have any amount of NHAP and any size of NHAP as described in various embodiments of this invention. In an embodiment, toothpaste has NHAP with particle size less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm or lesser. The NHAP in suspension is 20-50 nm in size. NHAP in suspension could be about 10 nm-50 nm, 10-30 nm, less than 20 nm, less than 10 nm or 10-50 nm. The size of NHAP in suspension could be any size as described in any embodiments of this invention. In an embodiment, composition contains dispersed, therein visible, palpable, agglomerated particles of a dental polishing agent that are substantially insoluble in the toothpaste and are easily reduced to impalpable particles of a dental polishing agent during toothbrushing. In an embodiment, polishing agents thus include insoluble phosphate salts, such as insoluble sodium metaphosphate, insoluble potassium metaphosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium orthophosphate, tricalcium phosphate, dicalcium phosphate dihydrate, anhydrous dicalcium phosphate and the like, calcium carbonate, magnesium carbonate, hydrated alumina, silica, zirconium silicate, aluminum silicate including calcined aluminum silicate and polymethyl methacrylate. In an embodiment, MOH hardness of at least 5 and particle size such as to be useful as a dental polishing agent (e.g., between 0.1 and 10 microns) are particularly suitable for use in the agglomerates. Representative of such materials are silica, zirconium silicate, aluminum silicate, calcined aluminum silicate, calcium silicate, silicon carbide, pumice, ilmenite (FeTiO3), CeO2, Fe2O3(hematite), SnO2, Topaz (aluminum hydroxy fluoro silicate) and TiO2, either natural or manufactured. When incorporated into a toothpaste, agglomerated particles of hard abrasive materials as defined above e.g., MOH hardness greater than 5 and particle size between 0.1 and 10 microns, make it possible to provide substantially increased cleaning and polishing properties to the formulation, without significantly increasing abrasion to the dental hard tissues, (enamel, dentin and cementum). In an embodiment, a product characterization of NHAP in toothpaste is shown in Table 1. TABLE 1Product characterization of NHAP in toothpaste.SECTION 1: PRODUCT IDENTIFICATIONTrade namenanoXIM•CarePasteSKU504102INCI nameHydroxyapatite (nano)IUPAC namePentacalcium hydroxidetriphosphateCAS number12167-74-7EC number235-330-6SynonymsHydroxyapatite(CAS No. 1306-06-5)Chemical formulaCa10(PO4)6(OH)2REACH ID number01-2119490075-38-0021SECTION 2: PRODUCT INFORMATIONLot numberF09-011Production dateSeptember 2020Expiry dateSeptember 2023Particle size<50 nmShelf life3 yearsPhysicalWhite aqueousappearancesuspensionSECTION 3: LOT CHARACTERIZATIONUnitAnalysisResultsSpecificationReference methodSolidswt %Lot20.320.0 ± 1.0Ph. Eur. 7th Ed.2.2.32.Hydroxyapatite,wt %Lot15.715.5 ± 0.5IT-07-NET*Ca10(PO4)6(OH)2Potassium Chloride, KC1wt %Lot4.54.5 ± 0.5IT-07-NET*Water, H2Owt %Lot79.780.0 ± 1.0IT-07-NET*pH @ 25° C.Lot10.210.0 ± 0.5IT-03-NET*Total heavy metals (as Pb)ppmLot<12≤20Ph. Eur. 7th Ed. 2.4.8.Total Aerobic Mesophiliccfu/gLot<3≤100ISO 11737-1:2006Microorganisms (bacteria,yeast & mold) In an embodiment, another product characterization of NHAP in toothpaste is shown in Table 2. TABLE 2Product characterization of NHAP in toothpasteSECTION 1: PRODUCT IDENTIFICATIONTrade namenanoXIM•CarePasteSKU504102INCI nameHydroxyapatite (nano)IUPAC namePentacalcium hydroxidetriphosphateCAS number12167-74-7EC number235-330-6SynonymsHydroxyapatite(CAS No. 1306-06-5)Chemical formulaCa10(PO4)6(OH)2REACH ID number01-2119490075-38-0021SECTION 2: PRODUCT INFORMATIONLot numberF11-015Production dateNovember 2020Expiry dateNovember 2023Particle size<50 nmShelf life3 yearsPhysicalWhite aqueousappearancesuspensionSECTION 3: LOT CHARACTERIZATIONUnitAnalysisResultsSpecificationReference methodSolidswt %Lot19.920.0 ± 1.0Ph. Eur. 7th Ed.2.2.32.Hydroxyapatite,wt %Lot15.415.5 ± 0.5IT-07-NET*Ca10(PO4)6(OH)2Potassium Chloride, KC1wt %Lot4.54.5 ± 0.5IT-07-NET*Water, H2Owt %Lot80.180.0 ± 1.0IT-07-NET*pH @ 25° C.ppmLot10.310.0 ± 0.5IT-03-NET*Total heavy metals (as Pb)Lot<12≤20Ph. Eur. 7th Ed. 2.4.8.Total Aerobic Mesophiliccfu/gLot<4≤100ISO 11737-1:2006Microorganisms (bacteria,yeast & mold) In an embodiment, tablets are 100% vegan, palm oil free, fluoride free and gluten free. In an embodiment, ingredients for tablets or paste are Finnish birch xylitol, ozonated aqua, organic coconut glycerin, organic coconut oil, organic cocoa butter, calcium glycerophosphate, sodium bicarbonate, betaine, menthol, organicMentha piperitaoil, potassium citrate, sodium anisate, zinc citrate, nanohydroxyapatite, xanthan gum, vegan vitamin K2 in MCT oil, vegan vitamin D3 in MCT oil, organic NZ manuka oil, organic NZ Totarol™. seasonal: erythritol, calcium carbonate, xylitol, natural coconut flavor, hydroxyapatite (nano), natural mango flavor, sodium bicarbonate, natural key lime flavor, guar gum, sodium cocoyl glutamate, zinc citrate, silicon dioxide, cellulose blend. In an embodiment, ingredients for paste are silica, sorbitol, glycerin, xylitol, hydroxyapatite, calcium carbonate, propanediol,Potassium cocoate, Stevia rebaudianaextract,Mentha arvensis(wild mint) oil,Mentha piperita(peppermint) oil,Cinnamomum cassia(cinnamon) bark extract,citrus Aurantium dulcis(orange) peel oil,Citrus limon(lemon) peel oil,melaleucaalternifolia (tea tree) oil, cellulose gum, sodium gluconate, menthol,Thymus vulgaris(thyme) extract, erythritol, xanthan gum,Eucalyptus globulusextract,Illicium verum(anise) extract. In an embodiment, ingredients for paste are aqua, hydrated silica, sorbitol, glycerin, xylitol, potassium nitrate, nanohydroxyapatite, magnesium aluminum silicate,Mentha piperitaoil, sodium lauroyl sarcosinate, xanthan gum, phenoxyethanol, potassium chloride, sodium sulfate, sodium saccharin, ci 77891. In an embodiment, ingredients for paste are water, vegetable glycerin, hydrated silica, sorbitol powder, silica, hydroxyapatite (nano), sodium benzoate, sodium lauroyl sarcosinate,Mentha piperitaessential (peppermint) oil,Mentha viridis(spearmint) oil,Illicium verum(star anise) oil,Gaultheria procumberis(wintergreen) oil, xylitol, xanthan gum,Stevia rebaudianaextract powder, methylsulfonylmethane,Aloe barbadensis(Aloe vera) leaf juice, sodium bicarbonate,Camellia sinensis(green tea) leaf extract,Cucumis sativus(cucumber) fruit extract,Persea gratissima(avocado) fruit extract,Mangifera indica(mango) fruit extract, menthol,Elettaria cardamomumminuscula seed (cardamom), potassium chloride. In an embodiment, ingredients for healthy gums toothpaste areAloe barbadensis(Aloe vera) leaf juice, vegetable glycerin (soy free), xylitol, hydrated silica (mineral), hydroxyapatite (mineral), calcium carbonate (mineral), and organicCocos. In an embodiment, ingredients for healthy smile toothpaste areAloe barbadensis(inner leaf) juice, hydroxyapatite (mineral), organic hemp seed oil, organicCocos nucifera(coconut) oil, vegetable glycerin (soy free), calcium ascorbate (vitamin c),Cinnamomum zeylanicum(cinnamon) oil,Eugenia caryophyllus(clove) oil,Ocimum sanctum(tulsi) leaf extract,Elettaria cardamomum(cardamom) oil,Azadirachta indica(neem) extract,Potassium cocoate(from organic coconut), xanthan gum (thickener),Stevia rebaudianaleaf/stem extractofficinalis(rosemary) oil, sodium cocoyl isethionate (from coconuts),Zingiber officinale(ginger) root extract. In an embodiment, ingredients for kids' anti-plaque toothpaste areAloe barbadensis(inner leaf) juice, vegetable glycerin, hydroxyapatite (mineral), calcium carbonate (mineral), hydrated silica (mineral),Cocos nucifera(coconut) oil,maranta. In an embodiment, ingredients for kids' mineral toothpaste areAloe barbadensis(inner fillet) leaf juice, vegetable glycerin (soy free), xylitol, hydrated silica (mineral), hydroxyapatite (mineral), calcium carbonate (mineral),Cocos nucifera. In an embodiment, ingredients for anti-plaque toothpaste are mint:Aloe barbadensis(Aloe vera) leaf juice, vegetable glycerin (soy free), hydroxyapatite (mineral), hydrated silica (mineral), xylitol, calcium carbonate (mineral), organicCocos nucifera(coconut oil),Mentha piperita(peppermint oil),Mentha spicata(spearmint leaf oil),Mentha arvensis(menthol crystals),Potassium cocoate(from coconut oil), sodium cocoyl isothionate (from coconut oil), calcium ascorbate (vitamin c),Azadirachta indica(neem extract),Vaccinium macrocarpon(cranberry fruit extract),Rosmarinus officinalis(rosemary leaf extract), xanthan gum (thickener),Stevia rebaudianaleaf/stem extract,Cinnamomum cassia(cinnamon bark extract),Illicium verum(anise extract). In an embodiment, ingredients for extreme whitening toothpaste—mint:Aloe barbadensis(Aloe vera) leaf juice, vegetable glycerin (soy free), hydroxyapatite (mineral), hydrated silica (mineral), xylitol, calcium carbonate (mineral), organicCocos nucifera(coconut oil),Mentha piperita(peppermint oil),Mentha spicata(spearmint leaf oil),Mentha arvensis(menthol crystals),Potassium cocoate(from coconut oil), sodium cocoyl isothionate (from coconut oil), calcium ascorbate (vitamin c),Melaleuca alternifolia(tea tree leaf oil), xanthan gum (thickener),Stevia rebaudianaleaf/stem extract,citrus Aurantium dulcis(orange peel oil),Citrus limon(lemon peel oil),Azadirachta indica(neem extract), activated (coconut charcoal),Illicium verum(anise extract), sodium chlorite. In an embodiment, ingredients for healthy gums toothpaste—mint:Aloe barbadensis(Aloe vera) leaf juice, vegetable glycerin (soy free), hydroxyapatite (mineral), hydrated silica (mineral), xylitol, calcium carbonate (mineral), organicCocos nucifera(coconut oil),Mentha piperita(peppermint oil),Mentha spicata(spearmint leaf oil),Mentha arvensis(menthol crystals),Potassium cocoate(from coconut oil), sodium cocoyl isothionate (from coconut oil), calcium ascorbate (vitamin c),Camellia sinensis(green tea leaf extract), xanthan gum (thickener),Azadirachta indica(neem extract),Stevia rebaudianaleaf/stem extract,Thymus vulgaris(thyme leaf oil),Cinnamomum cassia(cinnamon bark extract),Illicium verum(anise extract). In an embodiment, ingredients for sensitivity relief toothpaste—mint:Aloe barbadensis(Aloe vera) leaf juice, vegetable glycerin (soy free), hydroxyapatite (mineral), hydrated silica (mineral), xylitol, calcium carbonate (mineral), organicCocos nucifera(coconut oil), organic hemp seed oil,Mentha piperita(peppermint oil),Mentha spicata(spearmint leaf oil),Mentha arvensis(menthol crystals),Potassium cocoate(from coconut oil), sodium cocoyl isethionate (from coconut oil), calcium ascorbate (vitamin c),Chamomilla recutita matricaria(chamomile flower extract), xanthan gum (thickener),Stevia rebaudianaNed/stem extract,Cinnamomum cassia(cinnamon bark extract),Azadirachta indica(neem extract),Salvia officinalis(sage extract),Melaleuca alternifolia(tea tree leaf oil),Illicium verum(anise extract). In an embodiment, a composition of wipes comprises water, glycerin, xylitol, cetylpyridinium chloride, sodium levulinate, octanediol, ethylhexyl glycerin, grapefruit seed extract,Artemisia princepsextract. In an embodiment, a composition of wipes comprises water, xylitol, fruit extract, poly aminopropyl biguanide, citric acid, sodium benzoate. In an embodiment, a composition of wipes comprises water,Aloe barbadensisleaf juice, glycerin, hydrated silica, xylitol, sodium levulinate, sodium phytate, xanthan gum, potassium sorbate. sodium levulinate: a corn derived preservative that is 100% natural and eco-friendly. sodium phytate: a rice bran derivative that is a stabilizer. it is 100% natural and eco-friendly. In an embodiment, a composition of wipes comprises purified water, xylitol, glycerin, hydroxyethyl cellulose, sodium benzoate, citric acid, sodium citrate and natural grape flavor. In an embodiment, a composition of wipes comprises peppermint oil, menthol, methyl paraben, propyl paraben, PEG-400, sodium saccharine, starch. In an embodiment, a composition of wipes comprises peppermint oil, menthol, stevioside. In an embodiment, a composition of wipes comprises water/aqua/eau, alcohol denat, polysorbate 20, glycerin, sodium bicarbonate, flavor (aroma),Stevia rebaudianaextract,Vaccinium macrocarpon(cranberry) fruit extract. In an embodiment, a composition of wipes comprises water, sorbitol, glycerin, hydrogen peroxide, polysorbate 80,citrus Aurantium dulcis(orange) juice, calcium carbonate, citric acid, sodium bicarbonate, sodium chloride, disodium EDTA, sodium benzoate. In an embodiment, a composition of wipes comprises deionized water, potassium sorbate, carbopol, trolamine, organic vegetable glycerin, organic xanthan gum, oleth 20, organic coconut oil (Cocos nucifera), organic lemon myrtle (Backhousia citriodora), organic. In an embodiment, a composition could comprise one or more of xylitol, sodium bicarbonate, NHAP, coconut oil, peppermint oil, kava, glycerin, sodium lauryl sulfate, sodium sulfate, sodium lauroyl sarcosinate, sodium cocoyl glutamate,Potassium cocoate, silica, sodium benzoate, calcium carbonate, propanediol, potassium nitrate, potassium chloride, xanthan gum, sorbitol, erythritol,Stevia, aloeleaf juice, neem extract—Azadirachta indica, zinc citrate, silicon dioxide, guar gum, menthol, ozonated aqua, cocoa butter, calcium glycerophosphate, betaine, sodium anisate, K12 in MCT oil, D3 in MCT oil, manuka oil, Totarol™, diatomaceous earth, sodium chlorite. In an embodiment, a composition could comprise one or more of bamboo, cotton biodegradable, xylitol, coconut oil, NHAP, NaHCO3, peppermint oil, water, glycerin, cetylpyridinium chloride, sodium levulinate, octanediol, ethylhexyl glycerin, grapefruit seed extract,Artemisia princepsextract, fruit extract, poly aminopropyl biguanide, citric acid, sodium benzoate,Aloe barbadensisleaf juice, hydrated silica, sodium phytate, xanthan gum, potassium sorbate, hydroxyethyl cellulose, sodium citrate, natural flavor, alcohol denat, polysorbate 20, flavor (aroma), fruit extract,Stevia, menthol, stevioside, carbopol, trolamine, oleth 20, coconut charcoal, lemon myrtle organic, sorbitol, H2O2, calcium carbonate and phosphoric acid. In an embodiment, nanohydroxyapatite (NHAP) can be used in powder form with the particle size ranges from 30-50 micron. In an embodiment, a size range of NHAP particles as a powder form is selected from a value being either 30 micron, 35 micron, 40 micron, 45 micron, or 50 microns. The NHAP in powder or tablet version is most likely 30-50 microns in size (3,000-5,000 nm). In an embodiment, size of NHAP in powder or table is less than 30 micron, 20 micron, 15 micron, 10 micron, 0.1 micron, 0.08 micron, 0.07 micron, 0.06 micron or lesser. In an embodiment, another product characterization of NHAP in tooth powder is shown in Table 3. TABLE 3Characterization of NHAP in powderSECTION 1: PRODUCT IDENTIFICATIONTrade namenanoXIM•CarePasteSKU504202IUPAC namePentacalcium hydroxidetriphosphateCAS number12167-74-7EC number235-330-6SynonymsHydroxyapatite(CAS No. 1306-06-5)Chemical formulaCa10(PO4)6(OH)2REACH ID number01-2119490075-38-0021SECTION 2: PRODUCT INFORMATIONLot numberI466C5SDParticle sizeMicroparticlesPhysicalSpray dryer whiteappearanceand odourless powderSECTION 3: LOT CHARACTERIZATIONUnitAnalysisResultsSpecificationReference methodAssay (calculatedwt %Lot95.9≥90Ph. Forum 31(2): 2005on dry basis)Loss on dryingwt %Lot2.8≤5Ph. Eur. 7th Ed.2.2.32.Salts (K+; Cl−; Ca2+)wt %Lot0.2≤3Ph. Eur. 7th Ed.2.2.32.Particle size ≤ 100 μm%Lot99.9≥95Laser DiffractionTotal heavy metalsppmLot<12≤20Ph. Eur. 7th Ed. 2.4.8.(as Pb) FIG.1shows a particle size distribution of NHAP powder. In an embodiment, dental product further comprises fennel, licorice, CBD, kava (Piper methysticum), Lavender, Cacao, Camphor, Lalang oil, Pippali, Garlic, Tomar Beej, Sunthi, Babul Extract, Meswak Extract, calcium carbonate, magnesium, Hawaiian Fine Sea Salt, mustard seed Powder, noni, Essential Oils, bee propolis organic, Clove Cinnamon, Sodium Bentonite, Salt, Moringa, Activated Charcoal, Bentonite Clay, Cinnamon Oil, Clove Oil, Clove Powder, Matcha Powder, Kaolin Clay, Diomatceaus Earth, Neem, Ozone, Peelu, Turmeric, Wheat Grass, Peroxide, Sage,Stevia, Thieves Oil, Trace Mineral. The pH of composition with citric acid and baking soda is shown inFIG.4. The pH is measured using pH paper, which shows yellow and bluish color in the case of citric acid and baking soda, respectively, indicating their acidic and basic pH. Three conditions are depicted inFIG.5: i) Adding solely Citric Acid (CA) to the mixture results in an acidic pH, ii) adding 50 percent Baking Soda (BS) or 50 percent Baking Soda plus a drop of Citric Acid (CA) in a combination resulting in alkaline pH, iii) Adding 50 percent Citric Acid (CA) and 50 percent Baking Soda (BS) results in acidic pH. In an embodiment, floss comprises NHAP. In an embodiment, floss is made of ecofriendly vegan material which comprises fibers. Possible fibers which are not limited to Seaweed (Algae) & Cotton Floss, Bamboo Charcoal/Polyester, Corn, Hemp, Cotton, Silk or like. In an embodiment, tooth wipes are organic 100% cotton biodegradable 2×2 gauze. Most wipes on the market are water based, not oil based, as a result, more preventatives are required leading to exposure to harmful chemicals to the body. In an embodiment, tooth wipes are oil based. “Preservatives” signify a substance or a chemical that is added to products to prevent decomposition by microbial growth or by undesirable chemical changes. In an embodiment, preservatives such as but not limited to Chlorhexidine, Triclosan, Quaternary Ammonium Compounds (such as Benzalkonium Chloride) or Parabens (such as Methyl or Propyl Paraben) may be used in the compositions. The amount of preservative is typically in the range from 0 to about 1% wt. (w/v) such as 0.1-0.75%, such as 0.3 or 0.6% (w/v), such as 0.6 or 1% (w/v), approximately 0.3 to 0.5%. In an embodiment, composition is free of preservatives. “Shelf life” is the recommended maximum time for which products can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under expected (or specified) conditions of distribution, storage, and display. It means shelf life is the length of time that a commodity may be stored without becoming unfit for use, consumption, or sale. In an embodiment, the composition has a shelf life of more than 3 years. In another embodiment, shelf life of the composition is more than 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more. In an embodiment, growth of microorganisms in the composition is controlled using pH and KCl. Tooth wipes comprise coconut oil, nanohydroxyapatite, erythritol, peppermint oil and other flavoring agents. In an embodiment, tooth wipes compriseAloe vera. In an embodiment, adding a solution of 90% w/w Coconut Oil and 10% w/w NHAP solution to the wipe. In another embodiment, a solution has Oil (Natural Oil) about 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50%, whereas NHAP is about 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45% or 50% of a wipe. In another embodiment,Aloe Veracan be added in composition of tooth wipe. In an embodiment, tooth wipes have erythritol (safe for animals) and drops of peppermint oil for taste. In an embodiment, tooth wipe comprises NHAP. In an embodiment, tooth wipe has aqua, glycerin, xylitol, flavor, sodium benzoate, potassium sorbate, coconut oil, sodium bicarbonate, citric acid, NHAP and more. In an embodiment, tooth wipes can be used in combination with tooth floss. In an embodiment, tooth wipes and floss comprise NHAP. Table 4 shows a composition of tooth wipes according to one embodiment of this invention. TABLE 4Ingredients of tooth wipes.IngredientsRANGE %AQUAq.b.->100GLYCERIN1.0-3.0XYLITOL0.1-1.0FLAVORING AGENT0.1-1.0SODIUM BENZOATE0.1-0.5POTASSIUM SORBATE0.1-0.5COCOS NUCIFERA OIL<0.1SODIUM BICARBONATE<0.1CITRIC ACID<0.1NHAP<10% In an embodiment, tooth serum comprised nanohydroxyapatite to remineralize teeth and reduce sensitivity. In an embodiment, tooth serum comprisedAloe veraas carrier. In an embodiment, tooth serum comprised of 10% nanohydroxyapatite. In an embodiment, tooth serum has about 10%, 15%%, 20%, 25%, 30%, 35%, 40%, 50% or more NHAP in the composition. In an embodiment, the composition comprises erythritol instead of xylitol; thus the toothpaste, serum and wipe are all pet friendly. In an embodiment, tooth ribbon floss (wider floss), teeth whitening product, temporary tooth filling product, dental products with NHAP to be used in dental offices by dentists (pumice for cleaning teeth, serum to reduce sensitivity with high percentage NHAP, teeth whitening with NHAP), sterilizable and reusable floss holder. In an embodiment, NHAP is natural or synthetic. Natural NHAP could be from a plant source or an animal source. In an embodiment, NHAP is obtained from a plant source. In an embodiment, NHAP is synthetic. In an embodiment, NHAP is vegan. In an embodiment, dental product comprises natural and organic ingredients. Dental product does not contain glycerin, fluoride, and surfactants. In an embodiment, dental product comprises NHAP in suspension form and not in powder form. In an embodiment, dental product comprises NHAP in powder form. In one embodiment, other ingredients that may be used in the formulations of the dental composition include those conventionally used in toothpaste such as toothpaste bases, abrasives, carriers, flavorings, colorings, stabilizers, preservatives, viscosity enhancers, pH adjusters, sparkles, gelling agents, effervescent agents, thickeners, humectants, desensitizing agents, sensitivity agents, whitening agents, mucosal adhesives, bad breath agents, gingivitis agents, astringents, oxidizing agents, and the like. These may be used in the manner and quantity generally known in the art. In some embodiments, the powder formulation is in the form of an effervescent powder or an effervescent tablet. In some embodiments, the powder formulation further comprises a remineralization agent. In one embodiment, the remineralization agent is a nanomaterial comprising hydroxyapatite crystals on nanoparticles. In some embodiments, the powder formulation further comprises additional ingredients. At least some of the additional ingredients may comprise flavoring agents. In an embodiment, the powder formulation is in the form of a tablet or a beadlet. A beadlet is defined as a spherical, free-flowing granule with a narrow size distribution. In an embodiment, a beadlet is like a gel capsule with the toothpaste inside and it dissolves in the mouth. In an embodiment, a beadlet has a size of about 500 μm, about 1000 μm, about 2000 μm, about 4000 μm, about 6000 μm, about 8000 μm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 1 cm, about 1.5, about 2 cm or more. Antibacterial agents such as triclosan, zinc chloride, and zinc citrate may be used also in conventional amounts and often less than 5% wt./wt. (0.1 to 5%) and indeed less than 1% wt./wt. Where xylitol is used as a sweetener, if more than 2% is used, it can also act as an antimicrobial agent. Preservatives such as Phenoxyethanol may also be used in conventional amounts, usually less than 2% wt./wt. (0.1 to 2%) and indeed often less than 1%. In an embodiment, toothpaste comprises erythritol 24 g, calcium carbonate 16 g, sodium bicarbonate 4 g, NHAP suspension, 10 g, coconut oil 4 g, peppermint oil 2 g. The NHAP is taken from a stock suspension containing NHAP in 16.66% w/v. In some embodiments, the acid in the dental composition is selected from citric acid, tartaric acid, fumaric acid and malic acid, and combinations thereof. In one embodiment, the acid in the dental composition is citric acid. In an embodiment, powder is comprised of ingredients in a specific order. Starting with xylitol and sodium bicarbonate (baking soda), then add the nanohydroxyapatite suspension, then slowly add the coconut oil while mixing everything together and then add the peppermint oil at the end. The compositions of the embodiments herein are biocompatible, antibacterial, sterilizable, nontoxic, non-mutagenic, non-carcinogenic, radiopaque, impervious to moisture, and do not provoke any adverse immune response. In an embodiment, whitening agents comprise but are not limited to alumina, phosphates such as sodium hexametaphosphate, charcoal, and polyvinylpyrrolidone (PVP). In an embodiment, the composition comprises tooth whitening compositions, hydrogen peroxide (and its adducts or association complexes, such as carbamide peroxide and sodium percarbonate). the peroxide source is hydrogen peroxide, or a hydrogen peroxide precursor and the source of acetyl or functionally similar groups are C1-C5 molecule having between 1 to 5 labile C1-C5 acyl containing groups. Alternatively, in order to prevent premature reaction of the hydrogen peroxide or its precursor with the source of acetyl groups, an anhydrous formulation containing both the source of acetyl groups and hydrogen peroxide, or its precursor is provided. The hydrogen peroxide or its precursor, and the source of acetyl groups, upon placement against the stained tooth surface in the oral cavity, are activated by the aqueous content of the saliva to generate a peroxyacid, such as peroxyacetic acid. Alternatively, a composition may be manufactured having each of the hydrogen peroxide or its precursor and the source of acetyl groups as a separate and distinct component. The hydrogen peroxide precursor for use in connection with the present invention is preferably selected from the group consisting of carbamide peroxide, sodium percarbonate, sodium perborate, calcium peroxide, magnesium peroxide, sodium peroxide, and the anhydrous poly(vinyl pyrrolidone)/hydrogen peroxide complexes. It is contemplated that any compound which, when in contact with water, is capable of generating, converting to, or otherwise becoming hydrogen peroxide or peroxide anion, will have utility in the formulation of the present inventive compositions. For instance, it is possible to utilize other alkali metal percarbonates (such as potassium percarbonate), as well as enzymatic sources of hydrogen peroxide, such as glucose oxidase in combination with beta-D-glucose. Additional useful peroxide precursors will become apparent to those skilled in the art based upon the present disclosure. The peroxide precursor is present in the compositions of the present invention as they are applied directly to the tooth surface in an amount sufficient to result in a hydrogen peroxide concentration of from about 0.1 percent by weight to about 15 percent by weight. Higher levels of hydrogen peroxide may be used in conjunction with a supervised dental whitening procedure in which the soft tissue (i.e., the gingival and other mucosal surfaces) are physically isolated from the teeth being whitened. Hydrogen peroxide concentrations up to about 3 percent are acceptable for short-term (less than 60 minutes) incidental contact with soft tissue. Unexpectedly, the stable oral care compositions of the present invention result in the remineralization of teeth (that is, the formation of new hydroxyapatite) and the whitening of teeth (which can be immediate and predicted through the calcium salt nucleus of the source). As a result of the oral care composition coming into contact with the enamel and/or dentin of the teeth. Furthermore, after using the compositions of the present invention, the teeth are preferably less sensitive, and/or brighter, the same also being a direct result of the formation of hydroxyapatite in situ. The result was tested on primary and permanent dentition. In an embodiment, any of the formulations/composition disclosed in this specification may also be used for cosmetic products and leave-on and rinse-off cosmetic products for hair, skin, lips, face and nails. In an embodiment, any of the formulations/composition disclosed in this specification may also be used for polishing and buffing of other surfaces such as household fixtures, appliances and automobiles. Working Examples Use of Tooth Wipes on Primary Teeth Tooth wipes can be used to clean the teeth of a baby or child. These wipes were tested for six months on 3-year-old's baby's teeth, and the result showed that wipes eliminated all plaque visible to the naked eye from the teeth. Result of tooth wipes is shown inFIG.16. Use of Tooth Wipes on Permanent Teeth Tooth wipes were tested for six months on adult teeth to ensure that it could remove all the stained plaque, which it did. Tooth wipes were also tested on adult teeth for six months to determine if it could remove spoilage, and the results were positive. Tooth wipes can also help to reduce teeth sensitivity. Use of Toothpaste Tested on Permanent Dentition. Toothpaste was tested for six months on adult teeth to ensure that it could remove all the stained plaque, which it did. Toothpaste was also tested on adult teeth for six months to determine if it could remove spoilage, and the results were positive. Toothpaste can also help to reduce teeth sensitivity.FIG.3shows results of use of toothpaste according to this embodiment. Use of Tooth Paste on Primary Teeth Toothpaste can be used to clean the teeth of a baby or child. These pastes were tested for six months on 3-year-old's baby's teeth, and results showed that toothpaste eliminated all plaque visible to the naked eye from the teeth. Use of Tooth Floss on Primary Teeth Tooth floss can be used to clean the teeth of a baby or child. The floss was tested for six months on a 3-year-old baby's teeth, and the result showed that floss eliminated all plaque visible to the naked eye from the teeth. Use of Tooth Floss on Permanent Teeth Tooth floss was tested for six months on adult teeth to ensure that it could remove all the stained plaque, which it did. Tooth floss was also tested on adult teeth for six months to determine if it could remove spoilage, and the results were positive. Tooth floss can also help to reduce teeth sensitivity. REFERENCES All references, including granted patents and patent application publications, referred herein are incorporated herein by reference in their entirety.US20210069096A1—Enhanced Toothpaste and KitsUS20150238399A1—Tooth whitening compositionU.S. Pat. No. 1,045,633B2—Oral hygiene compositions and methodsUS20090263497A1—Production method for calcium phosphate nano-particles with high purity and their useU.S. Pat. No. 9,180,318B2—Stable oral care compositionsU.S. Pat. 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11857655 | DETAILED DESCRIPTION OF THE INVENTION Antimicrobial Particle The composition according to the invention comprises a modified clay particle comprising an antimicrobial compound, the modified clay particle comprising an asymmetric 1:1 or 2:1:1 clay particle having alternating tetrahedral and an octahedral sheets terminating with a tetrahedral and an octahedral sheet at exterior surface planes. Particle of 1:1 clay is preferred. The particle is prepared from a precursor with bipolar topospecific characteristics. Any chemical reaction or series of reactions wherein an antimicrobial compound is attached selectively to coordinating cations on the exterior plane of either the tetrahedral or the octahedral surface plane of asymmetric clay can be used to prepare the bipolar particle comprising an antimicrobial compound used in the present invention. In order to obtain a true bipolar antimicrobial particle it is preferred that the reaction is selective to only one of the exterior planes. By selective is meant that more than 50% of the total antimicrobial compound is present on one of the exterior planes, preferably more than 75%, more preferably than 80%, still more preferably than 90%, even more preferably than 95%, or even more than 99%. The bipolar particle comprising an antimicrobial compound used in the composition according to the invention can be prepared, e.g. by the process described in WO 2011/036031, incorporated herein by reference. According to the present invention, preferred 1:1 clays include kaolinite and serpentine subgroups of minerals. The species included within the kaolinite subgroup include but are not limited to kaolinite, dickite, halloysite and nacrite. The species within the serpentine subgroup include but are not limited to chrysolite, lizardite, and amesite. According to the present invention, preferred 2:1:1 clays include chlorite group of minerals. Chlorite is sometimes wrongly referred to as 2:2 clay by some mineralogists. The chlorite comprises tetrahedral-octahedral-tetrahedral sheets like 2:1 clays, with an extra weakly bound brucite like layer between tetrahedral layers. The tetrahedral sheet preferably comprises coordinating tetrahedral cations of silicon. The tetrahedral sheet may also comprise isomorphously substituted coordinating tetrahedral cations which are not silicon. Isomorphously substituted coordinating tetrahedral cations include, but are not limited to, cations of aluminum, iron or boron. The octahedral sheet preferably comprises coordinating octahedral cation of aluminum. The octahedral sheet may also comprise isomorphously substituted coordinating octahedral cations which are not aluminium. Isomorphously substituted coordinating octahedral cations include cations of magnesium or iron. The at least one antimicrobial compound is attached to the coordinating cation on an external surface plane of the clay particle. Preferably, no antimicrobial compound is attached to coordination cations of non-exterior tetrahedral or octahedral plane or on the interior side of the surface sheets. The antimicrobial compound may be attached to coordinating cations on the exterior side of the tetrahedral sheet or to the coordinating cations on the exterior side of the octahedral sheet. Preferably, the at least one antimicrobial compound is attached to the coordinating cations on the external surface of the octahedral surface plane. The at least one antimicrobial compound may be attached to coordinating cations on the exterior side of the same surface sheet or on the exterior side of each of the tetrahedral and the octahedral surface sheets. The antimicrobial compounds may be the same or different. Preferably, the antimicrobial compound attached to the coordinating cations on the exterior side of the tetrahedral surface sheet is preferably not identical to the compound attached to the coordinating cations on the exterior side of the octahedral surface sheet. Preferably, the modified clay particle has a clay:antimicrobial compound ratio is between 1:0.001 and 1:0.1, more preferably between 1:0.01 and 1:0.05, most preferably about 1:0.018. Antimicrobial Compound The antimicrobial compound is preferably selected from the group of quaternary ammonium salts, antimicrobial alcohols, antimicrobial phenols, antimicrobial organic acids/salts, or combinations thereof. Preferred antimicrobial alcohols include, but are not limited to phenoxy ethanol, benzyl alcohol, dichlorobenzyl alcohol, dimethyl oxazolidine, DMDM Hydantoin, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea, hexachlorophene. Preferred antimicrobial phenols include, for instance, triclosan, Thymol, dichlorophenol, 2-chloro-4-fluoro phenol, tetrafluorobenzoic acid, cresol, hexylresorcinol, microlides, etc. Preferred antimicrobial organic acids/salts are selected from benzoic acid/sodium benzoate, salicylic acid/sodium salicylate, sorbic acid/potassium sorbate, sodium hydroxymethyl glycinate, cyclohexane diacetic acid monoamide, chloronicotinic acids, succinic acid, peracetic acid or zinc pyrithione, ketoconazole, piroctone olamine (Octopirox®), or combinations thereof. Preferred quaternary ammonium salts are selected from the group comprising cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), cetyltrimethylammonium bromide (CTAB), benzalkonium chloride (BKC), benzethonium chloride (BZC), cetrimide, quaternium, polyhexamethylene BH. Further more preferred quaternary ammonium salts are selected from the group comprising CPC, CTAC, CTAB, BKC, or BZC. By quaterium is meant a compound having a quaternary ammonium group. In a preferred embodiment, the at least one antimicrobial compound is a quaternary ammonium salt, more preferably the at least one antimicrobial compound is cetylpyridinium chloride (CPC). The concentration of the antimicrobial compound in the antimicrobial composition will depend on the use of the composition. Accordingly, in some embodiments, the antimicrobial composition will comprise modified clay particles comprising the antimicrobial compound in a therapeutically effective amount. In another embodiments, the antimicrobial composition will comprise modified clay particles comprising the antimicrobial compound in a cosmetically effective amount. Typically, the composition comprises from 0.001 wt. % to 50 wt. % of the modified particle (La, the particle comprising the at least one antimicrobial compound), by total weight of the composition, more preferably 0.01 wt. % to 20 wt. %, even more preferably from 0.1 wt. % to 10 wt. %, most preferably from 1 wt. % to 5 wt. %, by total weight of the composition, even most preferably 0.2% to 3% by weight of the composition. Preferably, the antimicrobial compound is CPC, thus the particle is a CPC-clay particle, present in the composition in an amount between 0.01 wt. % to 50 wt. %, more preferably between 0.1 wt. % to 20 wt. %, even more preferably between 1 wt. % to 5 wt. %. Nonionic Triblock Copolymer Block copolymers are based on ethylene oxide and propylene oxide. Polyoxyethylene polyoxypropylene block copolymers (Poloxamers) are composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Suitable triblock copolymers are known by the trade names Pluronics, Synperonics and Kolliphor. Preferably, the polyoxyethylene polyoxypropylene block copolymers is present in an amount of 0.001-20 wt. %, more preferably 0.01-10 wt. %, even more preferably 0.1-10 wt. %, most preferably, 1-5 wt. %, by weight of the antimicrobial composition. Preferably, the copolymer is selected from Pluronic Polyols which are nonionic and may be represented by the general formula I: wherein a is an integer such that the hydrophobic base represented by (C3H6O) has a molecular weight of about 2750 to 4000, b is an integer such that the hydrophilic portion (moiety) represented by (C2H4O) 25 constitutes about 70-80% by weight of the copolymer. Pluronic Polyols of the F (solid flake or powder) type, with a hydrophobe of M.W. of about 2750 to 4000 and with from 70 to 80% hydrophilic polyoxyethylene groups form a gel at 18-25% by weight of the H2O2/Pluronic gel formulation. Examples of suitable Pluronic compounds are Pluronic F88, F98, F108 and F127. The most preferred gelling agent is Pluronic F127, which has a molecular weight of 4000 and contains 70% of the hydrophilic polyoxyethylene moiety. Preferably, the nonionic triblock copolymer is a poloxamer of general formula I wherein a is 101 and b is 56. In one embodiment, the composition is an oral care composition. Preferably, the oral care composition is selected from toothpaste, dentifrice (e.g. in gel or powder), tooth powder, topical oral gel, mouth rinse, denture product, mouth spray, lozenge, oral tablet, chewing gum, impregnated dental implement, dental floss, and combinations thereof. Pluronic compounds are preferably employed in oral care compositions according to the invention in an amount of 0.001-20%, more preferably 0.01-10%, even more preferably 0.1 wt. % to 5 wt. %, by weight of the composition. Preferably, these amounts of pluronic are used in a composition comprising a modified particle which is a CPC-clay particle. Antimicrobial Compositions The antimicrobial composition according to the invention is preferably an antimicrobial composition for personal care, such as a composition selected from oral care compositions, shampoos, deodorants (optionally comprising propellant), hand- or body-wash, etc. In one embodiment of the invention, the composition is present in creams (including UV-A and UV-B sunscreens), soaps, gels, deodorants, shampoos or hand- or body-wash compositions. Body-wash compositions are also known as detergent compositions. Deodorants, shampoos or hand- or body-wash compositions may comprise further conventional components, such as surfactants, humectants, fragrance, buffering agents, skin soothing agents, preservatives, UV-A or UV-B sunscreens, etc. Preferably, the composition comprises solvents such as ethyl alcohol, isopropanol, acetone, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether and mixtures thereof. The composition may also comprise a non-phenolic alcohol. The non-phenolic alcohol preferably selected from e.g. aliphatic alcohol, monohydric alcohol. The most preferred non-phenolic alcohol is monohydric alcohol. Preferably, the alcohol having straight or branched chain of carbon atoms preferably containing from 1 to 16, more preferably from 2 to 10, even more preferably from 3 to 8 carbon atoms. Illustrative examples of alcohol that may be used in the composition include methyl alcohol, ethyl alcohol, isopropyl alcohol, n-propyl alcohol, isobutyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol and mixtures thereof. Preferably, alcohol is selected from ethyl alcohol, isopropyl alcohol and mixtures thereof. Preferably, the composition comprises from 40 to 95%, more preferably from 45 to 90%, even more preferably from 50 to 85%, still more preferably 55 to 80%, yet more preferably from 60 to 75% and most preferably from 65 to 70%, by weight, of a non-phenolic alcohol. Advantageously, the composition may preferably comprise ingredients like vitamins, anti-acne actives, anti-wrinkle, anti-skin atrophy and skin repair actives, skin barrier repair actives, non-steroidal cosmetic soothing actives, artificial tanning agents and accelerators, sebum stimulators, sebum inhibitors, anti-oxidants, protease inhibitors, skin tightening agents, anti-itch ingredients, hair growth inhibitors, 5-alpha reductase inhibitors, desquamating enzyme enhancers, anti-glycation agents and mixtures thereof. The composition may preferably comprise powders like e.g. chalk, talc, fillers earth, kaolin, starch, gums, colloidal silica sodium polyacrylate, tetra alkyl and/or trialkyl aryl ammonium smectites, chemically modified magnesium aluminium silicate, organically modified montmorillonite clay, hydrated aluminium silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate and mixtures thereof. Preferably, the antimicrobial composition according to the invention further comprises surfactants. Preferably, the surfactants are selected from anionic, nonionic, cationic, amphoteric surfactants and mixtures thereof. More preferably, surfactants are selected from cationic surfactant, nonionic surfactant and mixtures thereof. Preferred anionic surfactants include soap, alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C8-C20 acyl isethionates, acyl glutamates, C8-C20 alkyl ether phosphates and mixtures thereof. Preferred nonionic surfactants are those with a C10-C20 fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene 30 oxide per mole of hydrophobe; C2-C10 alkyl phenols condensed with from 2 to 20 moles of alkylene oxide; mono- and di-fatty acid esters of ethylene glycol; fatty acid monoglyceride; sorbitan, mono- and di-C8-C20 fatty acids; block copolymers (ethylene oxide/propylene oxide); polyoxyethylene sorbitan and mixtures thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable nonionic surfactants. Preferred cationic surfactant include cetyl trimethylammonium bromide, benzalkonium halides that are also known as alkyldimethylbenzylammonium halides. Preferred cationic surfactant that may be used in the composition is benzalkonium chloride, also known as alkyldimethylbenzylammonium chloride. Preferred amphoteric surfactant include amide, betaine and amine oxide type. Particularly amphoteric surfactants include cocodiethanol amide and cocomonoethanol amide, cocoamidopropyl betaine and coco amido propyl amine oxide. A preferred amphoteric surfactant that may be used as a surfactant in the composition is cocoamidopropyl betaine. When incorporated in the composition, surfactants may be present in an amount from 1 to 80%, preferably from 3 to 60%, more preferably from 5 to 40%, even more preferably from 7 to 30%, further more preferably from 9 to 15% by weight of the composition. Preferably, the composition further comprises a cosmetically or pharmaceutically acceptable base. The cosmetically or pharmaceutically acceptable base is preferably in the form of a cream, lotion, gel or emulsion. Water and/or alcohol may also be used a cosmetically or pharmaceutically acceptable base. Alcohol may be a mono or polyhydric alcohol. Monohydric alcohols often are short chain, by which is meant that they contain up to 6 carbons, and in practice is most often ethanol or sometimes iso-propanol. Polyhydric alcohols commonly comprise ethylene or propylene glycol, or a homologue can be employed such as diethylene glycol. Preferably, the cosmetically or pharmaceutically acceptable base is present preferably from 10 to 99.9%, more preferably from 50 to 99%, even more preferably from 60 to 85% and further more preferably from 65 to 80% by weight of the composition. In a preferred embodiment, the antimicrobial composition is present in an oral care product. Accordingly, the composition according to the invention is preferably included in an oral care product selected from toothpaste, dentifrice, tooth powder, topical oral gel, mouth rinse, denture product, mouth spray, lozenge, oral tablet, chewing gum, impregnated dental implement, dental floss, and combinations thereof. More preferably, the oral care product is dental care product, such as tooth powder, toothpaste, impregnated dental implement, dental floss. Preferably, the composition comprises one or more orally acceptable component, such as abrasives, orally accepted actives (such as Fluor), teeth whiteners, fragrances, stabilizers, preservatives, among other conventional components to oral care compositions. Uses of the Antimicrobial Composition The present inventors have found that the antimicrobial composition according to the invention has an improved antimicrobial activity. Accordingly, the antimicrobial composition can be used for cosmetic (non-therapeutic) or therapeutic applications. a) Therapeutic Uses In one aspect, the antimicrobial composition according to the invention is for use in the prevention or treatment of a disease of a mammal. Diseases as used herein refer to diseases caused by micro-organisms on body surfaces (skin, nails, mucosa, etc.). In one embodiment the diseases are skin diseases such as selected from the group comprising acne, eczema, or bacterial or fungal infections on the scalp, nails or skin, such as those caused byPropionibacteriaspp.,Corynebacteriaspp.,Actinobacteriales, Staphylococcispp. (e.g.S. epidermidis),Lactobacilales, Clostridiales, proteobacteria, Flavobacteriales, Bacteriodales, Malasseziayeasts (e.g.Malassezia furfurandMalassezia globosa), among others. Preferably, the use is in the prevention or treatment of a disease in the oral cavity. Preferred diseases in the oral cavity are selected from caries, tartar, dental plaque, gum diseases, and combinations thereof, for instance diseases caused byActinomycesspp,Streptococcusspp (such asS. mutansandS. sobrinus),Lactobacillusssp. (such as,Lactobacillus acidophilus),Nocardiaspp.,Haemophilusspp,Capnocytophagaspp,Veillonellaspp,Neisseria, Fusobacterium, such asF. nucleatum, Treponemaspp,Tannerellassp., such asT. forsythensis, P. gingivalis, Aggregatibacter actinomycetemcomitans, and others. In a further aspect, the antimicrobial composition of the invention is for use in the removal of oral biofilm in a mammal subject. Typically, the particle comprising an antimicrobial compound is preferably incorporated in the composition for the cosmetic use in an amount from 0.05% to 10% by weight of the composition, more preferably from 0.1% to 10%, most preferably from 0.2% to 5% by weight of the composition. The clay:antimicrobial ratio is between 1:0.001 and 1:0.1, more preferably between 1:0.01 and 1:0.05, most preferably about 1:0.018. Preferably, the antimicrobial composition for the therapeutic uses according to the invention comprises a therapeutically effective amount of an antimicrobial compound. b) Cosmetic/Non-Therapeutic Uses The present invention provides cosmetic uses of the antimicrobial composition on body surfaces, such as skin, teeth, scalp, mucosa (gums), tongue, etc. The inventors have found an improved antimicrobial activity on micro-organisms normally found on said body surfaces and that may impair body appearance (such as teeth yellowing) or body odor, in particular mouth malodor. Preferably, the antimicrobial composition is for the cosmetic use in the prevention or treatment of halitosis caused by microorganisms in the oral cavity. Accordingly, in yet another aspect, the invention relates to a non-therapeutic method of treating the skin or oral cavity of a mammal, the method including the step of contacting the skin or oral cavity of a mammal with the antimicrobial composition according to the present invention. In another aspect, the invention relates to the use of an antimicrobial composition of the invention for reducing body malodor, preferably mouth malodor, and/or for reducing oral biofilm formation and/or for reducing tooth discoloration, in a mammal. The particle comprising an antimicrobial compound is preferably incorporated in the composition for the cosmetic use in an amount from 0.05% to 10% by weight of the composition, more preferably from 0.1% to 10%, most preferably from 0.2% to 5% by weight of the composition. EXAMPLES Example 1: Preparation of Modified Clay Particle To prepare the modified clay particle comprising an antimicrobial compound according to the invention, the following method was used: 5 g of Kaolinite (Super shine 90, EICL) was taken in a 500 ml of 0.1N HCl (Merck) solution and sonicated for 30 minutes. The pH is then increased to 9 by addition of NaOH (Merck) solution drop wise. To this 10 g CPC was added and the suspension was stirred over a magnetic stirrer (Spinpot) for 6 hours while maintaining the temperature of the solution at 75-80° C. The suspension was then washed with water for about 10 times to remove the excess CPC and a final ethanol (Les Alcools De Commerce) wash was given. The clay was then dried in a hot air oven. To determine that CPC was attached on the clay after reaction FTIR-spectroscopy method was utilized. The instrument used was Perkin Elmer instruments, Spectrum One FT-IR Spectrometer. Powder (diffuse reflectance) technique was utilized for this measurement. Clay as control and reacted clay of the invention were grounded with 50% w/w of KBr in a pestle and mortar and then IR was done on these powders. The IR spectrum of the reacted clay was compared against that of pure clay. New peaks were observed in the reacted clay at the wave numbers of 2926 cm-1, 2855 cm-1, 1487 cm-1 and 1466 cm-1. The peaks at 2926 cm-1 and 2855 cm-1 are due to the C—C stretching of the alkyl chain of the CPC, while the peaks at 1487 cm-1 and 1466 cm-1 are due to the ring carbon and nitrogen of the CPC. Example 2: Antimicrobial Activity of the Actives Antimicrobial activity of the compositions according to the invention has been compared with compositions comprising either only the modified clay particle comprising an antimicrobial compound (‘CPC’, prepared as in Ex. 1), or only a nonionic triblock copolymer (‘pluronic’). Salivary flora and actives (according to Table 1 below) were co-incubated overnight and at the end of incubation biofilm was stained with crystal violet. Detailed protocol as mentioned below: Treatment and Biofilm Formation Early morning saliva samples before brushing was collected from 4-5 people, pooled together and washed twice in saline. Absorbance was set to 0.2 OD620 nm in ultra-filtered tryptone yeast extract broth (2% sucrose) and used for experiments as mentioned in further steps. 2 ml of set culture was added into 24/12 well plate to which test actives at varying concentrations were added into each of the wells. The plate was incubated anaerobically overnight at 37° C. Staining Protocol At the end overnight incubation, decant the plate out over a biohazard bag to remove all the planktonic bacteria. Rinse the plate in a tray of water and decant the water out over the tray. This step was done once to remove the loosely adhered biofilm. Place the plate on a blotting paper/paper towel over the bench top. Stain all the test wells with 1 ml of 1% Crystal violet stain (CV) for 10 min. This step was done using a pipette. Decant the plate out over the biohazard discard bag to remove all the stain. Rinse the plate in a tray of water and pour the water out over the tray. This step was done thrice consecutively, in three separate trays of water. (Each tray procedure was repeated thrice-total 9 rinse). Cover the bench top with more blotting paper/paper towel and hit the plate against the bench top until all the wells are free of any liquid. This step was done to ensure that only CV remaining is bound to a biofilm at the bottom of a well. Leave the plate face up on the bench top at room temperature (23+2° C.) until it dries completely. Add 1 ml of 33% glacial acetic acid to the test wells to solubilize the biofilm bound CV stain. Allow the acetic acid to sit for 10 mins. Pipette up and down the mix of acetic acid and CV in the wells. Transfer 10 μl of above solution mix to 90 ul of 33% acetic acid in a well of flat bottom 96 well plate. Mix the solution well and absorbance is taken at 540 nm. All the test actives were done in duplicates. TABLE 1% BiofilmBacterial control1000.01% CPC-Clay830.001% Pluronic760.001% Pluronic + 0.01% CPC-Clay290.001% Pluronic + 0.005% CPC-Clay52 Example 3: Antimicrobial Activity of the Actives in Toothpaste Formulations Antimicrobial activity of the compositions according to the invention has been compared with compositions comprising either only the modified clay particle comprising an antimicrobial compound (‘CPC’, prepared as in Ex. 1), or only a nonionic triblock copolymer (‘pluronic’). The compositions (Table 2) were tested according to the following method. Pellicle coated HAP disc was treated with toothpaste base formulation topped up with actives for 2 mins, subjected to sterile water wash and challenged with salivary flora and incubated overnight. At the end of incubation, the discs were stained with Plaque disclosing dye (Plak-check, Vishal Dentocare PVT.LTD). The detailed method is as follows: Early morning saliva was collected from 5-6 volunteers and pooled. The pooled saliva was pelleted by centrifuging at 4000 g for 10 min to produce salivary pellicle (the supernatant was filter sterilized through 0.22 um filter and stored at 4° C.) and salivary flora (pellet was washed twice in 5 ml of 1×PBS (pH7.0) by centrifugation). The salivary flora was adjusted to 108 Log CFU/ml (0.2 OD620 nm) in ultra-filtered Tryptone yeast extract broth (nutrient media) containing 2% sucrose. Each sterile HAP was coated with salivary pellicle (discs were placed horizontally in 12 well plate) to allow the initial pellicle formation on the sterile HAP surface. The pellicle coated HAP disc was dip washed and challenged with 30% diluted toothpaste slurry topped up with actives for 2 mins. After 2 min of contact time, the treated HAP was dip washed thrice consecutively in 5 ml of sterile water. The treated HAP discs were challenged with salivary flora and incubated anaerobically at 37° C. for 22-24 h. At the end 22-24 h incubation, the disc was dip washed thrice in of sterile distilled water. The rinsed disc was immersed in plaque disclosing solution (Plak-Check containing erythrosin, from Vishal Dentocare Pvt Ltd) for 5 min. At the end of 5 min, the stained disc was dip washed thrice consecutively in 5 ml of sterile distilled water. The stained discs were dried at 25+3° C. for 1 hour. Once dried the stain on the discs was eluted using 1 ml of 0.1N NaOH. The absorbance of eluted solution was read at OD540 nm. Higher the absorbance reading, more the formed biofilm on the disc. The results are described in table 1 below. TABLE 2% BiofilmToothpaste base1000.5% CPC-Clay891.5% CPC-Clay84Pluronic 2%642% pluronic + 1.5% CPC-Clay28 | 26,442 |
11857656 | DETAILED DESCRIPTION The disclosure relates to systems, methods, and kits for altering the color of the hair. The systems, methods, and kits according to the disclosure surprisingly and unexpectedly provide improved color deposition to the hair that results in more vibrant and satisfying coloration to the hair. Systems It has been surprisingly and unexpectedly discovered that when hair is treated with a system comprising a pre-treatment composition comprising at least one amine-based compound, and a dyeing composition comprising a microtube-dye composite, improved color deposition onto the hair and more vibrant hair color can be achieved. Pre-Treatment Compositions The systems according to the disclosure comprise at least one pre-treatment composition comprising at least one non-surface active amine-based compound, and optionally at least one solvent. The non-surface active amine-based compounds useful in the pre-treatment compositions according to the disclosure have a molecular weight of less than about 10,000, such as less than about 8,000, less than about 6,000, less than about 5,000, less than about 4,000, less than about 3,000, or less than about 2,500. For example, the non-surface active amine-based compounds may, in various embodiments, have a molecular weight ranging from about 50 to about 10,000, from about 50 to about 9,000, from about 50 to about 8,000, from about 50 to about 7,000, from about 50 to about 6,000, from about 50 to about 5,000, from about 50 to about 4,000, from about 50 to about 3,000, from about 50 to about 2,000, from about 100 to about 10,000, from about 100 to about 9,000, from about 100 to about 8,000, from about 100 to about 7,000, from about 100 to about 6,000, from about 100 to about 5,000, from about 100 to about 4,000, from about 100 to about 3,000, or from about 100 to about 2,000. In some embodiments, the non-surface active amine-based compounds may have a molecular weight ranging from about 50 to about 7,500, from about 50 to about 2,500, from about 50 to about 1,500, from about 100 to about 7,500, from about 100 to about 2,500, or from about 100 to about 1,500. In various embodiments, useful non-surface active amine-based compounds have one or more nitrogen atoms in the main chain or backbone of the compound, i.e. at least one nitrogen atom other than as part of a side chain attached to the main chain or backbone of the compound. It should be understood that such compounds may include nitrogen atom(s) in one or more side chains, but that such compounds will also include one or more nitrogen atoms in the main chain or backbone of the compound. For example, amino acids such as arginine and/or lysine may be used. In some embodiments, the non-surface active amine-based compound may comprise, consist essentially of, or consist of arginine. In other embodiments, useful non-surface active amine-based compounds contain one or more imine groupings HN═C. For example, synthetic or natural polyamines may be chosen. In various embodiments polyalkyleneimines, such as branched or unbranched C2-C8 or C2-C5 polyalkyleneimines, may be chosen. For example, the amine-based compounds may be chosen from polyethyleneimine, polypropyleneimine, poly(allylamine), and/or polyvinylamine. In at least one embodiment, the amine-based compound comprises polyethyleneimine. As useful polyethyleneimines, mention may be made of the products available from BASF under the names LUPASOL or POLYIMIN, e.g. Lupasol® PS, Lupasol® PL, Lupasol® PR8515, Lupasol® G20, or Lupasol® G35. Optionally, dendrimers and derivatives of such polyalkyleneimines may also be used. In some embodiments, the non-surface active amine-based compound may comprise, consist essentially of, or consist of polyethyleneimine. In other embodiments, polyamino acids may be chosen, such as, for example polyarginine. Optionally, pre-treatment compositions according to the disclosure may comprise more than one non-surface active amine-based compound. In embodiments where more than one non-surface active amine-based compound is present, preferably at least one has a molecular weight of less than about 10,000, such as less than about 8,000, less than about 6,000, less than about 5,000, less than about 3,000, or less than about 2,500. The total amount of non-surface active amine-based compounds may range from about 0.001% to about 25% by weight, relative to the total weight of the pre-treatment composition. For example, in some embodiments, the total amount of amine-based compounds may range from about 0.001% to about 20%, such as about 0.001% to about 15%, about 0.001% to about 10%, 0.001% to about 9%, about 0.001% to about 8%, about 0.001% to about 7%, about 0.001% to about 6%, about 0.001% to about 5%, about 0.001% to about 4%, about 0.001% to about 3%, about 0.001% to about 2.5%, about 0.001% to about 2%, about 0.001% to about 1.5%, about 0.001% to about 1%, about 0.001% to about 0.5%, about 0.01% to about 25%, about 0.01% to about 20%, about 0.01% to about 15%, about 0.01% to about 10%, 0.01% to about 9%, about 0.01% to about 8%, about 0.01% to about 7%, about 0.01% to about 6%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2.5%, about 0.01% to about 2%, about 0.01% to about 1.5%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, 0.1% to about 9%, about 0.1% to about 8%, about 0.1% to about 7%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2.5%, about 0.1% to about 2%, about 0.1% to about 1.5%, about 0.1% to about 1%, or about 0.1% to about 0.5% by weight, relative to the total weight of the pre-treatment composition. In other embodiments, the total amount of non-surface active amine-based compounds ranges from about 0.2% to about 25%, about 0.2% to about 20%, about 0.2% to about 15%, about 0.2% to about 10%, about 0.2% to about 9%, about 0.2% to about 8%, about 0.2% to about 7%, about 0.2% to about 6%, about 0.2% to about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2% to about 2.5%, about 0.2% to about 2%, about 0.2% to about 1.5%, about 0.2% to about 1%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2.5%, about 0.5% to about 2%, about 0.5% to about 1.5%, or about 0.5% to about 1% by weight, relative to the total weight of the pre-treatment composition. The pre-treatment composition comprises at least one solvent, for example water, non-aqueous solvents, or a mixture thereof. In various embodiments, the solvent of the pre-treatment composition comprises, consists essentially of, or consists of water. Exemplary non-aqueous solvents include, for example, glycerin, C1-4alcohols, organic solvents, fatty alcohols, fatty ethers, fatty esters, polyols, glycols, vegetable oils, mineral oils, liposomes, laminar lipid materials, and mixtures thereof. As examples of organic solvents, non-limiting mentions can be made of monoalcohols and polyols such as ethyl alcohol, isopropyl alcohol, propyl alcohol, benzyl alcohol, and phenylethyl alcohol, or glycols or glycol ethers such as, for example, monomethyl, monoethyl and monobutyl ethers of ethylene glycol, propylene glycol or ethers thereof such as, for example, monomethyl ether of propylene glycol, butylene glycol, hexylene glycol, dipropylene glycol as well as alkyl ethers of diethylene glycol, for example monoethyl ether or monobutyl ether of diethylene glycol. The organic solvents can be volatile or non-volatile compounds. In certain embodiments, the pre-treatment composition comprises from about 60% to about 99.999% of a solvent, such as water, by weight. In certain embodiments, the pre-treatment composition comprises from about 75% to about 99.999% solvent by weight, such as from about 75% to about 99.99%, about 75% to about 99.9%, about 75% to about 99%, about 75% to about 98%, about 75% to about 97%, about 90% to about 99.9%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 95% to about 99.9%, about 95% to about 99%, about 95% to about 98%, or about 95% to about 97%, by weight of the pre-treatment composition. In various embodiments the systems comprise more than one pre-treatment composition, such as, for example, two or more pre-treatment compositions. In such embodiments, the non-surface active amine-based compound(s) present in the two or more pre-treatment compositions may be the same or different. Optionally, in such embodiments, at least one non-surface active amine-based compound has a molecular weight of less than about 10,000, such as less than about 8,000, less than about 6,000, less than about 5,000, less than about 3,000, or less than about 2,500. The pre-treatment composition(s) may comprise additional components. By way of example only, the pre-treatment composition may comprise pH adjusters, preservatives, humectants, oils, fragrances, etc. In various embodiments, the pre-treatment composition(s) have a pH of less than or equal to about 7, such as less than or equal to about 6, less than or equal to about 5, less than or equal to about 4, or less than or equal to about 3. For example, the pre-treatment composition may have a pH ranging from about 1 to about 7, such as from about 2 to about 6, from about 2.5 to about 5, or from about 3 to about 4. It has been discovered that pre-treatment compositions having a lower pH and/or comprising amine-based compounds with a lower molecular weight may provide surprising color-enhancement efficacy. Therefore, in certain embodiments, it may be preferable for the pre-treatment composition(s) to comprise at least one non-surface active amine-based compound with a molecular weight of less than about 10,000, such as less than about 8,000, less than about 6,000, less than about 5,000, less than about 3,000, or less than about 2,500, for example ranging from about 100 to about 10,000, from about 100 to about 7,500, from about 100 to about 5,000, or from about 100 to about 2,500, and for the pre-treatment composition to have a pH of less than or equal to 7, such as ranging from about 1 to about 5 or from about 2 to about 4. In other embodiments, it may be preferable for the pre-treatment composition to comprise at least one non-surface active amine-based compound with a molecular weight of less than about 10,000 or less than about 5,000, for example ranging from about 100 to about 5,000 or about 100 to about 2,500, and for the pre-treatment composition to have a pH of less than or equal to 7, such as ranging from about 1 to about 5 or from about 2 to about 4. Dyeing Composition The systems according to the disclosure comprise at least one dyeing composition comprising at least one microtube-dye composite, and optionally at least one solvent. The dye of the composite may include at least one anionic, cationic, nonionic, or natural direct dye, as well as mixtures thereof. The term “microtube” as used herein includes any tubular material having micron level dimensions or less (e.g., the length dimension of the tube being under about 1 mm), including nanotubes, or may refer to tubular structures having an outer diameter that is sub-micron and lengths under about 100 microns, such as under about 50 microns, or under about 10 microns. Various exemplary embodiments employ microtubes which are aluminosilicate in nature, such as halloysite and imogolite, or which are not aluminosilicate in nature, such as sepiolite or cylindrite. Exemplary and non-limiting microtubes include, for example halloysite (Al2Si2O5(OH)4) microtubes. Halloysite forms as small cylinders (nanotubes) that may, for example, have a wall thickness ranging from about 10 to about 15 atomic aluminosilicate sheets, an outer diameter ranging from about 50 to about 60 nm, an inner diameter ranging from about 12 to about 20 nm, and a length ranging from about 0.5 to about 10 μm, with an average length of about 1 μm. Their outer surface is mostly composed of —Si—O—Si— and the inner surface of —Al—OH, and hence those surfaces are oppositely charged at approximately neutral pH. In various embodiments of the disclosure, the microtubes comprise, consist essentially of, or consist of halloysite. The microtubes may be “loaded” with a hair dyeing agent, meaning that the dye agent is incorporated into the lumen of the microtube, in order to form the microtube-dye composite. The microtube-dye composite may be formed by methods known for loading microtubes (such as halloysite), for example as described in U.S. Pat. Nos. 8,507,056 and 10,799,439, and Abdullayev E. and Lvov Y., “Halloysite clay nanotubes as a ceramic ‘skeleton’ for functional biopolymer composites with sustained drug release,”J. Mater. Chem. B,1(23):2894-2903 (2013), all of which are incorporated herein by reference. By way of example, a hair dyeing agent may be dissolved in an appropriate solvent, such as water, a non-aqueous solvent, or a mixture thereof, to form a solution. The amount of dye may be chosen such that it is near or at the solubility limit of the dye in the solvent. In one exemplary embodiment, the solution may contain from about 1% to about 20%, such as from about 1% to about 15%, by weight, of the hair dyeing agent. In another embodiment, the solution may contain from about 1 to about 20 mg of dye per mL solvent, such as from about 1 to about 15 mg of dye per mL of solvent. An appropriate amount of the microtube component may be added to the dye solution, for example in powder form, to form a dispersion. The amount of the microtube component may, for example, be chosen to provide a weight ratio of dye:microtube ranging from about 1:1 to about 5:1, such as from about 1:1 to about 4:1, from about 1:1 to about 3:1, or from about 1.5:1 to about 2.5:1, such as about 2:1, which may enhance color. The dispersion may optionally be homogenized, sonicated, stirred, placed under vacuum, washed, and/or dried to provide microtubes loaded with the hair dyeing agent. For example, the dispersion may be sonicated for a period of time such as about 2-10 minutes, for example about 5 minutes, then mixed for a period of time such as about 10-60 minutes, for example about 30 minutes. The sonication and/or mixing steps can be repeated one or more times, and may be carried out under either ambient conditions, under vacuum and/or elevated temperature, or combinations thereof, until the microtubes are loaded with dye. Once the microtubes are loaded, the supernatant may be removed, e.g. by centrifuging, and the microtube-dye composite can be dried, for example in an oven at a temperature of at least 40° C., such as at least 45° C., for example about 50° C. In one embodiment, the solvent may comprise, consist essentially of, or consist of water. For example, the solvent may be water and may include a component for aiding dissolution of the dye chosen, for example sodium carbonate. In further embodiments, the solvent may comprise, consist essentially of, or consist of a non-aqueous solvent. Exemplary and non-limiting non-aqueous solvents that can be used for loading the hair dyeing agent into the microtubes include alkanediols (polyhydric alcohols) such as glycerin, 1,2,6-hexanetriol, trimethylolpropane, ethylene glycol, propylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, dipropylene glycol, 2-butene-1,4-diol, 2-ethyl-1,3-hexanediol, 2-methyl-2,4-pentanediol, caprylyl glycol, 1,2-hexanediol, 1,2-pentanediol, and 4-methyl-1,2-pentanediol; alkyl alcohols having 1 to 4 carbon atoms such as ethanol, methanol, butanol, propanol, and isopropanol; glycol ethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monomethyl ether acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol mono-n-propyl ether, ethylene glycol mono-iso-propyl ether, diethylene glycol mono-iso-propyl ether, ethylene glycol mono-n-butyl ether, ethylene glycol mono-t-butyl ether, diethylene glycol mono-t-butyl ether, 1-methyl-1-methoxybutanol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol mono-t-butyl ether, propylene glycol mono-n-propyl ether, propylene glycol mono-iso-propyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, dipropylene glycol mono-n-propyl ether, and dipropylene glycol mono-iso-propyl ether; 2-pyrrolidone, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, formamide, acetamide, dimethyl sulfoxide, sorbitan, acetine, diacetine, triacetine, sulfolane, acetone, and mixtures thereof. In certain embodiments, the solvent may comprise both water and a non-aqueous solvent, for example from about 1% to about 99% water mixed with about 1% to about 99% non-aqueous solvent. It is within the ability of those skilled in the art to choose the appropriate solvent and/or combination of solvents, and amounts thereof, in order to dissolve the hair dyeing agents useful according to the disclosure. In various embodiments, the internal and/or external surface of the microtube may be modified prior to loading with the hair dyeing agent, which may aid dye loading and/or the hair dyeing process. By way of example, the microtubes may be modified, e.g. with a surfactant such as sodium dodecyl sulphate, or may be made hydrophobic, which may be preferred when loading a hydrophobic dye. In one embodiment, the microtubes may be dispersed in a solution of anionic surfactant (e.g. at a weight ratio of about 1:1), optionally stirred and/or centrifuged, and optionally washed and/or dried, in order to produce microtubes modified with the anionic surfactant, having an increased net negative charge relative to unmodified microtubes. In another embodiment, the microtubes may be made hydrophobic, for example by coupling a silane coupling agent to hydroxyl groups present at the surface of the microtubes to increase the contact angle of the microtube, or by absorption of anionic amphiphile molecules into the positive lumen. In various embodiments, the contact angle may be increased to at least about 30°, such as at least about 50°, at least about 75°, at least about 100°, at least about 115°, such as about 120°. Exemplary and non-limiting silane coupling agents include (3-glycidyloxypropyl) trimethoxy silane (GTMS), 3-aminopropyltriethoxy silane (APTES), hexamethyldisilazane (HMDS), and octadecyltrimethoxy silane (ODTMS). By way of example, the microtubes may be sonicated with the silane coupling agent in a solvent, e.g. water, an organic solvent, or a mixture thereof, followed by refluxing at increased temperature, e.g. greater than about 50° C. or greater than about 75° C., such as about 85° C. In yet a further embodiment, the surface of the microtube may be selectively etched. For example, the inner surface of the halloysite lumen may be etched by treatment with acid, such as sulfuric acid, which may increase the diameter of the lumen. In one embodiment, the halloysite can be stirred in sulfuric acid (e.g. 1 M) at elevated temperature, e.g. greater than about 50° C. or greater than about 75° C., such as about 80° C., for a period of time such as at least 2 hours, at least 4 hours, at least 6 hours, or at least 8 hours. Such treatment can increase the loading capacity of the microtubes by 2, 3, 4, or even more times the pre-etching loading capacity. Useful hair dyeing agents according to the disclosure included direct dyes such as anionic, cationic, nonionic, and natural hair dyeing agents, as well as mixtures of any two or more thereof. In certain embodiments, the hair dyeing agents comprise, consist essentially of, or consist of natural hair dyeing agents. In various embodiments, the compositions are free or essentially free of oxidative hair dyeing agents. The term “anionic hair dyeing agent” is intended to mean any hair dyeing agent comprising in its structure at least one CO2R or SO3R substituent with R denoting a hydrogen atom or a cation originating from a metal or an amine, or an ammonium ion. By way of example, anionic hair dyeing agents may be chosen from acidic nitro direct dyes, acidic azo dyes, acidic azine dyes, acidic triarylmethane dyes, acidic indoamine dyes, acidic anthraquinone dyes, indigoid dyes, acidic natural dyes, and combinations thereof. In one exemplary embodiment, the anionic hair dyeing agent may be chosen from the diaryl anionic azo dyes of formula (II) or (III): wherein:R7, R8, R9, R10, R′7, R′8, R′9and R′10, which may be identical or different, represent a hydrogen atom or a group chosen from:alkyl;alkoxy, alkylthio;hydroxyl, mercapto;nitro, nitroso;R∘—C(X)—X′—, R∘—X′—C(X)—, R∘—X′—C(X)—X″— with R∘representing a hydrogen atom or an alkyl or aryl group; X, X′ and X″, which may be identical or different, representing an oxygen or sulfur atom, or NR with R representing a hydrogen atom or an alkyl group;(O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;(O)CO−—, M+with M+as defined previously;R″—S(O)2—, with R″ representing a hydrogen atom or an alkyl, aryl, (di)(alkyl)amino or aryl(alkyl)amino group; preferentially a phenylamino or phenyl group;R′″—S(O)2—X′— with R′″ representing an alkyl or optionally substituted aryl group, X′ as defined previously;(di)(alkyl)amino;aryl(alkyl)amino optionally substituted with one or more groups chosen from i) nitro; ii) nitroso; iii) (O)2S(O−), M+and iv) alkoxy, with M+as defined previously;optionally substituted heteroaryl; preferentially a benzothiazolyl group;cycloalkyl; in particular cyclohexyl;Ar—N═N— with Ar representing an optionally substituted aryl group; preferentially a phenyl optionally substituted with one or more alkyl, (O)2S(O—)—, M+ or phenylamino groups; oror alternatively two contiguous groups R7with R8or R8with R9or R9with R10together form a fused benzo group A′; and R′7with R′8or R′8with R′9or R′9with R′10together form a fused benzo group B′; with A′ and B′ optionally substituted with one or more groups chosen from i) nitro; ii) nitroso; iii) (O)2S(O—)—, M+; iv) hydroxyl; v) mercapto; vi) (di)(alkyl)amino; vii) R∘—C(X)—X′—; viii) R∘—X′—C(X)—; ix) R∘—X′—C(X)—X″—; x) Ar—N═N— and xi) optionally substituted aryl(alkyl)amino; with M+, R∘, X, X′, X″ and Ar previously defined; andW represents a sigma bond 6, an oxygen or sulfur atom, or a divalent radical i) —NR—, with R as defined previously, or ii) methylene —C(Ra)(Rb)—, with Raand Rb, which may be identical or different, representing a hydrogen atom or an aryl group, or alternatively Raand Rbform, with the carbon atom that bears them, a spiro cycloalkyl; preferentially, W represents a sulfur atom or Raand Rbtogether form a cyclohexyl;with the understanding that formulae (II) and (Ill) comprise at least one sulfonate radical (O)2S(O−)—, M+or one carboxylate radical (O)CO−—, M+on one of the rings A, A′, B, B′ or C; preferentially sodium sulfonate. As non-limiting examples of dyes of formula (II), mention may be made of Acid Red 1, Acid Red 4, Acid Red 13, Acid Red 14, Acid Red 18, Acid Red 27, Acid Red 28, Acid Red 32, Acid Red 33, Acid Red 35, Acid Red 37, Acid Red 40, Acid Red 41, Acid Red 42, Acid Red 44, Pigment red 57, Acid Red 68, Acid Red 73, Acid Red 135, Acid Red 138, Acid Red 184, Food Red 1, Food Red 13, Acid Orange 6, Acid Orange 7, Acid Orange 10, Acid Orange 19, Acid Orange 20, Acid Orange 24, Yellow 6, Acid Yellow 9, Acid Yellow 36, Acid Yellow 199, Food Yellow 3, Acid Violet 3, Acid Violet 7, Acid Violet 14, Acid Blue 113, Acid Blue 117, Acid Black 1, Acid Brown 4, Acid Brown 20, Acid Black 26, Acid Black 52, Food Black 1, Food Black 2 and Food yellow 3 or sunset yellow. As non-limiting examples of dyes of formula (III), mention may be made of Acid Red 111, Acid Red 134 and Acid yellow 38. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the pyrazolone anionic azo dyes of formulae (IV) and (V): wherein:R11, R12and R13, which may be identical or different, represent a hydrogen or halogen atom, an alkyl group or —(O)2S(O−), M+with M+as defined previously;R14represents a hydrogen atom, an alkyl group or a group —C(O)O—, M+ with M as defined previously;R15represents a hydrogen atom;R16represents an oxo group, in which case R′16is absent, or alternatively R15with R16together form a double bond;R17and R18, which may be identical or different, represent a hydrogen atom, or a group chosen from:(O)2S(O−)—, M+with M+as defined previously;Ar—O—S(O)2— with Ar representing an optionally substituted aryl group, preferentially a phenyl optionally substituted with one or more alkyl groups;R19and R20together form either a double bond, or a benzo group D′, which is optionally substituted;R′16, R′19and R′20, which may be identical or different, represent a hydrogen atom or an alkyl or hydroxyl group;R21represents a hydrogen atom or an alkyl or alkoxy group;Raand Rb, which may be identical or different, are as defined previously, preferentially Rarepresents a hydrogen atom and Rbrepresents an aryl group;Y represents either a hydroxyl group or an oxo group;represents a single bond when Y is an oxo group; and represents a double bond when Y represents a hydroxyl group;with the understanding that that formulae (IV) and (V) comprise at least one sulfonate radical (O)2S(O−)—, M+or one carboxylate radical C(O)O−—, M+on one of the rings D or E; preferentially sodium sulfonate. As non-limiting examples of dyes of formula (IV), mention may be made of Acid Red 195, Acid Yellow 23, Acid Yellow 27 and Acid Yellow 76. As a non-limiting example of a dye of formula (V), mention may be made of Acid Yellow 17. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the anthraquinone dyes of formulae (VI) and (VII): wherein:R22, R23, R24, R25, R26, and R27, which may be identical or different, represent a hydrogen or halogen atom, or a group chosen from:alkyl;hydroxyl, mercapto;alkoxy, alkylthio;optionally substituted aryloxy or arylthio, preferentially substituted with one or more groups chosen from alkyl and (O)2S(O−)—, M+with M+as defined previously;aryl(alkyl)amino optionally substituted with one or more groups chosen from alkyl and (O)2S(O−)—, M+with M+as defined previously;(di)(alkyl)amino;(di)(hydroxyalkyl)amino;(O)2S(O−)—, M+with M+as defined previously;Z′ represents a hydrogen atom or a group NR28R29with R28and R29, which may be identical or different, representing a hydrogen atom or a group chosen from:alkyl;polyhydroxyalkyl such as hydroxyethyl;aryl optionally substituted with one or more groups, more particularly i) alkyl such as methyl, n-dodecyl, n-butyl; ii) (O)2S(O−)—, M+with M+as defined previously; iii) R∘—C(X)—X′—, R∘—X′—C(X)—, R∘—X′—C(X)—X″— with R∘, X, X′ and X″ as defined previously, preferentially R∘represents an alkyl group;cycloalkyl; e.g. cyclohexyl;Z represents a group chosen from hydroxyl and NR′28R′29, with R′28and R′29, which may be identical or different, representing the same atoms or groups as R28and R29as defined previously;with the understanding that formulae (VI) and (VII) comprise at least one sulfonate radical (O)2S(O−)—, M+or a carboxylate radical C(O)O−—, M+; preferentially sodium sulfonate. As non-limiting examples of dyes of formula (VI), mention may be made of Acid Blue 25, Acid Blue 43, Acid Blue 62, Acid Blue 78, Acid Blue 129, Acid Blue 138, Acid Blue 140, Acid Blue 251, Acid Green 25, Acid Green 41, Acid Violet 42, Acid Violet 43, Mordant Red 3, and EXT violet No 2. As a non-limiting example of a dye of formula (VII), mention may be made of Acid Black 48. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the nitro dyes of formulae (VIII) and (IX): wherein:R30, R31and R32, which may be identical or different, represent a hydrogen or halogen atom, or a group chosen from:alkyl;alkoxy optionally substituted with one or more hydroxyl groups, alkylthio optionally substituted with one or more hydroxyl groups;hydroxyl, mercapto;nitro, nitroso;polyhaloalkyl;R∘—C(X)—X′—, R∘—X′—C(X)—, R∘—X′—C(X)—X′— with R∘, X, X′, and X″ as defined previously;(O)2S(O−)—, M+with M+as defined previously;(O)CO−—, M+with M+as defined previously;(di)(alkyl)amino;(di)(hydroxyalkyl)amino;heterocycloalkyl such as piperidino, piperazino or morpholino;Rcand Rd, which may be identical or different, represent a hydrogen atom or an alkyl group;W is as defined previously; for example W may represent an —NH— group;ALK represents a linear or branched divalent C1-C6alkylene group; more particularly, ALK represents a —CH2—CH2— group;n is 1 or 2;p represents an integer inclusively between 1 and 5;q represents an integer inclusively between 1 and 4;u is 0 or 1;when n is 1, J represents a nitro or nitroso group;when n is 2, J represents an oxygen or sulfur atom, or a divalent radical —S(O)m— with m representing an integer 1 or 2; for example, J represents a radical —SO2—;M′ represents a hydrogen atom or a cationic counterion; which may be present or absent, represents a benzo group optionally substituted with one or more R30groups as defined previously;it being understood that formulae (VIII) and (IX) comprise at least one sulfonate radical (O)2S(O−)—, M+or a carboxylate radical C(O)O−—, M; for example sodium sulfonate. As non-limiting examples of dyes of formula (VIII), mention may be made of Acid Brown 13 and Acid Orange 3. As non-limiting examples of dyes of formula (IX), mention may be made of Acid Yellow 1, the sodium salt of 2,4-dinitro-1-naphthol-7-sulfonic acid, 2-piperidino-5-nitrobenzenesulfonic acid, 2(4′-N,N(2″-hydroxyethyl)amino-2′-nitro)anilineethanesulfonic acid, 4-p-hydroxyethylamino-3-nitrobenzenesulfonic acid, and EXT D&C yellow 7. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the triarylmethane dyes of formula (X): wherein:R33, R34, R35and R36, which may be identical or different, represent a hydrogen atom or a group chosen from alkyl, optionally substituted aryl and optionally substituted arylalkyl; particularly an alkyl and benzyl group optionally substituted with a group (O)mS(O−)—, M with M+and m as defined previously;R37, R38, R39, R40, R41, R42, R43and R44, which may be identical or different, represent a hydrogen atom or group chosen from:alkyl;alkoxy, alkylthio;(di)(alkyl)amino;hydroxyl, mercapto;nitro, nitroso;R∘—C(X)—X′—, R∘—X′—C(X)—, R∘—X′—C(X)—X″— with R∘representing a hydrogen atom or an alkyl or aryl group; X, X′ and X″, which may be identical or different, representing an oxygen or sulfur atom, or NR with R representing a hydrogen atom or an alkyl group;(O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;(O)CO−—, M+with M+as defined previously;or alternatively two contiguous groups R41with R42or R42with R43or R43with R44together form a fused benzo group: I′; with I′ optionally substituted with one or more groups chosen from i) nitro; ii) nitroso; iii) (O)2S(O−)—, M+; iv) hydroxyl; v) mercapto; vi) (di)(alkyl)amino; vii) R∘—C(X)—X′—; viii) R∘—X′—C(X)—; ix) R∘—X′—C(X)—X″—; with M+, R∘, X, X′ and X″ as defined previously;with the understanding that at least one of the rings G, H, I or I′ comprises at least one sulfonate radical (O)2S(O−)— or a carboxylate radical —C(O)O—; for example sulfonate. In a preferred embodiment of formula (X), R37to R40represent a hydrogen atom, and R41to R44, which may be identical or different, represent a hydroxyl group or (O)2S(O−)—, M+; and when R43with R44together form a benzo group, it is preferentially substituted with an (O)2S(O−)— group. As non-limiting examples of dyes of formula (X), mention may be made of Acid Blue 1; Acid Blue 3; Acid Blue 7, Acid Blue 9; Acid Violet 49; Acid Green 3; Acid Green 5 and Acid Green 50. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the xanthene-based dyes of formula (XI): wherein:R45, R46, R47and R48, which may be identical or different, represent a hydrogen or halogen atom;R49, R50, R51and R52, which may be identical or different, represent a hydrogen or halogen atom, or a group chosen from:alkyl;alkoxy, alkylthio;hydroxyl, mercapto;nitro, nitroso;(O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;(O)CO−—, M+with M+as defined previously;G represents an oxygen or sulfur atom or a group NRewith Reas defined previously;L represents an alkoxide O−, M+; a thioalkoxide S−, M+or a group NRf, with Rfrepresenting a hydrogen atom or an alkyl group and M+as defined previously; M+is particularly sodium or potassium;L′ represents an oxygen or sulfur atom or an ammonium group: N+RfRg, with Rfand Rg, which may be identical or different, representing a hydrogen atom, an alkyl group or optionally substituted aryl; for example L′ represents an oxygen atom or a phenylamino group optionally substituted with one or more alkyl or (O)mS(O−)—, M+groups with m and M+as defined previously;Q and Q′, which may be identical or different, represent an oxygen or sulfur atom; andM+is as defined previously. As non-limiting examples of dyes of formula (XI), mention may be made of Acid Yellow 73; Acid Red 51; Acid Red 52; Acid Red 87; Acid Red 92; Acid Red 95 and Acid Violet 9. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the indole-based dyes of formula (XII): wherein:R53, R54, R55, R56, R57, R58, R59and R60, which may be identical or different, represent a hydrogen atom or group chosen from:alkyl;alkoxy, alkylthio;hydroxyl, mercapto;nitro, nitroso;R∘—C(X)—X′—, R∘—X′—C(X)—, R∘—X′—C(X)—X″— with R∘representing a hydrogen atom or an alkyl or aryl group; X, X′ and X″, which may be identical or different, representing an oxygen or sulfur atom, or NR with R representing a hydrogen atom or an alkyl group;(O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;(O)CO−—, M+with M+as defined previously;G represents an oxygen or sulfur atom or a group NRewith Reas defined previously;Riand Rh, which may be identical or different, represent a hydrogen atom or an alkyl group;it being understood that formula (XII) comprises at least one sulfonate radical (O)2S(O−)—, M+or a carboxylate radical —C(O)O—, M+; for example sodium sulfonate. As a non-limiting example of a dye of formula (XII), mention may be made of Acid Blue 74. In another exemplary embodiment, the anionic hair dyeing agent may be chosen from the quinoline-based dyes of formula (XIII): wherein:R61represents a hydrogen or halogen atom or an alkyl group;R62, R63, and R64, which may be identical or different, represent a hydrogen atom or a group (O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;or alternatively R61with R62, or R61with R64, together form a benzo group optionally substituted with one or more groups (O)2S(O−)—, M+with M+representing a hydrogen atom or a cationic counterion;it being understood that formula (XIII) comprises at least one sulfonate radical (O)2S(O−)—, for example sodium sulfonate. As non-limiting examples of dyes of formula (XIII), mention may be made of Acid Yellow 2, Acid Yellow 3 and Acid Yellow 5. Without limitation, exemplary anionic hair dyeing agents may be chosen from (C.I. 45380) Acid Red 87 (formula XI); (C.I. 10316) Sodium salt of 2,4-dinitro-1-naphthol-7-sulfonic acid (formula IX); (C.I. 10383) Acid Orange 3 (formula VIII); (C.I. 13015) Acid Yellow 9/Food Yellow 2 (formula II); (C.I. 14780) Direct Red 45/Food Red 13 (formula II); (C.I. 13711) Acid Black 52 (formula II); (C.I. 13065) Acid Yellow 36 (formula II); (C.I. 14700) Sodium salt of 1-hydroxy-2-(2′,4′-xylyl-5-sulfonatoazo)naphthalene-4-sulfonic acid/Food Red 1 (formula II); (C.I. 14720) Acid Red 14/Food Red 3/Mordant Blue 79 (formula II); (C.I. 14805) Sodium salt of 4-hydroxy-3-[(2-methoxy-5-nitrophenyl)diaza]-6-(phenylamino)naphthalene-2-sulfonic acid/Acid Brown 4 (formula II); (C.I. 15510) Acid Orange 7/Pigment Orange 17/Solvent Orange 4 (formula II); (C.I. 15985) Food Yellow 3/Pigment Yellow 104 (formula II); (C.I. 16185) Acid Red 27/Food Red 9 (formula II); (C.I. 16230) Acid Orange 10/Food Orange 4 (formula II); (C.I. 16250) Acid Red 44 (formula II); (C.I. 17200) Acid Red 33/Food Red 12 (formula II); (C.I. 15685) Acid Red 184 (formula II); (C.I. 19125) Acid Violet 3 (formula II); (C.I. 18055) Sodium salt of 1-hydroxy-2-(4′-acetamidophenylazo)-8-acetamidonaphthalene-3,6-disulfonic acid/Acid Violet 7/Food Red 11 (formula II); (C.I. 18130) Acid Red 135 (formula II); (C.I. 19130) Acid Yellow 27 (formula IV); (C.I. 19140) Acid Yellow 23/Food Yellow 4 (formula IV); (C.I. 20170) 4′-(sulfonato-2″,4″-dimethyl)bis(2,6-phenylazo)-1,3-dihydroxybenzene/Acid Orange 24 (formula II); (C.I. 20470) Sodium salt of 1-amino-2-(4′-nitrophenylazo)-7-phenylazo-8-hydroxy-naphthalene-3,6-disulfonic acid/Acid Black 1 (formula II); (C.I. 23266) (4-((4-methylphenyl)sulfonyloxy)phenylazo)-2,2′-dimethyl-4-((2-hydroxy-5,8-disulfonato) naphthylazo)biphenyl/Acid Red 111 (formula III); (C.I. 27755) Food Black 2 (formula II) (C.I. 25440) 1-(4′-sulfonatophenylazo)-4-((2″-hydroxy-3″-acetylamino-6″,8″-disulfonato)naphthylazo)-6-sulfonatonaphthalene (tetrasodium salt)/Food Black 1 (formula II), (C.I. 42090) Acid Blue 9 (formula X) (C.I. 60730) Acid Violet 43 (formula VI) (C.I. 61570) Acid Green 25 (formula VI), (C.I. 62045) Sodium salt of 1-amino-4-cyclohexylamino-9,10-anthraquinone-2-sulfonic acid/Acid Blue 62 (formula VI), (C.I. 62105) Acid Blue 78 (formula VI), (C.I. 14710) Sodium salt of 4-hydroxy-3-((2-methoxyphenyl)azo)-1-naphthalenesulfonic acid/Acid Red 4 (formula II); 2-Piperidino-5-nitrobenzenesulfonic acid (formula IX); 2-(4′-N,N-(2″-Hydroxyethyl)amino-2′-nitro)anilineethanesulfonic acid (formula IX); 4-β-Hydroxyethylamino-3-nitrobenzenesulfonic acid (formula IX); (C.I. 42640) Acid Violet 49 (formula X); (C.I. 42080) Acid Blue 7 (formula X); (C.I. 58005) Sodium salt of 1,2-dihydroxy-3-sulfoanthraquinone/Mordant Red 3 (formula VI); (C.I. 62055) Sodium salt of 1-amino-9,10-dihydro-9,10-dioxo-4-(phenylamino) 2-anthracenesulfonic acid/Acid Blue 25 (formula VI); or (C.I. 14710) Sodium salt of 4-hydroxy-3-((2-methoxyphenyl)azo)-1-naphthalenesulfonic acid/Acid Red 4 (formula II). Exemplary and non-limiting cationic dyes include the hydrazono cationic dyes of formulas (Va) and (V′a), the azo cationic dyes (Via) and (VI′a) and the diazo cationic dyes (VIIa) below: Het+—C(Ra)═N—N(Rb)—Ar, An−(Va)Het+—N(Ra)—N═C(Rb)—Ar, An−(V′a)Het+—N═N—Ar, An−(VIa)Ar+—N═N—Ar″, An−(VI′a) andHet+—N═N—Ar′—N═N—Ar, An−(VIIa)in which:Het+ represents a cationic heteroaryl radical, preferably bearing an endocyclic cationic charge, such as imidazolium, indolium or pyridinium, optionally substituted preferentially with one or more (C1-C8) alkyl groups such as methyl;Ar+ representing an aryl radical, such as phenyl or naphthyl, bearing an exocyclic cationic charge, preferentially ammonium, particularly tri(C1-C8)alkylammonium such as trimethylammonium;Ar represents an aryl group, especially phenyl, which is optionally substituted, preferentially with one or more electron-donating groups such as i) optionally substituted (C1-C8)alkyl, ii) optionally substituted (C1-C8)alkoxy, iii) (di)(C1-C8)(alkyl)amino optionally substituted on the alkyl group(s) with a hydroxyl group, iv) aryl(C1-C8)alkylamino, v) optionally substituted N—(C1-C8)alkyl-N-aryl(C1-C8)alkylamino or alternatively Ar represents a julolidine group;Ar′ is an optionally substituted divalent (hetero)arylene group such as phenylene, particularly para-phenylene, or naphthalene, which are optionally substituted, preferentially with one or more groups (C1-C8)alkyl, hydroxyl or (C1-C8)alkoxy;Ar″ is an optionally substituted (hetero)aryl group such as phenyl or pyrazolyl, which are optionally substituted, preferentially with one or more groups (C1-C8)alkyl, hydroxyl, (di)(C1-C8)(alkyl)amino, (C1-C8)alkoxy or phenyl;Ra and Rb, which may be identical or different, represent a hydrogen atom or a group (C1-C8)alkyl, which is optionally substituted, preferentially with a hydroxyl group;or alternatively the substituent Ra with a substituent of Het+ and/or Rb with a substituent of Ar and/or Ra with Rb form, together with the atoms that bear them, a (hetero)cycloalkyl;particularly, Ra and Rb represent a hydrogen atom or a group (C1-C4)alkyl, which is optionally substituted with a hydroxyl group; andAn- represents an anionic counter-ion such as mesylate or halide. For example, useful cationic dyes may be chosen from Basic Blue 6, Basic Blue 7, Basic Blue 9, Basic Blue 26, Basic Blue 41, Basic Blue 99, Basic Brown 4, Basic Brown 16, Basic Brown 17, Natural Brown 7, Basic Green, Basic Orange 31, 1, Basic Red 2, Basic Red 12 Basic Red 22, Basic Red 76 Basic Red 51, Basic Violet 1, Basic Violet 2, Basic Violet 3, Basic Violet 10, Basic Violet 14, Basic Yellow 57 and Basic Yellow 87. Non-limiting examples of nonionic hydrophobic direct dyes may be chosen from HC Blue No. 2, HC Blue No. 4, HC Blue No. 5, HC Blue No. 6, HC Blue No. 7, HC Blue No. 8, HC Blue No. 9, HC Blue No. 10, HC Blue No. 11, HC Blue No. 12, HC Blue No. 13, HC Blue 15, HC Blue No. 17, HC Brown No. 1, HC Brown No. 2, HC Green No. 1, HC Orange No. 1, HC Orange No. 2, HC Orange No. 3, HC Orange No. 5, HC Red BN, HC Red No. 1, HC Red No. 3, HC Red No. 7, HC Red No. 8, HC Red No. 9, HC Red No. 10, HC Red No. 11, HC Red No. 13, HC Red No. 54, HC Red No. 14, HC Violet BS, HC Violet No. 1, HC Violet No. 2, HC Yellow No. 2, HC Yellow No. 4, HC Yellow No. 5, HC Yellow No. 6, HC Yellow No. 7, HC Yellow No. 8, HC Yellow No. 9, HC Yellow No. 10, HC Yellow No. 11, HC Yellow No. 12, HC Yellow No. 13, HC Yellow No. 14, HC Yellow No. 15. Natural hair dyeing agents may also be chosen. As used herein, the term “natural” hair dyeing agents include dyes derived from natural materials (plant, mineral or animal origin), for instance extracts, ground material and decoctions, which have a greater or smaller concentration of dyes. Without being limiting, exemplary natural hair dyeing agents may be chosen from orceins, curcumin, indole derivatives such as isatin or indole-2,3-dione, indigoids including indigo, phthalocyanines, and porphyrins optionally complexed to a metal, glycosyl or non-glycosyl iridoids, chromene dyes, anthraquinone and naphthoquinone dyes such as lawsone or henna, juglone, spinulosin, chromene or chroman dyes, such as neoflavanols and neoflavanones, flavanols, and anthocyanidols. Use may also be made of extracts containing these natural dyes, for example plant extracts or poultices containing said dyes. In some embodiments, the dye comprises, consists essentially of, or consists of one or more natural dyes, preferably hydrophobic natural dyes. For example, the dye may comprise, consist essentially of, or consist of curcumin, indigo, or a mixture thereof. In various exemplary embodiments, the microtubes may be loaded with an amount of hair dyeing agent ranging from about 0.01% to about 50% by weight, based on the weight of the microtube prior to loading, such as about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 9%, about 0.1% to about 8%, about 0.1% to about 7%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.5% to about 50%, about 0.5% to about 45%, about 0.5% to about 40%, about 0.5% to about 35%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, or about 1% to about 2% by weight, based on the weight of the microtube prior to loading, including all subranges thereof. For example, in certain embodiments, the microtubes may be loaded with an amount of hair dyeing agent ranging from about 30% to about 50% by weight, based on the weight of the microtube prior to loading, such as about 35% to about 50%, about 40% to about 50%, about 30% to about 45%, about 35% to about 45%, or about 40% to about 45% by weight, based on the weight of the microtube prior to loading, including all subranges thereof. In other exemplary embodiments, the microtubes may be loaded with an amount of hair dyeing agent ranging from about 0.01% to about 10% by weight, based on the weight of the microtube prior to loading, such as about 0.01% to about 7.5%, about 0.01% to about 5%, about 0.01% to about 3.5%, about 0.1% to about 10%, about 0.1% to about 7.5%, about 0.1% to about 5%, about 0.1% to about 3.5%, about 1% to about 10%, about 1% to about 7.5%, about 1% to about 5%, or about 1% to about 3.5% by weight, based on the weight of the microtube prior to loading, including all subranges thereof. The dyeing composition typically comprises a solvent, in which the microtube-dye composite may be dispersed. The solvent may be chosen from water, non-aqueous solvents, or a mixture thereof. The solvent will advantageously be chosen so that it will not interfere with deposition of the microtube-dye composite on the hair, and that it will not damage or irritate the hair, scalp, and/or skin. In various embodiments, the solvent comprises, consists essentially of, or consists of water. Exemplary non-aqueous solvents include, for example, glycerin, C1-4alcohols, organic solvents, fatty alcohols, fatty ethers, fatty esters, polyols, glycols, vegetable oils, mineral oils, liposomes, laminar lipid materials, and mixtures thereof. As examples of organic solvents, non-limiting mentions can be made of monoalcohols and polyols such as ethyl alcohol, isopropyl alcohol, propyl alcohol, benzyl alcohol, and phenylethyl alcohol, or glycols or glycol ethers such as, for example, monomethyl, monoethyl and monobutyl ethers of ethylene glycol, propylene glycol or ethers thereof such as, for example, monomethyl ether of propylene glycol, butylene glycol, hexylene glycol, dipropylene glycol as well as alkyl ethers of diethylene glycol, for example monoethyl ether or monobutyl ether of diethylene glycol. The organic solvents can be volatile or non-volatile compounds. Further non-limiting examples of solvents which may be used include alkanediols (polyhydric alcohols) such as glycerin, 1,2,6-hexanetriol, trimethylolpropane, ethylene glycol, propylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, dipropylene glycol, 2-butene-1,4-diol, 2-ethyl-1,3-hexanediol, 2-methyl-2,4-pentanediol, caprylyl glycol, 1,2-hexanediol, 1,2-pentanediol, and 4-methyl-1,2-pentanediol; alkyl alcohols having 1 to 4 carbon atoms such as ethanol, methanol, butanol, propanol, and isopropanol; glycol ethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monomethyl ether acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol mono-n-propyl ether, ethylene glycol mono-iso-propyl ether, diethylene glycol mono-iso-propyl ether, ethylene glycol mono-n-butyl ether, ethylene glycol mono-t-butyl ether, diethylene glycol mono-t-butyl ether, 1-methyl-1-methoxybutanol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol mono-t-butyl ether, propylene glycol mono-n-propyl ether, propylene glycol mono-iso-propyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, dipropylene glycol mono-n-propyl ether, and dipropylene glycol mono-iso-propyl ether; 2-pyrrolidone, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, formamide, acetamide, dimethyl sulfoxide, sorbit, sorbitan, acetine, diacetine, triacetine, sulfolane, and mixtures thereof. The solvent may be present in the dyeing composition in an amount ranging from about 50% to about 99.99% by weight, relative to the total weight of the dyeing composition. For example, the total amount of solvent may range from about 80% to about 99%, about 80% to about 98%, about 80% to about 97%, about 80% to about 96%, about 80% to about 95%, about 80% to about 94%, about 80% to about 93%, about 80% to about 92%, about 80% to about 91%, or about 80% to 90% by weight, relative to the total weight of the dyeing composition. The dyeing composition may comprise additional components, as long as such additional components do not substantially interfere with the deposition of the microtube-dye composite onto the hair. By way of example only, the dyeing composition may comprise pH adjusters, preservatives, humectants, oils, fragrances, etc. In various embodiments, the dyeing composition has a pH of less than or equal to about 7, such as less than or equal to about 6, less than or equal to about 5, less than or equal to about 4, or less than or equal to about 3. For example, the pre-treatment composition may have a pH ranging from about 1 to about 7, such as from about 2 to about 6, from about 2.5 to about 5, or from about 3 to about 4. The microtube-dye composite may be present in the dyeing composition in an amount ranging from about 0.01% to about 15% by weight, based on the weight of the dyeing composition, such as about 0.1% to about 10%, about 0.1% to about 9%, about 0.1% to about 8%, about 0.1% to about 7%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.5% to about 10%, about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, or about 1% to about 2% by weight, based on the weight of the dyeing composition, including all subranges thereof. It should be understood that the microtube-dye composite included in the dyeing composition can include mixtures of different microtubes, mixtures of different dyes, or both. By way of example only, a first set of microtubes comprising halloysite may be loaded with one dye, a second set of microtubes comprising halloysite may be loaded with a second dye, and the first and second sets of microtube-dye composites may be included in the dyeing composition. As a further example, a first set of microtubes comprising halloysite may be loaded with one dye, a second set of microtubes comprising a structure other than halloysite may be loaded with a second dye, and the first and second sets of microtube-dye composites may be included in the dyeing composition. Methods It has been discovered that altering the color of the hair using systems according to the disclosure has the surprising and unexpected benefit of imparting improved color deposition and vibrancy. In particular, while it was previously discovered that microtube-dye composites can be used to impart color to hair with less damage to the hair and/or less skin and/or scalp irritation, systems and methods according to the disclosure provide surprisingly significant improvement of deposition of color and vibrancy compared to use of the microtube-dye composite alone. Methods according to the disclosure include treating the hair with the pre-treatment composition and treating the hair with the dyeing composition. In various methods according to the disclosure, the hair is first treated with the pre-treatment composition by applying the pre-treatment composition to the hair and optionally massaging or combing the composition throughout the hair to ensure complete coverage. The pre-treatment composition may be applied to the hair in any desired amount, for example up to about 10 grams of pre-treatment composition per gram of hair, such as up to about 5 grams per gram of hair, up to about 4 grams per gram of hair, up to about 3 grams per gram of hair, up to about 2 grams per gram of hair, or up to about 1 gram per gram of hair. The pre-treatment composition may optionally be left on the hair for a period of time, for example ranging up to about 60 minutes or up to about 45 minutes, such as about 30 seconds to about 60 minutes, about 1 minute to about 50 minutes, about 3 minutes to about 40 minutes, or about 5 minutes to about 30 minutes. The pre-treatment composition may optionally be partially, completely, or substantially completely removed from the hair after the leave-on period, and the hair may optionally be subsequently partially, completely, or substantially completely dried, e.g. with a blow dryer or hood or air dried. Alternatively, the hair may be partially, completely, or substantially completely dried, e.g. with a blow dryer or hood or air dried, without removing the pre-treatment composition from the hair, for example without rinsing the hair or without towel-drying the hair. Preferably, the hair is completely or substantially completely dried after a leave-in period without removing the pre-treatment composition from the hair, wherein the step of drying optionally includes the use of heat. The methods further include a step of applying the dyeing composition to the hair after the pre-treatment composition is applied to the hair and optionally dried, for example by massaging or combing the composition throughout the hair to ensure complete coverage. The dyeing composition, which includes a microtube-dye composite, e.g. a halloysite-dye composite, may be applied to the hair in any desired amount, for example up to about 10 grams of dyeing composition per gram of hair, such as up to about 5 grams per gram of hair, up to about 4 grams per gram of hair, up to about 3 grams per gram of hair, up to about 2 grams per gram of hair, or up to about 1 gram per gram of hair. The dyeing composition may be left on the hair for a period of time to achieve a desired coloration effect, for example ranging up to about 60 minutes or up to about 45 minutes, such as about 30 seconds to about 60 minutes, about 1 minute to about 50 minutes, about 3 minutes to about 40 minutes, or about 5 minutes to about 30 minutes. One skilled in the art will be able to determine an appropriate amount of time to leave the dyeing composition on the hair in order to achieve the desired effect. After a desired leave-in period, the dyeing composition may be rinsed from the hair, and the hair may optionally be washed, rinsed, dried, and/or styled in any conventional manner. In various embodiments, the above-described steps of applying a pre-treatment composition to the hair, optionally leaving the pre-treatment composition on the hair for a leave-in period, optionally removing the pre-treatment composition from the hair, applying a dyeing composition to the hair, optionally leaving the dyeing composition on the hair for a leave-in period, and optionally removing the dyeing composition from the hair may be repeated one or more times, with the same or different pre-treatment and/or dyeing composition(s). Thus, by way of example only, one method according to the disclosure may comprise applying a pre-treatment composition (1a) to the hair, leaving the pre-treatment composition (1a) on the hair for a leave-in period, removing the pre-treatment composition (1a) from the hair, applying a dyeing composition (1b) to the hair, leaving the dyeing composition (1b) on the hair for a leave-in period, and removing the dyeing composition (1b) from the hair. Another exemplary method according to the disclosure may comprise applying a pre-treatment composition (1a) to the hair, leaving the pre-treatment composition (1a) on the hair for a leave-in period, removing the pre-treatment composition (1a) from the hair, applying a dyeing composition (1b) to the hair, leaving the dyeing composition (1b) on the hair for a leave-in period, removing the dyeing composition (1b) from the hair, applying the pre-treatment composition (1a) to the hair a second time, leaving the pre-treatment composition (1a) on the hair for a leave-in period, removing the pre-treatment composition (1a) from the hair, applying the dyeing composition (1b) to the hair a second time, leaving the dyeing composition (1b) on the hair for a leave-in period, and removing the dyeing composition (1b) from the hair, where pre-treatment composition (1a) in the first application is identical to pre-treatment composition (1a) in the second application, and dyeing composition (1b) in the first application is identical to dyeing composition (1b) in the second application. Yet another exemplary method according to the disclosure may comprise applying a pre-treatment composition (1a) to the hair, leaving the pre-treatment composition (1a) on the hair for a leave-in period, removing the pre-treatment composition (1a) from the hair, applying a dyeing composition (1b) to the hair, leaving the dyeing composition (1b) on the hair for a leave-in period, removing the dyeing composition (1b) from the hair, applying a pre-treatment composition (2a) to the hair, leaving the pre-treatment composition (2a) on the hair for a leave-in period, removing the pre-treatment composition (2a) from the hair, applying a dyeing composition (2b) to the hair, leaving the dyeing composition (2b) on the hair for a leave-in period, and removing the dyeing composition (2b) from the hair, where pre-treatment compositions (1a) and (2a) are not identical, and hair dyeing compositions (1b) and (2b) are not identical. A further exemplary and non-limiting method according to the disclosure may comprise applying a pre-treatment composition (1a) to the hair, optionally drying the hair, applying a dyeing composition (1b) to the hair without first removing the pre-treatment composition (1a), optionally leaving the dyeing composition (1b) on the hair for a leave-in period, and removing the pre-treatment composition (1) and dyeing composition (1b) from the hair. Kits In a further embodiment, the disclosure relates to kits comprising the systems described herein. According to various embodiments, the kits may be multi-compartment or multi-container kits, where the compartments or containers are mutually separate. For example, the kits may comprise at least two compartments or containers, with a first compartment or container containing a pre-treatment composition according to the disclosure and a second compartment or container containing a dyeing composition according to the disclosure. In further embodiments, the kits may comprise at least three, at least four, or more compartments or containers. The compartments or containers of kits according to the disclosure can be in any configuration, without limitation. For example, they can be a bottle, a tube, a sachet, an ampoule, or any other container configured to contain the pre-treatment composition(s) and dyeing composition(s) mutually separately in the kit. Kits may optionally include additional compartments for additional components, such as, for example, additional pre-treatment compositions, additional dyeing compositions, shampoo compositions, and the like. Various exemplary embodiments of kits according to the disclosure comprise:a first compartment or container containing a pre-treatment composition comprising at least one amine-based compound and optionally at least one solvent; anda second compartment or container containing a dyeing composition comprising at least one microtube-dye composite and optionally at least one solvent, wherein, in the microtube-dye composite, the dye comprises at least one hair dyeing agent. In yet further exemplary embodiments, kits according to the disclosure comprise:a first compartment or container containing a pre-treatment composition comprising from about 0.01% to about 15% of at least one non-surface active amine-based compound and water,wherein the pre-treatment composition has a pH of less than or equal to about 7, such as from about 2 to about 6; anda second compartment or container containing a dyeing composition comprising from about 0.01% to about 15% of at least one halloysite-dye composite comprising at least one hair dyeing agent and water,wherein the dyeing composition has a pH of less than or equal to about 7, such as from about 2 to about 6. In various embodiments, kits such as those described above may optionally comprise additional compartments or containers, for example a third compartment or container containing a pre-treatment composition according to the disclosure different from that in the first compartment or container, and/or a fourth compartment or container containing a dyeing composition according to the disclosure different from that in the second compartment or container. It is to be understood that, in exemplary kits according to the disclosure, the pre-treatment composition(s) and dyeing composition(s) can be as described herein for various systems, methods, and examples, for example with regard to particular components and/or ranges thereof. Thus, in some embodiments, the pre-treatment and/or dyeing composition may not be present in the kit in a solvent, or may be present in the kit in a solvent but in concentrated form. For example, the pre-treatment and/or dyeing composition may be present in the kit in solid or powder form, and the user may mix the solid or powder with a solvent, such as water, prior to use. Alternatively, the pre-treatment and/or dyeing composition may be present in the form of a gel or thickened liquid that is to be mixed with a solvent, such as water, prior to use. In such embodiments, a kit with more than two compartments or containers may be envisioned. For example, a kit with three (and/or four) compartments or containers, where a third (and/or fourth) compartment or container includes a solvent, e.g. to mix with the pre-treatment composition and/or dyeing composition, may be chosen. Kits may also include additional components or compartments, such as, for example, instructions or an apparatus or tool for applying the pre-treatment and/or dyeing compositions onto the hair, e.g. an applicator brush, and/or a compartment for the same. As used herein, the terms “comprising,” “having,” and “including” (or “comprise,” “have,” and “include”) are used in their open, non-limiting sense. The terms “a,” “an,” and “the” are understood to encompass the plural as well as the singular. The term “and/or” should be understood to include both the conjunctive and the disjunctive. For example, “A and/or B” means “A and B” as well as “A or B,” and expressly covers instances of either without reference to the other. For example, “preventing and/or reducing” corrosion includes instances of preventing corrosion and reducing corrosion, as well as instances where corrosion is reduced but not prevented, etc. As used herein, the phrases “and mixtures thereof,” “and a mixture thereof,” “and combinations thereof,” “and a combination thereof,” “or mixtures thereof,” “or a mixture thereof,” “or combinations thereof,” and “or a combination thereof,” are used interchangeably to denote that the listing of components immediately preceding the phrase, such as “A, B, C, D, or mixtures thereof” signify that the component(s) may be chosen from A, from B, from C, from D, from A+B, from A+B+C, from A+D, from A+C+D, etc., without limitation on the variations thereof. Thus, the components may be used individually or in any combination thereof. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and/or reaction conditions are to be understood as being modified in all instances by the term “about,” meaning within +/−5% of the indicated number. All percentages, parts and ratios herein are based upon the total weight of the compositions of the present invention, unless otherwise indicated. As used herein, all ranges provided are meant to include every specific range within, and combination of sub ranges between, the given ranges. Thus, a range from 1-5, includes specifically 1, 2, 3, 4, and 5, as well as sub ranges such as 2-5, 3-5, 2-3, 2-4, 1-4, etc. All ranges and values disclosed herein are inclusive and combinable. For examples, any value or point described herein that falls within a range described herein can serve as a minimum or maximum value to derive a sub-range, etc. As used herein, “free” means that the component or property is not detectable using accepted methodologies, and “substantially” or “essentially” free means that the component or property, while detectable using accepted methodologies, is negligible. It is to be understood that the use of the terms “treat,” “treated,” “treatment,” and variations thereof is not intended to be limiting, but rather is merely intended to indicate that one or more compositions is applied to the hair, and optionally removed from the hair, as described herein. For example, hair that is “treated” with a pre-treatment composition according to the disclosure may have had the pre-treatment composition applied, and/or may have had the pre-treatment composition applied and removed, e.g. by rinsing or towel drying. As a further example, hair that is “treated” with a dyeing composition according to the disclosure may have had the dyeing composition applied, and/or may have had the dyeing composition applied and rinsed from the hair. As yet a further example, hair that is “treated” with a system according to the disclosure may have had the pre-treatment composition applied and optionally removed, and additionally may have had the dyeing composition applied and optionally rinsed from the hair. By “non-surface active amine” it is meant that the amine compounds are not amine-based surfactants in the compositions in which they are present. By way of non-limiting example only, in some embodiments, the “non-surface active amines” are not capable of depressing the surface tension of deionized water under standard conditions to a value of less than about 50 mN/m, when added to deionized water in a concentration by weight of 0.5-1%. All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls. The examples that follow serve to illustrate embodiments of the present disclosure without, however, being limiting in nature. It will be apparent to those skilled in the art that various modifications and variations can be made in the delivery system, compositions, and methods of the invention without departing from the spirit or scope of the invention. EXAMPLES Implementation of various non-limiting embodiments of the disclosure is demonstrated by way of the following Examples. In the Examples, the change in the color of hair is evaluated with the L*a*b* system, using Colorshot MS, where the change is determined by evaluating the color of the hair after treatment compared to the color of the hair before treatment. The change in color (ΔE) is defined as: The greater the value for ΔE, the greater the difference in color of treated hair relative to the color of the hair prior to treatment. Example 1—Microtube-Dye Composites The procedures of Examples 1A and 1B below were followed to prepare microtube-dye composites having the dye loadings shown in Table 1. TABLE 1CompositeDye loadingH-C(1.4)Halloysite-Curcumin1.4%H-C(3)Halloysite-Curcumin3%H-I(43)Halloysite-Indigo43% Example 1A—Halloysite-Curcumin Composites Curcumin was completely dissolved in acetone (10 mg dye per mL acetone) and unmodified halloysite was added (weight ratio of dye:halloysite ˜2:1) with stirring. The dispersion was sonicated for about five minutes and then vacuumed and stirred overnight. After centrifuging for about five minutes at 5000 rpm, the supernatant was discarded and the sample was dried under vacuum and crushed into a fine powder. Example 1B—Halloysite-Indigo Composites Indigo was completely dissolved in water (99.99% water with 0.01% sodium carbonate; 5 mg dye per mL solvent) and unmodified halloysite was added (weight ratio of dye:halloysite ˜2:1) with stirring. The dispersion was sonicated for about five minutes and then vacuumed and stirred overnight. After centrifuging for about five minutes at 5000 rpm, the supernatant was discarded and the sample was dried under vacuum and crushed into a fine powder. Example 2—Pre-treatment Compositions The pre-treatment compositions in Table 2 were prepared by dissolving the amine-based compound in water and adjusting the pH of the solution with sodium hydroxide and/or hydrochloric acid, as needed, to prepare aqueous compositions having the reported concentrations of amine-based compounds and pH values. TABLE 2Pre-treatmentMolecularcompositionAmine compoundweightpHConcentration2Apolyethyleneimine80033.33%2Bpolyethyleneimine130030.5%2Cpolyethyleneimine130033.33%2Dpolyethyleneimine1300310%2Epolyethyleneimine130073.33%2Fpolyethyleneimine1300113.33%2Gpolyethyleneimine200033.33%2Hpolyethyleneimine25,00033.33%2Iarginine174.233% Example 3—Dyeing Compositions The dyeing compositions in Table 3 were prepared by dispersing the specified halloysite-dye composites from Table 1 in water and adjusting the pH of the dispersion with sodium hydroxide and/or hydrochloric acid, as needed, to prepare aqueous compositions having the reported concentrations of halloysite-dye composites and pH values. TABLE 3DyeingHalloysite-dyecompositioncompositepHConcentration3AH-C(1.4)32.5%3BH-C(1.4)72.5%3CH-C(1.4)75%3DH-C(3)72.5%3EH-C(3)35%3FH-C(3)75%3GH-I(43)32.5%3HH-I(43)35%3IH-I(43)75% Example 4—Demonstration of Benefit of Pre-Treatment The following Examples 4-1 and 4-2 demonstrate the surprising improvement in color deposition on the hair using systems and methods according to the disclosure including pre-treatment with non-surface active amine-based compounds. Example 4-1—Color Imparted by Systems Comprising Pre-Treatment Compositions and Dyeing Compositions (Inventive) The color imparted to hair with systems 4A-4J comprising combinations of pre-treatment compositions prepared in Example 2 and dyeing compositions prepared in Example 3 as set forth in Table 4-1 was evaluated. TABLE 4-1Pre-treatmentDyeingSystemcompositioncompositionΔE4A2C3F27.24B2C3A15.94C2E3B13.24D2C3H43.14E2C3I39.74F2F3I39.64G2I3D21.24H2A3D19.34I2B3D20.44J2D3D17.3 The process for treating hair with inventive systems 4A-4J was as follows. First, swatches of 90% grey virgin hair were rinsed with ˜37° C. tap water (five passes), a commercial shampoo was applied and lathered for about 30 seconds, the swatch was allowed to rest for about one minute and then rinsed with tap water for about 30 seconds. The swatch was then allowed to air dry. Once the swatch was completely dry, the pre-treatment composition was applied to the swatch at a ratio of about 2 grams of pre-treatment composition per 1 gram of hair and massaged through the hair for about one minute. The swatch was then combed with a wide-tooth comb for five passes and allowed to rest at room temperature for approximately 30 minutes. The swatch was then dried with a hair dryer set on high. Once the swatch was completely dry, the dyeing composition was applied to the hair at a ratio of about 2 grams of dyeing composition per 1 gram of hair and massaged through the hair for about one minute. After a leave-in period of approximately 30 minutes at room temperature, the hair was rinsed with ˜37° C. tap water. The hair was then dried with a hair dryer set on high. The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for each of systems 4A-4J is shown in Table 4-1 andFIGS.1A-1D. Example 4-2—Color Imparted by Dyeing Compositions Without Pre-Treatment (Comparative) The color imparted to hair with dyeing compositions prepared in Example 3 and set forth in Table 4-2, without a pre-treatment step, was evaluated in order to determine the difference in color deposited on hair without a pre-treatment composition compared to color obtained with systems and methods including a pre-treatment composition. TABLE 4-2Pre-treatmentDyeingCompositioncompositioncompositionΔE4A′—3F18.84B′—3A12.34C′—3B9.84D′—3H40.44E′—3I31.74F′—3I31.74G′—3D12.2 The process for treating hair with comparative compositions 4A′-4G′ was as follows. First, swatches of 90% grey virgin hair were rinsed with ˜37° C. tap water (five passes), a commercial shampoo was applied and lathered for about 30 seconds, the swatch was allowed to rest for about one minute and then rinsed with tap water for about 30 seconds. The swatch was then allowed to air dry. Once the swatch was completely dry, the dyeing composition was applied to the hair at a ratio of about 2 grams of dyeing composition per 1 gram of hair and massaged through the hair for about one minute. After a leave-in period of approximately 30 minutes at room temperature, the hair was rinsed with ˜37° C. tap water. The hair was then dried with a hair dryer set on high. The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for each of compositions 4A′-4G′ is shown in Table 4-2 andFIGS.1A-1D. As seen inFIGS.1A-1D, the hair treated with inventive systems 4A-4J shows significantly greater change in color relative to the color of the hair prior to treatment, when compared to the change in color of hair treated with comparative compositions 4A′-4G′ not using a pre-treatment composition according to the disclosure. Example 4 thus demonstrates that hair colored according to the disclosure, i.e. treated with a pre-treatment composition according to the disclosure and subsequently dyed with a dyeing composition according to the disclosure, surprisingly and unexpectedly leads to enhanced color deposition and more vibrant hair colors compared to hair not pre-treated as described herein. Example 5—Demonstration of Benefit of Acidic pH The following Examples 5-1 and 5-2 demonstrate the surprising improvement in color deposition on the hair using systems and methods where the pre-treatment and/or dyeing composition(s) is(are) acidic. The process for treating hair with systems 5A-5F was the same as described in Example 4-1. Example 5-1—Color Imparted by Systems Comprising Acidic Pre-Treatment Compositions The color imparted to hair with systems 5A-5D comprising combinations of pre-treatment compositions prepared in Example 2 and dyeing compositions prepared in Example 3 as set forth in Table 5-1 was evaluated. TABLE 5-1Pre-treatmentpH pre-DyeingpHSystemcompositiontreatmentcompositiondyeingΔE5A2E73B713.25B2C33B714.95C2F113G340.45D2C33G343.1 The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with each of systems 5A-5D is shown in Table 5-1 andFIGS.2A-2B. As can be seen, the ΔE for hair treated with systems 5B and 5D (pre-treatment compositions having pH lower than 7) is greater than the ΔE for hair treated with systems 5A and 5C (identical systems but with corresponding pre-treatment compositions having pH of 7 or above). Example 5-1 thus demonstrates that systems according to the disclosure comprising pre-treatment compositions having lower pH surprisingly provide greater change in color compared to systems comprising pre-treatment compositions having higher pH. Example 5-2—Color Imparted by Systems Comprising Acidic Dyeing Compositions The color imparted to hair by systems 5E-5F comprising combinations of pre-treatment composition 2C of Example 2 and dyeing compositions 31 or 3H Example 3 as set forth in Table 5-2 was evaluated. TABLE 5-2Pre-treatmentpH pre-DyeingpHSystemcompositiontreatmentcompositiondyeingΔE5E2C33I739.75F2C33H343.1 The color change for both swatches was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with systems 5E-5F is shown in Table 5-2 andFIG.2C. As can be seen, the ΔE for hair treated with system 5F (dyeing composition having a pH lower than 7) is greater than the ΔE for hair treated with system 5E (identical system but with a dyeing composition having a pH of 7). Example 5-2 shows that systems according to the disclosure having dyeing compositions with a lower pH surprisingly provide greater change in color compared to systems comprising dyeing compositions having higher pH. Example 5 therefore demonstrates that hair colored according to the disclosure, treated with a pre-treatment composition having an acidic pH and subsequently dyed with a dyeing composition having an acidic pH, surprisingly and unexpectedly leads to enhanced color deposition and more vibrant hair color. Example 6—Demonstration of Benefit of Molecular Weight of Amine Compound The following Examples 6-1 and 6-2 demonstrate the surprising improvement in color deposition on the hair using systems and methods according to the disclosure. The process for treating hair with systems 6A-6C and C1 was the same as described in Example 4-1, and the process for treating hair with comparative compositions C2-C3 was the same as described in Example 4-2. Example 6-1— Comparison of Color Imparted by Systems Comprising Pre-Treatment Compositions Having Polyethyleneimine of Different Molecular Weights The color imparted to hair with systems according to the disclosure comprising a combination of pre-treatment composition 2C of Example 2 and dyeing composition 3B of Example 3, and with comparative system C1 comprising a combination of pre-treatment composition 2H of Example 2 and dyeing composition 3B of Example 3, as set forth in Table 6-1, was evaluated. The color imparted to hair by comparative composition C2 was also evaluated, without a pre-treatment step. TABLE 6-1Pre-MolecularDyeingtreatmentweight ofpH pre-compo-pHSystemcompositionaminetreatmentsitiondyeingΔE6A2C130033B714.9C12H25,00033B77C2———3B79.8 The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with systems 6A and C1, and composition C2, is shown in Table 6-1 andFIG.3A. As can be seen, the ΔE for hair treated with system C1 (pre-treatment composition with polyethyleneimine having a molecular weight of 25,000) is lower than that of either hair treated with system 6A (pre-treatment composition with polyethyleneimine having a molecular weight of 1300) or with dyeing composition C2 with no pre-treatment. Example 6-1 thus demonstrates that systems according to the disclosure comprising pre-treatment compositions having non-surface active amine-based compounds amine-based compounds with lower molecular weight surprisingly provide greater change in color compared to systems comprising pre-treatment compositions having amine-based compounds with higher molecular weight. Example 6-2—Comparison of Color Imparted by Systems Comprising Pre-Treatment Compositions Having Polyethyleneimine of Different Molecular Weights The color imparted to hair with systems 6B and 6C comprising combinations of pre-treatment compositions 2G or 2A of Example 2 and dyeing composition 3D of Example 3 as set forth in Table 6-2 was evaluated. The color imparted to hair by comparative composition C3 was also evaluated, without a pre-treatment step. TABLE 6-2Pre-MolecularDyeingtreatmentweight ofpH pre-compo-pHSystemcompositionaminetreatmentsitiondyeingΔE6B2G200033D715.56C2A80033D719.3C3———3D712.2 The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with each of systems 6B-6C, and composition C3, is shown in Table 6-2 andFIG.3B. As can be seen, the ΔE hair treated with system 6B (pre-treatment composition with polyethyleneimine having a molecular weight of 2000) is lower than that of hair treated with system 6C (pre-treatment composition with polyethyleneimine having a molecular weight of 800), but is higher that hair treated with dyeing composition C3 with no pre-treatment. Example 6-2 shows that systems according to the disclosure comprising pre-treatment compositions having non-surface active amine-based compounds with lower molecular weight surprisingly provide greater change in color compared to systems comprising pre-treatment compositions having amine-based compounds with higher molecular weight. As can be seen inFIG.3C, which shows the percent change in ΔE of hair treated with systems comprising pre-treatment compositions comprising non-surface active amine-based compounds followed by dyeing compositions according to the disclosure, as molecular weight of the non-surface active amine-based compound decreases, the ΔE surprisingly increases. Example 6 therefore demonstrates the surprising and unexpected improvement in color deposition on hair using systems having a pre-treatment step according to the disclosure. Example 7—Demonstration of Benefit of Amine-Based Pre-Treatment Compounds The following Examples 7-1 and 7-2 demonstrate the surprising improvement in color deposition on the hair using systems and methods including a pre-treatment composition comprising amine-based compounds. The process for treating hair with systems 7A-7B and C4-05 was the same as described in Example 4-1, and the process for treating hair with compositions C6-C7 was the same as described in Example 4-2. Example 7-1—Comparison of Color Imparted by Systems Comprising Pre-Treatment Compositions Having Amine-Based Compounds with Color Imparted by Pre-Treatment Compositions not Having Amine-Based Compounds The color imparted to hair by system 7A according to the disclosure comprising a combination of pre-treatment composition 2C of Example 2 and dyeing composition 3F of Example 3 was compared with the color imparted to hair by comparative system C4 comprising a combination of pre-treatment composition AA comprising acetic acid as the pre-treatment agent and dyeing composition 3F of Example 3, as set forth in Table 7-1. The color imparted to hair by comparative composition C6 was also evaluated, without a pre-treatment step. TABLE 7-1Pre-MolecularDyeingtreatmentweight ofpH pre-compo-pHSystemcompositionaminetreatmentsitiondyeingΔE7A2C130033F727.2C4AA*60.0533F718.2C6———3F718.8*AA is a 1% aqueous solution of acetic acid (molecular weight 60.05) having a pH of 3 The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with each of systems 7A and C4, and composition C6, is shown in Table 7-1 andFIG.4A. As can be seen, the ΔE for hair treated with inventive system 7A (pre-treatment composition with amine-based compound having a molecular weight of 1300) is greater than that of either hair treated with comparative system C4 (pre-treatment composition with non-amine-based compound having a molecular weight of 60.05) or with dyeing composition C6 with no pre-treatment. Notably, the ΔE for hair treated with acetic acid as the pre-treatment agent was lower than hair treated with composition C6 with no pre-treatment. Example 7-1 thus demonstrates that systems according to the disclosure comprising pre-treatment compositions having amine-based compounds surprisingly provide greater change in color compared to systems comprising pre-treatment compositions having non-amine-based compounds, even at the same pH. Example 7-2—Comparison of Color Imparted by Systems Comprising Pre-Treatment Compositions Having Amine-Based Compounds with Color Imparted by Pre-Treatment Compositions not Having Amine-Based Compounds The color imparted to hair by system 7B according to the disclosure comprising a combination of pre-treatment composition 2C of Example 2 and dyeing composition 3C of Example 3 was compared with color imparted to hair by comparative system C5 comprising a combination of pre-treatment composition TA comprising tannic acid as the pre-treatment agent and dyeing composition 3C of Example 3, as set forth in Table 7-2. The color imparted to hair by comparative composition C7 was also evaluated, without a pre-treatment step. TABLE 7-2Pre-MolecularDyeingtreatmentweight ofpH pre-compo-pHSystemcompositionaminetreatmentsitiondyeingΔE7B2C130033C716.4C5TA*1701.233C75.4C7———3C76.8*TA is a 3% aqueous solution of tannic acid (molecular weight 1701.2) having a pH of 3 The color change for each swatch was evaluated by determining the ΔE of the color of the hair after treatment compared to the color of the hair before treatment. The ΔE for hair treated with each of systems 7B and C5, and composition C7, is shown in Table 7-2 andFIG.4B. As can be seen, the ΔE for hair treated with inventive system 7B (pre-treatment composition with amine-based compound having a molecular weight of 1300) is greater than that of either hair treated with comparative system C5 (pre-treatment composition with non-amine-based compound having a molecular weight of 1701.2) or with dyeing composition C7 with no pre-treatment. Notably, the ΔE for hair treated with tannic acid as the pre-treatment agent was lower than hair treated with composition C7 with no pre-treatment. Example 7-2 shows that systems according to the disclosure comprising pre-treatment compositions having amine-based compounds surprisingly provide greater change in color compared to systems comprising pre-treatment compositions having non-amine-based compounds, even when the pre-treatment agents are of similar molecular weight and the pre-treatment compositions have the same pH. Example 7 therefore demonstrates the surprising and unexpected improvement in color deposition on hair using systems having a pre-treatment step with a non-surface active amine-based compound according to the disclosure. The above examples demonstrate that the systems, methods, and kits according to the disclosure surprisingly and unexpectedly provide improved color enhancement to hair relative to those not according to the disclosure. | 87,734 |
11857657 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates generally to the prevention and treatment of diseases and conditions mediated by binuclear enzymes. Specifically, the present invention includes a method for inhibiting the activity of an enzyme having a binuclear active site. Included in the present invention are novel compositions comprised of one or more diarylalkane(s). The diarylalkanes of the present invention can be isolated from one or more plant sources or can be obtained by organic synthesis. Further included in the present invention are methods for isolating these compounds from a natural source and methods for synthesizing these compounds. In one embodiment, the diarylalkanes are obtained by synthetic modification of a naturally occurring compound isolated from a plant source. Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided. Unless defined otherwise all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be noted that as used herein the term “a” or “an” entity refers to one or more of that entity; for example, a diarylalkane refers to one or more diarylalkanes. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. “Diarylalkanes” as used herein are a specific class of aromatic compounds having the following general structure: The present invention also includes a novel composition of matter comprised of one or more diarylalkanes, wherein said diarylalkanes are selected from the group of compounds represented by the following general structure: whereinAr1and Ar2are independently selected from the group consisting of a substituted 5- or 6-membered aromatic or heteroaromatic ring, wherein each 6-membered aromatic or heteroaromatic ring is independently substituted with 1-5 R′ groups (R′1—R′5), and each 5-membered aromatic or heteroaromatic ring is substituted with 1-4 R′ groups (R′1—R′4), except when Ar1and Ar2are both a 6-membered aromatic ring, i.e. a phenyl group at least one of Ar1and Ar2are substituted with 1-5 R′ groups (R′1—R′5), wherein at least 2 of said of R′1—R′5are not H whereinR′ is independently selected from the group consisting of —H, —OH, —SH, —OR, —CN, —SR, —NH2, —NHR, —NR2and X, and a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide(s) are independently selected from the group consisting of an aldopentose, methyl-aldopentose, aldohexose, ketohexose and chemical derivatives thereof; wherein R is an alkyl group having between 1-20 carbon atoms and X is a halogen, selected from the group consisting of Cl, Br, F and I;R6, and R7are independently selected from the group consisting of —H, —OH, —OR, —CN, —NHR, —NH2, and —X, wherein R is an alkyl group having between 1-20 carbon atoms and wherein X is a halogen, selected from the group consisting of Cl, Br, F, I; and n=1 to 10. In a preferred embodiment n=2-4. In one embodiment, said diarylalkanes and diarylalkanols are selected from the group of compounds represented by the following general structure: whereinR1, R2, R3, R4, R5R′1, R′2, R′3, R′4, and R′5are independently selected from the group consisting of —H, —OH, —SH, —OR, —CN, —SR, —NH2, —NHR, —NR2, X, and a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide(s) are independently selected from the group consisting of an aldopentose, methyl-aldopentose, aldohexose, ketohexose and chemical derivatives thereof; wherein R is an alkyl group having between 1-20 carbon atoms and X is a halogen, selected from the group consisting of Cl, Br, F, I, and wherein at least 2 of R1-R5or at least 2 of R′1—R′5are not H;R6, and R7are independently selected from the group consisting of —H, —OH, —OR, —CN, —NHR, —NH2, and —X, wherein R is an alkyl group having between 1-20 carbon atoms and wherein X is a halogen, selected from the group consisting of Cl, Br, F and I; andn=1 to 10. In a preferred embodiment n=2-4. “Diarylalkanols” as used herein are a specific type of “diarylalkanes” having at least one hydroxyl group (R6and/or R7=−OH) attached to the alkyl carbons between the two aromatic rings. “Binuclear enzyme” as used herein refers to an enzyme which has a binuclear active site, an example of which is tyrosinase which has two copper ions at its active site as discussed above. Binuclear enzymes include, but are not limited to tyrosinase, arginase, urease, cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctional carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotide reductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase, and bacterial phosphotriesterase. “Therapeutic” as used herein, includes prevention, treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals. “Pharmaceutically or therapeutically effective dose or amount” refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired. The precise dosage will vary according to a variety of factors, including but not limited to the age and size of the subject, the disease and the treatment being effected. “Placebo” refers to the substitution of the pharmaceutically or therapeutically effective dose or amount dose sufficient to induce a desired biological that may alleviate the signs, symptoms or causes of a disease with a non-active substance. A “host” or “patient” or “subject” is a living mammal, human or animal, for whom therapy is desired. The “host,” “patient” or “subject” generally refers to the recipient of the therapy to be practiced according to the method of the invention. It should be noted that the invention described herein may be used for veterinary as well as human applications and that the term “host” should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges can be determined as described below, taking into account the body weight of the animal. As used herein a “pharmaceutically acceptable carrier” refers to any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and which is not toxic to the host to which it is administered. Examples of “pharmaceutically acceptable carriers” include, but are not limited to, any of the standard pharmaceutical carriers such as a saline solution, i.e. Ringer's solution, a buffered saline solution, water, a dextrose solution, serum albumin, and other excipients and preservatives for tableting and capsulating formulations. The present invention includes a method for inhibiting the activity of an enzyme with a binuclear active site, referred to herein as a binuclear enzyme, said method comprising administering to a host in need thereof an effective amount of one or more diarylalkane(s), wherein said diarylalkanes are synthesized and/or isolated from a one or more plants. Examples of binuclear enzymes included within the scope of the present invention include, but are not limited to tyrosinase, arginase, urease, cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctional carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotide reductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase, and bacterial phosphotriesterase. In one embodiment, the binuclear enzyme is tyrosinase. The present invention also includes a method for the prevention and treatment of diseases and conditions related to the activity of binuclear enzymes. The method of prevention and treatment according to this invention comprises administering internally or topically to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s). Depending on the binuclear enzyme being inhibited the diarylalkane may be used as an anti-microbial, anti-fungal, anti-malaria, or anti-viral agent, a regulator for the production of nitric oxide as a means of controlling male and female sexual arousal, an anti-inflammatory drug, an antioxidant, a regulator of drug metabolism, for treatment and prevention of periodontal diseases, oral pre-cancerous conditions, oral cancers, and other oral malignancies, sensitive gums and teeth, sequelae, pulpitis, irritation, pain and inflammation caused by the physical implantation of oral dentures, trauma, injuries, bruxism and other minor wounds in mouth, on the gums or on the tongue, dental plague and calculus, tooth decalcification, proteolysis and caries (decay). and an inhibitor of the growth of cancers and solid tumors. The present invention further includes methods for the prevention and treatment of diseases and conditions related to the overproduction or uneven distribution of melanin, said method comprising administering internally or topically to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s). Diseases and conditions related to the overproduction or uneven distribution of melanin include, but not limited to suntan, hyper pigmentation spots caused by skin aging, liver diseases, thermal burns and topical wounds, skin pigmentation due to inflammatory conditions caused by fungal, microbial and viral infections, vitilago, carcinoma, melanoma, as well as other mammalian skin conditions. The method can also be used for preventing and treating skin darkening and damage resulting from exposure to ultraviolet (UV) radiation, chemicals, heat, wind and dry environments. Finally, the method can be used for preventing and treating wrinkles, saggy skin, lines and dark circles around the eyes, soothing sensitive skin and preventing and treating dermatitis and other allergy related conditions of the skin. In addition to their use for the prevention and treatment of the above described diseases and conditions of the skin, the therapeutic compositions described herein provide an efficacious composition that yields the benefit of smooth and youthful skin appearance with improved skin color, enhanced elasticity, reduced and delayed aging, enhanced youthful appearance and texture, and increased flexibility, firmness, smoothness and suppleness. By chelating with metal ions diarylalkanes also can be used to deliver essential metal ions into the blood stream of the host, and/or carry metal ions through the skin or blood/brain barrier, as well as, other membranes. In this embodiment, the method comprises administering to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s), together with the metal ion(s) to be delivered. In this capacity the diarylalkanes can be used to treat diseases and conditions including, but not limited to anemia and other iron deficiencies, inflammation; obesity and diabetes, periodontal diseases, oral pre-cancerous conditions, oral cancers, and other oral malignancies, sensitive gums and teeth, sequelae, pulpitis, irritation, pain and inflammation caused by the physical implantation of oral dentures, trauma, injuries, bruxism and other minor wounds in mouth, on the gums or on the tongue, dental plague and calculus, tooth decalcification, proteolysis and caries (decay), and viral infections. The metal ions are selected from the group including, but not limited to copper, iron, zinc, selenium, magnesium and other metal ions. In yet another embodiment, the dialkylalkanes can be used in the food industry to prevent browning and color changes in fruits, mushrooms and other food products. The diarylalkanes that can be used in accordance with the following include compounds illustrated by the general structure set forth above. The diarylalkanes of this invention may be obtained by synthetic methods or may be isolated from one or more families of plants selected from the group including, but not limited to Compositae, Fabaceae, Lauraceae, Leguminosae, Liliaceae, Loranthaceae, Moracea, and Myristicaceae. The diarylalkanes of this invention can be extracted, concentrated, and purified from the genera of high plants, including but not limited toAcacia, Broussonetia, Dianella, Helichrysum, Iryanthera, Knema, Lindera, Pterocarpus, Viscum, andXanthocercis. The diarylalkanes can be found in different parts of the plant, including but not limited to stems, stem barks, heart woods, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts. In a one embodiment, the diarylalkanes are isolated from a plant or plants in theBroussonetia, Dianella, andIryantheragenera. In another embodiment, the diarylalkanes of this invention are obtained by synthetic methods. Included in this invention is a method of synthesizing diarylalkanes and diarylalkanols said method comprising reducing a compound having the following general structure: whereinR1-R5and R′1—R′5and n are as defined above and wherein R6and R7together form one or more carbonyl group(s). The reducing agent can be selected from any known reducing agent for the reduction of ketones to alcohols including, but not limited to borohydrides, H2in the presence of a catalyst, NaH and LiAlH4. In one embodiment the reducing agent is NaBH4. In yet another embodiment, the diarylalkanes are obtained by synthetic modification of a naturally occurring compound isolated from a plant source. For example, the naturally occurring compound butein is isolated from a plant source, dehydrated and reduced to yield the corresponding diarylalkanol. In yet another embodiment, the diarylalkanes are obtained by the reaction of two appropriately substituted aromatic compounds. Feasible chemical reactions for synthesizing these compounds from two substituted aromatic compounds include, but are not limited to Aldol condensation between a substituted benzaldehyde and a substituted acetophenone; Claisen-Schmidt reaction or crossed aldol condensation between an aldehyde and a ketone; Grignard reaction using an organomagnesium halide of one substituted aromatic ring to link the second substituted aromatic ring through addition reaction to the carbonyl group on the molecule; Claisen rearrangement by an intra-molecular isomerization, in which an esterified phenol with appropriate substitution groups will be isomerized to link the second aromatic rings at the ortho-position of the phenol followed by a reducing reaction; and a Suzuki coupling reaction, in which two substituted aromatic rings are converted to arylboronic acids and then linked by an alkyl halide by using a carefully selected palladium catalyst. These reactions are well known to those of skill in the art and the conditions for such reactions can be determined using the information disclosed herein for the synthesis of these compounds. Note that throughout this application various citations are provided. Each of these citations is specifically incorporated herein by reference in its entirety. The present invention implements a strategy that combines a tyrosinase inhibition assay with a chemical dereplication process to identify active plant extracts and the particular compounds within those extracts that specifically inhibit the binuclear enzyme tyrosinase. As noted above, enzymes that inhibit tyrosinase may lead to a reduction in the production of melanin thereby effectively lightening the skin. A library of plant extracts was generated by extracting dry plant powders with an organic solvent, as described in Example 1. The tyrosinase inhibition assay was developed following a method reported by Jones et al. (2002) Pigment. Cell Res. 15:335, as described in Example 2. Using this assay, a total of 1144 plant extracts were screened for their ability to inhibit the activity of mushroom tyrosinase. This primary screen identified 20 plant extracts (1.75% hit rate) with potent tyrosinase inhibitory activity. Table 1 delineates percent inhibition of tyrosinase by four of these extracts isolated from four different genera. In order to efficiently identify active compounds from the active plant extracts, a high throughput fractionation process was used, as described in Example 3. Briefly, the active extracts were fractionated using a high throughput purification (HTP) system. Each of the fractions was then tested for its ability to inhibit tyrosinase activity as per the primary assay described in Example 2. After dereplication, using a combination of HPLC with PDA and MS detectors coupled with a structure database search and elimination of fractions that contained known tyrosinase inhibitors, such as polyphenols and chromones, a total of seven active extracts were chosen for bioassay guided large-scale isolation and purification as described in Examples 4-6, using the extracts ofBroussonetia kazinokiSieb. Et Zucc (Moraceae), andDianella ensifolia(L.) DC. (Liliaceae) for purposes of illustration. Example 4 describes the extraction, separation and purification of the novel diarylpropane: 1-(2-methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane (UP288) (1) fromBroussonetia kazinokiSieb. Et Zucc (Moraceae) (whole plant) using the general method set forth in Examples 1-3.FIG.4illustrates the HPLC/UV chromatogram of a HTP fraction that contains the UP288. The structure of the active compound UP288 was elucidated using a combination of spectroscopic methods as set forth in Example 4.FIG.5depicts the chemical structure and13C-NMR spectrum of UP288.FIG.6illustrates tyrosinase inhibitory dose response curves and IC50values for UP288 relative to kojic acid. The figure illustrates that UP288 (1) is as potent a tyrosinase inhibitor as kojic acid, having an IC50=24 μM. Surprisingly, two similar diarylalkanes were isolated and identified from a totally different family of plant—Dianella ensifolia(L.) DC. (Liliaceae), as described in Example 5.FIG.7depicts schematically the bioassay-guided isolation of these two active compounds (UP302a (2) and UP302b (3)) fromDianella ensifolia(P0389) (whole plant). With reference toFIG.7, it can be seen that only fifteen column fractions from a total of 264 collected samples exhibited potent inhibition of tyrosinase. A HPLC analysis (FIG.8) of the combined active fractions showed that active compounds were minor components in the best pool, which has already been heavily enriched. Laborious separation and purification efforts yielded two novel active compounds that have been fully characterized by NMR and other spectroscopic methods as illustrated in Example 5 andFIG.9as 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane (UP302a, IC50=0.24 μM) (2) and 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane (IP302b, IC50=1.2 μM) (3). Example 6 describes the large-scale isolation of UP302a (2), the most potent tyrosinase inhibitor, isolated fromDianella ensifolia(DE) (whole plant). With reference to Example 6, from 4.3 kg of dried biomass, a total of 30 mg of pure UP302a (2) was obtained after multiple column fractionations on silica gel, CG-161, and C-18 resins. The structure and biological function of the isolated compound were confirmed. Due to the low natural abundance of diarylalkanes/diarylalkanols methods to synthesize these biologically active compounds as an alternative commercial source of this class of compounds was developed. Example 7 describes a general method for the synthesis of diarylalkanes via the reduction of substituted chalcones. For purposes of illustration the reduction of 2,4-dihydroxyphenyl)-3′,4′-dimethoxyphenylchalcone (4) to 1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol (5) using sodium borohydride is described. However, as set forth in Example 7, a number of other diarylalkanes have been synthesized using this general method. All of the compounds synthesized showed high to moderate tyrosinase inhibitory activity. With respect to the general method described in Example 7, any other known reducing agents, can be used to effect this reduction, including, but are not limited to other borohydrides, H2in the presence of a catalyst, NaH and LiAlH4. Using the general reaction described in Example 7, several other substituted diarylpropanones have been converted to diarylpropanes and/or diarylpropanols as demonstrated in Examples 8, 9 and 10. Example 11 demonstrates the synthesis of a diarylpropanol using a flavonoid glycoside isolated from a natural source as the starting material. In another embodiment, the present invention includes methods for synthesizing this class of compounds by reaction of two appropriately substituted aromatic compounds. This embodiment is illustrated in Example 12, using the reaction of resorcinol with 3-methoxy-4-hydroxycinnamic acid for purposes of illustration. Feasible chemical reactions for synthesizing these compounds from two substituted aromatic compounds include, but are not limited to Aldol condensation between a substituted benzaldehyde and a substituted acetophenone; Claisen-Schmidt reaction or crossed aldol condensation between an aldehyde and a ketone; Grignard reaction using an organomagnesium halide of one substituted aromatic ring to link the second substituted aromatic ring through addition reaction to the carbonyl group on the molecule; Claisen rearrangement by an intra-molecular isomerization, in which an esterified phenol with appropriate substitution groups will be isomerized to link the second aromatic rings at the ortho-position of the phenol followed by a reducing reaction; and a Suzuki coupling reaction, in which two substituted aromatic rings are converted to arylboronic acids and then linked by an alkyl halide by using a carefully selected palladium catalyst. These reactions are well known to those of skill in the art and the conditions for such reactions can be determined using the information disclosed herein for the synthesis of these compounds. Example 13 sets forth the IC50values for a number of diarylalkanes and diarylalkanols synthesized according the methods of this invention. The compounds were evaluated using the general method described in Example 2. The IC50value of each sample was calculated using kinetics software to verify that the reaction was linear at a specified time and concentration. Using the methods described in Examples 7-12a total of 24 compounds were synthesized and evaluated for their ability to inhibit tyrosinase. The results are set forth in Table 2. With reference to Table 2, it can be seen that the IC50's of the synthetic diarylalkanols were comparable to the naturally occurring diarylpropanes. Thus, these two classes of compounds are capable of inhibiting tyrosinase to approximately the same extent. The most active diarylalkanes and/or diarylalkanols had three carbons between the two aromatic rings. Using the calculations described in Example 17, this structural feature was demonstrated to be critical in order to generate a parallel and superimposed intra-molecular conformations. However, diarylalkanols, which contained two and four carbons between the two aromatic rings, such as 1-(2,4-dihydroxyphenyl)-2-(4′-methoxyphenyl)-1-ethanol (IC50=77 μM) and 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethyl-buthane (IC50=700 μM) also were able to significantly inhibit tyrosinase activity. Using the method described in Example 2, the inhibition of tryosinase by UP302a (2) was evaluated using L-DOPA as the substrate as set forth in Example 14. The results are set forth inFIG.10. This study revealed that UP302a (2) is a powerful competitive inhibitor having a long lasting effect. Interestingly, tyrosinase activity was not resumed for several days after incubation with UP302a. In contrast, tyrosinase activity was totally restored after only 1 hour following incubation with kojic acid. Since two of the substituents on the aromatic rings of UP302a were methoxy groups, the inhibitor cannot be easily hydroxylated and/or oxidized. This may explain both the effectiveness and duration of the inhibitory activity of UP302a. Thus, it can be concluded that these compounds will have a long lasting effect. The efficacy of the claimed composition was also demonstrated by measuring the inhibition of melanin produced in an in vitro test on a B-16 cell line as described in Example 15. The results are set forth inFIG.11. The reduction of endogenous melanin by UP302a (2) was almost six fold greater than that of kojic acid. Additionally, inhibition of MSH induced melanin production by UP302a was also significantly greater than kojic acid. As expected, UP288 (1) was comparable to kojic acid in the B-16 cell line model. Example 16 describes an assay to assess the cytotoxicity of two diarylpropanes UP288 (1) and UP302a (2) relative to kojic acid. At a concentration of 250 μM, which was above IC50of all three tested compounds, the diarylpropanes demonstrated similar safety profiles to that of kojic acid. Example 17 describes the molecular modeling analyses performed to determine the most stable 3-D conformation of the active diarylalkanes and diarylalkanols. Molecular mechanics calculations were performed using Chem3D software. These calculations revealed that the most potent tyrosinase inhibitor—1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane (UP302a (2), IC50=0.24 μM) has a very unique 3-dimensional conformation with two the aromatic rings superimposed on each other as illustrated inFIG.12. The minimized total energy for the conformation is −4.7034 KJ/Mol and the distance between the two aromatic rings is 3.28 Å. The phenolic hydroxyl groups on the first aromatic ring are right above the two methoxy groups on the second aromatic ring with the distance between two oxygen atoms being as 2.99 Å and 3.16 Å, respectively as illustrated inFIG.14. The active site of the binuclear enzyme tyrosinase has two copper ions complexed to an oxygen molecule in a peroxide oxidation state [CuII—O2—CuII], which is key to the mechanism by which tyrosinase catalyzes the introduction of a hydroxyl group in the ortho position of the aromatic ring of a mono-phenol (such as tyrosine). (Decker et al. (2000) Angew. Chem. Int. Ed. 39:1591). The interactomic distances were reported as 3.56 Å for Cu—Cu. 1.90 Å for Cu—O and 1.41 Å for 0-0. (Kitajima et al. (1989) J. Am. Chem. Soc. 111:8975). The parallel conformation of 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane (UP302a, IC50=0.24 μM) will perfectly chelate with both copper ions of the [CuII—O2—CuII] complex from both the top and the bottom as illustrated inFIGS.13and14. This dual chelation by the inhibitor to both copper ions at the active site will totally block the access of the substrate, such as L-Dopa to the enzyme, thus effectively inhibiting the function of the protein. Using the same approach, the isolated and synthetic diarylalkanes and diarylalkanols listed in Table 2 were analyzed. The results of this analysis indicated that the compounds with twisted or non-parallel conformations possessed either no ability or only a weak ability to inhibit the activity of tyrosinase. From these studies it has been determined that the most effective diarylalkane inhibitors have two to three substituents on one aromatic ring and zero to multiple substituents on the second aromatic ring. The most favorable structures are those in which at least one aromatic ring is substituted in the 2 and 4-positions. Preferably the rings are 6-membered aromatic and/or heteroaromatic as demonstrated by two of the compounds isolated 1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8-yl)-1-propanol—IC50=72 μM and 3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propanol—IC50=225 μM. The compositions of this invention can be administered by any method known to one of ordinary skill in the art. The modes of administration include, but are not limited to, enteral (oral) administration, parenteral (intravenous, subcutaneous, and intramuscular) administration and topical application. The method of treatment according to this invention comprises administering internally or topically to a patient in need thereof a therapeutically effective amount of a diarylalkane or a mixture comprised of two or more diarylalkanes. The compositions of the present invention can be formulated as pharmaceutical compositions, which include other components such as a pharmaceutically and/or cosmetically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the host to be treated can tolerate. An excipient is an inert substance used as a diluent or vehicle for a therapeutic agent such as a diarylalkane or a mixture of diarylalkanes. Examples of such excipients include, but are not limited to water, buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, preservatives and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include, but are not limited to EDTA, disodium EDTA, BHA, BHT, vitamin C, vitamin E, sodium bisulfite, SnCl2, thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can be either liquid or solids, which can be taken up in a suitable liquid as a suspension or solution for administration. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. In one embodiment of the present invention, the composition can also include an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the biological response of a host to a specific bioactive agent. Suitable adjuvants include, but are not limited to, Freund's adjuvant, other bacterial cell wall components, aluminum, magnesium, copper, zinc, iron, calcium, and other metal ion based salts, silica, polynucleotides, toxoids, serum proteins, viral coat proteins, other bacterial-derived preparations, gamma interferon; block copolymer adjuvants; such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross, Ga.), Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated host. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols. In one embodiment, the composition is prepared as a controlled release formulation, which slowly releases the composition of the present invention into the host. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles will be known to those skilled in the art. Preferred controlled release formulations are biodegradable (i.e., bioerodible). The therapeutic agents of the instant invention are preferably administered topically by any suitable means, known to those of skill in the art for topically administering therapeutic compositions including, but not limited to as an ointment, gel, lotion, or cream base, or as a toothpaste, mouth-wash, or coated on dental flossing materials or as an emulsion, as a patch, dressing or mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Example 18 describes the preparation of two cream formulations with an active content at 0.01% and 0.1% of a pure and/or mixture of diarylalkane(s) in the total weight of the formula. Such topical application can be locally administered to any affected area, using any standard means known for topical administration. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient of the present invention. A therapeutic reagent of the present invention can be administered to any host, preferably to mammals, and more preferably to humans. The particular mode of administration will depend on the condition to be treated. In one embodiment, a suitable ointment is comprised of the desired concentration of a single diarylalkane or a mixture of two or more diarylalkanes, that is an efficacious, nontoxic quantity generally selected from the range of 0.001% to 100% based on the total weight of the topical formulation, from 65 to 100% (preferably 75 to 96%) of white soft paraffin, from 0 to 15% of liquid paraffin, and from 0 to 7% (preferably 3 to 7%) of lanolin or a derivative or synthetic equivalent thereof. In another embodiment the ointment may comprise a polyethylene-liquid paraffin matrix. In one embodiment, a suitable cream is comprised of an emulsifying system together with the desired concentration of a single diarylalkane or a mixture of two or more diarylalkanes as provided above. The emulsifying system is preferably comprised of from 2 to 10% of polyoxyethylene alcohols (e.g. the mixture available under the trademark Cetomacrogol™ 1000), from 10 to 25% of stearyl alcohol, from 20 to 60% of liquid paraffin, and from 10 to 65% of water; together with one or more preservatives, for example from 0.1 to 1% of N,N″-methylenebis[N′-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (available under the name Imidurea USNF), from 0.1 to 1% of alkyl 4-hydroxybenzoates (for example the mixture available from Nipa Laboratories under the trade mark Nipastat), from 0.01 to 0.1% of sodium butyl 4-hydroxybenzoate (available from Nipa Laboratories under the trade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol. In one embodiment, a suitable gel is comprised of a semi-solid system in which a liquid phase is constrained within a three dimensional polymeric matrix with a high degree of cross-linking The liquid phase may be comprised of water, together with the desired amount of a single diarylalkane or a mixture of two or more diarylalkanes, from 0 to 20% of water-miscible additives, for example glycerol, polyethylene glycol, or propylene glycol, and from 0.1 to 10%, preferably from 0.5 to 2%, of a thickening agent, which may be a natural product, selected from the group including, but not limited to tragacanth, pectin, carrageen, agar and alginic acid, or a synthetic or semi-synthetic compound, selected from the group including, but not limited to methylcellulose and carboxypolymethylene (carbopol); together with one or more preservatives, selected from the group including, but not limited to for example from 0.1 to 2% of methyl 4-hydroxybenzoate (methyl paraben) or phenoxyethanol-differential. Another suitable base, is comprised of the desired amount of a single diarylalkane or a mixture of diarylalkanes, together with from 70 to 90% of polyethylene glycol (for example, polyethylene glycol ointment containing 40% of polyethylene glycol 3350 and 60% of polyethylene glycol 400, prepared in accordance with the U.S. National Formulary (USNF)), from 5 to 20% of water, from 0.02 to 0.25% of an anti-oxidant (for example butylated hydroxytoluene), and from 0.005 to 0.1% of a chelating agent (for example ethylenediamine tetraacetic acid (EDTA)). The term soft paraffin as used above encompasses the cream or ointment bases white soft paraffin and yellow soft paraffin. The term lanolin encompasses native wool fat and purified wool fat. Derivatives of lanolin include in particular lanolins which have been chemically modified in order to alter their physical or chemical properties and synthetic equivalents of lanolin include in particular synthetic or semisynthetic compounds and mixtures which are known and used in the pharmaceutical and cosmetic arts as alternatives to lanolin and may, for example, be referred to as lanolin substitutes. One suitable synthetic equivalent of lanolin that may be used is the material available under the trademark Softisan™ known as Softisan 649. Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is a glycerine ester of natural vegetable fatty acids, of isostearic acid and of adipic acid; its properties are discussed by H. Hermsdorf in Fette, Seifen, Anstrichmittel, Issue No. 84, No. 3 (1982), pp. 3-6. The other substances mentioned hereinabove as constituents of suitable ointment or cream bases and their properties are discussed in standard reference works, for example pharmacopoeia. Cetomacrogol 1000 has the formula CH3(CH2)m(OCH2CH2)—OH, wherein m may be 15 or 17 and n may be 20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol. Nipastat is a mixture of methyl, ethyl, propyl and butyl 4-hydroxybenzoates. The compositions of the invention may be produced by conventional pharmaceutical techniques. Thus the aforementioned compositions, for example, may conveniently be prepared by mixing together at an elevated temperature, preferably 60-70° C., the soft paraffin, liquid paraffin if present, and lanolin or derivative or synthetic equivalent thereof. The mixture may then be cooled to room temperature, and, after addition of the hydrated crystalline calcium salt of mupirocin, together with the corticosteroid and any other ingredients, stirred to ensure adequate dispersion. Regardless of the manner of administration, the specific dose is calculated according to the approximate body weight of the host. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the scope of tasks routinely performed by them without undue experimentation, especially in light of the dosage information and assays disclosed herein. These dosages may be ascertained through use of the established assays for determining dosages utilized in conjunction with appropriate dose-response data. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLES Example 1 Preparation of Organic Extracts from Dry Plants Dried plant material was ground to a particle size of no larger than 2 mm and a portion (60 g) was transferred to an Erlenmeyer flask and extracted with 600 ml of methanol:dichloromethane (1:1). The mixture was shaken for one hour, filtered and the biomass was extracted again with methanol:dichloromethane (1:1) (600 ml). The organic extracts were combined and evaporated under vacuum to provide an organic extract from each plant material. Each extract (approximately 75 mg) was then dissolved in 1.5 ml DMSO to a concentration of 50 mg/ml, which was then stored in a −70° C. freezer. An aliquot of the extract solution was used for tyrosinase assay as described in Example 2. Example 2 Tyrosinase Inhibition Assay A tyrosinase inhibition assay was carried out using the method reported by Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, the conversion of L-Dopa, a substrate of tyrosinase, into dopachrome is followed by monitoring absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer, pH 6.8 (assay buffer) at 2000 U/ml and stored at −20° C. in 1 ml aliquots prior to use. For use in assays, stock enzyme solutions were thawed and diluted to 200 U/ml with assay buffer. A 2 mM working solution of substrate, L-DOPA, was prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted to 5 ml with assay buffer. The reaction mixture consisted of 0.050 ml 2 mM L-DOPA, 0.050 ml 200 U/ml mushroom tyrosinase and 0.050 ml inhibitor. Reaction volume was adjusted to 200 μl with assay buffer. Assays were performed in 96 well Falcon 3097 flat-bottom microtiter plates (Beckton Dickinson, N.J.). Appearance of dopachrome was measured with a WALLAC 1420 Multilable Counter (Turku, Finland). Average velocity was determined from linear enzyme rate as measured by change in absorbance (ΔA450) at 450 nm per minute. Percent inhibition of tyrosinase by test samples was determined by comparison of absorbance of samples versus control using formula (I): (Negative control absorption−sample absorption)/Negative control absorption×100 (1) The results are set forth in Table 1. TABLE 1Tyrosinase inhibitory activity of four plant extractsPlant LatinWeight ofPercent InhibitionName andthe Organicof TyrosinasePartsAmountExtract(concentration mg/ml)Broussonetica20 g1.1g68%kazinokiwhole(at 0.125 mg/ml)plantRhus chinensis20 g12.8g31%cecidiums(at 0.125 mg/ml)Polygonum20 g2.4g43%multiflorum(at 0.125 mg/ml)tubersDianella20 g1.7g57%ensifoliawhole(at 0.125 mg/ml)plant Example 3 HTP Fractionation of Active Plant Extracts Active organic extract (400 mg) was loaded onto a prepacked, normal phase, flash column. (2 cm ID×8.2 cm, 10 g silica gel). The column was eluted using a Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was dried under low vacuum and centrifugation. DMSO (1.5 mL) was used to dissolve the samples in each cell and a portion (100 μL) was taken for the tyrosinase inhibition assay in duplicate. Example 4 Extraction, Separation and Purification of 1-(2-Methoxy-4-Hydroxyphenyl)-3-(2′-Hydroxy-5′-Methoxyphenyl)-Propane (1) fromBroussonetia kazinoki(BK) (Whole Plant) Broussonetia kazinoki(100 g whole plant) was ground and extracted three times with 800 ml of MeOH:DCM (1:2). Dried extract (6 g) was fractionated using a silica gel column with gradient solvent elution of hexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 sets of 88 test tubes. LC/MS/PDA was utilized to check each of the fractions, which were then combined based on the similarity of their composition. The combined fractions were evaporated to remove solvent, dried and their tyrosinase inhibition activity measured as described in Example 2. It was found that fractions (P0346-HTP-F2-P0346-HTP-F4) were the most active and these fractions were combined and labeled as BK-F2-4. After solvent evaporation, BK-F2-4 was further separated on a pre-packed reverse phase column (C-18 column) using a water/MeOH gradient. Eighteen compound peaks were observed following separation. Fourteen reverse phase columns were performed and the similar fractions from each run were combined. One compound peak referred to as UP288 in the combined and enriched fraction showed strong tyrosinase inhibition activity (FIG.4). After separation and purification with preparative HPLC, 6 mg of 1-(2-methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane (UP288) (1) was obtained. The structure of this compound was elucidated using MS and NMR spectroscopy (1H,13C, HMQC and HMBC).FIG.5depicts the chemical structure and13C-NMR spectrum of UP288. UP288 is an inhibitor of tyrosinase having activity comparable with that of kojic acid with IC50value of 24 μM.FIG.6illustrates tyrosinase inhibitory dose response curves and IC50values for UP288 and kojic acid. 1-(2-Methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane (UP288). Yield 0.006% (purity >96%, HPLC); UV λmax: 281.0 nm; MS (Super Sound Ionization, Positive ion detection): m/z 289 (M+1, 100%);1H-NMR (400 MHz, (CD3)2SO): δ 1.70 (2H, m, CH2), 2.46 (4H, m, 2 CH2), 3.68 (3H, s, OCH3), 3.73 (3H, s, OCH3), 6.26 (1H, q, H-5), 6.35 (1H, d, H-3), 6.55 (1H, q, H-14), 6.65 (1H, d, H-13), 6.72 (1H, d, H-16), 6.86 (1H, d, H-6), 8.69 (1H, s, OH), 9.20 (1H, s, OH);13C-NMR (100 MHz, (CD3)2SO): Δ 28.5 (C-8), 31.6 (C-9), 34.5 (C-10), 55.0 (C-7), 55.6 (C-17), 98.9 (C-3), 106.4 (C-5), 112.4 (C-16), 115.2 (C-13), 119.7 (C-1), 119.8 (C-14), 120.3 (C-11), 120.4 (C-6), 132.9 (C-12), 144.6 (C-4), 147.2 (C-17) & 158.3 (C-7). Example 5 Extraction, Separation and Purification of 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,4′-Dihydroxyphenyl)-Propane (UP302a) (2) and 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,5′-Dihydroxyphenyl)-Propane (UP302b) (3) fromDianella ensifolia(P0389) (Whole Plant) Dianella ensifolia(P0389, 300 g whole plant) was ground and extracted three times 800 ml of MeOH:DCM (1:2). Dried extract (5 g) was fractionated using a silica gel column with gradient solvent elution of hexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 sets of 264 test tubes. LC/MS/PDA was utilized to check each of the fractions, which were then combined into 22 fractions based on the similarity of their composition. (FIG.7). The combined fractions were evaporated to remove solvent, dried and their tyrosinase inhibition activity measured as described in Example 2. It was found that fractions P0389-HTP-F12, P0389-HTP-F13 and P0389-HTP-F14 were the most active and these fractions were combined and relabeled as DE-F12-14. After solvent evaporation, DE-F12-14 was further separated on a pre-packed reverse phase column (RP-column) using a water/MeOH gradient. Two major and eleven minor compound peaks were observed following separation. The compounds corresponding to each of these peaks were isolated following 7 additional separations on RP-columns. All of the compounds collected were dried and tested for tyrosinase inhibitory activity. Two of the eleven minor peaks referred to as UP302a and UP302b, respectively, exhibited strong tyrosinase inhibitory activity. (FIG.8). After separation and purification, two active compounds were obtained: 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane (UP302a, 10 mg) (2) and 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane (UP302b, 6 mg) (3). The structures of these two compounds were elucidated using MS and NMR spectroscopy (1H,13C, gHSQC and HMBC).FIG.9depicts the gHSQC spectrum of UP302a. Tyrosinase inhibition assays showed that UP302a was the most potent inhibitor with an IC50of 0.24 μM, while UP302b has an IC50of 12 μM. 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane (UP302a) (2). Yield 0.02% (purity >98%, HPLC); UV λmax: 279.8 nm; MS (Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%);1H-NMR (400 MHz, (CD3)2SO): δ 1.70 (2H, m, CH2), 2.03 (3H, s, CH3), 2.43 (2H, m, CH2), 2.49 (2H, m, CH2), 3.58 (3H, s, OCH3), 3.73 (3H, s, OCH3), 6.11 (1H, q, H-16), 6.25 (1H, d, H-14), 6.65 (1H, d, H-5), 6.76 (1H, d, H-17), 6.97 (1H, d, H-6), 8.93 (1H, s, OH), 9.03 (1H, s, OH);13C-NMR (100 MHz, (CD3)2SO): δ 28.8 (C-9), 29.3 (C-11), 31.1 (C-10), 55.3 (C-7), 55.9 (C-8), 102.4 (C-14), 105.8 (C-16), 106.1 (C-5), 118.4 (C-1), 118.6 (C-12), 126.9 (C-3), 127.0 (C-6), 130.1 (C-17), 155.7 (C-13), 156.2 (C-15), 156.3 (C-4) and 156.8 (C-2). 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane (UP302b) (3). Yield 0.01% (purity >95%, HPLC); UV λmax: 279.8 nm; MS (Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%);1H-NMR (400 MHz, (CD3COCD3): δ 1.82 (2H, m, CH2), 2.07 (3H, s, CH3), 2.52 (2H, m, CH2), 2.56 (2H, m, CH2), 3.63 (3H, s, OCH3), 3.77 (3H, s, OCH3), 6.64 (1H, q, H-15), 6.72 (1H, d, H-14), 6.64 (1H, d, H-5), 6.70 (1H, d, H-17), 7.00 (1H, d, H-6), 7.65 (1H, s, OH), and 7.69 (1H, s, OH). Example 6 Large-Scale Isolation of 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,4′-Dihydroxyphenyl)-Propane (UP302a) (2) fromDianella ensifolia(DE) (Whole Plant) Dianella ensifolia(4.3 kg whole plant) was collected, ground and extracted three times using a percolation extractor with methanol as the solvent. The extracts were combined and evaporated to remove the methanol. The crude extract was then suspended in water and partitioned with DCM. The layers were separated and the DCM layer was evaporated to provide 60 g of material. LC-MS/PDA analysis of both layers revealed that the majority of the UP302a was present in the DCM layer with only a minor amount present in the water layer. The DCM extract was fractionated on three separate silica gel columns eluting with a gradient of hexane-ETOAC. A total of 15 sub-fractions were obtained and analyzed by HPLC-MS/PDA. Targeted compound (UP302a) was found in fractions 6 to 9, which were combined to yield a total of 3 g of enriched UP302a. The enriched UP302a was further separated on an open column packed with CG-161 resin eluting with a water-MeOH gradient. A total of 23 fractions were collected with the UP302a eluting in fractions 15 to 21. Fractions 15-21 were then combined and the solvent was evaporated to yield 700 mg of a solid, which was further purified by preparative HPLC on a C-18 column to generate 30 mg of UP302a. The structure, tyrosinase inhibitory activity and purity of the purified product was confirmed by NMR, enzyme inhibition assay and LC-MS/PDA. Example 7 Synthesis of Diarylalkanes by Sodium Borohydride Reduction of Substituted Chalcones A general method for the synthesis of diarylalkanes by sodium borohydride reduction of substituted chalcones is described below using the reduction of 2,4-dihydroxy)-3′,4′-dimethoxychalcone (4) for purposes of illustration. 2,4-Dihydroxy-3′,4′-dimethoxychalcone (4) (40 mg) was dissolved in 1-propanol (5 ml), followed by the addition of sodium borohydride (15 mg) and the mixture was allowed to react at room temperature for 2 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added and the mixture was heated at 80° C. for 5 minutes and cooled down. The mixture was then separated on a pre-packed C18column eluting with a MeOH/H2O gradient to provide 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol (5). The structure of compound (5) was confirmed by MS, UV spectroscopy, 1D and 2D1H-NMR. 1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol (5). Yield 60% (purity >98%, HPLC); UV λmax: 278.5 nm; MS (Super Sound Ionization, Positive ion detection): m/z 305 (M+1, 100%);1H-NMR (400 MHz, (CD3)2SO): δ 1.93 (2H, m, CH2), 2.60 (2H, m, CH2), 4.49 (1H, m, CH—OH), 3.78 (3H, s, OCH3), 3.80 (3H, s, OCH3), 6.28 (1H, q, H-5), 6.31 (1H, d, H-3), 6.98 (1H, d, H-6), 6.71 (1H, q, H-5′), 6.77 (1H, d, H-2′), 6.83 (1H, d, H-6′). Using the above-described general method the following compounds were reduced to their corresponding alcohols: 2,4-dihydroxy-2′-hydroxychalcone, 2′-hydroxy-4′-methoxy-2,4-dimethoxy-chalcone, 4′-hydroxy-4-hydroxy-chalcone, 2′,4′-dihydroxy-2-hydroxy-chalcone, 2′,4′-dihydroxy-3,4-dimethoxy-chalcone, 2′,4′,6′-trimethoxy-3,4-dimethoxy-chalcone and 2′-hydroxy-4′-methoxy-3,4,5-trimethoxy-chalcone to provide 1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol, 1-(4-hydroxyphenyl)-3-(4′-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(3′,4′-di-methoxyphenyl)-1-propanol, 1-(2,4,6-trimethoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol and 1-(2-hydroxy-4-methoxyphenyl)-3-(3′,4′,5′-trimethoxyphenyl)-1-propanol. Example 8 Synthesis of Substituted Diphenylpropanols by Sodium Borohydride Reduction of Substituted Diarylpropanones A general method for the synthesis of substituted diphenylpropanols by sodium borohydride reduction of substituted diarylpropanones is described below using the reduction of 1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanone (6) for purposes of illustration. 1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanone (6) (5 mg) was dissolved in 1-propanol (1 ml), followed by the addition of sodium borohydride (2 mg) and the mixture was allowed to react at room temperature for 5 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added to neutralize the excess sodium borohydride. The reaction mixture was then separated on a pre-packed C18column eluting with a MeOH/H2O gradient to provide 1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol (7). Following the above-described general synthetic procedure the following diarylalkane compounds were reduced: 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanone, 3-(5′-benzyloxy-4′-methoxy-2′-methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanone, 1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8′-yl)-1-propanone and 3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propenone to provide 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol, 3-(5′-benzyloxy-4′-methoxy-2′-methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxy-phenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8-yl)-1-propenol and 3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxy-phenyl)-1-propenol, respectively. Example 9 Synthesis of 1,3-Bis(2,4-Dimethoxyphenyl)-Propan-1,3-Diol (9) 1,3-Bis(2,4-dimethoxyphenyl)-propan-1,3-dione (8) (5 mg) was dissolved in 1-propanol (1 ml), followed by the addition of sodium borohydride (3 mg) and the mixture was allowed to react at room temperature for 3 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added to neutralize the excess sodium borohydride. The mixture was then separated on a pre-packed C18column eluting with a MeOH/H2O gradient to provide 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol (9). Example 10 Synthesis of 1-(2,4,6-Trihydroxyphenyl)-3-(3′-Hydroxy-4′-Methoxyphenyl)-1-Propanol (11) from Neohesperidin Neohesperidin is a glycoside of dihydrochalcone. A total weight of 100 mg of neohesperidin was suspended in 10 ml of 1 N HCl and heated at 80° C. for 2 hours. The hydrolyzed product (10) was cooled down and extracted with ethyl acetate (3×10 ml). The ethyl acetate layers were combined, evaporated to remove ethyl acetate and dissolved in 1-propanol (5 ml). Sodium borohydride (25 mg) was added to the propanol solution and stirred at room temperature for 2 hours. After the completion of the reaction, the mixture was separated on a pre-packed C18column eluting with a MeOH/H2O gradient to provide 1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol (11). Example 11 Extraction, Purification and Structure Modification of Butrin to Synthesize 142,4-Dihydroxyphenyl)-3-(3′,4′-Dihydroxyphenyl)-1-Propanol (14) Butrin is a high content flavanone-glycoside that has been extracted with methanol from the dried flowers ofButea frondosaand purified by multiple reverse phase column chromatographic separations. After removing sugars by hydrolysis with HCl, butin (12) was produced and purified by RP-HTP (1.5% yield from the whole plant). Butin was then treated with 10% sodium hydroxide at 80° C. to obtain butein (13), which was reduced with sodium borohydride to obtain 1-(2,4-dihydroxyphenyl)-3-(3′,4′-dihydroxyphenyl)-1-propanol (14) (IC50=250 nM). Example 12 Synthesis of 1-(2,4-Dihydroxyphenyl)-3-(3′-Methoxy-4′-Hydroxyphenyl)-1-Propanol (19) Resorcinol (15), 3-methoxy-4-hydroxy-cinnamic acid (16) and H2SO4(5%) were refluxed in THF for 4 hours to provide 7,4′-dihydroxy-3′-methoxy flavanone (17) (90% yield). The product, 7,4′-dihydroxy-3′-methoxy flavanone (17) was then treated with 10% sodium hydroxide at 80° C. for 1 hour, followed by reduction with sodium borohydride in propanol to provide, as confirmed LC-MS/PDA detection, 1-(2,4-dihydroxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol (19). The crude product exhibits quite strong tyrosinase inhibitory activity. The mixture was further purified by HTP. Example 13 IC50Measurements of Tyrosinase Inhibition by Synthetic Diarylalkanes Inhibition of tyrosinase by synthetic diarylalkanes was measured using the method described in Example 2. The IC50value of each sample was calculated using kinetics software to verify that the reaction was linear at a specified time and concentration. Using the methods described in Examples 7-12a total of 24 compounds were synthesized and evaluated for their ability to inhibit tyrosinase. The results are set forth in Table 2. TABLE 2IC50values of synthetic diarylalkanes and/or diarylalkanolsTyrosinaseInhibitionCompound Name(IC50)1-(2,4-dihydroxyphenyl)-3-(3′,4′-dihydroxyphenyl)-1-propanol0.5μm1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol0.85μm1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol0.7μm1-(2,4-dihydroxyphenyl)-3-(2′-methoxyphenyl)-1-propanol3μm1-(2,4-dihydroxyphenyl)-3-(4′-methoxyphenyl)-1-propanol6μm1-(2,4,6-trihydroxyphenyl)-3-(4′-aminophenyl)-1-propanol8μm1-(2,4-dihydroxyphenyl)-3-phenyl-1-propanol8μm1-(2,4-dihydroxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol8.5μm1-(2-hydroxy-4-methoxyphenyl)-3-(3′,4′,5′-trimethoxyphenyl)-1-propanol11μm1-(2-hydroxy-4-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol25μm1-(2-hydroxy-5-methoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol30μm1-(2,4-dihydroxyphenyl)-2-(4′-methoxyphenyl)-1-ethanol77μm1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-benzo(b)dioxocin-8′-yl)-72μm1-propanol3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-225μm1-propanol1-(4-hydroxyphenyl)-3-(4′-hydroxyphenyl)-1-propanol305μm1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol375μm1-(2,4-dihydroxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-ethanol431μm1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane700μm1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol1000μm1-(2,4-dihydroxyphenyl)-2-(2′,4′-dichlorophenyl)-1-ethanol1000μm1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol1200μm1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol1200μm1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol1200μm1-(2,4,6-trimethoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol1500μm Example 14 Enzyme Inhibition Kinetics Using the method described in Example 2, the inhibition of tryosinase was evaluated at different concentrations (0, 261, 522, 1044 nM) of an inhibitor (UP302a) using L-DOPA at concentrations of 0.75, 1.25, and 2.5 mM as the substrate. As shown inFIG.10, it was found that UP302a is a competitive inhibitor with potent and long lasting inhibitory effect. Tyrosinase activity was not resumed for several days after incubation with UP302a. In contrast, tyrosinase activity was totally restored after only 1 hour following incubation with kojic acid. Example 15 Inhibition of Melanin Production from B-16 Cell Line The inhibition of melanin production was evaluated using two different assays. In the first assay, the inhibition of melanin production was evaluated without induction by β-MSH; whereas in the second assay the inhibition of melanin production was measured with β-MSH induction in cell culture medium. Briefly, B16 F1 cells (ATCC #CRL-622) were grown to confluency and then seeded at 40,000 cells per well. The cells were allowed to attach overnight at 37° C. in a humidified environment containing 5% CO2. On day 2, inhibitors were added to the cells at concentrations ranging from 0-1000 μM in triplicate and allowed to incubate for four days. The amount of β-MSH required to induce melanin formation was determined by the addition of hormone at concentrations ranging from 0-1000 nM in ten-fold increments. Once the proper β-MSH concentration was determined, cells were seeded as above, allowed to attach overnight and then co-incubated with tyrosinase inhibitors at concentrations ranging from 0-1000 μM. Color development was visually monitored each morning. After the development of color, 200 μl of cell supernatant was removed from each well and absorbance was measured at 450 nm. The resulting readings were used to determine the IC50for melanin formation in the cell assay with and without β-MSH induction. For an initial comparison of cell toxicity, the 250 μM treated wells were used to perform a lactate dehydrogenase assay (LDH). LDH is a metabolic enzyme that leaks out of damaged or dead cells. The enzyme converts a chromophore in the presence of NAD+to yield a color change that can be monitored spectrophotometrically. The results of this assay revealed that all of the natural inhibitors tested (UP288, UP302a, and UP302b) are at least as good, if not better inhibitors than kojic acid. There were some differences in the IC50values under the two sets of conditions. Inhibition by kojic acid improved from an IC50of 170 μM for the endogenous experiment to an IC50of 67 μM in the induced experiment. Of the inhibitors tested relative to kojic acid, compound UP302b was only one that that showed an increase in IC50under the two sets of conditions increasing from an IC50of 5.2 μM to an IC50of 34 μM. The IC50's measured for inhibition of tyrosinase were relatively the same for all of the compounds tested with the exception of the two compounds UP302 and UP302b, which had low IC50's of 0.2 μM and 0.3 μM, respectively, compared to 28 μM and 5.2 μM in the endogenous assay and 40 μM and 34 μM in the induced assay. These differences may be due to decreased cell penetration by UP302a (2) and UP302b (3), as compared to the other inhibitors. This is overcome, however by the strength of their inhibition of the enzyme. Table 3 provides the results of these two assays for inhibitors UP288 and UP302a relative to kojic acid. Example 16 Cell Toxicity Assay The compound treated wells were used to perform a lactate dehydrogenase assay (LDH). LDH is a metabolic enzyme that leaks out of damaged or dead cells. The enzyme converts a chromophore in the presence of NAD′ to yield a color change that can be monitored spectrophotometrically. The cytotoxicity was examined at a concentration of 250 μM. At this concentration none of these compounds are significantly more cytotoxic than kojic acid. It should be noted however, that cytotoxicity at only one concentration (250 μM) was tested. As shown in the Table 3, UP288 (1) and UP302a (2) showed moderate cytotoxicity, which were comparable with kojic acid. TABLE 3Inhibition of mushroom tyrosinase and melanin formation in mouse B16F1 cells by isolated compounds and comparison of cell toxicityEndogenousMSH InducedTyrosinaseMelaninMelaninCellInhibitionInhibitionInhibitionToxicityCompoundIC50(μM)IC50(μM)IC50(μM)(LDH)UP28824.01081050.315UP302a0.2428400.265Kojic acid29170670.260 Example 17 Molecular Mechanics (MM2) Calculation Molecular mechanics calculations were performed using Chem3D software for purposes of energy minimization and determination of the most stable 3-D conformation. The following parameters were used: Step interval=2.0 fs, frame interval=10 fs, terminate after 10,000 steps, heating/cooling rate=1.000 Kcal/atom/PS, target temperature=3000K. Properties: pi bond orders and steric energy summary. All natural and synthetic compounds and other diarylalkane and diarylalkanol structures were analyzed. It was found that the most potent tyrosinase inhibitor—1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a (2), IC50=0.24 μM)—isolated from whole plants ofDianella ensifolia(L.) DC. has a very unique 3-dimensional conformation in which the two aromatic rings were superimposed on each other. The minimized total energy for the conformation is −4.7034 KJ/Mol. The distance between the two aromatic rings was 3.28 Å. The phenolic hydroxyl groups on the first aromatic ring were right above the two methoxyl groups on the second aromatic ring with the distance between two oxygen atoms being 2.99 and 3.16 Å, respectively as illustrated inFIGS.12-14. This intramolecular parallel conformation allows this compound to perfectly chelate both copper ions of the binuclear enzyme when it is in the peroxide form [CuII—O2—CuII] from both the top and the bottom. Example 18 Formulation of the Diarylalkane Composition into a Cream UP302a is comprised of a substituted diarylpropane as the major active component. These compounds are soluble in high polarity solvents including, but not limited to ethanol, propylene glycol and ethylene glycol. They can be formulated with a pharmaceutically and/or cosmetically acceptable excipient, an adjuvant, and/or a carrier. Examples of such excipients include, but are not limited to water, buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, preservatives and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles including, but not limited to fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include, but are not limited to suspensions containing viscosity enhancing agents, including, but not limited to sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives or preservatives, such as antioxidants that enhance color and chemical stability. UP302 also can be prepared in a liposome formulation to increase its skin penetration or as a controlled release formulation, which slowly releases the composition of the active ingredient into the host. UP302a is preferably administered topically as an ointment, gel, lotion, or cream base or as an emulsion, a patch, dressing or mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Such topical application can be locally administered to any affected area, using any standard means known for topical administration. UP302 can be administered to both humans and animals. A therapeutic composition of UP302a can be administered in a variety of unit dosage forms depending upon the method of administration and targeted indications. An efficacious, nontoxic quantity is generally recommended in the range of 0.01% to 5% based on total weight of the topical formulation. Two different concentrations of UP302a (0.01% and 0.5% by weight) were formulated in creams as illustrated in Tables 4 and 5. To prepare these creams the diarylalkane was dissolved in water at room temperature and homogenized in a blender until it was fully dispersed in solution (approximately 5 minutes) to yield a composition A. At room temperature and without stifling or agitating, Ultrez-21 carbomer was added to the homogenized solution by sprinkling it onto the surface and allowing it to fully wet (no white areas visible) and fall into the solution. With gentle stifling, the solution was then heated to 40° C. and glycerin was added and the composition was mixed for an additional 5 minutes to provide Composition B. At 40° C., Composition A is added to Composition B and the composition is mixed well until homogenous (approximately 5 minutes). The resulting emulsion is cooled to 30° C. and the pH adjusted to approximately 5.5 (5.3 to 5.7) by titrating with neutralizer while stifling with a stir bar and/or spatula. The emulsion became highly viscous due to the neutralization-induced conformational change of the carbomer. Upon stifling the emulsion will achieved a suitable viscosity for an emulsion cream. The composition was mixed until uniform, poured into clean storage vessels and stored at 2° C. to 8° C. TABLE 4Composition of 0.01% Diarylalkane CreamPhaseIngredient% (w/w)Weight (g)AqueousWater, Purified85.0012Diarylalkane (UP302a)0.010.0015Ultrez 21 Carbomer0.500.075Glycerin8.001.2OilPEG-7 Glyceryl Cocoate3.000.45Caprylic/Capric Triglyceride2.670.4PH NeutralizerSodium Hydroxide (18% w/v),0.000.0Molecular Biology GradeSUM7 Ingredients10015 TABLE 5Composition of 0.1% UP302 CreamPhaseIngredient% (w/w)Weight (g)AqueousWater, Purified84.0012.6Diarylalkane (UP302a)0.10.0015Ultrez 21 Carbomer0.500.075Glycerin8.001.2OilPEG-7 Glyceryl Cocoate3.000.45Caprylic/Capric Triglyceride2.670.4PH NeutralizerSodium Hydroxide (18% w/v),Molecular Biology GradeSUM7 Ingredients99.715 | 68,663 |
11857658 | DETAILED DESCRIPTION OF THE INVENTION In the following description of the present invention and the claims appended hereto, it is to be understood that all concentrations are by weight percent based on a total weight of the composition unless otherwise indicated. Where appropriate, the INCI (international Nomenclature of Cosmetic Ingredients) name of ingredients/components is used. The present invention is directed to a multi-functional cosmetic composition having an improved skin feel and appearance with good color intensity and color retention even after water exposure. The composition comprises the following components: a film former, a skin adhesion promoter, a color enhancer, and one or more colorants. It may further comprise a variety of different actives to provide desired functionality to the user. The unexpected synergy of the individual components together preserves color intensity and color retention even after water exposure so that skin appearance is continually enhanced. The cosmetic compositions of the present invention have the advantage of being durable, transfer-resistant, water resistant, color intense, and comfortable to the wearer. The cosmetic compositions can be formulated with pharmaceutically acceptable carrier components to provide different formulations as a lotion, cream gel, or spray. The film formers useful in the present invention protect the active ingredients and colorants from being removed from the skin by mechanical forces, i.e., rubbing. They also contribute to the water resistance of the final composition such that the actives and colorants are not easily washed off. In sunscreen compositions, the film formers are an important component to substantiate the claim of water resistance. Film formers useful in the present invention may comprise one or more polymers. The following are examples of suitable polymers, but the list is by no means limiting. Film former polymers may include acrylate copolymers such as those obtained by emulsion polymerization, and more specifically, acrylate/C12-22methacrylate copolymer polymerized from monomers of methacrylic acid, methyl methacrylate, butyl acrylate, and cetyl-eicosinyl methacrylate; and PPG-17/isophorone diisocyanate (IPDI)/dimethylol propionic acid (DMPA) copolymer. There are also aqueous polyurethane polymer dispersions such as Polyurethane-34 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, hexamethylene diisocyanate, ethylene diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid; Polyurethane-32 which is a sodium salt of polymerized monomers of polytetramethylene glycol, hexamethylene diisocyanate, (IPDI), ethylene diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid; Polyurethane-35 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, dicyclohexylmethane diisocyanate, ethylene diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid; and Polyurethane-48 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, isophorone diisocyanate, isophorone diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid. Also useful are vinylpyrrolidone (VP) and polyvinylpyrrolidone (PVP) crosspolymers, copolymers and/or interpolymers such as hydrolyzed wheat protein/PVP crosspolymers; maltodextrin/VP copolymer; butylated/PVP copolymer; VP/polycarbamylpolyglycol ester; VP/dimethylaminoethylmethacrylate/polycarbamyl polyglycol ester; and VP/dimethiconyl-acrylate/polycarbamyl polyglycol ester. One of skill in the art will understand that the above film formers may be used in combination to achieve the desired properties of the final cosmetic composition. In most preferred embodiments, the film former comprises acrylate/C12-22methacrylate copolymer; PPG-17/IPDI/DMPA copolymer; Polyurethane-34; VP/polycarbamyl polyglycol ester; or combinations thereof. These preferred film formers are commercially available as ALLIANZ® OPT available from Ashland Inc.; SOLTEX® OPT PG available from Dow Chemical Company; AVALURE® UR 450 available from Lubrizol Corp.; POLYDERM® PPG-17 available from Alzo International Inc.; BAYCUSAN® available from Bayer Material Science LLC; and PECOGEL® available from Phoenix Chemical, Inc. The film former is present in the cosmetic composition in an amount of at least about 0.01 to about 30.0 wt. %, based on a total weight of the cosmetic composition. Preferably, the film former is present in an amount of about 0.1 to about 10.0 wt. %, based on a total weight of the cosmetic composition. In a most preferred embodiment, the film former is present in an amount of about 0.5 to about 3.0 wt. %, based on a total weight of the cosmetic composition. The cosmetic compositions of the present invention further include a skin adhesion promoter that may comprise one or more polymers. The skin adhesion promoter theoretically entraps the active ingredients within its polymeric network to prevent the actives from migrating. It can also act as a plasticizer to soften the film former and works synergistically with the film forming component to enhance the water resistance of the final cosmetic composition. Depending upon the other components of the formulation, the skin adhesion promoter may be hydrophobic, hydrophilic, or a combination of both. One of skill in the art will understand that the foregoing skin adhesion promoters may be used in combination to achieve the desired properties of the final cosmetic composition. Hydrophobic skin adhesion promoter polymers useful in the present invention may include, but are not limited to, copolymers of monomers of a fatty acid and IPDI. Examples of such copolymers include polydiethylene glycol adipate/IPDI copolymer; hydrogenated castor Oil/IPDI copolymer; polyglyceryl-2 diisostearate/IPDI copolymer; glyceryl diricinoleate/IPDI copolymer; propylene glycol diricinoleate/IPDI copolymer; dimethiconol/IPDI copolymer (and) cyclomethicone; bis-PPG-15 dimethicone/IPDI copolymer. A useful saturated methylene diphenyldiisocyanate (SMDI) copolymer may be PPG-12/SMDI copolymer. Particular crosspolymers of adipic acid monomers are also useful. Exemplary of those crosspolymers of adipic acid monomers are adipic acid/diglycol crosspolymer; and trimethylpentanediol/adipic acid/glycerin crosspolymer. The hydrophobic skin adhesion promoters may also comprise polyesters such as Polyester-10 combined with propylene glycol dibenzoate as an emollient (herein referred to as Polyester-10 (and) propylene glycol dibenzoate), or Polyester-7 combined with neopentyl glycol polyester as an emollient (herein referred to as Polyester-7 (and) neopentylglycol polyester). Other copolymers useful as skin adhesion promoters may be VP or PVP copolymers such as VP/hexadecene copolymer, VP/eicosene copolymer, and tricontanyl PVP; (meth)acrylate copolymers such as acrylic acid/isobornylmethacrylate/isobutylmethacrylate copolymer/isododecane in isododecane, or behenyl methacrylate/t-butyl methacrylate copolymer; or hydrogenated olefinic polymers such as hydrogenated polyisobutene and copolymers thereof; or hydrogenated polycyclopentadiene. Gel polymers such as petrolatum (and) ethylene/propylene/styrene copolymer, butylene/ethylene/styrene copolymer are also contemplated herein. Preferred hydrophobic skin adhesion promoter polymers comprise polydiethylene glycol adipate/IPDI copolymer available as POLYDERM® PPI-PE from Alzo International Inc.; Polyester-7 (and) neopentylglycol polyester which is available as LEXFILM® Sun from Inolex Chemical Company; VP/eicosene copolymer available as GANEX®/ANTARON® V-220/V-220F from Ashland Inc.; hydrogenated polyisobutene (and) butylene/ethylene/styrene copolymer (and) ethylene/propylene/styrene copolymer available as PARLEAM® Gel from NOF Corp, individually or combinations thereof. Hydrophilic skin adhesion promoters useful in the present invention may be selected from, but are not limited to, the following IPDI copolymers such as PEG-40 hydrogenated castor oil/IPDI copolymer; PEG-200 hydrogenated castor oil/IPDI copolymer; glycereth-7/hydroxystearate/IPDI copolymer; bis-PPG-15 dimethicone/IPDI copolymer; and Polyurethane-18; individually or combinations thereof. Preferred hydrophilic skin adhesion promoter polymers are bis-PPG-15 dimethicone/IPDI copolymer available as POLYDERM® PPI-SI-WS from Alzo International Inc.; and Polyurethane-18 available as POLYDERM PE/PA also from Alzo International. Preferably, the skin adhesion promoter is present in a weight ratio of skin adhesion promoter to the photoactives of the cosmetic composition from about 1:150 to about 1:0.1, and preferably in a weight ratio of about 1:80 to about 1:1. Alternatively, the skin adhesion promoter is present in an amount of about 0.05 to about 15.0 wt. %, more preferably in an amount of about 0.1 to about 7.0 wt. %, and most preferably in an amount of about 0.1 to about 3.0 wt. %, all based on a total weight of the composition. The cosmetic compositions of the present invention further comprise a color enhancer. The color enhancer promotes uniform distribution of the colorants in the cosmetic composition for improved color intensity, and prevents the colorants from rubbing or transferring off the skin onto clothing and other substrates. It provides enhanced wetting of the colorants within the cosmetic formulation. It is preferred that the color enhancer comprises a material containing silicon. Such silicon-based color enhancers of the present invention are preferably selected from, but are not limited to, silicone resins, silicone crosspolymers, silicone copolymers, silicone elastomers, silicone resin elastomer gels, silica, fumed silica, amorphous silica, silica dimethyl silylates, hydrophobic modified silica, hydrated silica, silicates, and combinations thereof. Preferred color enhancers useful in the present invention comprise copolymers or crosspolymers of trimethylsiloxysilicate such as trimethylsiloxysilicate/polypropylsilsesquioxane copolymer; polymethylsilsesquioxane; polyphenylsilsesquioxane; C30-45alkyl dimethylsilyl polypropylsilsesquioxane. Dimethicone copolymers or crosspolymers such as dimethicone (and) polysilicone-11 crosspolymer; dimethicone (and) cetearyl dimethicone crosspolymer; dimethicone (and) dimethicone crosspolymer; stearoxymethicone/dimethicone copolymer; and/or dimethicone (and) vinyldimethyl/trimethylsiloxysilicate/dimethicone crosspolymer are all contemplated. Silicas useful as color enhancers include, but are not limited to, hydrated silica, fumed silica, pyrogenic silica, and/or hydrophobic modified silica. Kaolin may also be used a color enhancer as well. One of skill in the art knows that one or more color enhancers may be needed to formulate the desired properties of the final cosmetic composition. Preferred color enhancers are trimethylsiloxysilicate available as BELSIL® from Wacker Chemie AG, or the MQ line of resins by Dow Corning, as well as other suppliers; trimethylsiloxysilicate (and) polypropylsilsesquioxane, another MQ resin from Dow Corning; dimethicone (and) polysilicone-11 available as GRANSIL® DMG-3 from Grant Industries Inc. Preferred silicas are available as CAB-O-SIL® from Cabot Corp. or SYLOID® from WR Grace & Company. The color enhancers are preferably present in the cosmetic composition of the present invention in a weight ratio of color enhancer to colorants ranging from about 1:50 to about 1:0.05. Preferably, the weight ratio of color enhancer to colorants ranges from about 1:10 to about 1:0.1, and most preferably, the weight ratio ranges from about 1:4 to about 1:0.4. Alternatively, the color enhancer is preferably present in an amount of about 0.01 wt. % to about 10.0 wt. %, more preferably from about 0.05 wt. % to about 10.0 wt. %, and most preferably from about 0.05 wt. % to about 2.5 wt. %. Colorants provide a variety of color to the cosmetic compositions of the present invention to further enhance skin appearance. Such colorants are known to one of skill in the art, particularly in the cosmetic composition field, and may include, but are not limited to, hennas, caramels, malva extracts, hibiscus extracts, tyrosines, green teas, glyceraldehydes, ginsengs, erythruloses, ferric compounds (including iron oxide), annattos, ultramarine pigments, beta-carotenes, carmines, chrome oxides, titanium oxide, zinc oxide, D&C water-soluble dyes, bismuth compounds, FD&C water-soluble dyes, copper powders, guanines, walnut extracts, iron oxides, micas, and combinations thereof. Hennas, caramels, micas, pearl pigments, pearlescent pigments, or combinations thereof, are preferred. Pearlescent pigments are most preferred, and can be selected from white pearlescent pigments, such as mica covered with titanium or with bismuth oxychloride; colored pearlescent pigments, such as titanium oxide-coated mica with iron oxides, titanium oxide-coated mica with ferric blue or chromium oxide; or titanium oxide-coated mica with an organic pigment of the abovementioned type, and pearlescent pigments based on bismuth oxychloride. The colorants are present in the cosmetic compositions of the present invention from about 0.01 wt. % to about 20.0 wt, %, preferably from about 0.05 wt. % to about 10.0 wt. %, more preferably from about 0.05 wt. % to about 2.5 wt. %, and most preferably from about 0.1 wt. % to about 1.5 wt. %, of these skin coloring agents, all based on a total weight of the composition. The cosmetic compositions of the present invention may optionally include other active or inactive ingredients such as those selected from, but not limited to, cosmetically acceptable carriers, oils, sterols, amino acids, moisturizers, powders, ultraviolet absorbents or photoactives, colorants (including pigments and/or dyes) pH adjusters, perfumes, essential oils, cosmetic active ingredients, vitamins, essential fatty acids, sphingolipids, self-tanning compounds such as dihydroxyacetone (DHA) and erythruloses, sunscreens, excipients, fillers, emulsifying agents, antioxidants, surfactants, film formers, chelating agents, gelling agents, thickeners, emollients, humectants, moisturizers, minerals, viscosity and/or rheology modifiers, keratolytics, retinoids, hormonal compounds, alpha-hydroxy acids, alpha-keto acids, anti-mycobacterial agents, anti-fungal agents, anti-microbials, anti-virals, analgesics, anti-allergenic agents, H1 or H2 antihistamines, anti-inflammatory agents, anti-irritants, anti-neoplastics, immune system boosting agents, immune system suppressing agents, anti-acne agents, anesthetics, antiseptics, insect repellents, skin cooling compounds, skin protectants, skin penetration enhancers, exfoliants, lubricants, fragrances, colorants, staining agents, depigmenting agents, hypopigmenting agents, preservatives, stabilizers, pharmaceutical agents, photostabilizing agents, spherical powders and mixtures thereof. The cosmetic compositions of the present invention may also have other optional additives. For instance, one or more fragrances; plant extracts; absorbents; thickeners/rheology modifiers; salicylic acid; alpha and beta hydroxy acids; vitamins including vitamins A, C, and E; retinol and its derivatives; preservatives; or any mixtures thereof, may be included in the cosmetic compositions. Exemplary embodiments of the cosmetic compositions of the present invention may comprise about 0.01 wt. % to about 30.0 wt. %. of the water dispersible film former; about 0.05 wt. % to about 15.0 wt. % of the skin adhesion promoter; about 0.01 wt. % to about 20 wt. % colorants; and the color enhancer to colorants weight ratio is about 1:50 to about 1:0.05, based on a total weight of the composition. Another exemplary embodiment of the cosmetic composition of the present invention may comprise about 0.1 wt. % to about 10.0 wt. % of the water dispersible film former; about 0.05 wt, % to about 7.0 wt. % of the skin adhesion promoter; about 0.1 wt, % to about 5.0 wt. % colorants; and the color enhancer to colorants weight ratio is about 1:10 to about 1:0.1, based on a total weight of the composition. Yet another exemplary embodiment of the cosmetic composition of the present invention may comprise about 0.5 wt. % to about 3.0 wt. % of the film former; about 0.1 wt. % to about 3.0 wt. % of the skin adhesion promoter; about 0.1 wt. % to about 5.0 wt. % colorants; and the color enhancer to colorants weight ratio is about 1:4 to about 1:0.4, based on a total weight of the composition. In a most preferred embodiment, the cosmetic compositions of the present invention comprise sunscreen compositions by incorporating one or more photoactive agents into the composition. The one or more photoactives that can be used in the present invention must be capable of absorbing or blocking the harmful effects of ultraviolet radiation. In addition, they must be non-toxic and non-irritating when applied to the skin. Suitable photoactives that may be used in the sunscreen composition include, but are not limited to p-aminobenzoic acid and derivatives thereof; butyl methoxydibenzoylmethane; benzophenones; hydroxy-substituted benzophenones; methoxy-substituted benzophenones; benzophonone-1; benzophenone-2; benzophenone-3; benzophenone-4; benzophenone-6; benzophenone-8; benzophenone-12; methoxycinnamate; ethyl dihydroxypropyl-p-aminobenzoate; glyceryl-p-aminobenzoate; homosalate; methyl anthranilate; octocrylene; octyl dimethyl-p-aminobenzoate; octyl methoxycinnamate; octyl salicylate; 2 phenylbenzimidazole-5-sulphonic acid; triethanolamine salicylate; 3-(4-methylbenzylidene)-camphor; red petrolatum, 3-(4-methylbenzyldine)boran-2-one(methylbenzindinecamphor); benzotriazole; salicylates; phenylbenzimidazole-5-sulfonic acid; methylene bis-benzotriazolyl tetramethylbutyl phenol; avobenzone; 4-isopropyldibenzoylmethane; butylmethoxydibenzoylmethane; octisalate; oxybenzone; bis-ethylhexyloxyphenol methoxy triazine; 4-isopropyl-dibenzoylmethane; metal oxides; zinc oxide; octyltriethoxy silanol; titanium dioxide; alumina; triethoxy silane; and combinations thereof. The preferred sunscreen agents are avobenzone, benzophenone-3, octyl methoxycinnamate, octyl salicylate, homosalate, zinc oxide, octocrylene, titanium dioxide, or combinations thereof. The one or more sunscreen agents are included in a present composition at about 1.0 wt. % to about 40.0 wt. % based on a total weight of the composition. The amount and types of photoactives in the composition will vary in the above range depending on the sun protection factor (SPF) desired. The higher the SPF, the greater the total amount of photoactives. Preferably, the one or more photoactives are present in an amount of about 1 wt. % to about 35 wt. % to achieve a SPF of about 2 to about 50. In one embodiment of the present invention, the cosmetic composition includes about 0.1 wt. % to about 10.0 wt. % of one or more film formers selected from the group consisting of acrylate/C12-22methacrylate copolymer, PPG-17/IPDI/DMPA copolymer, Polyurethane-34, VP/carbamyl polyglycol ester, and combinations thereof; about 0.05 wt. % to about 15.0 wt. % of one or more skin adhesion promoters selected from the group consisting of polydiethylene glycol adipate/IPDI copolymer, Polyester-7 (and) neopentylglycol polyester, VP/eicosene copolymer, hydrogenated polyisobutene (and) butylene/ethylene/styrene copolymer (and) ethylene/propylene/styrene copolymer, bis-PPG-15 dimethicone/IPDI, Polyurethane-18, and combinations thereof; about 0.2 wt. % to about 2.5 wt. % of one or more color enhancers selected from the group consisting of trimethylsiloxysilicate, trimethylsiloxysilicate (and) polypropylsilsesquioxane resin, dimethicone (and) polysilicone-11, fumed silica, hydrated silica, and combinations thereof; and one or more colorants, wherein said composition is very water resistant for up to 80 minutes. In another embodiment of the present invention, the cosmetic composition is a sunscreen composition having an SPF of 4 comprising about 1.0 wt. % to about 3.0 wt. % of a film former; about 0.05 wt. % to about 1.0 wt. % of a skin adhesion promoter; about 0.2 wt. % to about 2.5 wt. % of a color enhancer; and about 2.0 wt. % of one or more photoactive agent. Preferably, the photoactives comprise octisalate, octocrylene, and avobenzone. In yet another embodiment of the present invention, the cosmetic composition is a sunscreen composition having an SPF of 8 comprising 1.0 wt. % to about 3.0 wt. % of a film former; about 0.05 wt. % to about 1.0 wt. % of a skin adhesion promoter; about 0.2 wt. % to about 2.5 wt. % of a color enhancer; and about 5.0 wt. % of one or more photoactive agents. Preferably, the photoactives comprise octisalate, octocrylene, and avobenzone. In yet another embodiment of the present invention, the cosmetic composition is a sunscreen composition having an SPF of 15 comprising 1.0 wt. % to about 3.0 wt. % of a film former; about 0.05 wt. % to about 1.0 wt. % of a skin adhesion promoter; about 0.2 wt. % to about 2.5 wt. % of a color enhancer; and about 8.0 wt. % of one or more photoactives. Preferably, the photoactives comprise octisalate, octocrylene, and avobenzone. In a particular embodiment of the present invention, the cosmetic composition is a sunscreen composition having an SPF of 30 comprising 1.0 wt. % to about 3.0 wt. % of a film former; about 0.05 wt. % to about 1.0 wt. % of a skin adhesion promoter; about 0.2 wt. % to about 2.5 wt. % of a color enhancer; and about 13.0 wt. % of one or more photoactives. Preferably, the photoactives comprise homosalate, octisalate, octocrylene, and avobenzone. One or more colorants may be included in the preceding sunscreen compositions in an amount of about 0.1 wt. % to about 5.0 wt. %. Self-tanners may also be incorporated as well. The cosmetic compositions may be prepared by using techniques and methods well known in the art. In general, all water soluble ingredients are placed in a main vessel such as thickeners, chelating agents, humectants, pH adjusters, water dispersible film formers, hydrophilic skin adhesion promoters, and/or water soluble actives. This water phase is mixed until uniform and heated to about 80° C. In a separate vessel, the oil soluble ingredients such as solvents, emollients, emulsifiers, emulsion stabilizers, preservatives, hydrophobic skin adhesion promoters, and/or oil soluble actives, including photoactives, are mixed until uniform and heated to about 80° C. The oil phase is added to the water phase, homogenized for 5 minutes until uniform, and the resultant mixture is cooled to room temperature. During the cooling period, the remaining ingredients are admixed therein. Any ingredients that are heat sensitive are added at this point. The cosmetic compositions of the present invention can then be formulated and packaged as a lotion, cream gel, spray, including alcohol-based sprays, or other acceptable carriers suitable for a cosmetic composition, in particular, a sunscreen composition. The following testing of inventive formulations and comparative formulations unexpectedly show that the synergistic effects of the water dispersible film former, skin adhesion promoters, and color enhancers work together to improve the color intensity and color retention of the cosmetic compositions of the present invention. An initial color reading of a subject's forearm skin was measured using a Konica Spectrophotometer CM-2600D with three sensors represented by numerical values plotted in a colorspace characterized by the brightness, L*, and the color coordinates a* and b*, as defined by the International Lighting Commission (Commission Internationale de l'Eclairage, or CIE) (1976). This initial reading is the baseline for each subject. Twenty-five milligrams of each sample was lightly spread within a 4 cm diameter circular test area on the subject's forearms. The sample was allowed to dry on the skin for 5 minutes. A second reading, ΔE1, was taken, representing the color difference between the baseline and after application of the sample. Samples showing good color intensity after application are marked with a positive sign “+.” Raw data was statistically analyzed using the Student t-test. Thereafter, the test area was subjected to a wet-rub method to evaluate the color retention of the samples on human skin after water exposure. This method simulates the effect of exposing the application area to water. After the initial application was dried, about 250 ml of tap water was used to completely wet the test area. A paper towel was placed on the top of the wet test area and a paint roller (6 inch×¾ inch nap) pre-wrapped with two pounds of waist/angle weight was rolled over the top of the paper towel to create friction between the forearm test area and the paper towel. After rolling back and forth 20 times, a third reading, ΔE2, was taken thereafter representing the difference in color between the initial application and after the wet-rub. Samples showing good color retention, when compared with the color intensity after the initial application and after the wet rub, are marked with a positive sign “+.” Raw data was statistically analyzed using the Student t-test. A lotion, Base Formula A, was prepared using methods known to one of skill in the art in formulating cosmetic compositions. Components of Base Formula A are listed in Table I below. Samples were prepared and tested with individual components of the cosmetic composition of the present invention and then compared with a cosmetic composition of the present invention highlighting the synergistic effect when the film former, skin adhesion promoter, and color enhancer are present in the same composition. TABLE IBase Formula A - LotionComponentwt. %*Deionized WaterQS. to 100Acrylates/C10-30alkyl acrylate crosspolymer0.2-0.4Disodium EDTA0.05-0.2Glycerin1.0-4.0Sodium Hydroxide0.08-0.2Diisopropyl adipate1.0-3.0Polyglyceryl-3 methylglucose distearate1.0-3.0Glyceryl stearate and PEG-100 stearate0.5-2.0Cetyl alcohol0.1-2.0Butters0.1-1.0Octocrylene1.0Avobenzone0.5Caramel and Pearls0.1-5.0Phenoxyethanol, methyl and propyl parabens0.3-1.5Vitamins0.01-0.1Botanical extracts and powders0.1-2.0Fragrance0.25*based on a total weight of the composition As shown in Table II, Samples 1 to 6 were prepared using Base Formula A as a base composition, and incorporating one or more of the individual components of the cosmetic composition of the present invention. Sample 7 is a composition of the present invention which demonstrates the unexpected synergy of the film former, skin adhesion promoter, and color enhancer when used concurrently in the base formulation. TABLE IILotion SamplesSample #Components1Base Formula A+6 wt. % snow white petrolatum USP2Base Formula A+6 wt. % snow white petrolatum USP+7 wt. % acrylates/C12-22methacrylate copolymer¥3Base Formula A+6 wt. % snow white petrolatum USP+7 wt. % polydiethyleneglycol adipate/IPDI copolymer4Base Formula A+6 wt. % snow white petrolatum USP+7 wt. % trimethylsiloxysilicate (CE)5Base Formula A+6 wt. % snow white petrolatum USP+3.5 wt. % acrylates/C12-22methacrylate copolymer+3.5 wt. % trimethylsilosilicate6Base Formula A+6 wt. % snow white petrolatum USP+3.5 wt. % acrylates/C12-22methacrylate copolymer+3.5 wt. % polydiethyleneglycol adipate/IPDI copolymer7Base Formula A+6 wt. % snow white petrolatum USP+3.5 wt. % acrylates/C12-22methacrylate copolymer+2.5 wt. % trimethylsilosiliate+1 wt. % polydiethyleneglycol adipate/IPDI copolymer Water resistance testing was conducted in accordance with 21 C.F.R. § 352.72(a). Samples 1 to 7 met or exceeded the 2011 U.S. Food & Drug Administration (FDA) water resistance guidelines for 80 minutes. Base Formula A lotion, the control, which did not contain any film formers, skin adhesion promoters, color enhancers, showed both poor initial color intensity and subsequently poor color retention after the wet rub. Samples 1, 3, 4 and 6 did not show significant improvement in skin color and color retention in comparison to the control. Samples 2 and 5 did not show significant improvement in color intensity, but did show significant improvement on color retention after wet rub in comparison to the control. Only Sample 7, the inventive composition, showed statistically significant improvements in both color intensity and color retention when compared to the performance of the control. Sample #ΔE1ΔE2% Color RetentionBase Formula A−−721−−692−−943−−594−−835−−886−−757*++87*p < 0.05 by Student t-test A second formulation, Base Formula B, was prepared using methods known to one of skill in the art in formulating cosmetic compositions. Components of Base Formula B, a low viscosity spray lotion, are listed in Table III below. Samples were prepared with individual components of the cosmetic composition of the present invention and then compared with a cosmetic composition of the present invention highlighting the synergistic effect when the film former, skin adhesion promoter, and color enhancer are present in the same composition. TABLE IIIBase Formula B - Low Viscosity Spray LotionComponentwt. %*Deionized WaterQS. to 100Xanthan Gum0.1-1.0Orange Fiber0.3-1.0Disodium EDTA0.05-0.2Glycerin0.5-4.0Sodium Hydroxide0.01-0.2Diisopropyl adipate1.0-3.0Polyglyceryl-3 methylglucose distearate1.0-3.0Glyceryl stearate and PEG-100 stearate0.5-2.0Cetyl alcohol0.1-2.0Octisalate2.0Octocrylene1.4Avobenzone1.2Caramel and Pearls0.1-5.0Phenoxyethanol, Methyl and Propyl parabens0.5-1.6Cocoa powders0.1-2.0Fragrance0.25*based on a total weight of the composition As shown in Table IV, Samples 8 and 9 were prepared using Base Formula B and incorporating one or more of the individual components of the cosmetic composition of the present invention. Sample 10 is a composition of the present invention which shows the unexpected synergy of the film former, skin adhesion promoter, and color enhancer when used concurrently in the base formulation. TABLE IVSpray Lotion SamplesSample #Components8Base Formula B+7 wt. % VP/eicosene copolymer9Base Formula B+7 wt. % PPG-17/IPDI/DMPA copolymer10Base Formula B+3.5 wt. % acrylates/C12-22methacrylate copolymer+2.5 wt. % trimethylsilosiliate+1 wt. % polydiethyleneglycol adipate/IPDI copolymer Water resistance testing was conducted in accordance with 21 C.F.R. § 352.72(a). Sample 10 met or exceeded the 2011 U.S. Food & Drug Administration (FDA) water resistance guidelines for 80 minutes. Sample #ΔE1ΔE2% Color Retention8−−879−−8010*−+99*p < 0.01 by Student t-test Sample 10, the inventive composition, unexpectedly showed significant improvements in initial color intensity and color retention after wet rub than both Samples 8 and 9. A third formulation Base Formula, C, was prepared using methods known to one of skill in the art in formulating cosmetic compositions. Components of Base Formula C, a cream gel, are listed in Table V below. Samples were prepared with individual components of the cosmetic composition of the present invention and then compared with a cosmetic composition of the present invention highlighting the synergistic effect when the film former, skin adhesion promoter, and color enhancer are present in the same composition. TABLE VBase Formula C - Cream GelComponentswt. %*Deionized WaterQS. to 100Acrylates/C10-30Alkyl acrylate crosspolymer0.5-1.5Hydroxyethylcellulose0.1-1.0Disodium EDTA0.05-0.2Glycerin0.5-4.0Water, Glycerin, Glyceryl Acrylate/Acrylic acid copolymer,1.0-5.0Propylene glycol (and) PVM/MA copolymerAminomethylpropanol and water0.5-1.0Polyglyceryl-4 laurate/Sebacate (and) Polyglyceryl-64.0-8.0caprylate/Caprate (and) waterOctisalate2.0Octocrylene1.4Avobenzone1.2Caramel and Pearls0.1-5.0Phenoxyethanol and Ethylhexyl glycerin1.0-2.0*based on a total weight of the composition As shown in Table VI, Samples 11 was prepared using Base Formula C as a cream gel and incorporating only a skin adhesion promoter. Sample 12 is a composition of the present invention which shows the unexpected synergy of the film former, skin adhesion promoter, and color enhancer when used concurrently in the base formulation. TABLE VICream Gel SamplesSample #Components11Base Formula C+7 wt. % bis-PEG-15 dimethicone/IPDI copolymer12Base Formula C+3.5 wt. % acrylates/C12-22methacrylate copolymer+2.5 wt. % trimethylsilosiliate+1 wt. % polydiethyleneglycol adipate/IPDI copolymer Sample 12, an inventive composition, showed statistically significant improvement in overall color intensity when compared to Sample 11. Sample #ΔE1ΔE2% Color Retention11−−5312*−+86*p < 0.05 by Student t-test To further evaluate the inventive composition of the present inventions, a low viscosity formulation of the present invention that includes the film former, skin adhesion promoter and color enhancer, similar to sample 10, was also evaluated by consumers for a two week period using the home-use test. In a total of 156 consumers, over 70% of respondents agreed that the inventive formulation enhanced their appearance such as providing a bronzed look, a beautiful sheen to their skin, natural looking color, as well as streak-free color. At least 73% of respondents agreed that the color did not rub off on their clothes. Over 75% of respondents agreed that this particular formulation felt good on the skin, did not leave the skin feeling greasy, and helped even out the skin tone. The above invention achieves the objects recited above. Inventive compositions of the present invention comprising a film former, skin adhesion promoter, and color enhancer are water resistant, and unexpectedly provide improved color intensity and color retention, with a non-greasy skin feel and appearance. While the present invention has been particularly described, in conjunction with specific preferred embodiments, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. It is therefore contemplated that the appended claims will embrace any such alternatives, modifications and variations as falling within the true scope and spirit of the present invention. | 34,188 |
11857659 | DETAILED DESCRIPTION OF THE INVENTION Unless stated otherwise, percentages (%) are meant to designate percent by weight of a composition. The present invention now has determined a way to improve the efficiency in depositing microcapsules on a substrate in rinse-off conditioning applications. What is referred to as improving deposition or improving deposition efficiency is the percentage of microcapsules that remains on a substrate during use, in particular that remains on a substrate after a rinsing step. Better deposition translates then into an improvement in the delivery performance of the active ingredient encapsulated, for instance the olfactive performance in the case of a perfume, meaning that the microcapsules are able to deliver long lasting perception of a fragrance. It has been surprisingly found that the full or partial replacement of quaternary ammonium salts by non quaternized conditioning ingredients and optionally by water soluble cationic conditioning polymers in a composition comprising cationically coated microcapsules could significantly improve the performance of those microcapsules in terms of deposition. A first object of the invention is therefore a rinse-off conditioner composition comprising:a core-shell microcapsules slurry comprising microcapsules having an oil-based core and a polymeric shell coated with at least one cationic polymer;up to 4 wt % by weight of at least one quaternary ammonium salt;from 0.25 to 15 wt % by weight of at least one non quaternized conditioning ingredient comprising an oil or a wax or a mixture thereof;less than 2% by weight of at least one water soluble cationic conditioning polymer;based on the total weight of the composition. Core-Shell Microcapsules A “core-shell microcapsule”, or the similar, in the present invention is meant to designate a capsule that has a particle size distribution in the micron range (e.g. a mean diameter (d(v, 0.5)) comprised between about 1 and 3000 μm) and comprises an external solid oligomer-based shell or a polymeric shell and an internal continuous phase enclosed by the external shell. The core-shell microcapsule according to the invention comprises an oil-based core. By “oil”, it is meant an organic phase that is liquid at about 20° C. which forms the core of the core-shell capsules. According to any one of the invention embodiments, said oil comprises an ingredient or composition selected amongst a perfume, perfume ingredient, flavour, flavour ingredient, nutraceuticals, cosmetic ingredient, sunscreen agent, insecticide, malodour counteracting substance, bactericide, fungicide, biocide actives, insect repellent or attractant, insect control agent, drug, agrochemical ingredient and mixtures thereof. According to a particular embodiment, said oil-based core comprises a perfume with another ingredient selected from the group consisting of nutraceuticals, cosmetics, insect control agents and biocide actives. According to a particular embodiment, the oil-based core comprises a perfume or flavour. According to a preferred embodiment, the oil-based core comprises a perfume. According to another embodiment, the oil-based core consists of a perfume. By “perfume oil” (or also “perfume”) what is meant here is an ingredient or composition that is a liquid at about 20° C. According to any one of the above embodiments said perfume oil can be a perfuming ingredient alone or a mixture of ingredients in the form of a perfuming composition. As a “perfuming ingredient” it is meant here a compound, which is used for the primary purpose of conferring or modulating an odour. In other words such an ingredient, to be considered as being a perfuming one, must be recognized by a person skilled in the art as being able to at least impart or modify in a positive or pleasant way the odor of a composition, and not just as having an odor. For the purpose of the present invention, perfume oil also includes combination of perfuming ingredients with substances which together improve, enhance or modify the delivery of the perfuming ingredients, such as perfume precursors, emulsions or dispersions, as well as combinations which impart an additional benefit beyond that of modifying or imparting an odor, such as long-lasting, blooming, malodour counteraction, antimicrobial effect, microbial stability, insect control. The nature and type of the perfuming ingredients present in the oil phase do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of its general knowledge and according to intended use or application and the desired organoleptic effect. In general terms, these perfuming ingredients belong to chemical classes as varied as alcohols, aldehydes, ketones, esters, ethers, acetates, nitriles, terpenoids, nitrogenous or sulphurous heterocyclic compounds and essential oils, and said perfuming co-ingredients can be of natural or synthetic origin. Many of these co-ingredients are in any case listed in reference texts such as the book by S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, N.J., USA, or its more recent versions, or in other works of a similar nature, as well as in the abundant patent literature in the field of perfumery. It is also understood that said ingredients may also be compounds known to release in a controlled manner various types of perfuming compounds. The perfuming ingredients may be dissolved in a solvent of current use in the perfume industry. The solvent is preferably not an alcohol. Examples of such solvents are diethyl phthalate, isopropyl myristate, Abalyn® (rosin resins, available from Eastman), benzyl benzoate, ethyl citrate, limonene or other terpenes, or isoparaffins. Preferably, the solvent is very hydrophobic and highly sterically hindered, like for example Abalyn® or benzyl benzoate. According to a particular embodiment, the solvent comprises low odour, high density materials like benzyl salicylate, cyclohexyl salicylate, hexyl salicylate. Preferably the perfume comprises less than 30% of solvent. More preferably the perfume comprises less than 20% and even more preferably less than 10% of solvent, all these percentages being defined by weight relative to the total weight of the perfume. Most preferably, the perfume is essentially free of solvent. The nature of the polymeric shell of the microcapsules of the invention can vary. As non-limiting examples, the shell can be made of a polymeric material selected from the group consisting of polyurea, polyurethane, polyamide, polyacrylate, polysiloxane, polycarbonate, polysulfonamide, urea formaldehyde, melamine formaldehyde resin, melamine urea resin, melamine glyoxal resin, gelatin/gum arabic shell wall, and mixtures thereof. The shell can also be hybrid, namely organic-inorganic such as a hybrid shell composed of at least two types of inorganic particles that are cross-linked, or yet a shell resulting from the hydrolysis and condensation reaction of a polyalkoxysilane macro-monomeric composition. According to an embodiment, the shell comprises an aminoplast copolymer, such as melamine-formaldehyde or urea-formaldehyde or cross-linked melamine formaldehyde or melamine glyoxal. According to a particular embodiment, the core-shell microcapsules are cross-linked melamine formaldehyde microcapsules obtainable by a process comprising the steps of:1) admixing a perfume oil with at least a polyisocyanate having at least two isocyanate functional groups to form an oil phase;2) dispersing or dissolving into water an aminoplast resin and optionally a stabilizer to form a water phase;3) adding the oil phase to the water phase to form an oil-in-water dispersion, wherein the mean droplet size is comprised between 1 and 100 microns, by admixing the oil phase and the water phase;4) performing a curing step to form the wall of said microcapsule; and5) optionally drying the final dispersion to obtain a dried core-shell microcapsule. This process is described in more details in WO 2013/092375 & WO 2015/110568, the contents of which are included by reference. According to another embodiment the shell is polyurea-based made from, for example but not limited to isocyanate-based monomers and amine-containing crosslinkers such as guanidine carbonate and/or guanazole. Preferred polyurea-based microcapsules comprise a polyurea wall which is the reaction product of the polymerisation between at least one polyisocyanate comprising at least two isocyanate functional groups and at least one reactant selected from the group consisting of an amine (for example a water soluble guanidine salt and guanidine); a colloidal stabilizer or emulsifier; and an encapsulated perfume. However, the use of an amine can be omitted. According to another embodiment, the shell is polyurethane-based made from, for example but not limited to polyisocyanate and polyols, polyamide, polyester, etc. According to a particular embodiment the colloidal stabilizer includes an aqueous solution of between 0.1% and 0.4% of polyvinyl alcohol, between 0.6% and 1% of a cationic copolymer of vinylpyrrolidone and of a quaternized vinylimidazol (all percentages being defined by weight relative to the total weight of the colloidal stabilizer). According to another embodiment, the emulsifier is an anionic or amphiphilic biopolymer preferably chosen from the group consisting of polyacrylate (and copolymers especially with acrylamide), gum arabic, soy protein, gelatin, sodium caseinate and mixtures thereof. According to a particular embodiment, the polyisocyanate is an aromatic polyisocyanate, preferably comprising a phenyl, a toluyl, a xylyl, a naphthyl or a diphenyl moiety. Preferred aromatic polyisocyanates are biurets and polyisocyanurates, more preferably a polyisocyanurate of toluene diisocyanate (commercially available from Bayer under the tradename Desmodur® RC), a trimethylol propane-adduct of toluene diisocyanate (commercially available from Bayer under the tradename Desmodur® L75), a trimethylol propane-adduct of xylylene diisocyanate (commercially available from Mitsui Chemicals under the tradename Takenate® D-110N). According to a particular embodiment, the polyisocyanate is a trimethylol propane-adduct of xylylene diisocyanate (commercially available from Mitsui Chemicals under the tradename Takenate® D-110N). The preparation of an aqueous dispersion/slurry of core-shell microcapsules is well known from a skilled person in the art. In one aspect, said microcapsule wall material may comprise any suitable resin and especially including melamine, glyoxal, polyurea, polyurethane, polyamide, polyester, etc. Suitable resins include the reaction product of an aldehyde and an amine, suitable aldehydes include, formaldehyde and glyoxal. Suitable amines include melamine, urea, benzoguanamine, glycoluril, and mixtures thereof. Suitable melamines include, methylol melamine, methylated methylol melamine, imino melamine and mixtures thereof. Suitable ureas include, dimethylol urea, methylated dimethylol urea, urea-resorcinol, and mixtures thereof. Suitable materials for making may be obtained from one or more of the following companies Solutia Inc. (St Louis, Mo. U.S.A.), Cytec Industries (West Paterson, N.J. U.S.A.), Sigma-Aldrich (St. Louis, Mo. U.S.A.). According to a particular embodiment, the core-shell microcapsule is a formaldehyde-free capsule. A typical process for the preparation of aminoplast formaldehyde-free microcapsules slurry comprises the steps of:1) preparing an oligomeric composition comprising the reaction product of, or obtainable by reacting togethera) a polyamine component in the form of melamine or of a mixture of melamine and at least one C1-C4compound comprising two NH2functional groups;b) an aldehyde component in the form of a mixture of glyoxal, a C4-62,2-dialkoxy-ethanal and optionally a glyoxalate, said mixture having a molar ratio glyoxal/C4-62,2-dialkoxy-ethanal comprised between 1/1 and 10/1; andc) a protic acid catalyst;2) preparing an oil-in-water dispersion, wherein the droplet size is comprised between 1 and 600 um, and comprising:i. an oil;ii. a water mediumiii. at least an oligomeric composition as obtained in step 1;iv. at least a cross-linker selected amongstA) C4-C12aromatic or aliphatic di- or tri-isocyanates and their biurets, triurets, trimmers, trimethylol propane-adduct and mixtures thereof; and/orB) a di- or tri-oxiran compounds of formulaA-(oxiran-2-ylmethyl)nwherein n stands for 2 or 3 and 1 represents a C2-C6group optionally comprising from 2 to 6 nitrogen and/or oxygen atoms;v. optionally a C1-C4compounds comprising two NH2functional groups;3) heating said dispersion;4) cooling said dispersion. This process is described in more details in WO 2013/068255, the content of which is included by reference. According to another embodiment, the shell of the microcapsule is polyurea- or polyurethane-based. Examples of processes for the preparation of polyurea and polyurethane-based microcapsule slurry are for instance described in WO2007/004166, EP2300146, EP2579976 the contents of which is also included by reference. Typically a process for the preparation of polyurea or polyurethane-based microcapsule slurry include the following steps:a) dissolving at least one polyisocyanate having at least two isocyanate groups in an oil to form an oil phase;b) preparing an aqueous solution of an emulsifier or colloidal stabilizer to form a water phase;c) adding the oil phase to the water phase to form an oil-in-water dispersion, wherein the mean droplet size is comprised between 1 and 500 μm, preferably between 5 and 50 μm;d) applying conditions sufficient to induce interfacial polymerisation and form microcapsules in form of a slurry. Cationic Coating Microcapsules present in the particular composition of the invention are coated with at least a cationic polymer. According to the invention, to form the cationic coating on microcapsules, the cationic polymer is added at some stage during the formation of the capsule slurry. In other words, the cationic coating is already present on microcapsules when they are added to the rinse-off conditioner composition and does not come from soluble cationic conditioning polymers or from the quaternary ammonium salts present in the composition. The microcapsule according to the invention is preferably anionic before coating with the cationic polymer as the preferred emulsifiers are negatively charged polymers. The coating of such an anionic microcapsule with a cationic polymer is well-known from a skilled person in the art. Cationic polymers are also well known to a person skilled in the art. Preferred cationic polymers have cationic charge densities of at least 0.5 meq/g, more preferably at least about 1.5 meq/g, but also preferably less than about 7 meq/g, more preferably less than about 6.2 meq/g. The cationic charge density of the cationic polymers may be determined by the Kjeldahl method as described in the US Pharmacopoeia under chemical tests for Nitrogen determination. The preferred cationic polymers are chosen from those that contain units comprising primary, secondary, tertiary and/or quaternary amine groups that can either form part of the main polymer chain or can be borne by a side substituent directly connected thereto. The weight average (Mw) molecular weight of the cationic polymer is preferably between 10,000 and 3.5M Dalton, more preferably between 50,000 and 2M Dalton. According to a particular embodiment, one will use cationic polymers based on acrylamide, methacrylamide, N-vinylpyrrolidone, quaternized N,N-dimethylaminomethacrylate, diallyldimethylammonium chloride, quaternized vinylimidazole (3-methyl-1-vinyl-1H-imidazol-3-ium chloride), vinylpyrrolidone, acrylamidopropyltrimonium chloride, cassia hydroxypropyltrimonium chloride, guar hydroxypropyltrimonium chloride or polygalactomannan 2-hydroxypropyltrimethylammonium chloride ether, starch hydroxypropyltrimonium chloride and cellulose hydroxypropyltrimonium chloride. Preferably copolymers shall be selected from the group consisting of polyquaternium-5, polyquaternium-6, polyquaternium-7, polyquaternium 10, polyquaternium-11, polyquaternium-16, polyquaternium-22, polyquaternium-28, polyquaternium-43, polyquaternium-44, polyquaternium-46, cassia hydroxypropyltrimonium chloride, guar hydroxypropyltrimonium chloride or polygalactomannan 2-hydroxypropyltrimethylammonium chloride ether, starch hydroxypropyltrimonium chloride and cellulose hydroxypropyltrimonium chloride. As specific examples of commercially available products, one may cite Salcare® SC60 (cationic copolymer of acrylamidopropyltrimonium chloride and acrylamide, origin: BASF) or Luviquat®, such as the PQ 11N, FC 550 or Style (polyquaternium-11 to 68 or quaternized copolymers of vinylpyrrolidone origin: BASF), or also the Jaguar® (C13S or C17, origin Rhodia). In the present invention “poly(acrylamidopropyltrimonium chloride-co-acrylamide” and “acrylamidopropyltrimonium chloride/acrylamide copolymer” are used indifferently. According to any one of the above embodiments of the invention, there is added an amount of cationic polymer comprised between about 0.25 and 2.0 wt %, or even between about 0.5 and 1.5 wt %, percentage being expressed on a w/w basis relative to the total weight of the microcapsule slurry. According to a particular embodiment, microcapsules are coated with a mixture of at least two cationic polymers. Such microcapsules are disclosed in WO 2017/001385 the content of which is also included by reference. Quaternary Ammonium Salts Quaternary ammonium conditioning agents that can be used in the present invention are well known to those skilled in the art. Examples of such compounds are described in US2006/0210509 ([19] to [32]). It has been found that a composition comprising a reduced amount of quaternary ammonium salts can be used to obtain a high deposition of microcapsules. Thus, according to the invention, the composition comprises up to 4%, preferably up to 3%, more preferably up to 1.5% by weight of quaternary ammonium salts. According to an embodiment, the composition comprises between 0 and 4 wt %, more preferably between 0 and 3 wt %, even more preferably between 0 and 1.5 wt % by weight of quaternary ammonium salts based on the total weigh of the composition. According to another embodiment, the composition comprises between 0.01 and 4 wt %, more preferably between 0.01 and 3 wt %, even more preferably between 0.01 and 1.5 wt %, by weight of quaternary ammonium salts based on the total weigh of the composition. According to a particular embodiment, the composition is free of quaternary ammonium salts. Quaternary ammonium salts of the present invention are preferably quaternary ammonium salts bearing at least one long alkyl chain having between 10 carbons and 24 carbons. As non-limiting examples, one may cite behentrimonium chloride, cetrimonium chloride, behentrimonium methosulfate, ester-containing quaternary ammonium salts such as monoesterquats, diesterquats and triesterquats and mixtures thereof. Surprisingly, it has been found that the partial or total substitution of quaternary ammonium salts with non-quarternized conditioning oils and optionally with water soluble cationic conditioning polymers and copolymers could improve the deposition of microcapsules. Non-Quaternized Conditioning Ingredients According to the invention, the non-quaternized conditioning ingredient is preferably hydrophobic or amphiphilic and comprises an oil or a wax or a mixture thereof. According to an embodiment, the non-quaternized conditioning ingredient is chosen in the group consisting of an oil, a wax and mixture thereof. According to the invention, the composition comprises between 0.25 and 15% by weight, preferably between 1 and 15%, more preferably between 3 and 15%, even more preferably between 5 and 15%, even more preferably between 6 and 15% by weight of non-quarternized conditioning ingredients based on the total weigh of the composition. Non quaternized conditioning ingredients can be chosen from the group consisting of polysiloxanes, aminosiloxanes, dimethicone copolyols, alkyl silicone copolymers mineral oil, organic oils such as Macadamia oil, Jojoba oil, sunflower oil, almond oil, olive oil, fatty alcohols such as lanolin alcohol and cetearyl alcohol, fatty acids such as stearic acid, lauric acid, and palmitic acid, fatty acid esters, fatty acid amides such as Bis-Ethyl(Isostearylimidazoline) Isostereamide (Keradyn™ HH), bee wax, and mixtures thereof. According to a particular embodiment, the non quaternized conditioning ingredient is chosen in the group consisting of stearate esters, cetearyl alcohol, jojoba oil, paraffin oil, bee wax, macadamia oil, lauric acid, olive oil, Bis-Ethyl(Isostearylimidazoline) Isostereamide, amodimethicone, dimethicone, and mixtures thereof. According to an embodiment, the at least one non quaternized conditioning ingredient is a mixture between cetearyl alcohol and at least one component chosen in the group consisting of jojoba oil, paraffin oil, bee wax, macadamia oil, lauric acid, olive oil, Bis-Ethyl(Isostearylimidazoline) Isostereamide and mixtures thereof. According to a particular embodiment, the non quaternized conditioning ingredient comprises Jojoba oil, preferably in combination with cetearyl alcohol. According to another particular embodiment, the non quaternized conditioning ingredient comprises paraffin oil, preferably in combination with cetearyl alcohol. According to another particular embodiment, the non quaternized conditioning ingredient comprises Bis-Ethyl(Isostearylimidazoline) Isostereamide (Keradyn™ HH), preferably in combination with cetearyl alcohol. Water Soluble Cationic Conditioning Polymers The composition of the invention can comprise water soluble cationic conditioning polymers and co-polymers preferably based on quarternized N,N-dimethylaminomethacrylate, diallyldimethylammonium chloride, quarternized vinylimidazole, vinylpyrrolidone, cassia hydroxypropyltrimonium chloride, guar hydroxypropyltrimonium chloride, polygalactomannan 2-hydroxypropyltrimonium chloride ether, starch hydroxypropyltrimonium chloride, cellulose hydroxypropyltrimoniumchloride, polyquaternium-5, polyquaternium-6, polyquaternium-7, polyquaternium-10, polyquaternium-11, polyquaternium-16, polyquaternium-22, polyquaternium-28, polyquaternium-43, polyquaternium-44, polyquaternium-46 and mixtures thereof and copolymers and terpolmers of the above with acrylic acid, methacrylic acid, acrylamide, methylacrylamide and N-vinylpyrrolidone. The composition comprises less than 2%, preferably less than 1% of water soluble cationic conditioning polymers and co-polymers, preferably from 0.1 to 1% by weight based on the total weigh of the composition. According to a particular embodiment, the composition is free of water soluble cationic conditioning polymers. According to a particular embodiment, the composition comprises:from 0.1 to 5 wt % of a core-shell microcapsules slurry comprising microcapsules having an oil-based core and a polymeric shell coated with at least one cationic polymer;up to 3 wt % by weight of at least one quaternary ammonium salt, preferably chosen in the group consisting of behentrimonium chloride, cetrimonium chloride, behentrimonium methosulfate, ester-containing quaternary ammonium salts such as monoesterquats, diesterquats and triesterquats and mixtures thereof;from 5 to 15%, preferably from 6 to 15 wt % by weight of at least one non quaternized conditioning ingredients, preferably chosen in the group consisting of stearate esters, cetearyl alcohol, jojoba oil, paraffin oil, bee wax, macadamia oil, lauric acid, olive oil, Bis-Ethyl(Isostearylimidazoline) Isostereamide, amodimethicone, dimethicone, and mixtures thereof;less than 1% by weight of water soluble cationic conditioning polymers, preferably chosen in the group consisting of Acrylamidopropyltrimonium Chloride/Acrylamide Copolymer, Guar Hydroxypropyltrimonium Chloride and mixture thereof;based on the total weight of the composition. According to an embodiment, the composition of the invention is free of anionic, amphoteric or zwitterionic surfactants. Other Ingredients The rinse-off conditioner composition of the invention may comprise one or more ingredients including those well-known in the art for use in rinse-off conditioner such viscosity modifiers, dyes, thickeners, solubilizers, foam boosters, perfumes. The composition may be in the form of a liquid, having a viscosity preferably comprised between 1500 and 30 000 cPs, preferably between 4000 and 30,000 cPs, more preferably between 5000 and 25,000 cPs. Viscosities were measured 24 hours after the sample preparation with a BROOKFIELD viscosimeter DV-II+at 20 rpm with spindle 5 at 25° C. According to an embodiment, the composition comprises a cosmetically acceptable aqueous phase that may be present at a level of from about 20% to about 95%, preferably from about 60% to about 85%. The cosmetically acceptable aqueous phase may be selected from the group consisting of water and water solutions of lower alkyl alcohols and polyhydric alcohols. The lower alkyl alcohols may be monohydric alcohols having 1 to 6 carbons. In an embodiment, the lower alkyl alcohols are ethanol and isopropanol. The polyhydric alcohols may be ethylene glycol, propylene glycol, hexylene glycol, glycerin, and propane diol. One may also cite Ethylene glycol ethers, polyethylene glycol ethers such as TWEEN-20 or BRIJ S20, polypropylene glycol ethers and polyethylene/polypropylene glycol ethers. According to an embodiment, the composition of the invention is in the form of a hair conditioner, preferably rinse-off conditioner, conditioning shampoo, skin conditioning rinse-off product such as in-shower lotion. Another object of the invention is the use of a composition as defined above for depositing microcapsules on a surface, preferably on hair and/or skin. Another object of the invention is a method for improving deposition of microcapsules on a surface, which comprises treating said surface with the composition of the invention as defined above. The invention will now be further described by way of examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples. EXAMPLES Deposition For the following examples, the analytical deposition of microcapsules onto hair was measured as described below. Fragranced microcapsules were added to the rinse-off compositions described in the invention at a dosage of 0.2% encapsulated perfume. The microcapsules contained perfume A described in Example 1, Table 1 and a UV tracer (Uvinul A Plus). For the quantification of deposition, the following procedure was used. A 500 mg mini brown Caucasian hair swatch was wet with 40 mL of tap water (39° C.). The excess water was gently squeezed out once and 0.1 mL of the conditioner formulation containing microcapsules loaded with a UV tracer were applied. The conditioner was distributed for 30 seconds with gentle rubbing between two fingers. The swatch was then rinsed with 100 mL tap water (39° C.) with 50 mL applied to each side of the swatch. The excess water was gently squeezed out and the hair swatch was then cut into a pre-weighed 20 mL scintillation vial. This process was repeated in triplicate and then the vials containing the cut hair were dried in a vacuum oven at 50-60° C. (100 Torr) for at least 5 hours. After the drying process, the vials were again weighed to determine the mass of hair in the vials. Controls were also prepared by adding 0.1 mL of the conditioner composition containing microcapsules to an empty vial. 8 mL of absolute ethanol were then added to each vial and they were subjected to 60 min of sonication. After sonication, the samples were filtered through a 0.45 μm PTFE filter and analysed with a HPLC using a UV detector. To determine the percentage of deposition of microcapsules from the conditioner compositions, the amount of Uvinul extracted from the hair samples was compared to the amount of Uvinul extracted from the control samples. For each deposition measurement, 3 repeat hair swatch samples were prepared and the deposition value is reported as the average value of the three samples. In case, the variation between the depositions measured on each swatch was higher than 5%, another 3 samples were prepared. A high deposition can be considered at measured values of 10% or above, ideally 25% or above. Viscosity Viscosities were measured 24 hours after the sample preparation with a BROOKFIELD viscosimeter DV-II+at 20 rpm with spindle 5 at 25° C. EXAMPLES Example 1 Preparation of Microcapsules Used in the Invention In a round bottom flask, melamine (0.91 g), 2,2-dimethoxyethanal (60 wt % in water, 1.37 g), glyoxal (40 wt % in water, 1.73 g) and 2-oxoacetic acid (50 wt % in water, 0.58 g) were dispersed in water (1.48 g) at RT. The pH value of the dispersion was controlled with sodium hydroxide (30 wt % in water, pH=9.5). The reaction mixture was heated at 45° C. for 25 minutes to give a solution. Then water (6.31 g) was added and the resin was stirred at 45° C. for 5 min. Resin was transferred in a 200 mL beaker. Guanazole (0.60 g) was dissolved in a solution of Ambergum 1221 (2 wt % in water, 27.04 g). The resulting solution was introduced into the beaker. An oil solution of Takenate D-110N (2.15 g) and a perfume oil (composition from TABLE 1) (29.56 g) was added into the aqueous solution. The biphasic reaction mixture was sheared with an Ultra-turrax at 21500 rpm for 2 min. Acetic acid was added to initiate the polycondensation (pH=5.35). The quality of the emulsion was controlled by light microscopy. The emulsion was transferred into a 200 mL Schmizo reactor and was heated at 45° C. for 1 h, then at 60° C. for 1 h and finally at 80° C. for 2 h. A solution of first cationic copolymer namely acrylamidopropyltrimonium chloride/acrylamide copolymer (Salcare SC60, origin BASF) (20 g, 3 wt % in water), and second cationic copolymer polygalactomannan 2-hydroxy propyltrimethylammonium chloride ether (Jaguar C13S, origin Rhodia) (11 g, 1 wt % in water), was then added and the reaction mixture was heated at 80° C. for 30 min. A solution of urea (6.25 g, 50 wt % in water) was finally added to the reaction mixture, which was heated at 80° C. for 30 min. The zeta potential measured for this capsule was measured to be −37 mV. TABLE 1Composition of perfume ARaw materialQuantity (g)Carbinol acetate2.20Citronellyl acetate16.5Linalyl acetate10.7Nopyle acetate8.0Terpinyl acetate2.10Verdyl acetate2.9Decanal0.1Hexylcinnamic aldehyde13.95Ethyl 2-methyl-pentanoate0.25Benzyl benzoate8.2Cyclogalbanate2.15Hedione ®1)11.95Hexyl isobutyrate2.60Nectalactone10.35Oxane0.1Rose oxide2)0.45Verdyl propionate4.35Beta-ionone0.50Williams ester1.252,4-Dimethyl-3-cyclohexene-1-1.40carbaldehyde1)Cyclopentaneacetic acid, 3-oxo-2-pentyl-, methyl ester; origin and trademark from Firmenich SA, Geneva, Switzerland2)4-methyl-2-(2-methyl1-propen-1-yl)tetrahydro-2H-pyran Example 2 Hair Conditioner Rinse-Off Composition According to the Invention Procedure: Aqueous solutions of Salcare SC 60 and Tylose H10 Y G4 were prepared and the ingredients in the order listed in Table 2 were mixed together. TABLE 2Hair Rinse-off compositions free of alkyl quat and comprisingamodimethicone as non-quaternized conditioning ingredient2-AIngredients%WATER DEIONIZEDq.s. 100%Acrylamidopropyltrimonium Chloride/40Acrylamide Copolymer *1)1% aqueous solutionHydroxyethylcellulose2)202% aqueous solution28% Amodimethicone** (&) Trideceth-63)1Methylchloroisothiazolinone0.1(&)Methylisothiazolinone4)Cationically coated microcapsules, described1in Example 1Total actives of cationic water-soluble0.4%polymers*Total actives of non-quaternized conditioning0.28%oils**Analytical deposition of microcapsules18.1%1)SALCARE SC 60, Ciba2)TYLOSE H10 Y G4, Shin Etsu3)MIRASIL ADM-E, Bluestar Silicone4)KATHON CG, Rohm and Haas Composition 2A shows good performance in terms of deposition. Example 3 Preparation of Hair Conditioner Rinse-Off Composition with Behentrimonium Chloride as Quaternary Ammonium Salt and Cetearyl Alcohol as Non-Quaternized Conditioning Ingredient Procedure: 1/Phase A: mix all ingredients till homogeneous and heat to 70-75° C. 2/Phase B: combine and melt all ingredients of phase B at 70-75° C. 3/At 70-75° C. slowly add Phase B into Phase A while mixing 4/Keep mixing until cooled down to 40° C. and add Phase C while agitating TABLE 3Hair Rinse-off compositions with Behentrimonium chloride as quaternary ammoniumsalt and Cetearyl Alcohol as non-quaternized conditioning ingredient3-A3-B3-CComparative 3-DComparative 3-EComparative 3-FIngredients%%%%%%AWATER DEIONIZED94.393.491.990.989.988.9Ethoxy (20) Stearyl111111Alcohol1)Behentrimonium01.53567Chloride2)*BCETEARYL4.644333ALCOHOL3)**CMethylchloroisothiazolinone (&)0.10.10.10.10.10.1Methylisothiazolinone4)Total actives of0.0%1.5%3.0%5.0%6.0%7.0%quaternary ammonium salt *Total actives of non-quaternized4.6%4.0%4.0%3.0%3.0%3.0%conditioning ingredients**Analytical deposition33.6%11.2%11.3%8.9%9.5%9.2%measured on hairViscosity (sp 5/20 rpm)1980 cPs3599 cPs5399 cPs2160 cPs3600 cPs4320 cPs1)BRIJ S20, solubiliser, Croda2)GENAMIN KDMP, Clariant3)Lanette O, BASF4)KATHON CG, Rohm and Haas TABLE 4continued: Hair Rinse-off compositions with Behentrimonium chloride as quaternaryammonium salt and Cetearyl Alcohol as non-quaternized conditioning ingredient3-G3-H3-I3-JIngredients%%%%AWATER DEIONIZED95.994.993.992.4Ethoxy (20) Stearyl Alcohol1)1111Behentrimonium Chloride2)*0001.5BCETEARYL ALCOHOL3)**3455CMethylchloroisothiazolinone0.10.10.10.1(&) Methylisothiazolinone4)Total actives of quaternary0.0%0.0%0.0%1.5%ammonium salt *Total actives of non-3.0%4.0%5.0%5.0%quaternized conditioningingredients**Analytical deposition of17.5%19.2%17.0%11.4%microcapsules measuredon hairViscosity (sp 5/20 rpm)1680 cPs2400 cPs3180 cPs5640 cPs1)BRIJ S20, solubiliser, Croda2)GENAMIN KDMP, Clariant3)Lanette O, BASF4)KATHON CG, Rohm and Haas Conclusion Compositions 3A, 3B, 3C, 3G, 3H and 3I show good performance in terms of deposition compared to comparative composition compositions 3D, 3E and 3F (that comprise an amount of quaternary ammonium salt greater than 4%). Both, the quaternary ammonium salt and the cetearyl alcohol participated in the final viscosity of the composition which is desired to be at 4000-5000 cPs or higher in order to provide a creamy aspect. This can be obtained at a quaternary ammonium salt dosage of 3 wt % and cetearyl alcohol dosage of 4% (3-C). Viscosity could be significantly increased at a low quaternary ammonium salt level of 1.5 wt % by increasing the amount of cetearyl alcohol (3-B vs. 3-J). Example 4 Hair Conditioner Rinse-Off Composition with Behentrimonium Methosulfate as Quaternary Ammonium Salt and Blends of Non-Quaternized Conditioning Ingredient Procedure: 1/Phase A: mix all ingredients till homogeneous and heat to 70-75° C. 2/Phase B: combine and melt all ingredients of phase B at 70-75° C. 3/At 70-75° C. slowly add Phase B into Phase A while mixing 4/Keep mixing until cooled down to 40° C. and add Phase C while agitating TABLE 5Hair Rinse-off compositions with Behentrimonium methosulfate as quaternaryammonium salt and blends of non-quaternized conditioning ingredients4-A4-B4-C4-DIngredients%%%%AWATER DEIONIZED93.393.494.492.9Ethoxy (20) Stearyl alcohol1)1111B55% Behentrimonium Methosulfate*01.51.54(&) 40% Cetyl Alcohol** (&) 5%Butylene Glycol-2)Bis-Ethyl(Isostearylimidazoline)1100Isostereamide**3)CETEARYL ALCOHOL**4)4.6443CMethylchloroisothiazolinone (&)0.10.10.10.1Methylisothiazolinone5)Total actives of quaternary ammonium0.0%0.8%0.8%2.2%salt *Total actives of non-quaternized6.1%5.7%4.7%4.8%conditioning ingredients**Analytical deposition of microcapsules,28.3%18.9%15.4%10.4%measured on hairViscosity (sp 5/20 rpm)9000 cPs5000 cPs7000 cPs2000 cPs1)BRIJ S20, Croda2)INCROQUAT BEHENYL TMS-50-PA- (MH), Croda3)KERADYN ™HH, Croda4)Lanette O, BASF5)KATHON CG, Rohm and Haas Compositions 4A-4D show good performance in terms of deposition. One can note that the addition of Bis-Ethyl(Isostearylimidazoline) Isostereamide (Keradyn™ HH) to cetearyl alcohol has a positive effect on deposition. Example 5 Hair Conditioner Rinse-Off Composition with Behentrimonium Chloride as Quaternary Ammonium Salt and Blends of Other Conditioning Ingredient Procedure: 1/Phase A: mix all ingredients till homogeneous and heat to 70-75° C. 2/Phase B: combine and melt all ingredients of phase B at 70-75° C. 3/At 70-75° C. slowly add Phase B into Phase A while mixing 4/Keep mixing until cooled down to 40° C. and add Phase C while agitating TABLE 6aHair Rinse-off compositions with Behentrimonium chloride asquaternary salt and blends of other conditioning ingredients55-A5-B5-C5-DIngredients%%%%%AWATER DEIONIZED93.490.989.490.989.4Ethoxy (20) Stearyl Alcohol1)11111Behentrimonium Chloride2)*1.501.501.5BPARAFFINE OIL***3)05500JOJOBA OIL***4)00055CETEARYL ALCOHOL***5)33333CMethylchloroisothiazolinone0.10.10.10.10.1(and)Methylisothiazolinone6)Total actives of quaternary1.5%0.0%1.5%0.0%1.5%ammonium salt *Total actives of non-3.0%8.0%8.0%8.0%8.0%quaternized conditioningingredients***Analytical deposition of10.7%20.0%34.0%18.2%31.8%microcapsules measured onhairViscosity (sp 5/20 rpm)3500 cps1980 cps2160 cps2790 cps6000 cps1)BRIJ S20, Croda2)GENAMIN KDMP, Clariant3)Savonol 40, Savita4)PNJ Deodorized Clear Jojoba, Purcell Jojoba International5)Lanette O, BASF6)KATHON CG, Room and Haas TABLE 6bHair Rinse-off compositions with Behentrimonium chloride as quaternaryammonium salt and blends of other conditioning ingredients5-E5-F5-G5-HIngredients%%%%AWATER DEIONIZED90.989.490.989.4Ethoxy (20) Stearyl Alcohol1)1111Behentrimonium Chloride2)*1.51.51.51.5BBees wax***3)5000Macadamia oil***4)0500Lauric acid***0050Olive oil***5)0005CETEARYL3333ALCOHOL***6)cMethylchloroisothiazolinone0.10.10.10.1(and)Methylisothiazolinone7)Total actives of quaternary1.5%1.5%1.5%1.5%ammonium salt *Total actives of non-8.0%8.0%8.0%8.0%quaternized conditioningingredients***Analytical deposition of13.6%11.3%14.4%11.9%microcapsules measured onhair1)BRIJ S20, Croda2)GENAMIN KDMP, Clariant3)Baerlocher4)FRUTAROM5)FLORIN AG6)Lanette O, BASF7)KATHON CG, Room and Haas Conclusion Compositions 5A to 5H present good performance in terms of deposition. A significant improvement of the deposition is observed when jojoba oil or paraffin oil is used as conditioning oils in addition to cetearyl alcohol. Example 6 Hair Conditioner Rinse-Off Composition with Mixtures of Quaternary Ammonium Salts and Blends of Non-Quaternized Conditioning Ingredients Procedure: 1/Phase A: disperse TYLOSE in the water till homogeneous, then add remaining ingredients while mixing and heat to 70-75° C. 2/Phase B: combine and melt all ingredients of phase B at 70-75° C. 3/At 70-75° C. slowly add Phase B into Phase A while mixing 4/Keep mixing until cooled down to 40° C. and add ingredients of Phase C while agitating TABLE 7Hair Rinse-off compositions with mixtures of alkyl quats and blends of non-quaternized conditioning ingredients6-A6-B6-C6-DComparative 6-EComparative 6-FIngredients%%%%%%AWATER DEIONIZED86.386.483.482.481.482.3Ethoxy (20) Stearyl alcohol1)1.51.511Hydroxyethylcellulose2)1.51.51.51.51.51.5Behentrimonium Chloride*3)1.5222.52.5Cetrimonium Chloride*4)1.51.2B55% Behentrimonium1144Methosulfate* (&) 40% CetylAlcohol** (&) 5% ButyleneGlycol5)Glycerol Stearate (&) PEG-2.62.53322100 Stearate**6)CETYL ALCOHOL**7)2Cetearyl Alcohol**8)434444STEARIC PALMITIC0.5ACIDS mixture**9)Polymethylsiloxane **10)2.5C50% Dimethicone ** (&)1.5C12-13 Pareth-4 (&) C12-13Pareth-23 (&) Salicylic Acidactives11)35% Amodimethicone** (&)2.53333Trideceth-12 (&) 5%Cetrimonium Chloride*12)28% Amodimethicone **1(&) Trideceth-6 actives13)PROPYLENE GLYCOL1Methylchloroisothiazolinone0.10.10.10.10.10.1(&) Methylisothiazolinone14)CITRIC ACID 10% aqueous0.50.50.50.4sol. till pH 3-3.5Total actives of quaternary1.5%1.6%2.7%2.7%4.9%6.1%ammonium salt *Total actives of non-quaternized9.9%8.9%8.5%8.8%8.9%8.9%conditioning ingredients**Analytical deposition of28.3%34.0%27.5%34.9%6.1%0.2%microcapsulesmeasured onhairViscosity (sp 5/20 rpm)12200 cPs15500 cPs22000 cPs21000 cPs20000 cPs15000 cPs1)BRIJ S20, Croda2)TYLOSE H10 Y G4, Shin Etsu3)GENAMIN KDM, Clariant4)GENAMIN CTAC, Clariant5)INCROQUAT BEHENYL TMS-50-PA- (MH), Croda6)ARLACEL 165, Croda7)Lanette 16, BASF8)Lanette O, BASF9)Berg + Schmidt10)DIMETHICONE 200 fluid 60000 cSt, Dow Corning11)XIAMETER MEM 169 1, Dow Corning12)XIAMETER MEM-949, Dow Corning13)MIRASIL ADM E, Bluestar Silicones14)KATHON CG, Rohm and Haas Conclusion The deposition of cationically coated microcapsules from Example 1 was strongly related to the amount of alkyl quats present in the composition. Up to 2.7 wt %, the deposition remained high around 30% (6-A; 6-B, 6-C; 6-D). With the increase of selected non-quaternized conditioning ingredients, the deposition could be increased above 30% (6-B; 6-D). At higher quaternary ammonium salt concentration of 4.9 wt %, the deposition dropped significantly (6-E) and even more significantly at quaternary ammonium salt concentration of 6.1 wt % (6-F). | 41,537 |
11857660 | DETAILED DESCRIPTION The disclosure relates to compositions and methods for altering the color or tone of the hair. The compositions comprise at least one amino acid, at least one carboxylic acid, monoethanolamine, and at least one hair coloring agent. The methods comprise applying the compositions according to the disclosure to the hair. Compositions Amino Acid The compositions according to the disclosure comprise at least one amino acid. As used herein, the term “amino acid” includes amino acids such as proteinogenic amino acids, amino sulfonic acids, and salts thereof. Amino acids are simple organic compounds containing both a carboxylic acid group (—COOH) and an amino group (—NH2). Amino sulfonic acids are simple organic compounds containing both a sulfonic acid group (—SO2OH) and an amino group (—NH2). Accordingly, the amino acids useful according to the disclosure may, in certain embodiments, be selected from compounds of Formula (I) and Formula (II): wherein:R represents a hydrogen atom, a linear or branched, preferably linear, C1-C5alkyl group, said alkyl group being optionally substituted with at least one group chosen from hydroxyl, —C(O)—OH, —S(O)2—OH, —C(O)—O−and M+, and S(O)2—O−and M+, with M+representing a cationic counter-ion such as an alkali metal, alkaline earth metal, or ammonium, andn is 0 or 1. The amino acids may be in their non-ionized form (I) and (II) or in their ionized or betaine form (I′) and (II′): wherein R and n are as defined above. The one or more amino acids may also be in their conjugate base form (Ib) and (IIb): wherein R and n are as defined above. Well-known amino acids include the twenty amino acids that form the proteins of living organisms (standard proteinogenic amino acids): alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The amino acids of the instant disclosure, however, are not limited to the standard proteinogenic amino acids. Non-limiting examples of amino sulfonic acids include aminomethane sulfonic acid, 2-aminoethane sulfonic acid (taurine), aminopropane sulfonic acid, aminobutane sulfonic acid, aminohexane sulfonic acid, aminoisopropyl sulfonic acid, aminododecyl sulfonic acid, aminobenzene sulfonic acid, aminotoulene sulfonic acid, sulfanilic acid, chlorosulfanilic acid, diamino benzene sulfonic acid, amino phenol sulfonic acid, amino propyl benzene sulfonic acid, amino hexyl benzene sulfonic acid, and a mixture thereof. In some cases, charged amino acids may be used. Non-limiting examples of charged amino acids include arginine, lysine, aspartic acid, and glutamic acid. In some cases, polar amino acids are useful. Non-limiting examples of polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan. In some cases, hydrophobic amino acids may be employed. Non-limiting examples of hydrophobic amino acids include alanine, isoleucine, leucine, phenylalanine, valine, proline, and glycine. In certain exemplary embodiments, compositions according to the disclosure include at least one amino acid selected from the group consisting of glycine, alanine, serine, beta-alanine, taurine, sodium glycinate, sodium alaninate, sodium serinate, lithium beta-alanine, sodium taurate, or combinations thereof. In further exemplary embodiments, compositions according to the disclosure include only amino acids, for example, those selected from the group consisting of aspartic acid, cysteine, glycine, lysine, methionine, proline, tyrosine, phenylalanine, carnitine, taurine, or a salt thereof. In one exemplary embodiment, the compositions include at least taurine. In a further embodiment, the only amino acid in the composition is taurine. In a further embodiment, the compositions include at least glycine. In yet a further embodiment, the only amino acid in the composition is glycine. In one embodiment, the compositions include both taurine and glycine. The total amount of the at least one amino acid may range from about 0.01% to about 10% by weight, relative to the total weight of the hair color toning composition. For example, in some embodiments, the total amount of the at least one amino acid may range from about 0.05% to about 5%, such as about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2.5%, about 0.1% to about 2.4%, about 0.1% to about 2.3%, about 0.1% to about 2.2%, about 0.1% to about 2.1%, about 0.1% to about 2%, about 0.1% to about 1.9%, about 0.1% to about 1.8%, about 0.1% to about 1.7%, about 0.1% to about 1.6%, about 0.1% to about 1.5%, about 0.1% to about 1.4%, about 0.1% to about 1.3%, about 0.1% to about 1.2%, about 0.1% to about 1.1%, about 0.1% to about 1%, about 0.1% to about 0.9%, about 0.1% to about 0.8%, about 0.1% to about 0.7%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, or about 0.1% to about 0.3% by weight, relative to the total weight of the hair color toning composition. In other embodiments, the total amount of the at least one amino acid ranges from about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2.5%, about 0.5% to about 2.4%, about 0.5% to about 2.3%, about 0.5% to about 2.2%, about 0.5% to about 2.1%, about 0.5% to about 2%, about 0.5% to about 1.9%, about 0.5% to about 1.8%, about 0.5% to about 1.7%, about 0.5% to about 1.6%, about 0.5% to about 1.5%, about 0.5% to about 1.4%, about 0.5% to about 1.3%, about 0.5% to about 1.2%, about 0.5% to about 1.1%, about 0.5% to about 1%, about 0.5% to about 0.9%, about 0.5% to about 0.8%, about 0.5% to about 0.7%, or about 0.5% to about 0.6% by weight, relative to the total weight of the hair color toning composition. The total amount of the at least one amino acid may, in certain embodiments, be about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, or about 0.35% by weight, relative to the total weight of the hair color toning composition. In yet further embodiments, the total amount of the amino acid may be about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1% about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7% about 1.8%, about 1.9%, or about 2% by weight, relative to the total weight of the hair color toning composition. It is to be understood that any of the above-recited numbers may provide an upper or lower boundary for a range of the total amount of the at least one amino acid. Carboxylic Acid The hair color toning compositions include at least one carboxylic acid. As used herein, the term “carboxylic acid” includes salts of carboxylic acids. In certain embodiments, the carboxylic acids include non-polymeric mono, di, and/or tricarboxylic acid which are organic compounds having one (mono), two (di), or three (tri) carboxylic acid groups (—COOH). The non-polymeric mono, di, and tricarboxylic acids, and/or salts thereof, typically have a molecular weight of less than about 500 g/mol, less than about 400 g/mol, or less than about 300 g/mol. Non-limiting examples of mono-carboxylic acids, or salts thereof, include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, entanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, lactic acid, a salt thereof, and a mixture thereof. In some cases, the hair color toning compositions include at least lactic acid and/or a salt thereof. Non-limiting examples of di-carboxylic acids and/or salts thereof include oxalic acid, malonic acid, malic acid, glutaric acid, citraconic acid, succinic acid, adipic acid, tartaric acid, fumaric acid, maleic acid, sebacic acid, azelaic acid, dodecanedioic acid, phthalic acid, isophthalic acid, terephthalic acid, 2,6-naphthalene dicarboxylic acid, a salt thereof, and a mixture thereof. In some cases, the hair color toning compositions include oxalic acid, malonic acid, malic acid, maleic acid, a salt thereof, or a mixture thereof. Non-limiting examples of tricarboxylic acids and salts thereof include citric acid, isocitric acid, aconitric acid, propane-1,2,3-tricarboxylic acid, benzene-1,3,5-tricarboxylic acid, a salt thereof, and a mixture thereof. In some instances, the hair color toning compositions include at least citric acid and/or a salt thereof. In one or more embodiments, the hair color toning composition comprises at least one carboxylic acid selected from the group consisting of oxalic acid, malonic acid, glutaric acid, succinic acid, adipic acid, glycolic acid, citric acid, tartaric acid, malic acid, sebacic acid, maleic acid, fumaric acid, benzoic acid, citraconic acid, aconitic acid, propane-1,2,3-tricarboxylic acid, trimesic acid, or combinations thereof. In one exemplary embodiment, the compositions include at least citric acid. In a further embodiment, the only carboxylic acid in the composition is citric acid. The total amount of the at least one carboxylic acid may range from about 0.01% to about 10% by weight, relative to the total weight of the hair color toning composition. For example, in some embodiments, the total amount of the at least one carboxylic acid may range from about 0.05% to about 5%, such as about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2.5%, about 0.1% to about 2.4%, about 0.1% to about 2.3%, about 0.1% to about 2.2%, about 0.1% to about 2.1%, about 0.1% to about 2%, about 0.1% to about 1.9%, about 0.1% to about 1.8%, about 0.1% to about 1.7%, about 0.1% to about 1.6%, about 0.1% to about 1.5%, about 0.1% to about 1.4%, about 0.1% to about 1.3%, about 0.1% to about 1.2%, about 0.1% to about 1.1%, about 0.1% to about 1%, about 0.1% to about 0.9%, about 0.1% to about 0.8%, about 0.1% to about 0.7%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, or about 0.1% to about 0.3% by weight, relative to the total weight of the hair color toning composition. In other embodiments, the total amount of the at least one carboxylic acid ranges from about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2.5%, about 0.5% to about 2.4%, about 0.5% to about 2.3%, about 0.5% to about 2.2%, about 0.5% to about 2.1%, about 0.5% to about 2%, about 0.5% to about 1.9%, about 0.5% to about 1.8%, about 0.5% to about 1.7%, about 0.5% to about 1.6%, about 0.5% to about 1.5%, about 0.5% to about 1.4%, about 0.5% to about 1.3%, about 0.5% to about 1.2%, about 0.5% to about 1.1%, about 0.5% to about 1%, about 0.5% to about 0.9%, about 0.5% to about 0.8%, about 0.5% to about 0.7%, or about 0.5% to about 0.6% by weight, relative to the total weight of the hair color toning composition. The total amount of the at least one carboxylic acid may, in certain embodiments, be about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, about 0.4%, about 0.41%, about 0.42%, about 0.43%, about 0.44%, about 0.45%, about 0.46%, about 0.47%, about 0.48%, about 0.49%, or about 0.5% by weight, relative to the total weight of the hair color toning composition. It is to be understood that any of the above-recited numbers may provide an upper or lower boundary for a range of the total amount of the at least one carboxylic acid. Monoethanolamine The hair color toning compositions according to the disclosure comprise monoethanolamine. The monoethanolamine may be present in the composition in an amount up to about 10%, such from about 0.001% up to about 10%, or from about 0.01% up to about 5%, up to about 4%, up to about 3.9%, up to about 3.8%, up to about 3.7%, up to about 3.6%, up to about 3.5%, up to about 3.4%, up to about 3.3%, up to about 3.2%, up to about 3.1%, up to about 3%, up to about 2.9%, up to about 2.8%, up to about 2.7%, up to about 2.6%, up to about 2.5%, up to about 2.4%, up to about 2.3%, up to about 2.2%, up to about 2.1%, up to about 2.0%, up to about 1.9%, up to about 1.8%, up to about 1.7%, up to about 1.6%, up to about 1.5%, up to about 1.4%, up to about 1.3%, up to about 1.2%, up to about 1.1%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or up to about 0.05% by weight, based on the weight of the composition. By way of example, the monoethanolamine may be present in an amount of about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, or about 3.5% by weight, based on the weight of the composition. Fatty Compounds Fatty compounds (also referred to interchangeably as “fatty substances”) may be included in one or more embodiments of the invention. In some embodiments, two or more fatty compounds may be included. In further embodiments, such fatty compounds may be a fatty compounds other than a fatty acid. As used herein, “fatty compound” means an organic compound insoluble in water at normal temperature (25° C.) and at atmospheric pressure (760 mmHg) (solubility below 5% and such as below 1% and further such as below 0.1%). Fatty compounds have in their structure a chain of at least two siloxane groups or at least one hydrocarbon chain having at least 6 carbon atoms. Moreover, fatty compounds are generally soluble in organic solvents in the same conditions of temperature and pressure, for example in chloroform, ethanol, benzene or decamethylcyclopentasiloxane. Fatty compounds are, for example, chosen from lower alkanes, fatty alcohols, esters of fatty acid, esters of fatty alcohol, oils such as mineral, vegetable, animal and synthetic non-silicone oils, non-silicone waxes and silicones. In some embodiments, the alcohols and esters have at least one linear or branched, saturated or unsaturated hydrocarbon group, comprising 6 to 30 carbon atoms, optionally substituted, for example, with at least one hydroxyl group (for example 1 to 4). If they are unsaturated, these compounds can have one to three, conjugated or unconjugated, carbon-carbon double bonds. With regard to the lower alkanes, in some embodiments, these have from 6 to 16 carbon atoms and are linear or branched, optionally cyclic. As examples, alkanes can be chosen from hexane and dodecane, isoparaffins such as isohexadecane and isodecane. Non-limiting examples of non-silicone oils usable in the composition of the disclosure, include: esters of a glycerol oligomer, in particular diglycerol esters, especially condensates of adipic acid and of glycerol, for which a portion of the hydroxyl groups of the glycerols has reacted with a mixture of fatty acids, such as stearic acid, capric acid, isostearic acid and 12-hydroxystearic acid, such as in particular those sold under the brand name Softisan 649 by Sasol; arachidyl propionate, sold under the brand name Waxenol 801 by Alzol; fatty acid triglycerides and their derivatives; pentaerythritol esters; esters of dimer diol and dimer diacid, if appropriate esterified on their free alcohol or acid functional group(s) by acid or alcohol radicals, in particular dimer dilinoleate esters; such esters can be chosen in particular from esters with the following INCI nomenclature: bis-behenyl/isostearyl/phytosteryl dimer dilinoleyl dimer dilinoleate (Plandool G), phytosteryl isostearyl dimer dilinoleate (Lusplan PI-DA or Lusplan PHY/IS-DA), phytosteryl/isostearyl/cetyl/stearyl/behenyl dimer dilinoleate (Plandool H or Plandool S), and their mixtures; mango butter, such as that sold under the reference Lipex 203 by AarhusKarlshamn; hydrogenated soybean oil, hydrogenated coconut oil, hydrogenated rapeseed oil or mixtures of hydrogenated vegetable oils, such as the soybean, coconut, palm and rapeseed hydrogenated vegetable oil mixture, for example the mixture sold under the reference Akogel® by AarhusKarlshamn (INCI name: Hydrogenated Vegetable Oil); shea butter, in particular that having the INCI name Butyrospermum Parkii Butter, such as that sold under the reference Sheasoft® by AarhusKarlshamn; cocoa butter, in particular that which is sold under the name CT Cocoa Butter Deodorized by Dutch Cocoa BV or that which is sold under the name Beurre De Cacao NCB HD703 758 by Barry Callebaut; shorea butter, in particular that which is sold under the name Dub Shorea T by Stearinerie Dubois; and their mixtures. According to a preferred embodiment, the fatty compound is chosen from hydrogenated vegetable oil, shea butter, cocoa butter, shorea butter, a soybean, coconut, palm and rapeseed hydrogenated vegetable oil mixture, and their mixtures, and more particularly those referenced above. Non-limiting examples of non-silicone oils usable in the composition of the disclosure, include: hydrocarbon oils of animal origin, such as perhydrosqualene; hydrocarbon oils of vegetable origin, such as liquid triglycerides of fatty acids having from 6 to 30 carbon atoms such as triglycerides of heptanoic or octanoic acids, or for example sunflower oil, maize oil, soya oil, cucurbit oil, grapeseed oil, sesame oil, hazelnut oil, apricot oil, macadamia oil, arara oil, sunflower oil, castor oil, avocado oil, triglycerides of caprylic/capric acids such as those sold by the company Stearineries Dubois or those sold under the names MIGLYOL® 810, 812 and 818 by the company Dynamit Nobel, jojoba oil, shea butter oil; hydrocarbons with more than 16 carbon atoms, linear or branched, of mineral or synthetic origin, such as paraffin oils, petroleum jelly, liquid paraffin, polydecenes, hydrogenated polyisobutene such as Parleam®. fluorinated, partially hydrocarbon oils; as fluorinated oils, non-limiting examples include perfluoromethylcyclopentane and perfluoro-1,3-dimethylcyclohexane, sold under the names “FLUTEC® PC1” and “FLUTEC® PC3” by the company BNFL Fluorochemicals; perfluoro-1,2-dimethylcyclobutane; perfluoroalkanes such as dodecafluoropentane and tetradecafluorohexane, sold under the names “PF 5050®” and “PF 5060®” by the 3M Company, or bromoperfluorooctyl sold under the name “FORALKYL®” by the company Atochem; nonafluoro-methoxybutane and nonafluoroethoxyisobutane; derivatives of perfluoromorpholine, such as 4-trifluoromethyl perfluoromorpholine sold under the name “PF 5052®” by the 3M Company. The non-silicone oils of the present invention may be employed in an amount of from about 0.5% to about 5% by weight, such as from about 1% to about 5.5% by weight, and further such as from about 1.5% to about 4% by weight, based on the total weight of the hair color composition of the present invention, including increments and ranges therein there between. The total amount of the non-silicone oils in the present invention may be employed in an amount of from about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, to about 5 percent by weight, including increments and ranges therein there between. As used herein, “fatty alcohol” refers to any alcohol with a carbon chain of C5 or greater, such as, for example, C8 or greater, C10 or greater, and C12 or greater. The at least one fatty alcohol may be chosen from, for example, C9-C11 alcohols, C12-C13 alcohols, C12-C15 alcohols, C12-C16 alcohols, C14-C15 alcohols, C12-C22 alcohols, arachidyl alcohol, behenyl alcohol, caprylic alcohol, cetearyl alcohol, cetyl alcohol, coconut alcohol, decyl alcohol, hydrogenated tallow alcohol, jojoba alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol, palm alcohol, palm kernel alcohol, stearyl alcohol, tallow alcohol, and tridecyl alcohol. As used herein, “alkoxylated fatty alcohol” refers to any fatty alcohol with a carbon chain of C5 or greater, as defined above, further comprising at least one alkoxy group. For example, the at least one alkoxylated fatty alcohol may have a carbon chain of C8 or greater, C10 or greater, and C12 or greater. Further, for example, the at least one alkoxylated fatty alcohol may be chosen from alkoxylated polymers (including co-, ter- and homo-polymers) derived from alcohols such as glycerol (e.g. polyglyceryl derived from four glycerol molecules). The at least one alkoxy group of the at least one alkoxylated fatty alcohol may, for example, be derived from an alkoxylation reaction carried out with alkylene oxide. Non-limiting examples of at least one alkoxylated fatty alcohol include any fatty alcohol comprising at least one polyethylene glycol ether and any fatty alcohol comprising at least one polypropylene glycol ether. Non-limiting examples of the at least one alkoxylated fatty alcohol include ceteareth-2, ceteareth-3, ceteareth-4, ceteareth-5, ceteareth-6, ceteareth-7, ceteareth-8, ceteareth-9, ceteareth-10, ceteareth-11, ceteareth-12, ceteareth-13, ceteareth-14, ceteareth-15, ceteareth-16, ceteareth-17, ceteareth-18, ceteareth-20, ceteareth-22, ceteareth-23, ceteareth-24, ceteareth-25, ceteareth-27, ceteareth-28, ceteareth-29, ceteareth-30, cetearel:h-33, ceteareth-34, ceteareth-40, ceteareth-50, ceteareth-55, ceteareth-60, ceteareth-80, ceteareth-100, laureth-1, laureth-2, laureth-3, laureth-4, laureth-5, laureth-6, laureth-7, laureth-8, laureth-9, laureth-10, laureth-11, laureth-12, laureth-13, laureth-14, laureth-15, lauretih-16, laureth-20, laureth-23, laureth-25, laureth-30, laureth-40, deceth-3, deceth-5, oleth-5, oleth-30, steareth-2, steareth-10, steareth-20, steareth-100, cetylsteareth-12, ceteareth-5, ceteareth-5, polyglyceryl 4-lauryl ether, polyglyceryl 4-oleyl ether, polyglyceryl 2-oleyl ether, polyglyceryl 2-cetyl ether, polyglyceryl 6-cetyl ether, polyglyceryl 6-oleylcetyl ether, polyglyceryl 6-octadecyl ether, C9-C11 pareth-3, C9-C11 pareth-6, C11-C15 pareth-3, C11-C15 pareth-5, C11-C15 pareth-12, C11-C15 pareth-20, C12-C15 pareth-9, C12-C15 pareth-12, and C22-C24 pareth-33. The fatty alcohols useful according to the disclosure include, but are not limited to, non-alkoxylated, saturated or unsaturated, linear or branched, and have from 6 to 30 carbon atoms and more particularly from 8 to 30 carbon atoms; For example, cetyl alcohol, stearyl alcohol and their mixture (cetylstearyl alcohol), octyldodecanol, 2-butyloctanol, 2-hexyldecanol, 2-undecylpentadecanol, oleic alcohol or linoleic alcohol. The exemplary non-silicone wax or waxes that can be used in the composition of the disclosure are chosen from carnauba wax, candelilla wax, and Alfa wax, paraffin wax, ozokerite, vegetable waxes such as olive wax, rice wax, hydrogenated jojoba wax or absolute waxes of flowers such as the essential wax of blackcurrant flower sold by the company BERTIN (France), animal waxes such as beeswaxes, or modified beeswaxes (cerabellina); other waxes or waxy raw materials usable according to the disclosure are, for example, marine waxes such as that sold by the company SOPHIM under reference M82, waxes of polyethylene or of polyolefins in general. The exemplary fatty acid esters are the esters of saturated or unsaturated, linear or branched C1-C26 aliphatic mono- or polyacids and of saturated or unsaturated, linear or branched C1-C26 aliphatic mono- or polyalcohols, the total number of carbons of the esters being, for example, greater than or equal to 10. Among the monoesters, non-limiting mentions can be made of dihydroabietyl behenate; octyldodecyl behenate; isocetyl behenate; cetyl lactate; C12-C15 alkyl lactate; isostearyl lactate; lauryl lactate; linoleyl lactate; oleyl lactate; (iso)stearyl octanoate; isocetyl octanoate; octyl octanoate; cetyl octanoate; decyl oleate; isocetyl isostearate; isocetyl laurate; isocetyl stearate; isodecyl octanoate; isodecyl oleate; isononyl isononanoate; isostearyl palmitate; methyl acetyl ricinoleate; myristyl stearate; octyl isononanoate; 2-ethylhexyl isononate; octyl palmitate; octyl pelargonate; octyl stearate; octyldodecyl erucate; oleyl erucate; ethyl and isopropyl palmitates, ethyl-2-hexyl palmitate, 2-octyldecyl palmitate, alkyl myristates such as isopropyl, butyl, cetyl, 2-octyldodecyl, mirystyl, stearyl myristate, hexyl stearate, butyl stearate, isobutyl stearate; dioctyl malate, hexyl laurate, and 2-hexyldecyl laurate. Further non-limiting mentions of esters can be made of the esters of C4-C22 di- or tricarboxylic acids and of C1-C22 alcohols and the esters of mono-, di- or tricarboxylic acids and of C2-C26 di-, tri-, tetra- or pentahydroxy alcohols. Even further non-limiting examples of esters include: diethyl sebacate; diisopropyl sebacate; diisopropyl adipate; di-n-propyl adipate; dioctyl adipate; diisostearyl adipate; dioctyl maleate; glyceryl undecylenate; octyldodecyl stearoyl stearate; pentaerythrityl monoricinoleate; pentaerythrityl tetraisononanoate; pentaerythrityl tetrapelargonate; pentaerythrityl tetraisostearate; pentaerythrityl tetraoctanoate; propylene glycol dicaprylate; propylene glycol dicaprate, tridecyl erucate; triisopropyl citrate; triisotearyl citrate; glyceryl trilactate; glyceryl trioctanoate; trioctyldodecyl citrate; trioleyl citrate, propylene glycol dioctanoate; neopentyl glycol diheptanoate; diethylene glycol diisanonate; glycol distearates; and polyethylene glycol distearates. Among the esters mentioned above, exemplary esters include ethyl, isopropyl, myristyl, cetyl, stearyl palmitates, ethyl-2-hexyl palmitate, 2-octyldecyl palmitate, alkyl myristates such as isopropyl, butyl, cetyl, 2-octyldodecyl myristate, hexyl stearate, butyl stearate, isobutyl stearate; dioctyl malate, hexyl laurate, 2-hexyldecyl laurate and isononyl isononanate, cetyl octanoate. The composition can also comprise, as fatty ester, esters and di-esters of sugars of C6-C30, such as C12-C22 fatty acids. “Sugar” as used in the disclosure means oxygen-containing hydrocarbon compounds that possess several alcohol functions, with or without aldehyde or ketone functions, and having at least 4 carbon atoms. These sugars can be monosaccharides, oligosaccharides or polysaccharides. As suitable sugars, non-limiting examples include sucrose, glucose, galactose, ribose, fucose, maltose, fructose, mannose, arabinose, xylose, lactose, and their derivatives, for example alkylated, such as methylated derivatives such as methylglucose. The esters of sugars and of fatty acids can, for example, be chosen from the esters or mixtures of esters of sugars described previously and of linear or branched, saturated or unsaturated C6-C30, such as C12-C22 fatty acids. If they are unsaturated, these compounds can have one to three, conjugated or unconjugated, carbon-carbon double bonds. The esters according to some embodiments can be chosen from mono-, di-, tri- and tetra-esters, polyesters and mixtures thereof. These esters can be for example oleate, laurate, palmitate, myristate, behenate, cocoate, stearate, linoleate, linolenate, caprate, arachidonates, or mixtures thereof such as the oleo-palmitate, oleo-stearate, palmito-stearate mixed esters. For example, the mono- and di-esters can be used, and such as the mono- or dioleate, stearate, behenate, oleopalmitate, linoleate, linolenate, oleostearate, of sucrose, of glucose or of methylglucose. Non-limiting mention can be made of the product sold under the name GLUCATE® DO by the company Amerchol, which is a dioleate of methylglucose. Exemplary esters or of mixtures of esters of sugar of fatty acid include: the products sold under the names F160, F140, F110, F90, F70, SL40 by the company Crodesta, denoting respectively the palmitostearates of sucrose formed from 73% of monoester and 27% of di- and tri-ester, from 61% of monoester and 39% of di-, tri-, and tetra-ester, from 52% of monoester and 48% of di-, tri-, and tetra-ester, from 45% of monoester and 55% of di-, tri-, and tetra-ester, from 39% of monoester and 61% of di-, tri-, and tetra-ester, and the mono-laurate of sucrose; the products sold under the name Ryoto Sugar Esters for example with the reference B370 and corresponding to the behenate of sucrose formed from 20% of monoester and 80% of di-triester-polyester; sucrose mono-di-palmito-stearate marketed by the company Goldschmidt under the name TEGOSOFT® PSE. Surfactants The hair color toning compositions comprise one or more surfactants selected from anionic surfactants, nonionic surfactants, amphoteric surfactants, and mixtures thereof. In at least one exemplary embodiment, the surfactant is a surfactant mixture comprising at least one amphoteric surfactant and at least one anionic surfactant. In a further embodiment, the surfactant is a surfactant mixture comprising at least one amphoteric surfactant and at least one non-ionic surfactant. In yet a further embodiment, the surfactant is a surfactant mixture comprising at least one non-ionic surfactant and at least one anionic surfactant. The total amount of the one or more surfactants included in the hair color toning compositions can vary, especially depending on the type of hair color toning composition in with they are contained. The total amount of the one or more surfactants typically ranges from about 0.1% to about 60% by weight, relative to the total weight of the hair color toning composition, including all ranges and subranges therebetween. In some cases, the total amount of the one or more surfactants ranges from about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1 to about 25%, about 0.1 to about 20%, about 0.1 to about 15%, about 0.1 to about 10%, about 0.1 to about 5%, about 0.5 to about 40%, about 0.5 to about 35%, about 0.5 to about 30%, about 0.5 to about 25%, about 0.5 to about 20%, about 0.5 to about 15%, about 0.5 to about 10%, about 0.5 to about 5%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight, relative to the total weight of the hair treatment composition. In further embodiments, the total amount of the one or more surfactants ranges from about trimester 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 55%, about 40% to about 50%, or about 40% to about 45% by weight, relative to the total weight of the hair color toning composition. Anionic Surfactants The term “anionic surfactant” means a surfactant comprising, as ionic or ionizable groups, only anionic groups. These anionic groups may optionally be chosen from the groups CO2H, CO2−, SO3H, SO3−, OSO3H, OSO3−O2PO2H, O2PO2H and O2PO22−. The hair color toning compositions may include one or more anionic surfactants. Non-limiting examples of anionic surfactants include alkyl sulfates, alkyl ether sulfates, acyl isethionates, acyl glycinates, acyl taurates, acyl amino acids, acyl sarcosinates, sulfosuccinates, sulfonates, and a mixture thereof, wherein the alkyl and acyl groups of all these compounds comprise from 6 to 24 carbon atoms. In some cases, anionic sulfate surfactants may be excluded from the one or more anionic surfactants. In such cases, the one or more anionic surfactants may be selected from the group consisting of acyl isethionates, acyl glycinates, acyl taurates, acyl amino acids, acyl sarcosinates, sulfosuccinates, sulfonates, and a mixture thereof, wherein the alkyl and acyl groups of all these compounds comprise from 6 to 24 carbon atoms. A more exhaustive list of anionic surfactants that may be included in the hair color toning compositions is provided later, under the heading “Anionic Surfactants.” The total amount of the one or more anionic surfactants may be about 1 to about 40% by weight, relative to the total weight of the hair color toning composition, including all ranges and subranges therebetween. Furthermore, the total amount of the one or more anionic surfactants may be about 1 to about 35%, about 1 to about 30%, about 5% to about 40%, about 5% to about 25%, about 5% to about 30%, about 10% to about 40%, about 10% to about 35%, or about 15% to about 40%. The anionic surfactant(s) that may be used may be alkyl sulfates, alkyl ether sulfates, alkylamido ether sulfates, alkylaryl polyether sulfates, monoglyceride sulfates, alkylsulfonates, alkylamide sulfonates, alkylarylsulfonates, alpha-olefin sulfonates, paraffin sulfonates, alkylsulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfoacetates, acylsarcosinates, acylglutamates, alkylsulfosuccinamates, acylisethionates and N-acyltaurates, salts of alkyl monoesters and polyglycoside-polycarboxylic acids, acyllactylates, salts of D-galactoside uronic acids, salts of alkyl ether carboxylic acids, salts of alkyl aryl ether carboxylic acids, and salts of alkylamido ether carboxylic acids; or the non-salified forms of all of these compounds, the alkyl and acyl groups of all of these compounds containing from 6 to 24 carbon atoms and the aryl group denoting a phenyl group. Some of these compounds may be oxyethylenated and then preferably comprise from 1 to 50 ethylene oxide units. For example, the anionic surfactant may be chosen from sodium olefin sulfonates, e.g. sodium C14-C16 olefin sulfonate. The salts of C6-C24alkyl monoesters of polyglycoside-polycarboxylic acids may be chosen from C6-C24alkyl polyglycoside-citrates, C6-C24alkyl polyglycoside-tartrates and C6-C24alkyl polyglycoside-sulfo succinates. When the anionic surfactant(s) are in salt form, they may be chosen especially from alkali metal salts such as the sodium or potassium salt and preferably the sodium salt, ammonium salts, amine salts and in particular amino alcohol salts, or alkaline-earth metal salts such as the magnesium salt. Examples of amino alcohol salts that may especially be mentioned include monoethanolamine, diethanolamine and triethanolamine salts, monoisopropanolamine, diisopropanolamine or triisopropanolamine salts, 2-amino-2-methyl-1-propanol salts, 2-amino-2-methyl-1,3-propanediol salts and tris(hydroxymethyl)aminomethane salts. Alkali metal or alkaline-earth metal salts and in particular the sodium or magnesium salts may be used. Use is also made of (C6-C24)alkyl sulfates, (C6-C24)alkyl ether sulfates, which are optionally ethoxylated, comprising from 2 to 50 ethylene oxide units, and a mixture thereof, in particular in the form of alkali metal salts or alkaline-earth metal salts, ammonium salts or amino alcohol salts. More preferentially, the anionic surfactant(s) are chosen from (C10-C20)alkyl ether sulfates, and in particular sodium lauryl ether sulfate. Non-Ionic Surfactants Non-ionic surfactants are compounds well known in themselves (see, e.g., in this regard, “Handbook of Surfactants” by M. R. Porter, Blackie & Son publishers (Glasgow and London), 1991, pp. 116-178), which is incorporated herein by reference in its entirety. The total amount of the one or more non-ionic surfactants may be about 1 to about 40% by weight, relative to the total weight of the hair color toning composition, including all ranges and subranges therebetween. Furthermore, the total amount of the one or more non-ionic surfactants may be about 1 to about 35%, about 1 to about 30%, about 5% to about 40%, about 5% to about 25%, about 5% to about 30%, about 10% to about 40%, about 10% to about 35%, or about 15% to about 40%. The non-ionic surfactant can be, for example, selected from alcohols, alpha-diols, alkylphenols and esters of fatty acids, these compounds being ethoxylated, propoxylated or glycerolated and having at least one fatty chain comprising, for example, from 8 to 18 carbon atoms, it being possible for the number of ethylene oxide or propylene oxide groups to range from 2 to 50, and for the number of glycerol groups to range from 1 to 30. Maltose derivatives may also be mentioned. Non-limiting mention may also be made of copolymers of ethylene oxide and/or of propylene oxide; condensates of ethylene oxide and/or of propylene oxide with fatty alcohols; polyethoxylated fatty amides comprising, for example, from 2 to 30 mol of ethylene oxide; polyglycerolated fatty amides comprising, for example, from 1.5 to 5 glycerol groups, such as from 1.5 to 4; ethoxylated fatty acid esters of sorbitan comprising from 2 to 30 mol of ethylene oxide; ethoxylated oils from plant origin; fatty acid esters of sucrose; fatty acid esters of polyethylene glycol; polyethoxylated fatty acid mono or diesters of glycerol (C6-C24)alkylpolyglycosides; N—(C6-C24)alkylglucamine derivatives, amine oxides such as (C10-C14)alkylamine oxides or N—(C10-C14)acylaminopropylmorpholine oxides; and a mixture thereof. The nonionic surfactants may preferably be chosen from polyoxyalkylenated or polyglycerolated nonionic surfactants. The oxyalkylene units are more particularly oxyethylene or oxypropylene units, or a mixture thereof, and are preferably oxyethylene units. Examples of oxyalkylenated nonionic surfactants that may be mentioned include: oxyalkylenated (C8-C24)alkylphenols, saturated or unsaturated, linear or branched, oxyalkylenated C8-C30alcohols, saturated or unsaturated, linear or branched, oxyalkylenated C8-C30amides, esters of saturated or unsaturated, linear or branched, C8-C30acids and of polyethylene glycols, polyoxyalkylenated esters of saturated or unsaturated, linear or branched, C8-C30acids and of sorbitol, saturated or unsaturated, oxyalkylenated plant oils, condensates of ethylene oxide and/or of propylene oxide, inter alia, alone or as mixtures. As examples of polyglycerolated nonionic surfactants, polyglycerolated C8-C40alcohols are preferably used. In particular, the polyglycerolated C8-C40alcohols correspond to the following formula: RO—[CH2—CH(CH2OH)—O]m—H or RO—[CH(CH2OH)—CH2O]m—H in which R represents a linear or branched C8-C40and preferably C8-C30alkyl or alkenyl radical, and m represents a number ranging from 1 to 30 and preferably from 1.5 to 10. As examples of compounds that are suitable in the context of the invention, mention may be made of lauryl alcohol containing 4 mol of glycerol (INCI name: Polyglyceryl-4 Lauryl Ether), lauryl alcohol containing 1.5 mol of glycerol, oleyl alcohol containing 4 mol of glycerol (INCI name: Polyglyceryl-4 Oleyl Ether), oleyl alcohol containing 2 mol of glycerol (INCI name: Polyglyceryl-2 Oleyl Ether), cetearyl alcohol containing 2 mol of glycerol, cetearyl alcohol containing 6 mol of glycerol, oleocetyl alcohol containing 6 mol of glycerol, and octadecanol containing 6 mol of glycerol. According to one of the embodiments according to the present invention, the nonionic surfactant may be selected from esters of polyols with fatty acids with a saturated or unsaturated chain containing for example from 8 to 24 carbon atoms, preferably 12 to 22 carbon atoms, and alkoxylated derivatives thereof, preferably with a number of alkyleneoxide of from 10 to 200, and more preferably from 10 to 100, such as glyceryl esters of a C8-C24, preferably C12-C22, fatty acid or acids and alkoxylated derivatives thereof, preferably with a number of alkyleneoxide of from 10 to 200, and more preferably from 10 to 100; polyethylene glycol esters of a C8-C24, preferably C12-C22, fatty acid or acids and alkoxylated derivatives thereof, preferably with a number of alkyleneoxide of from 10 to 200, and more preferably from 10 to 100; sorbitol esters of a C8-C24, preferably C12-C22, fatty acid or acids and alkoxylated derivatives thereof, preferably with a number of alkyleneoxide of from 10 to 200, and more preferably from 10 to 100; sugar (sucrose, glucose, alkylglycose) esters of a C8-C24, preferably C12-C22, fatty acid or acids and alkoxylated derivatives thereof, preferably with a number of alkyleneoxide of from 10 to 200, and more preferably from 10 to 100; ethers of fatty alcohols; ethers of sugar and a C8-C24, preferably C12-C22, fatty alcohol or alcohols; and a mixture thereof. Preferably, the nonionic surfactant may be a nonionic surfactant with an HLB of 18.0 or less, such as from 4.0 to 18.0, more preferably from 6.0 to 15.0 and furthermore preferably from 9.0 to 13.0. The HLB is the ratio between the hydrophilic part and the lipophilic part in the molecule. This term HLB is well known to those skilled in the art and is described in “The HLB system. A time-saving guide to emulsifier selection” (published by ICI Americas Inc., 1984). In some case, the nonionic surfactant is a fatty alkanolamide. Non-limiting examples of fatty alkanolamides that may be used include cocamide MEA, cocamide DEA, soyamide DEA, lauramide DEA, oleamide MIPA, stearamide MEA, myristamide DEA, stearamide DEA, oleylamide DEA, tallowamide DEA lauramide MIPA, tallowamide MEA, isostearamide DEA, isostearamide MEA, and a mixture thereof. Ampohoteric Surfactants Compositions according to the disclosure may comprise at least one amphoteric surfactant. Non-limiting examples of amphoteric surfactants useful in the compositions include, for example, optionally quaternized secondary or tertiary aliphatic amine derivatives, in which the aliphatic group is a linear or branched chain comprising from 8 to 22 carbon atoms, said amine derivatives containing at least one anionic group, for instance a carboxylate, sulfonate, sulfate, phosphate or phosphonate group. Mention may be made in particular of (C8-C20)alkylbetaines, sulfobetaines, (C8-C20)alkylsulfobetaines, (C8-C20)alkylamido(C1-C6)alkylbetaines, such as cocamidopropylbetaine, and (C8-C20)alkylamido(C1-C6)alkylsulfobetaines, and mixtures thereof. For example, mention may be made of compounds classified under the INCI names sodium cocoamphoacetate, sodium lauroamphoacetate, sodium caproamphoacetate and sodium capryloamphoacetate. Other compounds that may be chosen include disodium cocoamphodiacetate, disodium lauroamphodiacetate, disodium caproamphodiacetate, disodium capryloamphodiacetate, disodium cocoamphodipropionate, disodium lauroamphodipropionate, disodium caproamphodipropionate, disodium capryloamphodipropionate, lauroamphodipropionic acid and cocoamphodipropionic acid. Examples that may be mentioned include the cocoamphodiacetate sold by the company Rhodia under the trade name Miranol®. C2M Concentrate, the sodium cocoamphoacetate sold under the trade name Miranol Ultra C 32 and the product sold by the company Chimex under the trade name CHIMEXANE HA. In certain exemplary embodiments, the amphoteric surfactants are chosen from (C8-C20)alkylbetaines such as the one known under the INCI names coco-betaine, (C8-C20)alkylamido(C1-C6)alkylbetaines such as the one known under the INCI name cocamidopropylbetaine, and mixtures thereof. In one embodiment, the amphoteric surfactant is coco-betaine. The composition according to the invention may comprise the amphoteric surfactant(s) in an amount ranging from about 0.1% to about 10%, such as from about 0.5% to about 8%, from about 1% to about 5%, or from about 1% to about 3% by weight, relative to the total weight of the composition. Hair Coloring Agent The compositions according to the disclosure may comprise a hair coloring agent selected from the group consisting of oxidative dyes, direct dyes, or combinations thereof. In embodiments comprising oxidative dyes, the oxidative dyes are generally chosen from one or more oxidation bases optionally combined with one or more couplers. By way of example, the oxidation bases may be chosen from para-phenylenediamines, bis(phenyl)alkylenediamines, para-aminophenols, ortho-aminophenols and heterocyclic bases, and the addition salts thereof. Among the para-phenylenediamines that may be mentioned, for example, are para-phenylenediamine, para-toluenediamine, 2-chloro-para-phenylenediamine, 2,3-dimethyl-para-phenylenediamine, 2,6-dimethyl-para-phenylenediamine, 2,6-diethyl-para-phenylenediamine, 2,5-dimethyl-para-phenylenediamine, N,N-dimethyl-para-phenylenediamine, N,N-diethyl-para-phenylenediamine, N,N-dipropyl-para-phenylenediamine, 4-amino-N,N-diethyl-3-methylaniline, N,N-bis(β-hydroxyethyl)-para-phenylenediamine, 4-N,N-bis(β-hydroxyethyl)amino-2-methylaniline, 4-N,N-bis(β-hydroxyethyl)amino-2-chloroaniline, 2-β-hydroxyethyl-para-phenylenediamine, 2-methoxymethyl-para-phenylenediamine, 2-fluoro-para-phenylenediamine, 2-isopropyl-para-phenylenediamine, N-(β-hydroxypropyl)-para-phenylenediamine, 2-hydroxymethyl-para-phenylenediamine, N,N-dimethyl-3-methyl-para-phenylenediamine, N-ethyl-N-(β-hydroxyethyl)-para-phenylenediamine, N-(β,γ-dihydroxypropyl)-para-phenylenediamine, N-(4′-aminophenyl)-para-phenylenediamine, N-phenyl-para-phenylenediamine, 2-β-hydroxyethyloxy-para-phenylenediamine, 2-β-acetylaminoethyloxy-para-phenylenediamine, N-(β-methoxyethyl)-para-phenylenediamine, 4-aminophenylpyrrolidine, 2-thienyl-para-phenylenediamine, 2-β-hydroxyethylamino-5-aminotoluene and 3-hydroxy-1-(4′-aminophenyl)pyrrolidine, and the addition salts thereof with an acid. Among the para-phenylenediamines mentioned above, para-phenylenediamine, para-toluenediamine, 2-isopropyl-para-phenylenediamine, 2-β-hydroxyethyl-para-phenylenediamine, 2-β-hydroxyethyloxy-para-phenylenediamine, 2,6-dimethyl-para-phenylenediamine, 2,6-diethyl-para-phenylenediamine, 2,3-dimethyl-para-phenylenediamine, N,N-bis(β-hydroxyethyl)-para-phenylenediamine, 2-chloro-para-phenylenediamine and 2-β-acetylaminoethyloxy-para-phenylenediamine, and the addition salts thereof with an acid, are particularly preferred. Among the bis(phenyl)alkylenediamines that may be mentioned, for example, are N,N′-bis(β-hydroxyethyl)-N,N′-bis(4′-aminophenyl)-1,3-diaminopropanol, N,N′-bis(β-hydroxyethyl)-N,N′-bis(4′-aminophenyl)ethylenediamine, N,N′-bis(4-aminophenyl)tetramethylenediamine, N,N′-bis(β-hydroxyethyl)-N,N′-bis(4-aminophenyl)tetramethylenediamine, N,N′-bis(4-methylaminophenyl)tetramethylenediamine, N,N′-bis(ethyl)-N,N′-bis(4′-amino-3′-methylphenyl)ethylenediamine and 1,8-bis(2,5-diaminophenoxy)-3,6-dioxaoctane, and the addition salts thereof. Among the para-aminophenols that may be mentioned, for example, are para-aminophenol, 4-amino-3-methylphenol, 4-amino-3-fluorophenol, 4-amino-3-chlorophenol, 4-amino-3-hydroxymethylphenol, 4-amino-2-methylphenol, 4-amino-2-hydroxymethylphenol, 4-amino-2-methoxymethylphenol, 4-amino-2-aminomethylphenol, 4-amino-2-(β-hydroxyethylaminomethyl)phenol and 4-amino-2-fluorophenol, and the addition salts thereof with an acid. Among the ortho-aminophenols that may be mentioned, for example, are 2-aminophenol, 2-amino-5-methylphenol, 2-amino-6-methylphenol and 5-acetamido-2-aminophenol, and the addition salts thereof. Among the heterocyclic bases that may be mentioned, for example, are pyridine derivatives, pyrimidine derivatives and pyrazole derivatives. Among the pyridine derivatives that may be mentioned are the compounds described, for example, in patents GB 1 026 978 and GB 1 153 196, for instance 2,5-diaminopyridine, 2-(4-methoxyphenyl)amino-3-aminopyridine and 3,4-diaminopyridine, and the addition salts thereof. Other pyridine oxidation bases can include the 3-aminopyrazolo[1,5-a]pyridine oxidation bases or the addition salts thereof described, for example, in patent application FR 2 801 308. Examples that may be mentioned include pyrazolo[1,5-a]pyrid-3-ylamine, 2-acetylaminopyrazolo[1,5-a]pyrid-3-ylamine, 2-morpholin-4-ylpyrazolo[1,5-a]pyrid-3-ylamine, 3-aminopyrazolo[1,5-a]pyridine-2-carboxylic acid, 2-methoxypyrazolo[1,5-a]pyrid-3-ylamine, (3-aminopyrazolo[1,5-a]pyrid-7-yl)methanol, 2-(3-aminopyrazolo[1,5-a]pyrid-5-yl)ethanol, 2-(3-aminopyrazolo[1,5-a]pyrid-7-yl)ethanol, (3-aminopyrazolo[1,5-a]pyrid-2-yl)methanol, 3,6-diaminopyrazolo[1,5-a]pyridine, 3,4-diaminopyrazolo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine-3,7-diamine, 7-morpholin-4-ylpyrazolo[1,5-a]pyrid-3-ylamine, pyrazolo[1,5-a]pyridine-3,5-diamine, 5-morpholin-4-ylpyrazolo[1,5-a]pyrid-3-ylamine, 2-[(3-aminopyrazolo[1,5-a]pyrid-5-yl)(2-hydroxyethyl)amino]ethanol, 2-[(3-aminopyrazolo[1,5-a]pyrid-7-yl)(2-hydroxyethyl)amino]ethanol, 3-aminopyrazolo[1,5-a]pyridin-5-ol, 3-aminopyrazolo[1,5-a]pyridin-4-ol, 3-aminopyrazolo[1,5-a]pyridin-6-ol, 3-aminopyrazolo[1,5-a]pyridin-7-ol, 2-β-hydroxyethoxy-3-amino-pyrazolo[1,5-a]pyridine; 2-(4-dimethylpyperazinium-1-yl)-3-amino-pyrazolo[1,5-a]pyridine; and the addition salts thereof. More particularly, oxidation bases can be selected from 3-aminopyrazolo-[1,5-a]-pyridines and preferably substituted on carbon atom 2 by:(a) one (di)(C1-C6)(alkyl)amino group wherein said alkyl group can be substituted by at least one hydroxy, amino, imidazolium group;(b) one heterocycloalkyl group containing from 5 to 7 members chain, and from 1 to 3 heteroatoms, potentially cationic, potentially substituted by one or more (C1-C6)alkyl, such as di(C1-C4)alkylpiperazinium; or(c) one (C1-C6)alkoxy potentially substituted by one or more hydroxy groups such as α-hydroxyalkoxy, and the addition salts thereof. Among the pyrimidine derivatives that may be mentioned are the compounds described, for example, in the patents DE 2359399; JP 88-169571; JP 05-63124; EP 0770375 or patent application WO 96/15765, such as 2,4,5,6-tetraaminopyrimidine, 4-hydroxy-2,5,6-triaminopyrimidine, 2-hydroxy-4,5,6-triaminopyrimidine, 2,4-dihydroxy-5,6-diaminopyrimidine, 2,5,6-triaminopyrimidine and their addition salts and their tautomeric forms, when a tautomeric equilibrium exists. Among the pyrazole derivatives that may be mentioned are the compounds described in the patents DE 3843892, DE 4133957 and patent applications WO 94/08969, WO 94/08970, FR-A-2 733 749 and DE 195 43 988, such as 4,5-diamino-1-methylpyrazole, 4,5-diamino-1-(β-hydroxyethyl)pyrazole, 3,4-diaminopyrazole, 4,5-diamino-1-(4′-chlorobenzyl)pyrazole, 4,5-diamino-1,3-dimethylpyrazole, 4,5-diamino-3-methyl-1-phenylpyrazole, 4,5-diamino-1-methyl-3-phenylpyrazole, 4-amino-1,3-dimethyl-5-hydrazinopyrazole, 1-benzyl-4,5-diamino-3-methylpyrazole, 4,5-diamino-3-tert-butyl-1-methylpyrazole, 4,5-diamino-1-tert-butyl-3-methylpyrazole, 4,5-diamino-1-(β-hydroxyethyl)-3-methylpyrazole, 4,5-diamino-1-ethyl-3-methylpyrazole, 4,5-diamino-1-ethyl-3-(4′-methoxyphenyl)pyrazole, 4,5-diamino-1-ethyl-3-hydroxymethylpyrazole, 4,5-diamino-3-hydroxymethyl-1-methylpyrazole, 4,5-diamino-3-hydroxymethyl-1-isopropylpyrazole, 4,5-diamino-3-methyl-1-isopropylpyrazole, 4-amino-5-(2′-aminoethyl)amino-1,3-dimethylpyrazole, 3,4,5-triaminopyrazole, 1-methyl-3,4,5-triaminopyrazole, 3,5-diamino-1-methyl-4-methylaminopyrazole, 3,5-diamino-4-(β-hydroxyethyl)amino-1-methylpyrazole, and the addition salts thereof. 4,5-Diamino-1-(β-methoxyethyl)pyrazole may also be used. A 4,5-diaminopyrazole will preferably be used, and even more preferentially 4,5-diamino-1-(β-hydroxyethyl)pyrazole and/or a salt thereof. Pyrazole derivatives that may also be mentioned include diamino-N,N-dihydro-pyrazolopyrazolones and especially those described in patent application FR-A-2 886 136, such as the following compounds and the addition salts thereof: 2,3-diamino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 2-amino-3-ethylamino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 2-amino-3-isopropylamino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 2-amino-3-(pyrrolidin-1-yl)-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 4,5-diamino-1,2-dimethyl-1,2-dihydropyrazol-3-one, 4,5-diamino-1,2-diethyl-1,2-dihydropyrazol-3-one, 4,5-diamino-1,2-di-(2-hydroxyethyl)-1,2-dihydropyrazol-3-one, 2-amino-3-(2-hydroxyethyl)amino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 2-amino-3-dimethylamino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one, 2,3-diamino-5,6,7,8-tetrahydro-1H,6H-pyridazino[1,2-a]pyrazol-1-one, 4-amino-1,2-diethyl-5-(pyrrolidin-1-yl)-1,2-dihydropyrazol-3-one, 4-amino-5-(3-dimethylaminopyrrolidin-1-yl)-1,2-diethyl-1,2-dihydropyrazol-3-one, 2,3-diamino-6-hydroxy-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one. 4,5-Diamino-1-(β-hydroxyethyl)pyrazole and/or 2,3-diamino-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-one and/or a salt thereof will preferentially be used as heterocyclic bases. Compositions and/or systems according to embodiments of the disclosure may optionally further comprise one or more couplers advantageously chosen from those conventionally used in the dyeing or coloring of keratinous substrates. Among these couplers, mention may be made especially of meta-phenylenediamines, meta-aminophenols, meta-diphenols, naphthalene-based couplers and heterocyclic couplers, and also the addition salts thereof. Mention may be made, for example, of 2-methyl-5-aminophenol, 5-N-(ß-hydroxyethyl)amino-2-methylphenol, 3-aminophenol, 5-amino-6-chloro-o-cresol (3-amino-2-chloro-6-methylphenol), 1,3-dihydroxybenzene, 1,3-dihydroxy-2-methylbenzene, 4-chloro-1,3-dihydroxybenzene, 2,4-diamino-1-(ß-hydroxyethyloxy)benzene, 2-amino-4-(ß-hydroxyethylamino)-1-methoxybenzene, 1,3-diaminobenzene, 1,3-bis(2,4-diamino-phenoxy)propane, 3-ureidoaniline, 3-ureido-1-dimethylaminobenzene, sesamol, 1-ß-hydroxyethylamino-3,4-methylenedioxybenzene, α-naphthol, 2-methyl-1-naphthol, 6-hydroxyindole, 4-hydroxyindole, 4-hydroxy-N-methylindole, 2-amino-3-hydroxypyridine, 6-hydroxybenzomorpholine, 3,5-diamino-2,6-dimethoxypyridine, 1-N-(ß-hydroxyethyl)-amino-3,4-methylenedioxybenzene, 2,6-bis(ß-hydroxyethylamino)toluene, 6-hydroxy-indoline, 2,6-dihydroxy-4-methylpyridine, 1-H-3-methylpyrazol-5-one, 1-phenyl-3-methyl-pyrazol-5-one, 2,6-dimethylpyrazolo[1,5-b]-1,2,4-triazole, 2,6-dimethyl[3,2-c]-1,2,4-triazole and 6-methylpyrazolo[1,5-a]benzimidazole, the addition salts thereof with an acid, and mixtures thereof. In general, the addition salts of the oxidation bases and couplers that may be used in the context of the invention are especially selected from the addition salts with an acid such as the hydrochlorides, hydrobromides, sulfates, citrates, succinates, tartrates, lactates, tosylates, benzenesulfonates, phosphates and acetates. The oxidation base(s) may be present in an amount ranging from about 0.001% to 10% by weight, such as from about 0.005% to 5% by weight, relative to the total weight of the system or composition comprising the system in which it is present. The coupler(s), if they are present, may be present in an amount ranging from about 0.001% to 10% by weight, such as from about 0.005% to 5% by weight, relative to the total weight of the system or composition comprising the system in which it is present. Compositions according to embodiments of the disclosure may optionally comprise one or more synthetic or natural direct dyes, for example chosen from anionic and nonionic species, preferably cationic or nonionic species, either as sole dyes or in addition to the oxidation dye(s). Examples of suitable direct dyes that may be mentioned include azo direct dyes; (poly)methine dyes such as cyanins, hemicyanins and styryls; carbonyl dyes; azine dyes; nitro(hetero)aryl dyes; tri(hetero)arylmethane dyes; porphyrin dyes; phthalocyanin dyes, and natural direct dyes, alone or as mixtures. Preferably direct dyes are cationic direct dyes. Mention may be made of the hydrazono cationic dyes of formulas (Va) and (V′a), the azo cationic dyes (VIa) and (VI′a) and the diazo cationic dyes (VIIa) below: Het+-C(Ra)═N—N(Rb)—Ar, An−(Va)Het+-N(Rª)—N═C(Rb)—Ar, An−(V′a)Het+-N═N—Ar, An−(VIa)Ar+—N═N—Ar″, An−(VI′a) andHet+-N═N—Ar′—N═N—Ar, An−(VIIa) in which formulas (Va), (V′a), (VIa), (VI′a) and (VIIa):Het+represents a cationic heteroaryl radical, preferably bearing an endocyclic cationic charge, such as imidazolium, indolium or pyridinium, optionally substituted preferentially with one or more (C1-C8) alkyl groups such as methyl;Ar+representing an aryl radical, such as phenyl or naphthyl, bearing an exocyclic cationic charge, preferentially ammonium, particularly tri(C1-C8)alkylammonium such as trimethylammonium;Ar represents an aryl group, especially phenyl, which is optionally substituted, preferentially with one or more electron-donating groups such as i) optionally substituted (C1-C8)alkyl, ii) optionally substituted (C1-C8)alkoxy, iii) (di)(C1-C8)(alkyl)amino optionally substituted on the alkyl group(s) with a hydroxyl group, iv) aryl(C1-C8)alkylamino, v) optionally substituted N—(C1-C8)alkyl-N-aryl(C1-C8)alkylamino or alternatively Ar represents a julolidine group;Ar′ is an optionally substituted divalent (hetero)arylene group such as phenylene, particularly para-phenylene, or naphthalene, which are optionally substituted, preferentially with one or more groups (C1-C8)alkyl, hydroxyl or (C1-C8)alkoxy;Ar″ is an optionally substituted (hetero)aryl group such as phenyl or pyrazolyl, which are optionally substituted, preferentially with one or more groups (C1-C8)alkyl, hydroxyl, (di)(C1-C8)(alkyl)amino, (C1-C8)alkoxy or phenyl;Raand Rb, which may be identical or different, represent a hydrogen atom or a group (C1-C8)alkyl, which is optionally substituted, preferentially with a hydroxyl group; or alternatively the substituent Rawith a substituent of Het+and/or Rbwith a substituent of Ar and/or Rawith Rbform, together with the atoms that bear them, a (hetero)cycloalkyl; particularly, Raand Rbrepresent a hydrogen atom or a group (C1-C4)alkyl, which is optionally substituted with a hydroxyl group;An−represents an anionic counter-ion such as mesylate or halide. In particular, mention may be made of the azo and hydrazono cationic dyes bearing an endocyclic cationic charge of formulae (Va), (V′a) and (VIa) as defined previously. More particularly those of formulae (Va), (V′a) and (VIa) derived from the dyes described in patent applications WO 95/15144, WO 95/01772 and EP-714954. Preferentially, the cationic part is derived from the following derivatives: with:R1representing a (C1-C4) alkyl group such as methyl;R2and R3, which are identical or different, represent a hydrogen atom or a (C1-C4)alkyl group, such as methyl; andR4represents a hydrogen atom or an electron-donating group such as optionally substituted (C1-C8)alkyl, optionally substituted (C1-C8)alkoxy, or (di)(C1-C8)(alkyl)amino optionally substituted on the alkyl group(s) with a hydroxyl group; particularly, R4is a hydrogen atom,Z represents a CH group or a nitrogen atom, preferentially CH;An−represents an anionic counter-ion such as mesylate or halide. The dye of formulae (Va-1) and (VIa-1) can be chosen from Basic Red 51, Basic Yellow 87 and Basic Orange 31 or derivatives thereof: Among the natural direct dyes, mention may be made of lawsone, juglone, alizarin, purpurin, carminic acid, kermesic acid, purpurogallin, protocatechaldehyde, indigo, isatin, curcumin, spinulosin, apigenidin and orceins. Extracts or decoctions containing these natural dyes and in particular henna-based poultices or extracts may also be used. When they are present, the one or more direct dyes more particularly represent from about 0.001% to 10% by weight, such as from about 0.005% to 5% by weight, of the total weight of the system or composition comprising the system in which it is present. Solvent The hair color toning compositions according to the disclosure comprise a solvent. The solvent may be chosen from water, non-aqueous solvents, or combinations thereof. The solvent may be present in the hair color toning composition in an amount ranging from about 10% to about 95% by weight, relative to the total weight of the hair color toning composition. For example, the total amount of solvent may range from about 20% to about 90%, about 20% to about 85%, about 20% to 75%, about 20% to 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 30% to about 90%, about 30% to about 85%, about 30% to 75%, about 30% to 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 40% to about 90%, about 40% to about 85%, about 40% to 75%, about 40% to 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 50% to about 90%, about 50% to about 85%, about 50% to 75%, about 50% to 70%, about 50% to about 65%, or about 50% to about 60% by weight, relative to the total weight of the hair color toning composition. In some embodiments, the solvent comprises, consists essentially of, or consists of water. The total amount of water in the hair color toning compositions may vary depending on the type of composition and the desired consistency, viscosity, etc. In some embodiments, the total amount of water is about 10% to about 95% by weight, relative to the total weight of the hair color toning composition, including all ranges and subranges therebetween. For example, the total amount of water may be about 10% to about 90%, about 10% to about 85%, about 10% to 75%, about 10% to 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, or about 15% to about 30% by weight, relative to the total weight of the hair color toning composition. It may, in at least certain embodiments, be desirable to include water in an amount less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, or less than about 20% by weight, relative to the total weight of the hair color toning composition. For example, the water may be present in an amount ranging from about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, or about 15% to about 20% by weight, relative to the total weight of the hair color toning composition. In certain embodiments, the composition comprises, consists essentially of, or consists of non-aqueous solvents, for example, glycerin, C1-4alcohols, organic solvents, fatty alcohols, fatty ethers, fatty esters, polyols, glycols, vegetable oils, mineral oils, liposomes, laminar lipid materials, or any a mixture thereof. As examples of organic solvents, non-limiting mentions can be made of monoalcohols and polyols such as ethyl alcohol, isopropyl alcohol, propyl alcohol, benzyl alcohol, and phenylethyl alcohol, or glycols or glycol ethers such as, for example, monomethyl, monoethyl and monobutyl ethers of ethylene glycol, propylene glycol or ethers thereof such as, for example, monomethyl ether of propylene glycol, butylene glycol, hexylene glycol, dipropylene glycol as well as alkyl ethers of diethylene glycol, for example monoethyl ether or monobutyl ether of diethylene glycol. Other suitable examples of organic solvents are ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, propane diol, and glycerin. The organic solvents can be volatile or non-volatile compounds. Further non-limiting examples of solvents which may be used include alkanediols (polyhydric alcohols) such as glycerin, 1,2,6-hexanetriol, trimethylolpropane, ethylene glycol, propylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, dipropylene glycol, 2-butene-1,4-diol, 2-ethyl-1,3-hexanediol, 2-methyl-2,4-pentanediol, caprylyl glycol, 1,2-hexanediol, 1,2-pentanediol, and 4-methyl-1,2-pentanediol; alkyl alcohols having 1 to 4 carbon atoms such as ethanol, methanol, butanol, propanol, and isopropanol; glycol ethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monomethyl ether acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol mono-n-propyl ether, ethylene glycol mono-iso-propyl ether, diethylene glycol mono-iso-propyl ether, ethylene glycol mono-n-butyl ether, ethylene glycol mono-t-butyl ether, diethylene glycol mono-t-butyl ether, 1-methyl-1-methoxybutanol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol mono-t-butyl ether, propylene glycol mono-n-propyl ether, propylene glycol mono-iso-propyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, dipropylene glycol mono-n-propyl ether, and dipropylene glycol mono-iso-propyl ether; 2-pyrrolidone, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, formamide, acetamide, dimethyl sulfoxide, sorbit, sorbitan, acetine, diacetine, triacetine, sulfolane, and a mixture thereof. In some cases, the water-soluble solvent may be selected from the group consisting of one or more glycols, C1-4alcohols, glycerin, and a mixture thereof. In some cases, the water-soluble solvent is selected from the group consisting of hexylene glycol, proplene glycol, caprylyl glycol, glycerin, isopropyl alcohol, and a mixture thereof. Polyhydric alcohols are useful. Examples of polyhydric alcohols include glycerin, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 3-methyl-1,3-butanediol, 1,5-pentanediol, tetraethylene glycol, 1,6-hexanediol, 2-methyl-2,4-pentanediol, polyethylene glycol, 1,2,4-butanetriol, 1,2,6-hexanetriol, and a mixture thereof. Polyol compounds may also be used. Non-limiting examples include the aliphatic diols, such as 2-ethyl-2-methyl-1,3-propanediol, 3,3-dimethyl-1,2-butanediol, 2,2-diethyl-1,3-propanediol, 2-methyl-2-propyl-1,3-propanediol, 2,4-dimethyl-2,4-pentanediol, 2,5-dimethyl-2,5-hexanediol, 5-hexene-1,2-diol, and 2-ethyl-1,3-hexanediol, and a mixture thereof. The total amount of the non-aqueous solvents may vary, but in some cases ranges from about 0.01% to about 50% by weight, relative to the total weight of the composition. For example, the total amount of non-aqueous solvents may range from about 1% to about 50%, about 2% to about 50%, about 3% to 50%, about 4% to about 50%, about 5% to about 50%, 1% to about 40%, about 2% to about 40%, about 3% to 40%, about 4% to about 40%, about 5% to about 40%, about 1% to about 35%, about 2% to about 35%, about 3% to 35%, about 4% to about 35%, or about 5% to about 35% by weight, relative to the total weight of the composition. In certain embodiments, the total amount of non-aqueous solvents may range from about 1% to about 10%, about 2% to about 8%, about 3% to about 7%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, or about 30% to about 35% by weight, relative to the total weight of the composition. Thickening Agent In some embodiments, the hair color toning composition optionally further comprises a thickening agent. In other embodiments, the hair color toning composition does not comprise a thickening agent. The hair color toning compositions may contain one or more thickeners (also referred to as thickening agents or viscosity modifying agents). Classes of such agents include, but are not limited to, viscous liquids, such as polyethylene glycol, semisynthetic polymers, such as semisynthetic cellulose derivatives, synthetic polymers, such as carbomers, poloxamers, and acrylates/beheneth-25 methacrylate copolymer, acrylates copolymer, polyethyleneimines (e.g., PEI-10), naturally occurring polymers, such as acacia, tragacanth, alginates (e.g., sodium alginate), carrageenan, vegetable gums, such as xanthan gum, petroleum jelly, waxes, particulate associate colloids, such as bentonite, colloidal silicon dioxide, and microcrystalline cellulose, surfactants, such as PPG-2 hydroxyethyl coco/isostearamide, emulsifiers, such as disteareth-75 IPDI, and salts, such as sodium chloride, starches, such as hydroxypropyl starch phosphate, potato starch (modified or unmodified), celluloses such as hydroxyethylcellulose, guars such as hydroxypropyl guar, and a mixture thereof. In some cases, the thickening agents may include one or more associative thickening polymers such as anionic associative polymers, amphoteric associative polymers, cationic associative polymers, nonionic associative polymers, and a mixture thereof. A non-limiting example of an amphoteric associative polymer is acrylates/beheneth-25methacrylate copolymer, sold under the tradename NOVETHIX L-10 (Lubrizol). Non-limiting examples of anionic associative polymers include INCI name: acrylates copolymer, sold under the tradename CARBOPOL Aqua SF-1 (Lubrizol), INCI name: acrylates crosspolymer-4, sold under the tradename CARBOPOL Aqua SF-2 (Lubrizol), and a mixture thereof. The associative thickening polymers, for instance, the acrylates copolymer and/or the acrylates crosspolymer-4, may be neutralized in water or an aqueous solution with a neutralizing agent before the polymer is added into a hair color toning composition. In some embodiments, the thickener is chosen from hydroxyethylcellulose, cetyl hydroxyethylcellulose, or combinations thereof. The total amount of the one or more thickening agents may vary, but in some cases is about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 8%, about 0.1% to about 6%, about 0.15 to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.5% to about 10%, about 0.5% to about 8%, about 0.5% to about 5%, about 0.5% to about 2%, about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, or about 1% to about 5% by weight, relative to the total weight of the composition. pH Adjusters The hair color toning compositions according to the disclosure have a pH ranging from about 7 to about 10.4, such as from about 7.5 to about 10.4, about 8 to about 10.4, about 8.5 to about 10.4, about 9 to about 10.4, or about 9 to about 10. For example, the hair color toning compositions may have a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, 10.0, about 10.1, about 10.2, about 10.3, or about 10.4. The composition may therefore, optionally contain acid and alkali pH adjusters, which are well known in the art in the cosmetic treatment of keratin fibers, such as hair. Such pH adjusters include, but are not limited to, sodium metasilicate, silicate compounds, citric acid, ascorbic acid, and carbonate compounds. Additional Components The composition according to the disclosure may optionally comprise any auxiliary or additional component suitable for use in cosmetic compositions, and in particular suitable for hair color toning compositions. Such components may include, but are not limited to, dyes/pigments in addition to those listed above, silicone compounds, rheology modifying agents such as acrylic polymers, cationic, nonionic, amphoteric or zwitterionic surfactants or mixtures thereof, anionic, cationic, nonionic, amphoteric or zwitterionic polymers or mixtures, film forming agents or polymers, humectants and moisturizing agents, fatty substances other than the claimed fatty substances, emulsifying agents other than fatty substances, fillers, structuring agents, propellants, shine agents, antioxidants or reducing agents, penetrants, sequestrants, fragrances, buffers, dispersants, conditioning agents (e.g. plant extracts), for instance volatile or non-volatile, modified or unmodified silicones, ceramides, preserving agents, opacifiers, sunscreen agents, and antistatic agents. Methods It has been discovered that compositions according to the disclosure surprisingly impart improved properties to the hair, such as improved strength, shine, conditioning, feel, detangling, and/or combability, while also imparting excellent color and/or tone to the hair. Therefore, another aspect of the invention pertains to methods of using any of the compositions described herein by applying the compositions to the hair. In one embodiment, the method comprises applying the hair color toning compositions directly to hair. In further embodiments, the methods comprises mixing the hair color toning compositions with an oxidizing composition (also known as a developer composition) comprising one or more oxidizing agents to form a mixture, and applying the mixture to the hair. In embodiments comprising oxidizing agents, the at least one oxidizing agent may be chosen, for example, from peroxides, persulfates, perborates, percarbonates, alkali metal bromates, ferricyanides, peroxygenated salts, alkali metal carbonates, or a mixture thereof. Oxidizing agents that may also be used include at least one redox enzyme such as laccases, peroxidases, and 2-electron oxidoreductases, such as uricase, where appropriate in the presence of their respective donor or co-factor. Oxygen in the air may also be employed as an oxidizing agent. In one embodiment, the oxidizing agent can be hydrogen peroxide present in an aqueous solution whose titer may range from 1 to 40 volumes, such as from 5 to 40 volumes or such as from 5 to 20 volumes. In another embodiment, the oxidizing agent can be a persulfate and/or a monopersulfate such as, for example, potassium persulfate, sodium persulfate, ammonium persulfate, as well as mixtures thereof. In one embodiment, the oxidizing agents in the present disclosure are selected from hydrogen peroxide, potassium persulfate, sodium persulfate, and mixtures thereof. In certain embodiments, the oxidizing agent is hydrogen peroxide. In general, the oxidizing agent will be present in an amount ranging from about 0.05 to about 50% by weight, such as from about 0.1% to about 30% by weight, from about 0.1% to about 20% by weight, or from about 1% to about 10% by weight, based on the total weight of the developer composition or solution or system in which it is present. In some embodiments, the developer composition is aqueous or is in the form of an emulsion. The developer composition can contain at least one solvent, chosen from water, organic solvents, and mixtures thereof. In alternative embodiments, the developer composition is substantially anhydrous. The term “substantially anhydrous” means that the developer composition is either completely free of water or contains no appreciable amount of water, for example, no more than 5% by weight, or no more than 2% by weight, or no more than 1% by weight, based on the weight of the developer composition. It should be noted that this refers for example to bound water, such as the water of crystallization of the salts or traces of water absorbed by the raw materials used in the preparation of the compositions according to embodiments of the disclosure. When the developer composition is substantially anhydrous, the developer composition may comprise at least one solvent chosen from organic solvents. Suitable organic solvents for use in the developer composition include ethanol, isopropyl alcohol, propanol, benzyl alcohol, phenyl ethyl alcohol, glycols and glycol ethers, such as propylene glycol, hexylene glycol, ethylene glycol monomethyl, monoethyl or monobutyl ether, propylene glycol and its ethers, such as propylene glycol monomethyl ether, butylene glycol, dipropylene glycol, diethylene glycol alkyl ethers, such as diethylene glycol monoethyl ether and monobutyl ether, ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, propane diol, glycerin, hydrocarbons such as straight chain hydrocarbons, mineral oil, polybutene, hydrogenated polyisobutene, hydrogenated polydecene, polydecene, squalane, petrolatum, isoparaffins, and mixtures, thereof. The organic solvents for use in the developer composition can include volatile or non-volatile compounds. The organic solvent may, for example, be present in an amount ranging from about 0.5% to about 70% by weight, such as from about 2% to about 60% by weight, preferably from about 5 to about 50% by weight, relative to the total weight of the developer composition or system in which it is present. The developer composition may be in the form of a powder, gel, liquid, foam, lotion, cream, mousse, and emulsion. The pH of the developer composition can range from about 2 to about 12, such as from about 6 to about 11, and it may be adjusted to the desired value using acidifying/alkalizing agents that are well known in the art. In certain embodiments, the pH of the developer composition is below 7. The pH of the composition resulting from mixing together the hair color toning composition and the developer composition (i.e. the mixture) may range from about 6.0 to about 6.8, such as about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.4 to about 6.7, about 6.4 to about 6.6, or about 6.4 to about 6.5. For example, the pH of the mixture may be about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, or about 6.8. The hair color toning composition or the mixture may be left on the hair for a period of time sufficient to achieve the desired effect. For example, the hair composition or the mixture may be left on the hair for up to one hour, such as from about 3 minutes to about 45 minutes, from about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes. In further embodiments, the hair color toning composition or the mixture may be left on the hair for a period up to about 30 minutes, such as, for example, from about 1 to about 30 minutes, about 1 to about 10 minutes, or about 1 to about 5 minutes. One skilled in the art will be able to determine an appropriate amount of time to leave the hair color toning composition or the mixture on the hair in order to achieve the desired effect. If desired, the composition may, optionally, be shampooed and/or rinsed off the hair. Kits Another aspect of the invention pertains to kits which comprise any of the hair color toning compositions described herein for use in exemplary methods according to the disclosure. In some embodiments, the kit comprises a hair color toning composition according to the disclosure and a developer composition, as described above. The developer composition may be housed in a separate container from the hair color toning composition, and may then be mixed prior to application onto hair. According to at least one embodiment, the kits comprising the compositions according to the disclosure and the developer composition are free or substantially free of ammonia. As used herein, the terms “comprising,” “having,” and “including” (or “comprise,” “have,” and “include”) are used in their open, non-limiting sense. The terms “a,” “an,” and “the” are understood to encompass the plural as well as the singular. Thus, the term “a mixture thereof” or “a combination thereof” also relates to “mixtures thereof” and “combinations thereof.” Throughout the disclosure, the terms “a mixture thereof” and “a combination thereof” is used, following a list of elements as shown in the following example where letters A-F represent the elements: “one or more elements selected from the group consisting of A, B, C, D, E, F, and a mixture thereof.” The terms “a mixture thereof” or “a combination thereof” not require that the mixture include all of A, B, C, D, E, and F (although all of A, B, C, D, E, and F may be included). Rather, it indicates that a mixture of any two or more of A, B, C, D, E, and F can be included. In other words, it is equivalent to the phrase “one or more elements selected from the group consisting of A, B, C, D, E, F, and a mixture of any two or more of A, B, C, D, E, and F.” Likewise, the term “a salt thereof” also relates to “salts thereof.” Thus, where the disclosure refers to “an element selected from the group consisting of A, B, C, D, E, F, a salt thereof, and a mixture thereof,” it indicates that that one or more of A, B, C, D, and F may be included, one or more of a salt of A, a salt of B, a salt of C, a salt of D, a salt of E, and a salt of F may be include, or a mixture of any two of A, B, C, D, E, F, a salt of A, a salt of B, a salt of C, a salt of D, a salt of E, and a salt of F may be included. The salts, for example, the salts of the amino acids, the amino sulfonic acids, and the non-polymeric mono, di, and/or tricarboxylic acids, which are referred to throughout the disclosure may include salts having a counter-ion such as an alkali metal, alkaline earth metal, or ammonium counterion. This list of counterions, however, is non-limiting. The expression “one or more” means “at least one” and thus includes individual components as well as mixtures/combinations. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and/or reaction conditions are to be understood as being modified in all instances by the term “about,” meaning within +/−5% of the indicated number. All percentages, parts and ratios herein are based upon the total weight of the compositions of the present invention, unless otherwise indicated. “Keratinous substrates” as used herein, includes, but is not limited to keratin fibers such as hair and/or scalp on the human head. “Conditioning” as used herein means imparting to one or more hair fibers at least one property chosen from combability, moisture-retentivity, luster, shine, and/or softness. The state of conditioning can be evaluated by any means known in the art, such as, for example, measuring, and comparing, the ease of combability of the treated hair and of the untreated hair in terms of combing work (gm-in), and consumer perception. The term “treat” (and its grammatical variations) as used herein refers to the application of the compositions of the present disclosure onto the surface of keratinous substrates such as hair. The term “treat” (and its grammatical variations) as used herein also refers to contacting keratinous substrates such as hair with the compositions of the present disclosure. A “rinse off” product refers to a composition such as a hair color toning composition that is rinsed and/or washed with water either after or during the application of the composition onto the keratinous substrate, and before drying and/or styling said keratinous substrate. At least a portion, and typically most, of the composition is removed from the keratinous substrate during the rinsing and/or washing. The term “stable” as used herein means that the composition does not exhibit phase separation and/or crystallization for a period of time, for example, for at least 1 day (24 hours), one week, one month, or one year. “Volatile”, as used herein, means having a flash point of less than about 100° C. “Non-volatile”, as used herein, means having a flash point of greater than about 100° C. As used herein, all ranges provided are meant to include every specific range within, and combination of sub ranges between, the given ranges. Thus, a range from 1-5, includes specifically 1, 2, 3, 4 and 5, as well as sub ranges such as 2-5, 3-5, 2-3, 2-4, 1-4, etc. The term “substantially free” or “essentially free” as used herein means that there is less than about 5% by weight of a specific material added to a composition, based on the total weight of the compositions. Nonetheless, the compositions may include less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, or none of the specified material. All ranges and values disclosed herein are inclusive and combinable. For examples, any value or point described herein that falls within a range described herein can serve as a minimum or maximum value to derive a sub-range, etc. All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls. The following examples serve to illustrate embodiments of the present disclosure without, however, being limiting in nature. It will be apparent to those skilled in the art that various modifications and variations can be made in the delivery system, composition and methods of the invention without departing from the spirit or scope of the invention. Thus, it is intended that the present disclosure cover the modifications and variations that come within the scope of the appended claims and their equivalents. EXAMPLES Implementation of the present disclosure is demonstrated by way of the following non-limiting examples. Example 1—Hair Toner Formulations The following hair toner formulations according to the disclosure were prepared. INCI1A1B1C1DLAURYL ALCOHOLSURFACTANT2.500002.500002.500005.00000DECETH-3SURFACTANT9.000009.000009.0000010.0000COCAMIDE MIPASURFACTANT6.500006.500006.500007.20000SODIUM SULFITEACTIVE1.000001.000001.000001.00000COMPOUNDm-AMINOPHENOLDYE/PIGMENT0.014000.014000.014000.01400ETHANOLAMINEACTIVE0.500000.600000.560000.20000COMPOUNDSODIUM C14-16SURFACTANT22.500022.500022.500024.5000OLEFIN SULFONATE2,4-DIAMINO-DYE/PIGMENT0.009000.009000.009000.00900PHENOXYETHANOLHClERYTHORBIC ACIDACTIVE0.150000.150000.150000.15000COMPOUNDOLEYL ALCOHOLFATTY6.000006.000006.000006.00000COMPOUNDISOPROPYLSOLVENT10.0000010.0000010.00000—ALCOHOLPROPYLENESOLVENT7.000007.000007.000007.00000GLYCOLPPG-5-CETETH-10SURFACTANT0.900000.900000.900001.00000PHOSPHATEWHEAT AMINOVEGETAL0.500000.500000.500000.50000ACIDSEXTRACTPEG/PPG-4/12SILICON1.500001.500001.500001.50000DIMETHICONE4-AMINO-2-DYE/PIGMENT0.080000.080000.080000.08000HYDROXYTOLUENEFRAGRANCEFRAGRANCE0.300000.300000.300000.30000EDTAACTIVE0.058000.058000.058000.05800COMPOUNDCOCO-BETAINESURFACTANT2.500002.500002.500003.00000p-PHENYLENE-DYE/PIGMENT0.060000.060000.060000.06000DIAMINEPPG-2 BUTYLSOLVENT5.000005.000005.000005.00000ETHERTAURINEACTIVE0.200001.000000.500001.00000COMPOUNDCITRIC ACIDACTIVE0.200000.200000.200000.20000COMPOUNDDENATUREDSOLVENT———10.0000ALCOHOLWATERSOLVENTQ.S.Q.S.Q.S.Q.S.pH9.579.119.129.35 Example 2—Comparative Hair Toner Formulations The following comparative hair toner formulations were prepared. INCI2A2B2C2D2E2FLAURYL ALCOHOL2.500002.500002.500002.500002.500002.50000DECETH-39.000009.000009.000009.000009.000009.00000COCAMIDE MIPA6.500006.500006.500006.500006.500006.50000SODIUM SULFITE1.000001.000001.000001.000001.000001.00000m-AMINOPHENOL0.014000.014000.014000.014000.014000.01400ETHANOLAMINE0.760000.440000.590000.530000.410000.58000SODIUM C14-1622.5000022.5000022.5000022.5000022.5000022.50000OLEFIN SULFONATE2,4-DIAMINO-0.009000.009000.009000.009000.009000.00900PHENOXYETHANOLHClERYTHORBIC ACID0.150000.150000.150000.150000.150000.15000OLEYL ALCOHOL6.000006.000006.000006.000006.000006.00000ISOPROPYL10.0000010.0000010.0000010.0000010.0000010.00000ALCOHOLPROPYLENE7.000007.000007.000007.000007.000007.00000GLYCOLPPG-5-CETETH-100.900000.900000.900000.900000.900000.90000PHOSPHATEWHEAT AMINO0.500000.500000.500000.500000.500000.50000ACIDSPEG/PPG-4/121.500001.500001.500001.500001.500001.50000DIMETHICONE4-AMINO-2-0.080000.080000.080000.080000.080000.08000HYDROXYTOLUENEFRAGRANCE0.300000.300000.300000.300000.300000.30000EDTA0.058000.058000.058000.058000.058000.05800COCO-BETAINE2.500002.500002.500002.500002.500002.50000p-PHENYLENE-0.060000.060000.060000.060000.060000.06000DIAMINEPPG-2 BUTYL5.000005.000005.000005.000005.000005.00000ETHERTAURINE——————CITRIC ACID1.150000.570000.400000.28000—0.20000WATERQ.S.Q.S.Q.S.Q.S.Q.S.Q.S.pH9.529.769.9910.1610.419.88 Example 3—Comparative Hair Toner Formulations The following comparative hair toner formulations were prepared. INCI3A3B3C3DLAURYL ALCOHOL2.500002.500002.500002.50000DECETH-39.000009.000009.000009.00000COCAMIDE MIPA6.500006.500006.500006.50000SODIUM SULFITE1.000001.000001.000001.00000m-AMINOPHENOL0.014000.014000.014000.01400ETHANOLAMINE0.440000.440000.440000.44000SODIUM C14-1622.5000022.5000022.5000022.50000OLEFIN SULFONATE2,4-DIAMINO-0.009000.009000.009000.00900PHENOXYETHANOLHClERYTHORBIC ACID0.150000.150000.150000.15000OLEYL ALCOHOL6.000006.000006.000006.00000ISOPROPYL10.0000010.0000010.0000010.00000ALCOHOLPROPYLENE7.000007.000007.000007.00000GLYCOLPPG-5-CETETH-100.900000.900000.900000.90000PHOSPHATEWHEAT AMINO0.500000.500000.500000.50000ACIDSPEG/PPG-4/121.500001.500001.500001.50000DIMETHICONE4-AMINO-2-0.080000.080000.080000.08000HYDROXYTOLUENEFRAGRANCE0.300000.300000.300000.30000EDTA0.058000.058000.058000.05800COCO-BETAINE2.500002.500002.500002.50000p-PHENYLENE-0.060000.060000.060000.06000DIAMINEPPG-2 BUTYL5.000005.000005.000005.00000ETHERTAURINE————CITRIC ACID1.150000.570000.400000.28000WATERQ.S.Q.S.Q.S.Q.S.pH5.716.778.008.90 Example 4—Stability Testing of Hair Toner Formulations The following formulations were stored at either 25° C. or 45° C. and stability was evaluated visually, as set forth in the following table. FormulationTempLengthStability results1A45° C.2 monthsStable1B25° C.12 daysStable45° C.12 daysVery minor separation1C25° C.12 daysStable45° C.12 daysVery minor separation2A25° C.1 daySeparated3A45° C.11 daysSeparated2B25° C.5 daysSeparated3B45° C.11 daysSeparated2C45° C.5 daysVery minor separation3C45° C.20 daysStable2D45° C.5 daysVery minor separation3D45° C.2 monthsStable2E45° C.2 monthsStable2F45° C.2 monthsStable Example 5—Evaluation of Properties of Hair Treated with Formulations The formulations of Examples 1A, 1B, 2A-D, and 3A-D were mixed with 6.7V developer (1:1) and applied to swatches of hair that had been bleached, rinsed, and shampooed. The formulations were left on the hair for 20 minutes, and rinsed. The properties imparted to the treated hair were then evaluated by several panels while the hair was both wet and dry. Examples 1A and 1B, which have citric acid, ethanolamine, and taurine, were considered to provide good post-treatment wet detangling, combability, strength, and smoothness, and good dry hair strength, body, combability, and smooth look and feel. Examples 1A and 1B also provided excellent toning, color deposit, and shine to the hair. Examples 2C, 2E, and 2F were considered to provide good post-treatment wet detangling, combability, and smoothness, and good dry hair smooth look and feel, but were not considered to provide properties of wet or dry strength. The difference in toning and shine imparted to the hair was evaluated by comparing formulations 2A/3A; 2B/3B; 2C/3C; and 2D/3D. In each comparison, the hair swatch treated with the formulations of Example 2 had significantly greater toning, color deposit, and shine than those treated with the formulations of Example 3. Example 6—Evaluation of Strength of Treated Hair Hair treated with formulations 1D or 2E was subjected to miniature tensile testing (MTT) in order to evaluate the strength of the hair after treatment. The results of both break stress and break extension demonstrate statistically significant improvement in the strength of hair treated with inventive formulation 1D, compared to hair treated with comparative formulation 2E. StandardFormulationMeanDeviationCV(%)CI(95)CountBreak Stress1D123.220.530.16666.209422E105.920.080.18955.67948Break Extension %1D67.967.2120.10612.181422E61.086.5660.10751.85848 The above data confirms the panelists' conclusions described in Example 5, i.e. that the formulations according to the disclosure provide significantly better strength properties to the hair. The above examples demonstrate that the formulations according to the disclosure unexpectedly provide improved strength, smoothness, and feel to the hair, while achieving stability and superior toning, color deposit, and shine. | 94,285 |
11857661 | DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION The present invention relates to anti-dandruff compositions, and methods of using those compositions to treat or inhibit dandruff. The anti-dandruff compositions are useful for application to keratinous tissue or scalp surface and comprise an effective amount of a combination of anti-dandruff actives, and which inhibit, reduce, or eliminate dandruff symptoms arising from the proliferation ofMalassezia furfur. The anti-dandruff compositions may be in a wide variety of product forms that include, but are not limited to, solutions, suspensions, lotions, creams, gels, ointments, sprays, aerosols, shampoos, hair conditioners, pastes, foams, powders, mousses, wipes, strips, patches, hydrogels, film-forming products, and the like. The compositional form may follow from the particular dermatologically-acceptable carrier chosen. As used herein, the term “anti-dandruff composition” includes compositions that are applied to the hair and/or the skin underneath the hair, and comprise (or consist essentially of) at least one of various combinations of a punicalagin composition; a first monoterpenoid composition comprising (or consisting essentially of) p-cymene; and a second monoterpenoid composition comprising (or consisting essentially of) menthol; and a dermatologically-acceptable carrier, the combinations of which are effective to inhibitMalassezia furfurand treat dandruff. The term “topical application,” as used herein, means to apply or spread the compositions of the present invention onto the surface of the scalp from which mammalian hair grows. The term “dermatologically-acceptable,” as used herein, means that the compositions or components thereof so described are suitable for use in contact with mammalian keratinous or skin tissue without undue toxicity, incompatibility, instability, allergic response, and the like. The term “anti-dandruff active”, as used herein, refers to a single compound or a composition that possesses an ability to inhibit the proliferation ofMalassezia furfur, when present in a medium in an effective amount. The term “effective amount,” as used herein, means an amount of a compound or composition sufficient to reduce or inhibit the proliferation ofMalassezia furfur, reduce or inhibit the visible effects of dandruff caused by the proliferation ofMalassezia furfur, or reduce or inhibit scalp pruritus, by a statistically significant amount. The term “minimum inhibitory concentration,” also abbreviated as “MIC,” refers to the lowest concentration of the anti-dandruff active that will inhibit the growth ofMalassezia furfurafter incubation (at least 24 hours to 72 hours; 32.5° C.±2.5° C.) of a 1% agar solution mixed with an equal volume of an inoculated broth. Inhibition is defined as giving less than 20% growth compared to a control, as determined by spectrophotometric optical density (OD) reading at 620 nm, using the formula % of growth=[(ODproduct−OCabsorbance-control)]/(ODpositive growth-control). Anti-Dandruff Composition In accordance with embodiments of the present invention, the anti-dandruff composition comprises (or consists essentially of) an effective amount of a combination of at least two anti-dandruff actives selected from the group consisting of A) a punicalagin composition; B) a first monoterpenoid composition comprising (or consisting essentially of) p-cymene; and C) a second monoterpenoid composition comprising (or consisting essentially of) menthol; the combination of which is effective to inhibitMalassezia furfurand treat dandruff; and a dermatologically-acceptable carrier. A. Punicalagin Composition The term “punicalagin composition,” as used herein, refers to a composition comprising at least 0.1 weight percent (wt %) of punicalagin A, punicalagin B, or a combination of the anomers. For example, the punicalagin content of the punicalagin composition may be, on a dry weight basis, 0.2 wt %, 1 wt %, 5 wt %, 10 wt %, 20 wt %, 30 wt %, 50 wt %, 75 wt %, or more, or within a range between any two of the foregoing. In one embodiment, the punicalagin content of the punicalagin composition is, on a dry weight basis, 30 wt % or more. Punicalagin may be synthetic or may be derived from a botanical such asPunica granatum(pomegranate),Terminalia catappa(Indian-almond),Terminalia myriocarpa(East Indian almond), orCombretum molle(velvet bushwillow). In accordance with an embodiment, the punicalagin composition is derived fromPunica granatum. For example, the punicalagin composition may be a pomegranate extract. The term “pomegranate extract,” as used herein, refers to a product obtained by an extractive process from pomegranate (Punica granatum). Pomegranates are rich in antioxidants, with that activity being primarily attributable to polyphenols such as punicalagin A and B, punicalin, ellagic acid, gallic acid, etc. and often expressed in terms of gallic acid equivalents (GAE). While also present in pomegranate bark, seed, pulp, juice, and pericarp, the majority of the punicalagins and other ellagitannins, such as punicalin, is found in the skin (also called the rind or peel) of the fruit. Thus, in accordance with an embodiment, the solid pomegranate material may be a dried pomegranate rind or peel, which has been ground or flaked to reduce particle size thereby increasing the available surface area for extraction. In another embodiment, fresh pomegranate peels are utilized. Accordingly, in one embodiment, the pomegranate extract is a composition produced by a general method comprising the steps of: (a) contacting a solid pomegranate material with an aqueous extraction liquid (e.g., water or a mixture of a water-miscible organic solvent and water); and (b) filtering or centrifuging the resulting mixture to separate the extracted pomegranate solids from the aqueous fraction. The process may be repeated multiple times, and the combined aqueous fractions concentrated to remove the water-miscible organic solvent, water, and/or any other volatiles. The pomegranate extract can be standardized by dilution to a desired extent with an appropriate diluent. The selection of a suitable water-miscible organic solvent for the aqueous extraction liquid is not particularly limited, so long as the desired punicalagins are soluble therein. Non-limiting examples include methanol, ethanol, propanol, butanol, acetone, propylene glycol, butylene glycol, dimethylformamide, dimethyl sulfoxide, or combinations thereof. The volume ratio between the water-miscible organic solvent and water can vary between about 9:1 to about 1:9, for example about 7:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:7, or in a range between any two of the foregoing. In an embodiment, the aqueous extraction liquid is water. In another embodiment, the aqueous extraction liquid is an aqueous alcoholic solution. For example, the aqueous extraction liquid may be water, or it may comprise a 1:1 mixture of ethanol and water (i.e., 50% aqueous ethanol). The ratio of the mass of the extracting solution to the mass of pomegranate solids can vary depending on a variety of factors, such as vessel size, extraction efficiency, and the like. A suitable range of ratios includes, but is not limited to, about 1:1 to about 25:1, such as 2:1, 4:1, 6:1, 8:1, 10:1, 15:1, 20:1, or in a range between any two of the foregoing. The pomegranate extraction may be performed at a variety of pressures or temperatures. For example, the extraction can be conducted at ambient pressure and/or room temperature, or the extraction may be performed under pressure and/or at an elevated temperature. In one embodiment, the pomegranate extraction is performed near atmospheric pressure, and at a temperature within in a range from about 40° C. to about 100° C., such as about 50° C., about 60° C., about 70° C., about 80° C., or in a range between any two of the foregoing. Evaporation (e.g., lyophilization) of the extracting liquids provides a solid, pomegranate extract that is enriched in punicalagin. In accordance with an embodiment, the punicalagin composition comprises at least 0.1 wt % of the punicalagin anomers, on a dry weight basis of the extract. In accordance with another embodiment, the punicalagin composition comprises less than 5 wt % of ellagic acid, on a dry weight basis of the extract. In accordance with another embodiment, the punicalagin composition comprises a polyphenol content of at least 30 GAE (by Folin—Ciocalteu Method), on a dry weight basis of the extract. If desired, the punicalagin composition may be standardized to a target polyphenol content with an acceptable diluent. A non-limiting example of a suitable diluent is a polyol, such as glycerol. In one example, the punicalagin composition is diluted with glycerol to provide a standardized solution having a phenolic content of 12% GAE. In accordance with another embodiment, the pomegranate extract may be processed to further enrich the punicalagin concentration. For example, the pomegranate extract may be purified using chromatographic, precipitation, complexation, or other standard techniques to increase the punicalagin content of the extract. For example, the punicalagin content of the pomegranate extract may be, on a dry weight basis, 0.2 wt %, 1 wt %, 5 wt %, 10 wt %, 20 wt %, 30 wt %, 50 wt %, 75 wt %, or more, or within a range between any two of the foregoing. In one embodiment, the punicalagin content of the punicalagin composition is 30 wt % or more, on a dry weight basis of the extract. In one example, extraction of dried, flaked pomegranate rind (having a polyphenolic content of about 20-25 wt % as determined by Folin-Ciocalteu Method) is performed six times with 50% aqueous ethanol (liquid to solid mass ratio of about 4:1) at 60° C. to 70° C. The combined liquids were filtered, and then evaporated to near dryness to provide a solid material having a phenolic content of approximately 40-45% GAE. Analysis of the crude extract using High Performance Liquid Chromatography—Quadrupole Time of Flight Liquid (HPLC-QTOF) confirmed the presence of gallic acid, punicalagins A and B, punicalin, ellagic acid hexoside, ellagic acid pentoside, ellagic acid deoxyhexoside, ellagic acid, and galloyl HHDP hexoside. Quantification by HPLC revealed that ellagic acid was present in less than 5 wt %. The ellagic acid content may be further reduced using standard techniques, such as selective solvent treatments or column chromatography, to a level of 1 wt % or less, based on total weight of the punicalagin composition, where weight percentage is on a dry weight basis. In vitro Inhibition testing of the pomegranate extract againstMalassezia furfur, in accordance with the classic broth dilution protocol, revealed that the pomegranate extract possessed a minimum inhibition concentration (MIC) value of ≤0.1% againstMalassezia furfurCIP 1634.84. Inhibition testing on a substantially pure sample of the punicalagin anomers (A:B, 1:2; 93 wt % pure; part No. 00016983, Chromadex, Inc., Irvice, CA), yielded a MIC value of 0.05% againstMalassezia furfurCBS 1878. In comparison, ellagic acid (>95 wt %, prod. No. E2250, Sigma-Aldrich, St. Louis, MO) demonstrated a MIC value of 0.5% againstMalassezia furfurCBS 1878. In another embodiment, fresh peels are separated from the arils of whole pomegranates, and the peels, which may be subjected to size reduction, are mixed with water. The aqueous portions are collected and the solid residue pressed to expel residual aqueous fluid. This process may be repeated three or more times. The combined aqueous portions are centrifuged to remove suspended solids, and the aqueous portions passed through a packed column containing DIAION™ HP20 resin (Mitsubishi Chemical, Japan) to adsorb the polyphenols. Elution with 50% aqueous ethanol provided fractions rich in punicalagins, which were concentrated and spray dried to provide a solid pomegranate extract with a phenolic content of 70-75% GAE, with approximately 30 wt % punicalagins. A standardized solution was prepared by dilution with glycerol to a phenolic content of 12% GAE. In vitro inhibition testing of this standardized solution againstMalassezia furfurCBS 1878, in accordance with the classic broth dilution protocol, revealed a minimum inhibition concentration (MIC) value of ≤0.05%. Thus, it was observed that increasing the punicalagin content of the pomegranate extract further reduces its MIC value againstMalassezia furfur. B. First Monoterpenoid Composition The term “first monoterpenoid composition,” as used herein, refers to a composition comprising (or consisting essentially of) para-cymene. In an embodiment, the first monoterpenoid composition comprises (or consists essentially of) para-cymene and thymol. In another embodiment, the first monoterpenoid composition further comprises (or consists essentially of) para-cymene and gamma-terpinene. In yet another embodiment, the first monoterpenoid composition comprises (or consists essentially of) para-cymene, thymol, and gamma-terpinene. In an embodiment, the para-cymene content of the first monoterpenoid composition is at least 20 wt %. In an embodiment, the first monoterpenoid composition comprises 20-60 wt % gamma-terpinene, 20-60 wt % para-cymene, and 20-60 wt % thymol. In another embodiment, the combined masses of the gamma-terpinene, para-cymene, and thymol constitute at least 80 wt % of the first monoterpenoid composition. In yet another embodiment, the first monoterpenoid composition comprises gamma-terpinene, para-cymene, and thymol in a mass ratio of about 2:1:1, respectively. In another embodiment, the first monoterpenoid composition is derived from an ajowan (Trachyspermum ammisynCarum copticum) essential oil. As used herein, the term “essential oil,” refers to a concentrated hydrophobic liquid containing volatile aroma compounds from a botanical (plant) source, which may include the plant roots, leaves, stems, fruits, seeds, rinds, etc. Generally speaking, selection of the technique for obtaining an essential oil depends mainly on the starting material, i.e., the plant's original state, its characteristics, and the nature of its aroma compounds. The technique conditions the characteristics of the essential oil, in particular viscosity, color, solubility, volatility, and richness or poorness in certain constituents. Common techniques for obtaining essential oils include solvent or supercritical fluid extractions, cold pressing, or distillation, which can be carried out under a variety of conditions, e.g., dry distillation, hydrodistillation, steam distillation, etc. In accordance with an embodiment, the ajowan essential oil is obtained by steam distillation. Generally, steam distillation corresponds to the vaporization, in the presence of steam, of a substance that is not very miscible with water. The starting material together with water brought to boiling point (hydrodistillation), or with steam in a still (dry distillation). The steam entrains the essential oil vapor, which is condensed and recovered as a separate liquid phase from the water. In an embodiment, the ajowan essential oil is obtained by a typical steam distillation process. Ajowan seeds are ground and charged in an extraction vessel. The ground ajowan seeds are moistened first before applying direct steam. A typical steam distillation of ground ajowan seeds takes 10 hours or more until the desired recovery oil content is reached, and provides an oil yield of approximately 2 to 2.5 wt %, based on the weight of the ground seed starting material. Based on GC/MS analysis, the ajowan seed steam distilled essential oil includes the following listing of major components gamma-terpinene, thymol, and para-cymene. In vitro Inhibition testing of the ajowan essential oil againstMalassezia furfur, in accordance with the classic broth dilution protocol, revealed that the ajowan essential oil possessed a minimum inhibition concentration (MIC) value of 0.5% and 1.0% againstMalassezia furfurCIP 1634.84 andMalassezia furfurCBS 1878, respectively. In vitro inhibition testing of the major components of ajowan essential oil (i.e., gamma-terpinene, para-cymene, and thymol) revealed that gamma-terpinene, para-cymene, and thymol possessed MIC values of >1%, 0.5%, and 0.05%, respectively, againstMalassezia furfurCBS 1878. C. Second Monoterpenoid Composition The term “second monoterpenoid composition,” as used herein, refers to a composition comprising (or consisting essentially of) menthol. In an embodiment, the second monoterpenoid composition comprises (or consists essentially of) menthol, menthone, isomenthone, menthyl acetate, neomenthol, limonene, or combinations thereof (e.g. comprises—or consists essentially of—menthol, menthone, isomenthone, menthyl acetate, neomenthol and limonene). In another embodiment, the second monoterpenoid composition comprises 70-80 wt % menthol, 5-10 wt % menthone, 5-10 wt % isomenthone, 5-10 wt % menthyl acetate, 5-10 wt % neomenthol, and 1-5 wt % limonene. In yet another embodiment, the second monoterpenoid composition is derived from a corn mint (Mentha arvensis) essential oil. In a typical steam distillation of corn mint to produce an essential oil, semi-driedMentha arvensisis charged to a steam distillation vessel, packed well, and steam slowly applied to distill out the oil. Depending on a variety of factors, a typical steam distillation process of corn mint may take between 5 and 7 hours, and provides an oil yield of approximately 1.4 to 1.6 wt %, based on the weight of the semi-dried corn mint starting material. Based on GC/MS analysis, the corn mint essential oil product includes the following listing of components menthol, menthone, isomenthone, menthyl acetate, neomenthol, and limonene. In vitro Inhibition testing of the corn mint essential oil againstMalassezia furfur, in accordance with the classic broth dilution protocol, revealed that the corn mint essential oil possessed a minimum inhibition concentration (MIC) value of 0.5% againstMalassezia furfurCIP 1634.84, and a MIC value of 1% againstMalassezia furfurCBS 1878. As noted above, the anti-dandruff active portion of the anti-dandruff composition comprises (or consists essentially of) a combination of at least two of the three described anti-dandruff actives (i.e., the punicalagin composition, the first monoterpenoid composition, and the second monoterpenoid composition). The combinations are based on the additive or synergistic relationship between the individual anti-dandruff actives. Inhibition testing of the individual anti-dandruff actives to determine MIC levels followed the principles and general methods disclosed in C. Lens-Lisbonne, et al. “Methods for the evaluation of antibacterial activity of essential oils: applications to essences of thyme and cinnamon,” J. Pharm. Belg., 1987, 42(5): 297-302; and C. Lisbonne “Evaluation of bacteriostatic activity of essential oils and their components,” Doctoral Thesis, Pharmacy Faculty, Marseille, F R 1988. Interactions of bi-component mixtures (Fractional Inhibitory Concentration) were evaluated in accordance with the method described by J. L. Pons et al., “Evaluation of antimicrobial interactions between chlorhexidine, quaternary ammonium compounds, preservatives and excipients,” J. Appl. Bacteriol., 1992, 73, 395-400, to assess their additive or synergistic effects. In an embodiment, the punicalagin-containing pomegranate extract, the ajowan essential oil, and the corn mint essential oil, proved to inhibitMalassezia furfurin in vitro experiments at MIC values in a range from about 0.1% (w/v) to about 1.0% (w/v). Bi-component mixtures of the pomegranate extract (punicalagins content about 0.2 wt % on a dry weight basis) and the ajowan essential oil; and the ajowan essential oil and the corn mint essential oil demonstrated synergistic activities againstMalassezia furfur, whereas the bi-component mixture of the pomegranate extract (punicalagins content about 0.2 wt % on a dry weight basis) and the corn mint essential oil demonstrated an additive relationship. However, the bi-component mixture of a pomegranate extract having a punicalagins content of about 30 wt % on a dry weight basis and the corn mint essential oil demonstrated synergistic activities againstMalassezia furfur. Additional synergy testing of bi-component mixtures of the pomegranate extract having a punicalagins content of about 30 wt % on a dry weight basis with the major components of the ajowan essential oil (para-cymene, gamma-terpinene, and thymol) revealed that punicalagins and para-cymene demonstrated synergistic activities againstMalassezia furfur. Thus in accordance with an embodiment, the anti-dandruff composition comprises (or consists essentially of) a combination of the punicalagin composition and the second monoterpenoid composition, along with a dermatologically-acceptable carrier. In accordance with another embodiment, the anti-dandruff composition comprises (or consists essentially of) a combination of the punicalagin composition and the first monoterpenoid composition, along with a dermatologically-acceptable carrier. In accordance with yet another embodiment, the anti-dandruff composition comprises (or consists essentially of) a combination of the first and second monoterpenoid compositions, along with a dermatologically-acceptable carrier. And in accordance with yet another embodiment, the anti-dandruff composition comprises (or consists essentially of) a combination of the punicalagin composition, the first monoterpenoid composition, and the second monoterpenoid composition, along with a dermatologically-acceptable carrier. When used as one of the active components, the punicalagin composition is present in the anti-dandruff composition in an amount between about 0.1 wt % and 10 wt %. For example, the punicalagin composition (e.g. pomegranate extract) may comprise 0.1 wt %, 0.2 wt %, 0.5 wt %, 0.75 wt %, 1.0 wt %, 2.0 wt %, 5.0 wt %, 7.5 wt %, 10 wt % of the anti-dandruff composition, or in a range between any two of the foregoing. When used as one of the active components, the first monoterpenoid composition is present in the anti-dandruff composition in an amount between about 0.1 wt % and 5 wt %. For example, the first monoterpenoid composition (e.g. ajowan essential oil) may comprise 0.1 wt %, 0.2 wt %, 0.5 wt %, 0.75 wt %, 1.0 wt %, 2.0 wt %, 5.0 wt % of the anti-dandruff composition, or in a range between any two of the foregoing. When used as one of the active components, the second monoterpenoid composition is present in the anti-dandruff composition in an amount between about 0.1 wt % and 10 wt %. For example, the second monoterpenoid composition (e.g. corn mint essential oil) may comprise 0.1 wt %, 0.2 wt %, 0.5 wt %, 0.75 wt %, 1.0 wt %, 2.0 wt %, 5.0 wt %, 7.5 wt %, 10 wt % of the anti-dandruff composition, or in a range between any two of the foregoing. Wt % is based on the weight of the entire anti-dandruff composition. D. Dermatologically Acceptable Carrier The anti-dandruff compositions of the present invention comprise a dermatologically-acceptable carrier (which may be referred to as “carrier”) for the anti-dandruff actives composition. A suitable carrier is selected to yield a desired product form. Furthermore, the solubility or dispersibility of the components may dictate the form and character of the carrier. In one embodiment, the carrier is present at a level of from about 30 wt % to about 99 wt %, about 40 wt % to about 98 wt %, about 50 wt % to about 96 wt %, or, alternatively, from about 60 wt % to about 95 wt %, by weight of the composition. Wt % is based on the weight of the entire composition. The carrier can be in a wide variety of forms. Non-limiting examples include simple solutions (e.g., aqueous, organic solvent, or oil based), emulsions, and solid forms (e.g., gels, sticks, flowable solids, or amorphous materials). In certain embodiments, the carrier is an aqueous carrier, which may comprise water or natural botanical juices, such as aloe vera water. In certain embodiments, the dermatologically acceptable carrier is in the form of an emulsion. Emulsion may be generally classified as having a continuous aqueous phase (e.g., oil-in-water and water-in-oil-in-water) or a continuous oil phase (e.g., water-in-oil and oil-in-water-in-oil). The oil phase of the present invention may comprise silicone oils, non-silicone oils such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof. For emulsions, the aqueous phase comprises water, such as demineralized or distilled water, for example. Other acceptable carriers that may be used in the aqueous carrier include, but are not limited to alcohol or ether compounds, such as ethanol, glycerol, dipropylene glycol, propylene glycol, butylene glycol, 1,4-butanediol, 3-allyloxy-1,2-propanediol, dipropylene glycol n-butyl ether, 1,2-hexanediol, dimethyl isosorbide, 1,3-propanediol, 2,2′-thiodiethanol, and 1,6-hexanediol, or combinations thereof. The anti-dandruff compositions may have a pH ranging from about 3.0 to about 10.5, which may be measured by taking a direct pH measurement using a standard hydrogen electrode of the composition at 25° C. Accordingly, the pH of the anti-dandruff composition may be within the range from about 6 to about 9, for example. E. Optional Ingredients The anti-dandruff compositions of the present invention may be prepared in typical anti-dandruff formulations. They may be in the form of solutions, dispersion, emulsions, powders, talcs, encapsulated, spheres, spongers, solid dosage forms, foams, and other delivery mechanisms. The compositions of the embodiments of the present invention may be hair tonics, leave-on hair products such as conditioners, treatment, and styling products, rinse-off hair products such as conditioners, shampoos, and treatment products; and any other form that may be applied to the scalp. Accordingly, the anti-dandruff compositions may also include other common hair ingredients. The CTFA Cosmetic Ingredient Handbook, Tenth Edition (published by the Cosmetic, Toiletry, and Fragrance Association, Inc. (now called The Personal Care Products Council), Washington, D.C.) (2004), describes a wide variety of nonlimiting materials that can be added to the composition herein. Examples of these ingredient classes include, but are not limited to: abrasives, absorbents, fragrances, pigments, colorings/colorants, essential oils, skin sensates, astringents, anti-acne agents, anti-caking agents, antifoaming agents, antimicrobial agents, antioxidants, binders, biological additives, buffering agents, bulking agents, chelating agents, film formers, opacifying agents, pH adjusters, propellants, reducing agents, sequestrants, rheology modifiers, conditioning agents, emulsifiers, and surfactants In accordance with an embodiment, the anti-dandruff composition may be formulated as a hair care composition, such as a shampoo, a hair conditioner, or a shampoo-conditioner combination, which further include one or more of the following ingredients 1) surfactants (anionic, amphoteric/zwitterionic, non-ionic), 2) conditioning agents, 3) emulsifiers, 4) opacifiers, 5) thickeners, and 6) buffers. Accordingly, in one embodiment, the anti-dandruff composition of the invention may be formulated as an anti-dandruff hair care composition, with a shampoo matrix comprising at least one detersive surfactant selected from the group consisting of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, or a combination thereof. 1. Surfactants The hair care composition of the present invention may include a detersive surfactant, which provides cleaning performance to the composition. The detersive surfactant in turn may comprise an anionic surfactant, an amphoteric/zwitterionic surfactant, a non-ionic, or mixtures thereof. Various examples and descriptions of detersive surfactants are set forth in U.S. Pat. No. 6,649,155; U.S. Patent Application Publication No. 2008/0317698; and U.S. Patent Application Publication No. 2008/0206355, which are incorporated herein by reference in their entirety. The concentration of the detersive surfactant component in the hair care composition should be sufficient to provide the desired cleaning and lather performance, and generally ranges from about 2 wt % to about 50 wt %, from about 5 wt % to about 40 wt %, from about 8 wt % to about 35 wt %, or from about 10 wt % to about 30 wt %. Accordingly, the hair care composition may comprise a detersive surfactant in an amount of about 5 wt %, about 10 wt %, about 12 wt %, about 15 wt %, about 17 wt %, about 18 wt %, about 20 wt %, about 25 wt %, about 30 wt %, or in a range between any two of the foregoing, for example. Wt % is based on the weight of the entire composition. Anionic surfactants suitable for use in the compositions are the alkyl and alkyl ether sulfates. Other suitable anionic surfactants are the water-soluble salts of organic, sulfuric acid reaction products. Still other suitable anionic surfactants are the reaction products of fatty acids esterified with isethionic acid and neutralized with sodium hydroxide. Other similar anionic surfactants are described in U.S. Pat. Nos. 2,486,921; 2,486,922; and 2,396,278, which are incorporated herein by reference in their entirety. Exemplary anionic surfactants for use in the hair care composition include ammonium lauryl sulfate, ammonium laureth sulfate, triethylamine lauryl sulfate, triethylamine laureth sulfate, triethanolamine lauryl sulfate, triethanolamine laureth sulfate, monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate, diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauric monoglyceride sodium sulfate, sodium lauryl sulfate, sodium laureth sulfate, potassium lauryl sulfate, potassium laureth sulfate, sodium lauryl sarcosinate, sodium lauroyl sarcosinate, lauryl sarcosine, cocoyl sarcosine, ammonium cocoyl sulfate, ammonium lauroyl sulfate, sodium cocoyl sulfate, sodium lauroyl sulfate, potassium cocoyl sulfate, monoethanolamine cocoyl sulfate, sodium tridecyl benzene sulfonate, sodium dodecyl benzene sulfonate, sodium cocoyl isethionate and combinations thereof. In a further embodiment of the present invention, the anionic surfactant is sodium lauryl sulfate, sodium laureth sulfate, or a combination thereof. Suitable amphoteric/zwitterionic surfactants for use in the hair care composition herein include those which are known for use in hair care or other personal care cleansing. Concentrations of such surfactants range from about 0.5 wt % to about 20 wt %, and from about 1 wt % to about 10 wt %. Nonlimiting examples of suitable zwitterionic or amphoteric surfactants are described in U.S. Pat. Nos. 5,104,646 and 5,106,609, which are incorporated herein by reference in their entirety. Amphoteric detersive surfactants suitable for use in the hair care composition include those surfactants broadly described as derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be straight or branched chain and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms, and at least one contains an anionic group such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Zwitterionic detersive surfactants suitable for use in the hair care composition include those surfactants broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, in which the aliphatic radicals can be straight or branched chain, and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms and one contains an anionic group such as carboxy, sulfonate, sulfate, phosphate or phosphonate. Exemplary amphoteric and/or zwitterionic detersive surfactants for use in the present hair care composition include cocoamphoacetate, cocoamphodiacetate, lauroamphoacetate, lauroamphodiacetate, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, and mixtures thereof. Nonionic Surfactants: The shampoo compositions can comprise a nonionic surfactant. Nonionic surfactants include those compounds produced by condensation of alkylene oxide groups, hydrophilic in nature, with an organic hydrophobic compound, which may be aliphatic or alkyl aromatic in nature. Nonlimiting examples of nonionic surfactants for use in the shampoo compositions include the following: (1) polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 20 carbon atoms in either a straight chain or branched chain configuration, with ethylene oxide, the said ethylene oxide being present in amounts equal to from about 10 to about 60 moles of ethylene oxide per mole of alkyl phenol; (2) those derived from the condensation of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine products; (3) condensation products of aliphatic alcohols having from about 8 to about 18 carbon atoms, in either straight chain or branched chain configurations, with ethylene oxide, e.g., a coconut alcohol ethylene oxide condensate having from about 10 to about 30 moles of ethylene oxide per mole of coconut alcohol, the coconut alcohol fraction having from about 10 to about 14 carbon atoms; (4) long chain tertiary amine oxides of the formula [R1R2R3N→O] where R1contains an alkyl, alkenyl or monohydroxy alkyl radical of from about 8 to about 18 carbon atoms, from 0 to about 10 ethylene oxide moieties, and from 0 to about 1 glyceryl moiety, and R2and R3contain from about 1 to about 3 carbon atoms and from 0 to about 1 hydroxy group, e.g., methyl, ethyl, propyl, hydroxyethyl, or hydroxypropyl radicals; (5) long chain tertiary phosphine oxides of the formula [RR′R″P→O] where R contains an alkyl, alkenyl or monohydroxyalkyl radical ranging from about 8 to about 18 carbon atoms in chain length, from 0 to about 10 ethylene oxide moieties and from 0 to 1 glyceryl moieties and R′ and R″ are each alkyl or monohydroxyalkyl groups containing from about 1 to about 3 carbon atoms; (6) long chain dialkyl sulfoxides containing one short chain alkyl or hydroxy alkyl radical of from 1 to about 3 carbon atoms (usually methyl) and one long hydrophobic chain which include alkyl, alkenyl, hydroxy alkyl, or keto alkyl radicals containing from about 8 to about 20 carbon atoms, from 0 to about 10 ethylene oxide moieties and from 0 to 1 glyceryl moieties; (7) alkyl polysaccharide (APS) surfactants (e.g. alkyl polyglycosides), examples of which are described in U.S. Pat. No. 4,565,647, which is incorporated herein by reference in its entirety, and which discloses APS surfactants having a hydrophobic group with about 6 to about 30 carbon atoms and a polysaccharide (e.g., polyglycoside) as the hydrophilic group; optionally, there can be a polyalkylene-oxide group joining the hydrophobic and hydrophilic moieties; and the alkyl group (i.e., the hydrophobic moiety) can be saturated or unsaturated, branched or unbranched, and unsubstituted or substituted (e.g., with hydroxy or cyclic rings); and (8) polyoxyethylene alkyl ethers, having a general formula RO(CH2CH2)nH), and polyethylene glycol (PEG) glyceryl fatty esters, having a general formula R(O)OCH2CH(OH)CH2(OCH2CH2)nOH), wherein n is from 1 to about 200, preferably from about 20 to about 100, and R is an alkyl having from about 8 to about 22 carbon atoms. Certain nonionic surfactants can also function as foam stabilizers, viscosity control agents, or conditioning agents. Where included, the hair care composition may contain about 0.5 wt % to about 5.0 wt % nonionic surfactant, or about 0.75 wt % to about 2.0 wt %. Non limiting examples of other anionic, amphoteric/zwitterionic, nonionic, or optional additional surfactants suitable for use in the compositions are described in McCutcheon's, Emulsifiers and Detergents, 1989 Annual, published by M. C. Publishing Co., and U.S. Pat. Nos. 3,929,678, 2,658,072; 2,438,091; 2,528,378, which are incorporated herein by reference in their entirety. 2. Conditioning Agent In one embodiment of the present invention, the hair care compositions comprise one or more conditioning agents. Conditioning agents include materials that are used to give a particular conditioning benefit to hair and/or skin. The conditioning agents useful in the hair care compositions of the present invention typically comprise a water-insoluble, water-dispersible, non-volatile, liquid that forms emulsified, liquid particles. Suitable conditioning agents for use in the hair care composition are those conditioning agents characterized generally as silicones (e.g., silicone oils, cationic silicones, silicone gums, high refractive silicones, and silicone resins), organic conditioning oils (e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinations thereof, or those conditioning agents which otherwise form liquid, dispersed particles in the aqueous surfactant matrix. In an embodiment, one or more conditioning agents are present from about 0.01 wt % to about 10 wt %, from about 0.1 wt % to about 8 wt %, and from about 0.2 wt % to about 4 wt %, by weight of the entire composition. 3. Emulsifiers A variety of anionic emulsifiers can be used in the shampoo composition of the present invention as described below. The anionic emulsifiers include, by way of illustrating and not limitation, water-soluble salts of alkyl sulfates, alkyl ether sulfates, alkyl isethionates alkyl carboxylates, alkyl sulfosuccinates, alkyl succinamates, alkyl sulfate salts such as sodium dodecyl sulfate, alkyl sarcosinates, alkyl derivatives of protein hydrolyzates, acyl aspartates, alkyl or alkyl ether or alkylaryl ether phosphate esters, sodium dodecyl sulphate, phospholipids or lecithin, or soaps, sodium, potassium or ammonium stearate, oleate or palmitate, alkylarylsulfonic acid salts such as sodium dodecylbenzenesulfonate, sodium dialkylsulfosuccinates, dioctyl sulfosuccinate, sodium dilaurylsulfosuccinate, poly(styrene sulfonate) sodium salt, isobutylene-maleic anhydride copolymer, gum arabic, sodium alginate, carboxymethylcellulose, cellulose sulfate and pectin, poly(styrene sulfonate), carrageenan, pectic acid, tragacanth gum, almond gum and agar; semi-synthetic polymers such as carboxymethyl cellulose, sulfated cellulose, sulfated methylcellulose, carboxymethyl starch, phosphated starch, lignin sulfonic acid; and synthetic polymers such as maleic anhydride copolymers (including hydrolyzates thereof), polyacrylic acid, polymethacrylic acid, acrylic acid butyl acrylate copolymer or crotonic acid homopolymers and copolymers, vinylbenzenesulfonic acid or 2-acrylamido-2-methylpropanesulfonic acid homopolymers and copolymers, and partial amide or partial ester of such polymers and copolymers, carboxymodified polyvinyl alcohol, sulfonic acid-modified polyvinyl alcohol and phosphoric acid-modified polyvinyl alcohol, phosphated or sulfated tristyrylphenol ethoxylates. In addition, anionic emulsifiers that have acrylate functionality may also be used in the instant shampoo compositions. Anionic emulsifiers useful herein include, but aren't limited to: poly(meth)acrylic acid; copolymers of (meth)acrylic acids and its (meth)acrylates with C1-22 alkyl, C1-C8 alkyl, butyl; copolymers of (meth)acrylic acids and (meth)acrylamide; carboxyvinylpolymer; acrylate copolymers such as acrylate/C10-30 alkyl acrylate crosspolymer, acrylic acid/vinyl ester copolymer/acrylates/vinyl Isodecanoate crosspolymer, acrylates/palmeth-25 acrylate copolymer, acrylate/steareth-20 itaconate copolymer, and acrylate/cetech-20 itaconate copolymer; polystyrene sulphonate, copolymers of methacrylic acid and acrylamidomethylpropane sulfonic acid, and copolymers of acrylic acid and acrylamidomethylpropane sulfonic acid; carboxymethycellulose; carboxy guar; copolymers of ethylene and maleic acid; and acrylate silicone polymer. In an embodiment, the emulsifier, when present, ranges from about 0.01 wt % to about 5 wt %, by weight of the entire anti-dandruff hair care composition, or from about 0.1 wt % to about 4 wt %, or from about 0.1 wt % to about 3 wt %. Wt % is based on the weight of the entire composition. 4. Opacifiers Some formulations are often opacified by incorporating materials therein to achieve a cosmetically attractive pearl-like appearance, known as pearlescence. The opacifying or pearlescent materials include, but are not limited to, titanium dioxide coated mica, iron oxide coated mica, ethylene glycol mono-stearate, ethylene glycol distearate, polyethylene glycol distearate, bismuth oxychloride coated mica, myristyl myristate, guanine, glitter (polyester or metallic), and mixtures thereof. Other pearlescent materials can be found in U.S. Pat. Nos. 4,654,207 and 5,019,376, herein incorporated by reference. In an embodiment, the concentration of the opacifier, when present, ranges from about 0.01 wt % to about 5 wt %, by weight of the entire anti-dandruff hair care composition, or from about 0.1 wt % to about 3 wt %, or from about 0.1 wt % to about 2 wt %. Wt % is based on the weight of the entire composition. 5. Thickeners Thickeners or rheology modifiers include, but are not limited to, acrylamide/ammonium acrylate copolymer (and) polyisobutene (and) polysorbate 20; acrylamide/sodium acryloyldimethyl taurate copolymer/isohexadecane/polysorbate 80; acrylates copolymer; acrylates/beheneth-25 methacrylate copolymer; acrylates/C10-C30 alkyl acrylate crosspolymer; acrylates/steareth-20 itaconate copolymer; ammonium polyacrylate/Isohexadecane/PEG-40 castor oil; C12-16 alkyl PEG-2 hydroxypropylhydroxyethyl ethylcellulose (HM-EHEC); carbomer; crosslinked polyvinylpyrrolidone (PVP); dibenzylidene sorbitol; hydroxyethyl ethylcellulose (EHEC); hydroxypropyl methylcellulose (HPMC); hydroxypropylcellulose (HPC); methylcellulose (MC); methylhydroxyethyl cellulose (MEHEC); PEG-150/decyl alcohol/SMDI copolymer; PEG-150/stearyl alcohol/SMDI copolymer; polyacrylamide/C13-14 isoparaffin/laureth-7; polyacrylate 13/polyisobutene/polysorbate 20; polyacrylate crosspolymer-6; polyamide-3; polyquaternium-37 (and) hydrogenated polydecene (and) trideceth-6; polyurethane-39; sodium acrylate/acryloyldimethyltaurate/dimethylacrylamide; crosspolymer (and) isohexadecane (and) polysorbate 60; sodium polyacrylate, and combinations thereof. In an embodiment, the concentration of the rheology modifier, when present, ranges from about 0.01 wt % to about 7 wt %, by weight of the entire anti-dandruff hair care composition, or from about 0.1 wt % to about 5 wt %, or from about 0.2 wt % to about 4 wt %. Wt % is based on the weight of the entire composition. 6. Buffers The hair care compositions may have a pH ranging from about 3.0 to about 10, which may be stabilized by the presence of a buffer system. Suitable buffer solutions can be prepared using, for example, weak acid or weak base systems using citric acid, phosphoric acid, phthalic acid, glycine or mixtures thereof. In each case the proper buffering capacity is obtained by adjusting the final pH of the compositions to within the pH range indicated above. This may be done by using an acid (e.g., HCl, citric acid) or a base (e.g., NaOH, sodium citrate) as may be needed. The amount of buffer employed in the present compositions depends on the particular acid chosen but is generally from about 0.1 wt % to about 10 wt %, preferably from about 0.2 wt % to about 5 wt %. Wt % is based on the weight of the entire composition. Additional agents, such as benefit agents, may also be included in the hair care compositions. The benefit agent may comprise a material selected from the group consisting of perfumes; brighteners; enzymes; sensates (cooling or warming); attractants, antibacterial agents; dyes; pigments; bleaches; and mixtures thereof. It should be further appreciated that the inventive combination of natural anti-dandruff actives disclosed herein may also be used in combination with secondary scalp benefit agents, such as soluble and/or insoluble anti-dandruff actives. Such anti-dandruff actives include but are not limited to azoles, such as ketoconazole, econazole, climbazole, and elubiol; keratolytic agents such as salicylic acid; and zinc-containing layered (ZLM) materials, pyridinethione anti-dandruff particulates such as zinc pyrithione, coal tar, sulfur, charcoal, whitfield's ointment, castellani's paint, aluminum chloride, gentian violet, octopirox (piroctone olamine), ciclopirox olamine, undecylenic acid and it's metal salts, potassium permanganate, selenium sulphide, sodium thiosulfate, propylene glycol, urea preparations, griseofulvin, 8-Hydroxyquinoline ciloquinol, thiobendazole, thiocarbamates, haloprogin, polyenes, hydroxypyridone, morpholine, benzylamine, allylamines (such as terbinafine), Sensiva SC-50, Elestab HP-100, azelaic acid, lyticase, iodopropynyl butylcarbamate (IPBC), isothiazalinones such as octyl isothiazalinone, other natural oils, extracts, or compounds such as oil of bitter orange, tea tree oil, clove leaf oil, coriander, palmarosa, berberine, thyme red, cinnamon oil, cinnamic aldehyde, citronellic acid, hinokitol, ichthyol pale, and combinations thereof. In an embodiment, the concentration of the secondary scalp benefit agent ranges from about 0.01 wt % to about 5 wt %, by weight of the entire anti-dandruff hair care composition, or from about 0.1 wt % to about 3 wt %, or from about 0.1 wt % to about 2 wt %. Wt % is based on the weight of the entire composition. Alternatively, the inventive combination of natural anti-dandruff actives disclosed herein may also be void of any of the foregoing recited soluble and/or insoluble anti-dandruff actives. The anti-dandruff hair care compositions are generally prepared by conventional methods known in the art shampoo compositions. Such methods typically involve mixing of the ingredients in one or more steps to a relatively uniform state, with or without heating, cooling, application of vacuum, and the like. The compositions are prepared such as to optimize stability (physical stability, chemical stability, photostability) and/or delivery of the active materials. This optimization may include appropriate pH, and exclusion of materials that can complex or react with the active agent(s) and thus negatively impact stability or delivery. The anti-dandruff hair composition may be in a single phase or a single product, or the anti-dandruff hair composition may be in a separate phases or separate products. If two products are used, the products may be used together, at the same time or sequentially. Sequential use may occur in a short time period, such as immediately after the use of one product, or it may occur over a period of hours or days. Use of the Anti-dandruff Composition: According to yet another embodiment of the present invention, a method is provided for the treatment of a subject having dandruff and/or to prevent or inhibit the onset of dandruff symptoms associated with the proliferation of yeasts of theMalasseziagenus on a scalp of a subject. The method includes contacting the subject's scalp or keratinous tissue with an effective quantity of the anti-dandruff composition of the present invention. The anti-dandruff composition may be massaged onto the scalp and should remain in contact with the subjects scalp or keratinous tissue for a duration of at least 15 seconds or more. Depending on the formulation, the anti-dandruff composition may be a leave in or it may be rinsed out. Accordingly, in another embodiment, the method comprises topically applying an anti-dandruff composition comprising an effective amount of the anti-dandruff actives to a region of the subject's skin where inhibition ofMalassezia furfuris needed or wanted, where the anti-dandruff actives remain in contact with the region for a duration of 15 seconds or more; and then rinsed out. In still another embodiment, the method comprises applying the composition according to a regimen, wherein said regimen comprises: (a) cleansing the scalp to form a cleansed scalp; (b) topically applying the anti-dandruff composition to said cleansed scalp. The anti-dandruff composition may be used daily, weekly, or in a variety of regimens. The anti-dandruff composition may be used more than once a day, such as at night and in the morning. The product may be used after washing the hair (also on wet or dry hair), which may mean using the composition more than once per day on certain days or use only a few times per week. The anti-dandruff composition may be used three times per day, twice per day, once per day, six times per week, five times per week, four times per week, three times per week, two times per week, or one time per week. In some embodiments, the anti-dandruff composition is used four, five, six or seven times per week. According to another embodiment, the anti-dandruff composition is applied to at least once a day for at least about four weeks, or at least twice a day for at least about four weeks. According to another embodiment, the anti-dandruff composition is applied at least once a day for at least about eight weeks. The anti-dandruff composition may be used by males and females. Formulations and Examples The following are non-limiting examples of the present invention. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention, which would be recognized by one of ordinary skill in the art. In the examples, all concentrations are listed as weight percent, unless otherwise specified and may exclude minor materials such as diluents, filler, and so forth. The listed formulations, therefore, comprise the listed components and any minor materials associated with such components. As is apparent to one of ordinary skill in the art, the selection of these minors will vary depending on the physical and chemical characteristics of the particular ingredients selected to make the present invention as described herein. Anti-Dandruff Actives Extract ofPunica granatum:25 g of the flakedPunica granatumrind raw material (obtained from Pradip Agrotech Pvt Ltd.; Solapur, Maharashtra, India; phenolic content of 24.2 GAE/g) was extracted with 50% aqueous ethanol at 65° C. for 30 min and filtered to remove the undissolved solids from the filtrate. The undissolved solids were further extracted five more times with the same procedure as described above. The combined filtrates were filtered through a 10-13 micron filter cloth, evaporated in a rotary evaporator under vacuum to yield 13.98 g of dried solids having a phenolic content of 43.5 GAE/g (mass yield=55.92%). Based on LC/MS-QTOF analysis, this extract ofPunica granatumcontains gallic acid, punicalagin α, punicalagin β, punicalin, ellagic acid hexoside, ellagic acid pentoside, ellagic acid deoxyhexoside, ellagic acid, and galloyl HHDP hexoside. HPLC quantification against standards provided 3.9 wt % ellagic acid, 0.5 wt % gallic acid, 0.22 wt % punicalagin B, and 0.05 wt % punicalagin A. This unstandardized product (PE 1) can be diluted with a sufficient quantity of glycerol, and then filtered through a 400 micron metal sieve, to form a standardized solution having 12% GAE (PE 2). Alternatively, subjecting freshPunica granatumrind raw material to aqueous pressing and purification by column chromatography (DIAION™ HP20 resin, wash with water and elute with 50% aqueous ethanol), and spray drying yielded an unstandardized product with about 75 GAE and 30 wt % punicalagins, which was also dissolved in glycerol to form a standardized solution having 12% GAE (PE 3). Essential oil ofTrachyspermum ammi: The essential oil (EO) of Ajowan is produced by the steam distillation of ground, dried seeds ofTrachyspermum ammi(obtained from Shree Agro International; Unja, Gujarat, India). The ground seeds are charged to the vessel and moistened before applying steam. Direct steam is applied, and the distillation continued for a minimum of about 15 hours to obtain a total yield of Ajowan EO in about 2 wt % to about 2.5 wt %, based on the dried weight of the Ajowan seeds (AJ 1). The EO product is a liquid and has pale yellow to brownish yellow color. It has the characteristic odor of cyminic, with a thymolic after note. Analysis by GC-MS using an Agilent 5977A, equipped with GC-7890B and 7693 auto sampler, operated in electron impact (EI) mode (70 ev) revealed that the major components of Ajowan EO are gamma-terpinene (45.7%), p-cymene (22.0%), and thymol (23.2%), where % is FID area % from the GC-MS analysis. Ajowan EO may be obtained from Kancor Ingredients LTD (Kerala, India). Essential oil ofMentha arvensis: The essential oil (EO) of corn mint is produced by the steam distillation of the flowering herb ofMentha arvensis. The semi-dried corn mint leaves (obtained from Bareilly, Uttar Pradesh, India) are charged to a steam distillation vessel. Steam is slowly applied to distill out the EO and the process continued while monitoring the EO production. Average distillation time is approximately 5 to 7 hours, with a total yield of approximately 1.4 wt % to 1.6 wt %, based on the weight of the semi-dried corn mint leaves (CM 1). The corn mint EO product is a colorless or pale yellow liquid. Analysis by GC-MS revealed that the major constituent of corn mint essential oil is L-menthol (76.1%) and the other notable constituents include menthone (6.8%), isomenthone (3.6%), neomenthol (2.1%), menthyl acetate (2.1%), and limonene (1.7%), where % is FID area % from the GC-MS analysis. The corn mint EO may be obtained from Kancor Ingredients LTD (Kerala, India). Combinations: Based on their additive and synergistic anti-dandruff activities (discussed below), blends or mixtures of the ingredients, along with optional carrier(s) and other functional ingredients may be advantageously prepackaged and/or blended with a variety of hair care ingredients. TABLE 1ANTI-DANDRUFF SHAMPOO FORMULATIONSControlEx. 1Ex. 2Ex. 3Ex.IngredientTradenameABCDEDistilled watern/a64.2362.7363.1361.7061.9ACRYLATES/C10-30 ALKYLCARBOPOL ®0.200.20.20.190.19ACRYLATE CROSSPOLYMERETD 2020SODIUM HYDROXIDE 20% INn/a0.100.10.10.090.09WATERSODIUM LAURETH SULFATETEXAPON ®21.0021.0021.019.8119.8AND WATERN70COCAMIDE MIPACOMPERLAN ® IP0.10.10.10.090.09GLYCOL DISTEARATETEGIN ® BL 3152.002.002.001.891.89DISODIUMMIRANOL ®8.008.008.007.557.58COCOAMPHODIACETATEC2M CONC2.502.502.502.362.37DIMETHICONEXIAMETER ®PMX-200SILICONEFLUID 60000CSTHYDROXYPROPYL GUAR &JAGUAR ®0000.280.28HYDROXYPROPYL GUAR -C162HYDROXYPROPYLTRIMONIUMCHLORIDEGUAR -JAGUAR ®0.30.30.300HYDROXYPROPYLTRIMONIUMC13SCHLORIDEPPG-5-CETETH-20PROCETYL ™0.30.30.30.280.28AWSPE 201.671.671.671.67CM 101.00.510.5AJ 1000.100.1CITRIC ACID 20% IN WATERN/A0.100.10.10.090.09PEG-32PLURACARE ®00033E 1500Glycerinen/a1.170000100100100100100PE - pomegranate extract; AJ - ajowan essential oil; CM - corn mint essential oil; Part A - Shampoo chassis; Part B - anti-dandruff actives; Part C - pH or viscosity modifiers. Preparation of the anti-dandruff shampoo begins with dispersing the CARBOPOL® in 40% of water and then neutralizing to pH=6 with sodium hydroxide. JAGUAR® C162 is dispersed in the remaining water and then added to the CARBOPOL® solution. After the addition of TEXAPON® N70, the mixture is heated to 70° C., the solid raw materials are added, i.e., COMPERLAN®, TEGIN® BL315. When the solids were well dissolved, the mixture was allowed to cool down, and the other ingredients (including the anti-dandruff actives PE 2, CM 1, and AJ 1) added and mixed well until complete homogeneity. pH was adjusted to 5.65 with citric acid and viscosity adjusted with PLURACARE® E1500. Antimicrobial Activity and Interaction Testing Briefly, as is commonly known by those skilled in the art of the instant disclosure, assessment ofMalassezia furfurinhibition may be accomplished following standard and accepted practices, such as that described in U.S. Patent Application Publication No. 2008/0249186. Accordingly, an external testing laboratory performed a series of againstMalassezia furfurto evaluate antimicrobial activity and interactions (see Lens, et al. “Methods for the evaluation of antimicrobial activity of essential oils,” J. Pharm. Belg. 1987). Inhibitory activity testing was conducted by mixing 100 μl of various dilutions of the test product at a double concentration with an equal volume (100 μl) of a double strength Difco™ Sabouraud dextrose broth supplemented with olive oil, inoculated between 2 to 6×105cfu/mL. After incubation (24-72 hours, at 32.5° C.±2.5° C.), a spectrophotometric reading at 620 nm is performed and the percentage of growth calculated from optical density (OD) measuring each concentration with respect to a positive growth control according to the following equation: % of growth=[ODproduct−ODabsorbance-control)]/(ODpositive growth-control). Each concentration was tested in triplicate, and two assays were performed per product. The positive growth control consisted of 100 μl 1% agar solution mixed with 100 μl of inoculated broth. TABLE 2Minimum Inhibitory Concentrations againstM. furfur.SampleCommentTest 1Test 2M. furfurstrainPE 1MICRA 1506240.1%0.1%CIP 1634.84PE 2MICRA-L 1701050.1%0.1%CIP 1634.84PE 3MICRA-P0.05%0.05%CBS 1878CM 1OMAO 20065010.5%0.5%CIP 1634.84CM 1OMAO 20065011%1%CBS 1878AJ 1AJTEO 2048291%1%CIP 1634.84AJ 1AJTEO 2048290.5%0.5%CBS 1878PUPunicalagins A&B0.05%0.05%CBS 1878EAEllagic Acid0.5%0.5%CBS 1878Shampoo AControl, Table 110%—CIP 1634.84Shampoo BExample 1, Table 1≤5%≤1%CIP 1634.84Shampoo CExample 2, Table 1≤5%≤1%CIP 1634.84Shampoo FComparative≤5%—CIP 1634.84PE-pomegranate extract;AJ-ajowan essential oil;CM-corn mint essential oil;PU-punicalagins A&B;EA-ellagic acid;Comparative Shampoo F = HEAD & SHOULDERS ®, Procter & Gamble, UK.; Classic, Lot. No. (L) 60435395KF. Inhibition studies revealed that the shampoo formulations including a plurality of the nature-derived anti-dandruff actives of the present invention (i.e., Shampoos B and C) possessed enhance activity over the control (Shampoo A), and were approximately as effective as the comparative Shampoo F, which utilizes synthetic zinc pyrithione. Study of Associations. After obtaining the MIC levels for each of the anti-dandruff actives by broth dilution method, a checkerboard titration technique was performed to assess the synergistic activity of the combinations. In order to evaluate the activity of the combinations of the anti-dandruff actives, a Fractional Inhibitory Concentration index (FICindex) was calculated: (FICindex)=FICA+FICB, where FICAand FICBrepresent the minimum concentrations inhibiting theM. furfurgrowth for anti-dandruff actives A and B, respectively. And FICA=MICAcombination/MICAalone, and FICB=MICBcombination/MICBalone.] A mean FIC index was calculated based on the following equation: FICindex=FICA+FICB. Synergism is defined as FIC≤0.75; additive as FIC˜1; indifference as 1<FIC≤2; and antagonism is defined as FIC>2, as reported by J. L. Pons; N. Bonnaveiro; J. Chevalier; and A. Crémieux, “Evaluation of antimicrobial interactions between chlorhexidine, quaternary ammonium compounds, preservatives and excipients,” J. Appl. Bacteriology, 1992, 73(5), 395-400. TABLE 3Association testing of anti-dandruff active combinations.MIC in theSamplesMIC AMIC BassociationsFICABM.furfurstrainAlone %Alone %A %B %IndexConclusionPE 2AJ 1CIP 1634.840.210.0250.50.63SynergisticPE 1CM 1CIP 1634.840.20.50.10.251.00AdditiveCM 1AJ 1CIP 1634.840.510.06250.50.63SynergisticAJ 1PUCBS 18780.250.050.06250.01250.5SynergisticAJ 1EACBS 18780.250.50.250.251.5IndifferentPE 3AJ 1CBS 18780.050.250.01250.1250.75SynergisticPE 3CM 1CBS 18780.0510.01250.250.5SynergisticPE 3TMLCBS 18780.050.050.01250.051.25IndifferentPE 3GTPCBS 18780.05>10.050.1251.13IndifferentPE 3PCYCBS 18780.10.50.01250.250.625SynergisticPE - pomegranate extract; AJ - ajowan essential oil; CM - corn mint essential oil; TML - thymol; GTP - gamma-terpinene; PCY - para-cymene The association testing of the nature-derived anti-dandruff actives of the present invention utilizing the checkerboard titration technique revealed the synergistic activity of the combinations of a) the punicalagin composition (PE 2) and the first monoterpenoid composition (AJ 1); b) the second monoterpenoid composition (CM1) and the first monoterpenoid composition (AJ 1); and the first monoterpenoid composition (AJ 1) and punicalagins A & B (PU). The combination of the punicalagin composition (PE 1) and the second monoterpenoid composition (CM 1) was shown to be additive, whereas the combination of the first monoterpenoid composition (AJ 1) and ellagic acid (EA) was shown to be indifferent. The association testing of the major components of first monoterpenoid composition (i.e., thymol (TML), gamma-terpinene (GTP), and para-cymene (PCY)) and the pomegranate extract having a punicalagins content of about 30 wt % on a dry weight basis (PE 3) revealed that the bi-component mixture of PE 3 and para-cymene (PCY) demonstrated synergistic activities againstMalassezia furfur. The bi-component mixture of the punicalagin composition having a punicalagins content of at least 30 wt % on a dry weight basis (PE 3) and the second monoterpenoid composition (CM 1) was shown to be synergistic againstMalassezia furfur. Clinical Study Protocol: Over 80 prescreened subjects having dandruff symptoms participated in a 28 day blind study, prior to which subjects completed a 14-day wash-out period consisting of a daily wash using a neutral shampoo (Szampon Familijny®, Pollena-Savona Sp. z o. o.; Batches 170270, 160492). In the study, inventive Shampoo D (Table 1, Example 3; combination of pomegranate extract and corn mint EO) and inventive Shampoo E (Table 1, Example 4; combination of pomegranate extract, corn mint EO, and ajowan EO) were tested against comparative Shampoo F (HEAD & SHOULDERS®, Procter & Gamble, UK.; Classic, Lot. No. (L) 60435395KF, zinc pyrithione active). Prior to and after adhering to the required 28 day protocol, each scalp of each subject was assessed for adherent and non-adherent dandruff and given a score based on the qualitative dandruff criteria provided in Table 4. TABLE 4The clinical “total dandruff score” is the sumof the adherent and non-adherent dandruff.Qualitative Dandruff Scoring.ScoreNon-adherent dandruffAdherent dandruff0No dandruffNo squamae1A few dispersedA few disperseddandruff flakessquamae flakes2A small quantityA small quantity ofof dandruff flakessquamae dandruff3A moderate quantityA moderate quantityof dandruff flakesof squamae dandruff4A large quantityA large quantityof dandruffof dandruff5A very large quantityA very large quantityof dandruff flakesof squamae overthe whole scalp TABLE 5Total Dandruff Scoring Results.Non-adherentAdherentSamplescorescoreTotalShampoo D−1.23−0.86−2.09Shampoo E−1.01−0.87−1.87Shampoo F−1.77−1.37−3.13 After following the prescribed 28 day testing protocol, all the test products (Shampoos D-F) were very well tolerated on the cutaneous level, and demonstrated significant decrease in adherent, non-adherent, and total dandruff score (see Table 5). The inventive shampoos (Shampoo D and E), which utilize nature-based, naturally-derived anti-dandruff actives, were shown to be effective at treating dandruff without using the synthetic zinc pyrithione anti-dandruff active (which is used in comparative Shampoo F). Although not wishing to be limited by any particular theory, it is believed that topical application of the anti-dandruff actives in a dermatologically-acceptable carrier decreases the physical signs of dandruff by inhibiting the proliferation ofMalassezia furfur. Accordingly, topical application of the anti-dandruff compositions may be used to treat dandruff or used prophylactically to inhibit the onset of dandruff caused by the proliferation ofMalassezia furfur. All documents cited in the Detailed Description of Embodiments of the Invention are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “25° C.” is intended to mean “about 25° C.” While the present invention is illustrated by the description of one or more embodiments thereof, and while embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modification will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative product and method, and illustrative examples shown and described. Accordingly, departures from such details may be made without departing from the scope of the general inventive concept embraced by the following claims. | 65,830 |
11857662 | DETAILED DESCRIPTION OF THE INVENTION In the following description, it is understood that other embodiments may be utilized and structural and operational changes may be made without departure from the scope of the present embodiments disclosed herein. Definitions As used herein, the term “fatty” describes a long-chain hydrophobic portion of a compound made up of hydrogen and anywhere from 6 to 26 carbon atoms, which may be fully saturated or partially unsaturated, and optionally attached to a functional group such as a hydroxyl group or a carboxyl group. Fatty alcohols, fatty acids, fatty esters, fatty amides, and fatty hydrocarbon oils are examples of materials which contain a fatty portion. When the material contains a carbon-containing functional group, as is the case in a fatty acid which contains a carboxylic acid group (—COOH), the 6 to 26 carbon count refers to only the hydrophobic portion attached to the carbon-containing function group. Therefore, stearic acid, which has 18 carbons total, has a fatty portion with 17 carbon atoms. As used herein, “polyoxyalkylene” describes a polyether group derived from polymerization of one or more alkylene oxides having 2 to 4 carbon atoms, and specifically includes polyoxyethylene (derived from ethylene oxide), polyoxypropylene (derived from propylene oxide), and polyoxybutylene (derived from butylene oxide), as well as mixtures thereof. When referencing hair coloring compositions, the phrase “substantially free”, unless otherwise specified, describes an amount of a particular component present in the hair coloring composition being less than about 1 wt. %, preferably less than about 0.5 wt. %, more preferably less than about 0.1 wt. %, even more preferably less than about 0.05 wt. %, yet even more preferably 0 wt. %, relative to a total weight of the hair coloring composition. As used herein, the terms “optional” or “optionally” means that the subsequently described event(s) can or cannot occur or the subsequently described component(s) may or may not be present (e.g., 0 wt. %). Various hair coloring composition ingredients are listed throughout the present disclosure and are organized according to their primary or most desired function, benefit, or use. However, categorization of an ingredient under a particular function, benefit, or use is not meant to limit that ingredient to only that function, benefit, or use. For example, listing of benzyl alcohol as a preservative does not limit the usefulness of benzyl alcohol to only that of a preservative, since benzyl alcohol can also impart other beneficial attributes, such as acting as a fragrance and/or a solvent. When referencing “hair” or “keratin fibers” herein, it is to be recognized that hair exists on many different mammals (e.g., human) on many different body parts, and application of the hair coloring compositions herein is not limited to hair found on a specific body part. For example, the hair coloring compositions may be applied for dyeing of hair found on the head/scalp including the crown and side of the head, facial hair, and the like. Preferably, the hair being dyed is located on the head or scalp. Hair Coloring Composition The present disclosure is directed to hair coloring compositions that are substantially free of, preferably completely free of (0 wt. %) harsh oxidizing agents, are easy to apply without staining the scalp, and that provide rich, multi-tonal, natural-looking dyed hair. The hair coloring compositions may be used to dye hair of any color to a different shade or color, or preferably to restore gray hair to an original hair color, for example, yellow or brown. The hair coloring compositions therefore contain components which enable dyeing of keratin fibers without damaging the keratin fibers, as well as components which facilitate delivery of active ingredients and allow the hair coloring compositions to be easily applied at home to provide natural looking dyed hair. Such compositions generally include the following components: a dyestuff, which is preferably a mixture monosaccharides having 2 to 4 carbon atoms, a surfactant, a delivery agent, and optionally water, an organic solvent, a preservative, an acidulant, a conditioning agent, and a fragrance. In preferred embodiments, all components are compatible with the dyestuff (i.e., do not react with or cause the dyestuff to react) and are homogeneously dispersed or dissolved uniformly throughout the hair coloring composition. The hair coloring composition may be in a form chosen from a liquid, a solution, an emulsion, a cream, a gel, a paste, a mousse, a foam, or any other form that is suitable for topical application to keratin fibers. Preferably, the hair coloring composition is in the form of a foam, and is therefore easy to apply to hair follicles, including hair follicle roots, while minimizing contact with the scalp to avoid staining of the skin. Dyestuff To act as an effective hair dye (e.g., for gray hair), the hair coloring composition herein includes a “dyestuff”, which is any colored molecule that, when it is brought into contact with the keratin material, colors this material, or any non-colored molecule that, in contact with the keratin material, reacts with and colors the keratin material without the aid of an additional chemical agent, for example, without the aid of an oxidizing agent. The amount of dyestuff present in the hair coloring composition may vary depending on the color shade desired and the quantity and nature of the other components, however, the dyestuff is typically present in amounts of at least about 1 wt. %, preferably at least about 2 wt. %, more preferably at least about 3 wt. %, even more preferably at least about 4 wt. %, yet even more preferably at least about 5 wt. %, and up to about 35 wt. %, preferably up to about 25 wt. %, preferably up to about 20 wt. %, preferably up to about 15 wt. %, preferably up to about 10 wt. %, more preferably up to about 8 wt. %, even more preferably up to about 7 wt. %, yet even more preferably up to about 6 wt. %, based on a total weight of the hair coloring composition. In preferred embodiments, the dyestuff is a monosaccharide. Monosaccharides, such as dihydroxyacetone, react with amino acids naturally occurring in keratin materials and, by virtue of a Maillard reaction, form melanoids which produce a color change to the keratin material (Bobin et al. J. Soc. Cosmet. Chem., 35 pages 265-272, 1984; Maillard L. C., C. R. Acad. Sci. 154, 66-68, 1912—each incorporated herein by reference in its entirety). Advantageously, such color change is produced without the need for additional chemical agents (e.g., oxidizing agents) to drive the reaction. Different amino acids react with different monosaccharides differently to produce a variety of tones of coloration from yellow to brown. Any monosaccharide capable of reacting with amino acids found in keratin fibers (e.g., naturally occurring amino acids) to produce a colored keratin material, can be employed as the dyestuff herein. The monosaccharide may be an aldose having 2 to 6 carbon atoms, preferably 3 to 5 carbon atoms, more preferably 3 to 4 carbon atoms, even more preferably 3 carbon atoms, a ketose having 3 to 6 carbon atoms, preferably 4 to 5 carbon atoms, more preferably 3 to 4 carbon atoms, including mixtures such aldoses and/or ketoses. Exemplary monosaccharides include, but are not limited to, glycolaldehyde, glyceraldehyde, dihydroxyacetone (DHA), erythrulose, meso-tartaric aldehyde, glucose, gulose, xylose, fructose, ribose, arabinose, allose, talose, altrose, idose, mannose, galactose, and erythrose. Such monosaccharides produce natural looking color shades when applied to hair owing at least partially to the multi-tone color variations produced by reaction with various amino acids. Further advantages of employing monosaccharides as the dyestuff component herein, is that the monosaccharides are derivatives of, or are closely related to, physiologically harmless naturally occurring vegetable compounds, and the colors produced are integral with the keratin fibers themselves (Maillard reaction products) and are thus resistant to washing. In preferred embodiments, a mixture of monosaccharides is used, for example a mixture of a first monosaccharide and a second monosaccharide which is different from the first monosaccharide. The first monosaccharide is preferably a ketose having 2 to 4 carbon atoms, preferably three carbon atoms. In preferred embodiments, the first monosaccharide is dihydroxyacetone. The first monosaccharide may be present in amounts of from about 5 wt. %, preferably from about 5.5 wt. %, more preferably from about 6 wt. %, even more preferably from about 6.5 wt. %, and up to about 25 wt. %, preferably up to about 20 wt. %, preferably up to about 15 wt. %, preferably up to about 10 wt. %, more preferably up to about 9 wt. %, even more preferably up to about 8 wt. %, yet even more preferably up to about 7 wt. %, based on the total weight of the hair coloring composition. The second monosaccharide is preferably a ketose having 2 to 4 carbon atoms, preferably four carbon atoms. In preferred embodiments, the second monosaccharide is erythrulose. The second monosaccharide may be present in amounts of from about 0.001 wt. %, preferably from about 0.01 wt. %, more preferably from about 0.1 wt. %, even more preferably from about 0.5 wt. %, and up to about 10 wt. %, preferably up to about 8 wt. %, preferably up to about 6 wt. %, preferably up to about 4 wt. %, more preferably up to about 3 wt. %, even more preferably up to about 2 wt. %, yet even more preferably up to about 1 wt. %, based on the total weight of the hair coloring composition. The inventors have unexpectedly found that use of a single monosaccharide (e.g., dihydroxyacetone) often produces an unnatural dye result, for example an orange tone, but that a mixture of monosaccharides (e.g., dihydroxyacetone and erythrulose) provides a rich, long lasting dye with natural-looking multi-tonal colors. While the weight ratio of the first monosaccharide (e.g., dihydroxyacetone) to the second monosaccharide (e.g., erythrulose) may be varied depending on color shade, and the desired length of coloration, the weight ratio is typically from 10:1, preferably from 50:1, preferably from 100:1, preferably from 200:1, preferably from 300:1, more preferably from 400:1, even more preferably from 500:1, yet even more preferably from 600:1, and up to 1,000:1, preferably up to 900:1, more preferably up to 800:1, even more preferably up to 700:1. In preferred embodiments, the hair coloring compositions are substantially free of dyestuffs other monosaccharides, which includes being substantially free of, preferably completely free of (i.e., 0 wt. %) acid dyes, decoctions or extracts, direct dyes, and/or pigments which provide color. Alternatively, the hair coloring compositions may include other dyestuffs such as acid dyes, decoctions or extracts, direct dyes, and/or pigments in amounts listed previously. Examples of acid dyes which may be useful dyestuffs include, but are not limited to, Yellow No. 203 (D&C Yellow No. 10, color index (CI) given as CI 47005), Orange No. 205 (D&C Orange No. 4, CI 15510), Red No. 227 (D&C Red No. 33, CI 17200), Violet No. 401 (Ext. D&C Violet No. 2, CI 07301), and Black No. 401, CI 20470). Combinations of these acid dyes can be used, for example, Orange 4+ Yellow 10+ External Violet 2, Orange 4+ Red 33+ External Violet 2, Orange 4+ Red 33+ External Violet 2, or Orange 4+ Red 3. Extracts or decoctions may also be employed herein as dyestuffs, and are preferably extracts or decoctions from plant sources, for example, henna-based extracts, melanin, curcumin, caramels, malva extracts, hibiscus extracts, green teas, ginsengs, annattos, beta-carotenes, walnut extracts (e.g., black walnut),Menthe piperta, Melva silvestris, Cynara Scolymus, Theasinensis, Juglans regia, Lawsonia inermis, Castanea vulgaris, Asorum europaeum, Leonurus cardiac, Ballotafoetida, Ocimum basilicum, Stachys officinalis, Brunella vulgaris, Calamintha officinalis, Thymus vulgaris, Rosmarinus officinalis, Humulus lupulus, Vaccinium myrtillus, Arctotaphylosuva-ursi, Calluna vulgaris, Artemisian abisinthium, Artemisia vulgaris, Artemisia abrotonum, Artemisia glacialis, Artimesia mutellina, Artemisia spicata, Chamamelum nobile, Fraxinus excelsior, Syringa vulgaris, Jasminium grandijlorum, Lythrum salicaria, Althaea officinalis, Hysopus officinalis, Origanum majorana, Salvia officinalis, Melissa officinalis, Melittis melissophylum, Lavandula ojficinalis, Quercus robur, Fagus sylvatica, Nepta cataria, Origanum dictamus, Thymus serpyllum, Cichorium intybusL., andGymnema sylvestre. The hair coloring composition may include direct dyes as the dyestuff component. Various classes of direct dyes may be employed, such as indole-based dyes (e.g., isatin, 5,6-dihydroxy indole, indigo); pyrimidine-based dyes (e.g., alloxan); indane-based dyes (e.g., ninhydrin); nitrobenzene-based dyes (e.g., 1,4-diamino-2-nitrobenzene, 1-amino-2-nitro-4-β-hydroxyethylaminobenzene, 1-amino-2-nitro-4-bis(β-hydroxyethyl)aminobenzene, 1,4-bis(β-hydroxyethylamino)-2-nitrobenzene, 1,2-diamino-4-nitrobenzene, or the corresponding nitropyridine variants); quinone-based dyes (e.g., anthraquinone, 1-N-methylmorpholiniumpropylamino-4-hydroxyanthraquinone, 1-aminopropylamino-4-methylaminoanthraquinone, 1-aminopropylaminoanthraquinone, 5-β-hydroxyethyl-1,4-diaminoanthraquinone, 2-aminoethylaminoanthraquinone, 1,4-bis(β,γ-dihydroxypropylamino)anthraquinone, lawsone, juglone, alizarin, purpurin, carminic acid, kermesic acid, spinulosin, Disperse Red 15, Solvent Violet 13, Disperse Violet 1, Disperse Violet 4, Disperse Blue 1, Disperse Violet 8, Disperse Blue 3, Disperse Red 11, Disperse Blue 7, Basic Blue 22, Disperse Violet 15, Basic Blue 99); azo-based dyes (e.g., 1,3-dimethyl-2-[[4-(dimethylamino)phenyl]azo]-1H-imidazolium chloride, 1,3-dimethyl-2-[(4-aminophenyl]azo]-1H-imidazolium chloride, 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium methyl sulfate, Disperse Red 17, Basic Red 22, Basic Red 76, Basic Yellow 57, Basic Brown 16, Basic Brown 17, Disperse Black 9); azine-based dyes (e.g., Basic Blue 17, Basic Red 2); triarylmethane-based dyes (e.g., Basic Green 1, Basic Violet 3, Basic Violet 14, Basic Blue 7, Basic Blue 26, and those disclosed in EP 3034065 A1—which is incorporated herein by reference in its entirety; indoamine-based dyes (e.g., 2-β-hydroxyethylamino-5-[bis(β-4′-hydroxyethyl) amino]anilino-1,4-benzoquinone, 2-β-hydroxyethylamino-5-(2′-methoxy-4′-amino)anilino-1,4-benzoquinone, 3-N-(2′-chloro-4′-hydroxy)phenylacetamino-6-methoxy-1,4-benzoquinoneimine, 3-N-(3′-chloro-4′-methylamino)phenylureido-6-methyl-1,4-benzoquinoneimine, 3-[4′-N-(ethylcarbamylmethyl)amino]phenylureido-6-methyl-1,4-benzoquinoneimine); catechol-based dyes (e.g., purpurogallin, protocatechaldehyde); fluorescent dyes, such as those of the naphthalimide, cationic or non-cationic coumarin, xanthenodiquinolizine, azaxanthene, naphtholactam, azlactone, oxazine, thiazine, or dioxazine families, or polycationic fluorescent dyes of the azo, azomethine or methine families, for example those disclosed in US 2006/0005326—which is incorporated herein by reference in its entirety. The dyestuff may also be a pigment, such as white or colored pigments, lakes, and pearlescent agents or flakes. Exemplary pigments include, but are not limited to, titanium dioxide, which may or may not be surface-treated, zirconium oxide, cerium oxide, iron oxide or chromium oxide, manganese violet, ultramarine blue, chromium hydrate, ferric blue, and organic pigments (e.g., nitroso-, xanthene-, quinolone-, phthalocyanin-, isoindolinone-, isoindoline-, quinacridone-perinone-, perylene-, diketopyrrolopyrrole-, thioindigo-, dioxazine-, and quinophthalone-based organic pigments). Surfactant The hair coloring composition of the present disclosure may include one or more surfactants, which may be nonionic, amphoteric, anionic, or cationic surfactants, preferably nonionic surfactants, and may act as detergents, wetting agents, emulsifiers, foaming agents, and/or dispersants. The surfactant advantageously provides the hair coloring composition with good foamability and workability for ease of application, and may aid consistent application, coating, and delivery of the dyestuff to the hair follicles to provide the dyed hair with a long lasting, natural look, particularly when combined with the monosaccharide dyestuffs disclosed herein. The amount of surfactant present in the hair coloring composition may range from about 0.1 wt. %, preferably from about 0.5 wt. %, more preferably from about 1 wt. %, even more preferably from about 1.5 wt. %, yet even more preferably from about 2 wt. %, and up to about 10 wt. %, preferably up to about 5 wt. %, more preferably up to about 4.5 wt. %, even more preferably up to about 4 wt. %, yet even more preferably up to about 3 wt. %, based on the total weight of the hair coloring composition. The hair coloring compositions preferably include at least one nonionic surfactant, such as an alkyl pyranoside; a polyoxyalkylene ether of a fatty alcohol, a polyol, or an ester; an ethylene oxide/propylene oxide copolymer; and/or a fatty amide. Alkyl pyranoside surfactants are pyranose-based monosaccharides having a glycosidic bond to fatty alcohols having 6 to 26 carbon atoms, preferably 7 to 24 carbon atoms, more preferably 8 to 22 carbon atoms, even more preferably 9 to 20 carbon atoms, yet even more preferably 10 to 18 carbon atoms. The alkyl pyranoside may be formed by any combination of a pyranose-based monosaccharide, for example, allose, altrose, galactose, glucose, gulose, idose, mannose, and talose, as well as anhydro-, deoxy-, heteroatom-substituted-, or dehydro-variants thereof, with a fatty alcohol having 6 to 26 carbon atoms, either saturated or unsaturated, for example, 1-hexanol, 3-methyl-3-pentanol, 1-heptanol, 1-octanol, pelargonic alcohol, 1-decanol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, oleyl alcohol, nonadecyl alcohol, arachidyl alcohol, heneicosyl alcohol, behenyl alcohol, erucyl alcohol, lignoceryl alcohol, ceryl alcohol, as well as mixtures thereof (e.g., cetearyl alcohol, which is a mixture of cetyl and stearyl alcohols). Specific examples include, but are not limited to, hexyl glucoside, octyl glucoside, octyl galactoside, decyl glucoside, decyl galactoside, isodecyl glucoside, isoundecyl glucoside, lauryl glucoside, cetearyl glucoside, coco glucoside, and isotridecyl glucoside, as well as mixtures thereof, with decyl glucoside being the most preferred. The surfactant may also be one or more non-ionic surfactants of the following types: a polyoxyalkylene ether of a fatty alcohol, for example, laureth-3, ceteareth-6, ceteareth-11, ceteareth-15, ceteareth-16, ceteareth-17, ceteareth-18, ceteareth-20, ceteareth-23, ceteareth-25, ceteareth-27, ceteareth-28, ceteareth-30, isoceteth-20, laureth-9/myreth-9, and PPG-3 caprylyl ether, as well as mixtures thereof, preferably ceteareth-25 is used; a polyoxyalkylene ether of a polyol (e.g., glycerin, glucose, sorbitol, etc.), specific examples include, but are not limited to, glycereth-7 caprylate/caprate, glycereth-2 cocoate, PEG-7 glyceryl cocoate, glycereth-7, glycereth-7-triacetate, glycereth-5-lactate, glycereth-7-diisononanoate, methyl gluceth-10, and polysorbates such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80; and/or a polyoxyalkylene ether of an ester exemplified by PEG-14 laurate, PEG-15 laurate, PEG-20 laurate, PEG-32 laurate, PEG-75 laurate, and PEG-150 laurate, including mixtures thereof. Ethylene oxide/propylene oxide copolymers may also be included in the hair coloring compositions as nonionic surfactants, for example, PPG-12-buteth-16, PPG-3-buteth-5, PPG-5-buteth-7, PPG-7-buteth-10, PPG-9-buteth-12, PPG-12-buteth-16, PPG-15-buteth-20, PPG-20-buteth-30, PPG-28-buteth-35, and PPG-33-buteth-45. Examples of fatty amides or polyoxyalkylene fatty amides which may be used as nonionic surfactants herein include, but are not limited to, cocoamide DEA, cocamide MEA, cocamide MIPA, cocamidopropylamine oxide, PEG-6 cocamide, trideceth-2 carboxamide MEA, PEG-4 rapeseedamide, and the like. In preferred embodiments, the surfactant is a mixture of an alkyl pyranoside and a polyoxyalkylene ether of a fatty alcohol, for example in a weight ratio of from about 1:1, or from about 1.5:1, or from about 1.8:1, and up to about 3:1, or up to about 2:1. More preferably, the surfactant is a mixture of decyl glucoside and ceteareth-25, for example, in the above weight ratio ranges. Amphoteric surfactants are preferably not included in the hair coloring composition, but when present, can be selected from imidazoline sulfonates, carboxylates, or phosphates (e.g., alkyl hydroxy ethyl imidazoline sulfonates, disodium lauroamphodiacetate, sodium lauroampho PG-acetate phosphate), amidosultaines (e.g., lauramidopropyl hydroxysultaine, cocamidopropyl hydroxysultaine, oleamidopropyl hydroxysultaine), betaines (e.g., cocamidopropylphosphobetaine, lauric/myristic pyrophosphobetaine, cocamidopropyl betaine), and the like, as well as mixtures of such materials. The hair coloring compositions are preferably substantially free of anionic surfactants, however, when present, suitable anionic surfactants include, but are not limited to, sulfates of fatty alcohols or polyoxyalkylene ethers of fatty alcohols, phosphates of fatty alcohols or polyoxyalkylene ethers of fatty alcohols, sodium salts of fatty acids, acylamino acids, and lactylates. Specific examples of anionic surfactants that can be optionally included in the hair coloring compositions herein include, but are not limited to, sodium lauryl sulfate, sodium laureth sulfate, cetyl phosphate, sodium stearate, sodium behenoyl lactylate, sodium isostearoyl lactylate, sodium caproyl lactylate, sodium laureth-5 carboxylate, sodium laureth-6 carboxylate, sodium laureth-11 carboxylate, sodium stearate, dicetyl phosphate, ceteth-10-phosphate, sodium cocoyl taurate, sodium methyl cocoyl taurate, and sodium methyl oleoyl taurate. The hair coloring compositions are preferably substantially free of cationic surfactants. However, in some embodiments, cationic surfactants are present, such as protonated fatty amines, that is, fatty amine salts derived from primary, secondary or tertiary fatty amines in combination with an acid. For example, protonated fatty amines of stearamidopropyldimethylamine, stearamidopropyldiethylamine, stearamidoethyldiethylamine, stearamidoethyldimethylamine, palmitamidopropyldimethylamine, palmitamidopropyldiethylamine, palmitamidoethyldiethylamine, palmitamidoethyldimethylamine, behenamidopropyldimethylamine, behenamidopropyldiethylmine, behenamidoethyldiethylamine, behenamidoethyldimethylamine, arachidamidopropyldimethylamine, arachidamidopropyldiethylamine, arachidamidoethyldiethylamine, arachidamidoethyldimethylamine, and mixtures thereof Delivery Agent The hair coloring compositions of the present disclosure may include a delivery agent, which is a material capable of aiding the penetration of, and enhancing uniform delivery of, the dyestuff (and other components of the hair coloring composition) into the keratin fiber so that a deeper, longer-lasting, natural hair coloration can be achieved. In preferred embodiments, the delivery agent is a dianhydrohexitol or a derivative thereof, which includes monoalkoxy substituted dianhydrohexitols and dialkoxy substituted dianhydrohexitols. Dianhydrohexitols (i.e., 1,4;3,6-dianhydrohexitols) are by-products of the starch industry most often obtained by dehydration of D-hexitols. These chiral biomass-derived products exist as three main isomers, namely isosorbide (I), isomannide (II), and isoidide (III), depending on the configuration of the two hydroxyl groups (derived from D-glucose, D-mannose, and L-fructose, respectively), and any of these isomers or derivatives of any of these isomers may be employed as the delivery agent herein, including mixtures. In preferred embodiments, a monoalkoxy substituted dianhydrohexitol or a dialkoxy substituted dianhydrohexitol is used, which is a dianhydrohexitol where one hydroxyl group is substituted to form an alkoxy group or a dianhydrohexitol where both hydroxyl groups are substituted to form two alkoxy groups (which can be the same or different), respectively. As used herein, “alkoxy” substitution includes straight chain, branched, cyclic, or (poly)oxyalkylene-type, alkoxy groups, either saturated or unsaturated, having up to 10 carbon atoms, preferably up to 8 carbon atoms, preferably up to 4 carbon atoms, for example 1 to 4 carbon atoms, and specifically includes: acyclic alkoxy groups such as methoxy, ethoxy, propoxy, allyloxy, isopropoxy, butoxy, isobutoxy, secondary butoxy, tertiary butoxy, pentoxy, isopentoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy and decyloxy; cyclic alkoxy groups (having 3 to 10 carbon atoms) including cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and cycloheptyloxy groups; and (poly)oxyalkylene-type alkoxy groups such as polyoxyethylene (—O—(CH2—CH2—O)n—R), polyoxypropylene (—O—(CH2—CH(CH3)—O)n—R), and polyoxybutylene (—O—(CH2—CH(CH2CH3)—O)n—R) groups having up to 5 repeating units, i.e., n is from 1 to 5, wherein R is H or a C1to C3alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl). Preferably, the delivery agent is a C1to C4monoalkoxy substituted dianhydrohexitol, or a C1to C4dialkoxy substituted dianhydrohexitol, most preferably a C1to C4dialkoxy substituted dianhydrohexitol. In the case of a dialkoxy substituted dianhydrohexitol, the carbon count refers to a total number of carbon atoms for each alkoxy substituent, and thus “a C1to C4dialkoxy substituted dianhydrohexitol” refers to compounds where each alkoxy substituent has 1 to 4 carbon atoms, independently of the other alkoxy substituent. Examples of dianhydrohexitols or derivatives thereof suitable for use as delivery agents herein include, but are not limited to, isosorbide, isomannide, and isoidide, methyl isosorbide, dimethyl isosorbide, ethyl isosorbide, diethyl isosorbide, propyl isosorbide, dipropyl isosorbide, monoisopropyl isosorbide, diisopropyl isosorbide, methylethyl isosorbide, methylpropyl isosorbide, ethylpropyl isosorbide, butyl isosorbide, dibutyl isosorbide, isobutyl isosorbide, diisobutyl isosorbide, methylbutyl isosorbide, ethylbutyl isosorbide, propylbutyl isosorbide, methyl isomannide, methyl isoidide, dimethyl isomannide, and dimethyl isoidide. In preferred embodiments, the delivery agent is dimethyl isosorbide (a C1dialkoxy substituted dianhydrohexitol). In addition to aiding delivery of the dyestuff (and/or other components of the hair coloring composition), the unsubstituted and alkoxy substituted dianhydrohexitols disclosed herein are particularly advantageous because they are soluble in water, biologically harmless, and may enhance the health and appearance of hair follicles to which they are applied. Other acceptable delivery agents that may be used as delivery agents in lieu of, or in addition to the dianhydrohexitols or derivatives thereof, include pyrrolidinone, caprylyl pyrrolidone, N-methylpyrrolidone, lauryl pyrrolidone, propylene carbonate, 2-benzyloxyethanol, gamma-butyrolactone, phenylethanol, diethyl glycol-monoethylether, polyethylene glycols (e.g., PEG-4, PEG-6, PEG-8, PEG-10, PEG-12, PEG-32), isopentyldiol, ethoxydiglycol, and the like, as well as mixtures thereof. In some embodiments, the amount of delivery agent present in the hair coloring composition is from about 0.1 wt. %, preferably from about 0.5 wt. %, preferably from about 1 wt. %, preferably from about 1.5 wt. %, preferably from about 2 wt. %, and up to about 5 wt. %, preferably up to about 4 wt. %, preferably up to about 3 wt. %, preferably up to about 2.5 wt. %, based on a total weight of the hair coloring composition. Solvent In some embodiments, the hair coloring composition of the present disclosure is an aqueous composition or an oil-in-water (o/w) emulsion where the continuous phase is aqueous. Therefore, in preferred embodiments, the hair coloring composition further includes water in amounts of at least about 60 wt. %, more preferably at least about 65 wt. %, even more preferably at least about 70 wt. %, yet even more preferably at least about 75 wt. %, and up to about 95 wt. %, preferably up to about 90 wt. %, more preferably up to about 85 wt. %, even more preferably up to about 80 wt. %, based on a total weight of the hair coloring composition. In addition to water, the hair coloring composition may optionally include at least one organic solvent which may aid solubilization of components not sufficiently soluble in water, adjust the surface properties of the hair coloring composition for enhanced workability, foamability, and/or foam stability, or to generally provide a medium suitable for the dyeing operation. The at least one organic solvent, may be chosen from, for example, a C1to C4lower alkanol, for example, methanol, ethanol (e.g., denatured alcohol), isopropanol, butanol; polyols and polyol ethers, for example, glycol, 1,3-propanediol, 1,3-butanediol, 2-butoxyethanol, propylene glycol, butylene glycol, hexylene glycol, isoprene glycol, diethylene glycol, dipropylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol monomethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, as well as mixtures thereof. Preferably, the organic solvent is ethanol. When present, the organic solvent may be included in the hair coloring compositions in an amount ranging from about 0.5 wt. %, preferably from about 1 wt. %, more preferably from about 1.5 wt. %, even more preferably from about 2 wt. %, yet even more preferably from about 2.5 wt. %, and up to about 10 wt. %, preferably up to about 5 wt. %, preferably up to about 4.5 wt. %, more preferably up to about 4 wt. %, even more preferably up to about 3.5 wt. %, yet even more preferably up to about 3 wt. %, based on a total weight of the hair coloring composition. Preservative The hair coloring composition may optionally further include a preservative. For example, the preservative may be selected to kill bacteria that might otherwise be sustained or multiplied in the composition, or to prevent degradation or chemical breakdown (e.g., oxidative degradation) of the composition. Preservatives suitable for use in cosmetic formulations are well-known to those skilled in the art. In this respect, the preservative chosen may be varied depending on the particular components present in the hair coloring composition. Illustrative of suitable preservatives include ethylparaben, propylparaben, methylparaben, EDTA or salts thereof (such as disodium EDTA), phenoxyethanol, DMDM hydantoin, benzyl alcohol, ethyldibromoglutaronitrile-phenoxyethanol/polyquatemium-7 (Euxyl K-400, Calgon), imidazolidinyl urea, diazolidinyl urea, benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbic acid and the like, or combinations thereof. Preferably, the preservative is at least one of methylparaben, sodium benzoate, and benzyl alcohol, most preferably a mixture of these preservatives. When present, the preservative may be included herein in amounts of up to about 5 wt. %, preferably up to about 4 wt. %, preferably up to about 3 wt. %, preferably up to about 2 wt. %, preferably up to about 1 wt. %, for example from about 0.001 wt. % to about 3 wt. %, or 0.1 wt. % to about 2 wt. %, or 0.2 wt. % to about 1 wt. %, based on a total weight of the hair coloring composition. Acidulant The hair coloring compositions disclosed herein may be optionally formulated to include an acidulant for adjusting the pH to be more acidic/less alkaline. Additionally, depending on the chemical structure, the acidulant may act as a chelating agent and/or a buffering agent to neutralize minerals, enhance the activity of any preservatives present, and to stabilize active ingredients (e.g., the dyestuff). When the acidulant contains α-hydroxy acid functionality, the acidulant may also confer moisturizing and smoothing effects to the hair coloring composition. When present, the acidulant may be included herein in amounts of up to about 5 wt. %, preferably up to about 4 wt. %, preferably up to about 3 wt. %, preferably up to about 2 wt. %, preferably up to about 1 wt. %, for example from about 0.001 wt. % to about 3 wt. %, or 0.02 wt. % to about 2 wt. %, or 0.1 wt. % to about 1 wt. %, or 0.2 wt. % to about 0.5 wt. %, based on a total weight of the hair coloring composition. The pH of the hair coloring composition may be varied, but is preferably less than 5, for example, at least 2, preferably at least 2.5, more preferably at least 3, even more preferably at least 3.5, and up to 5, preferably up to 4.5, more preferably up to 4. The acidulant employed herein may be an inorganic acid or an organic acid, and specifically includes, but is not limited to, hydrochloric acid, orthophosphoric acid, sulfuric acid, carboxylic acids such as fumaric acid, acetic acid, and α-hydroxy acids such as tartaric acid, citric acid, and lactic acid, as well as mixtures thereof. Preferably citric acid is used. Conditioning Agent The hair coloring compositions of the present disclosure may also optionally include one or more conditioning agents, which may act as a moisturizer, emollient, occlusive agent, and/or humectant for the hair. Any suitable conditioning agent known to those of ordinary skill in the art may be employed herein. If included in the hair coloring composition, the amount of conditioning agent is typically less than about 15 wt. %, preferably less than about 10 wt. %, more preferably less than about 8 wt. %, or from about 0.1 wt. %, preferably from about 1 wt. %, more preferably from about 2 wt. %, even more preferably from about 4 wt. %, and up to about 10 wt. %, preferably up to about 8 wt. %, more preferably up to about 6 wt. %, even more preferably up to about 5 wt. %, based on a total weight of the hair coloring composition. In preferred embodiments, the conditioning agent employed herein is a monomeric polyol, preferably a monomeric polyol having at least three hydroxyl groups (e.g., glycerin, erythritol, pentaerythritol, threitol, arabitol, xylitol, ribitol). In most preferred embodiments, the conditioning agent is glycerin. The hair coloring compositions may be substantially free of, or alternatively may include, retinyl palmitate, petrolatum, gelatin, guar hydroxypropyl trimonium chloride, natural botanicals and extracts thereof such asChamomile recutita, Sambucus nigra, Primula veris, Helianthus annuus, Urtica dioica(i.e., nettle),Olea europaea(i.e., olive), aloe (e.g., barbadensis gel), kelp (e.g., sea kelp), phospholipids,), and the like, or combinations thereof. In some embodiments, the hair coloring compositions are substantially free of conditioning agents of the following types: a quaternary ammonium compound, a fatty material (e.g., fatty alcohols, fatty acids, fatty hydrocarbon oils, and fatty esters), and a silicone. However, such conditioning agents may optionally be included herein. Suitable quaternary ammonium compounds include cetyltrimethylammonium chloride, behenyltrimethylammonium chloride, behentrimonium methosulfate, cetylpyridinium chloride, tetramethylammonium chloride, tetraethylammonium chloride, octyltrimethylammonium chloride, dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octyldimethylbenzylammonium chloride, decyldimethylbenzylammonium chloride, stearyldimethylbenzylammonium chloride, didodecyldimethylammonium chloride, dioctadecyldimethylammonium chloride, tallowtrimethylammonium chloride, dihydrogenated tallow dimethyl ammonium chloride (e.g., Arquad 2HT-75, available from Akzo Nobel), cocotrimethylammonium chloride, PEG-2-oleammonium chloride, and the like, as well as the corresponding bromides or hydroxides thereof. Fatty materials which provide acceptable hair conditioning effects include fatty alcohols or corresponding carboxylic acids (i.e., fatty acid, for example, stearic acid), fatty hydrocarbon oils, and/or a fatty esters. Exemplary fatty alcohols have been listed previously, with preferred fatty alcohols being cetyl alcohol, stearyl alcohol, behenyl alcohol, and mixtures thereof (e.g., cetearyl alcohol). Fatty acids may include, but are not limited to, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. Fatty hydrocarbon oils include paraffin oil, mineral oil, saturated and unsaturated dodecane, saturated and unsaturated tridecane, saturated and unsaturated tetradecane, saturated and unsaturated pentadecane, saturated and unsaturated hexadecane, and the like, as well as mixtures thereof. Branched-chain isomers of these compounds, as well as higher chain length hydrocarbons, can also be used. Suitable fatty esters are characterized by having at least one fatty aliphatic chain derived from a fatty acid, a fatty alcohol, or both. Fatty esters herein may be monoesters of the formula R1COOR2in which at least one of R1and R2is an alkyl or alkenyl radical having 6 to 26 carbon atoms, preferably 7 to 24 carbon atoms, more preferably 8 to 22 carbon atoms, even more preferably 9 to 20 carbon atoms, yet even more preferably 10 to 18 carbon atoms, and where the sum of carbon atoms combined in R1and R2is from 7 to 52 carbon atoms, for example, cetyl octanoate and lauryl lactate. Diesters and triesters containing at least one fatty aliphatic portion can also be used. Particularly preferred fatty esters are mono-, di-, and tri-glycerides, more specifically the mono-, di-, and tri-esters of glycerol and at least one fatty acid, for example, glyceryl mono-, di-, or tri-stearate, cocoa butter, palm stearin, sunflower oil, soybean oil and coconut oil. Suitable silicone conditioning agents are polydiorganosiloxanes, in particular polydimethylsiloxanes (dimethicones), polydimethylsiloxanes having hydroxyl end groups (dimethiconols), amino-functional polydimethylsiloxanes (amodimethicones), polyoxyalkylene functionalized polydimethylsiloxanes (dimethicone copolyols), and mixtures thereof. Exemplary silicones include cyclomethicone, phenyltrimethicone, alkyl dimethicone, fluorinated silicones, dimethicone, PEG-3 dimethicone, PEG-7 dimethicone, PEG-8 dimethicone, PEG-9 dimethicone, PEG-10 dimethicone, PEG-12 dimethicone, PEG-14 dimethicone, PEG-17 dimethicone, PEG/PPG-3/10 dimethicone, PEG/PPG-4/12 dimethicone, PEG/PPG-6/11 dimethicone, PEG/PPG-8/14 dimethicone, PEG/PPG-14/4 dimethicone, PEG/PPG-15/15 dimethicone, PEG/PPG-16/2 dimethicone, PEG/PPG-17/18 dimethicone, PEG/PPG-18/18 dimethicone, PEG/PPG-19/19 dimethicone, PEG/PPG-20/6 dimethicone, PEG/PPG-20/15 dimethicone, PEG/PPG-20/20 dimethicone, PEG/PPG-20/23 dimethicone, PEG/PPG-20/29 dimethicone, PEG/PPG-22/23 dimethicone, PEG/PPG-22/24 dimethicone, PEG/PPG-23/6 dimethicone, PEG/PPG-25/25 dimethicone and PEG/PPG-27/27 dimethicone, and the like, and combinations thereof Fragrance The hair coloring compositions of the present disclosure may be optionally formulated to include one or more fragrances known to those of ordinary skill in the cosmetics arts to impart a pleasant scent or to help mask any malodorous components that may be present in the hair coloring compositions. Non-limiting examples of compounds used as fragrances herein include dihydrocitronellyl nitrile, 2,2,6-trimethylcyclohexane carboxylic acid ethyl ester (i.e., Thesaron, available from Takasago International Corporation), 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol racemic or optically active form, preferably (E)-(R)-form (i.e., Levosandol, available from Takasago International Corporation), 2,2,6-trimethylcyclohexyl-3-hexanol, cyclohexadecenone, 1-(2-methyl-2-propenyloxy)-2,2,4-trimethylpentan-3-ol, 1-phenyl-2,2,4-trimethyl-3-pentanone, 4,8-dimethyl-7-nonen-2-ol, 2-methyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol racemic or optically active form, preferably (E)-(R)-form, tri(cis-3-hexenyl) orthoformate, 4-ethoxy-2-methyl-butanethiol, 5-methoxy-3-methyl-3-pentanethiol, thioglycerin, dibutyl sulfide, thiogeraniol, thiocineol, limonenethiol, 2-methyl-4-propyl-1,3-oxathian, 4-methoxy-2-methyl-2-butanethiol, terpene hydrocarbons such as p-cymene, terpinolene, myrcene, and β-caryophylene, aldehydes such as heptanal, octanal, benzaldehyde, salicylic aldehyde, citronellal, α-hexylcinnamic aldehyde and lilial, esters such as methyl jasmonate, methyl dihydrojasmonate, γ-nonyllactone, γ-decalactone and coumarin, ethers such as anisole, p-cresyl methyl ether, β-naphthol methyl ether, and β-naphthol ethyl ether, ketones such as menthone, acetophenone, α-damascone, β-damascone, α-ionone, β-ionone, methyl ionone, irone, dihydrojasmone, cis-jasmone, muscone and civetone, alcohols such as cis-3-hexenol, heptanol, 2-octanol, benzyl alcohol, citronellol, geraniol, terpineol, tetrahydrogeraniol, anise alcohol, phenylethyl alcohol, phenoxy ethanol, santalol, sandalmysore core, bacdanol, ebanol, polysantol, and natural essential oils such as orange oil, lemon oil, lime oil, patchouli oil, cyprus oil, sandalwood oil, peppermint oil, spearmint oil, and anise oil, and the like, as well as mixtures thereof. In some embodiments, the fragrance is present in amounts of up to about 3 wt. %, preferably up to about 2 wt. %, preferably up to about 1 wt. %, preferably up to about 0.5 wt. %, preferably up to about 0.3 wt. %, preferably up to about 0.2 wt. %, preferably up to about 0.1 wt. %, preferably up to about 0.01 wt. %, based on a total weight of the hair coloring composition. Other Optional Ingredients Various optional ingredients frequently used in topical formulations such as propellants, vehicles, adjuvants, anti-aging components, proteins, rheology control agents, dispersants, thickeners, film-forming agents, sequestering agents, cleansing agents, vitamins, botanicals, and sunscreen agents, as well as other classes of materials whose presence may be cosmetically, medicinally or otherwise desirable, can also optionally be included at their conventional art-established usage levels. In preferred embodiments, the hair coloring compositions are substantially free of such optional ingredients, however, when included, non-limiting examples which can be used include film-forming and moisturizing materials such as hydrolyzed wheat protein/wheat oligosaccharides (e.g., Cropeptide W by Croda Inc.), hydrolyzed corn protein, hydrolyzed wheat gluten, hydrolyzed yeast protein, hydrolyzed vegetable protein, hydrolyzed soy protein, hydrolyzed rice protein, and hydrolyzed potato protein; cleansing agents and emollients such as polyethylene glycol derivatives of castor oil, for example, PEG-40 castor oil (Surfactol 365, available from Vertellus), PEG-45 castor oil, PEG-50 castor oil, PEG-60 castor oil, and PEG-100 castor oil; sunscreens or UV light absorbing compounds octyldimethyl PABA, benzophenone-4, DEA methoxycinnamate, 2-phenyl-benzimidazole-5-sulfonic acid, and triethanolamine salicylate; thickeners such as carbomer and C10-C30alkylacrylate cross-polymer; film forming polycationic polymers such as polyquaternium-1, polyquaternium-2, polyquaternium-4, polyquaternium-5, polyquaternium-6, polyquaternium-7, polyquaternium-8, polyquaternium-9, and polyquaternium-10; and thickening agents such as modified cellulose polymers, for example, hydroxyethyl cellulose and methyl cellulose. In preferred embodiments, the hair coloring composition includes 5 to 10 wt. % of a first monosaccharide having 2 to 4 carbon atoms (e.g., dihydroxyacetone), 0.001 to 0.5 wt. % of a second monosaccharide having 2 to 4 carbon atoms (e.g., erythrulose); 0.1 to 3 wt. % of a surfactant, which is a mixture of an alkyl pyranoside (e.g., decyl glucoside) and a polyoxyalkylene ether of a fatty alcohol (e.g., ceteareth-25); 0.1 to 1 wt. % of a delivery agent, which is a C1to C4dialkoxy substituted dianhydrohexitol (e.g., dimethyl isosorbide), and 70 to 85 wt. % water, with the balance optionally including one or more of an organic solvent (e.g., ethanol), a preservative (e.g., a mixture of methylparaben, sodium benzoate, and benzyl alcohol), an acidulant (e.g., citric acid), a conditioning agent (e.g., glycerin), and/or a fragrance. In preferred embodiments, the hair coloring composition includes 5 to 10 wt. % of a first monosaccharide having 2 to 4 carbon atoms (e.g., dihydroxyacetone), 0.001 to 0.5 wt. % of a second monosaccharide having 2 to 4 carbon atoms (e.g., erythrulose); 0.1 to 3 wt. % of a surfactant, which is a mixture of an alkyl pyranoside (e.g., decyl glucoside) and a polyoxyalkylene ether of a fatty alcohol (e.g., ceteareth-25); 0.1 to 1 wt. % of a delivery agent, which is a C1to C4dialkoxy substituted dianhydrohexitol (e.g., dimethyl isosorbide), 70 to 85 wt. % water, 1 to 3 wt. % of an organic solvent (e.g., ethanol), 0.001 to 0.3 wt. % of a preservative, which is a mixture of methylparaben, sodium benzoate, and benzyl alcohol, 0.001 to 0.2 wt. % of an acidulant (e.g., citric acid), 3 to 7 wt. % of a conditioning agent, which is a monomeric polyol having at least three hydroxyl groups (e.g., glycerin), and 0.01 to 0.5 wt. % of a fragrance. The hair coloring compositions herein can be prepared by any method known to those of ordinary skill in the art. By way of example, the hair coloring composition may be prepared by (i) mixing together all water soluble components in an appropriately sized vessel with water with optional heating (e.g., 40 to 90° C., preferably 50 to 80° C., more preferably 60 to 70° C.) and stirring until homogenous, (ii) in a separate vessel, mixing all oil phase ingredients, if any, with optional heating (e.g., 40 to 100° C., preferably 50 to 90° C., more preferably 60 to 80° C.) and stirring until homogeneous, (iii) mixing together the homogenous mixture from (i) with the homogenous mixture from (ii), if any, with optional heating (e.g., 40 to 100° C., preferably 50 to 90° C., more preferably 60 to 80° C.) and stirring to form the hair coloring composition. Alternatively, the hair coloring composition may be prepared by (i) mixing together all water soluble components except for the dyestuff component (e.g., the first and second monosaccharides having 2 to 4 carbon atoms) in an appropriately sized vessel with water with optional heating (e.g., 40 to 90° C., preferably 50 to 80° C., more preferably 60 to 70° C.) and stirring until homogenous, (ii) in a separate vessel, mixing all oil phase ingredients, if any, with optional heating (e.g., 40 to 100° C., preferably 50 to 90° C., more preferably 60 to 80° C.) and stirring until homogeneous, (iii) mixing together the homogenous mixture from (i) with the homogenous mixture from (ii), if any, with optional heating (e.g., 40 to 100° C., preferably 50 to 90° C., more preferably 60 to 80° C.) and stirring until a homogenous aqueous composition or uniform oil in water emulsion is formed, and (iv) adding the dyestuff component (e.g., the first and second monosaccharides having 2 to 4 carbon atoms) to the homogenous aqueous composition or uniform oil in water emulsion, once the homogenous aqueous composition or uniform oil in water emulsion has cooled to 40° C. or below, and stirring under conditions similar to above, to form the hair coloring composition. Once cooled, the hair coloring composition produced by either method may then be packaged, for example in a non-aerosol foam generating dispenser, for sale and/or distribution. Properties The hair coloring compositions of the present disclosure are stable for up to 4 years, for example from 24 months to 36 months, at room temperature or at sub-ambient temperatures without significant/rapid degradation of the active components or ingredient precipitation, which is known to sometimes occur with hair coloring compositions. The hair coloring compositions are thus suitable for long-term storage, distribution, and storage in between uses. The hair coloring compositions disclosed herein have acceptable viscosity and surface tension in a liquid state, which allows for facile processing, workability (e.g., foamability), and packaging, and provides suitable foam characteristics once converted into a foam. The viscosity of the hair coloring composition (as a liquid at 25° C.) is typically from about 1 mPa·s, preferably from about 2 mPa·s, more preferably from about 3 mPa·s, more preferably from about 5 mPa·s, even more preferably from about 10 mPa·s, yet even more from about 20 mPa·s, and up to about 300 mPa·s, preferably up to about 200 mPa·s, more preferably up to about 100 mPa·s, even more preferably up to about 50 mPa·s, yet even more preferably up to about 30 mPa·s. It is to be noted that the viscosity as referred to herein refers to a value obtained after rotating the liquid mixture at 60 rpm for one minute at 25° C. by a B-type rotational viscometer (model TV-10), manufactured by TOKYO KEIKI INC., with rotor No. 1 or 2. When the object to be measured has a viscosity of less than 100 mPa·s, the viscosity is measured using rotor No. 1, whereas when it has a viscosity of 100 to 499 mPa·s, the viscosity is measured using rotor No. 2. Measurement is made in a thermostat bath at 25° C. immediately after mixing, and temperature fluctuations caused by heat of reaction are negligible. In preferred embodiments, the hair coloring composition is foamable and is in the form of a foam when applied. The stability of foam depends upon an interaction between the foam and an interface between foams. The foam bursts as a liquid film becomes thinner from flowing downward due to gravity or as liquid generally flows due to curvature differences of the foam. If the thickness of the liquid film is in the range of about 5 to 15 nm, the liquid film cannot resist the pressure from the inside to burst. In general, foams generated from aqueous solutions lack repulsive forces of an electric double layer in a liquid film, have low stability by hydration, and low resistance against liquid film thinning due to gravitational/curvature effects, leading to poor foam stability. However, the addition of one or more surfactants in an appropriate amount lowers the surface tension thereby accelerating foam formation, maintaining liquid film thickness for longer periods of time, and delaying gravitational/curvature breakdown effects. Thus, the present inventors have discovered that the hair coloring compositions herein, in addition to providing desirable hair dyeing results, are also capable of being foamed into easily-applied and stable foams that resist dripping (i.e., conversion back into a liquid state) to aid hair application while preventing staining of the skin (e.g., the scalp). In preferred embodiments, the foams generated from the hair coloring compositions disclosed herein are continuously retained for at least 1 minute, preferably at least 2 minutes, more preferably at least 10 minutes, and up to 50 minutes. Method for Coloring Hair The present disclosure provides a method for coloring hair by applying the hair coloring composition, in one or more embodiments, onto the hair. In order to achieve an acceptable amount of coloration, a person who desires such coloration can apply evenly an effective amount of the hair coloring composition for an effective application time over an entire treatment area (e.g., total coverage of gray hair), or to particularly problematic spots (e.g., gray hair located on the side of the head or sideburns). Thus, the hair coloring compositions can be applied for subtle changes in hair color such as gray hair blending, or more dramatic effects such as total gray hair coverage. Further, the method herein produces natural looking dyed hair, and after dyeing, provides excellent fastness to shampooing, irrespective of the kind of shampoo used for shampooing. In hair coloring applications, the steadfastness of the color (i.e., the resistance to color fading after washing) is of greater importance than in skin coloring/tanning applications, since dyed hair is likely to be exposed to a greater number of washings per dyeing application when compared to dyed skin as skin cells are shed and renewed at a higher rate and thus have a lower life cycle than hair follicles. In a typical method, the hair coloring compositions as described above are topically applied to wet or dry hair. Preferably, the hair coloring compositions are applied to freshly shampooed and dried hair. The hair coloring compositions may be used as a single treatment to color the hair or applied in a progressive manner so that the hair color becomes more intense on subsequent applications until a desired coloration is reached. To avoid staining of skin, the hair coloring compositions may be applied with gloves or with a spreading instrument such as a comb or brush. Preferably, the hair coloring composition is applied uniformly to each hair fiber, from the root to the end of the hair fiber. The effective application time may range from 1 minute, preferably from 3 minutes, more preferably from 5 minutes, even more preferably from 10 minutes, and up to 60 minutes, preferably up to 50 minutes, more preferably up to 40 minutes, even more preferably up to 30 minutes, yet even more preferably up to 20 minutes, depending on whether or not heat is applied. Application times outside of these ranges may also be used to vary the degree of coloration, as desired. In preferred embodiments, the hair coloring compositions herein are applied to the hair in the form of a foam. Foam hair coloring compositions are easy to apply (for both complete coverage or for targeting problematic spots), and also help prevent unwanted staining of the skin and scalp that can be problematic with sprays and other topical forms, for example, by reducing the propensity of the compositions to drip. Any foaming method known to those of ordinary skill in the cosmetics arts can be used for foaming the hair coloring compositions of the present disclosure, but preferably a non-aerosol foam generating dispenser is used for discharging the hair coloring compositions as a foam. A non-aerosol foam generating dispenser is a device which is used to discharge the hair coloring composition in the form of a foam by mixing it with air without using a propellant, for example a dispenser actuated by manual mechanical pumping. In preferred embodiments, the method further involves applying heat to the hair at a dyeing temperature of 30 to 205° C. It has been surprisingly found that heat speeds up the color development with exemplary effective application times of less than 20 minutes, preferably less than 10 minutes, more preferably less than 5 minutes, and produces dyed hair with a deeper, richer color than colors produced in the absence of applied heat. In some embodiments, heat is applied by use of a hair dryer/blow dryer, for example, to achieve a dyeing temperature of from about 30° C., preferably from about 35° C., more preferably from about 40° C., and up to about 60° C., preferably up to about 55° C., more preferably up to about 50° C., even more preferably up to about 45° C. Therefore, common commercial/household hair dryers/blow dryers may be used as they would for normal operation for applying heat in the methods herein. While hair dryers or blow dryers capable of producing high temperatures (i.e., dyeing temperatures above 60° C.) may also be used, it is often difficult to apply high heat to hair in this fashion without burning the scalp. Therefore, in some embodiments, a hair iron (e.g., a straight iron, a curling iron, etc.) is used to apply high temperatures, that is, dyeing temperatures of more than 60° C., preferably more than 70° C., preferably more than 80° C., preferably more than 90° C., preferably more than 100° C., preferably more than 110° C., preferably more than 120° C., preferably more than 130° C., and up to 205° C., preferably up to 200° C., preferably up to 190° C., preferably up to 180° C., preferably up to 170° C., preferably up to 160° C., preferably up to 150° C., preferably up to 140° C. Thus, common commercial/household hair irons may be used as they would for normal operation for applying high heat in the methods herein. In some embodiments, after application of the hair coloring composition, and any optional heating, the hair is simply allowed to dry. In some embodiments, after application of the hair coloring composition, and after optional application of heat, the hair is rinsed and/or washed with shampoo to remove the hair coloring composition. Preferably, the hair coloration fully develops during the application process and prior to rinsing/washing, particularly when heat is applied. However, in some instances the color may continue to develop slightly, even after the hair coloring composition is removed by rinsing or shampooing. Even with the application of high heat, the methods herein are mild and do not damage keratin fibers during the dyeing process. To this end, it is preferable that the methods herein do not require the use of harsh chemicals, such as oxidizing agents, either together with the hair coloring compositions, or as a separate application. Oxidizing agents conventionally used for oxidative dyeing of keratin fibers are, for example, peroxides (e.g., hydrogen peroxide, benzyl peroxide, urea peroxide, etc.), bleaching agents (e.g., chlorine dioxide, hypochlorite, etc.), alkali metal bromates (e.g., sodium bromate), persalts such as perborates (e.g., sodium perborate) and persulfates (e.g., potassium persulfate), peracids (e.g., peracetic acid, pernonoic acid, nonylamidoperoxycaproic acid (NAPCA)), and oxidase enzymes such as peroxidases, 2-electron oxidoreductases (e.g., uricases) and 4-electron oxygenases (e.g., laccases). Preferably, the water used to wet and/or rinse the hair, either before or after application of the hair coloring composition, also contains less than 1 ppm of oxidizing agents (e.g., chlorine dioxide, hypochlorite, etc.), preferably less than 50 ppb. When one or more monosaccharides having 2 to 4 carbon atoms are employed as the dyestuff in the hair coloring composition, a two-component application system or “kit” is also envisioned which includes 1) the hair coloring composition comprising the monosaccharide(s) having 2 to 4 carbon atoms as discussed above, and 2) an amino acid composition configured to drive the Maillard reaction to increase the speed of color formation and/or to produce certain color tones in reaction with the monosaccharide from the first component. These two components may be part of an application kit and stored in separate containers. The amino acid composition may comprise any amino acid, but preferably includes one or more natural amino acids (i.e., 21 proteinogenic α-amino acids), most preferably one or more of serine, glycine, alanine, and glutamic acid. In preferred embodiments, the amount of amino acid(s) in the second component applied to the hair is at least 10 wt. %, preferably at least 20 wt. %, preferably at least 30 wt. %, preferably at least 40 wt. %, more preferably at least 50 wt. %, even more preferably at least 60 wt. %, yet even more preferably at least 70 wt. %, yet even more preferably at least 80 wt. %, and up to 100 wt. %, based on the total weight of monosaccharide(s) having 2 to 4 carbon atoms applied to the hair from the first component. The type of amino acid and the amount of amino acid present in the amino acid composition may be adjusted to vary the color produced. In addition to the amino acid(s), the amino acid composition may include water and optionally a surfactant, a delivery agent, an organic solvent, a preservative, an acidulant, a conditioning agent, a fragrance, and/or any other various ingredients disclosed herein in amounts similar to that of the hair coloring composition. In preferred embodiments, like the hair coloring composition, the amino acid composition is an aqueous mixture capable of being foamed for ease of application. To use the two-component application system, the amino acid composition may be mixed with the hair coloring composition of the disclosure at the time of use and the combined mixture may then be immediately applied to the hair. Alternatively, the amino acid composition is not premixed with the hair coloring composition. Instead, the two components are applied sequentially without intermediate rinsing, whereby the hair coloring composition is applied first followed by application of the amino acid composition (or vice versa, that is, the amino acid composition is applied first followed by application of the hair coloring composition). Following application of both the hair coloring composition and the amino acid composition, heat may then be applied at a dyeing temperature of 30 to 205° C. to speed the color formation and produce a deeper, richer color as discussed previously. The examples below are intended to further illustrate radiation curable inkjet inks and surface roughness properties and are not intended to limit the scope of the claims. EXAMPLES Example 1 Example Hair Coloring Composition RM in FinishedINCI NameProduct % wt./wt.Water73.418Glycerin5.00Methylparaben0.20Sodium Benzoate0.001Ceteareth-250.40Citric Acid0.02Decyl Glucoside (55%)0.75Dimethyl Isosorbide0.25Fragrance0.20Water11.00Erythrulose (80%)0.01Dihydroxyacetone6.25Alcohol Denat.2.50Benzyl Alcohol0.001INCI = International Nomenclature of Cosmetic IngredientsRM = raw material To prepare the hair coloring composition of Example 1, water and glycerin are added to a large vessel and heated to 75° C. Additional water soluble ingredients are added and mixed until homogenous. The mixture is cooled to 40° C. In a separate vessel dihydroxyacetone and erythrulose are mixed until clear. This premix is added to the large vessel and mixed for 15 minutes. Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out. As used herein the words “a” and “an” and the like carry the meaning of “one or more.” Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. All patents and other references mentioned above are incorporated in full herein by this reference, the same as if set forth at length. | 63,319 |
11857663 | While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments. DETAILED DESCRIPTION Provided herein are methods for producing pure and highly scalable silk protein fragment (SPF) mixture solutions that may be used across multiple industries for a variety of applications. The solutions are generated from raw pure intact silk protein material and processed in order to remove any sericin and achieve the desired weight average molecular weight (MW) and polydispersity of the fragment mixture. Select method parameters may be altered to achieve distinct final silk protein fragment characteristics depending upon the intended use. The resulting final fragment solution is pure silk protein fragments and water with PPM to non-detectable levels of process contaminants, levels acceptable in the pharmaceutical, medical and consumer cosmetic markets. The concentration, size and polydispersity of silk protein fragments in the solution may further be altered depending upon the desired use and performance requirements. In an embodiment, the pure silk fibroin based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 6 kDa to about 16 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the pure silk fibroin-based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 17 kDa to about 38 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the pure silk fibroin-based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 39 kDa to about 80 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the silk solutions of the present disclosure may be used to generate articles, such as silk films of various shapes and sizes by varying water content/concentration, or sold as a raw ingredient into the medical, consumer, or electronics markets. In an embodiment, the solutions may be used to generate articles, such as silk gels of varying gel and liquid consistencies by varying water content/concentration, or sold as a raw ingredient into the pharmaceutical, medical, consumer, or electronics markets. Depending on the silk solution utilized and the methods for casting the films or gels, various properties are achieved. The articles may be loaded with at least one therapeutic agent and/or at least one molecule. As used herein, the terms “substantially sericin free” or “substantially devoid of sericin” refer to silk fibers in which a majority of the sericin protein has been removed. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 10.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 9.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 8.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 7.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 6.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 5.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.05% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.1% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 1.0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 1.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 2.0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 2.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content between about 0.01% (w/w) and about 0.1% (w/w). In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.1% (w/w). In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.05% (w/w). In an embodiment, when a silk source is added to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes, a degumming loss of about 26 wt. % to about 31 wt. % is obtained. As used herein, the term “substantially homogeneous” may refer to pure silk fibroin based protein fragments that are distributed in a normal distribution about an identified molecular weight. As used herein, the term “substantially homogeneous” may refer to an even distribution of additive, for example vitamin C, throughout a composition of the present disclosure. As used herein, the term “substantially free of inorganic residuals” means that the composition exhibits residuals of 0.1% (w/w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.05% (w/w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.01% (w/w) or less. In an embodiment, the amount of inorganic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of inorganic residuals is ND to about 500 ppm. In an embodiment, the amount of inorganic residuals is ND to about 400 ppm. In an embodiment, the amount of inorganic residuals is ND to about 300 ppm. In an embodiment, the amount of inorganic residuals is ND to about 200 ppm. In an embodiment, the amount of inorganic residuals is ND to about 100 ppm. In an embodiment, the amount of inorganic residuals is between 10 ppm and 1000 ppm. As used herein, the term “substantially free of organic residuals” means that the composition exhibits residuals of 0.1% (w/w) or less. In an embodiment, substantially free of organic residuals refers to a composition that exhibits residuals of 0.05% (w/w) or less. In an embodiment, substantially free of organic residuals refers to a composition that exhibits residuals of 0.01% (w/w) or less. In an embodiment, the amount of organic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of organic residuals is ND to about 500 ppm. In an embodiment, the amount of organic residuals is ND to about 400 ppm. In an embodiment, the amount of organic residuals is ND to about 300 ppm. In an embodiment, the amount of organic residuals is ND to about 200 ppm. In an embodiment, the amount of organic residuals is ND to about 100 ppm. In an embodiment, the amount of organic residuals is between 10 ppm and 1000 ppm. Compositions of the present disclosure exhibit “biocompatibility” meaning that the compositions are compatible with living tissue or a living system by not being toxic, injurious, or physiologically reactive and not causing immunological rejection. Such biocompatibility can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. Compositions of the present disclosure are “hypoallergenic” meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. In an embodiment, a solution of the present disclosure is contacted with a therapeutic agent and/or a molecule prior to forming the article. In an embodiment, molecules include, but are not limited to, antioxidants and enzymes. In an embodiment, molecules include, but are not limited to, Selenium, Ubiquinone derivatives, Thiol-based antioxidants, Saccharide-containing antioxidants, Polyphenols, Botanical extracts, Caffeic acid, Apigenin, Pycnogenol, Resveratrol, Folic acid, Vitamin b12, Vitamin b6, Vitamin b3, Vitamin E, Vitamin C and derivatives thereof, Vitamin D, Vitamin A, Astaxathin, Lutein, Lycopene, Essential fatty acids (omegas 3 and 6), Iron, Zinc, magnesium, Flavonoids (soy, Curcumin, Silymarin, Pycnongeol), Growth factors, aloe, hyaluronic acid, extracellular matrix proteins, cells, nucleic acids, biomarkers, biological reagents, zinc oxide, benzyol peroxide, retnoids, titanium, allergens in a known dose (for sensitization treatment), essential oils including, but not limited to, lemongrass or rosemary oil, and fragrances. Therapeutic agents include, but are not limited to, small molecules, drugs, proteins, peptides and nucleic acids. In an embodiment, a silk film of the present disclosure includes a molecule that is a vitamin, such as vitamin C, vitamin A and vitamin E. In an embodiment, a solution of the present disclosure is contacted with an allergen of known quantity prior to forming the article. Allergens include but are not limited to milk, eggs, peanuts, tree nuts, fish, shellfish, soy and wheat. Known doses of allergen loaded within a silk article can be released at a known rate for controlled exposure allergy study, tests and sensitization treatment. In an embodiment, a solution of the present disclosure is used to create an article with microneedles by standard methods known to one in the art for controlled delivery of molecules or therapeutic agents to or through the skin. As used herein, the term “fibroin” includes silkworm fibroin and insect or spider silk protein. In an embodiment, fibroin is obtained fromBombyx mori. FIG.1is a flow chart showing various embodiments for producing pure silk fibroin-based protein fragments (SPFs) of the present disclosure. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure. As illustrated inFIG.1, step A, cocoons (heat-treated or non-heat-treated), silk fibers, silk powder or spider silk can be used as the silk source. If starting from raw silk cocoons fromBombyx mori, the cocoons can be cut into small pieces, for example pieces of approximately equal size, step B1. The raw silk is then extracted and rinsed to remove any sericin, step C1a. This results in substantially sericin free raw silk. In an embodiment, water is heated to a temperature between 84° C. and 100° C. (ideally boiling) and then Na2CO3(sodium carbonate) is added to the boiling water until the Na2CO3is completely dissolved. The raw silk is added to the boiling water/Na2CO3(100° C.) and submerged for approximately 15-90 minutes, where boiling for a longer time results in smaller silk protein fragments. In an embodiment, the water volume equals about 0.4× raw silk weight and the Na2CO3volume equals about 0.848× raw silk weight. In an embodiment, the water volume equals 0.1× raw silk weight and the Na2CO3volume is maintained at 2.12 g/L. This is demonstrated inFIG.62AandFIG.62B: silk mass (x-axis) was varied in the same volume of extraction solution (i.e., the same volume of water and concentration of Na2CO3) achieving sericin removal (substantially sericin free) as demonstrated by an overall silk mass loss of 26 to 31 percent (y-axis). Subsequently, the water dissolved Na2CO3solution is drained and excess water/Na2CO3is removed from the silk fibroin fibers (e.g., ring out the fibroin extract by hand, spin cycle using a machine, etc.). The resulting silk fibroin extract is rinsed with warm to hot water to remove any remaining adsorbed sericin or contaminate, typically at a temperature range of about 40° C. to about 80° C., changing the volume of water at least once (repeated for as many times as required). The resulting silk fibroin extract is a substantially sericin-depleted silk fibroin. In an embodiment, the resulting silk fibroin extract is rinsed with water at a temperature of about 60° C. In an embodiment, the volume of rinse water for each cycle equals 0.1 L to 0.2 L× raw silk weight. It may be advantageous to agitate, turn or circulate the rinse water to maximize the rinse effect. After rinsing, excess water is removed from the extracted silk fibroin fibers (e.g., ring out fibroin extract by hand or using a machine). Alternatively, methods known to one skilled in the art such as pressure, temperature, or other reagents or combinations thereof may be used for the purpose of sericin extraction. Alternatively, the silk gland (100% sericin free silk protein) can be removed directly from a worm. This would result in liquid silk protein, without any alteration of the protein structure, free of sericin. The extracted fibroin fibers are then allowed to dry completely.FIG.3is a photograph showing dry extracted silk fibroin. Once dry, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at a temperature between ambient and boiling, step C1b. In an embodiment, the solvent is a solution of Lithium bromide (LiBr) (boiling for LiBr is 140° C.). Alternatively, the extracted fibroin fibers are not dried but wet and placed in the solvent; solvent concentration can then be varied to achieve similar concentrations as to when adding dried silk to the solvent. The final concentration of LiBr solvent can range from 0.1 M to 9.3 M.FIG.63is a table summarizing the Molecular Weights of silk dissolved from different concentrations of Lithium Bromide (LiBr) and from different extraction and dissolution sizes. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature along with the concentration of dissolving solvent. Other solvents may be used including, but not limited to, phosphate phosphoric acid, calcium nitrate, calcium chloride solution or other concentrated aqueous solutions of inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed within the already heated solvent solution and then maintained at a temperature ranging from about 60° C. to about 140° C. for 1-168 hrs. In an embodiment, the silk fibers should be fully immersed within the solvent solution and then placed into a dry oven at a temperature of about 100° C. for about 1 hour. The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an effect on the time required to completely dissolve the fibroin and on the resulting molecular weight and polydispersity of the final SPF mixture solution. In an embodiment, silk solvent solution concentration is less than or equal to 20% w/v. In addition, agitation during introduction or dissolution may be used to facilitate dissolution at varying temperatures and concentrations. The temperature of the LiBr solution will provide control over the silk protein fragment mixture molecular weight and polydispersity created. In an embodiment, a higher temperature will more quickly dissolve the silk offering enhanced process scalability and mass production of silk solution. In an embodiment, using a LiBr solution heated to a temperature between 80° C.-140° C. reduces the time required in an oven in order to achieve full dissolution. Varying time and temperature at or above 60° C. of the dissolution solvent will alter and control the MW and polydispersity of the SPF mixture solutions formed from the original molecular weight of the native silk fibroin protein. Alternatively, whole cocoons may be placed directly into a solvent, such as LiBr, bypassing extraction, step B2. This requires subsequent filtration of silk worm particles from the silk and solvent solution and sericin removal using methods know in the art for separating hydrophobic and hydrophilic proteins such as a column separation and/or chromatography, ion exchange, chemical precipitation with salt and/or pH, and or enzymatic digestion and filtration or extraction, all methods are common examples and without limitation for standard protein separation methods, step C2. Non-heat treated cocoons with the silkworm removed, may alternatively be placed into a solvent such as LiBr, bypassing extraction. The methods described above may be used for sericin separation, with the advantage that non-heat treated cocoons will contain significantly less worm debris. Dialysis may be used to remove the dissolution solvent from the resulting dissolved fibroin protein fragment solution by dialyzing the solution against a volume of water, step E1. Pre-filtration prior to dialysis is helpful to remove any debris (i.e., silk worm remnants) from the silk and LiBr solution, step D. In one example, a 3 μm or 5 μm filter is used with a flow-rate of 200-300 mL/min to filter a 0.1% to 1.0% silk-LiBr solution prior to dialysis and potential concentration if desired. A method disclosed herein, as described above, is to use time and/or temperature to decrease the concentration from 9.3 M LiBr to a range from 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering creating a scalable process method. Alternatively, without the use of additional time or temperate, a 9.3 M LiBr-silk protein fragment solution may be diluted with water to facilitate debris filtration and dialysis. The result of dissolution at the desired time and temperate filtration is a translucent particle free room temperature shelf-stable silk protein fragment-LiBr solution of a known MW and polydispersity. It is advantageous to change the dialysis water regularly until the solvent has been removed (e.g., change water after 1 hour, 4 hours, and then every 12 hours for a total of 6 water changes). The total number of water volume changes may be varied based on the resulting concentration of solvent used for silk protein dissolution and fragmentation. After dialysis, the final silk solution maybe further filtered to remove any remaining debris (i.e., silk worm remnants). Alternatively, Tangential Flow Filtration (TFF), which is a rapid and efficient method for the separation and purification of biomolecules, may be used to remove the solvent from the resulting dissolved fibroin solution, step E2. TFF offers a highly pure aqueous silk protein fragment solution and enables scalability of the process in order to produce large volumes of the solution in a controlled and repeatable manner. The silk and LiBr solution may be diluted prior to TFF (20% down to 0.1% silk in either water or LiBr). Pre-filtration as described above prior to TFF processing may maintain filter efficiency and potentially avoids the creation of silk gel boundary layers on the filter's surface as the result of the presence of debris particles. Pre filtration prior to TFF is also helpful to remove any remaining debris (i.e., silk worm remnants) from the silk and LiBr solution that may cause spontaneous or long-term gelation of the resulting water only solution, step D. TFF, recirculating or single pass, may be used for the creation of water-silk protein fragment solutions ranging from 0.1% silk to 30.0% silk (more preferably, 0.1%-6.0% silk). Different cutoff size TFF membranes may be required based upon the desired concentration, molecular weight and polydispersity of the silk protein fragment mixture in solution. Membranes ranging from 1-100 kDa may be necessary for varying molecular weight silk solutions created for example by varying the length of extraction boil time or the time and temperate in dissolution solvent (e.g., LiBr). In an embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution and to create the final desired silk-to-water ratio. As well, TFF single pass, TFF, and other methods known in the art, such as a falling film evaporator, may be used to concentrate the solution following removal of the dissolution solvent (e.g., LiBr) (with resulting desired concentration ranging from 0.1% to 30% silk). This can be used as an alternative to standard HFIP concentration methods known in the art to create a water-based solution. A larger pore membrane could also be utilized to filter out small silk protein fragments and to create a solution of higher molecular weight silk with and/or without tighter polydispersity values.FIG.61is a table summarizing Molecular Weights for some embodiments of silk protein solutions of the present disclosure. Silk protein solution processing conditions were as follows: 100° C. extraction for 20 min, room temperature rinse, LiBr in 60° C. oven for 4-6 hours. TFF processing conditions for water-soluble films were as follows: 100° C. extraction for 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min.FIGS.67-78further demonstrate manipulation of extraction time, LiBr dissolution conditions, and TFF processing and resultant example molecular weights and polydispersities. These examples are not intended to be limiting, but rather to demonstrate the potential of specifying parameters for specific molecular weight silk fragment solutions. An assay for LiBr and Na2CO3detection was performed using an HPLC system equipped with evaporative light scattering detector (ELSD). The calculation was performed by linear regression of the resulting peak areas for the analyte plotted against concentration. More than one sample of a number of formulations of the present disclosure was used for sample preparation and analysis. Generally, four samples of different formulations were weighed directly in a 10 mL volumetric flask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and kept at 2-8° C. for 2 hours with occasional shaking to extract analytes from the film. After 2 hours the solution was diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask was transferred into HPLC vials and injected into the HPLC-ELSD system for the estimation of sodium carbonate and lithium bromide. The analytical method developed for the quantitation of Na2CO3and LiBr in silk protein formulations was found to be linear in the range 10-165 ng/mL, with RSD for injection precision as 2% and 1% for area and 0.38% and 0.19% for retention time for sodium carbonate and lithium bromide respectively. The analytical method can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein formulations. The final silk protein fragment solution, as shown inFIG.4, is pure silk protein fragments and water with PPM to undetectable levels of particulate debris and/or process contaminants, including LiBr and Na2CO3.FIG.55andFIG.58are tables summarizing LiBr and Na2CO3concentrations in solutions of the present disclosure. InFIG.55, the processing conditions included 100° C. extraction for 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min. TFF conditions including pressure differential and number of dia-filtration volumes were varied. InFIG.58, the processing conditions included 100° C. boil for 60 min, 60° C. rinse, LiBr in 60° C. oven for 4-6 hours. In an embodiment, a SPF composition of the present disclosure is not soluble in an aqueous solution due to the crystallinity of the protein. In an embodiment, a SPF composition of the present disclosure is soluble in an aqueous solution. In an embodiment, the SPFs of a composition of the present disclosure include a crystalline portion of about two-thirds and an amorphous region of about one-third. In an embodiment, the SPFs of a composition of the present disclosure include a crystalline portion of about one-half and an amorphous region of about one-half. In an embodiment, the SPFs of a composition of the present disclosure include a 99% crystalline portion and a 1% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 95% crystalline portion and a 5% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 90% crystalline portion and a 10% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 85% crystalline portion and a 15% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 80% crystalline portion and a 20% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 75% crystalline portion and a 25% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 70% crystalline portion and a 30% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 65% crystalline portion and a 35% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 60% crystalline portion and a 40% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 50% crystalline portion and a 50% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 40% crystalline portion and a 60% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 35% crystalline portion and a 65% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 30% crystalline portion and a 70% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 25% crystalline portion and a 75% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 20% crystalline portion and a 80% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 15% crystalline portion and a 85% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 10% crystalline portion and a 90% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 5% crystalline portion and a 90% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 1% crystalline portion and a 99% amorphous region. A unique feature of the SPF compositions of the present disclosure are shelf stability (they will not slowly or spontaneously gel when stored in an aqueous solution and there is no aggregation of fragments and therefore no increase in molecular weight over time), from 10 days to 3 years depending on storage conditions, percent silk, and number of shipments and shipment conditions. Additionally pH may be altered to extend shelf-life and/or support shipping conditions by preventing premature folding and aggregation of the silk. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 2 weeks at room temperature (RT). In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 4 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 6 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 8 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 10 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 12 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability ranging from about 4 weeks to about 52 weeks at RT. Table 1 below shows shelf stability test results for embodiments of SPF compositions of the present disclosure. TABLE 1Shelf Stability of SPF Compositions of thePresent Disclosure% SilkTemperatureTime to Gelation2RT4 weeks24C>9 weeks4RT4 weeks44C>9 weeks6RT2 weeks64C>9 weeks A known additive such as a vitamin (e.g., vitamin C) can be added to a SPF composition of the present disclosure to create a gel that is stable from 10 days to 3 years at room temperature (RT). Both examples, a SPF composition and the same with an additive, can be lyophilized for enhanced storage control ranging from 10 days to 10 years depending on storage and shipment conditions. The lyophilized silk powder can also be used as a raw ingredient in the medical, consumer, and electronic markets. Additionally, lyophilized silk powder can be resuspended in water, HFIP, or organic solution following storage to create silk solutions of varying concentrations, including higher concentration solutions than those produced initially. In another embodiment, the silk fibroin-based protein fragments are dried using a rototherm evaporator or other methods known in the art for creating a dry protein form containing less than 10% water by mass. Either the silk fragment-water solutions or the lyophilized silk protein fragment mixture can be sterilized following standard methods in the art not limited to filtration, heat, radiation or e-beam. It is anticipated that the silk protein fragment mixture, because of its shorter protein polymer length, will withstand sterilization better than intact silk protein solutions described in the art. Additionally, silk articles created from the SPF mixtures described herein may be sterilized as appropriate to application. For example, a silk film loaded with a molecule to be used in medical applications with an open wound/incision, may be sterilized standard methods such as by radiation or e-beam. FIG.2is a flow chart showing various parameters that can be modified during the process of producing a silk protein fragment solution of the present disclosure during the extraction and the dissolution steps. Select method parameters may be altered to achieve distinct final solution characteristics depending upon the intended use, e.g., molecular weight and polydispersity. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure. In an embodiment, a process for producing a silk protein fragment solution of the present disclosure includes forming pieces of silk cocoons from theBombyx morisilk worm; extracting the pieces at about 100° C. in a solution of water and Na2CO3for about 60 minutes, wherein a volume of the water equals about 0.4× raw silk weight and the amount of Na2CO3is about 0.848× the weight of the pieces to form a silk fibroin extract; triple rinsing the silk fibroin extract at about 60° C. for about 20 minutes per rinse in a volume of rinse water, wherein the rinse water for each cycle equals about 0.2 L× the weight of the pieces; removing excess water from the silk fibroin extract; drying the silk fibroin extract; dissolving the dry silk fibroin extract in a LiBr solution, wherein the LiBr solution is first heated to about 100° C. to create a silk and LiBr solution and maintained; placing the silk and LiBr solution in a dry oven at about 100° C. for about 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into mixture with desired molecular weight and polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to result in a 1% silk solution; and removing solvent from the solution using Tangential Flow Filtration (TFF). In an embodiment, a 10 kDa membrane is utilized to purify the silk solution and create the final desired silk-to-water ratio. TFF can then be used to further concentrate the pure silk solution to a concentration of 2% silk to water. Each process step from raw cocoons to dialysis is scalable to increase efficiency in manufacturing. Whole cocoons are currently purchased as the raw material, but pre-cleaned cocoons or non-heat treated cocoons, where worm removal leaves minimal debris, have also been used. Cutting and cleaning the cocoons is a manual process, however for scalability this process could be made less labor intensive by, for example, using an automated machine in combination with compressed air to remove the worm and any particulates, or using a cutting mill to cut the cocoons into smaller pieces. The extraction step, currently performed in small batches, could be completed in a larger vessel, for example an industrial washing machine where temperatures at or in between 60° C. to 100° C. can be maintained. The rinsing step could also be completed in the industrial washing machine, eliminating the manual rinse cycles. Dissolution of the silk in LiBr solution could occur in a vessel other than a convection oven, for example a stirred tank reactor. Dialyzing the silk through a series of water changes is a manual and time intensive process, which could be accelerated by changing certain parameters, for example diluting the silk solution prior to dialysis. The dialysis process could be scaled for manufacturing by using semi-automated equipment, for example a tangential flow filtration system. Varying extraction (i.e., time and temperature), LiBr (i.e., temperature of LiBr solution when added to silk fibroin extract or vice versa) and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, homogeneities, and colors (seeFIGS.5-32). Increasing the temperature for extraction, lengthening the extraction time, using a higher temperature LiBr solution at emersion and over time when dissolving the silk and increasing the time at temperature (e.g., in an oven as shown here, or an alternative heat source) all resulted in less viscous and more homogeneous solvent and silk solutions. While almost all parameters resulted in a viable silk solution, methods that allow complete dissolution to be achieved in fewer than 4 to 6 hours are preferred for process scalability. FIGS.5-10show photographs of four different silk extraction combinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr was prepared and allowed to sit at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in the 60° C. oven. Samples from each set were removed at 4, 6, 8, 12, 24, 168 and 192 hours. The remaining sample was photographed. FIGS.11-23show photographs of four different silk extraction combinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60° C., 80° C., 100° C. or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the 60° C. oven. Samples from each set were removed at 1, 4 and 6 hours. The remaining sample was photographed. FIGS.24-32show photographs of four different silk extraction combinations tested: Four different silk extraction combinations were used: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60° C., 80° C., 100° C. or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the oven at the same temperature of the LiBr. Samples from each set were removed at 1, 4 and 6 hours. 1 mL of each sample was added to 7.5 mL of 9.3 M LiBr and refrigerated for viscosity testing. The remaining sample was photographed. Molecular weight of the silk protein fragments may be controlled based upon the specific parameters utilized during the extraction step, including extraction time and temperature; specific parameters utilized during the dissolution step, including the LiBr temperature at the time of submersion of the silk in to the lithium bromide and time that the solution is maintained at specific temperatures; and specific parameters utilized during the filtration step. By controlling process parameters using the disclosed methods, it is possible to create SPF mixture solutions with polydispersity equal to or lower than 2.5 at a variety of different molecular weight ranging from 5 kDa to 200 kDa, more preferably between 10 kDa and 80 kDA. By altering process parameters to achieve silk solutions with different molecular weights, a range of fragment mixture end products, with desired polydispersity of equal to or less than 2.5 may be targeted based upon the desired performance requirements. For example, a lower molecular weight silk film containing a drug may have a faster release rate compared to a higher molecular weight film making it more ideal for a daily delivery vehicle in consumer cosmetics. Additionally, SPF mixture solutions with a polydispersity of greater than 2.5 can be achieved. Further, two solutions with different average molecular weights and polydispersities can be mixed to create combination solutions. Alternatively, a liquid silk gland (100% sericin free silk protein) that has been removed directly from a worm could be used in combination with any of the SPF mixture solutions of the present disclosure. Molecular weight of the pure silk fibroin-based protein fragment composition was determined using High Pressure Liquid Chromatography (HPLC) with a Refractive Index Detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC/SEC Software Version 3.3 (Agilent). Parameters were varied during the processing of raw silk cocoons into silk solution. Varying these parameters affected the MW of the resulting silk solution. Parameters manipulated included (i) time and temperature of extraction, (ii) temperature of LiBr, (iii) temperature of dissolution oven, and (iv) dissolution time. Molecular weight was determined with mass spec as shown inFIGS.64-80. Experiments were carried out to determine the effect of varying the extraction time.FIGS.64-70are graphs showing these results, and Tables 2-8 summarize the results. Below is a summary:A sericin extraction time of 30 minutes resulted in larger MW than a sericin extraction time of 60 minutesMW decreases with time in the oven140° C. LiBr and oven resulted in the low end of the confidence interval to be below a MW of 9500 Da30 min extraction at the 1 hour and 4 hour time points have undigested silk30 min extraction at the 1 hour time point resulted in a significantly high molecular weight with the low end of the confidence interval being 35,000 DaThe range of MW reached for the high end of the confidence interval was 18000 to 216000 Da (important for offering solutions with specified upper limit) TABLE 2The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 100° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageStdConfidenceTimeTimeMwdevIntervalPD301572471278035093933871.6360131520138711633854072.71304409732632142681176582.8760425082124810520598032.3830625604140510252639432.5060620980126210073436952.08 TABLE 3The effect of extraction time (30 min vs 60 min) on molecular weight ofsilk processed under the conditions of 100° C. Extraction Temperature,boiling Lithium Bromide (LiBr) and 60° C. Oven Dissolution or 4 hr.BoilAverageStdConfidenceSampleTimeMwdevIntervalPD30 min, 4 hr30496564580173061424782.8760 min, 4 hr6030042153611183807052.69 TABLE 4The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 60° C. Lithium Bromide (LiBr) and 60° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageStdConfidenceSampleTimeTimeMwdevIntervalPD30 min, 1 hr30158436222011538092.6360 min, 1 hr6013170011931842242.6630 min, 4 hr30461956.513337214631788472.8960 min, 4 hr60425578.524469979655642.56 TABLE 5The effect of extraction time (30 min vs 60 min) on molecular weight ofsilk processed under the conditions of 100° C. Extraction Temperature,80° C. Lithium Bromide (LiBr) and 80° C. Oven Dissolution for 6 hr.BoilAverageStdConfidenceSampleTimeMwdevIntervalPD30 min, 6 hr3063510186932157753.4060 min, 6 hr60251642389637657062.61 TABLE 6The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 80° C. Lithium Bromide (LiBr) and 60° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageStdConfidenceSampleTimeTimeMwdevIntervalPD30 min, 4 hr3045920214028190731837603.1060 min, 4 hr60426312.563710266674422.5630 min, 6 hr30646824180761212932.5960 min, 6 hr6062635310168683022.59 TABLE 7The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 60° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD30 min, 4 hr30447853197581159002.4260 min, 4 hr60425082124810520598042.3830 min, 6 hr306554218992191531603662.8960 min, 6 hr60620980126210073436942.08 TABLE 8The effect of extraction time (3 0 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 140° C. Lithium Bromide (LiBr) and 140° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD30 min, 4 hr3049024.511024493181272.0086560 min, 4 hr604155486954347622.235830 min, 6 hr306130215987283192.174960 min, 6 hr606108885364221002.0298 Experiments were carried out to determine the effect of varying the extraction temperature.FIG.71is a graph showing these results, and Table 9 summarizes the results. Below is a summary:Sericin extraction at 90° C. resulted in higher MW than sericin extraction at 100° C. extractionBoth 90° C. and 100° C. show decreasing MW over time in the oven TABLE 9The effect of extraction temperature (90° C. vs. 100° C.) onmolecular weight of silk processed under the conditions of 60 min.Extraction Temperature, 100° C. Lithium Bromide (LiBr) and 100° C.Oven Dissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD90° C., 4 hr604373084204133681041192.79100° C., 4 hr60425082124810520598042.3890° C., 6 hr60634224113512717921002.69100° C., 6 hr60620980126210073436942.08 Experiments were carried out to determine the effect of varying the Lithium Bromide (LiBr) temperature when added to silk.FIGS.72-73are graphs showing these results, and Tables 10-11 summarize the results. Below is a summary:No impact on MW or confidence interval (all CI˜10500-6500 Da)Studies illustrated that the temperature of LiBr-silk dissolution, as LiBr is added and begins dissolving, rapidly drops below the original LiBr temperature due to the majority of the mass being silk at room temp TABLE 10The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 60 min. ExtractionTime., 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).LiBrTempOvenAverageConfidenceSample(° C.)TimeMwStd devIntervalPD60° C. LiBr,6013170011931842232.661 hr100° C. LiBr,10012790720010735725522.601 hrRT LiBr, 4 hrRT429217108210789791192.7160° C. LiBr,6042557824459978655642.564 hr80° C. LiBr,8042631263710265674412.564 hr100° C. LiBr,100427681172911279679312.454 hrBoil LiBr,Boil430042153511183807042.694 hrRT LiBr, 6 hrRT626543189310783653322.4680° C. LiBr,8062635310167683012.596 hr100° C. LiBr,10062715091611020668892.466 hr TABLE 11The effect of Lithium Bromide LiBr) temperature on molecularweight of silk processed under the conditions of 30 min. ExtractionTime, 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).LiBrTempOvenAverageConfidenceSample(° C.)TimeMwStd devIntervalPD60° C. LiBr,6046195613336214631788472.894 hr80° C. LiBr,8045920214027190731837603.104 hr100° C. LiBr,100447853197571158992.424 hr80° C. LiBr,80646824180751212922.596 hr100° C. LiBr,1006554218991191521603662.896 hr Experiments were carried out to determine the effect of v oven/dissolution temperature.FIGS.74-78are graphs showing these results, and Tables 12-16 summarize the results. Below is a summary:Oven temperature has less of an effect on 60 min extracted silk than 30 min extracted silk. Without wishing to be bound by theory, it is believed that the 30 min silk is less degraded during extraction and therefore the oven temperature has more of an effect on the larger MW, less degraded portion of the silk.For 60° C. vs. 140° C. oven the 30 min extracted silk showed a very significant effect of lower MW at higher oven temp, while 60 min extracted silk had an effect but much lessThe 140° C. oven resulted in a low end in the confidence interval at ˜6000 Da TABLE 12The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 100° C. LithiumBromide (LiBr) (Oven/Dissolution Time was varied).BoilOven TempOvenAverageConfidenceTime(° C.)TimeMwStd devIntervalPD3060447853197581159002.42301004409732632142681176582.8730606554218992191531603662.8930100625604140510252639432.50 TABLE 13The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 100° C. LithiumBromide (LiBr) (Oven/Dissolution Time was varied).BoilOven TempOvenAverageConfidenceTime(° C.)TimeMwStd devIntervalPD606012790820010735725522.6060100131520138711633854072.716060427681173011279725522.6260100425082124810520598032.38606062715091611020668892.4660100620980126210073436952.08 TABLE 14The effect of oven/dissolution temperature on molecularweight of silk processed under the conditions of 100° C.Extraction Temperature, 60 min. Extraction Time, and 140° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied).BoilOven TempOvenAverageConfidenceTime(° C.)TimeMwStd devIntervalPD6060430042153611183807052.69601404155487255333222.14 TABLE 15The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 140° C. LithiumBromide (LiBr) (Oven/Dissolution Time was varied).OvenBoilTempOvenAverageConfidenceTime(° C.)TimeMwStd devIntervalPD30604496564580173061424782.87301404902511024493181272.01306065938311640176411998893.37301406130215987283192.17 TABLE 16The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 80° C. LithiumBromide (LiBr) (Oven/Dissolution Time was varied).BoilOven TempOvenAverageConfidenceTime(° C.)TimeMwStd devIntervalPD606042631363710266674422.566080430308429312279748062.47606062635310168683022.5960806251642389637657062.61 In an embodiment, the methods disclosed herein result in a solution with characteristics that can be controlled during manufacturing, including, but not limited to: MW—may be varied by changing extraction and/or dissolution time and temp (e.g., LiBr temperature), pressure, and filtration (e.g., size exclusion chromatography); Structure—removal or cleavage of heavy or light chain of the fibroin protein polymer; Purity—hot water rinse temperature for improved sericin removal or filter capability for improved particulate removal that adversely affects shelf stability of the silk fragment protein mixture solution; Color—the color of the solution can be controlled with, for example, LiBr temp and time; Viscosity; Clarity; and Stability of solution. The resultant pH of the solution is typically about 7 and can be altered using an acid or base as appropriate to storage requirements. The above-described SPF mixture solutions may be utilized to produce a pure silk protein fragment-film or pure silk protein fragment-gel for numerous applications (e.g., delivery of a drug, vitamin, antioxidant, etc. to the skin).FIG.33is a flow chart showing an embodiment for producing a silk film of the present disclosure from a silk solution of the present disclosure. In step A, a silk solution of the present disclosure is chosen, and then at least on molecule or therapeutic agent is added directly to the silk solution prior to gel or film processing, step B. When producing a silk film, the silk solution with additive(s) may be cast directly onto a shaped mold to achieve a unique film shape (e.g., silicone mold) or the silk solution may be cast as a sheet and then subsequently cut or punched into a variety of shapes, with a variety of cutting techniques, including, but not limited to cutting with a rotary blade or laser cutting for example (FIGS.83A and83B), depending upon the desired application, step C. If cast on a mold, for example silicone, the silicone mold may be heated on a laser-etched/patterned surface to create an impression that will be transferred to the final film. For example, the product logo could be transferred to the film, visible, but not palpable by hand, and used to show authenticity of the product. The concentration and/or mass of the final silk protein fragment film can be varied to control the film's degree of flexibility and conformity to different anatomical topographies. Altering the drying method for a silk film will also result in different final film characteristics. Applying airflow and/or heat impacts the properties of the film (e.g., brittleness, number of bubbles, curling, solubility, surface appearance), step D. Additionally, the percent moisture within the film at the time of packaging will impact stability over time with too much moisture resulting in yellowing of the films with time (FIGS.82A-82C). In some embodiments, films ideally may have between about 2 to about 20% water content at completion of drying. It was observed that greater moisture content than 20% in the films will decrease shelf life. If films are not dry enough (that is they have greater than 20% water content) before packaging, they will yellow over time (2+ weeks). It is advised that films are dried in an incubator until the relative humidity in the incubator is less than the relative humidity in the surrounding area and no greater than 36%. Ambient humidity will have an effect on the ability to remove moisture and therefore, a tactile/audio test can be used to determine whether films are ready for packaging. In an embodiment, the test includes removal of a film from the drying system, slightly bending one end of the film and releasing it. If the film feels and sounds similar to a piece of paper or thin plastic, it is considered dry. If the film has not completed drying, it will be pliable and will make no noise upon bending and release. In an embodiment, the film is flexible without the need for process additives such as glycerin, such that a film that is 2.5 cm wide by 10 cm long can be bent in half so that opposite ends of the film can touch one another without the film breaking or cracking. A film of this same size can be bent in half along the length of the film to create a 45-degree angle without breaking or cracking the film. The final silk protein fragment-film is pure with undetectable levels of particulate debris and/or process contaminants, including LiBr and Na2CO3. Alternatively, the final SPF mixture solution has less than 500 ppm process contaminants.FIG.56andFIG.57are tables summarizing LiBr and Na2CO3concentrations in films (2% silk films air dried at RT) of the present disclosure. InFIG.56, the processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours. InFIG.57, the processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours. In an embodiment, when producing a silk gel, an acid is used to help facilitate gelation. In an embodiment, when producing a silk gel that includes a neutral or a basic molecule and/or therapeutic agent, an acid can be added to facilitate gelation. In an embodiment, when producing a silk gel, increasing the pH (making the gel more basic) increases the shelf stability of the gel. In an embodiment, when producing a silk gel, increasing the pH (making the gel more basic) allows for a greater quantity of an acidic molecule to be loaded into the gel. In an embodiment, natural additives may be added to the silk gel to further stabilize additives. For example, trace elements such as selenium or magnesium or L-methoinine can be used. Further, light-block containers can be added to further increase stability. FIG.34summarizes an embodiment of parameters for a silk fragment-film drying study of the present disclosure.FIG.35is a graph showing silk fragment-film drying times (under various air flow and temperature conditions) based on the silk fragment-film drying study ofFIG.34. These studies indicate that airflow is an important parameter to consider for drying (i.e., samples in covered containers did not dry), temperature can be altered to alter drying rate (i.e., increased temperature results in a faster rate of water removal) and that a steady-state of moisture content within the films can be obtained with a variety of parameters (i.e., from 24 to 48 hours, mass is consistent in uncovered samples regardless of temperature). Of note, the final properties of the film, for example brittleness, will vary with drying conditions. Alternatively, film drying rate may be accelerated by the use of an additive in the SPF solution, such as a surfactant or oil. These additives may be used with or without heat to alter drying rate and final film physical properties. In an embodiment, the drying conditions of the SFP film are 24° C. in a forced air flow incubator for 12 to 48 hours depending on the number of films and ambient humidity. Under these drying conditions, a film that will not shrink more than 5 percent over time when stored in a foil pouch is created. Additionally, the film is homogeneous in composition and physical structure, with no sided-ness and an even distribution of additive, for example vitamin C, throughout. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 30% to about 100% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 35% to about 95% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 40% to about 90% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 45% to about 85% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 50% to about 80% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 55% to about 75% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 60% to about 70% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in a sealed airtight container or pouch that prevents light from contacting the film retaining about 80% to about 100% of its activity after 3 to 24 months of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in a sealed airtight container or pouch that prevents light from contacting the film retaining about 80% to about 100% of its activity after about 3 to about 60 months of storage. In an embodiment, the silk protein fragment-film may release between 50% to 90% of active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 50% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 60% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 70% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 80% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 90% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release between 10% to 100% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 10% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 20% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 30% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 40% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 50% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 60% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 70% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 80% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 90% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. It is believed that exposure to higher temperatures for a longer period of time may break down the silk protein into more versatile silk protein fragment mixtures and/or disrupt any silk protein tertiary and/or secondary silk protein structure that could adversely affect shelf stability and/or performance of resulting structures (e.g., gels, films, foams, etc.) as well as reduces the number of heavy chains within the silk protein. FIGS.36A and36Bshow two HPLC chromatograms from samples comprising vitamin C. The chromatogram on the left shows peaks from (1) a chemically stabilized sample of vitamin C at ambient conditions and (2) a sample of vitamin C taken after 1 hour at ambient conditions without chemical stabilization to prevent oxidation, where degradation products are visible. The chromatogram on the right shows peaks from two different embodiments of silk films of the present disclosure that were aged for at least 30 days at room temperature. No degradation products were visible.FIG.59is a table summarizing the vitamin C concentration in silk protein fragment-films (2% silk films air dried at RT) of the present disclosure. InFIG.59processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours.FIG.60is arable summarizing the stability of vitamin C in chemically stabilized solutions.FIGS.89A-89Bare tables summarizing vitamin C stability in SPF gels without chemical stabilizers as compared to chemically stabilized vitamin C in competitive anti-aging skincare products. A gel cast at 20% total vitamin C additive concentration did not gel. Without wishing to be bound by theory, it appears there is a relationship between vitamin C concentration, silk concentration, and gelation. An increase in vitamin C at a given concentration of silk will result in a longer time to gelation or inhibit gelation. This may be due to the vitamin C molecule physically blocking interaction between silk protein fragments or cross-linking of silk protein. In an embodiment, the molecule or molecules are stable and can be released over an extended time period. In an embodiment, release rate is controlled by the specific weight average molecular weight of the silk fibroin-based protein fragments used. In another embodiment, release rate is controlled by creation of a multi-layer structure. For example, multiple films can be cast and dried upon each other. Additionally, each layer can be formed using the same or different molecular weight compositions. In an embodiment, the degree of crystallinity of the protein structure is altered through film drying conditions, thereby controlling the release rate. The molecule or molecules may be released topically on the skin, subcutaneously following implantation, or locally or systemically through oral administration or implantation. In an embodiment, the molecule or molecules is released between 1 minutes and 20 minutes. In an embodiment, the molecule or molecules is released between 20 minutes and 60 minutes. In an embodiment, the molecule or molecules is released between 1 hour and 4 hours. In an embodiment, the molecule or molecules is released between 4 hours and 8 hours. In an embodiment, the molecule or molecules is released between 8 hours and 24 hours. In an embodiment, the molecule or molecules is released between 1 day and 7 days. In an embodiment, the molecule or molecules is released between 1 week and 4 weeks. In an embodiment, the molecule or molecules is released between 1 month and 3 months. In an embodiment, the molecule or molecules is released between 3 months and 6 months. In an embodiment, the molecule or molecules is released between 20 minutes and 6 months. In an embodiment, the molecule or molecules are stable at extreme temperature and humidity conditions. Films of the present disclosure comprised of about 20 kDA average weight average molecular weight silk fibroin based protein fragments and containing about 20% vitamin C by mass, were stored individually within foil pouches and exposed to temperature extremes. Foil pouches containing films were exposed to:Ambient conditions (time 0 films)“Extreme Cold” (−29° C.±2° C. for 72 hours), followed by “Hot Humid” (38° C. 2° C. at 85% Humidity±5% for 72 hours), and subsequently “Extreme Heat, Moderate Humidity” (60° C.±2° C. at 30% Humidity±5% for 6 hours) The amount of active vitamin C was measured using HPLC. All films were observed to support maintenance of vitamin C activity with exposure to extremes, as summarized in Table 17. TABLE 17Amount of active vitamin C in films under varying conditionsAverage Coneof vit C inStd.NConditionssample (mg/g)Dev4Time 0, ambient conditions184.9015.15161) −29° C. ± 2° C. for 72 hours193.9710.252) 38° C. ± 2° C. at 85% Humidity ±5% for 72 hours3) 60° C. ± 2° C. at 30% Humidity ±5% for 6 hours FIGS.37-45are photographs showing silk protein fragment-films of the present disclosure dried under various temperature, time and drying conditions. FIGS.46-54are photographs showing the dissolution, in water, of the formed silk protein fragment-films of the present disclosure under various temperature, time and drying conditions. The water solubility of films of the present disclosure may be varied by altering drying conditions. For example, drying a film to 20% humidity in a forced air incubator and then increasing ambient humidity to 50% for a period of hours and subsequently drying the film back to 20% humidity will result in an insoluble film. Under ordinary conditions where the humidity is steadily decreased, a water-soluble silk film is created. It is anticipated that the increase in humidity allowed the protein structure to be further mobilized in the film and further crystallized, resulting in a non-soluble film. Alternative methods in the art to create non-soluble films include the introduction of methanol. The films of the present disclosure are clearly differentiated from those films due to their solubility in water. The SFP gel articles described herein range from a hydrogel which can be injected or spread topically to a film-gel article that appears as a film and contains a minimal but controlled water content, thereby preventing crystallinity and allowing water solubility. In some embodiments, a composition of the present disclosure can further include skin penetration enhancers, including, but not limited to, sulfoxides (such as dimethylsulfoxide), pyrrolidones (such as 2-pyrrolidone), alcohols (such as ethanol or decanol), azones (such as laurocapram and 1-dodecylazacycloheptan-2-one), surfactants (including alkyl carboxylates and their corresponding acids such as oleic acid, fluoroalkylcarboxylates and their corresponding acids, alkyl sulfates, alkyl ether sulfates, docusates such as dioctyl sodium sulfosuccinate, alkyl benzene sulfonates, alkyl ether phosphates, and alkyl awl ether phosphates), glycols (such as propylene glycol), terpenes (such as limonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol, carveol, carvone, fenchone, and verbenone), and dimethyl isosorbide. Following are non-limiting examples of suitable ranges for various parameters in and for preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges of such parameters. In an embodiment, the percent silk in the solution is less than 30%. In an embodiment, the percent silk in the solution is less than 25%. In an embodiment, the percent silk in the solution is less than 20%. In an embodiment, the percent silk in the solution is less than 19%. In an embodiment, the percent silk in the solution is less than 18%. In an embodiment, the percent silk in the solution is less than 17%. In an embodiment, the percent silk in the solution is less than 16%. In an embodiment, the percent silk in the solution is less than 15%. In an embodiment, the percent silk in the solution is less than 14%. In an embodiment, the percent silk in the solution is less than 13%. In an embodiment, the percent silk in the solution is less than 12%. In an embodiment, the percent silk in the solution is less than 11%. In an embodiment, the percent silk in the solution is less than 10%. In an embodiment, the percent silk in the solution is less than 9%. In an embodiment, the percent silk in the solution is less than 8%. In an embodiment, the percent silk in the solution is less than 7%. In an embodiment, the percent silk in the solution is less than 6%. In an embodiment, the percent silk in the solution is less than 5%. In an embodiment, the percent silk in the solution is less than 4%. In an embodiment, the percent silk in the solution is less than 3%. In an embodiment, the percent silk in the solution is less than 2%. In an embodiment, the percent silk in the solution is less than 1%. In an embodiment, the percent silk in the solution is less than 0.9%. In an embodiment, the percent silk in the solution is less than 0.8%. In an embodiment, the percent silk in the solution is less than 0.7%. In an embodiment, the percent silk in the solution is less than 0.6%. In an embodiment, the percent silk in the solution is less than 0.5%. In an embodiment, the percent silk in the solution is less than 0.4%. In an embodiment, the percent silk in the solution is less than 0.3%. In an embodiment, the percent silk in the solution is less than 0.2%. In an embodiment, the percent silk in the solution is less than 0.1%. In an embodiment, the percent silk in the solution is greater than 0.1%. In an embodiment, the percent silk in the solution is greater than 0.2%. In an embodiment, the percent silk in the solution is greater than 0.3%. In an embodiment, the percent silk in the solution is greater than 0.4%. In an embodiment, the percent silk in the solution is greater than 0.5%. In an embodiment, the percent silk in the solution is greater than 0.6%. In an embodiment, the percent silk in the solution is greater than 0.7%. In an embodiment, the percent silk in the solution is greater than 0.8%. In an embodiment, the percent silk in the solution is greater than 0.9%. In an embodiment, the percent silk in the solution is greater than 1%. In an embodiment, the percent silk in the solution is greater than 2%. In an embodiment, the percent silk in the solution is greater than 3%. In an embodiment, the percent silk in the solution is greater than 4%. In an embodiment, the percent silk in the solution is greater than 5%. In an embodiment, the percent silk in the solution is greater than 6%. In an embodiment, the percent silk in the solution is greater than 7%. In an embodiment, the percent silk in the solution is greater than 8%. In an embodiment, the percent silk in the solution is greater than 9%. In an embodiment, the percent silk in the solution is greater than 10%. In an embodiment, the percent silk in the solution is greater than 11%. In an embodiment, the percent silk in the solution is greater than 12%. In an embodiment, the percent silk in the solution is greater than 13%. In an embodiment, the percent silk in the solution is greater than 14%. In an embodiment, the percent silk in the solution is greater than 15%. In an embodiment, the percent silk in the solution is greater than 16%. In an embodiment, the percent silk in the solution is greater than 17%. In an embodiment, the percent silk in the solution is greater than 18%. In an embodiment, the percent silk in the solution is greater than 19%. In an embodiment, the percent silk in the solution is greater than 20%. In an embodiment, the percent silk in the solution is greater than 25%. In an embodiment, the percent silk in the solution is between 0.1% and 30%. In an embodiment, the percent silk in the solution is between 0.1% and 25%. In an embodiment, the percent silk in the solution is between 0.1% and 20%. In an embodiment, the percent silk in the solution is between 0.1% and 15%. In an embodiment, the percent silk in the solution is between 0.1% and 10%. In an embodiment, the percent silk in the solution is between 0.1% and 9%. In an embodiment, the percent silk in the solution is between 0.1% and 8%. In an embodiment, the percent silk in the solution is between 0.1% and 7%. In an embodiment, the percent silk in the solution is between 0.1% and 6.5%. In an embodiment, the percent silk in the solution is between 0.1% and 6%. In an embodiment, the percent silk in the solution is between 0.1% and 5.5%. In an embodiment, the percent silk in the solution is between 0.1% and 5%. In an embodiment, the percent silk in the solution is between 0.1% and 4.5%. In an embodiment, the percent silk in the solution is between 0.1% and 4%. In an embodiment, the percent silk in the solution is between 0.1% and 3.5%. In an embodiment, the percent silk in the solution is between 0.1% and 3%. In an embodiment, the percent silk in the solution is between 0.1% and 2.5%. In an embodiment, the percent silk in the solution is between 0.1% and 2.0%. In an embodiment, the percent silk in the solution is between 0.1% and 2.4%. In an embodiment, the percent silk in the solution is between 0.5% and 5%. In an embodiment, the percent silk in the solution is between 0.5% and 4.5%. In an embodiment, the percent silk in the solution is between 0.5% and 4%. In an embodiment, the percent silk in the solution is between 0.5% and 3.5%. In an embodiment, the percent silk in the solution is between 0.5% and 3%. In an embodiment, the percent silk in the solution is between 0.5% and 2.5%. In an embodiment, the percent silk in the solution is between 1 and 4%. In an embodiment, the percent silk in the solution is between 1 and 3.5%. In an embodiment, the percent silk in the solution is between 1 and 3%. In an embodiment, the percent silk in the solution is between 1 and 2.5%. In an embodiment, the percent silk in the solution is between 1 and 2.4%. In an embodiment, the percent silk in the solution is between 1 and 2%. In an embodiment, the percent silk in the solution is between 20% and 30%. In an embodiment, the percent silk in the solution is between 0.1% and 6%. In an embodiment, the percent silk in the solution is between 6% and 10%. In an embodiment, the percent silk in the solution is between 6% and 8%. In an embodiment, the percent silk in the solution is between 6% and 9%. In an embodiment, the percent silk in the solution is between 10% and 20%. In an embodiment, the percent silk in the solution is between 11% and 19%. In an embodiment, the percent silk in the solution is between 12% and 18%. In an embodiment, the percent silk in the solution is between 13% and 17%. In an embodiment, the percent silk in the solution is between 14% and 16%. In an embodiment, the percent silk in the solution is 2.4%. In an embodiment, the percent silk in the solution is 2.0%. In an embodiment, the percent sericin in the solution is non-detectable to 30%. In an embodiment, the percent sericin in the solution is non-detectable to 5%. In an embodiment, the percent sericin in the solution is 1%. In an embodiment, the percent sericin in the solution is 2%. In an embodiment, the percent sericin in the solution is 3%. In an embodiment, the percent sericin in the solution is 4%. In an embodiment, the percent sericin in the solution is 5%. In an embodiment, the percent sericin in the solution is 10%. In an embodiment, the percent sericin in the solution is 30%. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 1 year. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 4 to 5 years. In an embodiment, the stability of a composition of the present disclosure is 10 days to 6 months. In an embodiment, the stability of a composition of the present disclosure is 6 months to 12 months. In an embodiment, the stability of a composition of the present disclosure is 12 months to 18 months. In an embodiment, the stability of a composition of the present disclosure is 18 months to 24 months. In an embodiment, the stability of a composition of the present disclosure is 24 months to 30 months. In an embodiment, the stability of a composition of the present disclosure is 30 months to 36 months. In an embodiment, the stability of a composition of the present disclosure is 36 months to 48 months. In an embodiment, the stability of a composition of the present disclosure is 48 months to 60 months. In an embodiment, a composition of the present disclosure includes pure silk fibroin based protein fragments having an average weight average molecular weight ranging from 6 kDa to 16 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 17 kDa to 38 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 39 kDa to 80 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 1 to 5 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 5 to 10 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 10 to 15 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 15 to 20 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 20 to 25 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 25 to 30 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 30 to 35 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 35 to 40 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 40 to 45 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 45 to 50 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 50 to 55 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin based protein fragments having an average weight average molecular weight ranging from 55 to 60 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 60 to 65 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 65 to 70 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 70 to 75 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 75 to 80 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 80 to 85 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 85 to 90 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 90 to 95 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 95 to 100 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 100 to 105 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 105 to 110 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 110 to 115 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 115 to 120 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 120 to 125 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 125 to 130 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin based protein fragments having an average weight average molecular weight ranging from 130 to 135 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 135 to 140 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 140 to 145 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 145 to 150 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 150 to 155 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 155 to 160 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 160 to 165 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 165 to 170 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 170 to 175 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 175 to 180 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 180 to 185 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 185 to 190 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 190 to 195 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 195 to 200 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 200 to 205 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin based protein fragments having an average weight average molecular weight ranging from 205 to 210 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 210 to 215 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 215 to 220 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 220 to 225 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 225 to 230 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 230 to 235 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 235 to 240 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 240 to 245 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 245 to 250 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 250 to 255 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 255 to 260 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 260 to 265 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 265 to 270 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 270 to 275 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 275 to 280 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin based protein fragments having an average weight average molecular weight ranging from 280 to 285 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 285 to 290 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 290 to 295 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 295 to 300 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 300 to 305 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 305 to 310 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 310 to 315 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 315 to 320 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 320 to 325 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 325 to 330 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 330 to 335 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 35 to 340 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 340 to 345 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 345 to 350 kDa. In an embodiment, a composition of the present disclosure having pure silk fibroin based protein fragments has a polydispersity ranging from about 1 to about 5.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1.5 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1 to about 1.5. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1.5 to about 2.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 2.0 to about 2.5. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments, has a polydispersity ranging from about is 2.0 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments, has a polydispersity ranging from about is 2.5 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has non-detectable levels of LiBr residuals. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 25 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 50 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 75 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 400 ppm to 500 ppm. In an embodiment, a composition of the present disclosure having pure silk fibroin based protein fragments, has non-detectable levels of Na2CO3residuals. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 400 ppm to 500 ppm. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 50 to 100%. In an embodiment, the water solubility of pure silk fibroin based protein fragments of the present disclosure is 60 to 100%. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 70 to 100%. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 80 to 100%. In an embodiment, the water solubility is 90 to 100%. In an embodiment, the silk fibroin-based fragments of the present disclosure are non-soluble in aqueous solutions. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 50 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 60 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 70 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 80 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 90 to 100%. In an embodiment, the silk fibroin-based fragments of the present disclosure are non-soluble in organic solutions. In an embodiment, the percent water content in gels of the present disclosure is 20% to 99.9%. In an embodiment, the percent water content in gels of the present disclosure is 20% to 25%. In an embodiment, the percent water content in gels of the present disclosure is 25% to 30%. In an embodiment, the percent water content in gels of the present disclosure is 30% to 35%. In an embodiment, the percent water content in gels of the present disclosure is 35% to 40%. In an embodiment, the percent water content in gels of the present disclosure is 40% to 45%. In an embodiment, the percent water content in gels of the present disclosure is 45% to 50%. In an embodiment, the percent water content in gels of the present disclosure is 50% to 55%. In an embodiment, the percent water content in gels of the present disclosure is 55% to 60%. In an embodiment, the percent water content in gels of the present disclosure is 60% to 65%. In an embodiment, the percent water in gel cosmetic gels of the present disclosure s is 65% to 70%. In an embodiment, the percent water content in gels of the present disclosure is 70% to 75%. In an embodiment, the percent water content in gels of the present disclosure is 75% to 80%. In an embodiment, the percent water content in gels of the present disclosure is 80% to 85%. In an embodiment, the percent water content in gels of the present disclosure is 85% to 90%. In an embodiment, the percent water content in gels of the present disclosure is 90% to 95%. In an embodiment, the percent water content in gels of the present disclosure is 95% to 99%. In an embodiment, the percent water content in films of the present disclosure is 20%. In an embodiment, the percent water content in films of the present disclosure is less than 20%. In an embodiment, the percent water content in films of the present disclosure is less than 18%. In an embodiment, the percent water content in films of the present disclosure is less than 16%. In an embodiment, the percent water content in films of the present disclosure is less than 14%. In an embodiment, the percent water content in films of the present disclosure is less than 12%. In an embodiment, the percent water content in films of the present disclosure is less than 10%. In an embodiment, the percent water content in films of the present disclosure is between about 2% and about 20%. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is greater than 84° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is less than 100° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 84° C. to 100° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 84° C. to 94° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 94° C. to 100° C. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXAMPLES Example 1. Development of a Silk Film of the Present Disclosure for Use in Fine Line Lifting Applications TABLE 18Film Recipe for Fine Line Lifting Film-FIG. 82A% SPF Mixture Solution of the2.4%Present DisclosureQuantity Vitamin C4:1 (silk:Vit C) (0.006 g/mL2.4% solution) 20%mL per film (2.5 cm by 10 cm)7.08 mLMass of silk per film:170 mgMass of 1-ascorbic acid per film:42.5 mgpH4.0 (when water is applied) Silk films (2.5 cm×10 cm) were manufactured according to methods disclosed herein varying process parameters so as to result in fine line lifting films. The silk films were given the name “PureProC™ film”, and can be packaged in a foil based package that is air tight and light proof. Table 18 provides details of the PureProC™ films used in a study of 32 individuals using the films for four (4) weeks. Biocompatibility and hypo-allergenicity of the films was observed. Further, no sensitization, toxicity, or immune response was observed.FIG.84is a graph summarizing the quantity of vitamin C in a daily dose (i.e., the average amount of product used to cover a 25 cm2area of skin) of PureProC™ and competitor products over a 30 day period.FIGS.85and86summarize resultant ease of use data and observed benefits within the first month of use. In an embodiment, PureProC™ films were removed by peeling the films off. In an embodiment, PureProC™ films were removed by using a wet cotton ball or similar removal pad. In an embodiment, PureProC™ films were removed by washing the area where the film is placed with a wash cloth. In an embodiment, PureProC™ film PureProC™ films were removed using water. The PureProC™ films can be shaped into strips for multiple areas of the face or larger pieces can be cut to fit target areas. In an embodiment, grips or backing(s) on the PureProC™ films can be included for ease of application. In an embodiment, a PureProC™ film of the present disclosure includes silk and vitamin C (20%). In an embodiment, a film of the present disclosure is soluble in water (insoluble border). In an embodiment, a film of the present disclosure is clear/transparent. In an embodiment, a film of the present disclosure has a pH=4 when water is applied. Films of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. Films of the present disclosure can be made with from about 1% to about 50% 1-ascorbic acid. Films of the present disclosure can adhere to skin with water. Films of the present disclosure can be spread on skin once water is applied. Films of the present disclosure can dry when humidity of drying equipment is 16-40% and below the humidity of the lab Example 2. Development of Silk Gels of the Present Disclosure TABLE 19Gel Samples - Silk gel formulations including additives, concentration of silk and additive,gelation conditions and gelation times.mLAmountSample2% silkMass Vit CRatioofTemp/Days toNamesolution(g)silk:Vit CAdditiveadditiveTreatmentGelation1100.045:01NoneNoneRT82100.082.5:1NoneNoneRT83100.21:01NoneNoneRT84100.41:02NoneNoneRT145100.81:04NoneNoneRTNone6100.045:01NoneNoneFridge~397100.082.5:1NoneNoneFridge~398100.21:01NoneNoneFridge~399100.41:02NoneNoneFridgeNone10100.81:04NoneNoneFridgeNone11100.21:01NoneNoneRT/Shake8vigorouslyO-1100.045:01NoneNone37° C.3OvenO-2100.045:01NoneNone50° C.2OvenO-3100.21:01NoneNone37° C.4OvenO-4100.21:01NoneNone50° C.3OvenM400.165:01NoneNoneRT5D400.165:01NoneNoneRT5E1100.045:01VitE1dropRT7E2100.045:01VitE3dropsRT7E3100NoneVitE1dropRTNoneE4100NoneVitE3dropsRTNoneL1100.045:01Lemon300uLRT6L2100.045:01Lemon Juice300uLRT6L3100.045:01Lemon Juice1000uLRT5L4100NoneLemon300uLRT6L5100NoneLemon Juice300uLRT7Jar 1200.085:01Lemon Juice2000uLRT5-7Jar 250.025:01Lemongrass1dropRT2-3OilR-1100.045:01Rosemary1dropRT7OilT-1100.045:01NoneNoneRT/Tube7RO-1100.045:01Rose Oil1dropRT6RO-210NoneNoneRose Oil1dropRTNone Ratio of Silk to Vitamin C Samples 1-10 were used to examine the effect of silk to vitamin C ratio on serum gelation. Samples 1-3 with less vitamin C gelled quicker than samples 4 and 5. All other conditions were kept constant. Samples 6-8 with less vitamin C gelled quicker than samples 9 and 10. All other conditions were kept constant. It is concluded that decreasing the ratio of silk to vitamin C (increasing the amount of vitamin C), will lengthen the time to gel creation. At ratios with small amounts of vitamin C, days to gel creation did not vary greatly. Physical Stimulation Samples 3 and 11 were used to examine the effect of physical stimulation on serum gelation. Each sample was prepared under the same conditions. Sample 11 was vigorously shaken for about 3 minutes after addition of vitamin C. Treatment of 3 and 11 was otherwise the same. The shaking resulted in bubbles but did not significantly change gel creation time. Temperature Treatment Samples 1, 3, 6, 8, O-1, O-2, O-3, and O-4 were used to examine the effect of temperature treatment on serum gelation time. Samples 1, 6, O-1, and O-2 were identical other than temperature treatment Samples 3, 8, O-3, and O-4 were identical other than temperature treatment. The two groups differed in silk to vitamin C ratio. Time to serum gelation was directly related to temperature treatment with a higher temperature resulting in quicker serum gelation. Solution Volume Samples 1, M and D were used to examine the effect of solution volume on serum gelation time. Samples M and D varied from sample 1 only by an increased solution volume. Samples M and D gelled in 5 days while sample 1 gelled in 8 days. Samples M and D were definitively noticed to be gelled on the day of gelling while sample 1 gelled over a weekend. Additives Samples E1, E2, E3, E4, L1, L2, L3, L4, L5, Jar 2, R1, RO-1 and RO-2 were used to examine the effect of additives on serum gelation time. Samples E1-4 contained Vitamin E. Only samples E1 and E2 contained vitamin C and only these two samples gelled. Vitamin E can be added to a solution to become a gel but it appears that another additive may be needed to create a gel. Samples L1-5 contained a form of lemon juice. Samples L1 and L4 had juice directly from a lemon while samples L2, L3 and L5 contained lemon juice from a plastic lemon container. Samples L4 and L5 did not have vitamin C while all others did. All samples gelled showing that lemon juice can create gel on its own. Amount of lemon juice and type of lemon juice had little effect on gelation time. Sample Jar 2 contained lemon grass oil which formed an albumen like substance when initially added. This sample also had vitamin C but gelation time was significantly quicker than with other vitamin C samples. Sample R1 contained rosemary oil, which seemed to be soluble, as well as vitamin C. The sample gelled in a similar time frame to other samples with only vitamin C. Samples RO-1 and RO-2 contained rose oil while only RO-1 had vitamin C. Only RO-1 gelled showing that rose oil will not create a gel quickly on its own. In both cases the rose oil was immiscible and visible as yellow bubbles. Aqueous silk fibroin based fragment solution and essential oils are immiscible liquids. In an embodiment, to increase the fragrance of the silk fibroin-based fragment solution, without entrapping oils within the solution, the solution is mixed with the essential oil with the use of a stir bar. The stir bar is rotated at a speed such that some turbulence is observed in the mixture, thus causing contact between the fragrant essential oil and the molecules in solution, adding a scent to the solution. Before casting of product from the solution, mixing may be stopped and the oil allowed to separate to the top of the solution. Dispensing from the bottom fraction of the solution into the final product allows for fragrance without visible essential oil within the final product. Alternatively, the silk fibroin-based solution and essential oil can be combined with or without additional ingredients and/or an emulsifier to create a composition containing both ingredients. In an embodiment, mixing of the solution as described above can reduce gelation time if the solution is used to create a gel formulation. Vessel Samples T1 and Jar 1 were used to examine the effect of casting vessel on serum gelation time. Jar 1 was cast in a glass jar while T1 was cast in an aluminum tube. Both samples gelled and did not affect serum gel time. Summary All treatments of silk solution for gel solution were in a conical tube at room temperature unless otherwise stated. The ratio of silk to vitamin C did affect the ability of a solution to gel as ratios above 1:2 did not gel and a 1:2 ratio took twice as long as other lower ratios (5:1, 2.5:1, 1:1). Temperature affected gel creation time with higher temperatures resulting in quicker gel times. 50° C. treatment gelled in as quick as 2 days, 37° C. treatment gelled in as quick as 3 days, room temperature treatment gelled in 5-8 days and storage in a refrigerator took at least 39 days to gel. The effects of additives on gel creation were dependent on the additive. Vitamin E, Rosemary Oil and Rose Oil all had no effect on gel creation. Each of these additives did not prevent gelation or affect the time to gelation. Each also required the presence of vitamin C to gel. Lemon juice from a fresh lemon, pre-squeezed lemon juice from a plastic lemon container and lemon grass oil did affect gel creation. Without wishing to be bound by theory, it is believed that the lower pH as a result of these additives is the reason the additives had an impact on decreasing gelation time. Both lemon juice types were able to cause gelation without the presence of vitamin C. This occurred in the same number of days as with vitamin C. The lemongrass oil was able to decrease the number of days to gelation to 2-3 days. All additives appeared soluble other than lemongrass oil and rose oil. Rose oil remained in yellow bubbles while the lemongrass oil was partially soluble and formed an albumen like chunk. In an embodiment, oils that are not fully soluble, can still be suspended within the gel as an additive. Physical stimulation by shaking, vessel the solution was cast into and solution volume did not affect gelation time.FIG.81is a graph representing the % Activity of Vitamin C in gels of the present disclosure. TABLE 20Concentration of vitamin C in various gelformulations.SampleConcentration ofWeightVitamin C (mg/g)Sample Info(mg)In SampleAverageRosemary685.73.25113.2657(Room3.2804Temperature6383.33363.3334storage)3.3332Lemongrass6462.86722.877(Room2.8868Temperature645.52.90512.9051storage)2.9052Rosemary645.23.90633.9147storage)(Room3.923Temperature;6493.94433.9374Foil Covered3.9305storage)Lemongrass630.13.82533.8274(Room3.8295Temperature;660.43.82833.8253Foil Covered3.8222storage)Rosemary672.45.16165.1484(Fridge, Foil5.1352Covered616.55.19845.201storage)5.2036Lemongrass640.55.18715.1824(Fridge, Foil5.1776Covered627.75.20985.2126storage)5.2154 Example 3. Development of Silk Gels of the Present Disclosure for Use as Smoothing Gel TABLE 21Lemongrass Gel% Silk Solution2%Quantity Vitamin C100 mg/15 mL solutionQuantity Lemongrass Oil20 uL/15 mL solution TABLE 22Rosemary Gel% Silk Solution2%Quantity Vitamin C100 mg/15 mL solutionQuantity Rosemary Oil20 uL/50 mL solution TABLE 23Lemongrass Gel (50 mL)% Silk Solution2%(60 minute boil, 25 kDA)Quantity Vitamin C12.82 mg/mL solution(ascorbyl glucoside)(641 mg total)Quantity Lemongrass Oil1.33 uL/mL solutionpH4 TABLE 24Rosemary Gel (50 mL)% Silk Solution2%(60 minute boil, 25 kDA)Quantity Vitamin C12.82 mg/mL solution(ascorbyl glucoside)(641 mg total)Quantity Rosemary Oil0.8 uL/mL solutionpH4 Gels of the present disclosure can be made with about 0.5% to about 8% silk solutions. Gels of the present disclosure can be made with ascorbyl glucoside at concentrations of about 0.67% to about 15% w/v. Gels of the present disclosure be clear/white in color. Gels of the present disclosure can have a consistency that is easily spread and absorbed by the skin. Gels of the present disclosure can produce no visual residue or oily feel after application. Gels of the present disclosure do not brown over time. Silk gels with essential oils were prepared by diluting a silk solution of the present disclosure to 2%. Vitamin C was added to the solution and allowed to dissolve. The essential oil was added, stirred and dissolved. The solution was aliquot into jars. A trial was conducted with 44 people on two formulations of the present disclosure, PureProC™ Rosemary Gel and PureProC™ Lemongrass Gel (FIGS.87and88). Respondents were asked to use each sample once a day for a week each. The majority of respondents applied the gel to the whole face. Other areas where it was most commonly applied included the forehead, under eyes and near mouth. The majority of respondents applied the gel during the morning (67%) with the balance 33% applying the gel in the evening. Ninety-eight (98%) of participants used the gel once a day during the test. Respondents were asked to describe in their own words how the gel felt when it was applied and how it felt during the 24 hours until the next application. Smooth, cool, and soft were the most often mentioned adjectives used to describe how the gel felt. Eighty percent (80%) of test participants gave a high score to interest in continuing to use the gel. Respondents were asked about what they did with their other products that were usually used on their face during the trial. The majority applied the gel first and then added the other products or applied the gel at night with no additional products. Only 14% of participants indicated that they eliminated one of their normal products while testing the gel. PureProC™ can be used in conjunction with or in replacement of other products. Additionally, sunscreen can be added to the gel or it may be dispensed from a pump instead of a jar. With repeated topical use, no skin irritation, rash, or signs of non-compatibility was observed. Biocompatibility and hypo-allergenicity of the gels was observed. Further, no sensitization, toxicity, or immune response was observed. Example 4. Silk Articles of the Present Disclosure Made from Silk Solutions of the Present Disclosure Silk solutions of various molecular weights and/or combinations of molecular weights can be optimized for specific applications. The following provides an example of this process but it not intended to be limiting in application or formulation. Three (3) silk solutions were utilized in standard silk structures in accordance with standard methods in the literature with the following results:Solution #1 is a silk concentration of 5.9%, average MW of 19.8 kDa and 2.2 PD (made with a 60 min boil extraction, 100 degree LiBr dissolution for 1 hr)Solution #2 is a silk concentration of 6.4% (made with a 30 min boil extraction, 60 degree LiBr dissolution for 4 hrs)Solution #3 is a silk concentration of 6.17% (made with a 30 min boil extraction, 100° C. LiBr dissolution for 1 hour) Films: Films were made in accordance with Rockwood et al (Nature Protocols; Vol. 6; No. 10; published on-line Sep. 22, 2011; doi:10.1038/nprot.2011.379). Briefly, 4 mL of 1% or 2% (wt/vol) aqueous silk solution was added into 100 mm Petri dish (Volume of silk can be varied for thicker or thinner films and is not critical) and allowed to dry overnight uncovered. The bottom of a vacuum desiccator was filled with water. Dry films were placed in the desiccator and vacuum applied, allowing the films to water anneal for 4 hours prior to removal from the dish. Films cast from solution #1 did not result in a structurally continuous film; the film was cracked in several pieces. These pieces of film dissolved in water in spite of the water annealing treatment. Egel: “Eger” is an electrogelation process as described in Rockwood et al. Briefly, 10 ml of aqueous silk solution is added to a 50 ml conical tube and a pair of platinum wire electrodes immersed into the silk solution. A 20 volt potential was applied to the platinum electrodes for 5 minutes, the power supply turned off and the gel collected. Solution #1 did not form an EGEL over the 5 minutes of applied electric current. Gelation: Solutions #2 and #3 were gelled in accordance with the published horseradish peroxidase (HRP) protocol. Behavior seemed typical of published solutions. Sonicated Gels: Gels were made following the sonication process in Rockwood et al. Briefly, 5 ml of silk solution was added to a 15 ml conical tube. The sonicating horn was immersed in the solution and the solution sonicated at 50% amplitude (21 W). Silk gels were made with 2%, 4% and 6% silk solutions. As compared to standard literature silk, Solutions #2 and #3 formed gels after a longer time, for example:Standard literature silk: 5-8 minSolution #2: 20 minSolution #3: 120 min Porous 3D scaffolds: Water based, salt leached scaffolds were made in accordance with the published methods of Rockwood. Salt with particle sizes of interest was prepared by stacking the sieves with the largest mesh on top and the smallest mesh on the bottom. Salt was added and sieves shaken vigorously collecting the salt. With a 5-ml syringe, 6% (wt/vol) fibroin solution was aliquoted into plastic containers, 2 ml per mold and 5-600 micron salt particles were slowly added on top of the fibroin solution in the mold while rotating the container so that the salt was uniform. The ratio of salt to silk in solution was maintained at 25:1. The container was gently tapped on the bench top to remove air bubbles, the cap closed and the solution allowed to settle overnight at room temperature. Once gelled, the lids were removed and the molds placed in a 2-liter beaker with ultrapure water (three containers per 2 liters of water). The beakers were transferred to a stir plate and stirred, changing the water 2-3 times per day for 2 d (4-6 washes in total). The scaffolds were removed from the molds and placed them in fresh water for an additional day. Solution #1 did not form a scaffold; it did not gel. Both solution #2 & #3 formed scaffolds. The scaffolds made with Solution #3 appear softer than the ones made with Solution #2, but both scaffolds were homogeneous. Example 5. Tangential Flow Filtration (TFF) to Remove Solvent from Dissolved Silk Solutions of the Present Disclosure A variety of % silk concentrations have been produced through the use of Tangential Flow Filtration (TFF). In all cases a 1% silk solution was used as the input feed. A range of 750-18,000 mL of 1% silk solution was used as the starting volume. Solution is diafiltered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, solution undergoes ultrafiltration to increase the concentration through removal of water. See examples below. 7.30% Silk Solution: A 7.30% silk solution was produced beginning with 30 minute extraction batches of 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 15,500 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 1300 mL. 1262 mL of 7.30% silk was then collected. Water was added to the feed to help remove the remaining solution and 547 mL of 3.91% silk was then collected. 6.44% Silk Solution: A 6.44% silk solution was produced beginning with 60 minute extraction batches of a mix of 25, 33, 50, 75 and 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35, 42, 50 and 71 g per batch of silk fibers were dissolved to create 20% silk in LiBr and combined. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 17,000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 3000 mL. 1490 mL of 6.44% silk was then collected. Water was added to the feed to help remove the remaining solution and 1454 mL of 4.88% silk was then collected 2.70% Silk Solution: A 2.70% silk solution was produced beginning with 60 minute extraction batches of 25 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35.48 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 1000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 300 mL. 312 mL of 2.7% silk was then collected. Example 6. Gel Vitamin C Derivatives of the Present Disclosure The purest form of vitamin C is L-ascorbic acid. There are a number of other derivatives of vitamin C that function like pure vitamin C after they are converted to L-ascorbic acid in the body. Vitamin C derivatives are being utilized to extend shelf life. Derivatives are stable forms of L-ascorbic acid and will not oxidize or lose stability. Table 25 below summarizes some vitamin C derivatives tested in the skin care products of the present disclosure: TABLE 25Derivatives ExploredSodium Ascorbyl Phosphate (Aromantic)Sodium Ascorbyl Phosphate (DSM)Magnesium Ascorbyl PhosphateAscorbic Acid-2-GlucosideAscorbyl Tetraisopalmitate The Tables inFIGS.89A-89Bsummarize embodiments of gels of the present disclosure. Ascorbic acid-2-glucoside was the most successful vitamin C derivative at gel formation with gel being formed in a 2% silk solution in 3 days. Sodium ascorbyl phosphate from DSM supplier formed a gel in a 2% silk solution after 28 days while the same molecule from Aromantic failed to create a gel. In all cases 100 mg of vitamin C derivative was mixed in 15 mL of 2% silk solution, and all gels had the same appearance as gels created with ascorbic acid. Gels were also cast with combinations of two vitamin C options. In each case, at least one of the vitamin C options was known to cause gelation (L-ascorbic acid or ascorbic acid-2-glucoside). All combination gels were able to gel at 1% total vitamin C additive concentration. A gel cast at 20% total vitamin C additive concentration did not gel. Without wishing to be bound by theory, it appears there is a relationship between vitamin C concentration, silk concentration, and gelation. An increase in vitamin C at a given concentration of silk will result in a longer time to gelation or inhibit gelation. This may be due to the vitamin C molecule physically blocking interaction between silk protein fragments or cross-linking of silk protein. Modification to pH may allow additional concentrations of vitamin C and derivatives thereof to be added. Ascorbyl tetraisopalmitate was not used in any gel forming formulation, as it was unable to dissolve or be dispersed in an aqueous silk solution. Ascorbyl tetraisopalmitate is a highly viscous, oil soluble liquid that might need the help of an emulsifier to possible dissolve in aqueous silk solution. Example 7. Film Vitamin C Derivatives of the Present Disclosure FIG.90is a table summarizing embodiments of films of the present disclosure. Sodium ascorbyl phosphate, magnesium ascorbyl phosphate and ascorbic acid glucoside could be cast in films with varying appearance. Sodium ascorbyl phosphate films were opaque and white with a textured top surface similar to plastic. Magnesium ascorbyl phosphate films were clear and cloudy with a textured top surface similar to plastic. Ascorbic acid-2-glucoside films were most similar to L-ascorbic acid films although slightly less pliable and slightly textured. All films were soluble with an insoluble border. In an embodiment, a film with an insoluble border can be made completely spreadable by punching a shape from the region contained within the soluble section. Example 8. Caffeine Films with Vitamin C of the Present Disclosure FIGS.91A-91Bare tables summarizing embodiments of caffeine films of the present disclosure. Films were cast with 0.5%, 1%, 2.5%, 5%, 10%, 15% and 20% caffeine and 20% or 25% vitamin C. All combinations formed films. 20% caffeine films had caffeine precipitate out. Films with 0.5%-2.5% were soluble. In an embodiment, a caffeine film of the present disclosure is used for reducing puffy eyes. Example 9. Caffeine Gels with Vitamin C of the Present Disclosure A silk gel with 2% silk and 100 mg L-ascorbic acid/15 mL solution was created with the addition of 50 mg caffeine/15 mL solution. The gel has the exact appearance of standard L-ascorbic acid gels. In an embodiment, a caffeine gel of the present disclosure is used for reducing puffy eyes. A range of essential oils can be used including, but not limited to, lemongrass, vanilla, geranium, and green tea. Example 10. Green Tea Gels with Vitamin C of the Present Disclosure Steps:Green Tea PrepHeat 250 mL water to boilSteep tea bag 2-3 minuteswith occasional stirremove tea bag and let coolGel SolutionUse TFF-10-0047 (3.71% silk)Prepdilute to 3% silk with waterdilute to 2% with green teaadd L-ascorbic acidGelGelation occurred like standardgel at room temperatureGreen/yellow colorGreen Tea scentSolution Spec:2% silk solution65 mL (35 ml of 3.71% silk, 8.3mL water, 21.66 mL green tea)0.43 gL-ascorbic acid FIG.92is a table summarizing an embodiment of a caffeine gel of the present disclosure. A silk gel with 2% silk and 100 mg L-ascorbic acid/15 mL solution was created with the addition of 50 mg caffeine/15 mL solution. The gel has the exact appearance of standard L-ascorbic acid gels. Example 11. Preservative Gels with Vitamin C of the Present Disclosure FIG.93is a table summarizing embodiments of preservative gels of the present disclosure. Silk gels were cast with standard 2% silk solution and 100 mg L-ascorbic acid/15 mL solution with the addition of a preservative and chelating agent. The preservative added was Verstatil SL by Kinetic (Water, Sodium Levulinate, Potassium Sorbate) at 1.5% and the chelating agent was Dermofeel-PA3 by Kinetic (Sodium Phytate) at 0.1%. The addition of preservatives extended gelation time to 7 days. Gel is being observed for discoloration and integrity with L-ascorbic acid and ascorbic acid-2-glucoside gel comparisons. Example 12. Chemical Peels of the Present Disclosure The primary variable investigated was the concentration of lactic acid and/or glycolic acid necessary to create a silk solution of a desired pH. In order to determine the relationship between concentration in silk and pH, 2% silk solutions (60 minute boil, 25 kDA) were titrated with glycolic and lactic acid and tested for pH with pH strips. See the following titration s/formulations below: TABLE 26Lactic Acid Peel 1: Initial solution:25 mL of 2% silk solution, pH = 7-8Quantity of LacticTotalAcid AddedLactic AcidpH100 μL100 μL3100 μL200 μL2100 μL300 μL1-2Time to gel: 3 days TABLE 27Lactic Acid Peel 2: Initial solution:25 mL of 2% silk solution, pH = 7-8Quantity of LacticTotalAcid AddedLactic AcidpH25 μL25 μL4Time to gel: >5 days TABLE 28Glycolic Acid Peel 1: Initial solution:25 mL of 2% silk solution, pH = 7-8Quantity of GlycolicTotalAcid AddedGlycolic AcidpH41 mg41 mg443.25 mg84.25 mg330.7 mg114.95 mg356.4 mg171.35 mg2-391.66 mg263.01 mg2171.35 mg434.4 mg1-2Time to gel: 3 days TABLE 29Glycolic Acid Peel 2: Initial solution:25 mL of 2% silk solution, pH = 7-8Quantity of LacticTotalAcid AddedLactic AcidpH41 mg41 mg4Time to gel: >5 days TABLE 30Lactic/Glycolic Acid Peel: Initial solution:25 mL of 2% silk solution, pH = 7-8TotalTotalLemon-Lactic AcidGlycolic AcidgrasspH150 μL200 mg33.3 μL2Time to gel: 3 days TABLE 31Lactic/Glycolic Acid Peel: Initial solution:30 mL of 2% silk solution, pH = 7-8% Silk Solution2%(60 minute boil, 25 kDA)Lactic Acid Concentration6 μL/mLGlycolic Acid Concentration8 mg/mLpH2Lemongrass Concentration1.33 μL/mL A peel of the present disclosure can have a % silk ranging from about 0.5% to about 8%. The pH of a peel of the present disclosure can be adjusted with varying quantities of lactic and glycolic acid. Peels can also be made with lactic acid only or glycolic acid only. A peel of the present disclosure can be clear/white in color. A peel of the present disclosure can have a gel consistency that is easily spread and absorbed by the skin. A peel of the present disclosure does not brown or change colors. In an embodiment, a chemical peel of the present disclosure can be applied weekly to reveal healthy, vibrant skin. In an embodiment, a chemical peel of the present disclosure can be applied weekly to diminish fine lines. In an embodiment, a chemical peel of the present disclosure can be applied weekly to firm the skin. Each formulation (after titration, if applicable) was applied as a liquid and as a gel and observed for look and feel. Peels of pH=4 (Lactic Acid Peel 2, Glycolic Acid peel 2) resulted in a minimal burning feeling after a few minutes of application, while peels of pH=˜2 (Lactic Acid Peel 1, Glycolic Acid Peel 1, Lactic/Glycolic Acid Peel) caused a slightly more intense burning feel. Little difference in degree of burning was felt between liquid and gel other than that the burning sensation was more delayed in the gel form. PH was maintained in the gel form and was confirmed by using a pH strip. Glycolic acid and lactic acid are both alpha hydroxy acids (AHA's) that are among the most commonly used peels for superficial peeling (outermost skin layer peeling). Chemical peels are intended to burn the top layers of the skin in a controlled manner, to remove superficial dermal layers and dead skin in order to improve appearance. AHAs are common in chemical peels due to low risk of adverse reactions and high control of strength (control pH and time applied). Glycolic acid is most commonly used and has a very small molecular size, enabling deep penetration into the epidermis. Lactic acid is another commonly used AHA and offers a more gentle peel with higher control due to its larger molecular size. Any number of chemicals known in the art that lower pH and are physical exfoliates can be used in place of AHAs. Example 13. Hydrating Serums of the Present Disclosure Variables include: concentration of silk in solution, concentration of HA, addition of vitamin C, and serum preparation method. Table 32 is a list of samples that were evaluated: TABLE 32Embodiments of serums of the present disclosure containingHA and Silk (60 minute boil, 25 kDA), with or without vitaminC, and with 20 uL/15 mL lemongrass essential oil (30 mL solution)HASilkVit CMethod(%)(%)(mg)ObservationHA added to water0.520White, slightly opaque, viscous liquidbefore dilution of1White/yellow, slightly opaque, viscous liquidsilk0.520Low viscosity, clear-white opaque with film ontop, some white residue when applied topicallyto skin1Slightly viscous, clear liquid with film on top0.510Slightly viscous, clear liquid with film on top1Smooth viscous liquid, no white residue whenapplied topically to skin0.50.50Moderately viscous liquid, clear1Smooth, clear, no white residue when appliedtopically to skin0.5235Non homogeneous mix of hard gel and viscousliquid1Non homogeneous mix of hard gel and viscousliquid1135Non homogeneous mix of hard gel and viscousliquid0.5Opaque, white liquid/non-viscous1435Separated mixture of hard gel and viscous liquid0Non homogeneous mix of hard gel and viscousliquid520Yellow, gelHA added to water1020Viscous jelly upon stirring with undissolved HAbefore dilution of5Very viscous jelly upon stirringsilk, stirred1Viscous jelly upon stirringvigorously0.5HA added to water120Non homogeneous thick, viscous jelly/gelbefore dilution of510silk, shakenHA added to water110Clear/slightly opaque, viscous liquid, smoothand let sit for 1 dayfeel, little to no white residue when appliedbefore dilution oftopically to skinsilkHA added to0.520Viscous, clear/white liquid varying indiluted silk1consistencysolution, stirred0.51Clear viscous liquid varying in consistency10.56White, opaque jelly varying in consistency1HA added to0.53.90White, slightly opaque, viscous liquiddiluted silk1solution, stirred0.5235White gel varying in consistency1 In an embodiment, a hydrating serum of the present disclosure protects the skin and seals in moisture with the power of silk fibroin based fragment proteins. In an embodiment, a hydrating serum of the present disclosure delivers moisture for immediate and long-term hydration throughout the day with concentrated hyaluronic acid. A range of essential oils can be used in a hydrating serum of the present disclosure including, but not limited to, lemongrass, vanilla, geranium, and green tea. In an embodiment, one or two drops of a hydrating serum of the present disclosure can be smoothed over the face and neck. In an embodiment, a hydrating serum of the present disclosure includes water, aqueous silk fibroin-based fragment solution, hyaluronic acid, and lemongrass oil. In an embodiment, the silk fibroin-based fragment protein in a hydrating serum of the present disclosure has the ability to stabilize and protect skin while sealing in moisture, all without the use of harsh chemical preservatives or synthetic additives. In an embodiment, the hyaluronic acid in a hydrating serum of the present disclosure nourishes skin and delivers moisture for lasting hydration. In an embodiment, the lemongrass essential oil in a hydrating serum of the present disclosure yields antioxidant and anti-inflammatory properties that support skin rejuvenation. In an embodiment, a hydrating serum of the present disclosure has a pH of about 6.0. Silk Fibroin-Based Fragment Solution Because silk fibroin-based fragment solution is both aqueous and able to entrap and deliver small molecules, the solution is able to deliver both water and hygroscopic HA molecules to the skin for hydration. A range in concentration of silk fibroin-based fragment compositions in solution from 0.5%-6.0% was tested for feasibility and product outcome. All concentrations tested were found to be feasible. Hyaluronic Acid Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in the hydrating serum due to its hygroscopic properties and ability to promote soft, hydrated skin. A range in concentration of hyaluronic acid in solution from 0.5%-10.0% was tested for feasibility and product outcome. All concentrations tested, with the exception of 10.0%, were found to be feasible. Feasibility was determined based on the ability to dissolve hyaluronic acid. Vitamin C and Derivatives Thereof Vitamin C (L-ascorbic acid) was tested as an ingredient in the hydrating serum. Initial vitamin C samples became a non-homogeneous mixture of gel and liquid. A follow-up trial with vitamin C resulted in a homogeneous, white, opaque, non-viscous liquid that was not quickly absorbed by the skin. In an embodiment, a vitamin C derivative that does not readily cause gelation, such as sodium ascorbyl phosphate, could be added up to the concentration at which it would no longer be soluble (for example, 0% to about 40%). In an embodiment, 20% sodium ascorbyl phosphate could be added. Vitamin C options that do cause gelation (L-ascorbic acid and ascorbyl glucoside) could be added at high concentrations (for example greater than about 10% up to about 50%) at which gelation is inhibited. Serum Creation Method Initial serums were created by the addition of HA to a silk fibroin based fragment solution followed by stirring. The HA appeared to stick together and was not dissolved until forcefully stirred. The mixing process was then changed so that HA was first dissolved in water and then immediately used to dilute a high concentration silk fibroin-based fragment solution (>4%) to the desired concentrations. The resulting serums were more homogeneous and had a desirable smooth, clear look and feel. Upon application to the skin, a white residue briefly appeared that could be rubbed in. In an alternate method formulations were created by dissolving HA in water and allowing it to sit for 1 day until complete dissolution was observed. The HA and water solution was then used to dilute a high concentration silk fibroin based fragment solution to the desired concentrations. The resulting serum was clear, smooth, homogeneous and left little to no white residue when applied. Example 14. UV Hydrating Serums of the Present Disclosure Variables tested include: concentration of HA, concentration of zinc oxide, concentration of titanium dioxide, addition of vitamin C, and serum preparation method. FIGS.94A-94Care tables summarizing embodiments of cosmetic serums of the present disclosure with varying additives and concentrations of components suitable for protection against ultraviolet radiation (UV). Table 33 provides an embodiment of a hydrating serum of the present disclosure with vitamin C. TABLE 33Embodiment of Hydrating serum ofthe present disclosure with vitamin C% Silk Solution1.0% w/v(60 minute boil, 25 kDA)Hyaluronic Acid0.75% w/v(sodium hyaluronate)Lemongrass Oil20 uL/15 mLsilk solutionSodium Ascorbyl Phosphate6 gLactic Acid1.2 mL A serum of the present disclosure can be made with from about 0.25% to about 10% sodium hyaluronate (increasing % results in more viscous serum). 0.5% to about 10% silk solutions can be used to prepare a serum of the present disclosure. A serum of the present disclosure can be clear and have a yellow tinted color. A serum of the present disclosure can have a pH=6. A serum of the present disclosure can have a lubricious texture that is rubbed in easily without residue. Concentration of HA: Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in the UV silk serum due to its hygroscopic properties and widely accepted use in cosmetic products to promote hydration of skin. 1%, 2.5% and 5% HA solutions were tested. With increasing HA %, the serum became more viscous and gel like. 1% HA was not feasible for the UV serum due to the fact that the UV additives (zinc oxide, titanium dioxide) are not water soluble and need to be dispersed. 1% HA was not viscous enough for dispersion and the UV additives precipitated out. 2.5% gave the best consistency based on preferred feel, texture and viscosity and was able to disperse the UV additives. 5% was a very thick, viscous serum. Concentration of Mineral Filters: Zinc Oxide and Titanium Dioxide: Zinc oxide and titanium dioxide were explored as UV additives that are considered safe. These additives mechanically protect from UV radiation by forming a physical reflective barrier on the skin. Both are not soluble in water and must be dispersed for the current aqueous solution. Zinc oxide concentration varied from 2.5%, 3.75%, 5%, 5.625%, 10%, 12% and 15%. Titanium dioxide concentrations varied from 1.25%, 1.875%, 3%, 5% and 10%. Increasing the concentration of UV additives resulted in minor increases of white residue and how well dispersed the additives were, however if mixed well enough the effects were negligible. Zinc oxide and titanium dioxide were mixed together into serums in order to achieve broad spectrum protection. Zinc oxide is a broad spectrum UV additive capable of protecting against long and short UV A and UV B rays. However titanium dioxide is better at UV B protection and often added with zinc oxides for best broad spectrum protection. Combinations included 3.75%/1.25% ZnO/TiO2, 5.625%/1.875% ZnO/TiO2, 12%/3% ZnO/TiO2, 15%/5% ZnO/TiO2. The 3.75%/1.25% ZnO/TiO2 resulted in spf 5 and the 5.625%/1.875% ZnO/TiO2 produced spf 8. Vitamin C: Sodium ascorbyl phosphate was used as a vitamin C source. Formulations were created with the vitamin C concentration equal to that in the silk gel (0.67%). Formulations were also created with 20% sodium ascorbyl phosphate which is soluble in water. Serum Preparation: The vitamin C (sodium ascorbyl phosphate) must first be dissolved in water. Sodium hyaluronate is then added to the water, mixed vigorously and left to fully dissolve. The result is a viscous liquid (depending on HA %). The viscosity of the HA solution allows even dispersion of the zinc oxide and titanium dioxide and therefore HA must be mixed before addition of UV additives. The zinc oxide and titanium dioxide are then added to the solution and mixed vigorously with the use of an electric blender. Silk solution is then added and mixed to complete the serum formulation. Chemical Filters: A UV serum of the present disclosure can include one, or a combination of two or more, of these active chemical filter ingredients: oxybenzone, avobenzone, octisalate, octocrylene, homosalate and octinoxate. A UV serum of the present disclosure can also include a combination of zinc oxide with chemical filters. In an embodiment, a UV serum of the present disclosure can be applied approximately 15 minutes before sun exposure to all skin exposed to sun, and can be reapplied at least every 2 hours. In an embodiment, a UV serum of the present disclosure includes water, zinc oxide, sodium hyaluronate, titanium dioxide, silk, and vitamin C or a vitamin C derivative such as sodium ascorbyl phosphate. In an embodiment, a UV serum of the present disclosure protects skin and seals in moisture with the power of silk protein. In an embodiment, a UV serum of the present disclosure improves skin tone, promotes collagen production and diminishes the appearance of wrinkles and fine lines with the antioxidant abilities of vitamin C. In an embodiment, a UV serum of the present disclosure delivers moisture for immediate and long-term hydration throughout the day with concentrated hyaluronic acid. In an embodiment, a UV serum of the present disclosure helps prevent sunburn with the combined action of zinc oxide and titanium dioxide. In an embodiment, a UV serum of the present disclosure is designed to protect, hydrate, and diminish fine lines while shielding skin from harsh UVA and UVB rays. In an embodiment, the silk protein in a UV serum of the present disclosure stabilizes and protects skin while sealing in moisture, without the use of harsh chemical preservatives or synthetic additives. In an embodiment, the vitamin C/derivative in a UV serum of the present disclosure acts as a powerful antioxidant that supports skin rejuvenation. In an embodiment, the sodium hyaluronate in a UV serum of the present disclosure nourishes the skin and delivers moisture for long-lasting hydration. In an embodiment, the zinc oxide and titanium dioxide in a UV serum of the present disclosure shields skin from harmful UVA and UVB rays. The silk protein stabilization matrix in a UV serum of the present disclosure protects the active ingredients from the air, to deliver their full benefits without the use of harsh chemicals or preservatives. The silk matrix also traps moisture within the skin furthering the hydrating effect of the sodium hyaluronate. Example 15. Dark Spot Films of the Present Disclosure To reduce the appearance of dark spots, a high concentration of vitamin C may be necessary to reverse the overproduction of melanin. In this example, a 40% vitamin C (1.5:1 silk to vitamin C) was studied. The size and shape of the film can be made appropriate to a targeted area, for example to a small circular film of diameter 1 in (2.54 cm). The dark spot film, or a similar film of the present disclosure, of varying vitamin C concentration (0-50%) can be applied as a hydrofilm. Skin can be wetted with water. The film is then applied to the wet area. Water is then applied to the top surface of the film to turn it into a gel. The gel can then be spread and gently massaged into the application area. Table 34 provides details of an embodiment of a hydrofilm of the present disclosure (with no insoluble border). TABLE 34An Embodiment of a hydrofilm of the present disclosure% Silk Solution2.56%(60 minute boil, 25 kDA)Quantity Vitamin C15.62 mg total (10 mg(l-ascorbic acid)in 1 in circle punch out)Volume of solution per mold2.44 mLFilm Size1.25 in diameter circle(7.917 cm{circumflex over ( )}2) A film of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. A film of the present disclosure can be made with from about 1% to about 50% 1-ascorbic acid. A film of the present disclosure is soluble in water (insoluble border is removed by punching out the center of the film). A film of the present disclosure can adhere to skin with water. A film of the present disclosure can be spread on skin once water is applied. A film of the present disclosure can be dried when the humidity of drying equipment is 16-40% and below the humidity of the lab. A film of the present disclosure can be clear/transparent. In an embodiment, a dark spot film of the present disclosure includes water, silk, and vitamin C (L-ascorbic acid). In an embodiment, a dark spot film of the present disclosure includes 40% vitamin C. In an embodiment, a dark spot film of the present disclosure reduces skin pigmentation and evens skin tone in a targeted area with daily use. Vitamin C can inhibit pigment transfer from pigment producing cells, called melanocytes, to skin surface cells with continual application. In an embodiment, a dark spot film of the present disclosure can be applied to clean, dampened skin for 20 minutes. In an embodiment, additional water can be applied to an adhered film. The silk protein stabilization matrix in a dark spot film of the present disclosure protects the active ingredients from the air, to deliver their full benefits without the use of harsh chemicals or preservatives, such as paraben and phthalate. Thus, a dark spot film of the present disclosure is paraben and phthalate-free. Table 35 provides details of an embodiment of a film of the present disclosure. TABLE 35An Embodiment of a Film of the Present Disclosure% Silk Solution (60 minute boil, 25 kDA)2.2%Surface area5.07cm{circumflex over ( )}2Volume of silk solution for casting1.56mLMass of silk per film:34mgMass of l-ascorbic acid per film:23mgConcentration of l-ascorbic acid in film:40%pH3 A 2.1% silk solution of the present disclosure (0.321 mL/cm{circumflex over ( )}2) to 2.4% silk solution of the present disclosure (0.282 mL/cm{circumflex over ( )}2) can been used to create dark spot films of the present disclosure with 34 mg of silk (6.7 mg/cm{circumflex over ( )}2). In an embodiment, a 2.2% silk solution of the present disclosure (60 minute boil, 25 kDA) is used to produce a film of the present disclosure. The % silk and volume of solution can vary to produce equivalent films. A dark spot film of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. A dark spot film of the present disclosure can be made with from about 15 to about 50% 1-ascorbic acid. A dark spot film of the present disclosure is soluble in water (insoluble border). A dark spot film of the present disclosure is clear/transparent. A dark spot film of the present disclosure has a pH=3 when water is applied. A dark spot film of the present disclosure can adhere to skin with water. A dark spot film of the present disclosure can dry when humidity of drying equipment is 16-40% and below the humidity of the lab Example 16. High Concentration Vitamin C Gels of the Present Disclosure High concentration vitamin C gels were pursued up to 20%. Vitamin C type, vitamin C concentration, % silk and pH were varied to increase the quantity of vitamin C in a gel. FIGS.95A-95Care tables summarizing embodiments of high concentration vitamin C gels of the present disclosure. The highest concentration of vitamin C to gel was a 15% ascorbic acid 2 glucoside gel with 3.8% silk solution after 12 days. 5 and 10% ascorbic acid-2-glucoside formulations with 2, 3 and 3.8% silk all gelled. For each group of % vitamin C, gelation first occurred in the 3.8% silk followed by the 3% and lastly the 2%. It appears that there is a relationship between vitamin C concentration, silk concentration and gelation. If a solution has too much vitamin C in relation to silk, gelation will be prevented. Therefore, in order to produce high concentration vitamin C gels, higher concentration silk is necessary. One sample was cast at 5.5% silk and 20% vitamin C but gelation did not occur and a higher % silk may be necessary. Samples were also brought to a pH of 2 with lactic acid in order to help induce gelation in 3% silk solutions with 10 or 20% vitamin C, however gelation did not occur in 12 days. Example 17. Microbiological Study of Gels of the Present Disclosure Contaminating micro-organisms in cosmetics may cause a spoilage of the product and, when pathogenic, they represent a serious health risk for consumers worldwide. The United States Pharmacopoeia (USP) Microbial Limits Test provides several methods for the determination of total microbial count for bacteria, yeast and mold. Various gels of the present disclosure were tested to evaluate the possible microbial contamination in three different states of their use (intact, in-use, ending product).FIG.96is a table summarizing the results of such testing. The samples of gel and water samples from carboys were analyzed for determination of CFU/mL (colony forming units per milliliter) of aerobic bacteria as well as yeast and mold. Samples were exposed to growth medium of Tryptic Soy Agar (TSA) for bacteria and Potato Dextrose Agar (PDA) for fungi (yeast/mold) at an exposure temperature of 23±3° C. Samples were incubated at 30.0±2° C. for 3 days (bacteria) and 5 days (Fungi). Samples were then observed for determination of colony-forming units/mL. The limit of detection for the assays was 10 CFU/ml or g for bacteria and fungi, and the values of <10 indicate that microorganisms could not be detected in the samples. Values of >1.00E+04 indicate that the microbial colonies are Too Numerous to Count in the dilutions plated. Example 18. UV Silk Foams and Liquids of the Present Disclosure In an embodiment, the vitamin C derivative sodium ascorbyl phosphate (DSM) was dissolved in water. Sodium hyaluronate (“HA”) was then added to the water, mixed vigorously, and left to fully dissolve. The result is a viscous liquid (depending on HA %). The viscosity of the HA solution allows even dispersion of the zinc oxide and titanium dioxide and therefore HA is typically mixed before addition of UV additives. The zinc oxide and titanium dioxide are added to the HA solution and mixed vigorously, for example with the use of an electric blender. 60 minute boiled (˜25 kDa) silk solution is then added and mixed to create a 1% silk formulation. Two formulations were created without the addition of sodium ascorbyl phosphate (samples “HU2” and “HU4”). For sample HU2, zinc oxide and titanium dioxide were added and mixed by blending with an electric blender and whisk. The result was a viscous white liquid (FIG.98andFIG.99). Silk was then added and blended with an electric blender and whisk. The solution became a creamy foam similar to shaving cream (FIG.97andFIG.100). Vitamin E in the form of dl-alpha tocopheryl acetate can be added to the solution to recover a viscous liquid texture that can be applied with a smooth even texture (FIG.98). With increasing the quantity of dl-alpha tocopheryl acetate, the formulation will become less foam-like and more of a smooth liquid or lotion texture. HU4 was split into two batches:FIG.99, batch 2 andFIG.100, batch 1. The first batch followed the same procedures to HU2 and became a foam. For the second batch of HU4, sodium ascorbyl phosphate was added and dissolved before adding any zinc, titanium or silk. The UV additives were then added by blending with an electric blender and whisk and created a standard white viscous liquid. Silk was then added with an electric blender and whisk. The result was slightly thicker viscous liquid than normally seen. Without wishing to be bound by theory, it appears the addition of sodium ascorbyl phosphate inhibits foaming. Without wishing to be bound by theory, it appears that whisking, as opposed to mixing or blending, creates a silk foam. TABLE 36Embodiments of UV Silk Foams and Liquids of the Present Disclosure% HAMassMassSodiumTotal(sodiumMassZnOTiO2AscorbylSampleVolume% silkhyaluronate)HA (g)% ZnO(g)% TiO2(g)Phosphate (g)HU25512.51.375126.631.65N/AHU427.513.50.9625123.330.8255.5Batch 1HU427.513.50.9625123.330.825N/ABatch 2 Example 19. Lyophilized Silk Powders of the Present Disclosure TABLE 37Embodiments of lyophilized silk powdersSilk SolutionTreatmentSoluble~60 kDa silk,lyophilize andno6% silk,cut with blenderpH = 7-8~60 kDa silk,lyophilize andno6% silk,cut with blenderpH = 10~25 kDa silk,lyophilize andyes6% silk,cut with blenderpH = 7-8~25 kDa silk,lyophilize andyes6% silk,cut with blenderpH = 10 The above silk solutions were transformed to a silk powder through lyophilization to remove bulk water and chopping to small pieces with a blender. pH was adjusted with sodium hydroxide. Low molecular weight silk (˜25 kDa) was soluble while high molecular weight silk (˜60 kDa) was not. The lyophilized silk powder can be advantageous for enhanced storage control ranging from 10 days to 10 years depending on storage and shipment conditions. The lyophilized silk powder can also be used as a raw ingredient in the pharmaceutical, medical, consumer, and electronic markets. Additionally, lyophilized silk powder can be re-suspended in water, HFIP, or an organic solution following storage to create silk solutions of varying concentrations, including higher concentration solutions than those produced initially. In an embodiment, aqueous pure silk fibroin-based protein fragment solutions of the present disclosure comprising 1%, 3%, and 5% silk by weight were each dispensed into a 1.8 L Lyoguard trays, respectively. All 3 trays were placed in a 12 ft2lyophilizer and a single run performed. The product was frozen with a shelf temperature of ≤−40° C. and held for 2 hours. The compositions were then lyophilized at a shelf temperature of −20° C., with a 3 hour ramp and held for 20 hours, and subsequently dried at a temperature of 30° C., with a 5 hour ramp and held for about 34 hours. Trays were removed and stored at ambient conditions until further processing. Each of the resultant lyophilized silk fragment compositions were able to dissolve in aqueous solvent and organic solvent to reconstitute silk fragment solutions between 0.1 wt % and 8 wt %. Heating and mixing were not required but were used to accelerate the dissolving rate. All solutions were shelf-stable at ambient conditions. In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 30 minute boil, has a molecular weight of about 57 kDa, a polydispersity of about 1.6, inorganic and organic residuals of less than 500 ppm, and a light amber color. In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 60 minute boil, has a molecular weight of about 25 kDa, a polydispersity of about 2.4, inorganic and organic residuals of less than 500 ppm, and a light amber color. A method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 6 kDa to about 16 kDa includes the steps of: degumming a silk source by adding the silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 60° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in an oven having a temperature of about 140° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk protein fragments, the aqueous solution comprising: fragments having an average weight average molecular weight ranging from about 6 kDa to about 16 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin-based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin-based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. A method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 17 kDa to about 38 kDa includes the steps of: adding a silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60° C. to about 100° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of pure silk fibroin-based protein fragments, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, wherein the aqueous solution of silk protein fragments comprises sodium carbonate residuals of between about 10 ppm and about 100 ppm, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises fragments having an average weight average molecular weight ranging from about 17 kDa to about 38 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin-based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin-based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. According to aspects illustrated herein, there is disclosed a method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 39 kDa to about 80 kDa, the method including the steps of: adding a silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of about 30 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60° C. to about 100° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of pure silk fibroin-based protein fragments, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, sodium carbonate residuals of between about 10 ppm and about 100 ppm, fragments having an average weight average molecular weight ranging from about 40 kDa to about 65 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin-based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin-based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin-based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin-based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While the methods of the present disclosure have been described in connection with the specific embodiments thereof, it will be understood that it is capable of further modification. Further, this application is intended to cover any variations, uses, or adaptations of the methods of the present disclosure, including such departures from the present disclosure as come within known or customary practice in the art to which the methods of the present disclosure pertain. | 173,599 |
11857664 | While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments. DETAILED DESCRIPTION Provided herein are methods for producing pure and highly scalable silk protein fragment (SPF) mixture solutions that may be used across multiple industries for a variety of applications. The solutions are generated from raw pure intact silk protein material and processed in order to remove any sericin and achieve the desired weight average molecular weight (MW) and polydispersity of the fragment mixture. Select method parameters may be altered to achieve distinct final silk protein fragment characteristics depending upon the intended use. The resulting final fragment solution is pure silk protein fragments and water with PPM to non-detectable levels of process contaminants, levels acceptable in the pharmaceutical, medical and consumer cosmetic markets. The concentration, size and polydispersity of silk protein fragments in the solution may further be altered depending upon the desired use and performance requirements. In an embodiment, the pure silk fibroin-based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 6 kDa to about 16 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the pure silk fibroin-based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 17 kDa to about 38 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the pure silk fibroin-based protein fragments in the solution are substantially devoid of sericin, have an average weight average molecular weight ranging from about 39 kDa to about 80 kDa, and have a polydispersity ranging from about 1.5 and about 3.0. In an embodiment, the silk solutions of the present disclosure may be used to generate articles, such as silk films of various shapes and sizes by varying water content/concentration, or sold as a raw ingredient into the medical, consumer, or electronics markets. In an embodiment, the solutions may be used to generate articles, such as silk gels of varying gel and liquid consistencies by varying water content/concentration, or sold as a raw ingredient into the pharmaceutical, medical, consumer, or electronics markets. Depending on the silk solution utilized and the methods for casting the films or gels, various properties are achieved. The articles may be loaded with at least one therapeutic agent and/or at least one molecule. As used herein, the terms “substantially sericin free” or “substantially devoid of sericin” refer to silk fibers in which a majority of the sericin protein has been removed. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 10.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 9.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 8.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 7.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 6.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.01% (w/w) and about 5.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.05% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.1% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 0.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 1.0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 1.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 2.0% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having between about 2.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content between about 0.01% (w/w) and about 0.1% (w/w). In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.1% (w/w). In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.05% (w/w). In an embodiment, when a silk source is added to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes, a degumming loss of about 26 wt. % to about 31 wt. % is obtained. As used herein, the term “substantially homogeneous” may refer to pure silk fibroin-based protein fragments that are distributed in a normal distribution about an identified molecular weight. As used herein, the term “substantially homogeneous” may refer to an even distribution of additive, for example vitamin C, throughout a composition of the present disclosure. As used herein, the term “substantially free of inorganic residuals” means that the composition exhibits residuals of 0.1% (w/w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.05% (w/w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.01% (w/w) or less. In an embodiment, the amount of inorganic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of inorganic residuals is ND to about 500 ppm. In an embodiment, the amount of inorganic residuals is ND to about 400 ppm. In an embodiment, the amount of inorganic residuals is ND to about 300 ppm. In an embodiment, the amount of inorganic residuals is ND to about 200 ppm. In an embodiment, the amount of inorganic residuals is ND to about 100 ppm. In an embodiment, the amount of inorganic residuals is between 10 ppm and 1000 ppm. As used herein, the term “substantially free of organic residuals” means that the composition exhibits residuals of 0.1% (w/w) or less. In an embodiment, substantially free of organic residuals refers to a composition that exhibits residuals of 0.05% (w/w) or less. In an embodiment, substantially free of organic residuals refers to a composition that exhibits residuals of 0.01% (w/w) or less. In an embodiment, the amount of organic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of organic residuals is ND to about 500 ppm. In an embodiment, the amount of organic residuals is ND to about 400 ppm. In an embodiment, the amount of organic residuals is ND to about 300 ppm. In an embodiment, the amount of organic residuals is ND to about 200 ppm. In an embodiment, the amount of organic residuals is ND to about 100 ppm. In an embodiment, the amount of organic residuals is between 10 ppm and 1000 ppm. Compositions of the present disclosure exhibit “biocompatibility” meaning that the compositions are compatible with living tissue or a living system by not being toxic, injurious, or physiologically reactive and not causing immunological rejection. Such biocompatibility can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. Compositions of the present disclosure are “hypoallergenic” meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. In an embodiment, a solution of the present disclosure is contacted with a therapeutic agent and/or a molecule prior to forming the article. In an embodiment, molecules include, but are not limited to, antioxidants and enzymes. In an embodiment, molecules include, but are not limited to, Selenium, Ubiquinone derivatives, Thiol-based antioxidants, Saccharide-containing antioxidants, Polyphenols, Botanical extracts, Caffeic acid, Apigenin, Pycnogenol, Resveratrol, Folic acid, Vitamin b12, Vitamin b6, Vitamin b3, Vitamin E, Vitamin C and derivatives thereof, Vitamin D, Vitamin A, Astaxathin, Lutein, Lycopene, Essential fatty acids (omegas 3 and 6), Iron, Zinc, magnesium, Flavonoids (soy, Curcumin, Silymarin, Pycnongeol), Growth factors, aloe, hyaluronic acid, extracellular matrix proteins, cells, nucleic acids, biomarkers, biological reagents, zinc oxide, benzyol peroxide, retnoids, titanium, allergens in a known dose (for sensitization treatment), essential oils including, but not limited to, lemongrass or rosemary oil, and fragrances. Therapeutic agents include, but are not limited to, small molecules, drugs, proteins, peptides and nucleic acids. In an embodiment, a silk film of the present disclosure includes a molecule that is a vitamin, such as vitamin C, vitamin A and vitamin E. In an embodiment, a solution of the present disclosure is contacted with an allergen of known quantity prior to forming the article. Allergens include but are not limited to milk, eggs, peanuts, tree nuts, fish, shellfish, soy and wheat. Known doses of allergen loaded within a silk article can be released at a known rate for controlled exposure allergy study, tests and sensitization treatment. In an embodiment, a solution of the present disclosure is used to create an article with microneedles by standard methods known to one in the art for controlled delivery of molecules or therapeutic agents to or through the skin. As used herein, the term “fibroin” includes silkworm fibroin and insect or spider silk protein. In an embodiment, fibroin is obtained fromBombyx mori. FIG.1is a flow chart showing various embodiments for producing pure silk fibroin-based protein fragments (SPFs) of the present disclosure. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure. As illustrated inFIG.1, step A, cocoons (heat-treated or non-heat-treated), silk fibers, silk powder or spider silk can be used as the silk source. If starting from raw silk cocoons fromBombyx mori, the cocoons can be cut into small pieces, for example pieces of approximately equal size, step B1. The raw silk is then extracted and rinsed to remove any sericin, step C1a. This results in substantially sericin free raw silk. In an embodiment, water is heated to a temperature between 84° C. and 100° C. (ideally boiling) and then Na2CO3(sodium carbonate) is added to the boiling water until the Na2CO3is completely dissolved. The raw silk is added to the boiling water/Na2CO3(100° C.) and submerged for approximately 15-90 minutes, where boiling for a longer time results in smaller silk protein fragments. In an embodiment, the water volume equals about 0.4× raw silk weight and the Na2CO3volume equals about 0.848× raw silk weight. In an embodiment, the water volume equals 0.1× raw silk weight and the Na2CO3volume is maintained at 2.12 g/L. This is demonstrated inFIG.62AandFIG.62B: silk mass (x-axis) was varied in the same volume of extraction solution (i.e., the same volume of water and concentration of Na2CO3) achieving sericin removal (substantially sericin free) as demonstrated by an overall silk mass loss of 26 to 31 percent (y-axis). Subsequently, the water dissolved Na2CO3solution is drained and excess water/Na2CO3is removed from the silk fibroin fibers (e.g., ring out the fibroin extract by hand, spin cycle using a machine, etc.). The resulting silk fibroin extract is rinsed with warm to hot water to remove any remaining adsorbed sericin or contaminate, typically at a temperature range of about 40° C. to about 80° C., changing the volume of water at least once (repeated for as many times as required). The resulting silk fibroin extract is a substantially sericin-depleted silk fibroin. In an embodiment, the resulting silk fibroin extract is rinsed with water at a temperature of about 60° C. In an embodiment, the volume of rinse water for each cycle equals 0.1 L to 0.2 L×raw silk weight. It may be advantageous to agitate, turn or circulate the rinse water to maximize the rinse effect. After rinsing, excess water is removed from the extracted silk fibroin fibers (e.g., ring out fibroin extract by hand or using a machine). Alternatively, methods known to one skilled in the art such as pressure, temperature, or other reagents or combinations thereof may be used for the purpose of sericin extraction. Alternatively, the silk gland (100% sericin free silk protein) can be removed directly from a worm. This would result in liquid silk protein, without any alteration of the protein structure, free of sericin. The extracted fibroin fibers are then allowed to dry completely.FIG.3is a photograph showing dry extracted silk fibroin. Once dry, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at a temperature between ambient and boiling, step C1b. In an embodiment, the solvent is a solution of Lithium bromide (LiBr) (boiling for LiBr is 140° C.). Alternatively, the extracted fibroin fibers are not dried but wet and placed in the solvent; solvent concentration can then be varied to achieve similar concentrations as to when adding dried silk to the solvent. The final concentration of LiBr solvent can range from 0.1 M to 9.3 M.FIG.63is a table summarizing the Molecular Weights of silk dissolved from different concentrations of Lithium Bromide (LiBr) and from different extraction and dissolution sizes. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature along with the concentration of dissolving solvent. Other solvents may be used including, but not limited to, phosphate phosphoric acid, calcium nitrate, calcium chloride solution or other concentrated aqueous solutions of inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed within the already heated solvent solution and then maintained at a temperature ranging from about 60° C. to about 140° C. for 1-168 hrs. In an embodiment, the silk fibers should be fully immersed within the solvent solution and then placed into a dry oven at a temperature of about 100° C. for about 1 hour. The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an effect on the time required to completely dissolve the fibroin and on the resulting molecular weight and polydispersity of the final SPF mixture solution. In an embodiment, silk solvent solution concentration is less than or equal to 20% w/v. In addition, agitation during introduction or dissolution may be used to facilitate dissolution at varying temperatures and concentrations. The temperature of the LiBr solution will provide control over the silk protein fragment mixture molecular weight and polydispersity created. In an embodiment, a higher temperature will more quickly dissolve the silk offering enhanced process scalability and mass production of silk solution. In an embodiment, using a LiBr solution heated to a temperature between 80° C.-140° C. reduces the time required in an oven in order to achieve full dissolution. Varying time and temperature at or above 60° C. of the dissolution solvent will alter and control the MW and polydispersity of the SPF mixture solutions formed from the original molecular weight of the native silk fibroin protein. Alternatively, whole cocoons may be placed directly into a solvent, such as LiBr, bypassing extraction, step B2. This requires subsequent filtration of silk worm particles from the silk and solvent solution and sericin removal using methods know in the art for separating hydrophobic and hydrophilic proteins such as a column separation and/or chromatography, ion exchange, chemical precipitation with salt and/or pH, and or enzymatic digestion and filtration or extraction, all methods are common examples and without limitation for standard protein separation methods, step C2. Non-heat treated cocoons with the silkworm removed, may alternatively be placed into a solvent such as LiBr, bypassing extraction. The methods described above may be used for sericin separation, with the advantage that non-heat treated cocoons will contain significantly less worm debris. Dialysis may be used to remove the dissolution solvent from the resulting dissolved fibroin protein fragment solution by dialyzing the solution against a volume of water, step E1. Pre-filtration prior to dialysis is helpful to remove any debris (i.e., silk worm remnants) from the silk and LiBr solution, step D. In one example, a 3 μm or 5 μm filter is used with a flow-rate of 200-300 mL/min to filter a 0.1% to 1.0% silk-LiBr solution prior to dialysis and potential concentration if desired. A method disclosed herein, as described above, is to use time and/or temperature to decrease the concentration from 9.3 M LiBr to a range from 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering creating a scalable process method. Alternatively, without the use of additional time or temperate, a 9.3 M LiBr-silk protein fragment solution may be diluted with water to facilitate debris filtration and dialysis. The result of dissolution at the desired time and temperate filtration is a translucent particle-free room temperature shelf-stable silk protein fragment-LiBr solution of a known MW and polydispersity. It is advantageous to change the dialysis water regularly until the solvent has been removed (e.g., change water after 1 hour, 4 hours, and then every 12 hours for a total of 6 water changes). The total number of water volume changes may be varied based on the resulting concentration of solvent used for silk protein dissolution and fragmentation. After dialysis, the final silk solution maybe further filtered to remove any remaining debris (i.e., silk worm remnants). Alternatively, Tangential Flow Filtration (TFF), which is a rapid and efficient method for the separation and purification of biomolecules, may be used to remove the solvent from the resulting dissolved fibroin solution, step E2. TFF offers a highly pure aqueous silk protein fragment solution and enables scalability of the process in order to produce large volumes of the solution in a controlled and repeatable manner. The silk and LiBr solution may be diluted prior to TFF (20% down to 0.1% silk in either water or LiBr). Pre-filtration as described above prior to TFF processing may maintain filter efficiency and potentially avoids the creation of silk gel boundary layers on the filter's surface as the result of the presence of debris particles. Pre-filtration prior to TFF is also helpful to remove any remaining debris (i.e., silk worm remnants) from the silk and LiBr solution that may cause spontaneous or long-term gelation of the resulting water only solution, step D. TFF, recirculating or single pass, may be used for the creation of water-silk protein fragment solutions ranging from 0.1% silk to 30.0% silk (more preferably, 0.1%-6.0% silk). Different cutoff size TFF membranes may be required based upon the desired concentration, molecular weight and polydispersity of the silk protein fragment mixture in solution. Membranes ranging from 1-100 kDa may be necessary for varying molecular weight silk solutions created for example by varying the length of extraction boil time or the time and temperate in dissolution solvent (e.g., LiBr). In an embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution and to create the final desired silk-to-water ratio. As well, TFF single pass, TFF, and other methods known in the art, such as a falling film evaporator, may be used to concentrate the solution following removal of the dissolution solvent (e.g., LiBr) (with resulting desired concentration ranging from 0.1% to 30% silk). This can be used as an alternative to standard HFIP concentration methods known in the art to create a water-based solution. A larger pore membrane could also be utilized to filter out small silk protein fragments and to create a solution of higher molecular weight silk with and/or without tighter polydispersity values.FIG.61is a table summarizing Molecular Weights for some embodiments of silk protein solutions of the present disclosure. Silk protein solution processing conditions were as follows: 100° C. extraction for 20 min, room temperature rinse, LiBr in 60° C. oven for 4-6 hours. TFF processing conditions for water-soluble films were as follows: 100° C. extraction for 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min.FIGS.67-78further demonstrate manipulation of extraction time, LiBr dissolution conditions, and TFF processing and resultant example molecular weights and polydispersities. These examples are not intended to be limiting, but rather to demonstrate the potential of specifying parameters for specific molecular weight silk fragment solutions. An assay for LiBr and Na2CO3detection was performed using an HPLC system equipped with evaporative light scattering detector (ELSD). The calculation was performed by linear regression of the resulting peak areas for the analyte plotted against concentration. More than one sample of a number of formulations of the present disclosure was used for sample preparation and analysis. Generally, four samples of different formulations were weighed directly in a 10 mL volumetric flask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and kept at 2-8° C. for 2 hours with occasional shaking to extract analytes from the film. After 2 hours the solution was diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask was transferred into HPLC vials and injected into the HPLC-ELSD system for the estimation of sodium carbonate and lithium bromide. The analytical method developed for the quantitation of Na2CO3and LiBr in silk protein formulations was found to be linear in the range 10-165 μg/mL, with RSD for injection precision as 2% and 1% for area and 0.38% and 0.19% for retention time for sodium carbonate and lithium bromide respectively. The analytical method can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein formulations. The final silk protein fragment solution, as shown inFIG.4, is pure silk protein fragments and water with PPM to undetectable levels of particulate debris and/or process contaminants, including LiBr and Na2CO3.FIG.55andFIG.58are tables summarizing LiBr and Na2CO3concentrations in solutions of the present disclosure. InFIG.55, the processing conditions included 100° C. extraction for 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min. TFF conditions including pressure differential and number of dia-filtration volumes were varied. InFIG.58, the processing conditions included 100° C. boil for 60 min, 60° C. rinse, LiBr in 60° C. oven for 4-6 hours. In an embodiment, a SPF composition of the present disclosure is not soluble in an aqueous solution due to the crystallinity of the protein. In an embodiment, a SPF composition of the present disclosure is soluble in an aqueous solution. In an embodiment, the SPFs of a composition of the present disclosure include a crystalline portion of about two-thirds and an amorphous region of about one-third. In an embodiment, the SPFs of a composition of the present disclosure include a crystalline portion of about one-half and an amorphous region of about one-half. In an embodiment, the SPFs of a composition of the present disclosure include a 99% crystalline portion and a 1% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 95% crystalline portion and a 5% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 90% crystalline portion and a 10% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 85% crystalline portion and a 15% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 80% crystalline portion and a 20% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 75% crystalline portion and a 25% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 70% crystalline portion and a 30% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 65% crystalline portion and a 35% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 60% crystalline portion and a 40% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 50% crystalline portion and a 50% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 40% crystalline portion and a 60% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 35% crystalline portion and a 65% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 30% crystalline portion and a 70% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 25% crystalline portion and a 75% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 20% crystalline portion and a 80% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 15% crystalline portion and a 85% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 10% crystalline portion and a 90% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 5% crystalline portion and a 90% amorphous region. In an embodiment, the SPFs of a composition of the present disclosure include a 1% crystalline portion and a 99% amorphous region. A unique feature of the SPF compositions of the present disclosure are shelf stability (they will not slowly or spontaneously gel when stored in an aqueous solution and there is no aggregation of fragments and therefore no increase in molecular weight over time), from 10 days to 3 years depending on storage conditions, percent silk, and number of shipments and shipment conditions. Additionally pH may be altered to extend shelf-life and/or support shipping conditions by preventing premature folding and aggregation of the silk. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 2 weeks at room temperature (RT). In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 4 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 6 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 8 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 10 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 12 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability ranging from about 4 weeks to about 52 weeks at RT. Table 1 below shows shelf stability test results for embodiments of SPF compositions of the present disclosure. TABLE 1Shelf Stability of SPF Compositions of the Present Disclosure% SilkTemperatureTime to Gelation2RT4 weeks24 C.>9 weeks4RT4 weeks44 C.>9 weeks6RT2 weeks64 C.>9 weeks A known additive such as a vitamin (e.g., vitamin C) can be added to a SPF composition of the present disclosure to create a gel that is stable from 10 days to 3 years at room temperature (RT). Both examples, a SPF composition and the same with an additive, can be lyophilized for enhanced storage control ranging from 10 days to 10 years depending on storage and shipment conditions. The lyophilized silk powder can also be used as a raw ingredient in the medical, consumer, and electronic markets. Additionally, lyophilized silk powder can be resuspended in water, HFIP, or organic solution following storage to create silk solutions of varying concentrations, including higher concentration solutions than those produced initially. In another embodiment, the silk fibroin-based protein fragments are dried using a rototherm evaporator or other methods known in the art for creating a dry protein form containing less than 10% water by mass. Either the silk fragment-water solutions or the lyophilized silk protein fragment mixture can be sterilized following standard methods in the art not limited to filtration, heat, radiation or e-beam. It is anticipated that the silk protein fragment mixture, because of its shorter protein polymer length, will withstand sterilization better than intact silk protein solutions described in the art. Additionally, silk articles created from the SPF mixtures described herein may be sterilized as appropriate to application. For example, a silk film loaded with a molecule to be used in medical applications with an open wound/incision, may be sterilized standard methods such as by radiation or e-beam. FIG.2is a flow chart showing various parameters that can be modified during the process of producing a silk protein fragment solution of the present disclosure during the extraction and the dissolution steps. Select method parameters may be altered to achieve distinct final solution characteristics depending upon the intended use, e.g., molecular weight and polydispersity. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure. In an embodiment, a process for producing a silk protein fragment solution of the present disclosure includes forming pieces of silk cocoons from theBombyx morisilk worm; extracting the pieces at about 100° C. in a solution of water and Na2CO3for about 60 minutes, wherein a volume of the water equals about 0.4× raw silk weight and the amount of Na2CO3is about 0.848× the weight of the pieces to form a silk fibroin extract; triple rinsing the silk fibroin extract at about 60° C. for about 20 minutes per rinse in a volume of rinse water, wherein the rinse water for each cycle equals about 0.2 L×the weight of the pieces; removing excess water from the silk fibroin extract; drying the silk fibroin extract; dissolving the dry silk fibroin extract in a LiBr solution, wherein the LiBr solution is first heated to about 100° C. to create a silk and LiBr solution and maintained; placing the silk and LiBr solution in a dry oven at about 100° C. for about 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into mixture with desired molecular weight and polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to result in a 1% silk solution; and removing solvent from the solution using Tangential Flow Filtration (TFF). In an embodiment, a 10 kDa membrane is utilized to purify the silk solution and create the final desired silk-to-water ratio. TFF can then be used to further concentrate the pure silk solution to a concentration of 2% silk to water. Each process step from raw cocoons to dialysis is scalable to increase efficiency in manufacturing. Whole cocoons are currently purchased as the raw material, but pre-cleaned cocoons or non-heat treated cocoons, where worm removal leaves minimal debris, have also been used. Cutting and cleaning the cocoons is a manual process, however for scalability this process could be made less labor intensive by, for example, using an automated machine in combination with compressed air to remove the worm and any particulates, or using a cutting mill to cut the cocoons into smaller pieces. The extraction step, currently performed in small batches, could be completed in a larger vessel, for example an industrial washing machine where temperatures at or in between 60° C. to 100° C. can be maintained. The rinsing step could also be completed in the industrial washing machine, eliminating the manual rinse cycles. Dissolution of the silk in LiBr solution could occur in a vessel other than a convection oven, for example a stirred tank reactor. Dialyzing the silk through a series of water changes is a manual and time intensive process, which could be accelerated by changing certain parameters, for example diluting the silk solution prior to dialysis. The dialysis process could be scaled for manufacturing by using semi-automated equipment, for example a tangential flow filtration system. Varying extraction (i.e., time and temperature), LiBr (i.e., temperature of LiBr solution when added to silk fibroin extract or vice versa) and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, homogeneities, and colors (seeFIGS.5-32). Increasing the temperature for extraction, lengthening the extraction time, using a higher temperature LiBr solution at emersion and over time when dissolving the silk and increasing the time at temperature (e.g., in an oven as shown here, or an alternative heat source) all resulted in less viscous and more homogeneous solvent and silk solutions. While almost all parameters resulted in a viable silk solution, methods that allow complete dissolution to be achieved in fewer than 4 to 6 hours are preferred for process scalability. FIGS.5-10show photographs of four different silk extraction combinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr was prepared and allowed to sit at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in the 60° C. oven. Samples from each set were removed at 4, 6, 8, 12, 24, 168 and 192 hours. The remaining sample was photographed. FIGS.11-23show photographs of four different silk extraction combinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60° C., 80° C., 100° C. or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the 60° C. oven. Samples from each set were removed at 1, 4 and 6 hours. The remaining sample was photographed. FIGS.24-32show photographs of four different silk extraction combinations tested: Four different silk extraction combinations were used: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60° C., 80° C., 100° C. or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the oven at the same temperature of the LiBr. Samples from each set were removed at 1, 4 and 6 hours. 1 mL of each sample was added to 7.5 mL of 9.3 M LiBr and refrigerated for viscosity testing. The remaining sample was photographed. Molecular weight of the silk protein fragments may be controlled based upon the specific parameters utilized during the extraction step, including extraction time and temperature; specific parameters utilized during the dissolution step, including the LiBr temperature at the time of submersion of the silk in to the lithium bromide and time that the solution is maintained at specific temperatures; and specific parameters utilized during the filtration step. By controlling process parameters using the disclosed methods, it is possible to create SPF mixture solutions with polydispersity equal to or lower than 2.5 at a variety of different molecular weight ranging from 5 kDa to 200 kDa, more preferably between 10 kDa and 80 kDA. By altering process parameters to achieve silk solutions with different molecular weights, a range of fragment mixture end products, with desired polydispersity of equal to or less than 2.5 may be targeted based upon the desired performance requirements. For example, a lower molecular weight silk film containing a drug may have a faster release rate compared to a higher molecular weight film making it more ideal for a daily delivery vehicle in consumer cosmetics. Additionally, SPF mixture solutions with a polydispersity of greater than 2.5 can be achieved. Further, two solutions with different average molecular weights and polydispersities can be mixed to create combination solutions. Alternatively, a liquid silk gland (100% sericin free silk protein) that has been removed directly from a worm could be used in combination with any of the SPF mixture solutions of the present disclosure. Molecular weight of the pure silk fibroin-based protein fragment composition was determined using High Pressure Liquid Chromatography (HPLC) with a Refractive Index Detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC/SEC Software Version 3.3 (Agilent). Parameters were varied during the processing of raw silk cocoons into silk solution. Varying these parameters affected the MW of the resulting silk solution. Parameters manipulated included (i) time and temperature of extraction, (ii) temperature of LiBr, (iii) temperature of dissolution oven, and (iv) dissolution time. Molecular weight was determined with mass spec as shown inFIGS.64-80. Experiments were carried out to determine the effect of varying the extraction time.FIGS.64-70are graphs showing these results, and Tables 2-8 summarize the results. Below is a summary:A sericin extraction time of 30 minutes resulted in larger MW than a sericin extraction time of 60 minutesMW decreases with time in the oven140° C. LiBr and oven resulted in the low end of the confidence interval to be below a MW of 9500 Da30 min extraction at the 1 hour and 4 hour time points have undigested silk30 min extraction at the 1 hour time point resulted in a significantly high molecular weight with the low end of the confidence interval being 35,000 DaThe range of MW reached for the high end of the confidence interval was 18000 to 216000 Da (important for offering solutions with specified upper limit) TABLE 2The effect of extraction time (30 min vs 60 min)on molecular weight of silk processed under theconditions of 100° C. Extraction Temperature,100° C. Lithium Bromide (LiBr) and 100° C. OvenDissolution (Oven/Dissolution Time was varied).BoilOvenAverageStdConfidenceTimeTimeMwdevIntervalPD301572471278035093933871.6360131520138711633854072.71304409732632142681176582.8760425082124810520598032.3830625604140510252639432.5060620980126210073436952.08 TABLE 3The effect of extraction time (30 min vs 60 min)on molecular weight of silk processed under theconditions of 100° C.Extraction Temperature, boiling LithiumBromide (LiBr) and 60° C. Oven Dissolution for 4 hr.BoilAverageStdConfidenceSampleTimeMwdevIntervalPD30 min, 4 hr30496564580173061424782.8760 min, 4 hr6030042153611183807052.69 TABLE 4The effect of extraction time (30 min vs 60 min)on molecular weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60° C. Lithium Bromide (LiBr)and 60° C. Oven Dissolution (Oven/Dissolution Time was varied).OvenAverageStdConfidenceSampleBoil TimeTimeMwdevIntervalPD30 min, 1 hr30158436222011538092.6360 min, 1 hr6013170011931842242.6630 min, 4 hr30461956.513337214631788472.8960 min, 4 hr60425578.524469979655642.56 TABLE 5The effect of extraction time (30 min vs 60 min)on molecular weight of silk processedunder the conditions of 100° C. Extraction Temperature, 80° C.Lithium Bromide (LiBr) and 80° C. Oven Dissolution for 6 hr.AverageStdConfidenceSampleBoil TimeMwdevIntervalPD30 min, 6 hr3063510186932157753.4060 min, 6 hr60251642389637657062.61 TABLE 6The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of100° C. Extraction Temperature, 80° C. Lithium Bromide (LiBr)and 60° C. Oven Dissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD30 min, 4 hr3045920214028190731837603.1060 min, 4 hr60426312.563710266674422.5630 min, 6 hr30646824180761212932.5960 min, 6 hr6062635310168683022.59 TABLE 7The effect of extraction time (30 min vs 60 min)on molecular weight of silk processed under the conditionsof 100° C. Extraction Temperature, 100° C. Lithium Bromide (LiBr)and 60° C. Oven Dissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD30 min, 4 hr30447853197581159002.4260 min, 4 hr60425082124810520598042.3830 min, 6 hr306554218992191531603662.8960 min, 6 hr60620980126210073436942.08 TABLE 8The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C.Extraction Temperature, 140° C. Lithium Bromide (LiBr) and140° C. Oven Dissolution (Oven/Dissolution Time was varied).BoilOvenAverageConfidenceSampleTimeTimeMwStd devIntervalPD30 min, 4 hr3049024.511024493181272.0086560 min, 4 hr604155486954347622.235830 min, 6 hr306130215987283192.174960 min, 6 hr606108885364221002.0298 Experiments were carried out to determine the effect of varying the extraction temperature.FIG.71is a graph showing these results, and Table 9 summarizes the results. Below is a summary:Sericin extraction at 90° C. resulted in higher MW than sericin extraction at 100° C. extractionBoth 90° C. and 100° C. show decreasing MW over time in the oven TABLE 9The effect of extraction temperature (90° C. vs. 100° C.) onmolecular weight of silk processed under the conditions of60 min. Extraction Temperature, 100° C. Lithium Bromide (LiBr) and100° C. Oven Dissolution (Oven/Dissolution Time was varied).SampleBoil TimeOven TimeAverage MwStd devConfidence IntervalPD90° C., 4 hr604373084204133681041192.79100° C., 4 hr60425082124810520598042.3890° C., 6 hr60634224113512717921002.69100° C., 6 hr60620980126210073436942.08 Experiments were carried out to determine the effect of varying the Lithium Bromide (LiBr) temperature when added to silk.FIGS.72-73are graphs showing these results, and Tables 10-11 summarize the results. Below is a summary:No impact on MW or confidence interval (all CI˜10500-6500 Da)Studies illustrated that the temperature of LiBr-silk dissolution, as LiBr is added and begins dissolving, rapidly drops below the original LiBr temperature due to the majority of the mass being silk at room temp TABLE 10The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 60 min.Extraction Time., 100° C. Extraction Temperature and60° C. Oven Dissolution (Oven/Dissolution Time was varied).LiBrTempOvenAverageConfidenceSample(° C.)TimeMwStd devIntervalPD60° C. LiBr,6013170011931842232.661 hr100° C. LiBr,10012790720010735725522.601 hrRT LiBr, 4 hrRT429217108210789791192.7160° C. LiBr,6042557824459978655642.564 hr80° C. LiBr,8042631263710265674412.564 hr100° C. LiBr,100427681172911279679312.454 hrBoil LiBr,Boil430042153511183807042.694 hrRT LiBr, 6 hrRT626543189310783653322.4680° C. LiBr,8062635310167683012.596 hr100° C. LiBr,10062715091611020668892.466 hr TABLE 11The effect of Lithium Bromide (LiBr) temperature onmolecular weight of silk processed under the conditionsof 30 min. Extraction Time, 100° C. Extraction Temperatureand 60° C. Oven Dissolution (Oven/Dissolution Time was varied).LiBrTempOvenAverageConfidenceSample(° C.)TimeMwStd devIntervalPD60° C. LiBr,6046195613336214631788472.894 hr80° C. LiBr,8045920214027190731837603.104 hr100° C. LiBr,100447853197571158992.424 hr80° C. LiBr,80646824180751212922.596 hr100° C. LiBr,1006554218991191521603662.896 hr Experiments were carried out to determine the effect of v oven/dissolution temperature.FIGS.74-78are graphs showing these results, and Tables 12-16 summarize the results. Below is a summary:Oven temperature has less of an effect on 60 min extracted silk than 30 min extracted silk. Without wishing to be bound by theory, it is believed that the 30 min silk is less degraded during extraction and therefore the oven temperature has more of an effect on the larger MW, less degraded portion of the silk.For 60° C. vs. 140° C. oven the 30 min extracted silk showed a very significant effect of lower MW at higher oven temp, while 60 min extracted silk had an effect but much lessThe 140° C. oven resulted in a low end in the confidence interval at ˜6000 Da TABLE 12The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 100° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied)OvenBoilTempOvenAverageStdConfidenceTime(° C.)TimeMwdevIntervalPD3060447853197581159002.42301004409732632142681176582.8730606554218992191531603662.8930100625604140510252639432.50 TABLE 13The effect of oven/dissolution temperature on molecularweight of silk processed under the conditions of 100° C.Extraction Temperature, 60 min. Extraction Time, and 100° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied).OvenBoilTempOvenAverageStdConfidenceTime(° C.)TimeMwdevIntervalPD606012790820010735725522.6060100131520138711633854072.716060427681173011279725522.6260100425082124810520598032.38606062715091611020668892.4660100620980126210073436952.08 TABLE 14The effect of oven/dissolution temperature on molecular weight ofsilk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 140° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied).BoilOvenOvenAverageConfidenceTimeTemp(° C.)TimeMwStd devIntervalPD6060430042153611183807052.69601404155487255333222.14 TABLE 15The effect of oven/dissolution temperature on molecular weight ofsilk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 140° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied).OvenBoilTempOvenAverageStdConfidenceTime(° C.)TimeMwdevIntervalPD30604496564580173061424782.87301404902511024493181272.01306065938311640176411998893.37301406130215987283192.17 TABLE 16The effect of oven/dissolution temperature on molecular weight ofsilk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 80° C.Lithium Bromide (LiBr) (Oven/Dissolution Time was varied).OvenBoilTempOvenAverageStdConfidenceTime(° C.)TimeMwdevIntervalPD606042631363710266674422.566080430308429312279748062.47606062635310168683022.5960806251642389637657062.61 In an embodiment, the methods disclosed herein result in a solution with characteristics that can be controlled during manufacturing, including, but not limited to: MW—may be varied by changing extraction and/or dissolution time and temp (e.g., LiBr temperature), pressure, and filtration (e.g., size exclusion chromatography); Structure—removal or cleavage of heavy or light chain of the fibroin protein polymer; Purity—hot water rinse temperature for improved sericin removal or filter capability for improved particulate removal that adversely affects shelf stability of the silk fragment protein mixture solution; Color—the color of the solution can be controlled with, for example, LiBr temp and time; Viscosity; Clarity; and Stability of solution. The resultant pH of the solution is typically about 7 and can be altered using an acid or base as appropriate to storage requirements. The above-described SPF mixture solutions may be utilized to produce a pure silk protein fragment-film or pure silk protein fragment-gel for numerous applications (e.g., delivery of a drug, vitamin, antioxidant, etc. to the skin).FIG.33is a flow chart showing an embodiment for producing a silk film of the present disclosure from a silk solution of the present disclosure. In step A, a silk solution of the present disclosure is chosen, and then at least on molecule or therapeutic agent is added directly to the silk solution prior to gel or film processing, step B. When producing a silk film, the silk solution with additive(s) may be cast directly onto a shaped mold to achieve a unique film shape (e.g., silicone mold) or the silk solution may be cast as a sheet and then subsequently cut or punched into a variety of shapes, with a variety of cutting techniques, including, but not limited to cutting with a rotary blade or laser cutting for example (FIGS.83A and83B), depending upon the desired application, step C. If cast on a mold, for example silicone, the silicone mold may be heated on a laser-etched/patterned surface to create an impression that will be transferred to the final film. For example, the product logo could be transferred to the film, visible, but not palpable by hand, and used to show authenticity of the product. The concentration and/or mass of the final silk protein fragment film can be varied to control the film's degree of flexibility and conformity to different anatomical topographies. Altering the drying method for a silk film will also result in different final film characteristics. Applying airflow and/or heat impacts the properties of the film (e.g., brittleness, number of bubbles, curling, solubility, surface appearance), step D. Additionally, the percent moisture within the film at the time of packaging will impact stability over time with too much moisture resulting in yellowing of the films with time (FIGS.82A-82C). In some embodiments, films ideally may have between about 2 to about 20% water content at completion of drying. It was observed that greater moisture content than 20% in the films will decrease shelf life. If films are not dry enough (that is they have greater than 20% water content) before packaging, they will yellow over time (2+ weeks). It is advised that films are dried in an incubator until the relative humidity in the incubator is less than the relative humidity in the surrounding area and no greater than 36%. Ambient humidity will have an effect on the ability to remove moisture and therefore, a tactile/audio test can be used to determine whether films are ready for packaging. In an embodiment, the test includes removal of a film from the drying system, slightly bending one end of the film and releasing it. If the film feels and sounds similar to a piece of paper or thin plastic, it is considered dry. If the film has not completed drying, it will be pliable and will make no noise upon bending and release. In an embodiment, the film is flexible without the need for process additives such as glycerin, such that a film that is 2.5 cm wide by 10 cm long can be bent in half so that opposite ends of the film can touch one another without the film breaking or cracking. A film of this same size can be bent in half along the length of the film to create a 45-degree angle without breaking or cracking the film. The final silk protein fragment-film is pure with undetectable levels of particulate debris and/or process contaminants, including LiBr and Na2CO3. Alternatively, the final SPF mixture solution has less than 500 ppm process contaminants.FIG.56andFIG.57are tables summarizing LiBr and Na2CO3concentrations in films (2% silk films air dried at RT) of the present disclosure. InFIG.56, the processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours. InFIG.57, the processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours. In an embodiment, when producing a silk gel, an acid is used to help facilitate gelation. In an embodiment, when producing a silk gel that includes a neutral or a basic molecule and/or therapeutic agent, an acid can be added to facilitate gelation. In an embodiment, when producing a silk gel, increasing the pH (making the gel more basic) increases the shelf stability of the gel. In an embodiment, when producing a silk gel, increasing the pH (making the gel more basic) allows for a greater quantity of an acidic molecule to be loaded into the gel. In an embodiment, natural additives may be added to the silk gel to further stabilize additives. For example, trace elements such as selenium or magnesium or L-methoinine can be used. Further, light-block containers can be added to further increase stability. FIG.34summarizes an embodiment of parameters for a silk fragment-film drying study of the present disclosure.FIG.35is a graph showing silk fragment-film drying times (under various air flow and temperature conditions) based on the silk fragment-film drying study ofFIG.34. These studies indicate that airflow is an important parameter to consider for drying (i.e., samples in covered containers did not dry), temperature can be altered to alter drying rate (i.e., increased temperature results in a faster rate of water removal) and that a steady-state of moisture content within the films can be obtained with a variety of parameters (i.e., from 24 to 48 hours, mass is consistent in uncovered samples regardless of temperature). Of note, the final properties of the film, for example brittleness, will vary with drying conditions. Alternatively, film drying rate may be accelerated by the use of an additive in the SPF solution, such as a surfactant or oil. These additives may be used with or without heat to alter drying rate and final film physical properties. In an embodiment, the drying conditions of the SFP film are 24° C. in a forced air flow incubator for 12 to 48 hours depending on the number of films and ambient humidity. Under these drying conditions, a film that will not shrink more than 5 percent over time when stored in a foil pouch is created. Additionally, the film is homogeneous in composition and physical structure, with no sided-ness and an even distribution of additive, for example vitamin C, throughout. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 30% to about 100% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 35% to about 95% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 40% to about 90% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 45% to about 85% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 50% to about 80% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 55% to about 75% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in light retaining about 60% to about 70% of its activity after 30 days of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in a sealed airtight container or pouch that prevents light from contacting the film retaining about 80% to about 100% of its activity after 3 to 24 months of storage. In an embodiment, the silk protein fragment-film may stabilize vitamin C and derivatives thereof at room temperature when stored in a sealed airtight container or pouch that prevents light from contacting the film retaining about 80% to about 100% of its activity after about 3 to about 60 months of storage. In an embodiment, the silk protein fragment-film may release between 50% to 90% of active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 50% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 60% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 70% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 80% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 90% active vitamin C and derivatives thereof within 20 mins when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release between 10% to 100% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 10% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 20% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 30% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 40% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 50% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 60% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 70% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 80% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. In an embodiment, the silk protein fragment-film may release at least 90% of active vitamin C and derivatives thereof within 5 mins to 8 hours when adhered to dampened skin. It is believed that exposure to higher temperatures for a longer period of time may break down the silk protein into more versatile silk protein fragment mixtures and/or disrupt any silk protein tertiary and/or secondary silk protein structure that could adversely affect shelf stability and/or performance of resulting structures (e.g., gels, films, foams, etc.) as well as reduces the number of heavy chains within the silk protein. FIGS.36A and36Bshow two HPLC chromatograms from samples comprising vitamin C. The chromatogram on the left shows peaks from (1) a chemically stabilized sample of vitamin C at ambient conditions and (2) a sample of vitamin C taken after 1 hour at ambient conditions without chemical stabilization to prevent oxidation, where degradation products are visible. The chromatogram on the right shows peaks from two different embodiments of silk films of the present disclosure that were aged for at least 30 days at room temperature. No degradation products were visible.FIG.59is a table summarizing the vitamin C concentration in silk protein fragment-films (2% silk films air dried at RT) of the present disclosure. InFIG.59processing conditions included 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours.FIG.60is a table summarizing the stability of vitamin C in chemically stabilized solutions.FIGS.89A-89Bare tables summarizing vitamin C stability in SPF gels without chemical stabilizers as compared to chemically stabilized vitamin C in competitive anti-aging skincare products. A gel cast at 20% total vitamin C additive concentration did not gel. Without wishing to be bound by theory, it appears there is a relationship between vitamin C concentration, silk concentration, and gelation. An increase in vitamin C at a given concentration of silk will result in a longer time to gelation or inhibit gelation. This may be due to the vitamin C molecule physically blocking interaction between silk protein fragments or cross-linking of silk protein. In an embodiment, the molecule or molecules are stable and can be released over an extended time period. In an embodiment, release rate is controlled by the specific weight average molecular weight of the silk fibroin-based protein fragments used. In another embodiment, release rate is controlled by creation of a multi-layer structure. For example, multiple films can be cast and dried upon each other. Additionally, each layer can be formed using the same or different molecular weight compositions. In an embodiment, the degree of crystallinity of the protein structure is altered through film drying conditions, thereby controlling the release rate. The molecule or molecules may be released topically on the skin, subcutaneously following implantation, or locally or systemically through oral administration or implantation. In an embodiment, the molecule or molecules is released between 1 minutes and 20 minutes. In an embodiment, the molecule or molecules is released between 20 minutes and 60 minutes. In an embodiment, the molecule or molecules is released between 1 hour and 4 hours. In an embodiment, the molecule or molecules is released between 4 hours and 8 hours. In an embodiment, the molecule or molecules is released between 8 hours and 24 hours. In an embodiment, the molecule or molecules is released between 1 day and 7 days. In an embodiment, the molecule or molecules is released between 1 week and 4 weeks. In an embodiment, the molecule or molecules is released between 1 month and 3 months. In an embodiment, the molecule or molecules is released between 3 months and 6 months. In an embodiment, the molecule or molecules is released between 20 minutes and 6 months. In an embodiment, the molecule or molecules are stable at extreme temperature and humidity conditions. Films of the present disclosure comprised of about 20 kDA average weight average molecular weight silk fibroin-based protein fragments and containing about 20% vitamin C by mass, were stored individually within foil pouches and exposed to temperature extremes. Foil pouches containing films were exposed to:Ambient conditions (time 0 films)“Extreme Cold” (−29° C.±2° C. for 72 hours), followed by “Hot Humid” (38° C. 2° C. at 85% Humidity±5% for 72 hours), and subsequently “Extreme Heat, Moderate Humidity” (60° C.±2° C. at 30% Humidity±5% for 6 hours) The amount of active vitamin C was measured using HPLC. All films were observed to support maintenance of vitamin C activity with exposure to extremes, as summarized in Table 17. TABLE 17Amount of active vitamin C in films under varying conditionsAverage Conc ofvit C in sampleNConditions(mg/g)Std. Dev4Time 0, ambient conditions184.9015.15161) −29° C. ± 2° C. for 72 hours193.9710.252) 38° C. ± 2° C. at 85% Humidity ±5% for 72 hours3) 60° C. ± 2° C. at 30% Humidity ±5% for 6 hours FIGS.37-45are photographs showing silk protein fragment-films of the present disclosure dried under various temperature, time and drying conditions. FIGS.46-54are photographs showing the dissolution, in water, of the formed silk protein fragment-films of the present disclosure under various temperature, time and drying conditions. The water solubility of films of the present disclosure may be varied by altering drying conditions. For example, drying a film to 20% humidity in a forced air incubator and then increasing ambient humidity to 50% for a period of hours and subsequently drying the film back to 20% humidity will result in an insoluble film. Under ordinary conditions where the humidity is steadily decreased, a water-soluble silk film is created. It is anticipated that the increase in humidity allowed the protein structure to be further mobilized in the film and further crystallized, resulting in a non-soluble film. Alternative methods in the art to create non-soluble films include the introduction of methanol. The films of the present disclosure are clearly differentiated from those films due to their solubility in water. The SFP gel articles described herein range from a hydrogel which can be injected or spread topically to a film-gel article that appears as a film and contains a minimal but controlled water content, thereby preventing crystallinity and allowing water solubility. In some embodiments, a composition of the present disclosure can further include skin penetration enhancers, including, but not limited to, sulfoxides (such as dimethylsulfoxide), pyrrolidones (such as 2-pyrrolidone), alcohols (such as ethanol or decanol), azones (such as laurocapram and 1-dodecylazacycloheptan-2-one), surfactants (including alkyl carboxylates and their corresponding acids such as oleic acid, fluoroalkylcarboxylates and their corresponding acids, alkyl sulfates, alkyl ether sulfates, docusates such as dioctyl sodium sulfosuccinate, alkyl benzene sulfonates, alkyl ether phosphates, and alkyl aryl ether phosphates), glycols (such as propylene glycol), terpenes (such as limonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol, carveol, carvone, fenchone, and verbenone), and dimethyl isosorbide. Following are non-limiting examples of suitable ranges for various parameters in and for preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges of such parameters. In an embodiment, the percent silk in the solution is less than 30%. In an embodiment, the percent silk in the solution is less than 25%. In an embodiment, the percent silk in the solution is less than 20%. In an embodiment, the percent silk in the solution is less than 19%. In an embodiment, the percent silk in the solution is less than 18%. In an embodiment, the percent silk in the solution is less than 17%. In an embodiment, the percent silk in the solution is less than 16%. In an embodiment, the percent silk in the solution is less than 15%. In an embodiment, the percent silk in the solution is less than 14%. In an embodiment, the percent silk in the solution is less than 13%. In an embodiment, the percent silk in the solution is less than 12%. In an embodiment, the percent silk in the solution is less than 11%. In an embodiment, the percent silk in the solution is less than 10%. In an embodiment, the percent silk in the solution is less than 9%. In an embodiment, the percent silk in the solution is less than 8%. In an embodiment, the percent silk in the solution is less than 7%. In an embodiment, the percent silk in the solution is less than 6%. In an embodiment, the percent silk in the solution is less than 5%. In an embodiment, the percent silk in the solution is less than 4%. In an embodiment, the percent silk in the solution is less than 3%. In an embodiment, the percent silk in the solution is less than 2%. In an embodiment, the percent silk in the solution is less than 1%. In an embodiment, the percent silk in the solution is less than 0.9%. In an embodiment, the percent silk in the solution is less than 0.8%. In an embodiment, the percent silk in the solution is less than 0.7%. In an embodiment, the percent silk in the solution is less than 0.6%. In an embodiment, the percent silk in the solution is less than 0.5%. In an embodiment, the percent silk in the solution is less than 0.4%. In an embodiment, the percent silk in the solution is less than 0.3%. In an embodiment, the percent silk in the solution is less than 0.2%. In an embodiment, the percent silk in the solution is less than 0.1%. In an embodiment, the percent silk in the solution is greater than 0.1%. In an embodiment, the percent silk in the solution is greater than 0.2%. In an embodiment, the percent silk in the solution is greater than 0.3%. In an embodiment, the percent silk in the solution is greater than 0.4%. In an embodiment, the percent silk in the solution is greater than 0.5%. In an embodiment, the percent silk in the solution is greater than 0.6%. In an embodiment, the percent silk in the solution is greater than 0.7%. In an embodiment, the percent silk in the solution is greater than 0.8%. In an embodiment, the percent silk in the solution is greater than 0.9%. In an embodiment, the percent silk in the solution is greater than 1%. In an embodiment, the percent silk in the solution is greater than 2%. In an embodiment, the percent silk in the solution is greater than 3%. In an embodiment, the percent silk in the solution is greater than 4%. In an embodiment, the percent silk in the solution is greater than 5%. In an embodiment, the percent silk in the solution is greater than 6%. In an embodiment, the percent silk in the solution is greater than 7%. In an embodiment, the percent silk in the solution is greater than 8%. In an embodiment, the percent silk in the solution is greater than 9%. In an embodiment, the percent silk in the solution is greater than 10%. In an embodiment, the percent silk in the solution is greater than 11%. In an embodiment, the percent silk in the solution is greater than 12%. In an embodiment, the percent silk in the solution is greater than 13%. In an embodiment, the percent silk in the solution is greater than 14%. In an embodiment, the percent silk in the solution is greater than 15%. In an embodiment, the percent silk in the solution is greater than 16%. In an embodiment, the percent silk in the solution is greater than 17%. In an embodiment, the percent silk in the solution is greater than 18%. In an embodiment, the percent silk in the solution is greater than 19%. In an embodiment, the percent silk in the solution is greater than 20%. In an embodiment, the percent silk in the solution is greater than 25%. In an embodiment, the percent silk in the solution is between 0.1% and 30%. In an embodiment, the percent silk in the solution is between 0.1% and 25%. In an embodiment, the percent silk in the solution is between 0.1% and 20%. In an embodiment, the percent silk in the solution is between 0.1% and 15%. In an embodiment, the percent silk in the solution is between 0.1% and 10%. In an embodiment, the percent silk in the solution is between 0.1% and 9%. In an embodiment, the percent silk in the solution is between 0.1% and 8%. In an embodiment, the percent silk in the solution is between 0.1% and 7%. In an embodiment, the percent silk in the solution is between 0.1% and 6.5%. In an embodiment, the percent silk in the solution is between 0.1% and 6%. In an embodiment, the percent silk in the solution is between 0.1% and 5.5%. In an embodiment, the percent silk in the solution is between 0.1% and 5%. In an embodiment, the percent silk in the solution is between 0.1% and 4.5%. In an embodiment, the percent silk in the solution is between 0.1% and 4%. In an embodiment, the percent silk in the solution is between 0.1% and 3.5%. In an embodiment, the percent silk in the solution is between 0.1% and 3%. In an embodiment, the percent silk in the solution is between 0.1% and 2.5%. In an embodiment, the percent silk in the solution is between 0.1% and 2.0%. In an embodiment, the percent silk in the solution is between 0.1% and 2.4%. In an embodiment, the percent silk in the solution is between 0.5% and 5%. In an embodiment, the percent silk in the solution is between 0.5% and 4.5%. In an embodiment, the percent silk in the solution is between 0.5% and 4%. In an embodiment, the percent silk in the solution is between 0.5% and 3.5%. In an embodiment, the percent silk in the solution is between 0.5% and 3%. In an embodiment, the percent silk in the solution is between 0.5% and 2.5%. In an embodiment, the percent silk in the solution is between 1 and 4%. In an embodiment, the percent silk in the solution is between 1 and 3.5%. In an embodiment, the percent silk in the solution is between 1 and 3%. In an embodiment, the percent silk in the solution is between 1 and 2.5%. In an embodiment, the percent silk in the solution is between 1 and 2.4%. In an embodiment, the percent silk in the solution is between 1 and 2%. In an embodiment, the percent silk in the solution is between 20% and 30%. In an embodiment, the percent silk in the solution is between 0.1% and 6%. In an embodiment, the percent silk in the solution is between 6% and 10%. In an embodiment, the percent silk in the solution is between 6% and 8%. In an embodiment, the percent silk in the solution is between 6% and 9%. In an embodiment, the percent silk in the solution is between 10% and 20%. In an embodiment, the percent silk in the solution is between 11% and 19%. In an embodiment, the percent silk in the solution is between 12% and 18%. In an embodiment, the percent silk in the solution is between 13% and 17%. In an embodiment, the percent silk in the solution is between 14% and 16%. In an embodiment, the percent silk in the solution is 2.4%. In an embodiment, the percent silk in the solution is 2.0%. In an embodiment, the percent sericin in the solution is non-detectable to 30%. In an embodiment, the percent sericin in the solution is non-detectable to 5%. In an embodiment, the percent sericin in the solution is 1%. In an embodiment, the percent sericin in the solution is 2%. In an embodiment, the percent sericin in the solution is 3%. In an embodiment, the percent sericin in the solution is 4%. In an embodiment, the percent sericin in the solution is 5%. In an embodiment, the percent sericin in the solution is 10%. In an embodiment, the percent sericin in the solution is 30%. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 1 year. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 4 to 5 years. In an embodiment, the stability of a composition of the present disclosure is 10 days to 6 months. In an embodiment, the stability of a composition of the present disclosure is 6 months to 12 months. In an embodiment, the stability of a composition of the present disclosure is 12 months to 18 months. In an embodiment, the stability of a composition of the present disclosure is 18 months to 24 months. In an embodiment, the stability of a composition of the present disclosure is 24 months to 30 months. In an embodiment, the stability of a composition of the present disclosure is 30 months to 36 months. In an embodiment, the stability of a composition of the present disclosure is 36 months to 48 months. In an embodiment, the stability of a composition of the present disclosure is 48 months to 60 months. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 6 kDa to 16 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 17 kDa to 38 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 39 kDa to 80 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 1 to 5 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 5 to 10 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 10 to 15 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 15 to 20 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 20 to 25 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 25 to 30 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 30 to 35 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 35 to 40 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 40 to 45 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 45 to 50 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 50 to 55 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 55 to 60 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 60 to 65 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 65 to 70 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 70 to 75 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 75 to 80 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 80 to 85 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 85 to 90 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 90 to 95 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 95 to 100 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 100 to 105 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 105 to 110 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 110 to 115 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 115 to 120 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 120 to 125 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 125 to 130 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 130 to 135 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 135 to 140 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 140 to 145 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 145 to 150 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 150 to 155 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 155 to 160 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 160 to 165 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 165 to 170 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 170 to 175 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 175 to 180 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 180 to 185 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 185 to 190 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 190 to 195 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 195 to 200 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 200 to 205 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 205 to 210 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 210 to 215 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 215 to 220 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 220 to 225 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 225 to 230 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 230 to 235 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 235 to 240 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 240 to 245 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 245 to 250 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 250 to 255 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 255 to 260 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 260 to 265 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 265 to 270 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 270 to 275 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 275 to 280 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 280 to 285 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 285 to 290 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 290 to 295 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 295 to 300 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 300 to 305 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 305 to 310 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 310 to 315 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 315 to 320 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 320 to 325 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 325 to 330 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 330 to 335 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 35 to 340 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 340 to 345 kDa. In an embodiment, a composition of the present disclosure includes pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from 345 to 350 kDa. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1 to about 5.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1.5 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1 to about 1.5. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 1.5 to about 2.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has a polydispersity ranging from about 2.0 to about 2.5. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments, has a polydispersity ranging from about is 2.0 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments, has a polydispersity ranging from about is 2.5 to about 3.0. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments has non-detectable levels of LiBr residuals. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 25 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 50 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 75 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 400 ppm to 500 ppm. In an embodiment, a composition of the present disclosure having pure silk fibroin-based protein fragments, has non-detectable levels of Na2CO3residuals. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the Na2CO3residuals in a composition of the present disclosure is 400 ppm to 500 ppm. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 50 to 100%. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 60 to 100%. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 70 to 100%. In an embodiment, the water solubility of pure silk fibroin-based protein fragments of the present disclosure is 80 to 100%. In an embodiment, the water solubility is 90 to 100%. In an embodiment, the silk fibroin-based fragments of the present disclosure are non-soluble in aqueous solutions. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 50 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 60 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 70 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 80 to 100%. In an embodiment, the solubility of pure silk fibroin-based protein fragments of the present disclosure in organic solutions is 90 to 100%. In an embodiment, the silk fibroin-based fragments of the present disclosure are non-soluble in organic solutions. In an embodiment, the percent water content in gels of the present disclosure is 20% to 99.9%. In an embodiment, the percent water content in gels of the present disclosure is 20% to 25%. In an embodiment, the percent water content in gels of the present disclosure is 25% to 30%. In an embodiment, the percent water content in gels of the present disclosure is 30% to 35%. In an embodiment, the percent water content in gels of the present disclosure is 35% to 40%. In an embodiment, the percent water content in gels of the present disclosure is 40% to 45%. In an embodiment, the percent water content in gels of the present disclosure is 45% to 50%. In an embodiment, the percent water content in gels of the present disclosure is 50% to 55%. In an embodiment, the percent water content in gels of the present disclosure is 55% to 60%. In an embodiment, the percent water content in gels of the present disclosure is 60% to 65%. In an embodiment, the percent water in gel cosmetic gels of the present disclosure s is 65% to 70%. In an embodiment, the percent water content in gels of the present disclosure is 70% to 75%. In an embodiment, the percent water content in gels of the present disclosure is 75% to 80%. In an embodiment, the percent water content in gels of the present disclosure is 80% to 85%. In an embodiment, the percent water content in gels of the present disclosure is 85% to 90%. In an embodiment, the percent water content in gels of the present disclosure is 90% to 95%. In an embodiment, the percent water content in gels of the present disclosure is 95% to 99%. In an embodiment, the percent water content in films of the present disclosure is 20%. In an embodiment, the percent water content in films of the present disclosure is less than 20%. In an embodiment, the percent water content in films of the present disclosure is less than 18%. In an embodiment, the percent water content in films of the present disclosure is less than 16%. In an embodiment, the percent water content in films of the present disclosure is less than 14%. In an embodiment, the percent water content in films of the present disclosure is less than 12%. In an embodiment, the percent water content in films of the present disclosure is less than 10%. In an embodiment, the percent water content in films of the present disclosure is between about 2% and about 20%. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is greater than 84° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is less than 100° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 84° C. to 100° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 84° C. to 94° C. In an embodiment, the extraction temperature during a method of preparing a composition of the present disclosure is 94° C. to 100° C. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXAMPLES Example 1. Development of a Silk Film of the Present Disclosure for Use in Fine Line Lifting Applications TABLE 18Film Recipe for Fine Line Lifting Film - FIG. 82A% SPF Mixture Solution of the2.4%Present DisclosureQuantity Vitamin C4:1 (silk:Vit C) (0.006 g/mL 2.4%solution) 20%mL per film (2.5 cm by 10 cm)7.08 mLMass of silk per film:170 mgMass of 1-ascorbic acid per film:42.5 mgpH4.0 (when water is applied) Silk films (2.5 cm×10 cm) were manufactured according to methods disclosed herein varying process parameters so as to result in fine line lifting films. The silk films were given the name “PureProC™ film”, and can be packaged in a foil based package that is air tight and light proof. Table 18 provides details of the PureProC™ films used in a study of 32 individuals using the films for four (4) weeks. Biocompatibility and hypo-allergenicity of the films was observed. Further, no sensitization, toxicity, or immune response was observed.FIG.84is a graph summarizing the quantity of vitamin C in a daily dose (i.e., the average amount of product used to cover a 25 cm2area of skin) of PureProC™ and competitor products over a 30 day period.FIGS.85and86summarize resultant ease of use data and observed benefits within the first month of use. In an embodiment, PureProC™ films were removed by peeling the films off. In an embodiment, PureProC™ films were removed by using a wet cotton ball or similar removal pad. In an embodiment, PureProC™ films were removed by washing the area where the film is placed with a wash cloth. In an embodiment, PureProC™ film PureProC™ films were removed using water. The PureProC™ films can be shaped into strips for multiple areas of the face or larger pieces can be cut to fit target areas. In an embodiment, grips or backing(s) on the PureProC™ films can be included for ease of application. In an embodiment, a PureProC™ film of the present disclosure includes silk and vitamin C (20%). In an embodiment, a film of the present disclosure is soluble in water (insoluble border). In an embodiment, a film of the present disclosure is clear/transparent. In an embodiment, a film of the present disclosure has a pH=4 when water is applied. Films of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. Films of the present disclosure can be made with from about 1% to about 50% 1-ascorbic acid. Films of the present disclosure can adhere to skin with water. Films of the present disclosure can be spread on skin once water is applied. Films of the present disclosure can dry when humidity of drying equipment is 16-40% and below the humidity of the lab Example 2. Development of Silk Gels of the Present Disclosure TABLE 19Gel Samples-Silk gel formulations including additives, concentrationof silk and additive, gelation conditions and gelation times.mL 2%MassRatioAmountSamplesilkVit Csilk:ofTemp/Days toNamesolution(g)VitCAdditiveadditiveTreatmentGelation1100.045:01NoneNoneRT82100.082.5:1NoneNoneRT83100.21:01NoneNoneRT84100.41:02NoneNoneRT145100.81:04NoneNoneRTNone6100.045:01NoneNoneFridge~397100.082.5:1NoneNoneFridge~398100.21:01NoneNoneFridge~399100.41:02NoneNoneFridgeNone10100.81:04NoneNoneFridgeNone11100.21:01NoneNoneRT/Shake8vigorouslyO-1100.045:01NoneNone37° C3OvenO-2100.045:01NoneNone50° C.2OvenO-3100.21:01NoneNone37° C.4OvenO-4100.21:01NoneNone50° C.3OvenM400.165:01NoneNoneRT5D400.165:01NoneNoneRT5E1100.045:01Vit E1 dropRT7E2100.045:01Vit E3 dropsRT7E3100NoneVit E1 dropRTNoneE4100NoneVit E3 dropsRTNoneL1100.045:01Lemon300 uLRT6L2100.045:01Lemon Juice300 uLRT6L3100.045:01Lemon Juice1000 uLRT5L4100NoneLemon300 uLRT6L5100NoneLemon Juice300 uLRT7Jar 1200.085:01Lemon Juice2000 uLRT5-7Jar 250.025:01Lemongrass1 dropRT2-3OilR-1100.045:01Rosemary1 dropRT7OilT-1100.045:01NoneNoneRT/Tube7RO-1100.045:01Rose Oil1 dropRT6RO-210NoneNoneRose Oil1 dropRTNone Ratio of Silk to Vitamin C Samples 1-10 were used to examine the effect of silk to vitamin C ratio on serum gelation. Samples 1-3 with less vitamin C gelled quicker than samples 4 and 5. All other conditions were kept constant. Samples 6-8 with less vitamin C gelled quicker than samples 9 and 10. All other conditions were kept constant. It is concluded that decreasing the ratio of silk to vitamin C (increasing the amount of vitamin C), will lengthen the time to gel creation. At ratios with small amounts of vitamin C, days to gel creation did not vary greatly. Physical Stimulation Samples 3 and 11 were used to examine the effect of physical stimulation on serum gelation. Each sample was prepared under the same conditions. Sample 11 was vigorously shaken for about 3 minutes after addition of vitamin C. Treatment of 3 and 11 was otherwise the same. The shaking resulted in bubbles but did not significantly change gel creation time. Temperature Treatment Samples 1, 3, 6, 8, O-1, O-2, O-3, and O-4 were used to examine the effect of temperature treatment on serum gelation time. Samples 1, 6, 0-1, and 0-2 were identical other than temperature treatment. Samples 3, 8, O-3, and O-4 were identical other than temperature treatment. The two groups differed in silk to vitamin C ratio. Time to serum gelation was directly related to temperature treatment with a higher temperature resulting in quicker serum gelation. Solution Volume Samples 1, M and D were used to examine the effect of solution volume on serum gelation time. Samples M and D varied from sample 1 only by an increased solution volume. Samples M and D gelled in 5 days while sample 1 gelled in 8 days. Samples M and D were definitively noticed to be gelled on the day of gelling while sample 1 gelled over a weekend. Additives Samples E1, E2, E3, E4, L1, L2, L3, L4, L5, Jar 2, R1, RO-1 and RO-2 were used to examine the effect of additives on serum gelation time. Samples E1-4 contained Vitamin E. Only samples E1 and E2 contained vitamin C and only these two samples gelled. Vitamin E can be added to a solution to become a gel but it appears that another additive may be needed to create a gel. Samples L1-5 contained a form of lemon juice. Samples L1 and L4 had juice directly from a lemon while samples L2, L3 and L5 contained lemon juice from a plastic lemon container. Samples L4 and L5 did not have vitamin C while all others did. All samples gelled showing that lemon juice can create gel on its own. Amount of lemon juice and type of lemon juice had little effect on gelation time. Sample Jar 2 contained lemon grass oil which formed an albumen like substance when initially added. This sample also had vitamin C but gelation time was significantly quicker than with other vitamin C samples. Sample R1 contained rosemary oil, which seemed to be soluble, as well as vitamin C. The sample gelled in a similar time frame to other samples with only vitamin C. Samples RO-1 and RO-2 contained rose oil while only RO-1 had vitamin C. Only RO-1 gelled showing that rose oil will not create a gel quickly on its own. In both cases the rose oil was immiscible and visible as yellow bubbles. Aqueous silk fibroin-based fragment solution and essential oils are immiscible liquids. In an embodiment, to increase the fragrance of the silk fibroin-based fragment solution, without entrapping oils within the solution, the solution is mixed with the essential oil with the use of a stir bar. The stir bar is rotated at a speed such that some turbulence is observed in the mixture, thus causing contact between the fragrant essential oil and the molecules in solution, adding a scent to the solution. Before casting of product from the solution, mixing may be stopped and the oil allowed to separate to the top of the solution. Dispensing from the bottom fraction of the solution into the final product allows for fragrance without visible essential oil within the final product. Alternatively, the silk fibroin-based solution and essential oil can be combined with or without additional ingredients and/or an emulsifier to create a composition containing both ingredients. In an embodiment, mixing of the solution as described above can reduce gelation time if the solution is used to create a gel formulation. Vessel Samples T1 and Jar 1 were used to examine the effect of casting vessel on serum gelation time. Jar 1 was cast in a glass jar while T1 was cast in an aluminum tube. Both samples gelled and did not affect serum gel time. SUMMARY All treatments of silk solution for gel solution were in a conical tube at room temperature unless otherwise stated. The ratio of silk to vitamin C did affect the ability of a solution to gel as ratios above 1:2 did not gel and a 1:2 ratio took twice as long as other lower ratios (5:1, 2.5:1, 1:1). Temperature affected gel creation time with higher temperatures resulting in quicker gel times. 50° C. treatment gelled in as quick as 2 days, 37° C. treatment gelled in as quick as 3 days, room temperature treatment gelled in 5-8 days and storage in a refrigerator took at least 39 days to gel. The effects of additives on gel creation were dependent on the additive. Vitamin E, Rosemary Oil and Rose Oil all had no effect on gel creation. Each of these additives did not prevent gelation or affect the time to gelation. Each also required the presence of vitamin C to gel. Lemon juice from a fresh lemon, pre-squeezed lemon juice from a plastic lemon container and lemon grass oil did affect gel creation. Without wishing to be bound by theory, it is believed that the lower pH as a result of these additives is the reason the additives had an impact on decreasing gelation time. Both lemon juice types were able to cause gelation without the presence of vitamin C. This occurred in the same number of days as with vitamin C. The lemongrass oil was able to decrease the number of days to gelation to 2-3 days. All additives appeared soluble other than lemongrass oil and rose oil. Rose oil remained in yellow bubbles while the lemongrass oil was partially soluble and formed an albumen like chunk. In an embodiment, oils that are not fully soluble, can still be suspended within the gel as an additive. Physical stimulation by shaking, vessel the solution was cast into and solution volume did not affect gelation time.FIG.81is a graph representing the % Activity of Vitamin C in gels of the present disclosure. TABLE 20Concentration of vitamin C in various gelformulations.SampleConcentration ofWeightVitamin C (mg/g)Sample Info(mg)In SampleAverageRosemary685.73.25113.2657(Room3.2804Temperature6383.33363.3334storage)3.3332Lemongrass6462.86722.877(Room2.8868Temperature645.52.90512.9051storage)2.9052Rosemary645.23.90633.9147(Room3.923Temperature;6493.94433.9374Foil Covered3.9305storage)Lemongrass630.13.82533.8274(Room3.8295Temperature;660.43.82833.8253Foil Covered3.8222storage)Rosemary672.45.16165.1484(Fridge, Foil5.1352Covered616.55.19845.201storage)5.2036Lemongrass640.55.18715.1824(Fridge, Foil5.1776Covered627.75.20985.2126storage)5.2154 Example 3. Development of Silk Gels of the Present Disclosure for Use as Smoothing Gel TABLE 21Lemongrass Gel% Silk Solution2%Quantity Vitamin C100 mg/15 mL solutionQuantity Lemongrass Oil20 uL/15 mL solution TABLE 22Rosemary Gel% Silk Solution2%Quantity Vitamin C100 mg/15 mL solutionQuantity Rosemary Oil20 uL/50 mL solution TABLE 23Lemongrass Gel (50 mL)% Silk Solution (60 minute boil, 25 kDA)2%Quantity Vitamin C (ascorbyl glucoside)12.82 mg/mL solution(641 mg total)Quantity Lemongrass Oil1.33 uL/mL solutionpH4 TABLE 24Rosemary Gel (50 mL)% Silk Solution (60 minute boil, 25 kDA)2%Quantity Vitamin C (ascorbyl glucoside)12.82 mg/mL solution(641 mg total)Quantity Rosemary Oil0.8 uL/mL solutionpH4 Gels of the present disclosure can be made with about 0.5% to about 8% silk solutions. Gels of the present disclosure can be made with ascorbyl glucoside at concentrations of about 0.67% to about 15% w/v. Gels of the present disclosure be clear/white in color. Gels of the present disclosure can have a consistency that is easily spread and absorbed by the skin. Gels of the present disclosure can produce no visual residue or oily feel after application. Gels of the present disclosure do not brown over time. Silk gels with essential oils were prepared by diluting a silk solution of the present disclosure to 2%. Vitamin C was added to the solution and allowed to dissolve. The essential oil was added, stirred and dissolved. The solution was aliquot into jars. A trial was conducted with 44 people on two formulations of the present disclosure, PureProC™ Rosemary Gel and PureProC™ Lemongrass Gel (FIGS.87and88). Respondents were asked to use each sample once a day for a week each. The majority of respondents applied the gel to the whole face. Other areas where it was most commonly applied included the forehead, under eyes and near mouth. The majority of respondents applied the gel during the morning (67%) with the balance 33% applying the gel in the evening. Ninety-eight (98%) of participants used the gel once a day during the test. Respondents were asked to describe in their own words how the gel felt when it was applied and how it felt during the 24 hours until the next application. Smooth, cool, and soft were the most often mentioned adjectives used to describe how the gel felt. Eighty percent (80%) of test participants gave a high score to interest in continuing to use the gel. Respondents were asked about what they did with their other products that were usually used on their face during the trial. The majority applied the gel first and then added the other products or applied the gel at night with no additional products. Only 14% of participants indicated that they eliminated one of their normal products while testing the gel. PureProC™ can be used in conjunction with or in replacement of other products. Additionally, sunscreen can be added to the gel or it may be dispensed from a pump instead of a jar. With repeated topical use, no skin irritation, rash, or signs of non-compatibility was observed. Biocompatibility and hypo-allergenicity of the gels was observed. Further, no sensitization, toxicity, or immune response was observed. Example 4. Silk Articles of the Present Disclosure Made from Silk Solutions of the Present Disclosure Silk solutions of various molecular weights and/or combinations of molecular weights can be optimized for specific applications. The following provides an example of this process but it not intended to be limiting in application or formulation. Three (3) silk solutions were utilized in standard silk structures in accordance with standard methods in the literature with the following results:Solution #1 is a silk concentration of 5.9%, average MW of 19.8 kDa and 2.2 PD (made with a 60 min boil extraction, 100 degree LiBr dissolution for 1 hr)Solution #2 is a silk concentration of 6.4% (made with a 30 min boil extraction, 60 degree LiBr dissolution for 4 hrs)Solution #3 is a silk concentration of 6.17% (made with a 30 min boil extraction, 100° C. LiBr dissolution for 1 hour) Films: Films were made in accordance with Rockwood et al (Nature Protocols; Vol. 6; No. 10; published on-line Sep. 22, 2011; doi:10.1038/nprot.2011.379). Briefly, 4 mL of 1% or 2% (wt/vol) aqueous silk solution was added into 100 mm Petri dish (Volume of silk can be varied for thicker or thinner films and is not critical) and allowed to dry overnight uncovered. The bottom of a vacuum desiccator was filled with water. Dry films were placed in the desiccator and vacuum applied, allowing the films to water anneal for 4 hours prior to removal from the dish. Films cast from solution #1 did not result in a structurally continuous film; the film was cracked in several pieces. These pieces of film dissolved in water in spite of the water annealing treatment. Egel: “Egel” is an electrogelation process as described in Rockwood et al. Briefly, 10 ml of aqueous silk solution is added to a 50 ml conical tube and a pair of platinum wire electrodes immersed into the silk solution. A 20 volt potential was applied to the platinum electrodes for 5 minutes, the power supply turned off and the gel collected. Solution #1 did not form an EGEL over the 5 minutes of applied electric current. Gelation: Solutions #2 and #3 were gelled in accordance with the published horseradish peroxidase (HRP) protocol. Behavior seemed typical of published solutions. Sonicated Gels: Gels were made following the sonication process in Rockwood et al. Briefly, 5 ml of silk solution was added to a 15 ml conical tube. The sonicating horn was immersed in the solution and the solution sonicated at 50% amplitude (21 W). Silk gels were made with 2%, 4% and 6% silk solutions. As compared to standard literature silk, Solutions #2 and #3 formed gels after a longer time, for example:Standard literature silk: 5-8 minSolution #2: 20 minSolution #3: 120 min Porous 3D Scaffolds: Water based, salt leached scaffolds were made in accordance with the published methods of Rockwood. Salt with particle sizes of interest was prepared by stacking the sieves with the largest mesh on top and the smallest mesh on the bottom. Salt was added and sieves shaken vigorously collecting the salt. With a 5-ml syringe, 6% (wt/vol) fibroin solution was aliquoted into plastic containers, 2 ml per mold and 5-600 micron salt particles were slowly added on top of the fibroin solution in the mold while rotating the container so that the salt was uniform. The ratio of salt to silk in solution was maintained at 25:1. The container was gently tapped on the bench top to remove air bubbles, the cap closed and the solution allowed to settle overnight at room temperature. Once gelled, the lids were removed and the molds placed in a 2-liter beaker with ultrapure water (three containers per 2 liters of water). The beakers were transferred to a stir plate and stirred, changing the water 2-3 times per day for 2 d (4-6 washes in total). The scaffolds were removed from the molds and placed them in fresh water for an additional day. Solution #1 did not form a scaffold; it did not gel. Both solution #2 & #3 formed scaffolds. The scaffolds made with Solution #3 appear softer than the ones made with Solution #2, but both scaffolds were homogeneous. Example 5. Tangential Flow Filtration (TFF) to Remove Solvent from Dissolved Silk Solutions of the Present Disclosure A variety of % silk concentrations have been produced through the use of Tangential Flow Filtration (TFF). In all cases a 1% silk solution was used as the input feed. A range of 750-18,000 mL of 1% silk solution was used as the starting volume. Solution is diafiltered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, solution undergoes ultrafiltration to increase the concentration through removal of water. See examples below. 7.30% Silk Solution: A 7.30% silk solution was produced beginning with 30 minute extraction batches of 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 15,500 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 1300 mL. 1262 mL of 7.30% silk was then collected. Water was added to the feed to help remove the remaining solution and 547 mL of 3.91% silk was then collected. 6.44% Silk Solution: A 6.44% silk solution was produced beginning with 60 minute extraction batches of a mix of 25, 33, 50, 75 and 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35, 42, 50 and 71 g per batch of silk fibers were dissolved to create 20% silk in LiBr and combined. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 17,000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 3000 mL. 1490 mL of 6.44% silk was then collected. Water was added to the feed to help remove the remaining solution and 1454 mL of 4.88% silk was then collected 2.70% Silk Solution: A 2.70% silk solution was produced beginning with 60 minute extraction batches of 25 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35.48 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 1000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 300 mL. 312 mL of 2.7% silk was then collected. Example 6. Gel Vitamin C Derivatives of the Present Disclosure The purest form of vitamin C is L-ascorbic acid. There are a number of other derivatives of vitamin C that function like pure vitamin C after they are converted to L-ascorbic acid in the body. Vitamin C derivatives are being utilized to extend shelf life. Derivatives are stable forms of L-ascorbic acid and will not oxidize or lose stability. Table 25 below summarizes some vitamin C derivatives tested in the skin care products of the present disclosure: TABLE 25Derivatives ExploredSodium Ascorbyl Phosphate (Aromantic)Sodium Ascorbyl Phosphate (DSM)Magnesium Ascorbyl PhosphateAscorbic Acid-2-GlucosideAscorbyl Tetraisopalmitate The Tables inFIGS.89A-89Bsummarize embodiments of gels of the present disclosure. Ascorbic acid-2-glucoside was the most successful vitamin C derivative at gel formation with gel being formed in a 2% silk solution in 3 days. Sodium ascorbyl phosphate from DSM supplier formed a gel in a 2% silk solution after 28 days while the same molecule from Aromantic failed to create a gel. In all cases 100 mg of vitamin C derivative was mixed in 15 mL of 2% silk solution, and all gels had the same appearance as gels created with ascorbic acid. Gels were also cast with combinations of two vitamin C options. In each case, at least one of the vitamin C options was known to cause gelation (L-ascorbic acid or ascorbic acid-2-glucoside). All combination gels were able to gel at 1% total vitamin C additive concentration. A gel cast at 20% total vitamin C additive concentration did not gel. Without wishing to be bound by theory, it appears there is a relationship between vitamin C concentration, silk concentration, and gelation. An increase in vitamin C at a given concentration of silk will result in a longer time to gelation or inhibit gelation. This may be due to the vitamin C molecule physically blocking interaction between silk protein fragments or cross-linking of silk protein. Modification to pH may allow additional concentrations of vitamin C and derivatives thereof to be added. Ascorbyl tetraisopalmitate was not used in any gel forming formulation, as it was unable to dissolve or be dispersed in an aqueous silk solution. Ascorbyl tetraisopalmitate is a highly viscous, oil soluble liquid that might need the help of an emulsifier to possible dissolve in aqueous silk solution. Example 7. Film Vitamin C Derivatives of the Present Disclosure FIG.90is a table summarizing embodiments of films of the present disclosure. Sodium ascorbyl phosphate, magnesium ascorbyl phosphate and ascorbic acid-2-glucoside could be cast in films with varying appearance. Sodium ascorbyl phosphate films were opaque and white with a textured top surface similar to plastic. Magnesium ascorbyl phosphate films were clear and cloudy with a textured top surface similar to plastic. Ascorbic acid-2-glucoside films were most similar to L-ascorbic acid films although slightly less pliable and slightly textured. All films were soluble with an insoluble border. In an embodiment, a film with an insoluble border can be made completely spreadable by punching a shape from the region contained within the soluble section. Example 8. Caffeine Films with Vitamin C of the Present Disclosure FIGS.91A-91Bare tables summarizing embodiments of caffeine films of the present disclosure. Films were cast with 0.5%, 1%, 2.5%, 5%, 10%, 15% and 20% caffeine and 20% or 25% vitamin C. All combinations formed films. 20% caffeine films had caffeine precipitate out. Films with 0.5%-2.5% were soluble. In an embodiment, a caffeine film of the present disclosure is used for reducing puffy eyes. Example 9. Caffeine Gels with Vitamin C of the Present Disclosure A silk gel with 2% silk and 100 mg L-ascorbic acid/15 mL solution was created with the addition of 50 mg caffeine/15 mL solution. The gel has the exact appearance of standard L-ascorbic acid gels. In an embodiment, a caffeine gel of the present disclosure is used for reducing puffy eyes. A range of essential oils can be used including, but not limited to, lemongrass, vanilla, geranium, and green tea. Example 10. Green Tea Gels with Vitamin C of the Present Disclosure Steps: Green Tea Prep Heat 250 mL water to boilSteep tea bag 2-3 minutes with occasionalstirremove tea bag and let coolGel SolutionPrep Use TFF-10-0047 (3.71% silk)dilute to 3% silk with waterdilute to 2% with green teaadd L-ascorbic acidGelation occurred like standard gel at roomGel temperatureGreen/yellow colorGreen Tea scentSolution Spec: 2% silk solution65 mL (35 ml of 3.71% silk, 8.3 mL water,21.66 mL green tea)0.43 g L-ascorbic acid FIG.92is a table summarizing an embodiment of a caffeine gel of the present disclosure. A silk gel with 2% silk and 100 mg L-ascorbic acid/15 mL solution was created with the addition of 50 mg caffeine/15 mL solution. The gel has the exact appearance of standard L-ascorbic acid gels. Example 11. Preservative Gels with Vitamin C of the Present Disclosure FIG.93is a table summarizing embodiments of preservative gels of the present disclosure. Silk gels were cast with standard 2% silk solution and 100 mg L-ascorbic acid/15 mL solution with the addition of a preservative and chelating agent. The preservative added was Verstatil SL by Kinetic (Water, Sodium Levulinate, Potassium Sorbate) at 1.5% and the chelating agent was Dermofeel-PA3 by Kinetic (Sodium Phytate) at 0.1%. The addition of preservatives extended gelation time to 7 days. Gel is being observed for discoloration and integrity with L-ascorbic acid and ascorbic acid-2-glucoside gel comparisons. Example 12. Chemical Peels of the Present Disclosure The primary variable investigated was the concentration of lactic acid and/or glycolic acid necessary to create a silk solution of a desired pH. In order to determine the relationship between concentration in silk and pH, 2% silk solutions (60 minute boil, 25 kDA) were titrated with glycolic and lactic acid and tested for pH with pH strips. See the following titrations/formulations below: TABLE 26Lactic Acid Peel 1: Initial solution: 25 mL of 2% silk solution, pH = 7-8Quantity of Lactic Acid AddedTotal Lactic AcidpH100 μL100 μL3100 μL200 μL2100 μL300 μL1-2Time to gel: 3 days TABLE 27Lactic Acid Peel 2: Initial solution: 25 mL of 2% silk solution, pH = 7-8Quantity of Lactic Acid AddedTotal Lactic AcidpH25 μL25 μL4Time to gel: >5 days TABLE 28Glycolic Acid Peel 1: Initial solution: 25 mL of 2% silk solution, pH = 7-8Quantity of Glycolic Acid AddedTotal Glycolic AcidpH41 mg41 mg443.25 mg84.25 mg330.7 mg114.95 mg356.4 mg171.35 mg2-391.66 mg263.01 mg2171.35 mg434.4 mg1-2Time to gel: 3 days TABLE 29Glycolic Acid Peel 2: Initial solution: 25 mL of 2% silk solution, pH = 7-8Quantity of Lactic Acid AddedTotal Lactic AcidpH41 mg41 mg4Time to gel: >5 days TABLE 30Lactic/Glycolic Acid Peel: Initial solution:25 mL of 2% silk solution, pH = 7-8Total Lactic AcidTotal Glycolic AcidLemongrasspH150 μL200 mg33.3 μL2Time to gel: 3 days TABLE 31Lactic/Glycolic Acid Peel: Initial solution:30 mL of 2% silk solution, pH = 7-8% Silk Solution (60 minute boil, 25 kDA)2%Lactic Acid Concentration6 μL/mLGlycolic Acid Concentration8 mg/mLpH2Lemongrass Concentration1.33 μL/mL A peel of the present disclosure can have a % silk ranging from about 0.5% to about 8%. The pH of a peel of the present disclosure can be adjusted with varying quantities of lactic and glycolic acid. Peels can also be made with lactic acid only or glycolic acid only. A peel of the present disclosure can be clear/white in color. A peel of the present disclosure can have a gel consistency that is easily spread and absorbed by the skin. A peel of the present disclosure does not brown or change colors. In an embodiment, a chemical peel of the present disclosure can be applied weekly to reveal healthy, vibrant skin. In an embodiment, a chemical peel of the present disclosure can be applied weekly to diminish fine lines. In an embodiment, a chemical peel of the present disclosure can be applied weekly to firm the skin. Each formulation (after titration, if applicable) was applied as a liquid and as a gel and observed for look and feel. Peels of pH=4 (Lactic Acid Peel 2, Glycolic Acid peel 2) resulted in a minimal burning feeling after a few minutes of application, while peels of pH=˜2 (Lactic Acid Peel 1, Glycolic Acid Peel 1, Lactic/Glycolic Acid Peel) caused a slightly more intense burning feel. Little difference in degree of burning was felt between liquid and gel other than that the burning sensation was more delayed in the gel form. PH was maintained in the gel form and was confirmed by using a pH strip. Glycolic acid and lactic acid are both alpha hydroxy acids (AHA's) that are among the most commonly used peels for superficial peeling (outermost skin layer peeling). Chemical peels are intended to burn the top layers of the skin in a controlled manner, to remove superficial dermal layers and dead skin in order to improve appearance. AHAs are common in chemical peels due to low risk of adverse reactions and high control of strength (control pH and time applied). Glycolic acid is most commonly used and has a very small molecular size, enabling deep penetration into the epidermis. Lactic acid is another commonly used AHA and offers a more gentle peel with higher control due to its larger molecular size. Any number of chemicals known in the art that lower pH and are physical exfoliates can be used in place of AHAs. Example 13. Hydrating Serums of the Present Disclosure Variables include: concentration of silk in solution, concentration of HA, addition of vitamin C, and serum preparation method. Table 32 is a list of samples that were evaluated: TABLE 32Embodiments of serums of the present disclosure containing HA andSilk (60 minute boil, 25 kDA), with or without vitamin C, and with20 uL/15 mL lemongrass essential oil (30 mL solution)HASilkVit CMethod(%)(%)(mg)ObservationHA added0.520White, slightly opaque, viscous liquidto water1White/yellow, slightly opaque, viscousbeforeliquiddilution0.520Low viscosity, clear-white opaque withof silkfilm on top, some white residue whenapplied topically to skin1Slightly viscous, clear liquid with filmon top0.510Slightly viscous, clear liquid with filmon top1Smooth viscous liquid, no white residuewhen applied topically to skin0.50.50Moderately viscous liquid, clear1Smooth, clear, no white residue whenapplied topically to skin0.5235Non homogeneous mix of hard gel andviscous liquid1Non homogeneous mix of hard gel andviscous liquid1135Non homogeneous mix of hard gel andviscous liquid0.5Opaque, white liquid/ non-viscous1435Separated mixture of hard gel andviscous liquid0Non homogeneous mix of hard gel andviscous liquid520Yellow, gelHA added1020Viscous jelly upon stirring withto waterundissolved HAbefore5Very viscous jelly upon stirringdilution of1Viscous jelly upon stirringsilk, stirred0.5vigorouslyHA added120Non homogeneous thick, viscousto waterjelly/gelbefore510dilutionof silk,shakenHA added110Clear/slightly opaque, viscous liquid,to watersmooth feel, little to no white residueand let sitwhen applied topically to skinfor 1 daybeforedilutionof silkHA added0.520Viscous, clear/white liquid varying into diluted1consistencysilk0.51Clear viscous liquid varying insolution,1consistencystirred0.56White, opaque jelly varying in1consistencyHA added0.53.90White, slightly opaque, viscous liquidto diluted1silk0.5235White gel varying in consistencysolution,1stirred In an embodiment, a hydrating serum of the present disclosure protects the skin and seals in moisture with the power of silk fibroin-based fragment proteins. In an embodiment, a hydrating serum of the present disclosure delivers moisture for immediate and long-term hydration throughout the day with concentrated hyaluronic acid. A range of essential oils can be used in a hydrating serum of the present disclosure including, but not limited to, lemongrass, vanilla, geranium, and green tea. In an embodiment, one or two drops of a hydrating serum of the present disclosure can be smoothed over the face and neck. In an embodiment, a hydrating serum of the present disclosure includes water, aqueous silk fibroin-based fragment solution, hyaluronic acid, and lemongrass oil. In an embodiment, the silk fibroin-based fragment protein in a hydrating serum of the present disclosure has the ability to stabilize and protect skin while sealing in moisture, all without the use of harsh chemical preservatives or synthetic additives. In an embodiment, the hyaluronic acid in a hydrating serum of the present disclosure nourishes skin and delivers moisture for lasting hydration. In an embodiment, the lemongrass essential oil in a hydrating serum of the present disclosure yields antioxidant and anti-inflammatory properties that support skin rejuvenation. In an embodiment, a hydrating serum of the present disclosure has a pH of about 6.0. Silk Fibroin-Based Fragment Solution Because silk fibroin-based fragment solution is both aqueous and able to entrap and deliver small molecules, the solution is able to deliver both water and hygroscopic HA molecules to the skin for hydration. A range in concentration of silk fibroin-based fragment compositions in solution from 0.5%-6.0% was tested for feasibility and product outcome. All concentrations tested were found to be feasible. Hyaluronic Acid Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in the hydrating serum due to its hygroscopic properties and ability to promote soft, hydrated skin. A range in concentration of hyaluronic acid in solution from 0.5%-10.0% was tested for feasibility and product outcome. All concentrations tested, with the exception of 10.0%, were found to be feasible. Feasibility was determined based on the ability to dissolve hyaluronic acid. Vitamin C and Derivatives Thereof Vitamin C (L-ascorbic acid) was tested as an ingredient in the hydrating serum. Initial vitamin C samples became a non-homogeneous mixture of gel and liquid. A follow-up trial with vitamin C resulted in a homogeneous, white, opaque, non-viscous liquid that was not quickly absorbed by the skin. In an embodiment, a vitamin C derivative that does not readily cause gelation, such as sodium ascorbyl phosphate, could be added up to the concentration at which it would no longer be soluble (for example, 0% to about 40%). In an embodiment, 20% sodium ascorbyl phosphate could be added. Vitamin C options that do cause gelation (L-ascorbic acid and ascorbyl glucoside) could be added at high concentrations (for example greater than about 10% up to about 50%) at which gelation is inhibited. Serum Creation Method Initial serums were created by the addition of HA to a silk fibroin-based fragment solution followed by stirring. The HA appeared to stick together and was not dissolved until forcefully stirred. The mixing process was then changed so that HA was first dissolved in water and then immediately used to dilute a high concentration silk fibroin-based fragment solution (>4%) to the desired concentrations. The resulting serums were more homogeneous and had a desirable smooth, clear look and feel. Upon application to the skin, a white residue briefly appeared that could be rubbed in. In an alternate method formulations were created by dissolving HA in water and allowing it to sit for 1 day until complete dissolution was observed. The HA and water solution was then used to dilute a high concentration silk fibroin-based fragment solution to the desired concentrations. The resulting serum was clear, smooth, homogeneous and left little to no white residue when applied. Example 14. UV Hydrating Serums of the Present Disclosure Variables tested include: concentration of HA, concentration of zinc oxide, concentration of titanium dioxide, addition of vitamin C, and serum preparation method. FIGS.94A-94Care tables summarizing embodiments of cosmetic serums of the present disclosure with varying additives and concentrations of components suitable for protection against ultraviolet radiation (UV). Table 33 provides an embodiment of a hydrating serum of the present disclosure with vitamin C. TABLE 33Embodiment of Hydrating serum of the present disclosure with vitamin C% Silk Solution (60 minute boil, 25 kDA)1.0%w/vHyaluronic Acid (sodium hyaluronate)0.75%w/vLemongrass Oil20 uL/15mLsilk solutionSodium Ascorbyl Phosphate6gLactic Acid1.2mL A serum of the present disclosure can be made with from about 0.25% to about 10% sodium hyaluronate (increasing % results in more viscous serum). 0.5% to about 10% silk solutions can be used to prepare a serum of the present disclosure. A serum of the present disclosure can be clear and have a yellow tinted color. A serum of the present disclosure can have a pH=6. A serum of the present disclosure can have a lubricious texture that is rubbed in easily without residue. Concentration of HA: Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in the UV silk serum due to its hygroscopic properties and widely accepted use in cosmetic products to promote hydration of skin. 1%, 2.5% and 5% HA solutions were tested. With increasing HA %, the serum became more viscous and gel like. 1% HA was not feasible for the UV serum due to the fact that the UV additives (zinc oxide, titanium dioxide) are not water soluble and need to be dispersed. 1% HA was not viscous enough for dispersion and the UV additives precipitated out. 2.5% gave the best consistency based on preferred feel, texture and viscosity and was able to disperse the UV additives. 5% was a very thick, viscous serum. Concentration of Mineral Filters: Zinc Oxide and Titanium Dioxide: Zinc oxide and titanium dioxide were explored as UV additives that are considered safe. These additives mechanically protect from UV radiation by forming a physical reflective barrier on the skin. Both are not soluble in water and must be dispersed for the current aqueous solution. Zinc oxide concentration varied from 2.5%, 3.75%, 5%, 5.625%, 10%, 12% and 15%. Titanium dioxide concentrations varied from 1.25%, 1.875%, 3%, 5% and 10%. Increasing the concentration of UV additives resulted in minor increases of white residue and how well dispersed the additives were, however if mixed well enough the effects were negligible. Zinc oxide and titanium dioxide were mixed together into serums in order to achieve broad spectrum protection. Zinc oxide is a broad spectrum UV additive capable of protecting against long and short UV A and UV B rays. However titanium dioxide is better at UV B protection and often added with zinc oxides for best broad spectrum protection. Combinations included 3.75%/1.25% ZnO/TiO2, 5.625%/1.875% ZnO/TiO2, 12%/3% ZnO/TiO2, 15%/5% ZnO/TiO2. The 3.75%/1.25% ZnO/TiO2 resulted in spf 5 and the 5.625%/1.875% ZnO/TiO2 produced spf 8. Vitamin C: Sodium ascorbyl phosphate was used as a vitamin C source. Formulations were created with the vitamin C concentration equal to that in the silk gel (0.67%). Formulations were also created with 20% sodium ascorbyl phosphate which is soluble in water. Serum Preparation: The vitamin C (sodium ascorbyl phosphate) must first be dissolved in water. Sodium hyaluronate is then added to the water, mixed vigorously and left to fully dissolve. The result is a viscous liquid (depending on HA %). The viscosity of the HA solution allows even dispersion of the zinc oxide and titanium dioxide and therefore HA must be mixed before addition of UV additives. The zinc oxide and titanium dioxide are then added to the solution and mixed vigorously with the use of an electric blender. Silk solution is then added and mixed to complete the serum formulation. Chemical Filters: A UV serum of the present disclosure can include one, or a combination of two or more, of these active chemical filter ingredients: oxybenzone, avobenzone, octisalate, octocrylene, homosalate and octinoxate. A UV serum of the present disclosure can also include a combination of zinc oxide with chemical filters. In an embodiment, a UV serum of the present disclosure can be applied approximately 15 minutes before sun exposure to all skin exposed to sun, and can be reapplied at least every 2 hours. In an embodiment, a UV serum of the present disclosure includes water, zinc oxide, sodium hyaluronate, titanium dioxide, silk, and vitamin C or a vitamin C derivative such as sodium ascorbyl phosphate. In an embodiment, a UV serum of the present disclosure protects skin and seals in moisture with the power of silk protein. In an embodiment, a UV serum of the present disclosure improves skin tone, promotes collagen production and diminishes the appearance of wrinkles and fine lines with the antioxidant abilities of vitamin C. In an embodiment, a UV serum of the present disclosure delivers moisture for immediate and long-term hydration throughout the day with concentrated hyaluronic acid. In an embodiment, a UV serum of the present disclosure helps prevent sunburn with the combined action of zinc oxide and titanium dioxide. In an embodiment, a UV serum of the present disclosure is designed to protect, hydrate, and diminish fine lines while shielding skin from harsh UVA and UVB rays. In an embodiment, the silk protein in a UV serum of the present disclosure stabilizes and protects skin while sealing in moisture, without the use of harsh chemical preservatives or synthetic additives. In an embodiment, the vitamin C/derivative in a UV serum of the present disclosure acts as a powerful antioxidant that supports skin rejuvenation. In an embodiment, the sodium hyaluronate in a UV serum of the present disclosure nourishes the skin and delivers moisture for long-lasting hydration. In an embodiment, the zinc oxide and titanium dioxide in a UV serum of the present disclosure shields skin from harmful UVA and UVB rays. The silk protein stabilization matrix in a UV serum of the present disclosure protects the active ingredients from the air, to deliver their full benefits without the use of harsh chemicals or preservatives. The silk matrix also traps moisture within the skin furthering the hydrating effect of the sodium hyaluronate. Example 15. Dark Spot Films of the Present Disclosure To reduce the appearance of dark spots, a high concentration of vitamin C may be necessary to reverse the overproduction of melanin. In this example, a 40% vitamin C (1.5:1 silk to vitamin C) was studied. The size and shape of the film can be made appropriate to a targeted area, for example to a small circular film of diameter 1 in (2.54 cm). The dark spot film, or a similar film of the present disclosure, of varying vitamin C concentration (0-50%) can be applied as a hydrofilm. Skin can be wetted with water. The film is then applied to the wet area. Water is then applied to the top surface of the film to turn it into a gel. The gel can then be spread and gently massaged into the application area. Table 34 provides details of an embodiment of a hydrofilm of the present disclosure (with no insoluble border). TABLE 34An Embodiment of a hydrofilm of the present disclosure% Silk Solution (60 minute boil, 25 kDA)2.56%Quantity Vitamin C15.62 mg total (10 mg(l-ascorbic acid)in 1 in circle punch out)Volume of solution per mold2.44 mLFilm Size1.25 in diameter circle(7.917 cm{circumflex over ( )}2) A film of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. A film of the present disclosure can be made with from about 1% to about 50% 1-ascorbic acid. A film of the present disclosure is soluble in water (insoluble border is removed by punching out the center of the film). A film of the present disclosure can adhere to skin with water. A film of the present disclosure can be spread on skin once water is applied. A film of the present disclosure can be dried when the humidity of drying equipment is 16-40% and below the humidity of the lab. A film of the present disclosure can be clear/transparent. In an embodiment, a dark spot film of the present disclosure includes water, silk, and vitamin C (L-ascorbic acid). In an embodiment, a dark spot film of the present disclosure includes 40% vitamin C. In an embodiment, a dark spot film of the present disclosure reduces skin pigmentation and evens skin tone in a targeted area with daily use. Vitamin C can inhibit pigment transfer from pigment producing cells, called melanocytes, to skin surface cells with continual application. In an embodiment, a dark spot film of the present disclosure can be applied to clean, dampened skin for 20 minutes. In an embodiment, additional water can be applied to an adhered film. The silk protein stabilization matrix in a dark spot film of the present disclosure protects the active ingredients from the air, to deliver their full benefits without the use of harsh chemicals or preservatives, such as paraben and phthalate. Thus, a dark spot film of the present disclosure is paraben and phthalate-free. Table 35 provides details of an embodiment of a film of the present disclosure. TABLE 35An Embodiment of a Film of the Present Disclosure% Silk Solution (60 minute boil, 25 kDA)2.2%Surface area5.07 cm{circumflex over ( )}2Volume of silk solution for casting1.56 mLMass of silk per film:34 mgMass of l-ascorbic acid per film:23 mgConcentration of l-ascorbic acid in film:40%pH3 A 2.1% silk solution of the present disclosure (0.321 mL/cm{circumflex over ( )}2) to 2.4% silk solution of the present disclosure (0.282 mL/cm{circumflex over ( )}2) can been used to create dark spot films of the present disclosure with 34 mg of silk (6.7 mg/cm{circumflex over ( )}2). In an embodiment, a 2.2% silk solution of the present disclosure (60 minute boil, 25 kDA) is used to produce a film of the present disclosure. The % silk and volume of solution can vary to produce equivalent films. A dark spot film of the present disclosure can be made with different combinations of % silk and volume to produce films with silk quantities of 3 mg/cm{circumflex over ( )}2 to 10 mg/cm{circumflex over ( )}2. A dark spot film of the present disclosure can be made with from about 15 to about 50% 1-ascorbic acid. A dark spot film of the present disclosure is soluble in water (insoluble border). A dark spot film of the present disclosure is clear/transparent. A dark spot film of the present disclosure has a pH=3 when water is applied. A dark spot film of the present disclosure can adhere to skin with water. A dark spot film of the present disclosure can dry when humidity of drying equipment is 16-40% and below the humidity of the lab Example 16. High Concentration Vitamin C Gels of the Present Disclosure High concentration vitamin C gels were pursued up to 20%. Vitamin C type, vitamin C concentration, % silk and pH were varied to increase the quantity of vitamin C in a gel. FIGS.95A-95Care tables summarizing embodiments of high concentration vitamin C gels of the present disclosure. The highest concentration of vitamin C to gel was a 15% ascorbic acid 2 glucoside gel with 3.8% silk solution after 12 days. 5 and 10% ascorbic acid-2-glucoside formulations with 2, 3 and 3.8% silk all gelled. For each group of % vitamin C, gelation first occurred in the 3.8% silk followed by the 3% and lastly the 2%. It appears that there is a relationship between vitamin C concentration, silk concentration and gelation. If a solution has too much vitamin C in relation to silk, gelation will be prevented. Therefore, in order to produce high concentration vitamin C gels, higher concentration silk is necessary. One sample was cast at 5.5% silk and 20% vitamin C but gelation did not occur and a higher % silk may be necessary. Samples were also brought to a pH of 2 with lactic acid in order to help induce gelation in 3% silk solutions with 10 or 20% vitamin C, however gelation did not occur in 12 days. Example 17. Microbiological Study of Gels of the Present Disclosure Contaminating micro-organisms in cosmetics may cause a spoilage of the product and, when pathogenic, they represent a serious health risk for consumers worldwide. The United States Pharmacopoeia (USP) Microbial Limits Test provides several methods for the determination of total microbial count for bacteria, yeast and mold. Various gels of the present disclosure were tested to evaluate the possible microbial contamination in three different states of their use (intact, in-use, ending product).FIG.96is a table summarizing the results of such testing. The samples of gel and water samples from carboys were analyzed for determination of CFU/mL (colony-forming units per milliliter) of aerobic bacteria as well as yeast and mold. Samples were exposed to growth medium of Tryptic Soy Agar (TSA) for bacteria and Potato Dextrose Agar (PDA) for fungi (yeast/mold) at an exposure temperature of 23±3° C. Samples were incubated at 30.0±2° C. for 3 days (bacteria) and 5 days (Fungi). Samples were then observed for determination of colony-forming units/mL. The limit of detection for the assays was 10 CFU/ml or g for bacteria and fungi, and the values of <10 indicate that microorganisms could not be detected in the samples. Values of >1.00E+04 indicate that the microbial colonies are Too Numerous to Count in the dilutions plated. Example 18. UV Silk Foams and Liquids of the Present Disclosure In an embodiment, the vitamin C derivative sodium ascorbyl phosphate (DSM) was dissolved in water. Sodium hyaluronate (“HA”) was then added to the water, mixed vigorously, and left to fully dissolve. The result is a viscous liquid (depending on HA %). The viscosity of the HA solution allows even dispersion of the zinc oxide and titanium dioxide and therefore HA is typically mixed before addition of UV additives. The zinc oxide and titanium dioxide are added to the HA solution and mixed vigorously, for example with the use of an electric blender. 60 minute boiled (˜25 kDa) silk solution is then added and mixed to create a 1% silk formulation. Two formulations were created without the addition of sodium ascorbyl phosphate (samples “HU2” and “HU4”). For sample HU2, zinc oxide and titanium dioxide were added and mixed by blending with an electric blender and whisk. The result was a viscous white liquid (FIG.98andFIG.99). Silk was then added and blended with an electric blender and whisk. The solution became a creamy foam similar to shaving cream (FIG.97andFIG.100). Vitamin E in the form of dl-alpha tocopheryl acetate can be added to the solution to recover a viscous liquid texture that can be applied with a smooth even texture (FIG.98). With increasing the quantity of dl-alpha tocopheryl acetate, the formulation will become less foam-like and more of a smooth liquid or lotion texture. HU4 was split into two batches:FIG.99, batch 2 andFIG.100, batch 1. The first batch followed the same procedures to HU2 and became a foam. For the second batch of HU4, sodium ascorbyl phosphate was added and dissolved before adding any zinc, titanium or silk. The UV additives were then added by blending with an electric blender and whisk and created a standard white viscous liquid. Silk was then added with an electric blender and whisk. The result was slightly thicker viscous liquid than normally seen. Without wishing to be bound by theory, it appears the addition of sodium ascorbyl phosphate inhibits foaming. Without wishing to be bound by theory, it appears that whisking, as opposed to mixing or blending, creates a silk foam. TABLE 36Embodiments of UV Silk Foams and Liquids of the Present Disclosure% HAMassSodiumTotal%(sodiumMassZnOMassAscorbylSampleVolumesilkhyaluronate)HA (g)% ZnO(g)% TiO2TiO2(g)Phosphate (g)HU25512.51.375126.631.65N/AHU427.513.50.9625123.330.8255.5Batch 1HU427.513.50.9625123.330.825N/ABatch 2 Example 19. Lyophilized Silk Powders of the Present Disclosure TABLE 37Embodiments of lyophilized silk powdersSilk SolutionTreatmentSoluble~60 kDa silk, 6% silk, pH = 8lyophilize and cut with blenderno~60 kDa silk, 6% silk, pH = 0lyophilize and cut with blenderno~25 kDa silk, 6% silk, pH = 8lyophilize and cut with blenderyes~25 kDa silk, 6% silk, pH = 0lyophilize and cut with blenderyes The above silk solutions were transformed to a silk powder through lyophilization to remove bulk water and chopping to small pieces with a blender. pH was adjusted with sodium hydroxide. Low molecular weight silk (˜25 kDa) was soluble while high molecular weight silk (˜60 kDa) was not. The lyophilized silk powder can be advantageous for enhanced storage control ranging from 10 days to 10 years depending on storage and shipment conditions. The lyophilized silk powder can also be used as a raw ingredient in the pharmaceutical, medical, consumer, and electronic markets. Additionally, lyophilized silk powder can be re-suspended in water, HFIP, or an organic solution following storage to create silk solutions of varying concentrations, including higher concentration solutions than those produced initially. In an embodiment, aqueous pure silk fibroin-based protein fragment solutions of the present disclosure comprising 1%, 3%, and 5% silk by weight were each dispensed into a 1.8 L Lyoguard trays, respectively. All 3 trays were placed in a 12 ft2lyophilizer and a single run performed. The product was frozen with a shelf temperature of ≤−40° C. and held for 2 hours. The compositions were then lyophilized at a shelf temperature of −20° C., with a 3 hour ramp and held for 20 hours, and subsequently dried at a temperature of 30° C., with a 5 hour ramp and held for about 34 hours. Trays were removed and stored at ambient conditions until further processing. Each of the resultant lyophilized silk fragment compositions were able to dissolve in aqueous solvent and organic solvent to reconstitute silk fragment solutions between 0.1 wt % and 8 wt %. Heating and mixing were not required but were used to accelerate the dissolving rate. All solutions were shelf-stable at ambient conditions. In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 30 minute boil, has a molecular weight of about 57 kDa, a polydispersity of about 1.6, inorganic and organic residuals of less than 500 ppm, and a light amber color. In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 60 minute boil, has a molecular weight of about 25 kDa, a polydispersity of about 2.4, inorganic and organic residuals of less than 500 ppm, and a light amber color. A method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 6 kDa to about 16 kDa includes the steps of: degumming a silk source by adding the silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 60° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in an oven having a temperature of about 140° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk protein fragments, the aqueous solution comprising: fragments having an average weight average molecular weight ranging from about 6 kDa to about 16 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin-based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin-based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin-based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin-based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. A method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 17 kDa to about 38 kDa includes the steps of: adding a silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60° C. to about 100° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of pure silk fibroin-based protein fragments, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, wherein the aqueous solution of silk protein fragments comprises sodium carbonate residuals of between about 10 ppm and about 100 ppm, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises fragments having an average weight average molecular weight ranging from about 17 kDa to about 38 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin-based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin-based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin-based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin-based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. According to aspects illustrated herein, there is disclosed a method for preparing an aqueous solution of pure silk fibroin-based protein fragments having an average weight average molecular weight ranging from about 39 kDa to about 80 kDa, the method including the steps of: adding a silk source to a boiling (100° C.) aqueous solution of sodium carbonate for a treatment time of about 30 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80° C. to about 140° C.; maintaining the solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60° C. to about 100° C. for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of pure silk fibroin-based protein fragments, wherein the aqueous solution of pure silk fibroin-based protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, sodium carbonate residuals of between about 10 ppm and about 100 ppm, fragments having an average weight average molecular weight ranging from about 40 kDa to about 65 kDa, and wherein the aqueous solution of pure silk fibroin-based protein fragments comprises a polydispersity of between about 1.5 and about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin-based protein fragments may comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin-based protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin-based protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin-based protein fragments may be lyophilized. The method may further comprise adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin-based protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid and citric acid. The method may further comprise adding hyaluronic acid or its salt form at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin-based protein fragments. The method may further comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin-based protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%. All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While the methods of the present disclosure have been described in connection with the specific embodiments thereof, it will be understood that it is capable of further modification. Further, this application is intended to cover any variations, uses, or adaptations of the methods of the present disclosure, including such departures from the present disclosure as come within known or customary practice in the art to which the methods of the present disclosure pertain. | 173,635 |
11857665 | DETAILED DESCRIPTION OF THE INVENTION As used herein, “active wt %” and “wt % (active)” refers to the amount of solid of an ingredient dissolved or suspended in water in the composition. As used herein, “ambient conditions” refers to a temperature of about 23 degrees Celsius (° C.) and 50% relative humidity (RH). As used herein, “Particle Size Distribution D50” refers to the diameter where fifty percent of the distribution has a smaller particle size. As used herein, “Particle Size Distribution D90” refers to the diameter where ninety percent of the distribution has a smaller particle size. As used herein, “separation” refers to the formation of a clear fluid layer at the top of a sample regardless of the uniformity of the particles in the sample below the clear fluid layer. Separation can include particle settling and/or syneresis. As used herein, “settling” refers to the falling of particles in a composition due to gravity (according to Stokes' Law) to the bottom of a container. Particle settling can be affected by the size of the particles and their agglomeration over time. As used herein, “static storage” refers to storage of a composition in the absence of vigorous or sustained vibration, agitation, or mixing prior to analysis or deposition of the composition. As used herein, “syneresis” means phase separation, i.e. extraction or expulsion of a liquid from a gel, in this case a weak colloidal gel. The particles in a composition exhibiting syneresis are still uniformly suspended below the clear fluid layer. As used herein, “ratio of (meth)acrylic acid homopolymer or a salt thereof to rheology modifier” refers to the ratio of the active wt % of the (meth)acrylic acid homopolymer or salt thereof divided by the active wt % of the rheology modifier. As used herein, “the storage modulus” or “G′” refers to the measure of the stored energy, representing the elastic portion of the composition. As used herein, “the loss modulus” or “G″” refers to the measure of the energy dissipated as heat, representing the viscous portion of the composition. As used herein, “weight percent as added” refers to the amount of the total active plus water as added to the composition. As used herein, “zeta potential” refers to the electrokinetic potential of the cosmetic ink composition. As used herein, the articles “a” and “an” are understood to mean one or more of the material that is claimed or described, for example, “a rheology modifier” or “an active”. All weights, measurements and concentrations herein are measured at ambient conditions unless otherwise specified. All percentages, parts, and ratios as used herein are based upon the total weight of the cosmetic ink composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the level as added in the composition, unless otherwise specified. Cosmetic inks can require a white or light colored particulate material comprising particles large enough to be visually perceptible to the human eye in order to create opacity to cover skin imperfections. However, printing a cosmetic ink composition comprising a particulate material having a particle size large enough to be visually perceptible can be a challenge for current inks, printers, and/or cartridges. These challenges are caused primarily by the settling of the large, dense particles and secondarily by the packing of the settled particles in the cartridge. Re-mixing, either by automated mixing or hand shaking, may not be compatible with consumer in-home or hand-held printing purposes because of the vigorous and repeated re-mixing needed to keep the particles uniformly suspended. Current opaque ink products can require repeated shaking, agitation, and/or rotation of the device and/or cartridge to keep the particles in a uniform suspension. This can be inconvenient to a consumer and is an additional step that consumers may not perform as instructed. If the particles are not re-dispersed, the cosmetic ink composition may not be uniform and/or the particles can become packed into the cartridge. Settling can be minimized by adding rheology modifiers to the ink. However, if the viscosity of the cosmetic ink composition is not well controlled, clogging and the speed of flow through the microfluidic channels and nozzles can make printing difficult. The use of dispersants in cosmetic ink compositions may help control agglomeration (i.e. the sticking of particles to one another) and hard packing of the particles; however, such formulations still require shaking to re-disperse the particles and do not exhibit particle suspension stability. Described herein is a cosmetic ink composition that can exhibit long-term particle suspension stability and a personal care device for depositing the cosmetic ink composition. In one aspect, “stable white ink” can refer to an ink substantially free from particle settling, where no agitation or mixing is required to use the ink for its application. In one aspect, the cosmetic ink composition described herein can be jetted onto any surface, preferably a keratinous surface including human skin and/or hair. The cosmetic ink composition can comprise a unique combination of a rheology modifier and a (meth)acrylic acid homopolymer or a salt thereof that can suspend a particulate material having particles large enough to be visually perceptible, yet can still be jettable. Careful balancing of the agglomeration of the particles and viscosity of the cosmetic ink composition can be used to inhibit particle settling, providing particle suspension stability beyond which has been previously reported without hindering high frequency printing capabilities via thermal and/or piezo inkjet printheads. One advantage to this is that little to no shaking, and/or agitation of the cosmetic ink composition by the consumer or automated mechanical processes before and/or during printing is needed to re-disperse the particles. This can make the cosmetic ink composition more user-friendly as it does not require the consumer to perform an additional step to re-disperse the particles and/or can eliminate the need for agitation or rotation systems, including automated systems, within the printing device, cartridge, printer servicing station, and/or docking station. Without being limited by theory, it is believed that the cosmetic ink composition can be stabilized using a (meth)acrylic acid homopolymer or a salt thereof to minimize and/or prevent particle agglomeration and a rheology modifier to introduce a secondary structure and build viscosity to suspend the particles in a weak colloidal gel. The colloidal gel can be strong enough to hold the particles in suspension when not printing (thus inhibiting particle settling), but weak enough to break up during printing. In particular, it was found that a cosmetic ink composition comprising a particulate material, such as TiO2, a rheology modifier selected from the group consisting of alkali swellable emulsion (“ASE”) polymers, hydrophobically modified alkali swellable emulsion (“HASE”) polymers, and combinations thereof, and a (meth)acrylic acid homopolymer or a salt thereof can be stable, yet still jettable using standard cartridges and nozzles in a thermal and/or piezo printer. It was found that the cosmetic ink composition described herein can have a first dynamic viscosity at a shear rate of 0.1 sec−1measured at 25° C. of greater than about 1,100 cP, which can prevent settling of the particles. It was further found that the cosmetic ink composition can have a second dynamic viscosity at a shear rate of 1,000 sec−1measured at 25° C. of less than about 100 cP, which can prevent clogging and speed of flow issues through the cartridge and/or nozzles. Without being limited by theory, it is believed that a shear rate of 0.1 sec−1can be representative of storage conditions, while a shear rate of 1,000 sec−1can be representative of printing conditions. In one aspect, the cosmetic ink composition need not be agitated or shaken to re-disperse the particles before and/or during use because the particles can remain in suspension over the shelf-life of the product. Cosmetic Ink Composition In one aspect, the cosmetic ink composition can be a skin care composition. The cosmetic ink composition can hide or camouflage a skin imperfection, such as hyperpigmentation, when deposited precisely and substantially only onto the skin imperfection. The cosmetic ink composition can be non-Newtonian, meaning the cosmetic ink composition can have a shear dependent viscosity and/or viscoelastic properties. The cosmetic ink composition can show shear thinning effects under the fluid ejection conditions in which the ink is moved between the cartridge and the printhead of an inkjet device. When the cosmetic ink composition is jetted, the shear rate can increase, resulting in a decrease in the viscosity. Thus, the cosmetic ink composition can be stored without particle settling, yet the viscosity and particle size are such that the cosmetic ink composition can still be printed. The cosmetic ink composition can comprise a particulate material, a (meth)acrylic acid homopolymer or a salt thereof, and a rheology modifier. In one aspect, the particulate material can be hydrophilic. In one aspect, the particulate material can be substantially coated with one or more ingredients to cause the particulate material to become more hydrophilic. As used herein, “substantially coated” can mean at least about 25%, preferably greater than about 50% surface coverage of the particulate material, more preferably greater than about 75%, most preferably greater than about 90%. Suitable coating ingredients that can render the particulate material hydrophilic in nature can include silica, alumina, polyethylene glycol (PEG) 12 dimethicone, phytic acid, sodium glycerophosphate, and combinations thereof. The particulate material can be substantially coated with one or more coating ingredients using techniques known in the art. One advantage to using a hydrophilic particulate material is that hydrophilic particulate material can be more easily dispersed in water. In one aspect, the particulate material can be titanium and/or iron oxide which has been substantially coated with silica and/or alumina. Suitable particulate materials can include pigments; encapsulated pigments; mica; clay; mixed metal oxide pigments; metal oxides such as iron oxide, titanium dioxide, zinc oxide, aluminum hydroxide, iron oxide, and combinations thereof; boron nitride; silica; talc; basic lead carbonate; magnesium silicate; baryte (BaSO4); calcium carbonate; pearlescent; colorants, including natural colorants and synthetic monomeric and polymeric colorants; dyes such as azo, indigoid, triphenylmethane, anthraquinone, and xanthine dyes which are designated as D&C and FD&C blues, browns, greens, oranges, reds, yellows, etc.; insoluble metallic salts of certified color additives, referred to as the Lakes; and combinations thereof. In one aspect, the particulate material can comprise titanium dioxide, iron oxide, and combinations thereof. In one aspect, the titanium dioxide and/or iron oxide can be readily dispersed in water. In one aspect, the titanium dioxide and/or iron oxide is not hydrophobically treated before use in the cosmetic ink composition because it may not be readily dispersed in water. Suitable particulate material can include slurries of titanium dioxide and iron oxide available from KOBO Products Inc (South Plainfield, NJ), or equivalents. In one aspect, the cosmetic ink composition comprises a white pigment. In one aspect, the cosmetic ink composition can have a white appearance. Alternatively, the cosmetic ink composition can have a white appearance with tints of red and/or yellow. Typical levels of particulate material for sufficient opacity to hide and/or camouflage skin imperfections can be around 30 active wt %. In one aspect, the cosmetic ink composition can comprise greater than about 15 active wt % particulate material, alternatively greater than about 20 active wt %, alternatively greater than about 30 active wt %. In one aspect, the cosmetic ink composition can comprise from about 1 to about 30 active wt % particulate material, alternatively from about 3 to about 25 active wt %, alternatively from about 5 to about 20 active wt %, alternatively from about 8 to about 18 active wt %. The particulate material can comprise particles having a Particle Size Distribution (PSD) D50 of about 100 nm to about 2,000 nm, alternatively from about 150 nm to about 1,000 nm, alternatively from about 200 nm to about 450 nm, alternatively from about 200 nm to about 350 nm. In one aspect, the particulate material can comprise particles having a PSD D90 of less than about 2 μm, alternatively less than about 1 μm. In one aspect, the particulate material can comprise particles having a PSD D90 of from about 700 to about 900 μm. Without being limited by theory, it is believed that if the particles are too big, they can clog the microfluidic channels of the cartridge and disrupt printing. One skilled in the art would understand that an acceptable particle size can vary depending on printhead die architecture. In one aspect, the particulate material can comprise any PSD so long as the particles can move through the microfluidic channels of the cartridge and/or the printhead without causing clogging. The Particle Size Distribution can be measured according to the Particle Size Distribution Method described hereafter. The particulate material can have a refractive index of between about 1.1 and about 5.0, alternatively from about 1.5 to about 4, alternatively from about 2 to about 3. The particulate material can have a density range of from about 1.5 to about 6 g/mL, alternatively from about 2 to about 4 g/mL. The cosmetic ink composition can comprise a rheology modifier. Rheology modifiers can assist in preventing settling by keeping the particles uniformly suspended such that little to no agitation of the cosmetic ink composition is needed. One preferred group of rheology modifiers are ASE polymers. ASE polymers contain a balance of hydrophilic (meth)acrylic acid monomers and hydrophobic (meth)acrylate ester monomers and can be supplied at high volume solids in liquid form. ASE polymers rely on a change from low to high pH (neutralization) to trigger thickening. The “trigger” is built into the polymer by creating an approximately 50:50 ratio of (meth)acrylic acid, which is soluble in water, and a (meth)acrylate ester, which is not soluble in water. When the acid is un-neutralized (low pH), the polymer is insoluble in water and does not thicken. When the acid is fully neutralized (high pH), the polymer becomes soluble and thickens. ASE polymers are supplied at low pH (<5) and maintain a low as-supplied viscosity (<100 cP) at solids of up to 35%. When subject to a pH of about 7 or higher, ASE polymers solubilize, swell, and thicken the composition through volume exclusion. The degree of thickening can be related to the molecular weight of the polymer. Because their performance depends on water absorption and swelling, ASE polymers tend to be very high in molecular weight, which allows them to thicken efficiently. The rheology profiles ASE polymers create are typically steeply shear-thinning (pseudoplastic), and thus ASE polymers are well suited to build high viscosity at very low shear rates. Different rheological characteristics can be achieved by manipulating the molecular weight, as well as the types and amounts of acid and ester monomers, of the polymer. In one aspect, the hydrophilic monomers of the ASE polymer can include (meth)acrylic acid and maleic acid. In one aspect, the hydrophobic monomers of the ASE polymer can include the esters of (meth)acrylic acid with C1- to C4-alcohols, in particular ethyl acrylate, butyl acrylate, and methyl methacrylate. In one aspect, the ASE polymer can be synthesized from 10-90 wt % of Hydrophilic Monomer A and 10-90 wt % of Hydrophobic Monomer B. The structure of Hydrophilic Monomer A and Hydrophobic Monomer B are shown below. wherein R1and R2are independently hydrogen or methyl; wherein R3is C1to C4alkyl. Yet another group of rheology modifier suitable for use in the cosmetic ink composition described herein are HASE polymers. These are tertiary polymers that build on the ASE polymer chemistry by adding a hydrophobic acrylic ester and/or vinyl ester monomer to the polymer composition. HASE polymers retain the pH dependent behavior of their ASE counterparts, but in addition to absorbing water, HASE polymers also thicken via hydrophobe association. This mechanism, known as associative thickening (i.e. associating with any hydrophobic moiety in the composition), offers performance properties over a wider range of shear levels and enables a wider range of rheology profiles than is possible with volume exclusion thickeners such as ASE and cellulosic compositions. The hydrophilic and hydrophobic monomers of the HASE polymers can be the same as described with respect to the ASE polymers. The associative monomer of the HASE polymer can be a monomer that shows a strong hydrophobic character. A preferred associative monomer is ester of (meth)acrylic acid with C8-C22alcohols. In one aspect, the HASE polymer can be synthesized from 10-90 wt % Hydrophilic Monomer A, 10-90 wt % Hydrophobic Monomer B, and 0.01 to 2 wt % Associative Monomer C. The structure of Associate Monomer C is shown below. wherein R4is hydrogen or methyl; wherein R5is C8to C22alkyl; wherein n is an integer from 0 to 50. Alternatively, the HASE polymer can be synthesized from 10-90 wt % Hydrophilic Monomer A, 10-90 wt % Hydrophobic Monomer B, and 0.01 to 2 wt % Associative Monomer D. The structure of Associative Monomer D is shown below. wherein R6is hydrogen or methyl; wherein R7is C8to C22alkyl. In one aspect, the associative monomer can be selected from the group consisting of steareth-20 methacrylate, beheneth-25 methacrylate, vinyl neodecanoate, and combinations thereof. In one aspect, more than one associative monomers can be used in the synthesis of the HASE polymer. In one aspect, ASE and HASE polymers can comprise a cross-linking agent. The cross-linking agent can contain at least two ethylenically unsaturated moieties, alternatively at least three ethylenically unsaturated moieties. Suitable cross-linking agents can include divinyl benzene, tetra allyl ammonium chloride, allyl acrylates, methacrylates, diacrylates, dimethacrylates of glycols and polyglycols, butadiene, 1,7-octadiene, allyl-acrylamides, allyl-methacrylamides, bisacrylamidoacetic acid, N,N′-methylene-bisacrylamide, polyol polyallylethers such as polyallylsaccharose and pentaerythrol triallylether, and mixtures thereof. In one aspect, the cross-linking agent can be present at a level of from about 25 to about 5,000 ppm, alternatively from about 50 to about 1,000 ppm, alternatively from about 100 to about 500 ppm. Another group of rheology modifiers are hydrophobically-modified ethylene oxide-based urethane (HEUR) polymers. Unlike ASE or HASE-type rheology modifiers, HEUR polymers are non-ionic and soluble at any pH. This solubility is due to the polymer's ethylene oxide backbone, which is water soluble and makes up the majority of the polymer structure. Thus, HEUR polymers require a hydrophobic moiety in the composition to interact with the ethylene oxide backbone to impart structure. The cosmetic ink composition can comprise a HEUR polymer. Alternatively, the cosmetic ink composition comprises little to no hydrophobic moieties and does not comprise a HEUR polymer. The rheology modifier can be a (meth)acrylate polymer, a (meth)acrylate copolymer, and mixtures thereof. The rheology modifier can be selected from the group consisting of ASE polymers, HASE polymers, and combinations thereof. Suitable HASE polymers can include ACULYN™ Excel; ACRYSOL™ TT615; ACULYN™ 22; ACULYN™ 88; (all available from The DOW Chemical Company, Lake Zurich, IL); and combinations thereof. Suitable ASE polymers can include Rheovis® 1125 (available from BASF Corporation, Charlotte, NC), ACULYN™ 33; ACULYN™ 38 (both available from The DOW Chemical Company, Lake Zurich, IL); and combinations thereof. The cosmetic ink composition can comprise an ASE polymer. Alternatively, the cosmetic ink composition can comprise an HASE polymer. The rheology modifier does not consist of a surfactant, an amine oxide, and/or a cellulosic ether. The cosmetic ink composition can comprise any amount of rheology modifier so long as the first dynamic viscosity of the cosmetic ink composition is greater than about 1,100 cP at a shear rate of 0.1 sec−1measured at 25° C. The cosmetic ink composition can comprise greater than about 0.30 active wt % rheology modifier, alternatively greater than about 0.40 active wt %, alternatively greater than about 0.50 active wt %. The cosmetic ink composition can comprise from about 0.30 to about 1 active wt % rheology modifier, alternatively from about 0.30 to about 0.80 active wt %, alternatively from about 0.40 to about 0.50 active wt %. Active wt % can be measured using standard High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) techniques. One advantage to keeping the level of rheology modifier within this range is that the viscosity of the cosmetic ink composition can be built such that the particles can be suspended in the composition. The particles can be suspended for about 11 days or more at 25° C., alternatively about 30 days or more at 25° C., alternatively for about 90 days or more at 25° C., alternatively for about 300 days or more at 25° C. Without being limited by theory, it is believed that at levels of rheology modifier below this range the particles may not be sufficiently suspended and settling may occur. If the level of rheology modifier is too high, the viscosity of the cosmetic ink composition may increase to a point that can impact jetting (i.e. the cosmetic ink composition may not shear thin enough for efficient printing). In one aspect, the cosmetic ink composition can be substantially free of neutral inorganic salts (as compared to an alkali salt base, like NaOH). Without being limited by theory, it is believed that neutral inorganic salts, such as calcium chloride or sodium chloride, can increase the ionic strength of the cosmetic ink composition and can disrupt the internal structure, thus impacting stability. It is known that HASE and/or ASE polymers become polyelectrolytes at high pHs. As pH increases, the carboxylic acids on the HASE and/or ASE polymers can be neutralized, generating ionic groups on the polymer chains that can produce electrostatic repulsion. These electrostatic repulsions can cause the polymer to expand and form an internal structure in the composition. It is believed that inorganic neutral salts can shield this electrostatic repulsion and can cause the HASE and/or ASE polymer to change structure, and thus its effectiveness in promoting stability. The cosmetic ink composition can comprise a (meth)acrylic acid homopolymer or a salt thereof. Non-limiting examples of acceptable salts can include sodium, potassium, ammonium, and mixtures thereof. The (meth)acrylic acid homopolymer or salt thereof can be a low molecular weight material that can act to control particle size and can help maintain a low viscosity in the cosmetic ink composition. The (meth)acrylic acid homopolymer or salt thereof does not greatly increase viscosity of the cosmetic ink composition. Non-limiting examples of suitable (meth)acrylic acid homopolymers or salt thereof can include sodium polyacrylate such as Darvan® 811D (available from RT Vanderbilt Holding Company Inc., Norwalk, CT), ammonium polyacrylate having a weight average molecular weight of about 3,500 daltons such as Darvan® 821A (available from RT Vanderbilt Holding Company Inc.), and combinations thereof. The (meth)acrylic acid homopolymer or salt thereof have a weight average molecular weight of less than about 20,000 daltons, preferably less than about 10,000 daltons, more preferably less than about 5,000 daltons. The cosmetic ink composition can comprise a (meth)acrylic acid homopolymer or salt thereof having a weight average molecular weight of from about 1,000 to about 20,000 daltons, alternatively from about 1,000 to about 10,000 daltons, alternatively from about 2,000 to about 5,000 daltons, alternatively from about 2,500 to about 4,000 daltons. Weight average molecular weight can be measured by standard High Performance Size-Exclusion Chromatography per ASTM method D5296-11 (Sep. 1, 2011). In one aspect, the (meth)acrylic acid homopolymer or salt thereof is not a film forming polymer. Without being limited by theory it is believed that the (meth)acrylic acid homopolymer or salt thereof will not form a film because of the low molecular weight. The cosmetic ink composition can comprise from about 0.01 to about 1 active wt % (meth)acrylic acid homopolymer or salt thereof, alternatively from about 0.10 to about 0.85 active wt %, alternatively from about 0.20 to about 0.75 active wt %, alternatively about 0.30 to about 0.65 active wt %. Without being limited by theory, it is believed that the (meth)acrylic acid homopolymer or salt thereof can control agglomeration, and thus the particle size, of the particulate material by creating a negative surface charge around the particles. Thus, the (meth)acrylic acid homopolymer or salt thereof can help to maintain a particle size that is compatible with printer cartridges and nozzles. Without being limited by theory, it is believed that a cosmetic ink composition comprising below 0.01 active wt % (meth)acrylic acid homopolymer or salt thereof may not have sufficient particle size control and/or the viscoelastic modulus may be too high to allow for reliable refill of the microfluidics. The ratio of the (meth)acrylic acid homopolymer or salt thereof to the rheology modifier can be less than about 1. The ratio of (meth)acrylic acid homopolymer or salt thereof to rheology modifier can be from about 0.10 to about 0.75, alternatively from about 0.30 to about 0.65. Without being limited by theory it is believed that if the level of (meth)acrylic acid homopolymer or salt thereof is greater than the level of rheology modifier, the rheology modifier may not be able to build the internal structure needed to suspend the particles. If the ratio of (meth)acrylic acid homopolymer or salt thereof to rheology modifier is too low, agglomeration may not be well controlled and the particle size may become too large to fit through printer nozzles, making printing difficult. It is believed that stability is inversely proportional to the level of (meth)acrylic acid homopolymer or salt thereof and directly proportional to the level of rheology modifier. The cosmetic ink composition can have a first dynamic viscosity of greater than about 1,100 cP at a shear rate of 0.1 sec−1measured at 25° C. and a second dynamic viscosity of less than about 100 cP at a shear rate of 1,000 sec−1measured at 25° C. The cosmetic ink composition can have a first dynamic viscosity of from about 1,100 cP to about 10,000 cP at a shear rate of 0.1 sec−1at 25° C., alternatively from about 1,500 cP to about 8,000 cP, alternatively from about 2,000 cP to about 5,000 cP. The cosmetic ink composition can have a second dynamic viscosity of from about 10 cP to about 100 cP at a shear rate of 1,000 sec−1at 25° C., alternatively from about 20 to about 80 cP. Viscosity can be measured according to the Viscosity Test Method described hereinafter. One advantage to having a first and second dynamic viscosity in this range is that at high shear rate, the cosmetic ink composition can drop to a viscosity that is similar to Newtonian inks, yet can still maintain a viscosity sufficient to suspend the particles when not printing. The cosmetic ink composition can have a first dynamic viscosity measured at a shear rate of 0.1 sec−1at 25° C. that is about 70% higher than the second dynamic viscosity of the cosmetic ink composition when measured at a shear rate of 1,000 sec−1at 25° C., alternatively about 80% higher, alternatively about 90% higher, alternatively about 95% higher. The cosmetic ink composition can have a first dynamic viscosity measured at a shear rate of 0.1 sec−1at 25° C. that is about 25 times greater than the second dynamic viscosity of the cosmetic ink composition when measured at a shear rate of 1,000 sec−1at 25° C., alternatively about 35 times greater, alternatively about 50 times greater, alternatively about 80 times greater. The cosmetic ink composition can have temperature dependent viscosity. Lower viscosity was observed at a shear rate of 1000 sec−1at an elevated temperature of about 70° C. The cosmetic ink composition can have a storage modulus (G′) of from about 2 to about 10, alternatively from about 3 to about 8, alternatively from about 4 to about 6. Without being limited by theory, it is believed that if the G′ of the cosmetic ink composition is greater than about 10, the decap or start up after idle time in the printhead may be difficult without intervention because the composition is too elastic. Storage modulus can be measured according to the Oscillatory Strain Sweep Method described hereafter. The cosmetic ink composition can have a loss modulus (G″) of from about 1 to about 5, alternatively from about 1.5 to about 4, alternatively from about 2 to about 3. Loss modulus can be measured according to the Oscillatory Strain Sweep Method described hereafter. The ratio of loss modulus to storage modulus, or tan(delta), is a useful representation of the extent of elasticity in a fluid. In this case, it can be a measure of the intrinsic stability of the cosmetic ink composition. When G″ is higher than G′, tan(delta) is greater than about 1 and indicates a viscous dominant fluid behavior. When G′ is higher than G″, tan(delta) is less than about 1 and indicates an elastic dominant fluid behavior. The cosmetic ink composition can have a tan(delta) of about 1. Alternatively, the cosmetic ink composition can have a tan(delta) of less than about 1, alternatively less than about 0.6. Without being limited by theory it is believed that when the tan(delta) is less than about 0.6, particle settling can be minimized and/or prevented. The cosmetic ink composition can have a tan(delta) of from about 0.2 to about 1, alternatively from about 0.4 to about 0.9, alternatively from about 0.6 to about 0.8. The cosmetic ink composition can have a zeta potential of about negative20or less, alternatively about negative30or less, alternatively greater than about positive20, alternatively greater than about positive30. The cosmetic ink composition can have a zeta potential of about negative20or less, or greater than about positive20. One advantage to having a zeta potential in this range is that the surface charge of the particles can be increased, thus preventing agglomeration of the particles. Zeta potential can be measured according to the Zeta Potential Test Method described hereafter. The cosmetic ink composition can have a neat pH of greater than about 7.5. The cosmetic ink composition can have a neat pH of about 7.5 to about 9.0, alternatively from about 7.5 to about 8.5. Without being limited by theory, it is believed that at a pH lower than about 7.5, syneresis can occur at a faster rate. It is believed that at a lower pH, the equilibrium between the carboxylic acid and carboxylate salts of the rheology modifier can be pushed toward the protonated acid and therefore are not available to suspend the particles. It is believed that as the pH increases, the larger the negative zeta potential becomes, thus preventing agglomeration of the particles. The cosmetic ink composition can comprise a buffering agent for adjusting the pH conditions. The buffering agent can be any basic excipient. In one aspect, the buffering agent can be a strong base, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and mixtures thereof. The neat pH of the cosmetic ink composition can be measured by standard methodology known to those skilled in the art. The cosmetic ink composition can have a surface tension of from about 25 to about 60 dyn/cm, alternatively from about 30 to about 55 dyn/cm, alternatively from about 40 to about 50 dyn/cm. The cosmetic ink composition can have an opacity of at least 0.2. In one aspect, the cosmetic ink composition can comprise an opacity of from about 0.2 to about 1, alternatively from about 0.25 to about 0.8, alternatively from about 0.3 to about 0.5. The cosmetic ink composition can be substantially free of particle settling. Substantially free of particle settling can mean that the variation between the weight % solids of the top and bottom of a sample of the cosmetic ink composition is less than 5% at ambient conditions at 4 days after formulation, alternatively less than about 3%, alternatively less than about 1%. Particle settling can be measured according to the Particle Settling Test Method described hereafter. The cosmetic ink composition can be substantially free of particle agglomeration. Substantially free of particle agglomeration can mean that the cosmetic ink composition exhibits less than about 25 nm of particle growth per month at ambient conditions, alternatively less than about 15 nm, alternatively less than about 10 nm. The rate of agglomeration can be determined by measuring particle size according to the Particle Size Distribution method described hereafter over a period of time. The cosmetic ink composition can have less than about 10% separation at 11 days after formulation at 25° C., alternatively less than about 5%, alternatively less than about 2%, alternatively less than about 1%. In one aspect, the cosmetic ink composition can have less than about 4 mm of separation at 11 days after formulation at 25° C., alternatively less than about 2 mm, alternatively less than about 1 mm, alternatively less than about 0.50 mm. Separation can be measured according to the Separation Test Method described hereafter. The cosmetic ink composition can have a shelf-life of about 1 month, alternatively about 3 months, alternatively about 6 months, alternatively about 12 months, alternatively about 18 months, alternatively about 24 months. As used herein, “shelf-life” means the amount of time the particles can remain uniformly suspended in the cosmetic ink composition at ambient conditions without the need for shaking or agitation. The cosmetic ink compositions may further comprise a humectant as a carrier or chassis for the other components in the cosmetic ink composition. An exemplary class of humectants can include polyhydric alcohols. Suitable polyhydric alcohols can include polyalkylene glycols and alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof; sorbitol; hydroxypropyl sorbitol; erythritol; threitol; pentaerythritol; xylitol; glucitol; mannitol; butylene glycol (e.g., 1,3-butylene glycol); pentylene glycol; hexane triol (e.g., 1,2,6-hexanetriol); glycerin; ethoxylated glycerine; propoxylated glycerine; and mixtures thereof. Other suitable humectants can include sodium 2-pyrrolidone-5-carboxylate; guanidine; glycolic acid and glycolate salts (e.g., ammonium and quaternary alkyl ammonium); lactic acid and lactate salts (e.g., ammonium and quaternary alkyl ammonium); aloe vera in any of its variety of forms (e.g., aloe vera gel); hyaluronic acid and derivatives thereof (e.g., salt derivatives such as sodium hyaluronate); lactamide monoethanolamine; acetamide monoethanolamine; urea; sodium pyroglutamate, water-soluble glyceryl poly(meth)acrylate lubricants (such as Hispagel®, available from BASF, Ludwigshafen, Germany); and mixtures thereof. The cosmetic ink composition can comprise from about 1 to about 40 active wt % humectant, alternatively from about 5% to about 35%, alternatively from about 10% to about 30%, alternatively from about 15% to about 20%. Alternatively, the cosmetic ink composition can comprise from about 20 to about 30 active wt % humectant. Without being limited by theory, it is believed that at a level of about 20 active wt % or more the humectant can help prevent drying and/or clogging of the nozzles and/or cartridge when the cosmetic ink composition is not being printed. In one aspect, the level of humectant is less than about 30 active wt % to promote fast dry times of the cosmetic ink composition on the skin. The cosmetic ink composition can be delivered alone or in the presence of a dermatologically-acceptable carrier. The phrase “dermatologically-acceptable carrier”, as used herein, means that the carrier is suitable for topical application to a keratinous tissue, has good aesthetic properties, is compatible with any additional components of the cosmetic ink composition, and/or will not cause any untoward safety or toxicity concerns. In one aspect, the cosmetic ink composition is safe for use on skin. In one aspect, the cosmetic ink composition does not comprise alkyds, celluloses, formaldehydes, phenolics, ketones, rubber resins, and combinations thereof because such ingredients may not be compatible with use on human skin. In one aspect, the cosmetic ink composition can be hypoallergenic. Water is by far the most common carrier, and is typically used in combination with other carriers. The carrier can be in a wide variety of forms. Non-limiting examples include simple solutions (water or oil based) or emulsions. The dermatologically acceptable carrier can be in the form of an emulsion. Emulsion may be generally classified as having a continuous aqueous phase (e.g., oil-in-water and water-in-oil-in-water) or a continuous oil phase (e.g., water-in-oil and oil-in-water-in-oil). The oil phase may comprise silicone oils, non-silicone oils such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof. For example, emulsion carriers can include, but are not limited to, continuous water phase emulsions such as silicone-in-water, oil-in-water, and water-in-oil-in-water emulsion and continuous oil phase emulsions such as water-in-oil and water-in-silicone emulsions, and oil-in-water-in-silicone emulsions. The cosmetic ink composition can comprise water, preferably deionized water. The cosmetic ink composition can comprise from about 40% to about 75% water, by weight of the cosmetic ink composition, alternatively from about 55% to about 70%, alternatively from about 60% to about 68%. Additionally, the cosmetic ink composition can optionally include anti-fungal and/or anti-bacterial components. Examples of anti-fungal and/or anti-bacterial components can include isothiazolinone such as methylisothiazolinone and methylchloroisothiazolinone. The cosmetic ink composition can optionally comprise a preservative. Non-limiting examples of suitable preservatives can include phenoxy ethanol, 1,2-Hexanediol, 1,2-Octanediol (commercially available as SymDiol® 68 from Symrise, AG, Branchburg, NJ), farnesol, 2-methyl 5-cyclohexypentanol, 1,2-decanediol, and combinations thereof. The cosmetic ink composition can comprise from about 0.01% to about 10% preservative, alternatively from about 0.1% to about 5%, alternatively from about 1% to about 3%, all by weight of the cosmetic ink composition. One advantage to including a preservative is that it can help to prevent microbial growth in the cosmetic ink composition, for instance if the cosmetic ink composition becomes contaminated with bacteria from the skin. The cosmetic ink composition may comprise a monohydric alcohol. The cosmetic ink composition can comprise about 50 ppm or more of a monohydric alcohol. The cosmetic ink composition can comprise from about 50 to about 10,000 ppm of a monohydric alcohol, alternatively from about 100 to about 5,000 ppm, alternatively from about 100 to about 1,000 ppm. Suitable monohydric alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol and 2-methyl-2-propanol. The cosmetic ink composition may comprise a safe and effective amount of one or more skin care actives (“active”) useful for regulating and/or improving the skin. “Safe and effective amount” means an amount of a compound or composition sufficient to induce a positive benefit but low enough to avoid serious side effects (i.e., provides a reasonable benefit to risk ratio within the judgment of a skilled artisan). A safe and effective amount of an active can be from about 1×10−6to about 25%, alternatively from about 0.0001 to about 20%, alternatively from about 0.01 to about 10%, alternatively from about 0.1 to about 5%, alternatively from about 0.2 to about 2%, all by weight of the cosmetic ink composition. Suitable skin care actives include, but are not limited to, vitamins (e.g., B3 compounds such as niacinamide, niacinnicotinic acid, and tocopheryl nicotinate; B5 compounds such as panthenol; vitamin A compounds and natural and/or synthetic analogs of Vitamin A, including retinoids, retinol, retinyl acetate, retinyl palmitate, retinoic acid, retinaldehyde, retinyl propionate, and carotenoids (pro-vitamin A); vitamin E compounds, or tocopherol, including tocopherol sorbate and tocopherol acetate; vitamin C compounds, including ascorbate, ascorbyl esters of fatty acids, and ascorbic acid derivatives such as magnesium ascorbyl phosphate and sodium ascorbyl phosphate; ascorbyl glucoside; and ascorbyl sorbate); peptides (e.g., peptides containing ten or fewer amino acids, their derivatives, isomers, and complexes with other species such as metal ions); sugar amines (e.g., N-acetyl-glucosamine); sunscreens; oil control agents; tanning actives; anti-acne actives; desquamation actives; anti-cellulite actives; chelating agents; skin lightening agents; flavonoids; protease inhibitors (e.g., hexamidine and derivatives); non-vitamin antioxidants and radical scavengers; salicylic acid; hair growth regulators; anti-wrinkle actives; anti-atrophy actives; minerals; phytosterols and/or plant hormones; tyrosinase inhibitors; N-acyl amino acid compounds; inositol; undecylenoyl phenylalanine; moisturizers; plant extracts; and derivatives of any of the aforementioned actives; and combinations thereof. The term “derivative” as used herein refers to structures which are not shown but which one skilled in the art would understand are variations of the basic compound. For example, removing a hydrogen atom from benzene and replacing it with a methyl group. In one aspect, the cosmetic ink composition can comprise peroxide, including hydrogen peroxide and/or benzoyl peroxide. In one aspect, the cosmetic ink composition can comprise a skin care active selected from the group consisting of niacinamide; inositol; undecylenoyl phenylalanine; and combinations thereof. In one aspect, the cosmetic ink composition is substantially free of latex polymer binders and/or a film forming polymers. In one aspect, the cosmetic ink composition comprises less than about 10% latex polymer binders and/or film forming polymers, alternatively less than about 1%, alternatively less than about 0.1%. Without being limited by theory, it is believed that latex polymer binders and/or film forming polymers can make printing can be difficult because these polymers can solidify after evaporation and irreversibly plug the nozzles. In one aspect, the cosmetic ink composition comprises from about 10% to about 30% solids. In one aspect, the cosmetic ink composition comprises less than 40% solids. Without being limited by theory, it is believed that at a solids level of greater than 40%, such as in prepaints or paints, printing can be difficult because a high level of solids may lead to irreversible nozzle clogging. In one aspect, the cosmetic ink composition can be removeable with water, alternatively with soap and water. Personal Care Device In one aspect, the cosmetic ink composition described herein can be applied to the skin using a hand-held personal care device. The personal care device can analyze the skin, identify skin imperfections, and deposit the cosmetic ink composition onto the identified skin imperfection in order to hide and/or camouflage the skin imperfection. An exemplary personal care device is described in U.S. Pat. No. 9,522,101. In one aspect, the personal care device can comprise a sensor configured to take at least one image of skin and a processor configured to calculate the average background lightness value of the image on a grey scale (lightness value on a grey scale is herein referred to as “L value”). Further, from the same image, a local L value can be calculated for individual pixels or a group of pixels. The processor can then compare the local L value to the background L value to identify skin imperfections. When a skin imperfection is identified, the processor can activate one or more nozzles to fire and dispense the cosmetic ink composition onto the skin imperfection. A skin imperfection is an area of skin where the absolute value of the difference between a local L value and the background L, this difference being defined as the measured delta L (“ΔLM”), is greater than a predetermined set delta L (“ΔLS”). The background L can be preset or calculated anywhere within the image. The image can be taken where the nozzles will fire the cosmetic ink composition. The background L can be the arithmetic average, median, or mean of a plurality of local Ls, which means the calculation can include all of the local Ls in the image, or a subset thereof. FIG.1, shows an exploded view of personal care device40. Physical spacer42of personal care device40is directly above skin surface18. Physical spacer42has a set, predetermined height a such that when it contacts skin surface18, the mechanical and electrical elements are all at a known distance from skin surface18. In one aspect, the height a is from about 1 mm to about 20 mm, alternatively from about 3 mm to about 15 mm, alternatively from about 4 mm to about 10 mm. The mechanical and electrical elements associated with personal care device40can include, but are not be limited to, light44, sensor46, nozzle array20which is embedded on cartridge die57which is attached to cartridge52. Cartridge die57can be made of silicon, glass, machinable glass ceramic, sapphire, alumina, printed wiring board substrates (for example, Liquid Crystal Polymer, polyimide etc.) within which nozzle array20can be formed. Nozzle array20can be in a linear configuration, multiple rows, off-set, sine wave, curved, circular, saw tooth arrangements, and combinations thereof. All of these elements can be enclosed within optional apparatus housing41. Light44can illuminate the area of skin surface18within physical spacer42such that sensor46has relatively constant illumination. Background lighting can affect sensor46as portions of physical spacer42lift off skin surface18and allow background light in and the illumination from light44to escape. Small deviations in illumination can be corrected for provided light44provides a relatively constant background illumination. In one aspect, physical spacer42can be opaque. Light44can be a LED, incandescent light, neon bulb based, or any other commercially available source of illumination. Light44can have constant illumination or adjustable illumination. For example, an adjustable light source might be useful if the background illumination is excessively bright or dark. Sensor46can be any component that is capable of obtaining a visual property of an area of skin surface. Non-limiting examples of sensors can include optical sensors, image capture devices, spectrophotometers, photonic measuring devices for wavelengths within the visible spectrum as well as those wavelengths above and below the visible spectrum which could measure sub-surface features, and combinations thereof. The image capture device can be any of a variety of commercially available devices such as a simple camera or a digital cmos camera chip. In one aspect, the image capture device can be a camera and the images can be taken or converted to a standard grey scale that is known in the art. It is understood that any numerical scale that measures lightness to darkness can be considered a “grey scale”. Moreover, as used herein, “grey scale” is intended to be a linear scale, or one band, or one visual attribute. For example, one “grey scale” visual attribute could be single wavelength or a narrow wavelength to define a specific visual color. Another example of one “grey scale” visual attribute could be a mix of wavelength numerical values averaged for each pixel making up the image, such as a true black, grey or white image from an RGB mixture. Sensor46can take a measurement of the L value of skin surface18and/or an image of skin surface18and can send it to processor50via image capture line48for analysis. The image may be analyzed for local L values, background L values, or both. Grey scale conversion can occur within the analytical processing capabilities of processor50. The comparison of background L to local L to determine the ΔLMoccurs within processor50, which can be a commercially available programmable chip, or other commercially available processing units. Processor50is generally referred to as a central processing unit (“CPU”). The CPU can be a single programmable chip like those found in consumer electronic devices such as a laptop computer, a cell phone, an electric razor, and the like. The CPU may comprise an Application Specific Integrated Circuit (ASIC), controller, Field Programmable Gate Array (FPGA), integrated circuit, microcontroller, microprocessor, processor, and the like. The CPU may also comprise memory functionality, either internal to the CPU as cache memory, for example Random Access Memory (RAM), Static Random Access Memory (SRAM), and the like, or external to the CPU, for example as Dynamic Random-Access Memory (DRAM), Read Only Memory (ROM), Static RAM, Flash Memory (e.g., Compact Flash or SmartMedia cards), disk drives, Solid State Disk Drives (SSD), or Internet Cloud storage. While it is anticipated that a remote CPU, either tethered to the personal care device or which communicates wirelessly, can be used, a local CPU within the personal care device is exemplified herein. Images can be taken in sequence or preferably continuously. The image capture device can take images at a speed of at least 4 frames per second, alternatively at least 100 frames per second, alternatively at least 200 frames per second, alternatively at least 600 frames per second. The CPU can process at a rate of 100 frames per second, alternatively greater than 200 frames per second, alternatively greater than 600 frames per second. The results of the image analysis, when compared to criteria pre-programmed into processor50, may result in a desired treatment of skin surface18. For instance, when the calculated ΔLMexceeds the pre-determined ΔLS, a signal is sent from processor50to cartridge52, via cartridge line51, to fire one or more nozzles21in nozzle array20and dispense the cosmetic ink composition. Power for cartridge52, light44, sensor46, processor50, and other mechanical and electrical elements that might be present can be supplied by power element54via one or more power lines55. Power element54can be turned off and on, which in turn turns personal care device40off and on, via power switch56which can be located anywhere on personal care device40, but is shown here on device cover58. Power element54may include energy storage functionality via a battery, a rechargeable battery, an electrochemical capacitor, a double-layer capacitor, a supercapacitor, a hybrid battery-capacitor system, and combinations thereof. FIG.2shows an exploded view of cartridge52comprising cartridge cap62and cartridge body64. Cartridge body64can include standpipe66which is typically enclosed within cartridge body64and defines nozzle outlet68. Optional filter70can help keep excessively large particles, and other debris out of nozzle array20. Filter70and nozzle array20can be on opposite sides of nozzle outlet68. Cosmetic ink composition74can be contained within cartridge body64. Foam core72can at least partially fill cartridge64and helps to regulate back pressure of cosmetic ink composition74. Back pressure can be regulated via bladders (not shown) and other methods known to the art. Foam core72shown here is just one example of how to help regulate the flow of cosmetic ink composition74to standpipe66through filter70and into nozzle array20. Connector78can provide the electrical power and signal to nozzle array20. Cosmetic ink composition74may be ejected from the cartridge52by piezoelectric means, thermal means, mechanical pumping means, or a combination of these. An exemplary cartridge for use herein can include cartridges described in Patent Application US 2002/0167566. There is no technical difference between an image used for background L values and those used for local L values, the difference is in the analysis of the image. Hence, the images are continually sent to the processor to calculate the L values and ΔLMvalues. By “sent” it is understood, that preferably at least 4 bits of data per pixel are transferred for each image, and preferably, this 4-bit (or more) packet of data is used in the calculation of each local L value. It is understood, that the background L can be calculated once in a treatment period and that value can be reused throughout the treatment period. Alternatively, it can be continually recalculated as long as the treatment process goes on. Moreover, there can be pre-programmed triggers to initiate a recalculation of the background L. Also, the background L may be retrieved from the processor memory to be used for the current background L. When the ΔLMexceeds the predetermined value, the cosmetic ink composition can be deposited onto at least a portion of the skin imperfection. In particular, the cosmetic ink composition can be deposited via an array of nozzles and the local L can be calculated along the length of, and in the firing range of, the array of nozzles. An individual nozzle may be fired to deposit the cosmetic ink composition, or multiple nozzles can be fired at the same time. The number of nozzles fired along the array of nozzles can be adjusted based on the size of the ΔLMand the size of the skin imperfection. Furthermore, the frequency of nozzle firing can be adjusted based on the ALM, with more droplets being fired in succession in response to larger ΔLMvalues. The personal care device may deposit the cosmetic ink composition in droplets having an average diameter of from about from about 0.1 μm to about 60 μm, alternatively from about 1 μm to about 50 μm, alternatively from about 5 μm to about 40 μm. Preferably, the cosmetic ink composition can be applied to the skin imperfection in a discontinuous pattern of discrete droplets. The cosmetic ink composition can be printed from a cartridge having a micro-electro-mechanical system (MEMS) that is different from typical consumer printing applications. It is known that the typical chamber height and nozzle plate thicknesses are from about 25 to about 50 μm since typical printing inks have a viscosity of less than about 10 cP. In one aspect, the cartridge can comprise a chamber height and nozzle plate thicknesses of from about 10 to about 20 μm, preferably from about 12 to about 17 μm. Without being limited by theory it is believed that the shorter chamber height and plate thickness can help minimize viscous loss. In addition, most consumer printing applications are optimized for printing at 10 kHz or more, so ink formulas and microfluidics are designed to achieve rapid refill. However, operating the cosmetic ink composition described herein at this frequency range can result in streaming and/or de-priming due to gulping of air. The cosmetic ink composition can be printed using the following start-up sequence: heating the substrate to about 60° C. for less than about 600 ms, firing the nozzles in a burst of from about 100 to about 500 fires at a frequency of about 300 to about 1000 Hz, and then maintaining the low shear condition with continuous 4 Hz firing. While it is possible the nozzles will start up with different algorithms, it is likely that the cosmetic ink composition would not be transitioned from its viscous at-rest state to a flowing state. Also described herein is a method for depositing the cosmetic ink composition onto skin. The method for depositing a cosmetic ink composition onto skin can comprise the steps of:a. providing a personal care device comprising one or more nozzles and a cartridge operatively associated with the one or more nozzles, wherein a cosmetic ink composition is disposed within the cartridge; andb. depositing the cosmetic ink composition onto a portion of skin, wherein the cosmetic ink composition is deposited in a discontinuous droplet pattern. More specifically, a method for depositing a cosmetic ink composition onto skin can comprise the steps of:a. providing a personal care device comprising an array of nozzles;b. providing a background lightness (L) value;c. obtaining a treatment image of skin and calculating at least one local L value of individual pixels or group of pixels within the treatment image;d. comparing the local L value to the background L value;e. identifying a skin deviation where the absolute value of the difference between the local L value and the background L value is greater than a predetermined set delta L value; and treating the skin deviation with a cosmetic ink composition; wherein the ink composition comprises a particulate material having a Particle Size Distribution D50 of about 100 nm to about 2,000 nm; a (meth)acrylic acid homopolymer or salt thereof having a weight average molecular weight of less than about 20,000 daltons; and a rheology modifier, wherein the rheology modifier is selected from the group consisting of alkali swellable emulsion polymers, hydrophobically modified alkali swellable emulsion polymers, and combinations thereof; and wherein the ink composition has a first dynamic viscosity of greater than about 1,100 cP at a shear rate of 0.1 sec−1measured at 25° C. and a second dynamic viscosity of less than about 100 cP at a shear rate of 1,000 sec−1measured at 25° C. Examples and Data The following data and examples, including comparative examples, are provided to help illustrate cosmetic ink compositions described herein. The exemplified compositions are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention. All parts, percentages, and ratios herein are by weight unless otherwise specified. A series of formulas were prepared in a fractional factorial study design to understand the effect of the level of (meth)acrylic acid homopolymer or salt thereof and rheology modifier in the formula. Examples 1-18 were made according to the procedure described hereafter. The level of Polyvinylpyrrolidone/vinyl acetate (PVP/VA) and 1,2 Hexanediol/Caprylyl Glycol (commercially available as Symdiol® from Symrise AG, Branchburg, NJ) was kept constant at 1%, respectively. The level of total particulate material, propylene glycol, (meth)acrylic acid homopolymer sodium polyacrylate (Darvan® 811D), and HASE type rheology modifier (ACULYN™ Excel, available from The Dow Chemical Company, Lake Zurich, IL) were varied. Example 4 is a control containing no (meth)acrylic acid homopolymer or salt thereof. The samples were stored in sealed glass jars at 50° C. until measurements were performed. Samples were tested to determine the optimal level of (meth)acrylic acid homopolymer or salt thereof and rheology modifier by measuring the amount of separation. The measurement of separation at 50° C. was used as a screening tool to understand which formulas could build sufficient internal structure to suspend the particles. Samples that exhibit a large change in particle size distribution and/or particle settling separation suggest that the formulations are unstable. Examples 1-18 were made according to the following formulas. Weight percent is shown as added. TABLE 1123456PhaseDescriptionWt. %Wt. %Wt. %Wt. %Wt. %Wt. %A75 wt % TiO2Slurry12.1812.1812.1812.1818.815.55(WPG75PFSP)1A45 wt % Iron Oxide1.781.781.781.782.760.81Slurry (WPG45GYSP)1A55 wt % Iron Oxide0.110.110.110.110.170.05Slurry (WPG55GRSP)1BDeionized Water60.0660.0657.5361.7350.9966.32BPuraGuard ™20.0020.0020.0020.0020.0020.00Propylene Glycol2B50 wt % PVP/VA1.001.001.001.001.001.00735W3in waterBSymdiol ® 6841.001.001.001.001.001.00C5 wt % Darvan ®2.202.204.200.002.202.20811D5in waterD15 wt % ACULYN ™1.671.672.202.203.073.07Excel6in water789101112PhaseDescriptionWt. %Wt. %Wt. %Wt. %Wt. %Wt. %A75 wt % TiO2Slurry15.5815.588.798.798.458.45(WPG75PFSP)1A45 wt % Iron Oxide2.282.281.291.291.241.24Slurry (WPG45GYSP)1A55 wt % Iron Oxide0.140.140.080.080.070.07Slurry (WPG55GRSP)1BDeionized Water46.8067.7456.6473.5879.6548.17BPuraGuard ™29.4710.5329.4710.533.4636.54Propylene Glycol2B50 wt % PVP/VA1.001.001.001.001.001.00735W3in waterBSymdiol ® 6841.001.001.001.001.001.00C5 wt % Darvan ®3.201.201.203.203.001.40811D5in waterD15 wt % ACULYN ™0.530.530.530.532.132.13Excel6in water131415161718PhaseDescriptionWt. %Wt. %Wt. %Wt. %Wt. %Wt. %A75 wt % TiO2Slurry21.9521.952.412.4115.9115.91(WPG75PFSP)1A45 wt % Iron Oxide3.223.220.350.352.332.33Slurry (WPG45GYSP)1A55 wt % Iron Oxide0.200.200.020.020.140.14Slurry (WPG55GRSP)1BDeionized Water55.2542.6962.7779.8337.9572.63BPuraGuard ™12.7227.2827.7812.7236.543.46Propylene Glycol2B50 wt % PVP/VA1.001.001.001.001.001.00735W3in waterBSymdiol ® 6841.001.001.001.001.001.00C5 wt % Darvan ®3.201.203.201.203.001.40811D5in waterD15 wt % ACULYN ™1.471.471.471.472.132.13Excel6in water1Supplied by KOBO Products Inc (South Plainfield, NJ).2Available from The Dow Chemical Company (Lake Zurich, IL).3Available from Ashland Specialty Chemical (Wilmington, DE).4Available from Symrise AG (Branchburg, NJ).5Sodium polyacrylate available from Vanderbilt Minerals LLC (Norwalk, CT).6Polyacrylate available from The Dow Chemical Company (Lake Zurich, IL). The table below shows the viscosity, PSD D50, and separation for each example. Properties such as viscosity, Particle Size Distribution (PSD), separation, loss modulus, and/or storage modulus can be used to evaluate whether the cosmetic ink composition can be stable and jettable. Viscosity was measured after approximately 1 week from formulation and PSD D50 was measured after formulation and again after 4 weeks. Viscosity was measured according to the Viscosity Test Method described hereafter. PSD was measured according to the Particle Size Distribution Method described hereafter. Separation was measured according to the Separation Test Method described hereafter. Separation is recorded in Table 2 as the number of days it took for the sample to reach 2 mm of separation, with the type of separation noted. TABLE 2FirstWt % (Active)DynamicPSD D50TotalViscosity(nm)SeparationParticulatePropyleneRheologySodium0.1 sec−1T = 4at 50° C.ExMaterialGlycolModifierPolyacrylatecPT = 0wksDaysType110.0020.000.250.1112302502301Syneresis210.0020.000.250.1114302602401Syneresis310.0020.000.330.2118102402705Syneresis410.0020.000.330.00294006701,00020Syneresis515.4420.000.460.11912028026024Syneresis64.5620.000.460.11495023024024Syneresis712.7929.470.080.161652602301Settling812.7910.530.080.061322302401Settling97.2129.470.080.06122402301Settling107.2110.530.080.1632702300Settling116.943.460.320.1511302402705Syneresis126.9436.540.320.07392023025014Syneresis1318.0212.720.220.165902402401Settling1418.0227.280.220.0627802302702Syneresis151.9827.280.220.163332602300Settling161.9812.720.220.063912502200Settling1713.0636.540.320.15288024024011Syneresis1813.063.460.320.07377027025015Syneresis It was found that the most stable formulas contained 0.32 active wt % rheology modifier or more. Examples 3-6, 11-12, and 17-18, which comprised 0.32 active wt % rheology modifier or greater, each had a first dynamic viscosity of greater than 1,100 cP and took 5-24 days to reach 2 mm of separation with syneresis observed, demonstrating that the formulas could build a sufficient internal structure to keep the particles in suspension. Without being limited by theory, it is believed that a sample demonstrating syneresis can still be stable because the particles are uniformly distributed below the clear fluid layer. In contrast, separation with settling in which the particles fall to the bottom of the sample may not be stable as the internal structure of the sample is not sufficient to suspend the particles. Examples 3 and 4 demonstrate the need for both the rheology modifier and the (meth)acrylic acid homopolymer sodium polyacrylate to achieve a viscosity and particle size suitable for jetting while maintaining particle suspension. Examples 3 and 4 comprised 0.33 active wt % rheology modifier and a constant level of total particulate material and propylene glycol. However, Example 3 had 0.21 active wt % (meth)acrylic acid homopolymer sodium polyacrylate while Example 4 had no (meth)acrylic acid homopolymer or salt thereof. Example 4 had better separation control as compared to Example 3; however, Example 4 exhibited a first dynamic viscosity of 29,400 cP and poor control of particle size over 4 weeks as demonstrated by the increase in PSD D50. It is believed that the particle size and viscosity of Example 4 would not be suitable for jetting. Examples 5 and 6 had a constant level of propylene glycol, rheology modifier, and (meth)acrylic acid homopolymer sodium polyacrylate, but varied the level of total particulate material. Example 5 had a higher level of total particulate material as compared to Example 4 and demonstrated a higher viscosity. However, both Example 5 and 6 took 24 days to reach 2 mm of separation. Without being limited by theory, it is believed that the level of (meth)acrylic acid homopolymer or salt thereof and rheology modifier needed for particle size control and particle suspension may not depend on the particulate material level. It is believed that as long as the rheology modifier is present with some amount of particulate material, a weak colloidal gel can be formed. In a separate experiment, different formulas were prepared to further assess the impact of the (meth)acrylic acid homopolymer or salt thereof and rheology modifier on the viscosity and separation of the cosmetic ink composition. Examples 19-22 were made according to the procedure described hereafter. Example 19 illustrates a cosmetic ink composition containing a (meth)acrylic acid homopolymer sodium polyacrylate (Darvan® 811D) and a HASE type rheology modifier (ACULYN™ Excel). Example 20 is a comparative example containing no rheology modifier. Example 21 is a comparative example containing no (meth)acrylic acid homopolymer or salt thereof. Example 22 is a comparative example containing a C12/C14 amine oxide as the rheology modifier. Examples 19-22 were made according to the following formulas. Weight percent is shown as added. TABLE 319202122PhaseDescriptionwt %wt %wt %wt %A75 wt % TiO2Slurry12.1811.5412.1815.81(WPG75PFSP)1A45 wt % Iron Oxide Slurry1.782.821.782.31(WPG45GYSP)1A55 wt % Iron Oxide Slurry0.1100.110(WPG55GRSP)1A45 wt % Iron Oxide Slurry00.1400.20(WPG45SIRSP)1BDeionized Water57.5360.3061.7353.98BPuraGuard ™ Propylene20.0020.0020.0022.00Glycol2B50 wt % PVP/VA 735W31.0001.000copolymer in waterBSymdiol ® 6841.001.001.000C5 wt % Darvan ® 811D54.204.2002.90in waterD15 wt % ACULYN ™ Excel62.2002.200in waterD15% C12/C14 Amine Oxide70002.801Supplied by KOBO Products Inc (South Plainfield, NJ).2Available from The Dow Chemical Company (Lake Zurich, IL).3Available from Ashland Specialty Chemical (Wilmington, DE).4Hexanediol/Caprylyl Glycol available from Symrise AG (Branchburg, NJ).5Sodium polyacrylate available from Vanderbilt Minerals LLC (Norwalk, CT).6Polyacrylate available from The Dow Chemical Company (Lake Zurich, IL).7Available from The Dow Chemical Company (Lake Zurich, IL). The samples were stored in sealed glass jars at 50° C. until measurements were performed. The table below shows the PSD, viscosity, tan(delta), and separation for each example. Example 22 was tested at a different time; however, the data are shown together for ease of comparison. PSD is measured according to the Particle Size Distribution Method described hereafter. Viscosity was measured according to the Viscosity Test Method described hereafter. Tan(delta) was calculated by using the storage modulus and loss modulus measured according to the Oscillatory Strain Sweep Method described hereafter. Separation was measured according to the Separation Test Method described hereafter. Separation is recorded in Table 4 as the number of days it took for the sample to reach 2 mm of separation, with the type of separation noted. TABLE 4RheologyFirstSecondModifierSodiumPSDPSDDynamicDynamicSeparation atwt %PolyacrylateD50D90ViscosityViscositytan50° C.Ex(active)wt % (active)(nm)(nm)0.1 s−1(cP)1000 s−1(cP)deltaDaysType190.330.213906601810350.625Syneresis2000.2139064012524.11<1Settling210.3301730356029400910.2120Syneresis220.420.1454440120011045.172Settling Example 19, which contained 0.33 active wt % HASE rheology modifier and 0.21 active wt % (meth)acrylic acid homopolymer sodium polyacrylate, had a first dynamic viscosity of 1,810 cP and a second dynamic viscosity of 35 cP, demonstrating that the formula had shear thinning behavior. In addition, Example 19 had agglomeration control as the PSD D90 was maintained under Finally, Example 19 had a tan(delta) below 1 and took 5 days to reach 2 mm of separation with syneresis observed. Comparative Example 20, which contained 0.21 active wt % (meth)acrylic acid homopolymer sodium polyacrylate and no rheology modifier, had a particle size distribution D90 under 1 μm. However, Comparative Example 20 had a first dynamic viscosity of only 125 cP and was not as shear thinning as compared to Example 19. Comparative Example 20 reached 2 mm of separation in less than 1 day with settling observed. It is believed that without the rheology modifier, the formula could not build enough viscosity to suspend the particles. It has been previously reported that the HASE rheology modifier ACULYN™ Excel can provide a thixotropic ink composition that is jettable. However, it was found that although Comparative Example 21, which contained 0.33 active wt % ACULYN™ Excel HASE rheology modifier and no (meth)acrylic acid homopolymer sodium polyacrylate, had shear thinning behavior and good separation control at 50° C., it had a PSD D50 4.4 times greater than Example 19, indicating poor particle size control. Comparative Example 21 had a PSD D50 of 1730 nm and a PSD D90 of 3560 nm. It is known that the microfluidic channels in ink cartridges can be very small, for example about from about 10 μm to about 25 μm. As such, it is believed that Comparative Example 21 would likely be difficult to refill in a standard printer ejection head because of the high viscosity and would likely clog the flow channels due to the presence of larger particles. It is also believed that the particles in Comparative Example 21 may not have the optimal size for opacity. Comparative Example 22 contained 0.42 active wt % C12/C14 amine oxide rheology modifier and 0.145 active wt % (meth)acrylic acid homopolymer sodium polyacrylate. It has been previously reported amine oxides may provide some yield stress and may help prevent settling of particles in a cosmetic ink composition. However, it was surprisingly found that C12/C14 amine oxide in Comparative Example 22 did not build viscosity to the same extent as Example 19 and reached 2 mm of separation in only 2 days with settling observed, demonstrating that C12/C14 in this formula is not able to build sufficient viscosity to suspend the particles. Rheology Modifier Study Different formulas were tested to assess the impact of rheology modifiers on particle settling. Base Formula 1 was prepared as described hereinafter. Various rheology modifiers (see Table 6) were then added to Base Formula 1 to form Examples 23-35. Propylene Glycol was added to Base Formula 1 as a control (Example 35). Examples 23-35 were prepared as described hereinafter. Approximately 30 mL of the resulting mixture was placed into a 40 mL vial and stored at ambient conditions. Base Formula 1 was made according to the following formula. Weight percent is shown as added. TABLE 5PhaseIngredientwt %A75 wt % TiO2Slurry (WPG75PFSP)122.50A45 wt % Iron Oxide Slurry (WPG45GYSP)13.30A55 wt % Iron Oxide Slurry (WPG55GRSP)10.20BDeionized Water46.00BPuraGuard ™ Propylene Glycol212.50B50 wt % Polyvinylpyrrolidone/vinyl acetate1.50(PVP) 735W3in waterBSymdiol ® 6841.00CDeionized Water10.00D5 wt % Darvan ® 811D5in water3.00Total100.001Supplied by KOBO Products Inc (South Plainfield, NJ).2Available from The Dow Chemical Company (Lake Zurich, IL).3Available from Ashland Specialty Chemical (Wilmington, DE).41, 2 Hexanediol/Caprylyl Glycol available from Symrise AG (Branchburg, NJ).5Sodium polyacrylate available from Vanderbilt Minerals LLC (Norwalk, CT). Examples 23-35 were held for 4 days at ambient conditions. Particle settling was assessed for each example by measuring weight percent solids at the top, middle, and bottom of the sample according to the Particle Settling Test Method described hereafter. Weight percent solids is recorded in table 6 as the solids remaining after drying the volatiles off. The table below summarizes the results. TABLE 6Weight Percent Solidswt %BottomMiddleTopExRheology Modifier(active)Chemical Type(wt %)(wt %)(wt %)23ACRYSOL ™ TT61520.2HASE45.01.01.024ACRYSOL ™ TT61520.4HASE14.314.45.525ACRYSOL ™ TT61520.6HASE18.016.315.926ACULYN ™ Excel60.4HASE/acrylates18.117.817.7copolymer27ACULYN ™ 3820.4ASE/acrylates57.11.31.8niodecanoate crosspolymer28ACRYSOL ™ TT93520.2HASE42.91.21.329ACRYSOL ™ TT93520.4HASE26.91.41.230ACRYSOL ™ TT93520.6HASE16.01.82.131Natrosol ™ 250 HHR80.4Hydroxyethyl cellulose24.42.61.232Stabylen 3090.2Acrylates/Vinyl21.910.33.2Isodecanoate Crosspolymer33Carbopol ® Ultrez 21100.2Hydrophobically modified17.912.04.1crosslinked polyacrylate34Aristoflex ® HMB110.5Ammonium43.23.92.6Acryloyldimethyltaurate/Beheneth-25 MethacrylateCrosspolymer35PuraGuard ™10.0Glycol20.914.92.0Propylene Glycol22Available from The Dow Chemical Company (Lake Zurich, IL).6Polyacrylate available from The Dow Chemical Company (Lake Zurich, IL).8Available from Ashland (Covington, KY).9Available from 3V Sigma (Georgetown, SC).10Available from Lubrizol (Wickliffe, OH).11Available from Clariant Corp. (Blue Ash, OH). It was found that Example 23, which contained 0.2 active wt % ACRYSOL™ TT615, did not build sufficient viscosity and particle settling took place over the 4 day period, as demonstrated by the differing weight percent solids content from top to bottom. However, it was found that as the concentration of ACRYSOL™ TT615 increased, particle settling stability increased. Examples 24 and 25, which comprised 0.4 and 0.6 active wt % ACRYSOL™ TT615, respectively, built sufficient viscosity and the particles did not significantly settle over the 4 day period, although some syneresis was observed. It was found that the weight percent solids content from top to bottom was consistent for Example 26, indicating that the particles did not significantly settle over the 4 day period. Examples 26 comprised 0.4 active wt % of the HASE polymer ACULYN™ Excel. It was found that the Example 28-30 formulations, which comprised the HASE class polymer ACRYSOL™ TT935, were not able to build sufficient viscosity within the composition and solids settling took place over the 4 day period. Without being limited by theory, it is believed that this particular HASE polymer may not have sufficient charge density and may have too much hydrophobicity to build the viscosity needed to maintain stability and limit particle settling. However, it is believed that at higher concentrations this rheology modifier may be able to thicken the cosmetic ink composition and build a sufficient viscosity to suspend the particles. Example 27, which comprised the ASE class polymer ACULYN™ 38 showed substantial particle settling over the 4 day period. It is believed that adjusting the level of ACULYN™ 38 ASE polymer can improve the suspension of solids and reduce the particle settling. Finally, Examples 31-35 are comparative examples which showed substantial variation in the solids content from top to bottom indicating that particle settling took place over the 4 day period. In a separate study, different formulas were tested to assess the impact of various types of ASE, HASE, and HEUR polymer rheology modifiers on viscosity and separation of the cosmetic ink composition. Examples 36-48 were prepared according to the procedure described hereinafter. Examples 36-48 were made according to the formulas in the tables below. Weight percent is shown as added. TABLE 73637383940PhaseIngredientwt %wt %wt %wt %wt %A75 wt % TiO2 Slurry18.4618.4615.0015.0015.00(WPG75PFSP)1A45 wt % Iron Oxide Slurry4.514.513.673.673.67(WPG45GYSP)1A45 wt % Iron Oxide Slurry0.290.290.220.220.22(WPG45SIRSP)1ADeionized Water10.0010.0010.0010.0010.00BDeionized Water37.837.1443.5140.5139.90BPuraGuard ™ Propylene Glycol223.0023.0023.0023.0023.00C5 wt % Darvan ® 811D52.602.602.604.004.00in waterD15 wt % ACRYSOL ™ TT61523.330000in waterD15 wt % Rheovis ® AS11251203.33000in waterD15 wt % ACULYN ™ Excel6002.0000in waterD15 wt % ACULYN ™ 222in0003.600waterD15 wt % ACULYN ™ 332in00003.93water4142434445PhaseIngredientwt %wt %wt %wt %wt %A75 wt % TiO2 Slurry15.0015.0015.0015.0015.00(WPG75PFSP)1A45 wt % Iron Oxide Slurry3.673.673.663.663.66(WPG45GYSP)1A45 wt % Iron Oxide Slurry0.220.220.220.220.22(WPG45SIRSP)1ADeionized Water10.0010.0010.0010.0010.00BDeionized Water40.3139.1139.6040.5237.72BPuraGuard ™ Propylene Glycol223.0023.0023.0023.0023.00C5 wt % Darvan ® 811D54.004.004.004.004.00in waterD15 wt % ACULYN ™ 3823.800000in waterD15 wt % ACULYN ™ 88205.00000in waterD15 wt % Rheovis ® 115212004.5300in waterD15 wt % Rheovis ® 1156120003.600in waterD15 wt % ACRYSOL ™ TT935200006.40in water464748PhaseIngredientwt %wt %wt %A75 wt % TiO2 Slurry (WPG75PFSP)115.0015.0015.00A45 wt % Iron Oxide Slurry (WPG45GYSP)13.663.663.66A45 wt % Iron Oxide Slurry (WPG45SIRSP)10.220.220.22ADeionized Water10.0010.0010.00BDeionized Water40.2632.1832.18BPuraGuard ™ Propylene Glycol223.0023.0023.00C5 wt % Darvan ® 811D5in water4.002.602.60D15 wt % ACRYSOL ™ DR3002solution3.8600D15 wt % ACULYN ™ 442in water012.400D15 wt % ACULYN ™ 46N2in water0012.471Supplied by KOBO Products Inc (South Plainfield, NJ).2Available from The Dow Chemical Company (Lake Zurich, IL).5Sodium polyacrylate available from Vanderbilt Minerals LLC (Norwalk, CT).6Polyacrylate available from The Dow Chemical Company (Lake Zurich, IL).12Available from BASF (Florham Park, NJ). The samples were stored in sealed glass jars at 25° C. or 40° C. until measurements were performed. The table below shows the viscosity, tan(delta), and separation measured 11 days after formulation for each example. Viscosity was measured according to the Viscosity Test Method described hereafter. Tan(delta) was calculated using the storage modulus and loss modulus measured according to the Oscillatory Strain Sweep Method described hereafter. Separation was measured according to the Separation Test Method described hereafter. TABLE 8SodiumRheology ModifierPolyacrylateViscositySeparation at 11 dayswt %wt %0.1 s−11000 s−1tan25° C.40° C.ExNameType(active)(active)(cP)(cP)(delta)(mm)(mm)Type36ACRYSOL ™ TT615HASE0.500.136290760.61116Syneresis37Rheovis ® AS1125ASE0.500.132040730.6823Syneresis38ACULYN ™ ExcelHASE0.300.133180370.5400none39ACULYN ™ 22HASE0.540.24670710.43014Syneresis40ACULYN ™ 33ASE0.590.24040630.4100none41ACULYN ™ 38ASE0.570.22280420.4901.5Syneresis42ACULYN ™ 88HASE0.750.24320710.500.51.5Syneresis43Rheovis ® 1152HASE0.680.23060800.953.525Syneresis44Rheovis ® 1156HASE0.540.21290691.772140Syneresis45ACRYSOL ™ TT935HASE0.960.2763611.893746Syneresis46ACRYSOL ™ DR300ASE0.580.2624431.644349.5Syneresis47ACULYN ™ 44HEUR1.860.13194223.304649.5Settling48ACULYN ™ 46NHEUR1.870.13460324.154852Settling For both ASE and HASE polymers, it is believed that the interaction of the anionic moiety of the rheology modifier with the particle surface and the (meth)acrylic acid homopolymer or salt thereof can promote stability. This forms a weak colloidal gel by introducing elasticity in the composition (as demonstrated by the storage modulus of the cosmetic ink composition) along with viscosity (as demonstrated by the loss modulus of the cosmetic ink composition). Particle suspension can be achieved for cosmetic ink compositions having a low ratio of G″ to G′, i.e. a tan(delta) of preferably less than about 1, more preferably less than about 0.60. It was found that Examples 36-43, which all had a first dynamic viscosity of greater than 1,100 cP and a tan(delta) of less than 1, had less than 4 mm of separation at 11 days at 25° C. with syneresis observed. Under accelerated temperature conditions, Examples 36-43 had 25 mm of separation or less at 11 days with syneresis observed. Examples 38 and 40 had no separation at 11 days under both temperature conditions tested. Although Example 44 had a first dynamic viscosity of 1,290 cP that falls within the preferred range, the tan(delta) was greater than 1 and 21 mm of separation was observed at 11 days. Examples 45 and 46 contained HASE and ASE type rheology modifiers, respectively. However, Examples 45 and 46 were not able to build a sufficient viscosity to suspend the particles, as demonstrated by the first dynamic viscosity of well below 1,100 cP and the mm of separation at both temperature conditions. Without being limited by theory, it is believed that these formulas were not able to form a strong enough colloidal gel to keep the particles in suspension because the formulations comprised too many hydrophobic moieties. Finally, Examples 47 and 48, which contained HEUR type rheology modifiers, had a first dynamic viscosity of less than 500 cP and a tan(delta) of well above 1. Examples 47 and 48 were not able to build sufficient viscosity to suspend the particles and had over 45 mm of separation at 11 days with particle settling observed under both temperature conditions. Not wishing to be bound by theory, it is believed that HEUR polymers may not be able to build sufficient elasticity and/or viscosity in the cosmetic ink composition to keep the particles suspended. Examples 1-22 were made according to the following procedure. First, the ingredients of Phase A were combined in an appropriate premix container and mixed for 30 minutes. The ingredients of Phase B were added into a main container. Phase B was mixed using a mixer with a propeller blade, such as a digital Eurostar 400® available from IKA® (Staufen im Breisgau, Germany) or equivalent, at low speed until the mixture was homogenous. The pH of the Phase B mixture was measured. Then, the contents of the premix container were transferred into the main container and mixed for 30 minutes. Approximately 10% of the water was withheld from Phase B and was used to wash the premix container and then added to the main container while mixing. Phase C was added to the main container. The mixing speed was increased to high speed and mixing continued for 10 minutes. Phase D was then added dropwise to the main container and the pH was maintained between 7.5-8.5 by adding 20% KOH. Homogeneity was ensured and the mixture was poured into a container, labeled, and stored at ambient conditions before use. Examples 23-35 were made according to the following procedure. First, Base Formula 1 was prepared. The ingredients of Phase A were combined in an appropriate premix container. The ingredients of Phase B were added into a main container. Phase B was mixed using a mixer with a propeller blade, such as a digital Eurostar 400® available from IKA® (Staufen im Breisgau, Germany), or equivalent, at low speed until the mixture was homogenous. While mixing, Phase A was added into the main container. Phase C was added to the premix container to wash out the container and then added to the main container while mixing continued. The mixing speed was increased to high speed and mixing continued for 10 minutes. Phase D was then added to the main container and mixed for 15 minutes to form Base Formula 1. Homogeneity was ensured and Base Formula 1 was poured into a container. In separate containers, the rheology modifiers were prepared as solution in water. Each rheology modifier was premixed in deionized water at 15 wt % and then added to Base Formula 1 at the active wt % levels described in table 6 while adjusting the pH to approximately 8 with 20% KOH. Examples 36-48 were prepared according to the following procedure. First, the ingredients of Phase A were combined in an appropriate premix container and mixed for 30 minutes. The ingredients of Phase B were added into a main container. Phase B was mixed using a mixer with a propeller blade, such as a digital Eurostar 400® available from IKA® (Staufen im Breisgau, Germany) or equivalent, at low speed until the mixture was homogenous. The pH of the Phase B mixture was measured. Then, the contents of the premix container were then transferred into the main container and mixed for 30 minutes. Phase C was added to the main container and mixed. The pH of the main container was measured. Phase D was added to the main container dropwise and the pH was maintained between 7.5-8.5 by adding 20% KOH solution. Homogeneity was ensured and the mixture was poured into a container, labeled, and stored at ambient conditions before use. Particle Size Distribution Method The particle size distribution is determined using a laser scattering particle size distribution analyzer. A suitable laser scattering particle size distribution analyzer can include a Horiba LA-950V2 (available from Horiba, Ltd., Kyoto, Japan). In this method, the principles of Mie and Fraunhofer scattering theories are used to calculate the size and distribution of particles suspended in a liquid. Results are normally displayed on a volume basis. The application of this method to pigments has been developed using a flow cell procedure. Samples are prepared by vortexing for 30 seconds with a Vortex Genie2to ensure there is no residue in the bottom of the sample vial. 200 mL of deionized (DI) water is added into the instrument reservoir and analyzed as a blank sample. A disposable micro pipet is used to dispense enough sample into the DI water in the instrument until the Transmittance is reduced from 100 down to 90±2%, approximately 250 μL. Results are reported as D50 or D90. Viscosity Test Method Viscosity is measured using a rheometer as a function of shear rate. A suitable rheometer can include an Ares M (available from TA Instruments, New Castle, DE), or equivalent. First, the samples and standards are equilibrated at room temperature prior to analysis. A 50 mm, 2 degree, cone and plate is zeroed prior to testing. While the sample is at 25° C.±0.5° C., the sample is tested. A shear sweep measurement is performed over a range of 0.1-1000 s−1to determine the shear thinning properties and viscosity at different shear rates. Separation Test Method Separation is measured by filing an 8-dram (1 oz., 25 mm×95 mm) screw cap glass vial to a height of 55 mm with the sample composition (as measured from the bottom of the vial to the top of the liquid composition). The vials are sealed and are placed in controlled temperature chambers. The vials are held static storage until measurements are performed. At each time point, the vial is carefully removed from the chamber without vigorous or prolonged agitation and observed for any visual signs of separation and the type of separation is noted as syneresis or settling. The amount of separation is determined by measuring the mm of clear fluid at the top of the sample with a digital caliper. Particle Settling Test Method Particle settling is measured as follows. An aluminum dish is weighed to determine the dish weight. A 1 gram aliquot of the sample from the top, middle, or bottom of the sample vial is added to the dish and weighed to determine the wet weight. “Top” means the surface of the sample in the vial. “Middle” means the middle of the vial. “Bottom” means the bottom of the vial. The dish with the sample aliquot is placed in an oven at 100° C. for one hour to evaporate the volatiles. The dish is removed and weighed again to get a dry weight. The weight % solids is calculated by the following equation: (dry weight−dish weight)/(wet weight−dish weight). Oscillatory Strain Sweep Method Oscillatory strain sweep is measured using a rheometer (such as an ARES-G2 available from TA Instruments, New Castle, DE), or equivalent. The samples and standards are allowed to equilibrate at ambient conditions prior to analysis. The rheometer is calibrated as disclosed in the operator's manual. The oscillatory strain sweep measurement is performed at a fixed angular frequency of 6.28 rad/s over a strain range of 0.0001-1 using a 40 mm 316SST (APS heat break) parallel plate at 25° C., with a 0.05 mm gap, to determine the storage and loss moduli. Zeta Potential Test Method Zeta potential is measured using a Zeta potential analyzer such as a NanoBrook ZetaPALS Potential Analyzer available from Brookhaven Instruments Corporation, Holtsville, NY, or equivalent. Zeta potential test samples are prepared by diluting the sample to 0.1 g/ml into deionized water. Zeta potential is measured on a Zeta potential analyzer using the Smoluchowski Zeta potential model with 5 runs and 10 cycles. After running a standard (BIZR3), the cells of the Zeta potential analyzer are loaded with 1 ml of test sample. Zeta potential is measured as a function of pH. Combinations A. A cosmetic ink composition comprising: from about 1% to about 30 active wt % of a particulate material having a Particle Size Distribution D50 of 100 nm to 2,000 nm; a (meth)acrylic acid homopolymer or salt thereof having a weight average molecular weight of less than 20,000 daltons; and a rheology modifier, wherein the rheology modifier is selected from the group consisting of alkali swellable emulsion polymers, hydrophobically modified alkali swellable emulsion polymers, and combinations thereof; wherein the cosmetic ink composition has a first dynamic viscosity of greater than 1,100 cP at a shear rate of 0.1 sec−1measured at 25° C. and a second dynamic viscosity of less than 100 cP at a shear rate of 1,000 sec−1measured at 25° C.B. The cosmetic ink composition of paragraph A wherein the cosmetic ink composition has a first dynamic viscosity of 1,100 cP to 10,000 cP at a shear rate of 0.1 sec−1measured at 25° C., preferably 1,500 cP to 8,000 cP, more preferably from 2,000 cP to 5,000 cP, and a second dynamic viscosity of from 10 to 100 cP at a shear rate of 1,000 sec−1measured at 25° C., preferably from 20 to 80 cP.C. The cosmetic ink composition of paragraph A or B, wherein the cosmetic ink composition comprises greater than 0.30 active wt % rheology modifier.D. The cosmetic ink composition of any of the preceding paragraphs comprising from 0.01 to 1 active wt % (meth)acrylic acid homopolymer or salt thereof, preferably from 0.10 to 0.85 active wt %, more preferably from 0.20 to 0.75 active wt %.E. The cosmetic ink composition of any of the preceding paragraphs wherein cosmetic ink composition comprises a ratio of (meth)acrylic acid homopolymer or salt thereof to rheology modifier of from 0.1 to 0.75, preferably from 0.30 to 0.65.F. The cosmetic ink composition of any of the preceding paragraphs wherein the cosmetic ink composition further comprises from 20 to 30 active wt % humectant.G. The cosmetic ink composition of any of the preceding paragraphs wherein the rheology modifier is an alkali swellable acrylic polymer emulsion.H. The cosmetic ink composition of any of the preceding paragraphs wherein the (meth)acrylic acid homopolymer or salt thereof is sodium polyacrylate.I. The cosmetic ink composition of any of the preceding paragraphs wherein the (meth)acrylic acid homopolymer or salt thereof has a weight average molecular weight of from 2,000 to 5,000 daltons.J. The cosmetic ink composition of any of the preceding paragraphs wherein the cosmetic ink composition has a neat pH of from 7.5 to 9.0.K. The cosmetic ink composition of any of the preceding paragraphs wherein the particulate material has a Particle Size Distribution D50 of from 150 nm to 1,000 nm, preferably from 200 nm to 500 nm, and more preferably from about 200 nm to about 350 nm.L. The cosmetic ink composition of any of the preceding paragraphs the wherein particulate material has a Particle Size Distribution D90 of from 700 nm to 900 nm.M. The cosmetic ink composition of any of the preceding paragraphs wherein the particulate material is selected from the group consisting of a pigment, a metal oxide, a colorant, a dye, a clay, and combinations thereof.N. The cosmetic ink composition of any of the preceding paragraphs wherein the cosmetic ink composition further comprises one or more skin care actives selected from the group consisting of niacinamide; inositol; undecylenoyl phenylalanine; and combinations thereof.O. The cosmetic ink composition of any of the preceding paragraphs wherein the cosmetic ink composition further comprises a preservative.P. The cosmetic ink composition of any of the preceding paragraphs further comprising a monohydric alcohol selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol and mixtures thereof. Values disclosed herein as ends of ranges are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each numerical range is intended to mean both the recited values and any integers within the range. For example, a range disclosed as “1 to 10” is intended to mean “1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.” The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.” Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. | 96,418 |
11857666 | DETAILED DESCRIPTION It is to be understood that the invention may assume various alternative orientations, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification are simply exemplary embodiments of the inventive concepts defined in the appended claims. Hence, specific dimensions and other physical characteristics relating to the embodiments disclosed herein are not to be considered as limiting, unless the claims expressly state otherwise. All ranges and parameters, including but not limited to percentages, parts, and ratios, disclosed herein are understood to encompass any and all sub-ranges assumed and subsumed therein, and every number between the endpoints. For example, a stated range of “1 to 10” should be considered to include any and all sub-ranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less (e.g., 1 to 6.1, or 2.3 to 9.4), and to each integer (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) contained within the range. In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made. The various embodiments of the urushiol treating compositions of the present disclosure may also be substantially free of any ingredient or feature described herein, provided that the remaining composition still contains all of the required ingredients or features as described herein. In this context, and unless otherwise specified, the term “substantially free” means that the selected composition contains less than a functional amount of the optional ingredient, typically less than 1%, including less than 0.5%, including less than 0.1%, and also including zero percent, by weight of such optional or selected essential ingredient. The compositions of the present disclosure described herein, including but not limited to compositions for washing urushiol off of skin, compositions for treating urushiol induced contact dermatitis and methods of treating urushiol on the skin and removing it as well as methods of treating urushiol induced contact dermatitis, and corresponding manufacturing methods may comprise, consist of, or consist essentially of the elements of the products as described herein, as well as any additional or optional element described herein or otherwise useful in topical wash product applications. Typically, the compositions of the present disclosure are free of pharmaceutically active ingredients/drug(s) or prodrugs, sodium chloride (NaCl)/granulated salt, any salt in granular form that would essentially operate as a exfoliating or scrubbing agent when the detergent compositions of the present disclosure are on the surface of a person's skin or clothing, and/or nonylphenol ethoxylate(s) such as Nonxyl-9. The detergent compositions typically do not include the foregoing components, but the claims of this application do not exclude them unless specifically indicated. “Consisting essentially of” in the context of the claims of this application limits the scope of a claim or claim element to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention as would be known by those of ordinary skill in the art whether or not such a composition is disclosed in the application or not as affecting the basic and novel characteristic. For example, in the case of a salt (NaCl), this ingredient, if added to compositions of the present disclosure containing the synergistic combination of C12 surfactants discussed herein, materially affects the surface tension and other performance characteristics of the surfactants of the present disclosure. To the extent that the term “includes” or “including” is used in the specification or the claims, it is intended to be inclusive in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed (e.g., A or B) it is intended to mean “A or B or both.” When the Applicant intends to indicate “only A or B but not both” then the term “only A or B but not both” will be employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. Also, to the extent that the terms “in” or “into” are used in the specification or the claims, it is intended to additionally mean “on” or “onto.” Typical Components of the Detergent/Cleaning Compositions of the Present Disclosure The overall detergent compositions of the present disclosure typically include water, more typically deionized water. Water can be included in an amount of from about 40-60 weight percent of the overall compositions. Water is a part of some of the raw material components of the detergent composition and added as a separate additive. The typical formulas for the detergent compositions of the present disclosure have water added independently in an amount of about 40-60 weight percent water as a separate and independent component. Water is also typically contributed by two other components of the compositions of the present disclosure. However, water is not necessarily required and can be utilized in lower amounts as well. Surprisingly, it is presently believed that the efficacy of the presently disclosed detergent compositions is in large part due to the use of a plurality of solely twelve carbon chain length surfactants (C12 surfactants) that work together to remove urushiol more effectively than other individual surfactants and typically at least as effectively as, if not more effectively than, prior overall compositions that included nonylphenol ethoxylates and/or pharmaceutically active ingredients. While C12-15 Pareth-9, which contains a mixture of C12, C13, C14 and C15 components, is a surfactant that may be used in the detergent compositions of the present disclosure, it is not a solely C12 surfactant ingredient because it has a range of surfactants in it. Two particular solely C12 surfactants that have been found to surprisingly provide synergistic results when combined with one another, in particular when the detergent composition is without a nonylphenol ethoxylate, which previously was thought to be a necessary component of many such compositions, include: SPAN 20™ (sorbitan laurate)(sorbitan monolaurate) and sodium lauroyl sarcosinate. SPAN 20™ is a sorbitan ester and is a biodegradable surfactant based on a natural fatty acid (lauric acid) and sugar alcohol sorbitol. This sorbitan ester is highly effective at forming oil in water emulsions, particularly when used with its ethoxylated derivative, TWEEN® 20. Sorbitan monolaurate is a non-ionic surfactant that is a mixture of esters formed from the fatty acid lauric acid and polyols derived from sorbitol, including sorbitan and isosorbide and has a chemical structure of C18H34O6. Sodium lauroyl sarcosinate (INCI), also known as sarkosyl, is an anionic surfactant derived from sarcosine. It is a sodium salt of lauroyl sarcosine. It generally conforms to the formula: The percentage of sodium lauroyl sarcosinate is generally a maximum of 1.5% of the ingredient itself. It is typically a white solid in pure form and produces a colorless to light yellow aqueous solution. When used, the sorbitan monolaurate is typically present in an amount of from about 0.5 percent to as much as about 50 percent by weight of the overall composition, more typically in an amount of about 2.0% or 10% by weight of the overall composition. The sodium lauroyl sarcosinate is typically added in the form of an aqueous solution with 30% of the solution being sodium lauroyl sarcosinate. Sodium lauroyl sarcosinate is typically present in slightly lower amounts than the amount of the sorbitan monolaurate in the overall detergent compositions of the present disclosure, but can be used in higher amounts as well. The sodium lauroyl sarcosinate and sorbitan monolaurate are present in a ratio based on the percent of active ingredient in the overall detergent composition in a ratio range of from 1 part sodium lauroyl sarcosinate to up to 2.5 parts sorbitan monolaurate. While lower amount of sodium lauroyl sarcosinate than sorbitan monolaurate are typically used, amounts of sodium lauroyl sarcosinate may be used in higher amounts as well including up to 10%, 15%, 20% or even 50% by weight of the overall detergent composition. The amount of sodium lauroyl sarcosinate is typically from about 1.0% to about 10% by weight of the overall composition. When higher amounts are included less water is typically added as a separate and independent component and possibly, but less preferentially, lower amounts of other ingredients/components employed. The detergent compositions of the present disclosure may include a quaternary ammonium salt such as Quaternium-15. It acts as an antimicrobial agent/preservative because it acts as a formaldehyde releaser. Any known preservative may be used instead of or in addition to the quaternary ammonium salt. Another component that is typically present in the compositions of the present disclosure is C12-15 Pareth-9. C12-15 Pareth-9 is a polyethylene glycol ether of a mixture of synthetic C12-15 fatty alcohols with an average of 9 moles of ethylene oxide. It is a non-ionic surfactant. It is a surfactant that has a component of C12 surfactant, but is not solely composed of a C12 surfactant. The detergent compositions of the present disclosure also typically contain a CARBOPOL®, which is a high molecular weight, hydrophilic, and cross-linked polyacrylic acid polymer. This physical hydrogel presents a three-dimensional polymer network that is swollen by water, and presents temporary, reversible inter-chain entanglements that are stronger when compared to chemical hydrogels. The particularly preferred CARBOPOL® of the present disclosure is CARBOPOL® 980 polymer, which is a white powder, cross-linked polyacrylic acid that is polymerized in a toxicologically-preferred co-solvent system. It is an extremely efficient rheology modifier capable of providing high viscosity and forms sparkling clear gels or hydro-alcoholic gels and creams. CARBOPOL® 980 has a viscosity of 40,000-60,000 cP (0.5% at pH 7.5) and a monomer molecular weight of 72.02 g/mol. The overall detergent composition is typically at a pH of from about 5.5 to 7.0, is typically un-fragranced, white to off-white and an opaque creamy lotion. When scrubbing beads are employed in the composition the scrubbing breads make the composition have a gritty texture. When one or more kinds of scrubbing beads are employed in connection with the compositions of the present disclosure, the beads are typically beads that are bio-degradable beads. However, polyethylene beads may also be employed as well. Sasol Ltd. DECORNEL® 300 synthetic wax polymer beads or Micro Powders Inc. SYNSCRUB® 50PC high molecular weight synthetic wax polymer beads or Low Density Polyethylene (LDPE) beads may be used. The synthetic wax used in DECORNEL® and SYNSCRUB® products is biodegradable. As such, this material is preferred over polyethylene beads, which have a very long degradation timetable comparatively—especially since the compositions of the present disclosure are skin washing compositions where the material is washed into the water treatment system/plumbing systems. The use of biodegradable beads in some form allows the detergent compositions of the present disclosure to completely biodegrade in ambient environment after 2 years. Additionally, the at least one type of bio-degradable beads of the present disclosure typically biodegrades in an ambient environment by 33.8% after 86 days according to the Organization for Economic Co-operation and Development Test Guideline 302 C entitled Inherent Biodegradability: Modified MITI Test (II). DECORNEL® 300 by Sasol acts as a consistency regulator, exfoliating and cleansing agent. It is a nonpolar, white, tasteless and odorless wax bead. It is a blend consisting predominantly of saturated n-alkanes, highly refined. It is available in 50-300 μm particle size. It is 100% hydrophobic and preservative-free. It is non-ionic and has no impact on the pH value of the overall composition when incorporated into compositions of the present disclosure. It offers consistent high quality and removes dead cells smoothly. It can outperform polyethylene beads in face, hand and foot scrubs as well as toothpaste. DECORNEL® 300 is synthesized from carbon monoxide and hydrogen in accordance with the Fischer-Tropsch method. It can replace plant waxes, hydrogenated fats and other hard base substances in products. As stated above, it does not influence the pH-value and can be applied in formulas with a pH-value of 3-11. It is a very pure product and inherently primarily biodegradable in the terms of the OECD Test 301-B/302-C. DECORNEL® 300 has a shelf life of 60 months. SYNSCRUB® 50PC is a synthetic wax powder. It is designed for use as an economical exfoliating agent and has an irregular particle shape which produces the same high performance as commonly used irregular particle shape polyethylene powders. It is, however, biodegradable. SYNSCRUB® 50PC has a maximum mesh side of 50 and a maximum particle size of 297 microns. Its density at 77° F. is 0.95. Another component of the typical detergent composition is a PEG-12 dimethicone, which belongs to the class of dimethyl-methyl(polyethyleneoxide) siloxanes. It is presently believed that any dimethyl-methyl(polyethyleneoxide) siloxane may be used or a plurality of dimethyl-methyl(polyethyleneoxide) siloxanes may be used in the detergent compositions of the present disclosure. When used, the PEG-12 dimethicone (or one or more dimethyl-methyl(polyethyleneoxide) siloxane) is typically present in an amount of from about 0.8% by weight to about 1.2% by weight of the overall detergent composition. PEG-12 dimethicone is a silicone glycol copolymer soluble in water, alcohol, and hydro-alcoholic systems. It acts as a surface tension depressant, wetting agent, emulsifier and foam builder. PEG-12 dimethicone gives a stable foam. The PEG-12 dimethicone is a silicone copolyol wetting agent. If a wetting agent is used, it must be soluble in water and not dispersible or insoluble, which could result in deposits that clog or block hair follicles into which urushiol has migrated. The wetting agent's hydrophilic-lipophilic balance is typically 10 or higher on the HLB scale from 0 to 20. The detergent compositions of the present disclosure also typically include disodium EDTA. Disodium EDTA is also known as Disodium ethylenediaminetetraacetic acid. Disodium EDTA is used as a chelating agent that sequesters a variety of polyvalent cations such as calcium. Chelating agents are chemical compounds that react with metal ions to form a stable, water-soluble complex. Chelating agents have a ring-like center which forms at least two bonds with the metal ion allowing it to be excreted. Disodium EDTA has a molecular weight of 338.22 g/mol and is a white crystalline powder. Disodium EDTA is typically included in the compositions of the present disclosure in an amount of from about 0.8% to 1.2% by weight of the overall composition. Finally, while not necessary, the detergent compositions of the present disclosure typically also include an amount of sodium hydroxide (25%). The sodium hydroxide is typically present in an amount of the overall composition to neutralize CARBOMER® and achieve a desirable pH. Possible Additional Ingredients It will be understood by one having ordinary skill in the art that construction of the described invention and other components is not limited to any specific material. Other exemplary embodiments disclosed herein may be formed from a wide variety of materials, unless described otherwise herein. The urushiol compositions and methods of treatment of the present disclosure may further comprise additional components that may modify the physical, chemical, aesthetic or processing characteristics of the formulas or serve as pharmaceutical or additional components when used in a targeted population. Non-limiting examples of such optional ingredients include preservatives, anti-oxidants, emulsifying agents, colorants, and related derivatives, thickening agents and stabilizers, and other additive or synergistic ingredients that will be appreciated in the art of urushiol treating composition formulation. However, the compositions of the present disclosure, as discuss above, will typically not include and are typically free of any nonylphenol ethoxylate, more typically any ethoxylate that is not part of the non-ionic polyethylene glycol ether of a mixture of synthetic C12-C15 fatty alcohols with an average of 9 moles of ethylene oxide; a granulated salt; or a pharmaceutically active component used to treat urushiol including, but not limited to neurokinin-1 (NK-1) antagonists, such as serlopiltant. Additionally, the detergent compositions of the present disclosure also typically an unbuffered detergent composition. The detergent compositions of the present disclosure also typically do not include any surfactant component that interferes with or alters the functional characteristics of the one or more solely C12 surfactants employed in the detergent compositions. It is also to be understood that variations and modifications can be made on the aforementioned detergent compositions and washing or cleaning methods without departing from the concepts of the present disclosure, and further it is to be understood that such concepts are intended to be covered by the following claims unless these claims by their language expressly state otherwise. When forming the detergent compositions of the present disclosure, a container is typically charged with the deionized water first, thereafter, the CARBOMER® is added using a lightning mixer or tri-blender. Once the CARBOMER® is completely hydrated the remaining ingredients are added. Typically, sodium hydroxide is added as the last ingredient. Each ingredient is mixed well between each addition. Use and Application of Detergent Composition to Wash/Clean Urushiol and Treat Urushiol Induced Contact Dermatitis In use, the detergent compositions of the present disclosure are applied directly or indirectly to the skin or clothing exposed to urushiol after the composition is hydrated, typically by wetting the composition in the user's hands for about 10 seconds until the product is worked into a paste form. The paste is typically a semisolid preparation intended for external application to the skin. Usually the paste is thick and does not melt at normal/ambient room temperature. The paste formed from the detergent composition should then be rubbed on the affected area of the skin for up to 3 minutes until there is no sign of itching (about 15 seconds is typical for mild to moderate reactions to urushiol). Thereafter, the area should be rinsed thoroughly. If itching returns, the process may be repeated any number of times. Typically, only a few treatments will be necessary. The methods of treating contact dermatitis and the methods of washing skin exposed to urushiol of the present disclosure typically include the above steps. | 19,630 |
11857667 | DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Given the number of various products on the market today and the myriad of different skin-types, a person is oftentimes at a loss to identify an appropriate product. Further, many current products include caustic agents that can irritate skin, leave the skin feeling dry, or leave the skin feeling oily. The inventors discovered a solution to counteract these issues. In particular, the solution concerns two separate combinations of ingredients obtained from natural sources that have been shown (see examples) to work on different skin types ranging from oily skin, dry skin, or normal skin. Further, subcombinations were found to work particularly well on a given skin type. These and other non-limiting aspects of the present invention are described in further detail below. A. Determining Skin-Type A first step it utilizing the compositions of the present invention can be to determine a user's skin type. It is well known in the cosmetic's field that there are three main skin types: (1) normal skin; (2) dry skin; and (3) oily skin. A fourth skin type is simply a combination of any one of normal, dry, or oily skin (e.g., normal/dry, normal/oily, oily/dry). There are also well-known methods for determining a person's skin type. For instance, normal skin can be identified as having a smooth texture and no greasy patches or flaky areas. Therefore, a product that can retain skin moisture in its present form can be used to maintain the appearance of normal skin. As for dry skin, it has a low level of sebum production from sebaceous glands and is prone to irritation or erythema. The appearance of dry skin has a parched look caused by the skin's inability to retain moisture. Oftentimes it feels “tight” and uncomfortable after washing and is prone to chapping, flaking, and cracking. Dry skin can be exacerbated by wind, extremes of temperature and air-conditioning, all of which cause the skin to flake, chap and feel tight. Dry skin typically has a dull appearance. Therefore, a product that deliver appropriate hydration and restore moisture to dry skin can be used to countreract the effects of dry skin. With respect to oily skin, such skin is shiny, thick and dull colored. It feels oily and has coarse pores and pimples and other unsightly blemishes due to overproduction of sebum from sebaceous glands and from clogged/blocked pores. In this regard, oily skin usually has oil producing sebaceous glands that are overactive and produce more oil than is needed. The oil oozes and gives the skin a greasy shine. The pores are enlarged and the skin has a coarse look. Therefore, a product that can help control skin surface oiliness while also cleansing clogged pores can be used to counteract the effects of oily skin. As noted above, combination skin is a combination of both oily, dry, and/or normal skin (e.g., normal/dry, oily/dry, normal/oily). For oily/dry skin, there is typically a greasy center panel consisting of nose, forehead and chin (also known as the “T-zone” of a person's face) and a dry panel consisting of cheeks, mouth and the areas around the eyes. Therefore, a product that can control the excess oil production in sebaceous glands in the T-zone while also hydrating the dry skin areas outside of the T-zone can be used for such oily/dry skin. Once a particular skin-type is identified, a person can then select an appropriate composition to correct or maintain the skin-type. B. Combination ofSilybum marianumand Luo Han Guo Fruit Extracts The inventors discovered that the combination ofSilybum marianumextract andMomordica grosvenorifruit extract was found to be effective on all skin-types of normal, dry, and oily skin. Milk thistle (Silybum marianum) is a plant native to Southern Europe and Asia. It is known for producing red to purple flowers, shiny pale green leaves with white veins, and fruit. TheSilybum marianumextract of the present invention is a hydroalcohlic (water and alcohol denat) extract that includes silymarin as an active ingredient (silymarin is a mixture of flavanonol derivatives that includes silibine, silicristine, silidianin, isosolibine, and isosilicristine). The fruit portion ofSilybum marianumincludes silymarin. TheSilybum marianumextract can be obtained from the fruit portion of this plant by mascerating the fruit pulp and then subjecting the pulp to a hydroalcoholic solution of water and SD alcohol 39-C (alcohol denat.) to obtain the extract. The extract can then filtered and packaged for storage or be added to a composition of the present invention. In addition to this extraction process,Silybum marianumextract can be purchased from Provital S.A (SPAIN) under the trade names PRONALEN SILYMARIN HSC or PRONALEN SILYMARIN SPE. Luo han guo (Momordica grosvenori) is a perennial vine that grows 3-5 meters long with narrow heart shaped leaves and green round fruit 5-7 cm in diameter. This plant is native to southern China. The fruit has been used as a natural food sweetener in China for several decades. The luo han guo extract of the present invention can be obtained from the fruit portion of this plant by macerating the fruit pulp and then subjecting the pulp to a hydroglycolic solution of water, glycerin, and preservatives to obtain the extract. The extract can then filtered and packaged for storage or be added to a composition of the present invention. In addition to this extraction process, luo han guo fruit extract can be purchased from Carrubba Inc., Milford, Connecticut (USA). Data also suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: CGRP expression; Cyclo-oxygenase 1 and 2 activity; FAAH activity; lipoxygenase activity; TNF-α expression; IL-2, 8, and 10 activity; angiogenin expression; INF-γ expression; IL 12p40 expression; and tyrosinase activity. This combination also has anti-oxidative properties, which can be used to prevent or at the very least reduce oxidative damage to skin cells. This combination also can increase ICAM-1 expression in skin cells. Example 2 provides a more detailed account of these data. 1. Dry Skin The addition ofLinum usitatissimumseed extract and hydrolyzed algin to theSilybum marianumand luo han guo fruit extracts was found to work well on dry skin. Flax seed (Linum usitatissimum(Linseed)) is an annual, biennial or perennial herb that can reach 3 feet in height. It includes a slender stem, lance-shaped leaves, and can produce ski-blue flowers and oily brown seeds. This plant is native to Europe and Asia. The flax seed extract of the present invention can be obtained from the seed portion of this plant by macerating the seed and then subjecting the seed to a hydroglycolic solution of water and glycerin to obtain the extract. The extract can then filtered and packaged for storage or be added to a composition of the present invention. In addition to this extraction process, flax seed extract can be purchased from Carrubba Inc., Milford, Connecticut (USA). Hydrolyzed algin can be obtained fromLaminaria digitata, which is a brown alga, that is found in the littoral zone of bodies of water. The hydrolyzed algin is an aqueous solution of an oligosaccharide that can be produced by controlled enzymatic depolymerization of membranous polysaccharides fromLaminaria digitata. The structure of the oligosaccharide is a chain of 2 uronic acids: mannuronic and guluronic, which can be illustrated as follows: In addition to this production process, hydrolyzed algin can be purchased from Barnet Products Corp., Englewood Cliffs, New Jersey (USA) under the trade name PHYKO AL-PF. Data also suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: CGRP expression; Cyclo-oxygenase 1 and 2 activity; FAAH activity; lipoxygenase activity; TNF-α expression; IL-2, 6, 8, and 10 expression; angiogenin expression; INF-γ expression; IL 12p40 expression; tyrosinase activity; MMP2, 3, and 9 activity; and melanogenesis activity. This combination also has anti-oxidative properties, which can be used to prevent or at the very least reduce oxidative damage to skin cells. This combination also has the ability to increase laminin expression and ICAM-1 expression in skin cells and can also be used as an involucrin reporter. Example 2 provides a more detailed account of these data. 2. Oily Skin The addition ofPsidium guajavafruit extract andKunzea ericoidesleaf extract toSilybum marianumand luo han guo fruit extracts was found to work well on oily skin. Guava orPsidium guajavais an evergreen tree or shrub that can reach 6 to 25 feet in height. It produces green leaves, fragrant white flowers, and fruit. The fruit is pear-shaped and 3 to 6 cm in length. When ripe, the skin of the fruit has a reddish-yellow color. This plant is native to the region spanning Mexico to northern South America. The fruit portion of guava is used in the context of the present invention to obtain the extract. The guava fruit extract of the present invention can be produced by macerating the fruit pulp and then subjecting the pulp to a hydroglycolic solution of water and glycerin to obtain the extract. The extract can then be filtered and packaged for storage. In addition to this extraction process, guava fruit extract of the present invention can be purchased from Carrubba Inc., Milford, Connecticut (USA). Kanuka orKunzea ericoidesis a tree that can reach up to 30 meters in height. The leaves have an oval shape and the flowers are white. This plant is native to Australia and New Zealand. TheKunzea ericoidesleaf extract of the present invention can be obtained from the leaf portion of this plant by macerating the leaf and then subjecting the leaf to an aqueous extraction process. The extract can then be filtered, placed in a butylene glycol solution, and packaged for storage or be added to a composition of the present invention. In addition to this extraction process, kanuka leaf extract can be purchased from Southern Cross Botanicals, New South Wales (AUSTRALIA) under the trade name ABACROSS KANUKA BG. Data also suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: CGRP expression; Cyclo-oxygenase 1 and 2 activity; FAAH activity; lipoxygenase activity; TNF-α expression; IL-2, 8, and 10 expression; angiogenin expression; INF-γ expression; IL 12p40 expression; tyrosinase activity; and elastase activity. This combination also has anti-oxidative properties, which can be used to prevent or at the very least reduce oxidative damage to skin cells. Further, this combination also has the ability to increase ICAM-1 expression and collagen production in skin cells. Example 2 provides a more detailed account of these data. 3. Normal Skin The addition ofPlumeria albaflower extract andNymphea giganteaflower extract toSilybum marianumand luo han guo fruit extracts was found to work well on normal skin. Plumeria alba(Frangipani) is a large evergreen shrub with narrow elongated leaves and large white followers that have a yellow center. It is native to Central America and the Caribbean. The frangipani flower extract of the present invention can be obtained from the flower portion of this plant by macerating the flower and then subjecting the flower to an aqueous extraction process. The extract can then be filtered, placed in a butylene glycol solution, and packaged for storage or be added to a composition of the present invention. In addition to this extraction process, frangipani flower extract can be purchased from Southern Cross Botanicals, New South Wales (AUSTRALIA) under the trade name ABACROSS FRANGIPANI FLOWER BG. Nymphea gigantea(Giant Water Lily) is a tropical plant that is native to the tropical and subtropical regions of Australia. This plant can produce large (up to 25 cm) blue-white flowers that emerge from the water and large circular leaves that grow up to 75 cm in diameter. TheNymphea giganteaflower extract of the present invention can be obtained from the flower portion of this plant by macerating the flower and then subjecting the flower to an aqueous extraction process. The extract can then be filtered, placed in a butylene glycol solution, and packaged for storage or be added to a composition of the present invention. In addition to this extraction process, frangipani flower extract can be purchased from Southern Cross Botanicals, New South Wales (AUSTRALIA) under the trade name ABACROSS WATER LILY BG. Data also suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: CGRP expression; Cyclo-oxygenase 1 and 2 activity; FAAH activity; lipoxygenase activity; TNF-α expression; IL-2, 6, 8, and 10 expression; angiogenin expression; INF-γ expression; IL 12p40 expression; tyrosinase activity; MMP2, 3, and 9 activity; and melanogenesis activity. This combination also has anti-oxidative properties, which can be used to prevent or at the very least reduce oxidative damage to skin cells. This combination also has the ability to increase laminin and ICAM-1 expression and collagen production in skin cells and can also be used as an involucrin reporter. Example 2 provides a more detailed account of these data. C. Combination ofSilybum marianumand Gorgonian Extracts The inventors discovered that the combination ofSilybum marianumextract andPseudopterogorgia elisabethaeextract was found to be effective on all skin-types of normal, dry, and oily skin. Silybum marianumextract is described above. Gorgonian extract is a marine extract derived fromPseudopterogorgia elisabethae(or Sea Whip) plant.Pseudopterogorgia elisabethaecan be harvested from the Atlantic Ocean. Gorgonian extract can be prepared by macerating thePseudopterogorgia elisabethaeplant and then subjecting the macerated plant with butylenes glycol, or caprylic/capric triglyceride, or pentylene glycol. One of the active ingredients in Gorgonian extract can be pseudopterosins (e.g., pseudopterosin A). In addition to this extraction process, Gorgonian extract can be purchased from Lipo Chemicals Inc., Paterson, New Jersey (USA) under the trade names GORGONIAN EXTRACT BG, GORGONIAN EXTRACT GC, or GORGONIAN PTG. In some instances, this combination can also includeHelianthus annuus(sunflower) seed extract. The sunflower is a plant that produces bright yellow sunflowers. Seeds are contained within the flower. It is native to North and South America. Sunflower seed extract is the extract of the seeds of the sunflower, can be purchased from Silab (France) under the trade names ANTIGLYSKIN™, ASIDERILIKE 2™, BIOHAIR™, MX023-COMMUCELL™, GLYCALINE™, HELIOXINE™, MX016 SENSIKIN™, or RETICALMINE™. 1. Dry Skin The addition ofLinum usitatissimumseed extract and hydrolyzed algin to theSilybum marianumand gorgonian extracts was found to work well on dry skin. Both of these extracts are described above. 2. Oily Skin The addition ofPsidium guajavafruit extract andSpiraea ulmariaextract to theSilybum marianumand gorgonian extracts was found to work well on oily skin.Psidium guajavafruit extract is described above. Spiraea ulmaria(Meadow Sweet) is a perennial herb that is native throughout most of Europe and Western Asia. Extracts obtained from the leaf can be purchased from Gattefosse (Canada) under the trade name CYTOBIO ULMAIRE™.Spiraea ulmariaextract obtained from the whole plant can be purchased from Active Concepts (USA) under the trade name ACB MEADOWSWEET EXTRACT™, ACB MEADOWSWEET EXTRACT 20%™, from Silab (France) under the trade names DERMAPUR™, SEBONORMINE™, and SEBOREGUL™, or from Phytocos (France) under the trade names COMPLEXE AMINCISSANT LP1™, COMPLEXE AMINCISSANT SGLP™, EXTRAIT d'ULMAIRE LP1™, and EXTRAIT d'ULMAIRE SGLP™.Spiraea ulmariaextract obtained from the flower can be purchased from Indena S.A. (France) under the trade name SWEET SUPEXTRAT™ or from Greentech S.A. (France) under the trade names PHYTELENE COMPLEX EGX 250™, PHYTELENE OF QUEEN MEADOW EG 213 LIQUID™, and PHYTELENE OF ULMAIRE EG 213 LIQUID™, and SLIMMING™.Spiraea ulmariaextract obtained from the root can purchased from Active Organics (USA) under the trade names ACTIPHYTE OF MEADOWSWEET PG50™, CO ACTIPHYTE OF MEADOWSWEET AJ™, CO ACTIPHYTE OF MEADOWSWEET AL™, CO ACTIPHYTE OF MEADOWSWEET GL™, CO ACTIPHYTE OF MEADOWSWEET LIPO O™, CO ACTIPHYTE OF MEADOWSWEET LIPO RS™, CO ACTIPHYTE OF MEADOWSWEET LIPO S™, and CO ACTIPHYTE OF MEADOWSWEET LIPO SUN™. 3. Normal Skin The addition ofPlumeria albaflower extract,Euterpe oleraceaefruit extract, andCamellia sinensisleaf extract to theSilybum marianumand gorgonian extracts was found to work well on normal skin.Plumeria albaflower extract is described above. Euterpe oleracea(acai) is a plant that is native to Brazil. It produces dark purple fruit. Extract from the fruit of acai can be purchased from Southern Cross Botanicals Pty Ltd (NSW Australia), Amax NutraSource (USA) under the trade name ACAI FRUIT EXTRACT™, from Assessa-Industria (Brazil) under the trade name FRULIX TF ACAI™, or from Centroflora Group Botucatu (Brazil) under the trade name ACAI BERRY EXTRACT™. Camellia sinensisWith respect toCamellia sinensisextract, theCamellia sinensisplant is native to China, and is a flowing plant. The extract can be obtained from the whole plant or parts of said plant. In particular instances, it is from the leaf, root, flower, or seed of said extract and in particular, the leaf. In particular instances theCamellia sinensisextract can include a polyphenol compound such as epigallocatechin gallate.Camellia sinensisextract, whether from the whole plant or parts of said plant, is commercially available from a wide range of sources (see, e.g., CTFA, Volume 1, pages 400-07, which is incorporated by reference). D. Combination ofSilybum marianum, Luo Han Guo Fruit, and Gorgonian Extracts Descriptions ofSilybum marianum, luo han guo, and gorgonian extracts are described above. Data suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: CGRP expression; Cyclo-oxygenase 1 and 2 activity; FAAH activity; lipoxygenase activity; TNF-α expression; IL-2, 8, and 10 expression; angiogenin expression; INF-γ expression; IL 12p40 expression; and tyrosinase activity. This combination also has anti-oxidative properties, which can be used to prevent or at the very least reduce oxidative damage to skin cells. This combination also can increase ICAM-1 expression in skin cells. Example 2 provides a more detailed account of these data. E. Combination of Flax Seed and Hydrolyzed Algin Extracts Descriptions of flax seed and hydrolyzed algin extracts are described above. Data suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells the following: MMP 2, 3, and 9 activity; CGRP expression; TNF-α expression; IL 6, 8, and 10 expression; IL12p40 expression, and melanogenesis activity. Further, this combination also has the ability to increase laminin production in skin cells and also to act as an involucrin reporter. Example 2 provides a more detailed account of these data. F. Combination of Frangipani Flower andNymphaea giganteaFlower Extracts Descriptions of frangipani flower and water lily extracts are described above. Data suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells TNF-α expression. Further, this combination also has anti-oxidative properties and has the ability to increase collagen production in skin cells. Example 2 provides a more detailed account of these data. G. Combination of Guava Fruit and Kanuka Leaf Extracts Descriptions of guava fruit and kanuka leaf extracts are described above. Data suggests that the combination of these ingredients in a topical skin formulation can be used to treat a wide variety of skin conditions by reducing in skin cells TNF-α expression and reducing elastase activity. Further, this combination also has anti-oxidative properties and has the ability to increase collagen production in skin cells. Example 2 provides a more detailed account of these data. H. Determining Skin-Type The primary skin types of humans are normal skin, dry skin, oily skin, and combination skin. Normal skin typically has an even tone, soft, a smooth texture, with no visible pores or blemishes, and no greasy patches or flaky areas. Therefore, a product that can retain skin moisture in its present form can be used to maintain the appearance of normal skin. Dry skin usually has a low level of sebum and can be prone to irritation. The appearance of dry skin is usually a parched look caused by the skin's inability to retain moisture. Oftentimes it feels “tight” and uncomfortable after washing and is prone to chapping, flaking, and cracking. Dry skin can be exacerbated by wind, extremes of temperature and air-conditioning, all of which cause the skin to flake, chap and feel tight. Dry skin typically has a dull appearance. Therefore, a product that deliver appropriate hydration and restore moisture to dry skin can be used to countreract the effects of dry skin. Oily skin is typically shiny, thick and dull colored. It typically feels oily and has coarse pores and pimples and other unsightly blemishes. Oily skin usually has oil producing sebaceous glands that are overactive and produce more oil than is needed. The oil oozes and gives the skin a greasy shine. The pores are enlarged and the skin has a coarse look. Therefore, a product that can help control skin surface oiliness while also cleansing clogged pores can be used to counteract the effects of oily skin. Combination skin is a combination of both oily and dry skin. Usually, there is a greasy center panel consisting of nose, forehead and chin (also known as the “T-zone” of a person's face) and a dry panel consisting of cheeks, mouth and the areas around the eyes. Therefore, a product that can help control the excess oil production in sebaceous glands in the T-zone while also hydrating the dry skin areas outside of the T-zone can be used. I. Compositions of the Present Invention It is contemplated that the compositions of the present invention can include any amount of the ingredients. The compositions can also include any number of combinations of additional ingredients described throughout this specification (e.g., pigments, or additional cosmetic or pharmaceutical ingredients). The concentrations of the any ingredient within the compositions can vary. In non-limiting embodiments, for example, the compositions can comprise, consisting essentially of, or consist of, in their final form, for example, at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%, 0.0021%, 0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%, 0.0035%, 0.0036%, 0.0037%, 0.0038%, 0.0039%, 0.0040%, 0.0041%, 0.0042%, 0.0043%, 0.0044%, 0.0045%, 0.0046%, 0.0047%, 0.0048%, 0.0049%, 0.0050%, 0.0051%, 0.0052%, 0.0053%, 0.0054%, 0.0055%, 0.0056%, 0.0057%, 0.0058%, 0.0059%, 0.0060%, 0.0061%, 0.0062%, 0.0063%, 0.0064%, 0.0065%, 0.0066%, 0.0067%, 0.0068%, 0.0069%, 0.0070%, 0.0071%, 0.0072%, 0.0073%, 0.0074%, 0.0075%, 0.0076%, 0.0077%, 0.0078%, 0.0079%, 0.0080%, 0.0081%, 0.0082%, 0.0083%, 0.0084%, 0.0085%, 0.0086%, 0.0087%, 0.0088%, 0.0089%, 0.0090%, 0.0091%, 0.0092%, 0.0093%, 0.0094%, 0.0095%, 0.0096%, 0.0097%, 0.0098%, 0.0099%, 0.0100%, 0.0200%, 0.0250%, 0.0275%, 0.0300%, 0.0325%, 0.0350%, 0.0375%, 0.0400%, 0.0425%, 0.0450%, 0.0475%, 0.0500%, 0.0525%, 0.0550%, 0.0575%, 0.0600%, 0.0625%, 0.0650%, 0.0675%, 0.0700%, 0.0725%, 0.0750%, 0.0775%, 0.0800%, 0.0825%, 0.0850%, 0.0875%, 0.0900%, 0.0925%, 0.0950%, 0.0975%, 0.1000%, 0.1250%, 0.1500%, 0.1750%, 0.2000%, 0.2250%, 0.2500%, 0.2750%, 0.3000%, 0.3250%, 0.3500%, 0.3750%, 0.4000%, 0.4250%, 0.4500%, 0.4750%, 0.5000%, 0.5250%, 0.0550%, 0.5750%, 0.6000%, 0.6250%, 0.6500%, 0.6750%, 0.7000%, 0.7250%, 0.7500%, 0.7750%, 0.8000%, 0.8250%, 0.8500%, 0.8750%, 0.9000%, 0.9250%, 0.9500%, 0.9750%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or any range derivable therein, of at least one of the ingredients that are mentioned throughout the specification and claims. In non-limiting aspects, the percentage can be calculated by weight or volume of the total composition. A person of ordinary skill in the art would understand that the concentrations can vary depending on the addition, substitution, and/or subtraction of ingredients in a given composition. J. Vehicles The compositions of the present invention can be incorporated into all types of vehicles. Non-limiting examples include emulsions (e.g., water-in-oil, water-in-oil-in-water, oil-in-water, silicone-in-water, water-in-silicone, oil-in-water-in-oil, oil-in-water-in-silicone emulsions), creams, lotions, solutions (both aqueous and hydro-alcoholic), anhydrous bases (such as lipsticks and powders), gels, and ointments. Variations and other appropriate vehicles will be apparent to the skilled artisan and are appropriate for use in the present invention. In certain aspects, it is important that the concentrations and combinations of the compounds, ingredients, and agents be selected in such a way that the combinations are chemically compatible and do not form complexes which precipitate from the finished product. K. Cosmetic Products and Articles of Manufacture The composition of the present invention can also be used in many cosmetic products including, but not limited to, lip sticks, lip balms, lip glosses, sunscreen products, sunless skin tanning products, hair products, finger nail products, moisturizing creams, skin benefit creams and lotions, softeners, day lotions, gels, ointments, foundations, night creams, cleansers, toners, masks, or other known cosmetic products or applications. Additionally, the cosmetic products can be formulated as leave-on or rinse-off products. In certain aspects, the compositions of the present invention are stand-alone products. L. Additional Ingredients In addition to the guava fruit extract compositions of the present invention can include additional ingredients such as cosmetic ingredients and pharmaceutical active ingredients. Non-limiting examples of these additional ingredients are described in the following subsections. 1. Cosmetic Ingredients The CTFA International Cosmetic Ingredient Dictionary and Handbook (2004 and 2008) describes a wide variety of non-limiting cosmetic ingredients that can be used in the context of the present invention. Examples of these ingredient classes include: fragrances (artificial and natural), dyes and color ingredients (e.g., Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C blue no. 4, D&C green no. 5, D&C orange no. 4, D&C red no. 17, D&C red no. 33, D&C violet no. 2, D&C yellow no. 10, and D&C yellow no. 11), adsorbents, lubricants, solvents, moisturizers (including, e.g., emollients, humectants, film formers, occlusive agents, and agents that affect the natural moisturization mechanisms of the skin), water-repellants, UV absorbers (physical and chemical absorbers such as paraaminobenzoic acid (“PABA”) and corresponding PABA derivatives, titanium dioxide, zinc oxide, etc.), essential oils, vitamins (e.g. A, B, C, D, E, and K), trace metals (e.g. zinc, calcium and selenium), anti-irritants (e.g. steroids and non-steroidal anti-inflammatories), botanical extracts (e.g. aloe vera, chamomile, cucumber extract,Ginkgo biloba, ginseng, and rosemary), anti-microbial agents, antioxidants (e.g., BHT and tocopherol), chelating agents (e.g., disodium EDTA and tetrasodium EDTA), preservatives (e.g., methylparaben and propylparaben), pH adjusters (e.g., sodium hydroxide and citric acid), absorbents (e.g., aluminum starch octenylsuccinate, kaolin, corn starch, oat starch, cyclodextrin, talc, and zeolite), skin bleaching and lightening agents (e.g., hydroquinone and niacinamide lactate), humectants (e.g., sorbitol, urea, and manitol), exfoliants, waterproofing agents (e.g., magnesium/aluminum hydroxide stearate), skin conditioning agents (e.g., aloe extracts, allantoin, bisabolol, ceramides, dimethicone, hyaluronic acid, and dipotassium glycyrrhizate). Non-limiting examples of some of these ingredients are provided in the following subsections. a. UV Absorption Agents UV absorption agents that can be used in combination with the compositions of the present invention include chemical and physical sunblocks. Non-limiting examples of chemical sunblocks that can be used include para-aminobenzoic acid (PABA), PABA esters (glyceryl PABA, amyldimethyl PABA and octyldimethyl PABA), butyl PABA, ethyl PABA, ethyl dihydroxypropyl PABA, benzophenones (oxybenzone, sulisobenzone, benzophenone, and benzophenone-1 through 12), cinnamates (octyl methoxycinnamate, isoamyl p-methoxycinnamate, octylmethoxy cinnamate, cinoxate, diisopropyl methyl cinnamate, DEA-methoxycinnamate, ethyl diisopropylcinnamate, glyceryl octanoate dimethoxycinnamate and ethyl methoxycinnamate), cinnamate esters, salicylates (homomethyl salicylate, benzyl salicylate, glycol salicylate, isopropylbenzyl salicylate, etc.), anthranilates, ethyl urocanate, homosalate, octisalate, dibenzoylmethane derivatives (e.g., avobenzone), octocrylene, octyl triazone, digalloy trioleate, glyceryl aminobenzoate, lawsone with dihydroxyacetone, ethylhexyl triazone, dioctyl butamido triazone, benzylidene malonate polysiloxane, terephthalylidene dicamphor sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexyl benzoate, bis diethylamino hydroxybenzoyl benzoate, bis benzoxazoylphenyl ethylhexylimino triazine, drometrizole trisiloxane, methylene bis-benzotriazolyl tetramethylbutyiphenol, and bis-ethylhexyloxyphenol methoxyphenyltriazine, 4-methylbenzylidenecamphor, and isopentyl 4-methoxycinnamate. Non-limiting examples of physical sunblocks include, kaolin, talc, petrolatum and metal oxides (e.g., titanium dioxide and zinc oxide). b. Moisturizing Agents Non-limiting examples of moisturizing agents that can be used with the compositions of the present invention include amino acids, chondroitin sulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol polymers, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose, mannitol, natural moisturizing factor, PEG-15 butanediol, polyglyceryl sorbitol, salts of pyrollidone carboxylic acid, potassium PCA, propylene glycol, sodium glucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol. Other examples include acetylated lanolin, acetylated lanolin alcohol, alanine, algae extract, aloe barbadensis, aloe-barbadensis extract, aloe barbadensis gel,Althea officinalisextract, apricot (Prunus armeniaca) kernel oil, arginine, arginine aspartate,Arnica montanaextract, aspartic acid, avocado (Persea gratissima) oil, barrier sphingolipids, butyl alcohol, beeswax, behenyl alcohol, beta-sitosterol, birch (Betula alba) bark extract, borage (Borago officinalis) extract, butcherbroom (Ruscus aculeatus) extract, butylene glycol,Calendula officinalisextract,Calendula officinalisoil, candelilla (Euphorbia cerifera) wax, canola oil, caprylic/capric triglyceride, cardamon (Elettaria cardamomum) oil, carnauba (Copernicia cerifera) wax, carrot (Daucus carota sativa) oil, castor (Ricinus communis) oil, ceramides, ceresin, ceteareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (Anthemis nobilis) oil, cholesterol, cholesterol esters, cholesteryl hydroxystearate, citric acid, clary (Salvia sclarea) oil, cocoa (Theobroma cacao) butter, coco-caprylate/caprate, coconut (Cocos nucifera) oil, collagen, collagen amino acids, corn (Zea mays) oil, fatty acids, decyl oleate, dimethicone copolyol, dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythrityl hexacaprylate/hexacaprate, DNA, erythritol, ethoxydiglycol, ethyl linoleate,Eucalyptus globulusoil, evening primrose (Oenothera biennis) oil, fatty acids, geranium maculatum oil, glucosamine, glucose glutamate, glutamic acid, glycereth-26, glycerin, glycerol, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape (Vitis vinifera) seed oil, hazel (Corylus americana) nut oil, hazel (Corylus avellana) nut oil, hexylene glycol, hyaluronic acid, hybrid safflower (Carthamus tinctorius) oil, hydrogenated castor oil, hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm glyceride, hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated tallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin, hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, isocetyl stearate, isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate, isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl neopentanoate, jasmine (Jasminum officinale) oil, jojoba (Buxus chinensis) oil, kelp, kukui (Aleurites moluccana) nut oil, lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid, lanolin alcohol, lanolin oil, lanolin wax, lavender (Lavandula angustifolia) oil, lecithin, lemon (Citrus medica limonum) oil, linoleic acid, linolenic acid,Macadamia ternifolianut oil, maltitol, matricaria (Chamomilla recutita) oil, methyl glucose sesquistearate, methylsilanol PCA, mineral oil, mink oil, mortierella oil, myristyl lactate, myristyl myristate, myristyl propionate, neopentyl glycol dicaprylate/dicaprate, octyldodecanol, octyldodecyl myristate, octyldodecyl stearoyl stearate, octyl hydroxystearate, octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive (Olea europaea) oil, orange (Citrus aurantium dulcis) oil, palm (Elaeis guineensis) oil, palmitic acid, pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach (Prunus persica) kernel oil, peanut (Arachis hypogaea) oil, PEG-8 C12-18 ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-20 methyl glucose sesquistearate, PEG40 sorbitan peroleate, PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG40 stearate, PEG-50 stearate, PEG-100 stearate, PEG-150 stearate, pentadecalactone, peppermint (Mentha piperita) oil, petrolatum, phospholipids, polyamino sugar condensate, polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, propylene glycol, propylene glycol dicaprylate/dicaprate, propylene glycol dioctanoate, propylene glycol dipelargonate, propylene glycol laurate, propylene glycol stearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate, retinol, retinyl palmitate, rice (Oryza sativa) bran oil, RNA, rosemary (Rosmarinus officinalis) oil, rose oil, safflower (Carthamus tinctorius) oil, sage (Salvia officinalis) oil, sandalwood (Santalum album) oil, serine, serum protein, sesame (Sesamum indicum) oil, shea butter (Butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, soluble collagen, sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol, soybean (Glycine soja) oil, sphingolipids, squalane, squalene, stearamide MEA-stearate, stearic acid, stearoxy dimethicone, stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower (Helianthus annuus) seed oil, sweet almond (Prunus amygdalus dulcis) oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water, waxes, wheat (Triticum vulgare) germ oil, and ylang ylang (Cananga odorata) oil. c. Antioxidants Non-limiting examples of antioxidants that can be used with the compositions of the present invention include acetyl cysteine, ascorbic acid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanol pectinate, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, t-butyl hydroquinone, cysteine, cysteine HCl, diamylhydroquinone, di-t-butylhydroquinone, dicetyl thiodipropionate, dioleyl tocopheryl methylsilanol, disodium ascorbyl sulfate, distearyl thiodipropionate, ditridecyl thiodipropionate, dodecyl gallate, erythorbic acid, esters of ascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters, hydroquinone, isooctyl thioglycolate, kojic acid, magnesium ascorbate, magnesium ascorbyl phosphate, methylsilanol ascorbate, natural botanical anti-oxidants such as green tea or grape seed extracts, nordihydroguaiaretic acid, octyl gallate, phenylthioglycolic acid, potassium ascorbyl tocopheryl phosphate, potassium sulfite, propyl gallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite, sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxide dismutase, sodium thioglycolate, sorbityl furfural, thiodiglycol, thiodiglycolamide, thiodiglycolic acid, thioglycolic acid, thiolactic acid, thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12, tocophereth-18, tocophereth-50, tocopherol, tocophersolan, tocopheryl acetate, tocopheryl linoleate, tocopheryl nicotinate, tocopheryl succinate, and tris(nonylphenyl)phosphite. d. Structuring Agents In other non-limiting aspects, the compositions of the present invention can include a structuring agent. Structuring agent, in certain aspects, assist in providing rheological characteristics to the composition to contribute to the composition's stability. In other aspects, structuring agents can also function as an emulsifier or surfactant. Non-limiting examples of structuring agents include stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmitic acid, the polyethylene glycol ether of stearyl alcohol having an average of about 1 to about 21 ethylene oxide units, the polyethylene glycol ether of cetyl alcohol having an average of about 1 to about 5 ethylene oxide units, and mixtures thereof. e. Emulsifiers In certain aspects of the present invention, the compositions do not include an emulsifier. In other aspects, however, the compositions can include one or more emulsifiers. Emulsifiers can reduce the interfacial tension between phases and improve the formulation and stability of an emulsion. The emulsifiers can be nonionic, cationic, anionic, and zwitterionic emulsifiers (See McCutcheon's (1986); U.S. Pat. Nos. 5,011,681; 4,421,769; 3,755,560). Non-limiting examples include esters of glycerin, esters of propylene glycol, fatty acid esters of polyethylene glycol, fatty acid esters of polypropylene glycol, esters of sorbitol, esters of sorbitan anhydrides, carboxylic acid copolymers, esters and ethers of glucose, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether phosphates, fatty acid amides, acyl lactylates, soaps, TEA stearate, DEA oleth-3 phosphate, polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soya sterol, steareth-2, steareth-20, steareth-21, ceteareth-20, PPG-2 methyl glucose ether distearate, ceteth-10, polysorbate 80, cetyl phosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate, polysorbate 60, glyceryl stearate, PEG-100 stearate, and mixtures thereof. f. Silicone Containing Compounds In non-limiting aspects, silicone containing compounds include any member of a family of polymeric products whose molecular backbone is made up of alternating silicon and oxygen atoms with side groups attached to the silicon atoms. By varying the —Si—O— chain lengths, side groups, and crosslinking, silicones can be synthesized into a wide variety of materials. They can vary in consistency from liquid to gel to solids. The silicone containing compounds that can be used in the context of the present invention include those described in this specification or those known to a person of ordinary skill in the art. Non-limiting examples include silicone oils (e.g., volatile and non-volatile oils), gels, and solids. In certain aspects, the silicon containing compounds includes a silicone oils such as a polyorganosiloxane. Non-limiting examples of polyorganosiloxanes include dimethicone, cyclomethicone, polysilicone-11, phenyl trimethicone, trimethylsilylamodimethicone, stearoxytrimethylsilane, or mixtures of these and other organosiloxane materials in any given ratio in order to achieve the desired consistency and application characteristics depending upon the intended application (e.g., to a particular area such as the skin, hair, or eyes). A “volatile silicone oil” includes a silicone oil have a low heat of vaporization, i.e. normally less than about 50 cal per gram of silicone oil. Non-limiting examples of volatile silicone oils include: cyclomethicones such as Dow Corning 344 Fluid, Dow Corning 345 Fluid, Dow Corning 244 Fluid, and Dow Corning 245 Fluid, Volatile Silicon 7207 (Union Carbide Corp., Danbury, Conn.); low viscosity dimethicones, i.e. dimethicones having a viscosity of about 50 cst or less (e.g., dimethicones such as Dow Corning 200-0.5 cst Fluid). The Dow Corning Fluids are available from Dow Corning Corporation, Midland, Michigan. Cyclomethicone and dimethicone are described in the Third Edition of the CTFA Cosmetic Ingredient Dictionary (incorporated by reference) as cyclic dimethyl polysiloxane compounds and a mixture of fully methylated linear siloxane polymers end-blocked with trimethylsiloxy units, respectively. Other non-limiting volatile silicone oils that can be used in the context of the present invention include those available from General Electric Co., Silicone Products Div., Waterford, N.Y. and SWS Silicones Div. of Stauffer Chemical Co., Adrian, Michigan. g. Essential Oils Essential oils include oils derived from herbs, flowers, trees, and other plants. Such oils are typically present as tiny droplets between the plant's cells, and can be extracted by several method known to those of skill in the art (e.g., steam distilled, enfleurage (i.e., extraction by using fat), maceration, solvent extraction, or mechanical pressing). When these types of oils are exposed to air they tend to evaporate (i.e., a volatile oil). As a result, many essential oils are colorless, but with age they can oxidize and become darker. Essential oils are insoluble in water and are soluble in alcohol, ether, fixed oils (vegetal), and other organic solvents. Typical physical characteristics found in essential oils include boiling points that vary from about 160° to 240° C. and densities ranging from about 0.759 to about 1.096. Essential oils typically are named by the plant from which the oil is found. For example, rose oil or peppermint oil are derived from rose or peppermint plants, respectively. Non-limiting examples of essential oils that can be used in the context of the present invention include sesame oil, macadamia nut oil, tea tree oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil, thyme oil, pimento berries oil, rose oil, anise oil, balsam oil, bergamot oil, rosewood oil, cedar oil, chamomile oil, sage oil, clary sage oil, clove oil, cypress oil, eucalyptus oil, fennel oil, sea fennel oil, frankincense oil, geranium oil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lavender oil, lemon oil, lemongrass oil, lime oil, mandarin oil, marjoram oil, myrrh oil, neroli oil, orange oil, patchouli oil, pepper oil, black pepper oil, petitgrain oil, pine oil, rose otto oil, rosemary oil, sandalwood oil, spearmint oil, spikenard oil, vetiver oil, wintergreen oil, or ylang ylang. Other essential oils known to those of skill in the art are also contemplated as being useful within the context of the present invention. h. Thickening Agents Thickening agents, including thickener or gelling agents, include substances which that can increase the viscosity of a composition. Thickeners includes those that can increase the viscosity of a composition without substantially modifying the efficacy of the active ingredient within the composition. Thickeners can also increase the stability of the compositions of the present invention. In certain aspects of the present invention, thickeners include hydrogenated polyisobutene or trihydroxystearin, or a mixture of both. Non-limiting examples of additional thickening agents that can be used in the context of the present invention include carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, and gums. Examples of carboxylic acid polymers include crosslinked compounds containing one or more monomers derived from acrylic acid, substituted acrylic acids, and salts and esters of these acrylic acids and the substituted acrylic acids, wherein the crosslinking agent contains two or more carbon-carbon double bonds and is derived from a polyhydric alcohol (see U.S. Pat. Nos. 5,087,445; 4,509,949; 2,798,053; CTFA International Cosmetic Ingredient Dictionary, Fourth edition, 1991, pp. 12 and 80). Examples of commercially available carboxylic acid polymers include carbomers, which are homopolymers of acrylic acid crosslinked with allyl ethers of sucrose or pentaerytritol (e.g., Carbopol™ 900 series from B. F. Goodrich). Non-limiting examples of crosslinked polyacrylate polymers include cationic and nonionic polymers. Examples are described in U.S. Pat. Nos. 5,100,660; 4,849,484; 4,835,206; 4,628,078; 4,599,379). Non-limiting examples of polyacrylamide polymers (including nonionic polyacrylamide polymers including substituted branched or unbranched polymers) include polyacrylamide, isoparaffin and laureth-7, multi-block copolymers of acrylamides and substituted acrylamides with acrylic acids and substituted acrylic acids. Non-limiting examples of polysaccharides include cellulose, carboxymethyl hydroxyethylcellulose, cellulose acetate propionate carboxylate, hydroxyethylcellulose, hydroxyethyl ethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl hydroxyethylcellulose, microcrystalline cellulose, sodium cellulose sulfate, and mixtures thereof. Another example is an alkyl substituted cellulose where the hydroxy groups of the cellulose polymer is hydroxyalkylated (preferably hydroxy ethylated or hydroxypropylated) to form a hydroxyalkylated cellulose which is then further modified with a C10-C30straight chain or branched chain alkyl group through an ether linkage. Typically these polymers are ethers of C10-C30straight or branched chain alcohols with hydroxyalkylcelluloses. Other useful polysaccharides include scleroglucans comprising a linear chain of (1-3) linked glucose units with a (1-6) linked glucose every three unit. Non-limiting examples of gums that can be used with the present invention include acacia, agar, algin, alginic acid, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluroinic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, sodium carboyxmethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and mixtures thereof. i. Preservatives Non-limiting examples of preservatives that can be used in the context of the present invention include quaternary ammonium preservatives such as polyquaternium-1 and benzalkonium halides (e.g., benzalkonium chloride (“BAC”) and benzalkonium bromide), parabens (e.g., methylparabens and propylparabens), phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. 2. Pharmaceutical Ingredients Pharmaceutical active agents are also contemplated as being useful with the compositions of the present invention. Non-limiting examples of pharmaceutical active agents include anti-acne agents, agents used to treat rosacea, analgesics, anesthetics, anorectals, antihistamines, anti-inflammatory agents including non-steroidal anti-inflammatory drugs, antibiotics, antifungals, antivirals, antimicrobials, anti-cancer actives, scabicides, pediculicides, antineoplastics, antiperspirants, antipruritics, antipsoriatic agents, antiseborrheic agents, biologically active proteins and peptides, burn treatment agents, cauterizing agents, depigmenting agents, depilatories, diaper rash treatment agents, enzymes, hair growth stimulants, hair growth retardants including DFMO and its salts and analogs, hemostatics, kerotolytics, canker sore treatment agents, cold sore treatment agents, dental and periodontal treatment agents, photosensitizing actives, skin protectant/barrier agents, steroids including hormones and corticosteroids, sunburn treatment agents, sunscreens, transdermal actives, nasal actives, vaginal actives, wart treatment agents, wound treatment agents, wound healing agents, etc. M. Kits Kits are also contemplated as being used in certain aspects of the present invention. For instance, compositions of the present invention can be included in a kit. A kit can include a container. Containers can include a bottle, a metal tube, a laminate tube, a plastic tube, a dispenser, a pressurized container, a barrier container, a package, a compartment, a lipstick container, a compact container, cosmetic pans that can hold cosmetic compositions, or other types of containers such as injection or blow-molded plastic containers into which the dispersions or compositions or desired bottles, dispensers, or packages are retained. The kit and/or container can include indicia on its surface. The indicia, for example, can be a word, a phrase, an abbreviation, a picture, or a symbol. The containers can dispense a pre-determined amount of the composition. In other embodiments, the container can be squeezed (e.g., metal, laminate, or plastic tube) to dispense a desired amount of the composition. The composition can be dispensed as a spray, an aerosol, a liquid, a fluid, or a semi-solid. The containers can have spray, pump, or squeeze mechanisms. A kit can also include instructions for employing the kit components as well the use of any other compositions included in the container. Instructions can include an explanation of how to apply, use, and maintain the compositions. EXAMPLES The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 In Vivo Data The combination ofSilybum marianumextract andMomordica grosvenorifruit extract was found through in vivo studies on women (one week study) to be effective on skin-types of normal, dry, and oily (data not shown). TheSilybum marianumextract was a hydroalcoholic extract and theMomordica grosvenorifruit extract was a hydroglycolic extract. The addition of the following combinations were found to work particularly well with certain skin-types (data not shown):Dry Skin: The addition ofLinum usitatissimumseed extract and hydrolyzed algin was found to work well on dry skin. TheLinum usitatissimumseed extract was a hydroglycolic extract and the hydrolyzed algin was obtained fromLaminaria digitata.Oily Skin: The addition ofPsidium guajavafruit extract andKunzea ericoidesleaf extract was found to work well on oily skin. ThePsidium guajavafruit extract was a hydroglycolic fruit extract and theKunzea ericoidesleaf extract was an aqueous extract.Normal Skin: The addition ofPlumeria albaflower extract andNymphea giganteaflower extract was found to work well on normal skin. ThePlumeria albaflower extract was an aqueous extract and wherein theNymphea giganteaflower extract was an aqueous extract. The combination ofSilybum marianumextract andPseudopterogorgia elisabethaeextract was found through in vivo studies on women (one week study) to be effective on skin-types of normal, dry, and oily (data not shown). TheSilybum marianumextract was a hydroalcoholic extract and thePseudopterogorgia elisabethaeextract was a butylene glycol extract. The addition of the following combinations were found to work particularly well with certain skin-types (data not shown):Dry Skin: The addition ofLinum usitatissimumseed extract and hydrolyzed algin was found to work well on dry skin. TheLinum usitatissimumseed extract was a hydroglycolic extract and the hydrolyzed algin was obtained fromLaminaria digitata.Oily Skin: The addition ofPsidium guajavafruit extract andSpiraea ulmariaextract extract was found to work well on oily skin.Psidium guajavafruit extract was a hydroglycolic fruit extract.Normal Skin: The addition ofPlumeria albaflower extract,Euterpe oleraceaefruit extract, andCamellia sinensisleaf extract was found to work well on normal skin. ThePlumeria albaflower extract was an aqueous extract. Example 2 In Vitro Data Table 1 includes data concerning the combination ofSilybum marianumextract and luo han guo fruit extract. TABLE 1SilybummarianumLuo Han GuoAssayExtract*Extract*CGRP Expression−51%−Cyclo-oxygenase 1 activity−65%−inhibitionCyclo-oxygenase 2 activity−59%−inhibitionFAAH inhibition−41%−Lipoxygenase activity−30%−TNF-α expression−50%−IL 8 expression−26.47%−IL-2 expression−24.34%−Angiogenin expression−31.54%−ICAM-1 expression+2.47%−IFN-γ expression−43.59%−IL 10 expression−39.84%−IL12p40 expression−37.79%−Anti-oxidant activity (ORAC)**+135%−Anti-oxidant activity (TEAC)**+87%−Lipid peroxidation (cell-based)DCRS**−Mushroom tyrosinase inhibition−50%−*PRONALEN SILYMARIN HSC from Provital S.A. (SPAIN) was used to obtain data; Luo han guo fruit extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain the data.**ORAC activity of positive control; TEAC activity of positive control; DCRS exogenous and endogenous peroxide. Table 2 includes data concerning the combination ofSilybum marianumextract, luo han guo fruit extract, and gorgonian extract. TABLE 2SilybumLuo HanmarianumGuoGorgonianAssayExtract*Extract*Extract*CGRP Expression−51%−Cyclo-oxygenase 1 activity−65%−inhibitionCyclo-oxygenase 2 activity−59%−−50%inhibitionFAAH inhibition−41%−Lipoxygenase activity−30%−TNF-α expression−50%−−81%IL 8 expression−26.47%−IL-2 expression−24.34%−Angiogenin expression−31.54%−ICAM-1 expression+2.47%−IFN-γ expression−43.59%−IL 10 expression−39.84%−IL12p40 expression−37.79%−Anti-oxidant activity (ORAC)**+135%−Anti-oxidant activity (TEAC)**+87%−+86%Lipid peroxidation (cell-based)DCRS**−Mushroom tyrosinase inhibition−50%−*PRONALEN SILYMARIN HSC from Provital S.A. (SPAIN) was used to obtain data; Luo han guo fruit extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; Gorgonian Extract BG from Lipo Chemicals, Inc., Paterson, New Jersey (USA) was used to obtain data.**ORAC activity of positive control; TEAC activity of positive control; DCRS exogenous and endogenous peroxide. Table 3 includes data concerning the combination ofSilybum marianumextract, luo han guo fruit extract, flax seed extract, and hydrolyzed algin extract. TABLE 3SilybumLuo HanFlaxHydrolyzedmarianumGuoSeedAlginAssayExtract*Extract*Extract*ExtractCGRP Expression−51%−−−66%Cyclo-oxygenase 1−65%−−−activityCyclo-oxygenase 2−59%−−−activityFAAH inhibition−41%−−−Lipoxygenase activity−30%−−−TNF-α expression−50%−−−21%IL 6 expression−−−−70%IL 8 expression−26.47%−−−33%IL-2 expression−24.34%−−−Angiogenin expression−31.54%−−−ICAM-1 expression+2.47%−−−IFN-γ expression−43.59%−−−IL 10 expression−39.84%−−−40%IL12p40 expression−37.79%−−−32%Anti-oxidant activity+135%−−−(ORAC)**Anti-oxidant activity+87%−−−(TEAC)**Lipid peroxidationDCRS**−−−(cell-based)Mushroom tyrosinase−50%−−−inhibitionInvolucrin reporter−−++−Laminin ELISA−−+168%−MMP2 inhibition−−−27%−MMP3 inhibition−−−52%−MMP9 inhibition−−−15%−B16 pigmentation−−−−24.50%*PRONALEN SILYMARIN HSC from Provital S.A. (SPAIN) was used to obtain data; Luo han guo fruit extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; Flax Seed Extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; PHYKO AL-PF from Barnet Products Corp., Englewood Cliffs, New Jersey (USA), was used to obtain data.**ORAC activity of positive control; TEAC activity of positive control; DCRS exogenous and endogenous peroxide. Table 4 includes data concerning the combination ofSilybum marianumextract, luo han guo fruit extract, frangipani flower extract, andNymphaea giganteaflower extract. TABLE 4SilybumLuo HanFrangipaniNymphaeamarianumGuoFlowerGiganteaAssayExtract*Extract*Extract*ExtractCGRP Expression−51%−−−Cyclo-oxygenase 1−65%−−−activityCyclo-oxygenase 2−59%−−−activityFAAH inhibition−41%−−−Lipoxygenase activity−30%−−−TNF-α expression−50%−−89%−60%IL 8 expression−26.47%−−−IL-2 expression−24.34%−−−Angiogenin expression−31.54%−−−ICAM-1 expression+2.47%−−−IFN-γ expression−43.59%−−−IL 10 expression−39.84%−−−IL12p40 expression−37.79%−−−Anti-oxidant activity+135%−+30%+38%(ORAC)**Anti-oxidant activity+87%−−−(TEAC)**Lipid peroxidationDCRS**−−−(cell-based)Mushroom tyrosinase−50%−−−inhibitionCollagen II ELISA−−−+43%*PRONALEN SILYMARIN HSC from Provital S.A. (SPAIN) was used to obtain data; Luo han guo fruit extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; ABACROSS FRANGIPANI FLOWER BG from Southern Cross Botanicals, New South Wales (AUSTRALIA) was used to obtain data; ABACROSS WATER LILY BG from Southern Cross Botanicals, New South Wales (AUSTRALIA) was used to obtain data. Table 5 includes data concerning the combination ofSilybum marianumextract, luo han guo fruit extract, guava fruit extract, and kanuka leaf extract. TABLE 5SilybumLuo HanGuavaKanukamarianumGuoFruitLeafAssayExtract*Extract*Extract*Extract*CGRP expression−51%−−−Cyclo-oxygenase−65%−−−1 activityCyclo-oxygenase−59%−−−2 activityFAAH inhibition−41%−−−Lipoxygenase activity−30%−−−TNF-α expression−50%−−89%−63%IL 8 expression−26.47%−−−IL-2 expression−24.34%−−−Angiogenin expression−31.54%−−−ICAM-1 expression+2.47%−−−IFN-γ expression−43.59%−−−IL 10 expression−39.84%−−−IL12p40 expression−37.79%−−−Anti-oxidant activity+135%−+30%−(ORAC)**Anti-oxidant activity+87%−−+99%(TEAC)**Lipid peroxidationDCRS**−−−(cell-based)Mushroom tyrosinase−50%−−−inhibitionCollagen Il ELISA−−−+78%Elastase inhibition−−−−35%*PRONALEN SILYMARIN HSC from Provital S.A. (SPAIN) was used to obtain data; Luo han guo fruit extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; Guava Fruit Extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; ABACROSS KANUKA BG from Southern Cross Botanicals, New South Wales (AUSTRALIA) was used to obtain data. Table 6 includes data concerning the combination of flax seed and hydrolyzed algin extracts. TABLE 6Hydrolyzed AlginAssayFlax Seed Extract*Extract*Involucrin reporter++−Laminin ELISA+168%−MMP2 inhibition−27%−MMP3 inhibition−52%−MMP9 inhibition−15%−CGRP expression−−66%TNF-α expression−−21%IL-6 expression−−70%IL-8 expression−−33%IL-10 expression−−40%IL12p40 expression−−32%B16 pigmentation−−24.50%*Flax Seed Extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data;PHYKO AL-PF from Barnet Products Corp., Englewood Cliffs, New Jersey (USA), was used to obtain data. Table 7 includes data concerning the combination of frangipani flower andNymphaea giganteaflower extracts. TABLE 7Frangipani FlowerNymphaea giganteaAssayExtract*Flower Extract*TNF-α expression−89%−60%Anti-Oxidant Capacity+30%+38%(ORAC)**Collagen I ELISA−+43%*ABACROSS FRANGIPANI FLOWER BG from Southern Cross Botanicals, New South Wales (Australia) was used to obtain data; ABACROSS WATER LILY BG from Southern Cross Botanicals, New South Wales (Austmlia) was used to obtain data.**ORAC activity of positive control. Table 8 includes data concerning the combination of guava fruit extract and kanuka leaf extract. TABLE 8Guava FruitKanuka LeafAssayExtract *Extract*TNF-α expression−−63%Anti-Oxidant Capacity (TEAC)**−+99%Collagen I ELISA−+78%Elastase inhibition−−35%*Guava Fruit Extract from Carrubba Inc., Milford, Connecticut (USA) was used to obtain data; ABACROSS KANUKA BG from Southern Cross Botanicals, New South Wales (AUSTRALIA) was used to obtain data. Example 3 Formulations The Tables 9-19 compositions are non-limiting compositions that can be used in the context of the present invention. TABLE 9*Ingredient% Concentration (by weight)Phase AWaterq.sXanthum gum0.1M-paraben0.15P-paraben0.1Citric acid0.01Phase BCetyl alcohol4.0Glyceryl stearate + PEG 1004.0Octyl palmitate4.0Dimethicone1.0Tocopheryl acetate0.2Phase CExtract(s)**2.0*Sprinkle Xanthum gum in water and mix for 10 min. Subsequently, add all ingredients in phase A and heat to 70-75° C. Add all items in phase B to separate beaker and heat to 70-75° C. Mix phases A and B at 70-75° C. Continue mixing and allow composition to cool to 30° C. Subsequently, add phase C ingredient while mixing.**Extracts refers to the extracts and combinations of extracts described in Examples 1-8 and throughout the specification. TABLE 10*Ingredient% Concentration (by weight)Phase AWaterq.sM-paraben0.2P-paraben0.1Na2 EDTA0.1Shea butter4.5Petrolatum4.5Glycerin4.0Propylene Glycol2.0Finsolve TN2.0Phase BSepigel 3052.0Phase CExtract(s)**2.0*Add ingredients in phase A to beaker and heat to 70-75° C. while mixing. Subsequently, add the phase B ingredient with phase A and cool to 30° C. with mixing. Subsequently, add phase C ingredient while mixing.**Extracts refers to the extracts and combinations of extracts described in Examples 1-8 and throughout the specification. TABLE 11*Ingredient% Concentration (by weight)Water**q.s.Petrolatum4-6Glycerin2-4Glyceryl Stearate2-4PEG-6 Caprylic/Capric Glycerides2-4Shea Butter2-4Cetyl Alcohol1-2Stearyl Alcohol0.5 to 2Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**70-80% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 12*Ingredient% Concentration (by weight)Water**q.s.Sunflower seed oil7-10Glycerin3-7Cetearyl ethylhexanoate3-5Dicaprylyl carbonate1-3Glyceryl isostearate1-3Glyceryl stearate1-3PEG-80.5-2Stearic acid0.5-2Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**70-80% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 13*Ingredient% Concentration (by weight)Water**q.s.TEA-Lauryl Sulfate7-10Glycerin2-5Propylene glycol1-3Cocamidopropyl betaine1-3Sodium methyl cocoyl taurate1-3Dimethicone0.5-2Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**70-80% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 14*Ingredient% Concentration (by weight)Water**q.s.Butylene glycol5-10Glycerin3-7PEG-323-7Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**75-85% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 15*Ingredient% Concentration (by weight)Water**q.s.Butylene glycol3-5Glycerin3-5PEG-323-5Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**80-90% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 16*Ingredient% Concentration (by weight)Water**q.s.Butylene glycol1-3Glycerin1-3Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**90-96% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 17*Ingredient% Concentration (by weight)Water**q.s.Cetearyl ethylhexanoate5-10Glycerin3-7Caprylic/Capric triglyceride3-5Butylene glycol2-5Sunflower Seed Oil2-5Glyceryl Stearate1-3Isostearyl alcohol1-3Petrolatum1-3Stearic Acid0.5-2Betaine0.5-2Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**60-70% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 18*Ingredient% Concentration (by weight)Water**q.s.Glycerin3-7Butylene glycol3-5Cetearyl Ethylhexanoate3-5Caprylic/Capric triglyceride1-3Glyceryl Stearate1-3Dimethicone1-3Betaine0.5-2Isostearyl alcohol0.5-2Stearic acid0.5-2Extract(s)**0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**70-80% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. TABLE 19*Ingredient% Concentration (by weight)Water**q.s.Glycerin3-5Isododecane3-5Butylene glycol3-5Dimethicone1-3Betaine0.5-2Extract(s)***0.001-5*Formulation can be prepared by mixing the ingredients in a beaker under heat 70-75° C. until homogenous. Subsequently, the formulation can be cooled to standing room temperature (20-25° C.).**75-85% w/w of water works well.***Extracts refers to the extracts and combinations of extracts described throughout this specification can be used. In particular, any of the combinations discussed in Example 1 can be used to create products for all skin or for dry skin or for oily skin or for normal skin or for combination skin. Also, the 0.001-5% references the total amount of said extracts or the amount of each extract individually in the formulation. Example 4 Assays Used to Obtain Data B16 Pigmentation Assay: Melanogenesis is the process by which melanocytes produce melanin, a naturally produced pigment that imparts color to skin, hair, and eyes. Inhibiting melanogenesis is beneficial to prevent skin darkening and lighten dark spots associated with aging. This bioassay utilizes B16-F1 melanocytes (ATCC), an immortalized mouse melanoma cell line, to analyze the effect of compounds on melanogenesis. The endpoint of this assay is a spectrophotometric measurement of melanin production and cellular viability. B16-F1 melanocytes, cultivated in standard DMEM growth medium with 10% fetal bovine serum (Mediatech) at 37° C. in 10% CO2, were treated with each of the extracts identified in Tables 1-8 for 6 days. Following incubation, melanin secretion was measured by absorbance at 405 nm and cellular viability was quantified. Collagen Stimulation Assay: Collagen is an extracellular matrix protein critical for skin structure. Increased synthesis of collagen helps improve skin firmness and elasticity. This bioassay analyzes the effect of extracts on the production of procollagen peptide (a precursor to collagen) by human epidermal fibroblasts. The endpoint of this assay is a spectrophotometric measurement that reflects the presence of procollagen peptide and cellular viability. The assay employs the quantitative sandwich enzyme immunoassay technique whereby a monoclonal antibody specific for procollagen peptide has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any procollagen peptide □present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for procollagen peptide is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of procollagen peptide bound in the initial step using a microplate reader for detection at 450 nm. The color development is stopped and the intensity of the color is measured. Subconfluent normal human adult epidermal fibroblasts (Cascade Biologics) cultivated in standard DMEM growth medium with 10% fetal bovine serum (Mediatech) at 37° C. in 10% CO2, were treated with each of the extracts identified in Tables 1-8 for 3 days. Following incubation, cell culture medium was collected and the amount of procollagen peptide secretion quantified using a sandwhich enzyme linked immuno-sorbant assay (ELISA) from Takara (#MK101). Tumor Necrosis Factor Alpha (TNF-α) Assay: The prototype ligand of the TNF superfamily, TNF-α, is a pleiotropic cytokine that plays a central role in inflammation. Increase in its expression is associated with an up regulation in pro-inflammatory activity. This bioassay analyzes the effect of extracts on the production of TNF-α by human epidermal keratinocytes. The endpoint of this assay is a spectrophotometric measurement that reflects the presence of TNF-α and cellular viability. The assay employs the quantitative sandwich enzyme immunoassay technique whereby a monoclonal antibody specific for TNF-α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α □present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-α is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TNF-α bound in the initial step using a microplate reader for detection at 450 nm. The color development is stopped and the intensity of the color is measured. Subconfluent normal human adult keratinocytes (Cascade Biologics) cultivated in EpiLife standard growth medium (Cascade Biologics) at 37° C. in 5% CO2, were treated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Sigma Chemical, #P1585-1MG) and each of the extracts identified in Tables 1-8 for 6 hours. PMA has been shown to cause a dramatic increase in TNF-α secretion which peaks at 6 hours after treatment. Following incubation, cell culture medium was collected and the amount of TNF-α secretion quantified using a sandwhich enzyme linked immuno-sorbant assay (ELISA) from R&D Systems (#DTA00C). Antioxidant (AO) assay: An in vitro bioassay that measures the total anti-oxidant capacity of an extract. The assay relies on the ability of antioxidants in the sample to inhibit the oxidation of ABTS® (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS®.+ by metmyoglobin. The antioxidant system of living organisms includes enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; macromolecules such as albumin, ceruloplasmin, and ferritin; and an array of small molecules, including ascorbic acid, α-tocopherol, β-carotene, reduced glutathione, uric acid, and bilirubin. The sum of endogenous and food-derived antioxidants represents the total antioxidant activity of the extracellular fluid. Cooperation of all the different antioxidants provides greater protection against attack by reactive oxygen or nitrogen radicals, than any single compound alone. Thus, the overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual components, as it considers the cumulative effect of all antioxidants present in plasma and body fluids. The capacity of the antioxidants in the sample to prevent ABTS oxidation is compared with that of Trolox, a water-soluble tocopherol analogue, and is quantified as molar Trolox equivalents. Anti-Oxidant capacity kit #709001 from Cayman Chemical (Ann Arbor, Michigan USA) was used as an in vitro bioassay to measure the total anti-oxidant capacity of each of the extracts identified in Tables 1-8. The protocol was followed according to manufacturer recommendations. The assay relied on antioxidants in the sample to inhibit the oxidation of ABTS® (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS®.+ by metmyoglobin. The capacity of the antioxidants in the sample to prevent ABTS oxidation was compared with that Trolox, a water-soluble tocopherol analogue, and was quantified as a molar Trolox equivalent. ORAC Assay: Oxygen Radical Absorption (or Absorbance) Capacity (ORAC) of the aromatic skin-active ingredients and compositions can also be assayed by measuring the antioxidant activity of such ingredients or compositions. This assay can quantify the degree and length of time it takes to inhibit the action of an oxidizing agent such as oxygen radicals that are known to cause damage cells (e.g., skin cells). The ORAC value of the aromatic skin-active ingredients and compositions can be determined by methods known to those of ordinary skill in the art (see U.S. Publication Nos. 2004/0109905 and 2005/0163880; Cao et al. (1993)), all of which are incorporated by reference). In summary, the assay described in Cao et al. (1993) measures the ability of antioxidant compounds in test materials to inhibit the decline of B-phycoerythrm (B-PE) fluorescence that is induced by a peroxyl radical generator, AAPH. The extracts identified in Tables 1-8 were subjected to this assay. Mushroom tyrosinase activity assay: In mammalian cells, tyrosinase catalyzes two steps in the multi-step biosynthesis of melanin pigments from tyrosine (and from the polymerization of dopachrome). Tyrosinase is localized in melanocytes and produces melanin (aromatic quinone compounds) that imparts color to skin, hair, and eyes. Purified mushroom tyrosinase (Sigma) was incubated with its substrate L-Dopa (Fisher) in the presence or absence of each of the extracts in Tables 1-8. Pigment formation was evaluated by colorimetric plate reading at 490 nm. The percent inhibition of mushroom tyrosinase activity was calculated compared to non-treated controls to determine the ability of test extracts to inhibit the activity of purified enzyme. Test extract inhibition was compared with that of kojic acid (Sigma). Matrix Metalloproteinase Enzyme Activity (MMP3; MMP9) Assay: An in vitro matrix metalloprotease (MMP) inhibition assay. MMPs are extracellular proteases that play a role in many normal and disease states by virtue of their broad substrate specificity. MMP3 substrates include collagens, fibronectins, and laminin; while MMP9 substrates include collagen VII, fibronectins and laminin. Using Colorimetric Drug Discovery kits from BioMol International for MMP3 (AK-400) and MMP-9 (AK-410), this assay is designed to measure protease activity of MMPs using a thiopeptide as a chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoylRG-OC2H5)5,6. The MMP cleavage site peptide bond is replaced by a thioester bond in the thiopeptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5′-dithiobis(2-nitrobenzoic acid), Ellman's reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm (ε=13,600 M-1 cm-1 at pH 6.0 and above 7). The extracts identified in Tables 1-8 were subjected to this assay. Cyclooxygenase (COX) Assay: An in vitro cyclooxygenase-1 and -2 (COX-1, -2) inhibition assay. COX is a bifunctional enzyme exhibiting both cyclooxygenase and peroxidase activities. The cyclooxygenase activity converts arachidonic acid to a hydroperoxy endoperoxide (Prostaglandin G2; PGG2) and the peroxidase component reduces the endoperoxide (Prostaglandin H2; PGH2) to the corresponding alcohol, the precursor of prostaglandins, thromboxanes, and prostacyclins. This COX Inhibitor screening assay measures the peroxidase component of cyclooxygenases. The peroxidase activity is assayed colorimetrically by monitoring the appearance of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). This inhibitor screening assay includes both COX-1 and COX-2 enzymes in order to screen isozyme-specific inhibitors. The Colormetric COX (ovine) Inhibitor screening assay (#760111, Cayman Chemical), was used to analyze the effects of each of the extracts identified in Tables 1-8 on the activity of purified cyclooxygnase enzyme (COX-1 or COX-2). According to manufacturer instructions, purified enzyme, heme and test extracts were mixed in assay buffer and incubated with shaking for 15 min at room temperature. Following incubation, arachidonic acid and colorimetric substrate were added to initiate the reaction. Color progression was evaluated by colorimetric plate reading at 590 nm. The percent inhibition of COX-1 or COX-2 activity was calculated compared to non-treated controls to determine the ability of test extracts to inhibit the activity of purified enzyme. Lipoxygenase (LO) Assay: An in vitro lipoxygenase (LO) inhibition assay. LOs are non-heme iron-containing dioxygenases that catalyze the addition of molecular oxygen to fatty acids. Linoleate and arachidonate are the main substrates for LOs in plants and animals. Arachadonic acid may then be converted to hydroxyeicosotrienenoic (HETE) acid derivatives, that are subsequently converted to leukotirenes, potent inflammatory mediators. This assay provides an accurate and convenient method for screening lipoxygenase inhibitors by measuring the hydroperoxides generated from the incubation of a lipoxygenase (5-, 12-, or 15-LO) with arachidonic acid. The Colorimetric LO Inhibitor screening kit (#760700, Cayman Chemical) was used to determine the ability of each of the extracts identified in Tables 1-8 to inhibit enzyme activity. Purified 15-lipoxygenase and test extracts were mixed in assay buffer and incubated with shaking for 10 min at room temperature. Following incubation, arachidonic acid was added to initiate the reaction and mixtures incubated for an additional 10 min at room temperature. Colorimetric substrate was added to terminate catalysis and color progression was evaluated by fluorescence plate reading at 490 nm. The percent inhibition of lipoxyganse activity was calculated compared to non-treated controls to determine the ability of test extracts to inhibit the activity of purified enzyme. Elastase Assay: EnzChek® Elastase Assay (Kit #E-12056) from Molecular Probes (Eugene, Oregon USA) was used as an in vitro enzyme inhibition assay for measuring inhibition of elastase activity for each of the extracts identified in Tables 1-8. The EnzChek kit contains soluble bovine neck ligament elastin that has been labeled with dye such that the conjugate's fluorescence is quenched. The non-fluorescent substrate can be digested by elastase or other proteases to yield highly fluorescent fragments. The resulting increase in fluorescence can be monitored with a fluorescence microplate reader. Digestion products from the elastin substrate have absorption maxima at ˜505 nm and fluorescence emission maxima at ˜515 nm. The peptide, chloromethyl ketone, is used as a selective, collective inhibitor of elastase when utilizing the EnzChek Elastase Assay Kit for screening for elastase inhibitors. Example 5 Additional Assays Additional assays that can be used to determine the efficacy of any one of the compositions disclosed throughout the specification and claims can be determined by methods known to those of ordinary skill in the art. The following are non-limiting assays that can be used in the context of the present invention. It should be recognized that other testing procedures can be used, including, for example, objective and subjective procedures. Oil Control Assay: An assay to measure reduction of sebum secretion from sebaceous glands and/or reduction of sebum production from sebaceous glands can be assayed by using standard techniques known to those having ordinary skill in the art. In one instance, the forehead can be used. A composition of the present invention can be applied to one portion of the forehead once or twice daily for a set period of days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days), while another portion of the forehead is not treated with the composition. After the set period of days expires, then sebum secretion can be assayed by application of fine blotting paper to the treated and untreated forehead skin. This is done by first removing any sebum from the treated and untreated areas with moist and dry cloths. Blotting paper can then be applied to the treated and untreated areas of the forehead, and an elastic band can be placed around the forehead to gently press the blotting paper onto the skin. After 2 hours the blotting papers can be removed, allowed to dry and then transilluminated. Darker blotting paper correlates with more sebum secretion (or lighter blotting paper correlates with reduced sebum secretion. Erythema Assay: An assay to measure the reduction of skin redness can be evaluated using a Minolta Chromometer. Skin erythema may be induced by applying a 0.2% solution of sodium dodecyl sulfate on the forearm of a subject. The area is protected by an occlusive patch for 24 hrs. After 24 hrs, the patch is removed and the irritation-induced redness can be assessed using the a* values of the Minolta Chroma Meter. The a* value measures changes in skin color in the red region. Immediately after reading, the area is treated with a composition of the present invention. Repeat measurements are taken at regular intervals to determine the formula's ability to reduce redness and irritation. Skin Moisture/Hydration Assay: Skin moisture/hydration benefits can be measured by using impedance measurements with the Nova Dermal Phase Meter. The impedance meter measures changes in skin moisture content. The outer layer of the skin has distinct electrical properties. When skin is dry it conducts electricity very poorly. As it becomes more hydrated increasing conductivity results. Consequently, changes in skin impedance (related to conductivity) can be used to assess changes in skin hydration. The unit can be calibrated according to instrument instructions for each testing day. A notation of temperature and relative humidity can also be made. Subjects can be evaluated as follows: prior to measurement they can equilibrate in a room with defined humidity (e.g., 30-50%) and temperature (e.g., 68-72° C.). Three separate impedance readings can be taken on each side of the face, recorded, and averaged. The T5 setting can be used on the impedance meter which averages the impedance values of every five seconds application to the face. Changes can be reported with statistical variance and significance. Skin Clarity and Reduction in Freckles and Age Spots Assay: Skin clarity and the reduction in freckles and age spots can be evaluated using a Minolta Chromometer. Changes in skin color can be assessed to determine irritation potential due to product treatment using the a* values of the Minolta Chroma Meter. The a* value measures changes in skin color in the red region. This is used to determine whether a composition is inducing irritation. The measurements can be made on each side of the face and averaged, as left and right facial values. Skin clarity can also be measured using the Minolta Meter. The measurement is a combination of the a*, b, and L values of the Minolta Meter and is related to skin brightness, and correlates well with skin smoothness and hydration. Skin reading is taken as above. In one non-limiting aspect, skin clarity can be described as L/C where C is chroma and is defined as (a2+b2)1/2. Skin Dryness, Surface Fine Lines, Skin Smoothness, and Skin Tone Assay: Skin dryness, surface fine lines, skin smoothness, and skin tone can be evaluated with clinical grading techniques. For example, clinical grading of skin dryness can be determined by a five point standard Kligman Scale: (0) skin is soft and moist; (1) skin appears normal with no visible dryness; (2) skin feels slightly dry to the touch with no visible flaking; (3) skin feels dry, tough, and has a whitish appearance with some scaling; and (4) skin feels very dry, rough, and has a whitish appearance with scaling. Evaluations can be made independently by two clinicians and averaged. Clinical Grading of Skin Tone Assay: Clinical grading of skin tone can be performed via a ten point analog numerical scale: (10) even skin of uniform, pinkish brown color. No dark, erythremic, or scaly patches upon examination with a hand held magnifying lens. Microtexture of the skin very uniform upon touch; (7) even skin tone observed without magnification. No scaly areas, but slight discolorations either due to pigmentation or erythema. No discolorations more than 1 cm in diameter; (4) both skin discoloration and uneven texture easily noticeable. Slight scaliness. Skin rough to the touch in some areas; and (1) uneven skin coloration and texture. Numerous areas of scaliness and discoloration, either hypopigmented, erythremic or dark spots. Large areas of uneven color more than 1 cm in diameter. Evaluations were made independently by two clinicians and averaged. Clinical Grading of Skin Smoothness Assay: Clinical grading of skin smoothness can be analyzed via a ten point analog numerical scale: (10) smooth, skin is moist and glistening, no resistance upon dragging finger across surface; (7) somewhat smooth, slight resistance; (4) rough, visibly altered, friction upon rubbing; and (1) rough, flaky, uneven surface. Evaluations were made independently by two clinicians and averaged. Skin Smoothness and Wrinkle Reduction Assay With Methods Disclosed in Packman et al. (1978): Skin smoothness and wrinkle reduction can also be assessed visually by using the methods disclosed in Packman et al. (1978). For example, at each subject visit, the depth, shallowness and the total number of superficial facial lines (SFLs) of each subject can be carefully scored and recorded. A numerical score was obtained by multiplying a number factor times a depth/width/length factor. Scores are obtained for the eye area and mouth area (left and right sides) and added together as the total wrinkle score. Skin Firmness Assay with a Hargens Ballistometer: Skin firmness can be measured using a Hargens ballistometer, a device that evaluates the elasticity and firmness of the skin by dropping a small body onto the skin and recording its first two rebound peaks. The ballistometry is a small lightweight probe with a relatively blunt tip (4 square mm-contact area) was used. The probe penetrates slightly into the skin and results in measurements that are dependent upon the properties of the outer layers of the skin, including the stratum corneum and outer epidermis and some of the dermal layers. Skin Softness/Suppleness Assay with a Gas Bearing Electrodynamometer: Skin softness/suppleness can be evaluated using the Gas Bearing Electrodynamometer, an instrument that measures the stress/strain properties of the skin. The viscoelastic properties of skin correlate with skin moisturization. Measurements can be obtained on the predetermined site on the cheek area by attaching the probe to the skin surface with double-stick tape. A force of approximately 3.5 gm can be applied parallel to the skin surface and the skin displacement is accurately measured. Skin suppleness can then be calculated and is expressed as DSR (Dynamic Spring Rate in gm/mm). Appearance of Lines and Wrinkles Assay with Replicas: The appearance of lines and wrinkles on the skin can be evaluated using replicas, which is the impression of the skin's surface. Silicone rubber like material can be used. The replica can be analyzed by image analysis. Changes in the visibility of lines and wrinkles can be objectively quantified via the taking of silicon replicas form the subjects' face and analyzing the replicas image using a computer image analysis system. Replicas can be taken from the eye area and the neck area, and photographed with a digital camera using a low angle incidence lighting. The digital images can be analyzed with an image processing program and the are of the replicas covered by wrinkles or fine lines was determined. Surface Contour of the Skin Assay with a Profilometer/Stylus Method: The surface contour of the skin can be measured by using the profilometer/Stylus method. This includes either shining a light or dragging a stylus across the replica surface. The vertical displacement of the stylus can be fed into a computer via a distance transducer, and after scanning a fixed length of replica a cross-sectional analysis of skin profile can be generated as a two-dimensional curve. This scan can be repeated any number of times along a fix axis to generate a simulated 3-D picture of the skin. Ten random sections of the replicas using the stylus technique can be obtained and combined to generate average values. The values of interest include Ra which is the arithmetic mean of all roughness (height) values computed by integrating the profile height relative to the mean profile height. Rt which is the maximum vertical distance between the highest peak and lowest trough, and Rz which is the mean peak amplitude minus the mean peak height. Values are given as a calibrated value in mm. Equipment should be standardized prior to each use by scanning metal standards of know values. Ra Value can be computed by the following equation: Ra=Standardize roughness; lm=the traverse (scan) length; and y=the absolute value of the location of the profile relative to the mean profile height (x-axis). MELANODERM™ Assay: In other non-limiting aspects, the efficacy of the compositions of the present invention can be evaluated by using a skin analog, such as, for example, MELANODERM™. Melanocytes, one of the cells in the skin analog, stain positively when exposed to L-dihydroxyphenyl alanine (L-DOPA), a precursor of melanin. The skin analog, MELANODERM™, can be treated with a variety of bases containing the compositions and whitening agents of the present invention or with the base alone as a control. Alternatively, an untreated sample of the skin analog can be used as a control. All of the skin-active ingredients, compositions, or methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of the present disclosure. While the skin-active ingredients, compositions, or methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the skin-active ingredients, compositions, or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. | 96,225 |
11857668 | DETAILED DESCRIPTION In the following detailed description of the invention, numerous details, examples, and embodiments of the invention are described. However, it will be clear and apparent to one skilled in the art that the invention is not limited to the embodiments set forth and that the invention can be adapted for any of several applications. The composition of the present disclosure may be used as an underarm toner for eliminating body odor and may comprise the following elements. This list of possible constituent elements is intended to be exemplary only, and it is not intended that this list be used to limit the composition of the present application to just these elements. Persons having ordinary skill in the art relevant to the present disclosure may understand there to be equivalent elements that may be substituted within the present disclosure without changing the essential function or operation of the composition. The various elements of the present disclosure may be related in the following exemplary fashion. It is not intended to limit the scope or nature of the relationships between the various elements, and the following examples are presented as illustrative examples only. By way of example, some embodiments of the present disclosure include an underarm toner for eliminating or reducing odor-causing bacteria, wherein the underarm toner comprises nature-derived ingredients. More specifically, the underarm toner may comprise rose water (i.e., rosa damascena), water, aloe leaf juice, calendula, lemon balm, glycerin, ylang-ylang, lactic acid,lactobacillusand coconut fruit extract,lactobacillusferment, watermelon fruit extract, chamomile extract, a probiotic blend, grapefruit seed extract, sandalwood oil, lavender oil, rosemary oil, clary sage oil, eucalyptus oil, lemon peel oil, red radish root, geranium oil, and cinnamon leaf oil, wherein the underarm toner maintains the skin's pH level at, for example, about 4 to about 5 to prevent the odor-causing bacteria from growing and proliferating. In a particular embodiment, the underarm toner of the present disclosure may comprise about 19 to about 23 volume % (vol. %) rosa damascena, about 15 to about 19 vol. % water, about 7 to about 9 vol. % aloe leaf juice, about 1 to about 3 vol. % calendula, about 1 to about 3 vol. % lemon balm, about 1 to about 3 vol. % glycerin, about 1 to about 2 vol. % ylang ylang, about 3 to about 5 vol. % lactic acid, about 2 to about 4 vol. %lactobacillusand coconut fruit extract, about 2 to about 4 vol. %lactobacillusferment, about 0.3 to about 0.6 vol. % watermelon fruit extract, about 0.3 to about 0.6 vol. % chamomile extract, about 0.08 to about 0.09 vol. % probiotic blend, about 0.2 to about 0.4 vol. % grapefruit seed oil, about 0.01 to about 0.02 vol. % sandalwood oil, about 0.001 to about 0.01 vol. % lavender oil, about 0.001 o about 0.01 vol. % rosemary oil, about 0.001 to about 0.01 vol. % clary sage oil, about 0.01 to about 0.02 vol. % eucalyptus oil, about 0.001 to about 0.01 vol. % lemon peel oil, about 0.001 to about 0.01 vol. % geranium oil, about 0.01 to about 0.02 vol. % cinnamon leaf oil, and about 0.5 to about 0.75 vol. % red radish root. In embodiments, the probiotic blend may compriseBacillus coagulans, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei, andStreptococcus thermophiles. For example, in a particular embodiment, the probiotic blend may comprise each of the ingredients combined in equal volumes. Thus, the volume ratio of each ingredient to each other ingredient may be about 1:1. In embodiments, the probiotic blend may increase the activity and growth of beneficial skin microbiota. To create the underarm toner of the present disclosure, the ingredients may simply be combined and blended to emulsify the toner. To use the underarm toner of the present disclosure, a user may apply 2 to 3 sprays of the toner under each arm and gently wipe off the excess liquid with a cotton round or a tissue. The user may then follow with their favorite deodorant. Because the combination of the ingredients in the toner creates a pH level of about 4 to about 5, such as 4.5, the body odor-causing bacterial is eliminated, leaving the underarm skin at a slightly acidic pH. As such, bacteria is not able to grow for at least 24 hours. Because it is the bacteria that creates the body odor and because using the underarm toner of the present disclosure will free the underarm of the bacteria, when deodorant is applied on top of the skin treated with the toner, the user will stay fresh all day or longer. As evidenced by the above description of the toner, the underarm toner of the present disclosure is free of aluminum, alcohol, and coconut oil, each of which can have negative health effects when applied to the underarm. The above-described embodiments of the invention are presented for purposes of illustration and not of limitation. While these embodiments of the invention have been described with reference to numerous specific details, one of ordinary skill in the art will recognize that the invention can be embodied in other specific forms without departing from the spirit of the invention. Thus, one of ordinary skill in the art would understand that the invention is not to be limited by the foregoing illustrative details, but rather is to be defined by the appended claims. | 5,445 |
11857669 | DETAILED DESCRIPTION The present disclosure is directed to various methods and formulations for treating diseases of the gastrointestinal tract with an JAK inhibitor. For example, in an embodiment, a method of treating a disease of the gastrointestinal tract in a subject comprises administering to the subject a pharmaceutical formulation comprising an JAK inhibitor wherein the pharmaceutical formulation is released in the subject's gastrointestinal tract proximate to one or more sites of disease. For example, in an embodiment, the pharmaceutical formulation comprises a therapeutically effective amount of an JAK inhibitor. In some embodiments, the formulation is contained in an ingestible device, and the device releases the formulation at a location proximate to the site of disease. The location of the site of disease may be predetermined. For example, an ingestible device, the location of which within the GI tract can be accurately determined as disclosed herein, may be used to sample one or more locations in the GI tract and to detect one or more analytes, including markers of the disease, in the GI tract of the subject. A pharmaceutical formulation may be then administered via an ingestible device and released at a location proximate to the predetermined site of disease. The release of the formulation may be triggered autonomously, as further described herein. The following disclosure illustrates aspects of the formulations and methods embodied in the claims. Formulations, including Pharmaceutical Formulations As used herein, a “formulation” of an JAK inhibitor may refer to either the JAK inhibitor in pure form, such as, for example, a lyophilized JAK inhibitor, or a mixture of the JAK inhibitor with one or more physiologically acceptable carriers, excipients or stabilizers. Thus, therapeutic formulations or medicaments can be prepared by mixing the JAK inhibitor having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) antibody; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX<®>, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases. Exemplary lyophilized formulations are described in U.S. Pat. No. 6,267,958. Aqueous formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer. A formulation of an JAK inhibitor as disclosed herein, e.g., sustained-release formulations, can further include a mucoadhesive agent, e.g., one or more of polyvinyl pyrolidine, methyl cellulose, sodium carboxyl methyl cellulose, hydroxyl propyl cellulose, carbopol, a polyacrylate, chitosan, a eudragit analogue, a polymer, and a thiomer. Additional examples of mucoadhesive agents that can be included in a formulation with an JAK inhibitor are described in, e.g., Peppas et al.,Biomaterials17(16):1553-1561, 1996; Kharenko et al.,Pharmaceutical Chemistry J.43(4):200-208, 2009; Salamat-Miller et al.,Adv. Drug Deliv. Reviews57(11):1666-1691, 2005; Bernkop-Schnurch,Adv. Drug Deliv. Rev.57(11):1569-1582, 2005; and Harding et al.,Biotechnol. Genet. Eng. News16(1):41-86, 1999. In some embodiments, components of a formulation may include any one of the following components, or any combination thereof: Acacia, Alginate, Alginic Acid, Aluminum Acetate, an antiseptic, Benzyl Alcohol, Butyl Paraben, Butylated Hydroxy Toluene, an antioxidant. Citric acid, Calcium carbonate, Candelilla wax, a binder, Croscarmellose sodium, Confectioner sugar, Colloidal silicone dioxide, Cellulose, Carnuba wax, Corn starch, Carboxymethylcellulose calcium, Calcium stearate, Calcium disodium EDTA, Chelation agents, Copolyvidone, Castor oil hydrogenated, Calcium hydrogen phosphate dehydrate, Cetylpyridine chloride, Cysteine HCl, Crosspovidone, Dibasic Calcium Phosphate, Disodium hydrogen phosphate, Dimethicone, Erythrosine Sodium, Ethyl Cellulose, Gelatin, Glyceryl monooleate, Glycerin, Glycine, Glyceryl monostearate, Glyceryl behenate, Hydroxy propyl cellulose, Hydroxyl propyl methyl cellulose, Hypromellose, HPMC Pthalate, Iron oxides or ferric oxide, Iron oxide yellow, Iron oxide red or ferric oxide, Lactose (hydrous or anhydrous or monohydrate or spray dried), Magnesium stearate, Microcrystalline cellulose, Mannitol, Methyl cellulose, Magnesium carbonate, Mineral oil, Methacrylic acid copolymer, Magnesium oxide, Methyl paraben, PEG, Polysorbate 80, Propylene glycol, Polyethylene oxide, Propylene paraben, Polaxamer 407 or 188 or plain, Potassium bicarbonate, Potassium sorbate, Potato starch, Phosphoric acid, Polyoxy 140 stearate, Sodium starch glycolate, Starch pregelatinized, Sodium crossmellose, Sodium lauryl sulfate, Starch, Silicon dioxide, Sodium benzoate, Stearic acid, Sucrose base for medicated confectionery, a granulating agent, Sorbic acid, Sodium carbonate, Saccharin sodium, Sodium alginate, Silica gel, Sorbiton monooleate, Sodium stearyl fumarate, Sodium chloride, Sodium metabisulfite, Sodium citrate dehydrate, Sodium starch, Sodium carboxy methyl cellulose, Succinic acid, Sodium propionate, Titanium dioxide, Talc, Triacetin, Triethyl citrate. Accordingly, in some embodiments of the method of treating a disease as disclosed herein, the method comprises administering to the subject a pharmaceutical composition that is a formulation as disclosed herein. In some embodiments the formulation is a dosage form, which may be, as an example, a solid form such as, for example, a capsule, a tablet, a sachet, or a lozenge; or which may be, as an example, a liquid form such as, for example, a solution, a suspension, an emulsion, or a syrup. In some embodiments, the formulation is not comprised in an ingestible device. In some embodiments wherein the formulation is not comprised in an ingestible device, the formulation may be suitable for oral administration. The formulation may be, for example, a solid dosage form or a liquid dosage form as disclosed herein. In some embodiments wherein the formulation is not comprised in an ingestible device, the formulation may be suitable for rectal administration. The formulation may be, for example, a dosage form such as a suppository or an enema. In embodiments where the formulation is not comprised in an ingestible device, the formulation releases the JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease. Such localized release may be achieved, for example, with a formulation comprising an enteric coating. Such localized release may be achieved, an another example, with a formulation comprising a core comprising one or more polymers suitable for controlled release of an active substance. A non-limiting list of such polymers includes: poly(2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, poly(ethylene glycol), poly(2-aminoethyl methacrylate), (2-hydroxypropyl)methacrylamide, poly(β-benzyl-1-aspartate), poly(N-isopropylacrylamide), and cellulose derivatives. In some embodiments, the formulation is comprised in an ingestible device as disclosed herein. In some embodiments wherein the formulation is comprised in an ingestible device, the formulation may be suitable for oral administration. The formulation may be, for example, a solid dosage form or a liquid dosage form as disclosed herein. In some embodiments the formulation is suitable for introduction and optionally for storage in the device. In some embodiments the formulation is suitable for introduction and optionally for storage in a reservoir comprised in the device. In some embodiments the formulation is suitable for introduction and optionally for storage in a reservoir comprised in the device. Thus, in some embodiments, provided herein is a reservoir comprising a therapeutically effective amount of an JAK inhibitor, wherein the reservoir is configured to fit into an ingestible device. In some embodiments, the reservoir comprising a therapeutically effective amount of an JAK inhibitor is attachable to an ingestible device. In some embodiments, the reservoir comprising a therapeutically effective amount of an JAK inhibitor is capable of anchoring itself to the subject's tissue. As an example, the reservoir capable of anchoring itself to the subject's tissue comprises silicone. As an example, the reservoir capable of anchoring itself to the subject's tissue comprises polyvinyl chloride. In some embodiments the formulation is suitable for introduction in a spray catheter, as disclosed herein. The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other. For instance, the formulation may further comprise another JAK inhibitor or a chemotherapeutic agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the JAK inhibitor, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated JAK inhibitors remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. Pharmaceutical formulations may contain one or more JAK inhibitors. The pharmaceutical formulations may be formulated in any manner known in the art. In some embodiments the formulations include one or more of the following components: a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811, incorporated by reference herein in its entirety). The formulations can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required, proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Controlled release of the JAK inhibitor can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.). In some embodiments, the JAK inhibitor is present in a pharmaceutical formulation within the device. In some embodiments, the JAK inhibitor is present in solution within the device. In some embodiments, the JAK inhibitor is present in a suspension in a liquid medium within the device. In some embodiments, the JAK inhibitor is present as a pure, powder (e.g., lyophilized) form of the JAK inhibitor. Definitions: By “ingestible”, it is meant that the device can be swallowed whole. “Gastrointestinal inflammatory disorders” are a group of chronic disorders that cause inflammation and/or ulceration in the mucous membrane. These disorders include, for example, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, indeterminate colitis and infectious colitis), mucositis (e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis and proctitis), necrotizing enterocolitis and esophagitis. “Inflammatory Bowel Disease” or “IBD” is a chronic inflammatory autoimmune condition of the gastrointestinal (GI) tract. The GI tract can be divided into four main different sections, the oesophagus, stomach, small intestine and large intestine or colon. The small intestine possesses three main subcompartments: the duodenum, jejunum and ileum. Similarly, the large intestine consists of six sections: the cecum, ascending colon, transverse colon, ascending colon, sigmoid colon, and the rectum. The small intestine is about 6 m long, its diameter is 2.5 to 3 cm and the transit time through it is typically 3 hours. The duodenum has a C-shape, and is 30 cm long. Due to its direct connection with the stomach, it is physically more stable than the jejunum and ileum, which are sections that can freely move. The jejunum is 2.4 m in length and the ileum is 3.6 m in length and their surface areas are 180 m2and 280 m2respectively. The large intestine is 1.5 m long, its diameter is between 6.3 and 6.5 cm, the transit time though this section is 20 hours and has a reduced surface area of approximately 150 m2. The higher surface area of the small intestine enhances its capacity for systemic drug absorption. The etiology of IBD is complex, and many aspects of the pathogenesis remain unclear. The treatment of moderate to severe IBD poses significant challenges to treating physicians, because conventional therapy with corticosteroids and immunomodulator therapy (e.g., azathioprine, 6 mercaptopurine, and methotrexate administered via traditional routes such as tablet form, oral suspension, or intravenously) is associated with side effects and intolerance and has not shown proven benefit in maintenance therapy (steroids). Monoclonal antibodies targeting tumor necrosis factor alpha (TNF-a), such as infliximab (a chimeric antibody) and adalimumab (a fully human antibody), are currently used in the management of CD. Infliximab has also shown efficacy and has been approved for use in UC. However, approximately 10%-20% of patients with CD are primary nonresponders to anti TNF therapy, and another ˜20%-30% of CD patients lose response over time (Schnitzler et al., Gut 58:492-500 (2009)). Other adverse events (AEs) associated with anti TNFs include elevated rates of bacterial infection, including tuberculosis, and, more rarely, lymphoma and demyelination (Chang et al., Nat Clin Pract Gastroenterol Hepatology 3:220 (2006); Hoentjen et al., World J. Gastroenterol. 15(17):2067 (2009)). No currently available therapy achieves sustained remission in more than 20%-30% of IBD patients with chronic disease (Hanauer et al, Lancet 359: 1541-49 (2002); Sandborn et al, N Engl J Med 353: 1912-25 (2005)). In addition, most patients do not achieve sustained steroid-free remission and mucosal healing, clinical outcomes that correlate with true disease modification. Although the cause of IBD remains unknown, several factors such as genetic, infectious and immunologic susceptibility have been implicated. IBD is much more common in Caucasians, especially those of Jewish descent. The chronic inflammatory nature of the condition has prompted an intense search for a possible infectious cause. Although agents have been found which stimulate acute inflammation, none has been found to cause the chronic inflammation associated with IBD. The hypothesis that IBD is an autoimmune disease is supported by the previously mentioned extraintestinal manifestation of IBD as joint arthritis, and the known positive response to IBD by treatment with therapeutic agents such as adrenal glucocorticoids, cyclosporine and azathioprine, which are known to suppress immune response. In addition, the GI tract, more than any other organ of the body, is continuously exposed to potential antigenic substances such as proteins from food, bacterial byproducts (LPS), etc. A chronic inflammatory autoimmune condition of the gastrointestinal (GI) tract presents clinically as either ulcerative colitis (UC) or Crohn's disease (CD). Both IBD conditions are associated with an increased risk for malignancy of the GI tract. “Crohn's disease” (“CD”) is a chronic transmural inflammatory disease with the potential to affect any part of the entire GI tract, and UC is a mucosal inflammation of the colon. Both conditions are characterized clinically by frequent bowel motions, malnutrition, and dehydration, with disruption in the activities of daily living. CD is frequently complicated by the development of malabsorption, strictures, and fistulae and may require repeated surgery. UC, less frequently, may be complicated by severe bloody diarrhea and toxic megacolon, also requiring surgery. The most prominent feature Crohn's disease is the granular, reddish-purple edematous thickening of the bowel wall. With the development of inflammation, these granulomas often lose their circumscribed borders and integrate with the surrounding tissue. Diarrhea and obstruction of the bowel are the predominant clinical features. As with ulcerative colitis, the course of Crohn's disease may be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease is not curable by resection of the involved segment of bowel. Most patients with Crohn's disease require surgery at some point, but subsequent relapse is common and continuous medical treatment is usual. Crohn's disease may involve any part of the alimentary tract from the mouth to the anus, although typically it appears in the ileocolic, small-intestinal or colonic-anorectal regions. Histopathologically, the disease manifests by discontinuous granulomatomas, crypt abscesses, fissures and aphthous ulcers. The inflammatory infiltrate is mixed, consisting of lymphocytes (both T and B cells), plasma cells, macrophages, and neutrophils. There is a disproportionate increase in IgM- and IgG-secreting plasma cells, macrophages and neutrophils. To date, the primary outcome measure in Crohn's Disease clinical trials is the Crohn's Disease Activity Index (CDAI), which has served as the basis for approval of multiple drug treatments, including for example, vedolizumab and natalizumab. The CDAI was developed by regressing clinician global assessment of disease activity on eighteen potential items representing patient reported outcomes (PROs) (i.e. abdominal pain, pain awakening patient from sleep, appetite), physical signs (i.e. average daily temperature, abdominal mass), medication use (i.e. loperamide or opiate use for diarrhea) and a laboratory test (i.e. hematocrit). Backward stepwise regression analysis identified eight independent predictors which are the number of liquid or soft stools, severity of abdominal pain, general well-being, occurrence of extra-intestinal symptoms, need for anti diarrheal drugs, presence of an abdominal mass, hematocrit, and body weight. The final score is a composite of these eight items, adjusted using regression coefficients and standardization to construct an overall CDAI score, ranging from 0 to 600 with higher score indicating greater disease activity. Widely used benchmarks are: CDAI <150 is defined as clinical remission, 150 to 219 is defined as mildly active disease, 220 to 450 is defined as moderately active disease, and above 450 is defined as very severe disease (Best W R, et al., Gastroenterology 77:843-6, 1979). Vedolizumab and natalizumab have been approved on the basis of demonstrated clinical remission, i.e. CDAI <150. Although the CDAI has been in use for over 40 years, and has served as the basis for drug approval, it has several limitations as an outcome measure for clinical trials. For example, most of the overall score comes from the patient diary card items (pain, number of liquid bowel movements, and general well-being), which are vaguely defined and not standardized terms (Sandler et al., J. Clin. Epidemiol 41:451-8, 1988; Thia et al., Inflamm Bowel Dis 17: 105-11, 2011). In addition, measurement of pain is based on a four-point scale rather than an updated seven-point scale. The remaining 5 index items contribute very little to identifying an efficacy signal and may be a source of measurement noise. Furthermore, concerns have been raised about poor criterion validity for the CDAI, a reported lack of correlation between the CDAI and endoscopic measures of inflammation (which may render the CDAI as a poor discriminator of active CD and irritable bowel syndrome) and high reported placebo rates (Korzenik et al., N Engl J Med. 352:2193-201, 2005; Sandborn W J, et al., N Engl J Med 353: 1912-25, 2005; Sandborn W J, et al., Ann Intern 19; 146:829-38, 2007, Epub 2007 Apr. 30; Kim et al., Gastroenterology 146: (5 supplement 1)S-368, 2014). It is, thus, generally recognized that additional or alternative measures of CD symptoms are needed, such as new PRO tools or adaptations of the CDAI to derive a new PRO. The PRO2 and PRO3 tools are such adaptations of the CDAI and have been recently described in Khanna et al., Aliment Pharmacol. Ther. 41: 77-86, 2015. The PRO2 evaluates the frequency of loose/liquid stools and abdominal pain {Id). These items are derived and weighted accordingly from the CDAI and are the CDAI diary card items, along with general well-being, that contribute most to the observed clinical benefit measured by CDAI (Sandler et al., J. Clin. Epidemiol 41:451-8, 1988; Thia et al., Inflamm Bowel Dis 17: 105-11, 2011; Kim et al., Gastroenterology 146: (5 supplement 1)S-368, 2014). The remission score of <11 is the CDAI-weighted sum of the average stool frequency and pain scores in a 7-day period, which yielded optimum sensitivity and specificity for identification of CDAI remission (score of <150) in a retrospective data analysis of ustekinumab induction treatment for moderate to severe CD in a Phase II clinical study (Gasink C, et al., abstract, ACG Annual Meeting 2014). The PRO2 was shown to be sensitive and responsive when used as a continuous outcome measure in a retrospective data analysis of MTX treatment in active CD (Khanna R, et al., Inflamm Bowel Dis 20: 1850-61, 2014) measured by CDAI. Additional outcome measures include the Mayo Clinic Score, the Crohn disease endoscopic index of severity (CDEIS), and the Ulcerative colitis endoscopic index of severity (UCEIS). Additional outcome measures include Clinical remission, Mucosal healing, Histological healing (transmural), MRI or ultrasound for measurement or evaluation of bowel wall thickness, abscesses, fistula and histology. An additional means of assessing the extent and severity of Crohn's Disease is endoscopy. Endoscopic lesions typical of Crohn's disease have been described in numerous studies and include, e.g., aphthoid ulcerations, “punched-out ulcers,” cobblestoning and stenosis. Endoscopic evaluation of such lesions was used to develop the first validated endoscopic score, the Crohn's Disease Endoscopic Index of Severity (CDEIS) (Mary et al., Gut 39:983-9, 1989). More recently, because the CDEIS is time-consuming, complicated and impractical for routine use, a Simplified Endoscopic Activity Score for Crohn's Disease (SES-CD) was developed and validated (Daperno et al., Gastrointest. Endosc. 60(4):505-12, 2004). The SES-CD consists of four endoscopic variables (size of ulcers, proportion of surface covered by ulcers, proportion of surface with any other lesions (e.g., inflammation), and presence of narrowings [stenosis]) that are scored in five ileocolonic segments, with each variable, or assessment, rated from 0 to 3. To date, there is no cure for CD. Accordingly, the current treatment goals for CD are to induce and maintain symptom improvement, induce mucosal healing, avoid surgery, and improve quality of life (Lichtenstein G R, et al., Am J Gastroenterol 104:465-83, 2009; Van Assche G, et al., J Crohns Colitis. 4:63-101, 2010). The current therapy of IBD usually involves the administration of antiinflammatory or immunosuppressive agents, such as sulfasalazine, corticosteroids, 6-mercaptopurine/azathioprine, or cyclosporine, all of which are not typically delivered by localized release of a drug at the site or location of disease. More recently, biologics like TNF-alpha inhibitors and IL-12/IL-23 blockers, are used to treat IBD. If anti-inflammatory/immunosuppressive/biologic therapies fail, colectomies are the last line of defense. The typical operation for CD not involving the rectum is resection (removal of a diseased segment of bowel) and anastomosis (reconnection) without an ostomy. Sections of the small or large intestine may be removed. About 30% of CD patients will need surgery within the first year after diagnosis. In the subsequent years, the rate is about 5% per year. Unfortunately, CD is characterized by a high rate of recurrence; about 5% of patients need a second surgery each year after initial surgery. Refining a diagnosis of inflammatory bowel disease involves evaluating the progression status of the diseases using standard classification criteria. The classification systems used in IBD include the Truelove and Witts Index (Truelove S. C. and Witts, L. J. Br Med J. 1955; 2: 1041-1048), which classifies colitis as mild, moderate, or severe, as well as Lennard-Jones. (Lennard-Jones J E. Scand J Gastroenterol Suppl 1989; 170:2-6) and the simple clinical colitis activity index (SCCAI). (Walmsley et. al. Gut. 1998; 43:29-32) These systems track such variables as daily bowel movements, rectal bleeding, temperature, heart rate, hemoglobin levels, erythrocyte sedimentation rate, weight, hematocrit score, and the level of serum albumin. There is sufficient overlap in the diagnostic criteria for UC and CD that it is sometimes impossible to say which a given patient has; however, the type of lesion typically seen is different, as is the localization. UC mostly appears in the colon, proximal to the rectum, and the characteristic lesion is a superficial ulcer of the mucosa; CD can appear anywhere in the bowel, with occasional involvement of stomach, esophagus and duodenum, and the lesions are usually described as extensive linear fissures. In approximately 10-15% of cases, a definitive diagnosis of ulcerative colitis or Crohn's disease cannot be made and such cases are often referred to as “indeterminate colitis.” Two antibody detection tests are available that can help the diagnosis, each of which assays for antibodies in the blood. The antibodies are “perinuclear anti-neutrophil antibody” (pANCA) and “anti-Saccharomyces cervisiaeantibody” (ASCA). Most patients with ulcerative colitis have the pANCA antibody but not the ASCA antibody, while most patients with Crohn's disease have the ASCA antibody but not the pANCA antibody. However, these two tests have shortcomings as some patients have neither antibody and some Crohn's disease patients may have only the pANCA antibody. A third test, which measures the presence and accumulation of circulating anti-microbial antibodies—particularly flagellin antibodies, has proven to be useful for detecting susceptibility to Crohn's Disease before disease development. See Choung, R. S., et al. “Serologic microbial associated markers can predict Crohn's disease behaviour years before disease diagnosis.” Alimentary pharmacology & therapeutics 43.12 (2016): 1300-1310. “Ulcerative colitis (UC)” afflicts the large intestine. The course of the disease may be continuous or relapsing, mild or severe. The earliest lesion is an inflammatory infiltration with abscess formation at the base of the crypts of Lieberkuhn. Coalescence of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply, leading to ulceration. Symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose discharges consisting mainly of blood, pus and mucus with scanty fecal particles. A total colectomy may be required for acute, severe or chronic, unremitting ulcerative colitis. The clinical features of UC are highly variable, and the onset may be insidious or abrupt, and may include diarrhea, tenesmus and relapsing rectal bleeding. With fulminant involvement of the entire colon, toxic megacolon, a life-threatening emergency, may occur. Extraintestinal manifestations include arthritis, pyoderma gangrenoum, uveitis, and erythema nodosum. The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (for example, full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific, trispecific etc. antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be human, humanized and/or affinity matured. “Antibody fragments” comprise only a portion of an intact antibody, where in certain embodiments, the portion retains at least one, and typically most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). “Treatment regimen” refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication. “Effective treatment regimen” refers to a treatment regimen that will offer beneficial response to a patient receiving the treatment. “Effective amount” refers to an amount of drug that offers beneficial response to a patient receiving the treatment. For example, an effective amount may be a Human Equivalent Dose (HED). “Dispensable”, with reference to any substance, refers to any substance that may be released from an ingestible device as disclosed herein, or from a component of the device such as a reservoir. For example, a dispensable substance may be an JAK inhibitor, and/or a formulation comprising an JAK inhibitor. “Patient response” or “patient responsiveness” can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of disease progression, including slowing down and complete arrest; (2) reduction in the number of disease episodes and/or symptoms; (3) reduction in lesional size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing down or complete stopping) of disease spread; (6) decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; (7) relief, to some extent, of one or more symptoms associated with the disorder; (8) increase in the length of disease-free presentation following treatment; and/or (9) decreased mortality at a given point of time following treatment. The term “responsiveness” refers to a measurable response, including complete response (CR) and partial response (PR). As used herein, “complete response” or “CR” means the disappearance of all signs of inflammation or remission in response to treatment. This does not necessarily mean the disease has been cured. “Partial response” or “PR” refers to a decrease of at least 50% in the severity of inflammation, in response to treatment. A “beneficial response” of a patient to treatment with a therapeutic agent and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for or suffering from a gastrointestinal inflammatory disorder from or as a result of the treatment with the agent. Such benefit includes cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from or as a result of the treatment with the agent. As used herein, “non-response” or “lack of response” or similar wording means an absence of a complete response, a partial response, or a beneficial response to treatment with a therapeutic agent. “A patient maintains responsiveness to a treatment” when the patient's responsiveness does not decrease with time during the course of a treatment. A “symptom” of a disease or disorder (e.g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease) is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease. Inhibitory Agents of Janus Kinase (JAK) Activity and/or Expression The term “JAK inhibitor” refers to an agent which decreases the expression of Janus kinase 1 (JAK1), JAK2, JAK3, or non-receptor protein tyrosine kinase 2 (TYK-2) and/or the kinase activity of at least one of JAK1, JAK2, JAK3, and TYK-2. In some embodiments, the JAK inhibitor decreases the expression of JAK1. In some embodiments, the JAK inhibitor decreases the expression of JAK2. In some embodiments, the JAK inhibitor decreases the expression of JAK3. In some embodiments, the JAK inhibitor decreases the expression of TYK-2. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK1. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK2. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK3. In some embodiments, the JAK inhibitor decreases the kinase activity of TYK-2. In some embodiments, the JAK inhibitor is a decreases the kinase activity of JAK1, JAK2, JAK3, and TYK2. In some embodiments, the JAK inhibitor decreases the kinase activity of two or more (e.g., 3 or 4) of: JAK1, JAK2, JAK3 and TYK2. In some embodiments, the JAK inhibitor decreases the kinase activity of a single JAK isoform (e.g., JAK1, JAK2, JAK3, or TYK2). In some embodiments, the JAK inhibitor decreases the kinase activity of JAK1 and JAK2. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK1 and JAK3. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK2 and JAK3. In some embodiments, the JAK inhibitor decreases the kinase activity of JAK1, JAK2 and JAK3. In some embodiments, a JAK inhibitory agent is an inhibitory nucleic acid or a small molecule. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid, a ribozyme, a small interfering RNA, a small hairpin RNA, or a microRNA. Examples of aspects of these different inhibitory nucleic acids are described below. Any of the examples of inhibitory nucleic acids that can decrease expression of a JAK1, JAK2, JAK3, or TYK2 mRNA in a mammalian cell can be synthesized in vitro. Inhibitory nucleic acids that can decrease the expression of JAK1, JAK2, JAK3, or TYK2 mRNA expression in a mammalian cell include antisense nucleic acid molecules, i.e., nucleic acid molecules whose nucleotide sequence is complementary to all or part of a JAK1, JAK2, JAK3, or TYK2 mRNA (e.g., complementary to all or a part of any one of SEQ ID NOs: 1-15). Human JAK1 mRNA Variant 1(SEQ ID NO: 1)1ggggcgggac gggaggcggt gcgtcgctga gcgcaggccg cggcggccgc ggagtatcct61ggagctgcag acagtgcggg cctgcgccca gtcccggctg tcctcgccgc gacccctcct121cagccctggg cgcgcgcacg ctggggcccc gcggggctgg ccgcctagcg agcctgccgg181tcgaccccag ccagcgcagc gacggggcgc tgcctggccc aggcgcacac ggaagtgcgc241ttctctgaag tagctttgga aagtagagaa gaaaatccag tttgcttctt ggagaacact301ggacagctga ataaatgcag tatctaaata taaaagagga ctgcaatgcc atggctttct361gtgctaaaat gaggagctcc aagaagactg aggtgaacct ggaggcccct gagccagggg421tggaagtgat cttctatctg tcggacaggg agcccctccg gctgggcagt ggagagtaca481cagcagagga actgtgcatc agggctgcac aggcatgccg tatctctcct ctttgtcaca541acctctttgc cctgtatgac gagaacacca agctctggta tgctccaaat cgcaccatca601ccgttgatga caagatgtcc ctccggctcc actaccggat gaggttctat ttcaccaatt661ggcatggaac caacgacaat gagcagtcag tgtggcgtca ttctccaaag aagcagaaaa721atggctacga gaaaaaaaag attccagatg caacccctct ccttgatgcc agctcactgg781agtatctgtt tgctcaggga cagtatgatt tggtgaaatg cctggctcct attcgagacc841ccaagaccga gcaggatgga catgatattg agaacgagtg tctagggatg gctgtcctgg901ccatctcaca ctatgccatg atgaagaaga tgcagttgcc agaactgccc aaggacatca961gctacaagcg atatattcca gaaacattga ataagtccat cagacagagg aaccttctca1021ccaggatgcg gataaataat gttttcaagg atttcctaaa ggaatttaac aacaagacca1081tttgtgacag cagcgtgtcc acgcatgacc tgaaggtgaa atacttggct accttggaaa1141ctttgacaaa acattacggt gctgaaatat ttgagacttc catgttactg atttcatcag1201aaaatgagat gaattggttt cattcgaatg acggtggaaa cgttctctac tacgaagtga1261tggtgactgg gaatcttgga atccagtgga ggcataaacc aaatgttgtt tctgttgaaa1321aggaaaaaaa taaactgaag cggaaaaaac tggaaaataa acacaagaag gatgaggaga1381aaaacaagat ccgggaagag tggaacaatt tttcttactt ccctgaaatc actcacattg1441taataaagga gtctgtggtc agcattaaca agcaggacaa caagaaaatg gaactgaagc1501tctcttccca cgaggaggcc ttgtcctttg tgtccctggt agatggctac ttccggctca1561cagcagatgc ccatcattac ctctgcaccg acgtggcccc cccgttgatc gtccacaaca1621tacagaatgg ctgtcatggt ccaatctgta cagaatacgc catcaataaa ttgcggcaag1681aaggaagcga ggaggggatg tacgtgctga ggtggagctg caccgacttt gacaacatcc1741tcatgaccgt cacctgcttt gagaagtctg agcaggtgca gggtgcccag aagcagttca1801agaactttca gatcgaggtg cagaagggcc gctacagtct gcacggttcg gaccgcagct1861tccccagctt gggagacctc atgagccacc tcaagaagca gatcctgcgc acggataaca1921tcagcttcat gctaaaacgc tgctgccagc ccaagccccg agaaatctcc aacctgctgg1981tggctactaa gaaagcccag gagtggcagc ccgtctaccc catgagccag ctgagtttcg2041atcggatcct caagaaggat ctggtgcagg gcgagcacct tgggagaggc acgagaacac2101acatctattc tgggaccctg atggattaca aggatgacga aggaacttct gaagagaaga2161agataaaagt gatcctcaaa gtcttagacc ccagccacag ggatatttcc ctggccttct2221tcgaggcagc cagcatgatg agacaggtct cccacaaaca catcgtgtac ctctatggcg2281tctgtgtccg cgacgtggag aatatcatgg tggaagagtt tgtggaaggg ggtcctctgg2341atctcttcat gcaccggaaa agcgatgtcc ttaccacacc atggaaattc aaagttgcca2401aacagctggc cagtgccctg agctacttgg aggataaaga cctggtccat ggaaatgtgt2461gtactaaaaa cctcctcctg gcccgtgagg gcatcgacag tgagtgtggc ccattcatca2521agctcagtga ccccggcatc cccattacgg tgctgtctag gcaagaatgc attgaacgaa2581tcccatggat tgctcctgag tgtgttgagg actccaagaa cctgagtgtg gctgctgaca2641agtggagctt tggaaccacg ctctgggaaa tctgctacaa tggcgagatc cccttgaaag2701acaagacgct gattgagaaa gagagattct atgaaagccg gtgcaggcca gtgacaccat2761catgtaagga gctggctgac ctcatgaccc gctgcatgaa ctatgacccc aatcagaggc2821ctttcttccg agccatcatg agagacatta ataagcttga agagcagaat ccagatattg2881tttcagaaaa aaaaccagca actgaagtgg accccacaca ttttgaaaag cgcttcctaa2941agaggatccg tgacttggga gagggccact ttgggaaggt tgagctctgc aggtatgacc3001ccgaagggga caatacaggg gagcaggtgg ctgttaaatc tctgaagcct gagagtggag3061gtaaccacat agctgatctg aaaaaggaaa tcgagatctt aaggaacctc tatcatgaga3121acattgtgaa gtacaaagga atctgcacag aagacggagg aaatggtatt aagctcatca3181tggaatttct gccttcggga agccttaagg aatatcttcc aaagaataag aacaaaataa3241acctcaaaca gcagctaaaa tatgccgttc agatttgtaa ggggatggac tatttgggtt3301ctcggcaata cgttcaccgg gacttggcag caagaaatgt ccttgttgag agtgaacacc3361aagtgaaaat tggagacttc ggtttaacca aagcaattga aaccgataag gagtattaca3421ccgtcaagga tgaccgggac agccctgtgt tttggtatgc tccagaatgt ttaatgcaat3481ctaaatttta tattgcctct gacgtctggt cttttggagt cactctgcat gagctgctga3541cttactgtga ttcagattct agtcccatgg ctttgttcct gaaaatgata ggcccaaccc3601atggccagat gacagtcaca agacttgtga atacgttaaa agaaggaaaa cgcctgccgt3661gcccacctaa ctgtccagat gaggtttatc aacttatgag gaaatgctgg gaattccaac3721catccaatcg gacaagcttt cagaacctta ttgaaggatt tgaagcactt ttaaaataag3781aagcatgaat aacatttaaa ttccacagat tatcaagtcc ttctcctgca acaaatgccc3841aagtcatttt ttaaaaattt ctaatgaaag aagtttgtgt tctgtccaaa aagtcactga3901actcatactt cagtacatat acatgtataa ggcacactgt agtgcttaat atgtgtaagg3961acttcctctt taaatttggt accagtaact tagtgacaca taatgacaac caaaatattt4021gaaagcactt aagcactcct ccttgtggaa agaatatacc accatttcat ctggctagtt4081caccatcaca actgcattac caaaagggga tttttgaaaa cgaggagttg accaaaataa4141tatctgaaga tgattgcttt tccctgctgc cagctgatct gaaatgtttt gctggcacat4201taatcataga taaagaaaga ttgatggact tagccctcaa atttcagtat ctatacagta4261ctagaccatg cattcttaaa atattagata ccaggtagta tatattgttt ctgtacaaaa4321atgactgtat tctctcacca gtaggactta aactttgttt ctccagtggc ttagctcctg4381ttcctttggg tgatcactag cacccatttt tgagaaagct ggttctacat ggggggatag4441ctgtggaata gataatttgc tgcatgttaa ttctcaagaa ctaagcctgt gccagtgctt4501tcctaagcag tataccttta atcagaactc attcccagaa cctggatgct attacacatg4561cttttaagaa acgtcaatgt atatcctttt ataactctac cactttgggg caagctattc4621cagcactggt tttgaatgct gtatgcaacc agtctgaata ccacatacgc tgcactgttc4681ttagagggtt tccatactta ccaccgatct acaagggttg atccctgttt ttaccatcaa4741tcatcaccct gtggtgcaac acttgaaaga cccggctaga ggcactatgg acttcaggat4801ccactagaca gttttcagtt tgcttggagg tagctgggta atcaaaaatg tttagtcatt4861gattcaatgt gaacgattac ggtctttatg accaagagtc tgaaaatctt tttgttatgc4921tgtttagtat tcgtttgata ttgttacttt tcacctgttg agcccaaatt caggattggt4981tcagtggcag caatgaagtt gccatttaaa tttgttcata gcctacatca ccaaggtctc5041tgtgtcaaac ctgtggccac tctatatgca ctttgtttac tctttataca aataaatata5101ctaaagactt tacatgcaHuman JAK1 mRNA Variant 2(SEQ ID NO: 2)1agaagcggag cgtatacgga ggaggcggga tgcatttctg catcgagcgc acaaagttat61ctaaaacagt tcatgctgct gaaaacctcc ttcctggcag atgtccctca accctactgg121tgcctggctt ctgagacaca cgcttctctg aagtagcttt ggaaagtaga gaagaaaatc181cagtttgctt cttggagaac actggacagc tgaataaatg cagtatctaa atataaaaga241ggactgcaat gccatggctt tctgtgctaa aatgaggagc tccaagaaga ctgaggtgaa301cctggaggcc cctgagccag gggtggaagt gatcttctat ctgtcggaca gggagcccct361ccggctgggc agtggagagt acacagcaga ggaactgtgc atcagggctg cacaggcatg421ccgtatctct cctctttgtc acaacctctt tgccctgtat gacgagaaca ccaagctctg481gtatgctcca aatcgcacca tcaccgttga tgacaagatg tccctccggc tccactaccg541gatgaggttc tatttcacca attggcatgg aaccaacgac aatgagcagt cagtgtggcg601tcattctcca aagaagcaga aaaatggcta cgagaaaaaa aagattccag atgcaacccc661tctccttgat gccagctcac tggagtatct gtttgctcag ggacagtatg atttggtgaa721atgcctggct cctattcgag accccaagac cgagcaggat ggacatgata ttgagaacga781gtgtctaggg atggctgtcc tggccatctc acactatgcc atgatgaaga agatgcagtt841gccagaactg cccaaggaca tcagctacaa gcgatatatt ccagaaacat tgaataagtc901catcagacag aggaaccttc tcaccaggat gcggataaat aatgttttca aggatttcct961aaaggaattt aacaacaaga ccatttgtga cagcagcgtg tccacgcatg acctgaaggt1021gaaatacttg gctaccttgg aaactttgac aaaacattac ggtgctgaaa tatttgagac1081ttccatgtta ctgatttcat cagaaaatga gatgaattgg tttcattcga atgacggtgg1141aaacgttctc tactacgaag tgatggtgac tgggaatctt ggaatccagt ggaggcataa1201accaaatgtt gtttctgttg aaaaggaaaa aaataaactg aagcggaaaa aactggaaaa1261taaacacaag aaggatgagg agaaaaacaa gatccgggaa gagtggaaca atttttctta1321cttccctgaa atcactcaca ttgtaataaa ggagtctgtg gtcagcatta acaagcagga1381caacaagaaa atggaactga agctctcttc ccacgaggag gccttgtcct ttgtgtccct1441ggtagatggc tacttccggc tcacagcaga tgcccatcat tacctctgca ccgacgtggc1501ccccccgttg atcgtccaca acatacagaa tggctgtcat ggtccaatct gtacagaata1561cgccatcaat aaattgcggc aagaaggaag cgaggagggg atgtacgtgc tgaggtggag1621ctgcaccgac tttgacaaca tcctcatgac cgtcacctgc tttgagaagt ctgagcaggt1681gcagggtgcc cagaagcagt tcaagaactt tcagatcgag gtgcagaagg gccgctacag1741tctgcacggt tcggaccgca gcttccccag cttgggagac ctcatgagcc acctcaagaa1801gcagatcctg cgcacggata acatcagctt catgctaaaa cgctgctgcc agcccaagcc1861ccgagaaatc tccaacctgc tggtggctac taagaaagcc caggagtggc agcccgtcta1921ccccatgagc cagctgagtt tcgatcggat cctcaagaag gatctggtgc agggcgagca1981ccttgggaga ggcacgagaa cacacatcta ttctgggacc ctgatggatt acaaggatga2041cgaaggaact tctgaagaga agaagataaa agtgatcctc aaagtcttag accccagcca2101cagggatatt tccctggcct tcttcgaggc agccagcatg atgagacagg tctcccacaa2161acacatcgtg tacctctatg gcgtctgtgt ccgcgacgtg gagaatatca tggtggaaga2221gtttgtggaa gggggtcctc tggatctctt catgcaccgg aaaagcgatg tccttaccac2281accatggaaa ttcaaagttg ccaaacagct ggccagtgcc ctgagctact tggaggataa2341agacctggtc catggaaatg tgtgtactaa aaacctcctc ctggcccgtg agggcatcga2401cagtgagtgt ggcccattca tcaagctcag tgaccccggc atccccatta cggtgctgtc2461taggcaagaa tgcattgaac gaatcccatg gattgctcct gagtgtgttg aggactccaa2521gaacctgagt gtggctgctg acaagtggag ctttggaacc acgctctggg aaatctgcta2581caatggcgag atccccttga aagacaagac gctgattgag aaagagagat tctatgaaag2641ccggtgcagg ccagtgacac catcatgtaa ggagctggct gacctcatga cccgctgcat2701gaactatgac cccaatcaga ggcctttctt ccgagccatc atgagagaca ttaataagct2761tgaagagcag aatccagata ttgtttcaga aaaaaaacca gcaactgaag tggaccccac2821acattttgaa aagcgcttcc taaagaggat ccgtgacttg ggagagggcc actttgggaa2881ggttgagctc tgcaggtatg accccgaagg ggacaataca ggggagcagg tggctgttaa2941atctctgaag cctgagagtg gaggtaacca catagctgat ctgaaaaagg aaatcgagat3001cttaaggaac ctctatcatg agaacattgt gaagtacaaa ggaatctgca cagaagacgg3061aggaaatggt attaagctca tcatggaatt tctgccttcg ggaagcctta aggaatatct3121tccaaagaat aagaacaaaa taaacctcaa acagcagcta aaatatgccg ttcagatttg3181taaggggatg gactatttgg gttctcggca atacgttcac cgggacttgg cagcaagaaa3241tgtccttgtt gagagtgaac accaagtgaa aattggagac ttcggtttaa ccaaagcaat3301tgaaaccgat aaggagtatt acaccgtcaa ggatgaccgg gacagccctg tgttttggta3361tgctccagaa tgtttaatgc aatctaaatt ttatattgcc tctgacgtct ggtcttttgg3421agtcactctg catgagctgc tgacttactg tgattcagat tctagtccca tggctttgtt3481cctgaaaatg ataggcccaa cccatggcca gatgacagtc acaagacttg tgaatacgtt3541aaaagaagga aaacgcctgc cgtgcccacc taactgtcca gatgaggttt atcaacttat3601gaggaaatgc tgggaattcc aaccatccaa tcggacaagc tttcagaacc ttattgaagg3661atttgaagca cttttaaaat aagaagcatg aataacattt aaattccaca gattatcaag3721tccttctcct gcaacaaatg cccaagtcat tttttaaaaa tttctaatga aagaagtttg3781tgttctgtcc aaaaagtcac tgaactcata cttcagtaca tatacatgta taaggcacac3841tgtagtgctt aatatgtgta aggacttcct ctttaaattt ggtaccagta acttagtgac3901acataatgac aaccaaaata tttgaaagca cttaagcact cctccttgtg gaaagaatat3961accaccattt catctggcta gttcaccatc acaactgcat taccaaaagg ggatttttga4021aaacgaggag ttgaccaaaa taatatctga agatgattgc ttttccctgc tgccagctga4081tctgaaatgt tttgctggca cattaatcat agataaagaa agattgatgg acttagccct4141caaatttcag tatctataca gtactagacc atgcattctt aaaatattag ataccaggta4201gtatatattg tttctgtaca aaaatgactg tattctctca ccagtaggac ttaaactttg4261tttctccagt ggcttagctc ctgttccttt gggtgatcac tagcacccat ttttgagaaa4321gctggttcta catgggggga tagctgtgga atagataatt tgctgcatgt taattctcaa4381gaactaagcc tgtgccagtg ctttcctaag cagtatacct ttaatcagaa ctcattccca4441gaacctggat gctattacac atgcttttaa gaaacgtcaa tgtatatcct tttataactc4501taccactttg gggcaagcta ttccagcact ggttttgaat gctgtatgca accagtctga4561ataccacata cgctgcactg ttcttagagg gtttccatac ttaccaccga tctacaaggg4621ttgatccctg tttttaccat caatcatcac cctgtggtgc aacacttgaa agacccggct4681agaggcacta tggacttcag gatccactag acagttttca gtttgcttgg aggtagctgg4741gtaatcaaaa atgtttagtc attgattcaa tgtgaacgat tacggtcttt atgaccaaga4801gtctgaaaat ctttttgtta tgctgtttag tattcgtttg atattgttac ttttcacctg4861ttgagcccaa attcaggatt ggttcagtgg cagcaatgaa gttgccattt aaatttgttc4921atagcctaca tcaccaaggt ctctgtgtca aacctgtggc cactctatat gcactttgtt4981tactctttat acaaataaat atactaaaga ctttacatgc aHuman JAK1 mRNA Variant 3(SEQ ID NO: 3)1atctatcaca tggcagagat agaataaaaa cagaaaaatg gcgacggtca cgttgtggcg61agccttgctg cgtcattaga taatcctcat gcaaatagcg ggaagaacaa aggaagggga121gcccgggacc cccgggggcg cagcgcttct ctgaagtagc tttggaaagt agagaagaaa181atccagtttg cttcttggag aacactggac agctgaataa atgcagtatc taaatataaa241agaggactgc aatgccatgg ctttctgtgc taaaatgagg agctccaaga agactgaggt301gaacctggag gcccctgagc caggggtgga agtgatcttc tatctgtcgg acagggagcc361cctccggctg ggcagtggag agtacacagc agaggaactg tgcatcaggg ctgcacaggc421atgccgtatc tctcctcttt gtcacaacct ctttgccctg tatgacgaga acaccaagct481ctggtatgct ccaaatcgca ccatcaccgt tgatgacaag atgtccctcc ggctccacta541ccggatgagg ttctatttca ccaattggca tggaaccaac gacaatgagc agtcagtgtg601gcgtcattct ccaaagaagc agaaaaatgg ctacgagaaa aaaaagattc cagatgcaac661ccctctcctt gatgccagct cactggagta tctgtttgct cagggacagt atgatttggt721gaaatgcctg gctcctattc gagaccccaa gaccgagcag gatggacatg atattgagaa781cgagtgtcta gggatggctg tcctggccat ctcacactat gccatgatga agaagatgca841gttgccagaa ctgcccaagg acatcagcta caagcgatat attccagaaa cattgaataa901gtccatcaga cagaggaacc ttctcaccag gatgcggata aataatgttt tcaaggattt961cctaaaggaa tttaacaaca agaccatttg tgacagcagc gtgtccacgc atgacctgaa1021ggtgaaatac ttggctacct tggaaacttt gacaaaacat tacggtgctg aaatatttga1081gacttccatg ttactgattt catcagaaaa tgagatgaat tggtttcatt cgaatgacgg1141tggaaacgtt ctctactacg aagtgatggt gactgggaat cttggaatcc agtggaggca1201taaaccaaat gttgtttctg ttgaaaagga aaaaaataaa ctgaagcgga aaaaactgga1261aaataaacac aagaaggatg aggagaaaaa caagatccgg gaagagtgga acaatttttc1321ttacttccct gaaatcactc acattgtaat aaaggagtct gtggtcagca ttaacaagca1381ggacaacaag aaaatggaac tgaagctctc ttcccacgag gaggccttgt cctttgtgtc1441cctggtagat ggctacttcc ggctcacagc agatgcccat cattacctct gcaccgacgt1501ggcccccccg ttgatcgtcc acaacataca gaatggctgt catggtccaa tctgtacaga1561atacgccatc aataaattgc ggcaagaagg aagcgaggag gggatgtacg tgctgaggtg1621gagctgcacc gactttgaca acatcctcat gaccgtcacc tgctttgaga agtctgagca1681ggtgcagggt gcccagaagc agttcaagaa ctttcagatc gaggtgcaga agggccgcta1741cagtctgcac ggttcggacc gcagcttccc cagcttggga gacctcatga gccacctcaa1801gaagcagatc ctgcgcacgg ataacatcag cttcatgcta aaacgctgct gccagcccaa1861gccccgagaa atctccaacc tgctggtggc tactaagaaa gcccaggagt ggcagcccgt1921ctaccccatg agccagctga gtttcgatcg gatcctcaag aaggatctgg tgcagggcga1981gcaccttggg agaggcacga gaacacacat ctattctggg accctgatgg attacaagga2041tgacgaagga acttctgaag agaagaagat aaaagtgatc ctcaaagtct tagaccccag2101ccacagggat atttccctgg ccttcttcga ggcagccagc atgatgagac aggtctccca2161caaacacatc gtgtacctct atggcgtctg tgtccgcgac gtggagaata tcatggtgga2221agagtttgtg gaagggggtc ctctggatct cttcatgcac cggaaaagcg atgtccttac2281cacaccatgg aaattcaaag ttgccaaaca gctggccagt gccctgagct acttggagga2341taaagacctg gtccatggaa atgtgtgtac taaaaacctc ctcctggccc gtgagggcat2401cgacagtgag tgtggcccat tcatcaagct cagtgacccc ggcatcccca ttacggtgct2461gtctaggcaa gaatgcattg aacgaatccc atggattgct cctgagtgtg ttgaggactc2521caagaacctg agtgtggctg ctgacaagtg gagctttgga accacgctct gggaaatctg2581ctacaatggc gagatcccct tgaaagacaa gacgctgatt gagaaagaga gattctatga2641aagccggtgc aggccagtga caccatcatg taaggagctg gctgacctca tgacccgctg2701catgaactat gaccccaatc agaggccttt cttccgagcc atcatgagag acattaataa2761gcttgaagag cagaatccag atattgtttc agaaaaaaaa ccagcaactg aagtggaccc2821cacacatttt gaaaagcgct tcctaaagag gatccgtgac ttgggagagg gccactttgg2881gaaggttgag ctctgcaggt atgaccccga aggggacaat acaggggagc aggtggctgt2941taaatctctg aagcctgaga gtggaggtaa ccacatagct gatctgaaaa aggaaatcga3001gatcttaagg aacctctatc atgagaacat tgtgaagtac aaaggaatct gcacagaaga3061cggaggaaat ggtattaagc tcatcatgga atttctgcct tcgggaagcc ttaaggaata3121tcttccaaag aataagaaca aaataaacct caaacagcag ctaaaatatg ccgttcagat3181ttgtaagggg atggactatt tgggttctcg gcaatacgtt caccgggact tggcagcaag3241aaatgtcctt gttgagagtg aacaccaagt gaaaattgga gacttcggtt taaccaaagc3301aattgaaacc gataaggagt attacaccgt caaggatgac cgggacagcc ctgtgttttg3361gtatgctcca gaatgtttaa tgcaatctaa attttatatt gcctctgacg tctggtcttt3421tggagtcact ctgcatgagc tgctgactta ctgtgattca gattctagtc ccatggcttt3481gttcctgaaa atgataggcc caacccatgg ccagatgaca gtcacaagac ttgtgaatac3541gttaaaagaa ggaaaacgcc tgccgtgccc acctaactgt ccagatgagg tttatcaact3601tatgaggaaa tgctgggaat tccaaccatc caatcggaca agctttcaga accttattga3661aggatttgaa gcacttttaa aataagaagc atgaataaca tttaaattcc acagattatc3721aagtccttct cctgcaacaa atgcccaagt cattttttaa aaatttctaa tgaaagaagt3781ttgtgttctg tccaaaaagt cactgaactc atacttcagt acatatacat gtataaggca3841cactgtagtg cttaatatgt gtaaggactt cctctttaaa tttggtacca gtaacttagt3901gacacataat gacaaccaaa atatttgaaa gcacttaagc actcctcctt gtggaaagaa3961tataccacca tttcatctgg ctagttcacc atcacaactg cattaccaaa aggggatttt4021tgaaaacgag gagttgacca aaataatatc tgaagatgat tgcttttccc tgctgccagc4081tgatctgaaa tgttttgctg gcacattaat catagataaa gaaagattga tggacttagc4141cctcaaattt cagtatctat acagtactag accatgcatt cttaaaatat tagataccag4201gtagtatata ttgtttctgt acaaaaatga ctgtattctc tcaccagtag gacttaaact4261ttgtttctcc agtggcttag ctcctgttcc tttgggtgat cactagcacc catttttgag4321aaagctggtt ctacatgggg ggatagctgt ggaatagata atttgctgca tgttaattct4381caagaactaa gcctgtgcca gtgctttcct aagcagtata cctttaatca gaactcattc4441ccagaacctg gatgctatta cacatgcttt taagaaacgt caatgtatat ccttttataa4501ctctaccact ttggggcaag ctattccagc actggttttg aatgctgtat gcaaccagtc4561tgaataccac atacgctgca ctgttcttag agggtttcca tacttaccac cgatctacaa4621gggttgatcc ctgtttttac catcaatcat caccctgtgg tgcaacactt gaaagacccg4681gctagaggca ctatggactt caggatccac tagacagttt tcagtttgct tggaggtagc4741tgggtaatca aaaatgttta gtcattgatt caatgtgaac gattacggtc tttatgacca4801agagtctgaa aatctttttg ttatgctgtt tagtattcgt ttgatattgt tacttttcac4861ctgttgagcc caaattcagg attggttcag tggcagcaat gaagttgcca tttaaatttg4921ttcatagcct acatcaccaa ggtctctgtg tcaaacctgt ggccactcta tatgcacttt4981gtttactctt tatacaaata aatatactaa agactttaca tgcaHuman JAK1 mRNA Variant 4(SEQ ID NO: 4)1atctatcaca tggcagagat agaataaaaa cagaaaaatg gcgacggtca cgttgtggcg61agccttgctg cgtcattaga taatcctcat gcaaatagcg ggaagaacaa aggaagggga121gcccgggacc cccgggggcg caggatccgg cgggaggagt ctaagaggag gaggcggcgg181tgccggagga ggaggaggag ggagggagaa gagaggaaga ccggagtccc cgcggcggcg241gcggtccgga gagagggcga gccccgcgcg gcgccgggga ccgggcgcta ccacgaggcc301gggacgctgg agtctgggtt atctaaaaca gttcatgctg ctgaaaacct ccttcctggc361agatgtccct caaccctact ggtgcctggc ttctgagaca cacgcttctc tgaagtagct421ttggaaagta gagaagaaaa tccagtttgc ttcttggaga acactggaca gctgaataaa481tgcagtatct aaatataaaa gaggactgca atgccatggc tttctgtgct aaaatgagga541gctccaagaa gactgaggtg aacctggagg cccctgagcc aggggtggaa gtgatcttct601atctgtcgga cagggagccc ctccggctgg gcagtggaga gtacacagca gaggaactgt661gcatcagggc tgcacaggca tgccgtatct ctcctctttg tcacaacctc tttgccctgt721atgacgagaa caccaagctc tggtatgctc caaatcgcac catcaccgtt gatgacaaga781tgtccctccg gctccactac cggatgaggt tctatttcac caattggcat ggaaccaacg841acaatgagca gtcagtgtgg cgtcattctc caaagaagca gaaaaatggc tacgagaaaa901aaaagattcc agatgcaacc cctctccttg atgccagctc actggagtat ctgtttgctc961agggacagta tgatttggtg aaatgcctgg ctcctattcg agaccccaag accgagcagg1021atggacatga tattgagaac gagtgtctag ggatggctgt cctggccatc tcacactatg1081ccatgatgaa gaagatgcag ttgccagaac tgcccaagga catcagctac aagcgatata1141ttccagaaac attgaataag tccatcagac agaggaacct tctcaccagg atgcggataa1201ataatgtttt caaggatttc ctaaaggaat ttaacaacaa gaccatttgt gacagcagcg1261tgtccacgca tgacctgaag gtgaaatact tggctacctt ggaaactttg acaaaacatt1321acggtgctga aatatttgag acttccatgt tactgatttc atcagaaaat gagatgaatt1381ggtttcattc gaatgacggt ggaaacgttc tctactacga agtgatggtg actgggaatc1441ttggaatcca gtggaggcat aaaccaaatg ttgtttctgt tgaaaaggaa aaaaataaac1501tgaagcggaa aaaactggaa aataaacaca agaaggatga ggagaaaaac aagatccggg1561aagagtggaa caatttttct tacttccctg aaatcactca cattgtaata aaggagtctg1621tggtcagcat taacaagcag gacaacaaga aaatggaact gaagctctct tcccacgagg1681aggccttgtc ctttgtgtcc ctggtagatg gctacttccg gctcacagca gatgcccatc1741attacctctg caccgacgtg gcccccccgt tgatcgtcca caacatacag aatggctgtc1801atggtccaat ctgtacagaa tacgccatca ataaattgcg gcaagaagga agcgaggagg1861ggatgtacgt gctgaggtgg agctgcaccg actttgacaa catcctcatg accgtcacct1921gctttgagaa gtctgagcag gtgcagggtg cccagaagca gttcaagaac tttcagatcg1981aggtgcagaa gggccgctac agtctgcacg gttcggaccg cagcttcccc agcttgggag2041acctcatgag ccacctcaag aagcagatcc tgcgcacgga taacatcagc ttcatgctaa2101aacgctgctg ccagcccaag ccccgagaaa tctccaacct gctggtggct actaagaaag2161cccaggagtg gcagcccgtc taccccatga gccagctgag tttcgatcgg atcctcaaga2221aggatctggt gcagggcgag caccttggga gaggcacgag aacacacatc tattctggga2281ccctgatgga ttacaaggat gacgaaggaa cttctgaaga gaagaagata aaagtgatcc2341tcaaagtctt agaccccagc cacagggata tttccctggc cttcttcgag gcagccagca2401tgatgagaca ggtctcccac aaacacatcg tgtacctcta tggcgtctgt gtccgcgacg2461tggagaatat catggtggaa gagtttgtgg aagggggtcc tctggatctc ttcatgcacc2521ggaaaagcga tgtccttacc acaccatgga aattcaaagt tgccaaacag ctggccagtg2581ccctgagcta cttggaggat aaagacctgg tccatggaaa tgtgtgtact aaaaacctcc2641tcctggcccg tgagggcatc gacagtgagt gtggcccatt catcaagctc agtgaccccg2701gcatccccat tacggtgctg tctaggcaag aatgcattga acgaatccca tggattgctc2761ctgagtgtgt tgaggactcc aagaacctga gtgtggctgc tgacaagtgg agctttggaa2821ccacgctctg ggaaatctgc tacaatggcg agatcccctt gaaagacaag acgctgattg2881agaaagagag attctatgaa agccggtgca ggccagtgac accatcatgt aaggagctgg2941ctgacctcat gacccgctgc atgaactatg accccaatca gaggcctttc ttccgagcca3001tcatgagaga cattaataag cttgaagagc agaatccaga tattgtttca gaaaaaaaac3061cagcaactga agtggacccc acacattttg aaaagcgctt cctaaagagg atccgtgact3121tgggagaggg ccactttggg aaggttgagc tctgcaggta tgaccccgaa ggggacaata3181caggggagca ggtggctgtt aaatctctga agcctgagag tggaggtaac cacatagctg3241atctgaaaaa ggaaatcgag atcttaagga acctctatca tgagaacatt gtgaagtaca3301aaggaatctg cacagaagac ggaggaaatg gtattaagct catcatggaa tttctgcctt3361cgggaagcct taaggaatat cttccaaaga ataagaacaa aataaacctc aaacagcagc3421taaaatatgc cgttcagatt tgtaagggga tggactattt gggttctcgg caatacgttc3481accgggactt ggcagcaaga aatgtccttg ttgagagtga acaccaagtg aaaattggag3541acttcggttt aaccaaagca attgaaaccg ataaggagta ttacaccgtc aaggatgacc3601gggacagccc tgtgttttgg tatgctccag aatgtttaat gcaatctaaa ttttatattg3661cctctgacgt ctggtctttt ggagtcactc tgcatgagct gctgacttac tgtgattcag3721attctagtcc catggctttg ttcctgaaaa tgataggccc aacccatggc cagatgacag3781tcacaagact tgtgaatacg ttaaaagaag gaaaacgcct gccgtgccca cctaactgtc3841cagatgaggt ttatcaactt atgaggaaat gctgggaatt ccaaccatcc aatcggacaa3901gctttcagaa ccttattgaa ggatttgaag cacttttaaa ataagaagca tgaataacat3961ttaaattcca cagattatca agtccttctc ctgcaacaaa tgcccaagtc attttttaaa4021aatttctaat gaaagaagtt tgtgttctgt ccaaaaagtc actgaactca tacttcagta4081catatacatg tataaggcac actgtagtgc ttaatatgtg taaggacttc ctctttaaat4141ttggtaccag taacttagtg acacataatg acaaccaaaa tatttgaaag cacttaagca4201ctcctccttg tggaaagaat ataccaccat ttcatctggc tagttcacca tcacaactgc4261attaccaaaa ggggattttt gaaaacgagg agttgaccaa aataatatct gaagatgatt4321gcttttccct gctgccagct gatctgaaat gttttgctgg cacattaatc atagataaag4381aaagattgat ggacttagcc ctcaaatttc agtatctata cagtactaga ccatgcattc4441ttaaaatatt agataccagg tagtatatat tgtttctgta caaaaatgac tgtattctct4501caccagtagg acttaaactt tgtttctcca gtggcttagc tcctgttcct ttgggtgatc4561actagcaccc atttttgaga aagctggttc tacatggggg gatagctgtg gaatagataa4621tttgctgcat gttaattctc aagaactaag cctgtgccag tgctttccta agcagtatac4681ctttaatcag aactcattcc cagaacctgg atgctattac acatgctttt aagaaacgtc4741aatgtatatc cttttataac tctaccactt tggggcaagc tattccagca ctggttttga4801atgctgtatg caaccagtct gaataccaca tacgctgcac tgttcttaga gggtttccat4861acttaccacc gatctacaag ggttgatccc tgtttttacc atcaatcatc accctgtggt4921gcaacacttg aaagacccgg ctagaggcac tatggacttc aggatccact agacagtttt4981cagtttgctt ggaggtagct gggtaatcaa aaatgtttag tcattgattc aatgtgaacg5041attacggtct ttatgaccaa gagtctgaaa atctttttgt tatgctgttt agtattcgtt5101tgatattgtt acttttcacc tgttgagccc aaattcagga ttggttcagt ggcagcaatg5161aagttgccat ttaaatttgt tcatagccta catcaccaag gtctctgtgt caaacctgtg5221gccactctat atgcactttg tttactcttt atacaaataa atatactaaa gactttacat5281gcaHuman JAK1 mRNA Variant 5(SEQ ID NO: 5)1atctatcaca tggcagagat agaataaaaa cagaaaaatg gcgacggtca cgttgtggcg61agccttgctg cgtcattaga taatcctcat gcaaatagcg ggaagaacaa aggaagggga121gcccgggacc cccgggggcg caggatccgg cgggaggagt ctaagaggag gaggcggcgg181tgccggagga ggaggaggag ggagggagaa gagaggaaga ccggagtccc cgcggcggcg241gcggtccgga gagagggcga gccccgcgcg gcgccgggga ccgggcgcta ccacgaggcc301gggacgctgg agtctgggcg cttctctgaa gtagctttgg aaagtagaga agaaaatcca361gtttgcttct tggagaacac tggacagctg aataaatgca gtatctaaat ataaaagagg421actgcaatgc catggctttc tgtgctaaaa tgaggagctc caagaagact gaggtgaacc481tggaggcccc tgagccaggg gtggaagtga tcttctatct gtcggacagg gagcccctcc541ggctgggcag tggagagtac acagcagagg aactgtgcat cagggctgca caggcatgcc601gtatctctcc tctttgtcac aacctctttg ccctgtatga cgagaacacc aagctctggt661atgctccaaa tcgcaccatc accgttgatg acaagatgtc cctccggctc cactaccgga721tgaggttcta tttcaccaat tggcatggaa ccaacgacaa tgagcagtca gtgtggcgtc781attctccaaa gaagcagaaa aatggctacg agaaaaaaaa gattccagat gcaacccctc841tccttgatgc cagctcactg gagtatctgt ttgctcaggg acagtatgat ttggtgaaat901gcctggctcc tattcgagac cccaagaccg agcaggatgg acatgatatt gagaacgagt961gtctagggat ggctgtcctg gccatctcac actatgccat gatgaagaag atgcagttgc1021cagaactgcc caaggacatc agctacaagc gatatattcc agaaacattg aataagtcca1081tcagacagag gaaccttctc accaggatgc ggataaataa tgttttcaag gatttcctaa1141aggaatttaa caacaagacc atttgtgaca gcagcgtgtc cacgcatgac ctgaaggtga1201aatacttggc taccttggaa actttgacaa aacattacgg tgctgaaata tttgagactt1261ccatgttact gatttcatca gaaaatgaga tgaattggtt tcattcgaat gacggtggaa1321acgttctcta ctacgaagtg atggtgactg ggaatcttgg aatccagtgg aggcataaac1381caaatgttgt ttctgttgaa aaggaaaaaa ataaactgaa gcggaaaaaa ctggaaaata1441aacacaagaa ggatgaggag aaaaacaaga tccgggaaga gtggaacaat ttttcttact1501tccctgaaat cactcacatt gtaataaagg agtctgtggt cagcattaac aagcaggaca1561acaagaaaat ggaactgaag ctctcttccc acgaggaggc cttgtccttt gtgtccctgg1621tagatggcta cttccggctc acagcagatg cccatcatta cctctgcacc gacgtggccc1681ccccgttgat cgtccacaac atacagaatg gctgtcatgg tccaatctgt acagaatacg1741ccatcaataa attgcggcaa gaaggaagcg aggaggggat gtacgtgctg aggtggagct1801gcaccgactt tgacaacatc ctcatgaccg tcacctgctt tgagaagtct gagcaggtgc1861agggtgccca gaagcagttc aagaactttc agatcgaggt gcagaagggc cgctacagtc1921tgcacggttc ggaccgcagc ttccccagct tgggagacct catgagccac ctcaagaagc1981agatcctgcg cacggataac atcagcttca tgctaaaacg ctgctgccag cccaagcccc2041gagaaatctc caacctgctg gtggctacta agaaagccca ggagtggcag cccgtctacc2101ccatgagcca gctgagtttc gatcggatcc tcaagaagga tctggtgcag ggcgagcacc2161ttgggagagg cacgagaaca cacatctatt ctgggaccct gatggattac aaggatgacg2221aaggaacttc tgaagagaag aagataaaag tgatcctcaa agtcttagac cccagccaca2281gggatatttc cctggccttc ttcgaggcag ccagcatgat gagacaggtc tcccacaaac2341acatcgtgta cctctatggc gtctgtgtcc gcgacgtgga gaatatcatg gtggaagagt2401ttgtggaagg gggtcctctg gatctcttca tgcaccggaa aagcgatgtc cttaccacac2461catggaaatt caaagttgcc aaacagctgg ccagtgccct gagctacttg gaggataaag2521acctggtcca tggaaatgtg tgtactaaaa acctcctcct ggcccgtgag ggcatcgaca2581gtgagtgtgg cccattcatc aagctcagtg accccggcat ccccattacg gtgctgtcta2641ggcaagaatg cattgaacga atcccatgga ttgctcctga gtgtgttgag gactccaaga2701acctgagtgt ggctgctgac aagtggagct ttggaaccac gctctgggaa atctgctaca2761atggcgagat ccccttgaaa gacaagacgc tgattgagaa agagagattc tatgaaagcc2821ggtgcaggcc agtgacacca tcatgtaagg agctggctga cctcatgacc cgctgcatga2881actatgaccc caatcagagg cctttcttcc gagccatcat gagagacatt aataagcttg2941aagagcagaa tccagatatt gtttcagaaa aaaaaccagc aactgaagtg gaccccacac3001attttgaaaa gcgcttccta aagaggatcc gtgacttggg agagggccac tttgggaagg3061ttgagctctg caggtatgac cccgaagggg acaatacagg ggagcaggtg gctgttaaat3121ctctgaagcc tgagagtgga ggtaaccaca tagctgatct gaaaaaggaa atcgagatct3181taaggaacct ctatcatgag aacattgtga agtacaaagg aatctgcaca gaagacggag3241gaaatggtat taagctcatc atggaatttc tgccttcggg aagccttaag gaatatcttc3301caaagaataa gaacaaaata aacctcaaac agcagctaaa atatgccgtt cagatttgta3361aggggatgga ctatttgggt tctcggcaat acgttcaccg ggacttggca gcaagaaatg3421tccttgttga gagtgaacac caagtgaaaa ttggagactt cggtttaacc aaagcaattg3481aaaccgataa ggagtattac accgtcaagg atgaccggga cagccctgtg ttttggtatg3541ctccagaatg tttaatgcaa tctaaatttt atattgcctc tgacgtctgg tcttttggag3601tcactctgca tgagctgctg acttactgtg attcagattc tagtcccatg gctttgttcc3661tgaaaatgat aggcccaacc catggccaga tgacagtcac aagacttgtg aatacgttaa3721aagaaggaaa acgcctgccg tgcccaccta actgtccaga tgaggtttat caacttatga3781ggaaatgctg ggaattccaa ccatccaatc ggacaagctt tcagaacctt attgaaggat3841ttgaagcact tttaaaataa gaagcatgaa taacatttaa attccacaga ttatcaagtc3901cttctcctgc aacaaatgcc caagtcattt tttaaaaatt tctaatgaaa gaagtttgtg3961ttctgtccaa aaagtcactg aactcatact tcagtacata tacatgtata aggcacactg4021tagtgcttaa tatgtgtaag gacttcctct ttaaatttgg taccagtaac ttagtgacac4081ataatgacaa ccaaaatatt tgaaagcact taagcactcc tccttgtgga aagaatatac4141caccatttca tctggctagt tcaccatcac aactgcatta ccaaaagggg atttttgaaa4201acgaggagtt gaccaaaata atatctgaag atgattgctt ttccctgctg ccagctgatc4261tgaaatgttt tgctggcaca ttaatcatag ataaagaaag attgatggac ttagccctca4321aatttcagta tctatacagt actagaccat gcattcttaa aatattagat accaggtagt4381atatattgtt tctgtacaaa aatgactgta ttctctcacc agtaggactt aaactttgtt4441tctccagtgg cttagctcct gttcctttgg gtgatcacta gcacccattt ttgagaaagc4501tggttctaca tggggggata gctgtggaat agataatttg ctgcatgtta attctcaaga4561actaagcctg tgccagtgct ttcctaagca gtataccttt aatcagaact cattcccaga4621acctggatgc tattacacat gcttttaaga aacgtcaatg tatatccttt tataactcta4681ccactttggg gcaagctatt ccagcactgg ttttgaatgc tgtatgcaac cagtctgaat4741accacatacg ctgcactgtt cttagagggt ttccatactt accaccgatc tacaagggtt4801gatccctgtt tttaccatca atcatcaccc tgtggtgcaa cacttgaaag acccggctag4861aggcactatg gacttcagga tccactagac agttttcagt ttgcttggag gtagctgggt4921aatcaaaaat gtttagtcat tgattcaatg tgaacgatta cggtctttat gaccaagagt4981ctgaaaatct ttttgttatg ctgtttagta ttcgtttgat attgttactt ttcacctgtt5041gagcccaaat tcaggattgg ttcagtggca gcaatgaagt tgccatttaa atttgttcat5101agcctacatc accaaggtct ctgtgtcaaa cctgtggcca ctctatatgc actttgttta5161ctctttatac aaataaatat actaaagact ttacatgcaHuman JAK1 mRNA Variant 6(SEQ ID NO: 6)1ggggcgggac gggaggcggt gcgtcgctga gcgcaggccg cggcggccgc ggagtatcct61ggagctgcag acagtgcggg cctgcgccca gtcccggctg tcctcgccgc gacccctcct121cagccctggg cgcgcgcacg ctggggcccc gcggggctgg ccgcctagcg agcctgccgg181tcgaccccag ccagcgcagc gacggggcgc tgcctggccc aggcgcacac ggaagtgtta241tctaaaacag ttcatgctgc tgaaaacctc cttcctggca gatgtccctc aaccctactg301gtgcctggct tctgagacac acgcttctct gaagtagctt tggaaagtag agaagaaaat361ccagtttgct tcttggagaa cactggacag ctgaataaat gcagtatcta aatataaaag421aggactgcaa tgccatggct ttctgtgcta aaatgaggag ctccaagaag actgaggtga481acctggaggc ccctgagcca ggggtggaag tgatcttcta tctgtcggac agggagcccc541tccggctggg cagtggagag tacacagcag aggaactgtg catcagggct gcacaggcat601gccgtatctc tcctctttgt cacaacctct ttgccctgta tgacgagaac accaagctct661ggtatgctcc aaatcgcacc atcaccgttg atgacaagat gtccctccgg ctccactacc721ggatgaggtt ctatttcacc aattggcatg gaaccaacga caatgagcag tcagtgtggc781gtcattctcc aaagaagcag aaaaatggct acgagaaaaa aaagattcca gatgcaaccc841ctctccttga tgccagctca ctggagtatc tgtttgctca gggacagtat gatttggtga901aatgcctggc tcctattcga gaccccaaga ccgagcagga tggacatgat attgagaacg961agtgtctagg gatggctgtc ctggccatct cacactatgc catgatgaag aagatgcagt1021tgccagaact gcccaaggac atcagctaca agcgatatat tccagaaaca ttgaataagt1081ccatcagaca gaggaacctt ctcaccagga tgcggataaa taatgttttc aaggatttcc1141taaaggaatt taacaacaag accatttgtg acagcagcgt gtccacgcat gacctgaagg1201tgaaatactt ggctaccttg gaaactttga caaaacatta cggtgctgaa atatttgaga1261cttccatgtt actgatttca tcagaaaatg agatgaattg gtttcattcg aatgacggtg1321gaaacgttct ctactacgaa gtgatggtga ctgggaatct tggaatccag tggaggcata1381aaccaaatgt tgtttctgtt gaaaaggaaa aaaataaact gaagcggaaa aaactggaaa1441ataaacacaa gaaggatgag gagaaaaaca agatccggga agagtggaac aatttttctt1501acttccctga aatcactcac attgtaataa aggagtctgt ggtcagcatt aacaagcagg1561acaacaagaa aatggaactg aagctctctt cccacgagga ggccttgtcc tttgtgtccc1621tggtagatgg ctacttccgg ctcacagcag atgcccatca ttacctctgc accgacgtgg1681cccccccgtt gatcgtccac aacatacaga atggctgtca tggtccaatc tgtacagaat1741acgccatcaa taaattgcgg caagaaggaa gcgaggaggg gatgtacgtg ctgaggtgga1801gctgcaccga ctttgacaac atcctcatga ccgtcacctg ctttgagaag tctgagcagg1861tgcagggtgc ccagaagcag ttcaagaact ttcagatcga ggtgcagaag ggccgctaca1921gtctgcacgg ttcggaccgc agcttcccca gcttgggaga cctcatgagc cacctcaaga1981agcagatcct gcgcacggat aacatcagct tcatgctaaa acgctgctgc cagcccaagc2041cccgagaaat ctccaacctg ctggtggcta ctaagaaagc ccaggagtgg cagcccgtct2101accccatgag ccagctgagt ttcgatcgga tcctcaagaa ggatctggtg cagggcgagc2161accttgggag aggcacgaga acacacatct attctgggac cctgatggat tacaaggatg2221acgaaggaac ttctgaagag aagaagataa aagtgatcct caaagtctta gaccccagcc2281acagggatat ttccctggcc ttcttcgagg cagccagcat gatgagacag gtctcccaca2341aacacatcgt gtacctctat ggcgtctgtg tccgcgacgt ggagaatatc atggtggaag2401agtttgtgga agggggtcct ctggatctct tcatgcaccg gaaaagcgat gtccttacca2461caccatggaa attcaaagtt gccaaacagc tggccagtgc cctgagctac ttggaggata2521aagacctggt ccatggaaat gtgtgtacta aaaacctcct cctggcccgt gagggcatcg2581acagtgagtg tggcccattc atcaagctca gtgaccccgg catccccatt acggtgctgt2641ctaggcaaga atgcattgaa cgaatcccat ggattgctcc tgagtgtgtt gaggactcca2701agaacctgag tgtggctgct gacaagtgga gctttggaac cacgctctgg gaaatctgct2761acaatggcga gatccccttg aaagacaaga cgctgattga gaaagagaga ttctatgaaa2821gccggtgcag gccagtgaca ccatcatgta aggagctggc tgacctcatg acccgctgca2881tgaactatga ccccaatcag aggcctttct tccgagccat catgagagac attaataagc2941ttgaagagca gaatccagat attgtttcag aaaaaaaacc agcaactgaa gtggacccca3001cacattttga aaagcgcttc ctaaagagga tccgtgactt gggagagggc cactttggga3061aggttgagct ctgcaggtat gaccccgaag gggacaatac aggggagcag gtggctgtta3121aatctctgaa gcctgagagt ggaggtaacc acatagctga tctgaaaaag gaaatcgaga3181tcttaaggaa cctctatcat gagaacattg tgaagtacaa aggaatctgc acagaagacg3241gaggaaatgg tattaagctc atcatggaat ttctgccttc gggaagcctt aaggaatatc3301ttccaaagaa taagaacaaa ataaacctca aacagcagct aaaatatgcc gttcagattt3361gtaaggggat ggactatttg ggttctcggc aatacgttca ccgggacttg gcagcaagaa3421atgtccttgt tgagagtgaa caccaagtga aaattggaga cttcggttta accaaagcaa3481ttgaaaccga taaggagtat tacaccgtca aggatgaccg ggacagccct gtgttttggt3541atgctccaga atgtttaatg caatctaaat tttatattgc ctctgacgtc tggtcttttg3601gagtcactct gcatgagctg ctgacttact gtgattcaga ttctagtccc atggctttgt3661tcctgaaaat gataggccca acccatggcc agatgacagt cacaagactt gtgaatacgt3721taaaagaagg aaaacgcctg ccgtgcccac ctaactgtcc agatgaggtt tatcaactta3781tgaggaaatg ctgggaattc caaccatcca atcggacaag ctttcagaac cttattgaag3841gatttgaagc acttttaaaa taagaagcat gaataacatt taaattccac agattatcaa3901gtccttctcc tgcaacaaat gcccaagtca ttttttaaaa atttctaatg aaagaagttt3961gtgttctgtc caaaaagtca ctgaactcat acttcagtac atatacatgt ataaggcaca4021ctgtagtgct taatatgtgt aaggacttcc tctttaaatt tggtaccagt aacttagtga4081cacataatga caaccaaaat atttgaaagc acttaagcac tcctccttgt ggaaagaata4141taccaccatt tcatctggct agttcaccat cacaactgca ttaccaaaag gggatttttg4201aaaacgagga gttgaccaaa ataatatctg aagatgattg cttttccctg ctgccagctg4261atctgaaatg ttttgctggc acattaatca tagataaaga aagattgatg gacttagccc4321tcaaatttca gtatctatac agtactagac catgcattct taaaatatta gataccaggt4381agtatatatt gtttctgtac aaaaatgact gtattctctc accagtagga cttaaacttt4441gtttctccag tggcttagct cctgttcctt tgggtgatca ctagcaccca tttttgagaa4501agctggttct acatgggggg atagctgtgg aatagataat ttgctgcatg ttaattctca4561agaactaagc ctgtgccagt gctttcctaa gcagtatacc tttaatcaga actcattccc4621agaacctgga tgctattaca catgctttta agaaacgtca atgtatatcc ttttataact4681ctaccacttt ggggcaagct attccagcac tggttttgaa tgctgtatgc aaccagtctg4741aataccacat acgctgcact gttcttagag ggtttccata cttaccaccg atctacaagg4801gttgatccct gtttttacca tcaatcatca ccctgtggtg caacacttga aagacccggc4861tagaggcact atggacttca ggatccacta gacagttttc agtttgcttg gaggtagctg4921ggtaatcaaa aatgtttagt cattgattca atgtgaacga ttacggtctt tatgaccaag4981agtctgaaaa tctttttgtt atgctgttta gtattcgttt gatattgtta cttttcacct5041gttgagccca aattcaggat tggttcagtg gcagcaatga agttgccatt taaatttgtt5101catagcctac atcaccaagg tctctgtgtc aaacctgtgg ccactctata tgcactttgt5161ttactcttta tacaaataaa tatactaaag actttacatg caHuman JAK1 mRNA Variant 7(SEQ ID NO: 7)1agaagcggag cgtatacgga ggaggcggga tgcatttctg catcgagcgc acaaagcgct61tctctgaagt agctttggaa agtagagaag aaaatccagt ttgcttcttg gagaacactg121gacagctgaa taaatgcagt atctaaatat aaaagaggac tgcaatgcca tggctttctg181tgctaaaatg aggagctcca agaagactga ggtgaacctg gaggcccctg agccaggggt241ggaagtgatc ttctatctgt cggacaggga gcccctccgg ctgggcagtg gagagtacac301agcagaggaa ctgtgcatca gggctgcaca ggcatgccgt atctctcctc tttgtcacaa361cctctttgcc ctgtatgacg agaacaccaa gctctggtat gctccaaatc gcaccatcac421cgttgatgac aagatgtccc tccggctcca ctaccggatg aggttctatt tcaccaattg481gcatggaacc aacgacaatg agcagtcagt gtggcgtcat tctccaaaga agcagaaaaa541tggctacgag aaaaaaaaga ttccagatgc aacccctctc cttgatgcca gctcactgga601gtatctgttt gctcagggac agtatgattt ggtgaaatgc ctggctccta ttcgagaccc661caagaccgag caggatggac atgatattga gaacgagtgt ctagggatgg ctgtcctggc721catctcacac tatgccatga tgaagaagat gcagttgcca gaactgccca aggacatcag781ctacaagcga tatattccag aaacattgaa taagtccatc agacagagga accttctcac841caggatgcgg ataaataatg ttttcaagga tttcctaaag gaatttaaca acaagaccat901ttgtgacagc agcgtgtcca cgcatgacct gaaggtgaaa tacttggcta ccttggaaac961tttgacaaaa cattacggtg ctgaaatatt tgagacttcc atgttactga tttcatcaga1021aaatgagatg aattggtttc attcgaatga cggtggaaac gttctctact acgaagtgat1081ggtgactggg aatcttggaa tccagtggag gcataaacca aatgttgttt ctgttgaaaa1141ggaaaaaaat aaactgaagc ggaaaaaact ggaaaataaa cacaagaagg atgaggagaa1201aaacaagatc cgggaagagt ggaacaattt ttcttacttc cctgaaatca ctcacattgt1261aataaaggag tctgtggtca gcattaacaa gcaggacaac aagaaaatgg aactgaagct1321ctcttcccac gaggaggcct tgtcctttgt gtccctggta gatggctact tccggctcac1381agcagatgcc catcattacc tctgcaccga cgtggccccc ccgttgatcg tccacaacat1441acagaatggc tgtcatggtc caatctgtac agaatacgcc atcaataaat tgcggcaaga1501aggaagcgag gaggggatgt acgtgctgag gtggagctgc accgactttg acaacatcct1561catgaccgtc acctgctttg agaagtctga gcaggtgcag ggtgcccaga agcagttcaa1621gaactttcag atcgaggtgc agaagggccg ctacagtctg cacggttcgg accgcagctt1681ccccagcttg ggagacctca tgagccacct caagaagcag atcctgcgca cggataacat1741cagcttcatg ctaaaacgct gctgccagcc caagccccga gaaatctcca acctgctggt1801ggctactaag aaagcccagg agtggcagcc cgtctacccc atgagccagc tgagtttcga1861tcggatcctc aagaaggatc tggtgcaggg cgagcacctt gggagaggca cgagaacaca1921catctattct gggaccctga tggattacaa ggatgacgaa ggaacttctg aagagaagaa1981gataaaagtg atcctcaaag tcttagaccc cagccacagg gatatttccc tggccttctt2041cgaggcagcc agcatgatga gacaggtctc ccacaaacac atcgtgtacc tctatggcgt2101ctgtgtccgc gacgtggaga atatcatggt ggaagagttt gtggaagggg gtcctctgga2161tctcttcatg caccggaaaa gcgatgtcct taccacacca tggaaattca aagttgccaa2221acagctggcc agtgccctga gctacttgga ggataaagac ctggtccatg gaaatgtgtg2281tactaaaaac ctcctcctgg cccgtgaggg catcgacagt gagtgtggcc cattcatcaa2341gctcagtgac cccggcatcc ccattacggt gctgtctagg caagaatgca ttgaacgaat2401cccatggatt gctcctgagt gtgttgagga ctccaagaac ctgagtgtgg ctgctgacaa2461gtggagcttt ggaaccacgc tctgggaaat ctgctacaat ggcgagatcc ccttgaaaga2521caagacgctg attgagaaag agagattcta tgaaagccgg tgcaggccag tgacaccatc2581atgtaaggag ctggctgacc tcatgacccg ctgcatgaac tatgacccca atcagaggcc2641tttcttccga gccatcatga gagacattaa taagcttgaa gagcagaatc cagatattgt2701ttcagaaaaa aaaccagcaa ctgaagtgga ccccacacat tttgaaaagc gcttcctaaa2761gaggatccgt gacttgggag agggccactt tgggaaggtt gagctctgca ggtatgaccc2821cgaaggggac aatacagggg agcaggtggc tgttaaatct ctgaagcctg agagtggagg2881taaccacata gctgatctga aaaaggaaat cgagatctta aggaacctct atcatgagaa2941cattgtgaag tacaaaggaa tctgcacaga agacggagga aatggtatta agctcatcat3001ggaatttctg ccttcgggaa gccttaagga atatcttcca aagaataaga acaaaataaa3061cctcaaacag cagctaaaat atgccgttca gatttgtaag gggatggact atttgggttc3121tcggcaatac gttcaccggg acttggcagc aagaaatgtc cttgttgaga gtgaacacca3181agtgaaaatt ggagacttcg gtttaaccaa agcaattgaa accgataagg agtattacac3241cgtcaaggat gaccgggaca gccctgtgtt ttggtatgct ccagaatgtt taatgcaatc3301taaattttat attgcctctg acgtctggtc ttttggagtc actctgcatg agctgctgac3361ttactgtgat tcagattcta gtcccatggc tttgttcctg aaaatgatag gcccaaccca3421tggccagatg acagtcacaa gacttgtgaa tacgttaaaa gaaggaaaac gcctgccgtg3481cccacctaac tgtccagatg aggtttatca acttatgagg aaatgctggg aattccaacc3541atccaatcgg acaagctttc agaaccttat tgaaggattt gaagcacttt taaaataaga3601agcatgaata acatttaaat tccacagatt atcaagtcct tctcctgcaa caaatgccca3661agtcattttt taaaaatttc taatgaaaga agtttgtgtt ctgtccaaaa agtcactgaa3721ctcatacttc agtacatata catgtataag gcacactgta gtgcttaata tgtgtaagga3781cttcctcttt aaatttggta ccagtaactt agtgacacat aatgacaacc aaaatatttg3841aaagcactta agcactcctc cttgtggaaa gaatatacca ccatttcatc tggctagttc3901accatcacaa ctgcattacc aaaaggggat ttttgaaaac gaggagttga ccaaaataat3961atctgaagat gattgctttt ccctgctgcc agctgatctg aaatgttttg ctggcacatt4021aatcatagat aaagaaagat tgatggactt agccctcaaa tttcagtatc tatacagtac4081tagaccatgc attcttaaaa tattagatac caggtagtat atattgtttc tgtacaaaaa4141tgactgtatt ctctcaccag taggacttaa actttgtttc tccagtggct tagctcctgt4201tcctttgggt gatcactagc acccattttt gagaaagctg gttctacatg gggggatagc4261tgtggaatag ataatttgct gcatgttaat tctcaagaac taagcctgtg ccagtgcttt4321cctaagcagt atacctttaa tcagaactca ttcccagaac ctggatgcta ttacacatgc4381ttttaagaaa cgtcaatgta tatcctttta taactctacc actttggggc aagctattcc4441agcactggtt ttgaatgctg tatgcaacca gtctgaatac cacatacgct gcactgttct4501tagagggttt ccatacttac caccgatcta caagggttga tccctgtttt taccatcaat4561catcaccctg tggtgcaaca cttgaaagac ccggctagag gcactatgga cttcaggatc4621cactagacag ttttcagttt gcttggaggt agctgggtaa tcaaaaatgt ttagtcattg4681attcaatgtg aacgattacg gtctttatga ccaagagtct gaaaatcttt ttgttatgct4741gtttagtatt cgtttgatat tgttactttt cacctgttga gcccaaattc aggattggtt4801cagtggcagc aatgaagttg ccatttaaat ttgttcatag cctacatcac caaggtctct4861gtgtcaaacc tgtggccact ctatatgcac tttgtttact ctttatacaa ataaatatac4921taaagacttt acatgcaHuman JAK1 mRNA Variant 8(SEQ ID NO: 8)1ggggcgggac gggaggcggt gcgtcgctga gcgcaggccg cggcggccgc ggagtatcct61ggagctgcag acagtgcggg cctgcgccca gtcccggctg tcctcgccgc gacccctcct121cagccctggg cgcgcgcacg ctggggcccc gcggggctgg ccgcctagcg agcctgccgg181tcgaccccag ccagcgcagc gacggggcgc tgcctggccc aggcgcacac ggaagtgcgc241ttctctgaag tagctttgga aagtagagaa gaaaatccag tttgcttctt ggagaacact301ggacagctga ataaatgcag tatctaaata taaaagagga ctgcaatgcc atggctttct361gtgctaaaat gaggagctcc aagaagactg aggtgaacct ggaggcccct gagccagggg421tggaagtgat cttctatctg tcggacaggg agcccctccg gctgggcagt ggagagtaca481cagcagagga actgtgcatc agggctgcac aggcatgccg tatctctcct ctttgtcaca541acctctttgc cctgtatgac gagaacacca agctctggta tgctccaaat cgcaccatca601ccgttgatga caagatgtcc ctccggctcc actaccggat gaggttctat ttcaccaatt661ggcatggaac caacgacaat gagcagtcag tgtggcgtca ttctccaaag aagcagaaaa721atggctacga gaaaaaaaag attccagatg caacccctct ccttgatgcc agctcactgg781agtatctgtt tgctcaggga cagtatgatt tggtgaaatg cctggctcct attcgagacc841ccaagaccga gcaggatgga catgatattg agaacgagtg tctagggatg gctgtcctgg901ccatctcaca ctatgccatg atgaagaaga tgcagttgcc agaactgccc aaggacatca961gctacaagcg atatattcca gaaacattga ataagtccat cagacagagg aaccttctca1021ccaggatgcg gataaataat gttttcaagg atttcctaaa ggaatttaac aacaagacca1081tttgtgacag cagcgtgtcc acgcatgacc tgaaggtgaa atacttggct accttggaaa1141ctttgacaaa acattacggt gctgaaatat ttgagacttc catgttactg atttcatcag1201aaaatgagat gaattggttt cattcgaatg acggtggaaa cgttctctac tacgaagtga1261tggtgactgg gaatcttgga atccagtgga ggcataaacc aaatgttgtt tctgttgaaa1321aggaaaaaaa taaactgaag cggaaaaaac tggaaaataa acacaagaag gatgaggaga1381aaaacaagat ccgggaagag tggaacaatt tttcttactt ccctgaaatc actcacattg1441taataaagga gtctgtggtc agcattaaca agcaggacaa caagaaaatg gaactgaagc1501tctcttccca cgaggaggcc ttgtcctttg tgtccctggt agatggctac ttccggctca1561cagcagatgc ccatcattac ctctgcaccg acgtggcccc cccgttgatc gtccacaaca1621tacagaatgg ctgtcatggt ccaatctgta cagaatacgc catcaataaa ttgcggcaag1681aaggaagcga ggaggggatg tacgtgctga ggtggagctg caccgacttt gacaacatcc1741tcatgaccgt cacctgcttt gagaagtctg aggtgcaggg tgcccagaag cagttcaaga1801actttcagat cgaggtgcag aagggccgct acagtctgca cggttcggac cgcagcttcc1861ccagcttggg agacctcatg agccacctca agaagcagat cctgcgcacg gataacatca1921gcttcatgct aaaacgctgc tgccagccca agccccgaga aatctccaac ctgctggtgg1981ctactaagaa agcccaggag tggcagcccg tctaccccat gagccagctg agtttcgatc2041ggatcctcaa gaaggatctg gtgcagggcg agcaccttgg gagaggcacg agaacacaca2101tctattctgg gaccctgatg gattacaagg atgacgaagg aacttctgaa gagaagaaga2161taaaagtgat cctcaaagtc ttagacccca gccacaggga tatttccctg gccttcttcg2221aggcagccag catgatgaga caggtctccc acaaacacat cgtgtacctc tatggcgtct2281gtgtccgcga cgtggagaat atcatggtgg aagagtttgt ggaagggggt cctctggatc2341tcttcatgca ccggaaaagc gatgtcctta ccacaccatg gaaattcaaa gttgccaaac2401agctggccag tgccctgagc tacttggagg ataaagacct ggtccatgga aatgtgtgta2461ctaaaaacct cctcctggcc cgtgagggca tcgacagtga gtgtggccca ttcatcaagc2521tcagtgaccc cggcatcccc attacggtgc tgtctaggca agaatgcatt gaacgaatcc2581catggattgc tcctgagtgt gttgaggact ccaagaacct gagtgtggct gctgacaagt2641ggagctttgg aaccacgctc tgggaaatct gctacaatgg cgagatcccc ttgaaagaca2701agacgctgat tgagaaagag agattctatg aaagccggtg caggccagtg acaccatcat2761gtaaggagct ggctgacctc atgacccgct gcatgaacta tgaccccaat cagaggcctt2821tcttccgagc catcatgaga gacattaata agcttgaaga gcagaatcca gatattgttt2881cagaaaaaaa accagcaact gaagtggacc ccacacattt tgaaaagcgc ttcctaaaga2941ggatccgtga cttgggagag ggccactttg ggaaggttga gctctgcagg tatgaccccg3001aaggggacaa tacaggggag caggtggctg ttaaatctct gaagcctgag agtggaggta3061accacatagc tgatctgaaa aaggaaatcg agatcttaag gaacctctat catgagaaca3121ttgtgaagta caaaggaatc tgcacagaag acggaggaaa tggtattaag ctcatcatgg3181aatttctgcc ttcgggaagc cttaaggaat atcttccaaa gaataagaac aaaataaacc3241tcaaacagca gctaaaatat gccgttcaga tttgtaaggg gatggactat ttgggttctc3301ggcaatacgt tcaccgggac ttggcagcaa gaaatgtcct tgttgagagt gaacaccaag3361tgaaaattgg agacttcggt ttaaccaaag caattgaaac cgataaggag tattacaccg3421tcaaggatga ccgggacagc cctgtgtttt ggtatgctcc agaatgttta atgcaatcta3481aattttatat tgcctctgac gtctggtctt ttggagtcac tctgcatgag ctgctgactt3541actgtgattc agattctagt cccatggctt tgttcctgaa aatgataggc ccaacccatg3601gccagatgac agtcacaaga cttgtgaata cgttaaaaga aggaaaacgc ctgccgtgcc3661cacctaactg tccagatgag gtttatcaac ttatgaggaa atgctgggaa ttccaaccat3721ccaatcggac aagctttcag aaccttattg aaggatttga agcactttta aaataagaag3781catgaataac atttaaattc cacagattat caagtccttc tcctgcaaca aatgcccaag3841tcatttttta aaaatttcta atgaaagaag tttgtgttct gtccaaaaag tcactgaact3901catacttcag tacatataca tgtataaggc acactgtagt gcttaatatg tgtaaggact3961tcctctttaa atttggtacc agtaacttag tgacacataa tgacaaccaa aatatttgaa4021agcacttaag cactcctcct tgtggaaaga atataccacc atttcatctg gctagttcac4081catcacaact gcattaccaa aaggggattt ttgaaaacga ggagttgacc aaaataatat4141ctgaagatga ttgcttttcc ctgctgccag ctgatctgaa atgttttgct ggcacattaa4201tcatagataa agaaagattg atggacttag ccctcaaatt tcagtatcta tacagtacta4261gaccatgcat tcttaaaata ttagatacca ggtagtatat attgtttctg tacaaaaatg4321actgtattct ctcaccagta ggacttaaac tttgtttctc cagtggctta gctcctgttc4381ctttgggtga tcactagcac ccatttttga gaaagctggt tctacatggg gggatagctg4441tggaatagat aatttgctgc atgttaattc tcaagaacta agcctgtgcc agtgctttcc4501taagcagtat acctttaatc agaactcatt cccagaacct ggatgctatt acacatgctt4561ttaagaaacg tcaatgtata tccttttata actctaccac tttggggcaa gctattccag4621cactggtttt gaatgctgta tgcaaccagt ctgaatacca catacgctgc actgttctta4681gagggtttcc atacttacca ccgatctaca agggttgatc cctgttttta ccatcaatca4741tcaccctgtg gtgcaacact tgaaagaccc ggctagaggc actatggact tcaggatcca4801ctagacagtt ttcagtttgc ttggaggtag ctgggtaatc aaaaatgttt agtcattgat4861tcaatgtgaa cgattacggt ctttatgacc aagagtctga aaatcttttt gttatgctgt4921ttagtattcg tttgatattg ttacttttca cctgttgagc ccaaattcag gattggttca4981gtggcagcaa tgaagttgcc atttaaattt gttcatagcc tacatcacca aggtctctgt5041gtcaaacctg tggccactct atatgcactt tgtttactct ttatacaaat aaatatacta5101aagactttac atgcaHuman JAK2 mRNA Variant 1(SEQ ID NO: 9)1ctgcaggaag gagagaggaa gaggagcaga agggggcagc agcggacgcc gctaacggcc61tccctcggcg ctgacaggct gggccggcgc ccggctcgct tgggtgttcg cgtcgccact121tcggcttctc ggccggtcgg gcccctcggc ccgggcttgc ggcgcgcgtc ggggctgagg181gctgctgcgg cgcagggaga ggcctggtcc tcgctgccga gggatgtgag tgggagctga241gcccacactg gagggccccc gagggcccag cctggaggtc gttcagagcc gtgcccgtcc301cggggcttcg cagaccttga cccgccgggt aggagccgcc cctgcgggct cgagggcgcg361ctctggtcgc ccgatctgtg tagccggttt cagaagcagg caacaggaac aagatgtgaa421ctgtttctct tctgcagaaa aagaggctct tcctcctcct cccgcgacgg caaatgttct481gaaaaagact ctgcatggga atggcctgcc ttacgatgac agaaatggag ggaacatcca541cctcttctat atatcagaat ggtgatattt ctggaaatgc caattctatg aagcaaatag601atccagttct tcaggtgtat ctttaccatt cccttgggaa atctgaggca gattatctga661cctttccatc tggggagtat gttgcagaag aaatctgtat tgctgcttct aaagcttgtg721gtatcacacc tgtgtatcat aatatgtttg ctttaatgag tgaaacagaa aggatctggt781atccacccaa ccatgtcttc catatagatg agtcaaccag gcataatgta ctctacagaa841taagatttta ctttcctcgt tggtattgca gtggcagcaa cagagcctat cggcatggaa901tatctcgagg tgctgaagct cctcttcttg atgactttgt catgtcttac ctctttgctc961agtggcggca tgattttgtg cacggatgga taaaagtacc tgtgactcat gaaacacagg1021aagaatgtct tgggatggca gtgttagata tgatgagaat agccaaagaa aacgatcaaa1081ccccactggc catctataac tctatcagct acaagacatt cttaccaaaa tgtattcgag1141caaagatcca agactatcat attttgacaa ggaagcgaat aaggtacaga tttcgcagat1201ttattcagca attcagccaa tgcaaagcca ctgccagaaa cttgaaactt aagtatctta1261taaatctgga aactctgcag tctgccttct acacagagaa atttgaagta aaagaacctg1321gaagtggtcc ttcaggtgag gagatttttg caaccattat aataactgga aacggtggaa1381ttcagtggtc aagagggaaa cataaagaaa gtgagacact gacagaacag gatttacagt1441tatattgcga ttttcctaat attattgatg tcagtattaa gcaagcaaac caagagggtt1501caaatgaaag ccgagttgta actatccata agcaagatgg taaaaatctg gaaattgaac1561ttagctcatt aagggaagct ttgtctttcg tgtcattaat tgatggatat tatagattaa1621ctgcagatgc acatcattac ctctgtaaag aagtagcacc tccagccgtg cttgaaaata1681tacaaagcaa ctgtcatggc ccaatttcga tggattttgc cattagtaaa ctgaagaaag1741caggtaatca gactggactg tatgtacttc gatgcagtcc taaggacttt aataaatatt1801ttttgacttt tgctgtcgag cgagaaaatg tcattgaata taaacactgt ttgattacaa1861aaaatgagaa tgaagagtac aacctcagtg ggacaaagaa gaacttcagc agtcttaaag1921atcttttgaa ttgttaccag atggaaactg ttcgctcaga caatataatt ttccagttta1981ctaaatgctg tcccccaaag ccaaaagata aatcaaacct tctagtcttc agaacgaatg2041gtgtttctga tgtaccaacc tcaccaacat tacagaggcc tactcatatg aaccaaatgg2101tgtttcacaa aatcagaaat gaagatttga tatttaatga aagccttggc caaggcactt2161ttacaaagat ttttaaaggc gtacgaagag aagtaggaga ctacggtcaa ctgcatgaaa2221cagaagttct tttaaaagtt ctggataaag cacacagaaa ctattcagag tctttctttg2281aagcagcaag tatgatgagc aagctttctc acaagcattt ggttttaaat tatggagtat2341gtgtctgtgg agacgagaat attctggttc aggagtttgt aaaatttgga tcactagata2401catatctgaa aaagaataaa aattgtataa atatattatg gaaacttgaa gttgctaaac2461agttggcatg ggccatgcat tttctagaag aaaacaccct tattcatggg aatgtatgtg2521ccaaaaatat tctgcttatc agagaagaag acaggaagac aggaaatcct cctttcatca2581aacttagtga tcctggcatt agtattacag ttttgccaaa ggacattctt caggagagaa2641taccatgggt accacctgaa tgcattgaaa atcctaaaaa tttaaatttg gcaacagaca2701aatggagttt tggtaccact ttgtgggaaa tctgcagtgg aggagataaa cctctaagtg2761ctctggattc tcaaagaaag ctacaatttt atgaagatag gcatcagctt cctgcaccaa2821agtgggcaga attagcaaac cttataaata attgtatgga ttatgaacca gatttcaggc2881cttctttcag agccatcata cgagatctta acagtttgtt tactccagat tatgaactat2941taacagaaaa tgacatgtta ccaaatatga ggataggtgc cctggggttt tctggtgcct3001ttgaagaccg ggatcctaca cagtttgaag agagacattt gaaatttcta cagcaacttg3061gcaagggtaa ttttgggagt gtggagatgt gccggtatga ccctctacag gacaacactg3121gggaggtggt cgctgtaaaa aagcttcagc atagtactga agagcaccta agagactttg3181aaagggaaat tgaaatcctg aaatccctac agcatgacaa cattgtaaag tacaagggag3241tgtgctacag tgctggtcgg cgtaatctaa aattaattat ggaatattta ccatatggaa3301gtttacgaga ctatcttcaa aaacataaag aacggataga tcacataaaa cttctgcagt3361acacatctca gatatgcaag ggtatggagt atcttggtac aaaaaggtat atccacaggg3421atctggcaac gagaaatata ttggtggaga acgagaacag agttaaaatt ggagattttg3481ggttaaccaa agtcttgcca caagacaaag aatactataa agtaaaagaa cctggtgaaa3541gtcccatatt ctggtatgct ccagaatcac tgacagagag caagttttct gtggcctcag3601atgtttggag ctttggagtg gttctgtatg aacttttcac atacattgag aagagtaaaa3661gtccaccagc ggaatttatg cgtatgattg gcaatgacaa acaaggacag atgatcgtgt3721tccatttgat agaacttttg aagaataatg gaagattacc aagaccagat ggatgcccag3781atgagatcta tatgatcatg acagaatgct ggaacaataa tgtaaatcaa cgcccctcct3841ttagggatct agctcttcga gtggatcaaa taagggataa catggctgga tgaaagaaat3901gaccttcatt ctgagaccaa agtagattta cagaacaaag ttttatattt cacattgctg3961tggactatta ttacatatat cattattata taaatcatga tgctagccag caaagatgtg4021aaaatatctg ctcaaaactt tcaaagttta gtaagttttt cttcatgagg ccaccagtaa4081aagacattaa tgagaattcc ttagcaagga ttttgtaaga agtttcttaa acattgtcag4141ttaacatcac tcttgtctgg caaaagaaaa aaaatagact ttttcaactc agctttttga4201gacctgaaaa aattattatg taaattttgc aatgttaaag atgcacagaa tatgtatgta4261tagtttttac cacagtggat gtataatacc ttggcatctt gtgtgatgtt ttacacacat4321gagggctggt gttcattaat actgttttct aatttttcca tagttaatct ataattaatt4381acttcactat acaaacaaat taagatgttc agataattga ataagtacct ttgtgtcctt4441gttcatttat atcgctggcc agcattataa gcaggtgtat acttttagct tgtagttcca4501tgtactgtaa atatttttca cataaaggga acaaatgtct agttttattt gtataggaaa4561tttccctgac cctaaataat acattttgaa atgaaacaag cttacaaaga tataatctat4621tttattatgg tttcccttgt atctatttgt ggtgaatgtg ttttttaaat ggaactatct4681ccaaattttt ctaagactac tatgaacagt tttcttttaa aattttgaga ttaagaatgc4741caggaatatt gtcatccttt gagctgctga ctgccaataa cattcttcga tctctgggat4801ttatgctcat gaactaaatt taagcttaag ccataaaata gattagattg ttttttaaaa4861atggatagct cattaagaag tgcagcaggt taagaatttt ttcctaaaga ctgtatattt4921gaggggtttc agaattttgc attgcagtca tagaagagat ttatttcctt tttagagggg4981aaatgaggta aataagtaaa aaagtatgct tgttaatttt attcaagaat gccagtagaa5041aattcataac gtgtatcttt aagaaaaatg agcatacatc ttaaatcttt tcaattaagt5101ataaggggtt gttcgttgtt gtcatttgtt atagtgctac tccactttag acaccatagc5161taaaataaaa tatggtgggt tttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg5221tgttatttat acaaaactta aaatacttgc tgttttgatt aaaaagaaaa tagtttctta5281ctttaHuman JAK2 mRNA Variant 2(SEQ ID NO: 10)1attcggggag actgcaggcc aaccgggagg ctgagttcga agctagcagg gcggcgaagc61cagtgtcgcc cgcggcgttg agaagacggt gtggccccgg agagggtgga gacaactgtg121acgggcttcc cggctgcccg aagtgggagt ggtgtggggc tgcaggaagg agagaggaag181aggagcagaa gggggcagca gcggacgccg ctaacggcct ccctcggcgc tgacaggctg241ggccggcgcc cggctcgctt gggtgttcgc gtcgccactt cggcttctcg gccggtcggg301cccctcggcc cgggcttgcg gcgcgcgtcg gggctgaggg ctgctgcggc gcagggagag361gcctggtcct cgctgccgag ggatgtgagt gggagctgag cccacactgg agggcccccg421agggcccagc ctggaggtcg ttcagagccg tgcccgtccc ggggcttcgc agaccttgac481ccgccgggtt tcagaagcag gcaacaggaa caagatgtga actgtttctc ttctgcagaa541aaagaggctc ttcctcctcc tcccgcgacg gcaaatgttc tgaaaaagac tctgcatggg601aatggcctgc cttacgatga cagaaatgga gggaacatcc acctcttcta tatatcagaa661tggtgatatt tctggaaatg ccaattctat gaagcaaata gatccagttc ttcaggtgta721tctttaccat tcccttggga aatctgaggc agattatctg acctttccat ctggggagta781tgttgcagaa gaaatctgta ttgctgcttc taaagcttgt ggtatcacac ctgtgtatca841taatatgttt gctttaatga gtgaaacaga aaggatctgg tatccaccca accatgtctt901ccatatagat gagtcaacca ggcataatgt actctacaga ataagatttt actttcctcg961ttggtattgc agtggcagca acagagccta tcggcatgga atatctcgag gtgctgaagc1021tcctcttctt gatgactttg tcatgtctta cctctttgct cagtggcggc atgattttgt1081gcacggatgg ataaaagtac ctgtgactca tgaaacacag gaagaatgtc ttgggatggc1141agtgttagat atgatgagaa tagccaaaga aaacgatcaa accccactgg ccatctataa1201ctctatcagc tacaagacat tcttaccaaa atgtattcga gcaaagatcc aagactatca1261tattttgaca aggaagcgaa taaggtacag atttcgcaga tttattcagc aattcagcca1321atgcaaagcc actgccagaa acttgaaact taagtatctt ataaatctgg aaactctgca1381gtctgccttc tacacagaga aatttgaagt aaaagaacct ggaagtggtc cttcaggtga1441ggagattttt gcaaccatta taataactgg aaacggtgga attcagtggt caagagggaa1501acataaagaa agtgagacac tgacagaaca ggatttacag ttatattgcg attttcctaa1561tattattgat gtcagtatta agcaagcaaa ccaagagggt tcaaatgaaa gccgagttgt1621aactatccat aagcaagatg gtaaaaatct ggaaattgaa cttagctcat taagggaagc1681tttgtctttc gtgtcattaa ttgatggata ttatagatta actgcagatg cacatcatta1741cctctgtaaa gaagtagcac ctccagccgt gcttgaaaat atacaaagca actgtcatgg1801cccaatttcg atggattttg ccattagtaa actgaagaaa gcaggtaatc agactggact1861gtatgtactt cgatgcagtc ctaaggactt taataaatat tttttgactt ttgctgtcga1921gcgagaaaat gtcattgaat ataaacactg tttgattaca aaaaatgaga atgaagagta1981caacctcagt gggacaaaga agaacttcag cagtcttaaa gatcttttga attgttacca2041gatggaaact gttcgctcag acaatataat tttccagttt actaaatgct gtcccccaaa2101gccaaaagat aaatcaaacc ttctagtctt cagaacgaat ggtgtttctg atgtaccaac2161ctcaccaaca ttacagaggc ctactcatat gaaccaaatg gtgtttcaca aaatcagaaa2221tgaagatttg atatttaatg aaagccttgg ccaaggcact tttacaaaga tttttaaagg2281cgtacgaaga gaagtaggag actacggtca actgcatgaa acagaagttc ttttaaaagt2341tctggataaa gcacacagaa actattcaga gtctttcttt gaagcagcaa gtatgatgag2401caagctttct cacaagcatt tggttttaaa ttatggagta tgtgtctgtg gagacgagaa2461tattctggtt caggagtttg taaaatttgg atcactagat acatatctga aaaagaataa2521aaattgtata aatatattat ggaaacttga agttgctaaa cagttggcat gggccatgca2581ttttctagaa gaaaacaccc ttattcatgg gaatgtatgt gccaaaaata ttctgcttat2641cagagaagaa gacaggaaga caggaaatcc tcctttcatc aaacttagtg atcctggcat2701tagtattaca gttttgccaa aggacattct tcaggagaga ataccatggg taccacctga2761atgcattgaa aatcctaaaa atttaaattt ggcaacagac aaatggagtt ttggtaccac2821tttgtgggaa atctgcagtg gaggagataa acctctaagt gctctggatt ctcaaagaaa2881gctacaattt tatgaagata ggcatcagct tcctgcacca aagtgggcag aattagcaaa2941ccttataaat aattgtatgg attatgaacc agatttcagg ccttctttca gagccatcat3001acgagatctt aacagtttgt ttactccaga ttatgaacta ttaacagaaa atgacatgtt3061accaaatatg aggataggtg ccctggggtt ttctggtgcc tttgaagacc gggatcctac3121acagtttgaa gagagacatt tgaaatttct acagcaactt ggcaagggta attttgggag3181tgtggagatg tgccggtatg accctctaca ggacaacact ggggaggtgg tcgctgtaaa3241aaagcttcag catagtactg aagagcacct aagagacttt gaaagggaaa ttgaaatcct3301gaaatcccta cagcatgaca acattgtaaa gtacaaggga gtgtgctaca gtgctggtcg3361gcgtaatcta aaattaatta tggaatattt accatatgga agtttacgag actatcttca3421aaaacataaa gaacggatag atcacataaa acttctgcag tacacatctc agatatgcaa3481gggtatggag tatcttggta caaaaaggta tatccacagg gatctggcaa cgagaaatat3541attggtggag aacgagaaca gagttaaaat tggagatttt gggttaacca aagtcttgcc3601acaagacaaa gaatactata aagtaaaaga acctggtgaa agtcccatat tctggtatgc3661tccagaatca ctgacagaga gcaagttttc tgtggcctca gatgtttgga gctttggagt3721ggttctgtat gaacttttca catacattga gaagagtaaa agtccaccag cggaatttat3781gcgtatgatt ggcaatgaca aacaaggaca gatgatcgtg ttccatttga tagaactttt3841gaagaataat ggaagattac caagaccaga tggatgccca gatgagatct atatgatcat3901gacagaatgc tggaacaata atgtaaatca acgcccctcc tttagggatc tagctcttcg3961agtggatcaa ataagggata acatggctgg atgaaagaaa tgaccttcat tctgagacca4021aagtagattt acagaacaaa gttttatatt tcacattgct gtggactatt attacatata4081tcattattat ataaatcatg atgctagcca gcaaagatgt gaaaatatct gctcaaaact4141ttcaaagttt agtaagtttt tcttcatgag gccaccagta aaagacatta atgagaattc4201cttagcaagg attttgtaag aagtttctta aacattgtca gttaacatca ctcttgtctg4261gcaaaagaaa aaaaatagac tttttcaact cagctttttg agacctgaaa aaattattat4321gtaaattttg caatgttaaa gatgcacaga atatgtatgt atagttttta ccacagtgga4381tgtataatac cttggcatct tgtgtgatgt tttacacaca tgagggctgg tgttcattaa4441tactgttttc taatttttcc atagttaatc tataattaat tacttcacta tacaaacaaa4501ttaagatgtt cagataattg aataagtacc tttgtgtcct tgttcattta tatcgctggc4561cagcattata agcaggtgta tacttttagc ttgtagttcc atgtactgta aatatttttc4621acataaaggg aacaaatgtc tagttttatt tgtataggaa atttccctga ccctaaataa4681tacattttga aatgaaacaa gcttacaaag atataatcta ttttattatg gtttcccttg4741tatctatttg tggtgaatgt gttttttaaa tggaactatc tccaaatttt tctaagacta4801ctatgaacag ttttctttta aaattttgag attaagaatg ccaggaatat tgtcatcctt4861tgagctgctg actgccaata acattcttcg atctctggga tttatgctca tgaactaaat4921ttaagcttaa gccataaaat agattagatt gttttttaaa aatggatagc tcattaagaa4981gtgcagcagg ttaagaattt tttcctaaag actgtatatt tgaggggttt cagaattttg5041cattgcagtc atagaagaga tttatttcct ttttagaggg gaaatgaggt aaataagtaa5101aaaagtatgc ttgttaattt tattcaagaa tgccagtaga aaattcataa cgtgtatctt5161taagaaaaat gagcatacat cttaaatctt ttcaattaag tataaggggt tgttcgttgt5221tgtcatttgt tatagtgcta ctccacttta gacaccatag ctaaaataaa atatggtggg5281ttttgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttattta tacaaaactt5341aaaatacttg ctgttttgat taaaaagaaa atagtttctt actttaHuman JAK2 mRNA Variant 3(SEQ ID NO: 11)1attcggggag actgcaggcc aaccgggagg ctgagttcga agctagcagg gcggcgaagc61cagtgtcgcc cgcggcgttg agaagacggc aaatgttctg aaaaagactc tgcatgggaa121tggcctgcct tacgatgaca gaaatggagg gaacatccac ctcttctata tatcagaatg181gtgatatttc tggaaatgcc aattctatga agcaaataga tccagttctt caggtgtatc241tttaccattc ccttgggaaa tctgaggcag attatctgac ctttccatct ggggagtatg301ttgcagaaga aatctgtatt gctgcttcta aagcttgtgg tatcacacct gtgtatcata361atatgtttgc tttaatgagt gaaacagaaa ggatctggta tccacccaac catgtcttcc421atatagatga gtcaaccagg cataatgtac tctacagaat aagattttac tttcctcgtt481ggtattgcag tggcagcaac agagcctatc ggcatggaat atctcgaggt gctgaagctc541ctcttcttga tgactttgtc atgtcttacc tctttgctca gtggcggcat gattttgtgc601acggatggat aaaagtacct gtgactcatg aaacacagga agaatgtctt gggatggcag661tgttagatat gatgagaata gccaaagaaa acgatcaaac cccactggcc atctataact721ctatcagcta caagacattc ttaccaaaat gtattcgagc aaagatccaa gactatcata781ttttgacaag gaagcgaata aggtacagat ttcgcagatt tattcagcaa ttcagccaat841gcaaagccac tgccagaaac ttgaaactta agtatcttat aaatctggaa actctgcagt901ctgccttcta cacagagaaa tttgaagtaa aagaacctgg aagtggtcct tcaggtgagg961agatttttgc aaccattata ataactggaa acggtggaat tcagtggtca agagggaaac1021ataaagaaag tgagacactg acagaacagg atttacagtt atattgcgat tttcctaata1081ttattgatgt cagtattaag caagcaaacc aagagggttc aaatgaaagc cgagttgtaa1141ctatccataa gcaagatggt aaaaatctgg aaattgaact tagctcatta agggaagctt1201tgtctttcgt gtcattaatt gatggatatt atagattaac tgcagatgca catcattacc1261tctgtaaaga agtagcacct ccagccgtgc ttgaaaatat acaaagcaac tgtcatggcc1321caatttcgat ggattttgcc attagtaaac tgaagaaagc aggtaatcag actggactgt1381atgtacttcg atgcagtcct aaggacttta ataaatattt tttgactttt gctgtcgagc1441gagaaaatgt cattgaatat aaacactgtt tgattacaaa aaatgagaat gaagagtaca1501acctcagtgg gacaaagaag aacttcagca gtcttaaaga tcttttgaat tgttaccaga1561tggaaactgt tcgctcagac aatataattt tccagtttac taaatgctgt cccccaaagc1621caaaagataa atcaaacctt ctagtcttca gaacgaatgg tgtttctgat gtaccaacct1681caccaacatt acagaggcct actcatatga accaaatggt gtttcacaaa atcagaaatg1741aagatttgat atttaatgaa agccttggcc aaggcacttt tacaaagatt tttaaaggcg1801tacgaagaga agtaggagac tacggtcaac tgcatgaaac agaagttctt ttaaaagttc1861tggataaagc acacagaaac tattcagagt ctttctttga agcagcaagt atgatgagca1921agctttctca caagcatttg gttttaaatt atggagtatg tgtctgtgga gacgagaata1981ttctggttca ggagtttgta aaatttggat cactagatac atatctgaaa aagaataaaa2041attgtataaa tatattatgg aaacttgaag ttgctaaaca gttggcatgg gccatgcatt2101ttctagaaga aaacaccctt attcatggga atgtatgtgc caaaaatatt ctgcttatca2161gagaagaaga caggaagaca ggaaatcctc ctttcatcaa acttagtgat cctggcatta2221gtattacagt tttgccaaag gacattcttc aggagagaat accatgggta ccacctgaat2281gcattgaaaa tcctaaaaat ttaaatttgg caacagacaa atggagtttt ggtaccactt2341tgtgggaaat ctgcagtgga ggagataaac ctctaagtgc tctggattct caaagaaagc2401tacaalttta tgaagatagg catcagcttc ctgcaccaaa gtgggcagaa ttagcaaacc2461ttataaataa ttgtatggat tatgaaccag atttcaggcc ttctttcaga gccatcatac2521gagatcttaa cagtttgttt actccagatt atgaactatt aacagaaaat gacatgttac2581caaatatgag gataggtgcc ctggggtttt ctggtgcctt tgaagaccgg gatcctacac2641agtttgaaga gagacatttg aaatttctac agcaacttgg caagggtaat tttgggagtg2701tggagatgtg ccggtatgac cctctacagg acaacactgg ggaggtggtc gctgtaaaaa2761agcttcagca tagtactgaa gagcacctaa gagactttga aagggaaatt gaaatcctga2821aatccctaca gcatgacaac attgtaaagt acaagggagt gtgctacagt gctggtcggc2881gtaatctaaa attaattatg gaatatttac catatggaag tttacgagac tatcttcaaa2941aacataaaga acggatagat cacataaaac ttctgcagta cacatctcag atatgcaagg3001gtatggagta tcttggtaca aaaaggtata tccacaggga tctggcaacg agaaatatat3061tggtggagaa cgagaacaga gttaaaattg gagattttgg gttaaccaaa gtcttgccac3121aagacaaaga atactataaa gtaaaagaac ctggtgaaag tcccatattc tggtatgctc3181cagaatcact gacagagagc aagttttctg tggcctcaga tgtttggagc tttggagtgg3241ttctgtatga acttttcaca tacattgaga agagtaaaag tccaccagcg gaatttatgc3301gtatgattgg caatgacaaa caaggacaga tgatcgtgtt ccatttgata gaacttttga3361agaataatgg aagattacca agaccagatg gatgcccaga tgagatctat atgatcatga3421cagaatgctg gaacaataat gtaaatcaac gcccctcctt tagggatcta gctcttcgag3481tggatcaaat aagggataac atggctggat gaaagaaatg accttcattc tgagaccaaa3541gtagatttac agaacaaagt tttatatttc acattgctgt ggactattat tacatatatc3601attattatat aaatcatgat gctagccagc aaagatgtga aaatatctgc tcaaaacttt3661caaagtttag taagtttttc ttcatgaggc caccagtaaa agacattaat gagaattcct3721tagcaaggat tttgtaagaa gtttcttaaa cattgtcagt taacatcact cttgtctggc3781aaaagaaaaa aaatagactt tttcaactca gctttttgag acctgaaaaa attattatgt3841aaattttgca atgttaaaga tgcacagaat atgtatgtat agtttttacc acagtggatg3901tataatacct tggcatcttg tgtgatgttt tacacacatg agggctggtg ttcattaata3961ctgttttcta atttttccat agttaatcta taattaatta cttcactata caaacaaatt4021aagatgttca gataattgaa taagtacctt tgtgtccttg ttcatttata tcgctggcca4081gcattataag caggtgtata cttttagctt gtagttccat gtactgtaaa tatttttcac4141ataaagggaa caaatgtcta gttttatttg tataggaaat ttccctgacc ctaaataata4201cattttgaaa tgaaacaagc ttacaaagat ataatctatt ttattatggt ttcccttgta4261tctatttgtg gtgaatgtgt tttttaaatg gaactatctc caaatttttc taagactact4321atgaacagtt ttcttttaaa attttgagat taagaatgcc aggaatattg tcatcctttg4381agctgctgac tgccaataac attcttcgat ctctgggatt tatgctcatg aactaaattt4441aagcttaagc cataaaatag attagattgt tttttaaaaa tggatagctc attaagaagt4501gcagcaggtt aagaattttt tcctaaagac tgtatatttg aggggtttca gaattttgca4561ttgcagtcat agaagagatt tatttccttt ttagagggga aatgaggtaa ataagtaaaa4621aagtatgctt gttaatttta ttcaagaatg ccagtagaaa attcataacg tgtatcttta4681agaaaaatga gcatacatct taaatctttt caattaagta taaggggttg ttcgttgttg4741tcatttgtta tagtgctact ccactttaga caccatagct aaaataaaat atggtgggtt4801ttgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gttatttata caaaacttaa4861aatacttgct gttttgatta aaaagaaaat agtttcttac tttaHuman JAK2 mRNA Variant 4(SEQ ID NO: 12)1gggagtggtg tggggctgca ggaaggagag aggaagagga gcagaagggg gcagcagcgg61acgccgctaa cggcctccct cggcgctgac aggctgggcc ggcgcccggc tcgcttgggt121gttcgcgtcg ccacttcggc ttctcggccg gtcgggcccc tcggcccggg cttgcggcgc181gcgtcggggc tgagggctgc tgcggcgcag ggagaggcct ggtcctcgct gccgagggat241gtgagtggga gctgagccca cactggaggg cccccgaggg cccagcctgg aggtcgttca301gagccgtgcc cgtcccgggg cttcgcagac cttgacccgc cgggtaggag ccgcccctgc361gggctcgagg gcgcgctctg gtcgcccgat ctgtgtagcc ggcaaatgtt ctgaaaaaga421ctctgcatgg gaatggcctg ccttacgatg acagaaatgg agggaacatc cacctcttct481atatatcaga atggtgatat ttctggaaat gccaattcta tgaagcaaat agatccagtt541cttcaggtgt atctttacca ttcccttggg aaatctgagg cagattatct gacctttcca601tctggggagt atgttgcaga agaaatctgt attgctgctt ctaaagcttg tggtatcaca661cctgtgtatc ataatatgtt tgctttaatg agtgaaacag aaaggatctg gtatccaccc721aaccatgtct tccatataga tgagtcaacc aggcataatg tactctacag aataagattt781tactttcctc gttggtattg cagtggcagc aacagagcct atcggcatgg aatatctcga841ggtgctgaag ctcctcttct tgatgacttt gtcatgtctt acctctttgc tcagtggcgg901catgattttg tgcacggatg gataaaagta cctgtgactc atgaaacaca ggaagaatgt961cttgggatgg cagtgttaga tatgatgaga atagccaaag aaaacgatca aaccccactg1021gccatctata actctatcag ctacaagaca ttcttaccaa aatgtattcg agcaaagatc1081caagactatc atattttgac aaggaagcga ataaggtaca gatttcgcag atttattcag1141caattcagcc aatgcaaagc cactgccaga aacttgaaac ttaagtatct tataaatctg1201gaaactctgc agtctgcctt ctacacagag aaatttgaag taaaagaacc tggaagtggt1261ccttcaggtg aggagatttt tgcaaccatt ataataactg gaaacggtgg aattcagtgg1321tcaagaggga aacataaaga aagtgagaca ctgacagaac aggatttaca gttatattgc1381gttttaccta atattattga tgtcagtatt aagcaagcaa accaagaggg ttcaaatgaa1441agccgagttg taactatcca taagcaagat ggtaaaaatc tggaaattga acttagctca1501ttaagggaag ctttgtcttt cgtgtcatta attgatggat attatagatt aactgcagat1561gcacatcatt acctctgtaa agaagtagca cctccagccg tgcttgaaaa tatacaaagc1621aactgtcatg gcccaatttc gatggatttt gccattagta aactgaagaa agcaggtaat1681cagactggac tgtatgtact tcgatgcagt cctaaggact ttaataaata ttttttgact1741tttgctgtcg agcgagaaaa tgtcattgaa tataaacact gtttgattac aaaaaatgag1801aatgaagagt acaacctcag tgggacaaag aagaacttca gcagtcttaa agatcttttg1861aattgttacc agatggaaac tgttcgctca gacaatataa ttttccagtt tactaaatgc1921tgtcccccaa agccaaaaga taaatcaaac cttctagtct tcagaacgaa tggtgtttct1981gatgtaccaa cctcaccaac attacagagg cctactcata tgaaccaaat ggtgtttcac2041aaaatcagaa atgaagattt gatatttaat gaaagccttg gccaaggcac ttttacaaag2101atttttaaag gcgtacgaag agaagtagga gactacggtc aactgcatga aacagaagtt2161cttttaaaag ttctggataa agcacacaga aactattcag agtctttctt tgaagcagca2221agtatgatga gcaagctttc tcacaagcat ttggttttaa attatggagt atgtgtctgt2281ggagacgaga atattctggt tcaggagttt gtaaaatttg gatcactaga tacatatctg2341aaaaagaata aaaattgtat aaatatatta tggaaacttg aagttgctaa acagttggca2401tgggccatgc attttctaga agaaaacacc cttattcatg ggaatgtatg tgccaaaaat2461attctgctta tcagagaaga agacaggaag acaggaaatc ctcctttcat caaacttagt2521gatcctggca ttagtattac agttttgcca aaggacattc ttcaggagag aataccatgg2581gtaccacctg aatgcattga aaatcctaaa aatttaaatt tggcaacaga caaatggagt2641tttggtacca ctttgtggga aatctgcagt ggaggagata aacctctaag tgctctggat2701tctcaaagaa agctacaatt ttatgaagat aggcatcagc ttcctgcacc aaagtgggca2761gaattagcaa accttataaa taattgtatg gattatgaac cagatttcag gccttctttc2821agagccatca tacgagatct taacagtttg tttactccag attatgaact attaacagaa2881aatgacatgt taccaaatat gaggataggt gccctggggt tttctggtgc ctttgaagac2941cgggatccta cacagtttga agagagacat ttgaaatttc tacagcaact tggcaagggt3001aattttggga gtgtggagat gtgccggtat gaccctctac aggacaacac tggggaggtg3061gtcgctgtaa aaaagcttca gcatagtact gaagagcacc taagagactt tgaaagggaa3121attgaaatcc tgaaatccct acagcatgac aacattgtaa agtacaaggg agtgtgctac3181agtgctggtc ggcgtaatct aaaattaatt atggaatatt taccatatgg aagtttacga3241gactatcttc aaaaacataa agaacggata gatcacataa aacttctgca gtacacatct3301cagatatgca agggtatgga gtatcttggt acaaaaaggt atatccacag ggatctggca3361acgagaaata tattggtgga gaacgagaac agagttaaaa ttggagattt tgggttaacc3421aaagtcttgc cacaagacaa agaatactat aaagtaaaag aacctggtga aagtcccata3481ttctggtatg ctccagaatc actgacagag agcaagtttt ctgtggcctc agatgtttgg3541agctttggag tggttctgta tgaacttttc acatacattg agaagagtaa aagtccacca3601gcggaattta tgcgtatgat tggcaatgac aaacaaggac agatgatcgt gttccatttg3661atagaacttt tgaagaataa tggaagatta ccaagaccag atggatgccc agatgagatc3721tatatgatca tgacagaatg ctggaacaat aatgtaaatc aacgcccctc ctttagggat3781ctagctcttc gagtggatca aataagggat aacatggctg gatgaaagaa atgaccttca3841ttctgagacc aaagtagatt tacagaacaa agttttatat ttcacattgc tgtggactat3901tattacatat atcattatta tataaatcat gatgctagcc agcaaagatg tgaaaatatc3961tgctcaaaac tttcaaagtt tagtaagttt ttcttcatga ggccaccagt aaaagacatt4021aatgagaatt ccttagcaag gattttgtaa gaagtttctt aaacattgtc agttaacatc4081actcttgtct ggcaaaagaa aaaaaataga ctttttcaac tcagcttttt gagacctgaa4141aaaattatta tgtaaatttt gcaatgttaa agatgcacag aatatgtatg tatagttttt4201accacagtgg atgtataata ccttggcatc ttgtgtgatg ttttacacac atgagggctg4261gtgttcatta atactgtttt ctaatttttc catagttaat ctataattaa ttacttcact4321atacaaacaa attaagatgt tcagataatt gaataagtac ctttgtgtcc ttgttcattt4381atatcgctgg ccagcattat aagcaggtgt atacttttag cttgtagttc catgtactgt4441aaatattttt cacataaagg gaacaaatgt ctagttttat ttgtatagga aatttccctg4501accctaaata atacattttg aaatgaaaca agcttacaaa gatataatct attttattat4561ggtttccctt gtatctattt gtggtgaatg tgttttttaa atggaactat ctccaaattt4621ttctaagact actatgaaca gttttctttt aaaattttga gattaagaat gccaggaata4681ttgtcatcct ttgagctgct gactgccaat aacattcttc gatctctggg atttatgctc4741atgaactaaa tttaagctta agccataaaa tagattagat tgttttttaa aaatggatag4801ctcattaaga agtgcagcag gttaagaatt ttttcctaaa gactgtatat ttgaggggtt4861tcagaatttt gcattgcagt catagaagag atttatttcc tttttagagg ggaaatgagg4921taaataagta aaaaagtatg cttgttaatt ttattcaaga atgccagtag aaaattcata4981acgtgtatct ttaagaaaaa tgagcataca tcttaaatct tttcaattaa gtataagggg5041ttgttcgttg ttgtcatttg ttatagtgct actccacttt agacaccata gctaaaataa5101aatatggtgg gttttgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttattt5161atacaaaact taaaatactt gctgttttga ttaaaaagaa aatagtttct tactttaHuman JAK3 mRNA(SEQ ID NO: 13)1cacacaggaa ggagccgagt gggactttcc tctcgctgcc tcccggctct gcccgccctt61cgaaagtcca gggtccctgc ccgctaggca agttgcactc atggcacctc caagtgaaga121gacgcccctg atccctcagc gttcatgcag cctcttgtcc acggaggctg gtgccctgca181tgtgctgctg cccgctcggg gccccgggcc cccccagcgc ctatctttct cctttgggga241ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc agcggcatcc tgcctgtgta301ccactccctc tttgctctgg ccacggagga cctgtcctgc tggttccccc cgagccacat361cttctccgtg gaggatgcca gcacccaagt cctgctgtac aggattcgct tttacttccc421caattggttt gggctggaga agtgccaccg cttcgggcta cgcaaggatt tggccagtgc481tatccttgac ctgccagtcc tggagcacct ctttgcccag caccgcagtg acctggtgag541tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt gagtgtctca gcctggccgt601gttggacctg gcccggatgg cgcgagagca ggcccagcgg ccgggagagc tgctgaagac661tgtcagctac aaggcctgcc tacccccaag cctgcgcgac ctgatccagg gcctgagctt721cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc ctgcgccgcg tggccgcctg781ccaggcagac cggcactcgc tcatggccaa gtacatcatg gacctggagc ggctggatcc841agccggggcc gccgagacct tccacgtggg cctccctggg gcccttggtg gccacgacgg901gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc tggacccagg gagaacagga961ggtcctccag cccttctgcg actttccaga aatcgtagac attagcatca agcaggcccc1021gcgcgttggc ccggccggag agcaccgcct ggtcactgtt accaggacag acaaccagat1081tttagaggcc gagttcccag ggctgcccga ggctctgtcg ttcgtggcgc tcgtggacgg1141ctacttccgg ctgaccacgg actcccagca cttcttctgc aaggaggtgg caccgccgag1201gctgctggag gaagtggccg agcagtgcca cggccccatc actctggact ttgccatcaa1261caagctcaag actgggggct cacgtcctgg ctcctatgtt ctccgccgca gcccccagga1321ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc cttggtcctg attataaggg1381ctgcctcatc cggcgcagcc ccacaggaac cttccttctg gttggcctca gccgacccca1441cagcagtctt cgagagctcc tggcaacctg ctgggatggg gggctgcacg tagatggggt1501ggcagtgacc ctcacttcct gctgtatccc cagacccaaa gaaaagtcca acctgatcgt1561ggtccagaga ggtcacagcc cacccacatc atccttggtt cagccccaat cccaatacca1621gctgagtcag atgacatttc acaagatccc tgctgacagc ctggagtggc atgagaacct1681gggccatggg tccttcacca agatttaccg gggctgtcgc catgaggtgg tggatgggga1741ggcccgaaag acagaggtgc tgctgaaggt catggatgcc aagcacaaga actgcatgga1801gtcattcctg gaagcagcga gcttgatgag ccaagtgtcg taccggcatc tcgtgctgct1861ccacggcgtg tgcatggctg gagacagcac catggtgcag gaatttgtac acctgggggc1921catagacatg tatctgcgaa aacgtggcca cctggtgcca gccagctgga agctgcaggt1981ggtcaaacag ctggcctacg ccctcaacta tctggaggac aaaggcctgc cccatggcaa2041tgtctctgcc cggaaggtgc tcctggctcg ggagggggct gatgggagcc cgcccttcat2101caagctgagt gaccctgggg tcagccccgc tgtgttaagc ctggagatgc tcaccgacag2161gatcccctgg gtggcccccg agtgtctccg ggaggcgcag acacttagct tggaagctga2221caagtggggc ttcggcgcca cggtctggga agtgtttagt ggcgtcacca tgcccatcag2281tgccctggat cctgctaaga aactccaatt ttatgaggac cggcagcagc tgccggcccc2341caagtggaca gagctggccc tgctgattca acagtgcatg gcctatgagc cggtccagag2401gccctccttc cgagccgtca ttcgtgacct caatagcctc atctcttcag actatgagct2461cctctcagac cccacacctg gtgccctggc acctcgtgat gggctgtgga atggtgccca2521gctctatgcc tgccaagacc ccacgatctt cgaggagaga cacctcaagt acatctcaca2581gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc tatgacccgc taggcgacaa2641tacaggtgcc ctggtggccg tgaaacagct gcagcacagc gggccagacc agcagaggga2701ctttcagcgg gagattcaga tcctcaaagc actgcacagt gatttcattg tcaagtatcg2761tggtgtcagc tatggcccgg gccgccagag cctgcggctg gtcatggagt acctgcccag2821cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc ctcgatgcca gccgcctcct2881tctctattcc tcgcagatct gcaagggcat ggagtacctg ggctcccgcc gctgcgtgca2941ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag gcacacgtca agatcgctga3001cttcggccta gctaagctgc tgccgcttga caaagactac tacgtggtcc gcgagccagg3061ccagagcccc attttctggt atgcccccga atccctctcg gacaacatct tctctcgcca3121gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc ttcacctact gcgacaaaag3181ctgcagcccc tcggccgagt tcctgcggat gatgggatgt gagcgggatg tccccgccct3241ctgccgcctc ttggaactgc tggaggaggg ccagaggctg ccggcgcctc ctgcctgccc3301tgctgaggtt cacgagctca tgaagctgtg ctgggcccct agcccacagg accggccatc3361attcagcgcc ctgggccccc agctggacat gctgtggagc ggaagccggg ggtgtgagac3421tcatgccttc actgctcacc cagagggcaa acaccactcc ctgtcctttt catagctcct3481gcccgcagac ctctggatta ggtctctgtt gactggctgt gtgaccttag gcccggagct3541gcccctctct gggcctcaga ggccttatga gggtcctcta cttcaggaac acccccatga3601cattgcattt gggggggctc ccgtggcctg tagaatagcc tgtggccttt gcaatttgtt3661aaggttcaag acagatgggc atatgtgtca gtggggctct ctgagtcctg gcccaaagaa3721gcaaggaacc aaatttaaga ctctcgcatc ttcccaaccc cttaagccct ggccccctga3781gtttcctttt ctgtctctct ctttttattt tttttatttt tatttttatt tttgagacag3841agcctcgctc tgttacccag ggtggagtgc agtggtgcga tctcggctca gtgcaacctc3901tgcttcccag gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggt3961gtgcaccacc acacccggct aatttttttt atttttaata gagatgaggt ttcaccatga4021tggccaggct gatctcgaac tcctaacctc aagtgatcct cccacctcag cctcccaaag4081tgttggaata ataggcatga gccactgcac ccaggctttt ttttttttaa atttattatt4141attattttta agagacagga tcttgctacg ttgcccaggc tggtcttgaa ctcctgggct4201acagtgatcc tcctgcctta tcctcctaaa tagctgggac tacagcacct agttttgagt4261ttcctgtctt atttccaatg gggacattca tgtagctttt tttttttttt tttttttgag4321acggagtctc gctctgtcgc ccaggctgga gtacagtggc gcaatctagg ctcactgcaa4381gctccgcctc ctgggttcac accattctct cgcctcagcc tcccaagtag ctgggactac4441aggcgcccgc caccacaccc ggctaatttt ttgtattttt agtagagacg gggtttcacc4501ttgttagcca ggatggtttc catctcctga cctcgtgatc tgcccgtctc ggcctcccaa4561agtgctggga ttacaggcat gagccactgc gcccggccct catgtagctt taaatgtatg4621atctgacttc tgctccccga tctctgtttc tctggaggaa gccaaggaca agagcagttg4681ctgtggctgg gactctgcct tttaggggag cccgtgtatc tctttgggat cctgaaaggg4741ggcaggaaag gctggggtcc cagtccaccc taatggtatc tgagtgtcct agggcttcag4801ttttcccacc tgtccaatgg gaccctttct gtcctcaccc tacaaggggc acaaagggat4861gacaccaaac ctggcaggaa cttttcacgc aatcaaggga aggaaaggca ttcctggcag4921agggaacagc atgccaagcg tgagaaggct cagagtaagg aggttaagag cccaagtatt4981ggagcctaca gttttgcccc ttccatgcag tgtgacagtg ggcaagttcc tttccctctc5041tgggtctcag ttctgtcccc tgcaaaatgg tcagagctta ccccttggct gtgcagggtc5101aactttctga ctggtgagag ggattctcat gcaggttaag cttctgctgc tcctcctcac5161ctgcaaagct tttctgccac ttttgcctcc ttggaaaact cttatccatc tctcaaaact5221ccagctacca catccttgca gccttccctc atataccccc actactactg tagccctgtc5281cttccctcca gccccactct ggccctgggg ctggggaagt gtctgtgtcc agctgtctcc5341cctgacctca gggttccttg ggggctgggc tgaggcctca gtacagaggg ggctctggaa5401atgtttgttg actgaataaa ggaattcagt ggaaaaaaaa aaaaaaaaaHuman JAK3 mRNA(SEQ ID NO: 14)1ccctctgacc aggactgagg ggctttttct ctctgtgccc caggcaagtt gcactcatta61tggaattccg gcggcccgct aggcaagttg cactcatggc acctccaagt gaagagacgc121ccctgatccc tcagcgttca tgcagcctct tgtccacgga ggctggtgcc ctgcatgtgc181tgctgcccgc tcgggccccg gggccccccc agcgcctatc tttctccttt ggggaccact241tggctgagga cctgtgcgtg caggctgcca aggccagcgg catcctgcct gtgtaccact301ccctctttgc tctggccacg gaggacctgt cctgctggtt ccccccgagc cacatcttct361ccgtggagga tgccagcacc caagtcctgc tgtacaggat tcgcttttac ttccccaatt421ggtttgggct ggagaagtgc caccgcttcg ggctacgcaa ggatttggcc agtgctatcc481ttgacctgcc agtcctggag cacctctttg cccagcaccg cagtgacctg gtgagtgggc541gcctccccgt gggcctcagt ctcaaggagc agggtgagtg tctcagcctg gccgtgttgg601acctggcccg gatggcgcga gagcaggccc agcggccggg agagctgctg aagactgtca661gctacaaggc ctgcctaccc ccaagcctgc gcgacctgat ccagggcctg agcttcgtga721cgcggagggc tattcggagg acggtgcgca gagccctgcc gcgcgtggcc gcctgccagg781cagaccggca ctcgctcatg gccaagtaca tcatggacct ggagcggctg gatccagccg841gggccgccga gaccttccac gtgggcctcc ctggggccct tggtggccac gacgggctgg901ggctgctccg cgtggctggt gacggcggca tcgcctggac ccagggagaa caggaggtcc961tccagccctt ctgcgacttt ccagaaatcg tagacattag catcaagcag gccccgcgcg1021ttggcccggc cggagagcac cgcctggtca ctgttaccag gacagacaac cagattttag1081aggccgagtt cccagggctg cccgaggctc tgtcgttcgt ggcgctcgtg gacggctact1141tccggctgac cacggactcc cagcacttct tctgcaagga ggtggcaccg ccgaggctgc1201tggaggaagt ggccgagcag tgccacggcc ccatcactct ggactttgcc atcaacaagc1261tcaagactgg gggctcacgt cctggctcct atgttctccg ccgcagcccc caggactttg1321acagcttcct cctcactgtc tgtgtccaga acccccttgg tcctgattat aagggctgcc1381tcatccggcg cagccccaca ggaaccttcc ttctggttgg cctcagccga ccccacagca1441gtcttcgaga gctcctggca acctgctggg atggggggct gcacgtagat ggggtggcag1501tgaccctcac ttcctgctgt atccccagac ccaaagaaaa gtccaacctg atcgtggtcc1561agagaggtca cagcccaccc acatcatcct tggttcagcc ccaatcccaa taccagctga1621gtcagatgac atttcacaag atccctgctg acagcctgga gtggcatgag aacctgggcc1681atgggtcctt caccaagatt taccggggct gtcgccatga ggtggtggat ggggaggccc1741gaaagacaga ggtgctgctg aaggtcatgg atgccaagca caagaactgc atggagtcat1801tcctggaagc agcgagcttg atgagccaag tgtcgtaccg gcatctcgtg ctgctccacg1861gcgtgtgcat ggctggagac agcaccatgg tgcaggaatt tgtacacctg ggggccatag1921acatgtatct gcgaaaacgt ggccacctgg tgccagccag ctggaagctg caggtggtca1981aacagctggc ctacgccctc aactatctgg aggacaaagg cctgccccat ggcaatgtct2041ctgcccggaa ggtgctcctg gctcgggagg gggctgatgg gagcccgccc ttcatcaagc2101tgagtgaccc tggggtcagc cccgctgtgt taagcctgga gatgctcacc gacaggatcc2161cctgggtggc ccccgagtgt ctccgggagg cgcagacact tagcttggaa gctgacaagt2221ggggcttcgg cgccacggtc tgggaagtgt ttagtggcgt caccatgccc atcagtgccc2281tggatcctgc taagaaactc caatlltatg aggaccggca gcagctgccg gcccccaagt2341ggacagagct ggccctgctg attcaacagt gcatggccta tgagccggtc cagaggccct2401ccttccgagc cgtcattcgt gacctcaata gcctcatctc ttcagactat gagctcctct2461cagaccccac acctggtgcc ctggcacctc gtgatgggct gtggaatggt gcccagctct2521atgcctgcca agaccccacg atcttcgagg agagacacct caagtacatc tcacagctgg2581gcaagggcaa ctttggcagc gtggagctgt gccgctatga cccgctagcc cacaatacag2641gtgccctggt ggccgtgaaa cagctgcagc acagcgggcc agaccagcag agggactttc2701agcgggagat tcagatcctc aaagcactgc acagtgattt cattgtcaag tatcgtggtg2761tcagctatgg cccgggccgg ccagagctgc ggctggtcat ggagtacctg cccagcggct2821gcttgcgcga cttcctgcag cggcaccgcg cgcgcctcga tgccagccgc ctccttctct2881attcctcgca gatctgcaag ggcatggagt acctgggctc ccgccgctgc gtgcaccgcg2941acctggccgc ccgaaacatc ctcgtggaga gcgaggcaca cgtcaagatc gctgacttcg3001gcctagctaa gctgctgccg cttgacaaag actactacgt ggtccgcgag ccaggccaga3061gccccatttt ctggtatgcc cccgaatccc tctcggacaa catcttctct cgccagtcag3121acgtctggag cttcggggtc gtcctgtacg agctcttcac ctactgcgac aaaagctgca3181gcccctcggc cgagttcctg cggatgatgg gatgtgagcg ggatgtcccc gccctctgcc3241gcctcttgga actgctggag gagggccaga ggctgccggc gcctcctgcc tgccctgctg3301aggttcacga gctcatgaag ctgtgctggg cccctagccc acaggaccgg ccatcattca3361gcgccctggg cccccagctg gacatgctgt ggagcggaag ccgggggtgt gagactcatg3421ccttcactgc tcacccagag ggcaaacacc actccctgtc cttttcatag ctcctgcccg3481cagacctctg gattaggtct ctgttgactg gctgtgtgac cttaggcccg gagctgcccc3541tctctgggcc tcagaggcct tatgagggtc ctctacttca ggaacacccc catgacattg3601catttggggg ggctcccgtg gcctgtagaa tagcctgtgg cctttgcaat ttgttaaggt3661tcaagacaga tgggcatatg tgtcagtggg gctctctgag tcctggccca aagaagcaag3721gaaccaaatt taagactctc gcatcttccc aaccccttaa gccctggccc cctgagtttc3781cttttctcgt ctctctcttt ttattttttt tatttttatt tttatttttg agacagagcc3841tcgctcgtta cccagggtgg agtgcagtgg tagcgatctc ggctcacagt gcaacctctg3901cttcccaggt tcaagcgatt ctcctgcctc agcctcccga gtagctggga ttacaggtgt3961gcaccaccac acccggctaa ttttttttat ttttaataga gatgaggttt caccatgatg4021gccaggctga tctcgaactc ctaacctcaa gtgatcctcc caccHuman TYK2 mRNA(SEQ ID NO: 15)1aagcagtagc tacccgcggg agcggggagg ggtccgggtt cgagcttgtg ttcccccgga61agggtgagtc tggacgcggg cgcggaagga gcgcggccgg aggtcctcag gaagaagccg121cggggactgg ctgcgcttga caggctgcac ttggatggga gcacctggtg cctcgggact181gctccgatgc ccgggtctgt gctgaatgtg taatatgcgg aactatattg aaacattaca241accatctttt gatggcaaca ccctgaggac ctcccttttc cagatgggga aactgaggcc301cagaattgct aagtggcttg cttgagttga cacagggagc tccaggactc accctcagct361gagccacctg ccgggagcat gcctctgcgc cactggggga tggccagggg cagtaagccc421gttggggatg gagcccagcc catggctgcc atgggaggcc tgaaggtgct tctgcactgg481gctggtccag gcggcgggga gccctgggtc actttcagtg agtcatcgct gacagctgag541gaagtctgca tccacattgc acataaagtt ggtatcactc ctccttgctt caatctcttt601gccctcttcg atgctcaggc ccaagtctgg ttgcccccaa accacatcct agagatcccc661agagatgcaa gcctgatgct atatttccgc ataaggtttt atttccggaa ctggcatggc721atgaatcctc gggaaccggc tgtgtaccgt tgtgggcccc caggaaccga ggcatcctca781gatcagacag cacaggggat gcaactcctg gacccagcct catttgagta cctctttgag841cagggcaagc atgagtttgt gaatgacgtg gcatcactgt gggagctgtc gaccgaggag901gagatccacc actttaagaa tgagagcctg ggcatggcct ttctgcacct ctgtcacctc961gctctccgcc atggcatccc cctggaggag gtggccaaga agaccagctt caaggactgc1021atcccgcgct ccttccgccg gcatatccgg cagcacagcg ccctgacccg gctgcgcctt1081cggaacgtct tccgcaggtt cctgcgggac ttccagccgg gccgactctc ccagcagatg1141gtcatggtca aatacctagc cacactcgag cggctggcac cccgcttcgg cacagagcgt1201gtgcccgtgt gccacctgag gctgctggcc caggccgagg gggagccctg ctacatccgg1261gacagtgggg tggcccctac agaccctggc cctgagtctg ctgctgggcc cccaacccac1321gaggtgctgg tgacaggcac tggtggcatc cagtggtggc cagtagagga ggaggtgaac1381aaggaggagg gttctagtgg cagcagtggc aggaaccccc aagccagcct gtttgggaag1441aaggccaagg ctcacaaggc agtcggccag ccggcagaca ggccgcggga gccactgtgg1501gcctacttct gtgacttccg ggacatcacc cacgtggtgc tgaaagagca ctgtgtcagc1561atccaccggc aggacaacaa gtgcctggag ctgagcttgc cttcccgggc tgcggcgctg1621tccttcgtgt cgctggtgga cggctatttc cgcctgacgg ccgactccag ccactacctg1681tgccacgagg tggctccccc acggctggtg atgagcatcc gggatgggat ccacggaccc1741ctgctggagc catttgtgca ggccaagctg cggcccgagg acggcctgta cctcattcac1801tggagcacca gccaccccta ccgcctgatc ctcacagtgg cccagcgtag ccaggcacca1861gacggcatgc agagcttgcg gctccgaaag ttccccattg agcagcagga cggggccttc1921gtgctggagg gctggggccg gtccttcccc agcgttcggg aacttggggc tgccttgcag1981ggctgcttgc tgagggccgg ggatgactgc ttctctctgc gtcgctgttg cctgccccaa2041ccaggagaaa cctccaatct catcatcatg cggggggctc gggccagccc caggacactc2101aacctcagcc agctcagctt ccaccgggtt gaccagaagg agatcaccca gctgtcccac2161ttgggccagg gcacaaggac caacgtgtat gagggccgcc tgcgagtgga gggcagcggg2221gaccctgagg agggcaagat ggatgacgag gaccccctcg tgcctggcag ggaccgtggg2281caggagctac gagtggtgct caaagtgctg gaccctagtc accatgacat cgccctggcc2341ttctacgaga cagccagcct catgagccag gtctcccaca cgcacctggc cttcgtgcat2401ggcgtctgtg tgcgcggccc tgaaaatatc atggtgacag agtacgtgga gcacggaccc2461ctggatgtgt ggctgcggag ggagcggggc catgtgccca tggcttggaa gatggtggtg2521gcccagcagc tggccagcgc cctcagctac ctggagaaca agaacctggt tcatggtaat2581gtgtgtggcc ggaacatcct gctggcccgg ctggggttgg cagagggcac cagccccttc2641atcaagctga gtgatcctgg cgtgggcctg ggcgccctct ccagggagga gcgggtggag2701aggatcccct ggctggcccc cgaatgccta ccaggtgggg ccaacagcct aagcaccgcc2761atggacaagt gggggtttgg cgccaccctc ctggagatct gctttgacgg agaggcccct2821ctgcagagcc gcagtccctc cgagaaggag catttctacc agaggcagca ccggctgccc2881gagccctcct gcccacagct ggccacactc accagccagt gtctgaccta tgagccaacc2941cagaggccat cattccgcac catcctgcgt gacctcaccc ggctgcagcc ccacaatctt3001gctgacgtct tgactgtgaa cccggactca ccggcgtcgg accctacggt tttccacaag3061cgctatttga aaaagatccg agatctgggc gagggtcact tcggcaaggt cagcttgtac3121tgctacgatc cgaccaacga cggcactggc gagatggtgg cggtgaaagc cctcaaggca3181gactgcggcc cccagcaccg ctcgggctgg aagcaggaga ttgacattct gcgcacgctc3241taccacgagc acatcatcaa gtacaagggc tgctgcgagg accaaggcga gaagtcgctg3301cagctggtca tggagtacgt gcccctgggc agcctccgag actacctgcc ccggcacagc3361atcgggctgg cccagctgct gctcttcgcc cagcagatct gcgagggcat ggcctatctg3421cacgcgcagc actacatcca ccgagaccta gccgcgcgca acgtgctgct ggacaacgac3481aggctggtca agatcgggga ctftggccta gccaaggccg tgcccgaagg ccacgagtac3541taccgcgtgc gcgaggatgg ggacagcccc gtgttctggt atgccccaga gtgcctgaag3601gagtataagt tctactatgc gtcagatgtc tggtccttcg gggtgaccct gtatgagctg3661ctgacgcact gtgactccag ccagagcccc cccacgaaat tccttgagct cataggcatt3721gctcagggtc agatgacagt tctgagactc actgagttgc tggaacgagg ggagaggctg3781ccacggcccg acaaatgtcc ctgtgaggtc tatcatctca tgaagaactg ctgggagaca3841gaggcgtcct ttcgcccaac cttcgagaac ctcataccca ttctgaagac agtccatgag3901aagtaccaag gccaggcccc ttcagtgttc agcgtgtgct gaggcacaat ggcagccctg3961cctgggagga ctggaccagg cagtggctgc agagggagcc tcctgctccc tgctccagga4021tgaaaccaag agggggatgt cagcctcacc cacaccgtgt gccttactcc tgtctagaga4081ccccacctct gtgaacttat ttttctttct tggccgtgag cctaaccatg atcttgaggg4141acccaacatt tgtaggggca ctaatccagc ccttaaatcc cccagcttcc aaacttgagg4201cccaccatct ccaccatctg gtaataaact catgttttct ctgctggaaa aaaaaaaaaa4261aa Inhibitory Nucleic Acids An antisense nucleic acid molecule can be complementary to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a JAK1, JAK2, JAK3, or TYK2 protein. Non-coding regions (5′ and 3′ untranslated regions) are the 5′ and 3′ sequences that flank the coding region in a gene and are not translated into amino acids. Based upon the sequences disclosed herein, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense nucleic acids to target a nucleic acid encoding a JAK1, JAK2, JAK3, or TYK2 protein described herein. Antisense nucleic acids targeting a nucleic acid encoding a JAK1, JAK2, JAK3, or TYK2 protein can be designed using the software available at the Integrated DNA Technologies website. An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides or more in length. An antisense oligonucleotide can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate an antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). The antisense nucleic acid molecules described herein can be prepared in vitro and administered to a mammal, e.g., a human. Alternatively, they can be generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a JAK1, JAK2, JAK3, or TYK2 protein to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarities to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense nucleic acid molecules can be delivered to a mammalian cell using a vector (e.g., a lentivirus, a retrovirus, or an adenovirus vector). An antisense nucleic acid can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual, β-units, the strands run parallel to each other (Gaultier et al.,Nucleic Acids Res.15:6625-6641, 1987). The antisense nucleic acid can also comprise a 2′-O-methylribonucleotide (Inoue et al.,Nucleic Acids Res.15:6131-6148, 1987) or a chimeric RNA-DNA analog (Inoue et al.,FEBS Lett215:327-330, 1987). Another example of an inhibitory nucleic acid is a ribozyme that has specificity for a nucleic acid encoding a JAK1, JAK2, JAK3, or TYK2 protein (e.g., specificity for a JAK1, JAK2, JAK3, or TYK2 mRNA, e.g., specificity for any one of SEQ ID NOs: 1-15). Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach,Nature334:585-591, 1988)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a JAK1, JAK2, JAK3, or TYK2 mRNA can be designed based upon the nucleotide sequence of any of the JAK1, JAK2, JAK3, or TYK2 mRNA sequences disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a JAK1, JAK2, JAK3, or TYK2 mRNA (see, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742). Alternatively, a JAK1, JAK2, JAK3, or TYK2 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al.,Science261:1411-1418, 1993. An inhibitory nucleic acid can also be a nucleic acid molecule that forms triple helical structures. For example, expression of a JAK1, JAK2, JAK3, or JAK4 polypeptide can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the JAK1, JAK2, JAK3, or TYK2 polypeptide (e.g., the promoter and/or enhancer, e.g., a sequence that is at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of the transcription initiation start state) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene, Anticancer Drug Des. 6(6):569-84, 1991; Helene,Ann. N.Y. Acad. Sci.660:27-36, 1992; and Maher, Bioassays 14(12):807-15, 1992. In various embodiments, inhibitory nucleic acids can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see, e.g., Hyrup et al.,Bioorganic Medicinal Chem.4(1):5-23, 1996). Peptide nucleic acids (PNAs) are nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs allows for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols (see, e.g., Perry-O'Keefe et al.,Proc. Natl. Acad. Sci. U.S.A.93:14670-675, 1996). PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation. The synthesis of PNA-DNA chimeras can be performed as described in Finn et al.,Nucleic Acids Res.24:3357-63, 1996. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5′ end of DNA (Mag et al.,Nucleic Acids Res.17:5973-88, 1989). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.,Nucleic Acids Res.24:3357-63, 1996). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,Bioorganic Med. Chem. Lett.5:1119-11124, 1975). In some embodiments, the inhibitory nucleic acids can include other appended groups such as peptides, or agents facilitating transport across the cell membrane (see, Letsinger et al.,Proc. Natl. Acad. Sci. U.S.A.86:6553-6556, 1989; Lemaitre et al.,Proc. Natl. Acad. Sci. U.S.A.84:648-652, 1989; and WO 88/09810). In addition, the inhibitory nucleic acids can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al.,Bio/Techniques6:958-976, 1988) or intercalating agents (see, e.g., Zon, Pharm. Res., 5:539-549, 1988). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. Another means by which expression of a JAK1, JAK2, JAK3, or TYK2 mRNA can be decreased in a mammalian cell is by RNA interference (RNAi). RNAi is a process in which mRNA is degraded in host cells. To inhibit an mRNA, double-stranded RNA (dsRNA) corresponding to a portion of the gene to be silenced (e.g., a gene encoding a JAK1, JAK2, JAK3, or TYK2 polypeptide) is introduced into a mammalian cell. The dsRNA is digested into 21-23 nucleotide-long duplexes called short interfering RNAs (or siRNAs), which bind to a nuclease complex to form what is known as the RNA-induced silencing complex (or RISC). The RISC targets the homologous transcript by base pairing interactions between one of the siRNA strands and the endogenous mRNA. It then cleaves the mRNA about 12 nucleotides from the 3′ terminus of the siRNA (see Sharp et al.,Genes Dev.15:485-490, 2001, and Hammond et al.,Nature Rev. Gen.2:110-119, 2001). RNA-mediated gene silencing can be induced in a mammalian cell in many ways, e.g., by enforcing endogenous expression of RNA hairpins (see, Paddison et al.,Proc. Natl. Acad. Sci. U.S.A.99:1443-1448, 2002) or, as noted above, by transfection of small (21-23 nt) dsRNA (reviewed in Caplen,Trends Biotech.20:49-51, 2002). Methods for modulating gene expression with RNAi are described, e.g., in U.S. Pat. No. 6,506,559 and US 2003/0056235, which are hereby incorporated by reference. Standard molecular biology techniques can be used to generate siRNAs. Short interfering RNAs can be chemically synthesized, recombinantly produced, e.g., by expressing RNA from a template DNA, such as a plasmid, or obtained from commercial vendors, such as Dharmacon. The RNA used to mediate RNAi can include synthetic or modified nucleotides, such as phosphorothioate nucleotides. Methods of transfecting cells with siRNA or with plasmids engineered to make siRNA are routine in the art. The siRNA molecules used to decrease expression of a JAK1, JAK2, JAK3, or TYK2 mRNA can vary in a number of ways. For example, they can include a 3′ hydroxyl group and strands of 21, 22, or 23 consecutive nucleotides. They can be blunt ended or include an overhanging end at either the 3′ end, the 5′ end, or both ends. For example, at least one strand of the RNA molecule can have a 3′ overhang from about 1 to about 6 nucleotides (e.g., 1-5, 1-3, 2-4, or 3-5 nucleotides (whether pyrimidine or purine nucleotides) in length. Where both strands include an overhang, the length of the overhangs may be the same or different for each strand. To further enhance the stability of the RNA duplexes, the 3′ overhangs can be stabilized against degradation (by, e.g., including purine nucleotides, such as adenosine or guanosine nucleotides or replacing pyrimidine nucleotides by modified analogues (e.g., substitution of uridine 2-nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNAi). Any siRNA can be used in the methods of decreasing a JAK1, JAK2, JAK3, or TYK2 mRNA, provided it has sufficient homology to the target of interest (e.g., a sequence present in any one of SEQ ID NOs: 1-15, e.g., a target sequence encompassing the translation start site or the first exon of the mRNA). There is no upper limit on the length of the siRNA that can be used (e.g., the siRNA can range from about 21 base pairs of the gene to the full length of the gene or more (e.g., about 20 to about 30 base pairs, about 50 to about 60 base pairs, about 60 to about 70 base pairs, about 70 to about 80 base pairs, about 80 to about 90 base pairs, or about 90 to about 100 base pairs). Non-limiting examples of JAK inhibitors that are short interfering RNAs (siRNAs) are described in Cook et al.,Blood123:2826-2837, 2014. Non-limiting examples of JAK inhibitors that are short hairpin RNAs (shRNAs) are described in Koppikar et al.,Nature489(7414):155-159, 2012). In certain embodiments, a therapeutically effective amount of an inhibitory nucleic acid targeting a nucleic acid encoding a JAK1, JAK2, JAK3, or TYK2 protein can be administered to a subject (e.g., a human subject) in need thereof. In some embodiments, the inhibitory nucleic acid can be about 10 nucleotides to about 40 nucleotides (e.g., about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 10 to about 15 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, or 40 nucleotides) in length. One skilled in the art will appreciate that inhibitory nucleic acids may comprise at least one modified nucleic acid at either the 5′ or 3′end of DNA or RNA. As is known in the art, the term “thermal melting point (Tm)” refers to the temperature, under defined ionic strength, pH, and inhibitory nucleic acid concentration, at which 50% of the inhibitory nucleic acids complementary to the target sequence hybridize to the target sequence at equilibrium. In some embodiments, an inhibitory nucleic acid can bind specifically to a target nucleic acid under stingent conditions, e.g., those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In some embodiments of any of the inhibitory nucleic acids described herein, the inhibitory nucleic acid binds to a target nucleic acid (e.g., a nucleic acid encoding JAK1, JAK2, JAK3, or TYK2) with a Tmof greater than 20° C., greater than 22° C., greater than 24° C., greater than 26° C., greater than 28° C., greater than 30° C., greater than 32° C., greater than 34° C., greater than 36° C., greater than 38° C., greater than 40° C., greater than 42° C., greater than 44° C., greater than 46° C., greater than 48° C., greater than 50° C., greater than 52° C., greater than 54° C., greater than 56° C., greater than 58° C., greater than 60° C., greater than 62° C., greater than 64° C., greater than 66° C., greater than 68° C., greater than 70° C., greater than 72° C., greater than 74° C., greater than 76° C., greater than 78° C., or greater than 80° C., e.g., as measured in phosphate buffered saline using a UV spectrophotometer. In some embodiments of any of the inhibitor nucleic acids described herein, the inhibitory nucleic acid binds to a target nucleic acid (e.g., a nucleic acid encoding a JAK1, JAK2, JAK3, or TYK2) with a Tmof about 20° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., about 32° C., about 30° C., about 28° C., about 26° C., about 24° C., or about 22° C. (inclusive); about 22° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., about 32° C., about 30° C., about 28° C., about 26° C., or about 24° C. (inclusive); about 24° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., about 32° C., about 30° C., about 28° C., or about 26° C. (inclusive); about 26° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., about 32° C., about 30° C., or about 28° C. (inclusive); about 28° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., about 32° C., or about 30° C. (inclusive); about 30° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., about 34° C., or about 32° C. (inclusive); about 32° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., about 36° C., or about 34° C. (inclusive); about 34° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., about 38° C., or about 36° C. (inclusive); about 36° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., about 40° C., or about 38° C. (inclusive); about 38° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., about 42° C., or about 40° C. (inclusive); about 40° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., about 44° C., or about 42° C. (inclusive); about 42° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., about 46° C., or about 44° C. (inclusive); about 44° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., about 48° C., or about 46° C. (inclusive); about 46° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., about 50° C., or about 48° C. (inclusive); about 48° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., about 52° C., or about 50° C. (inclusive); about 50° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., about 54° C., or about 52° C. (inclusive); about 52° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., about 56° C., or about 54° C. (inclusive); about 54° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., about 58° C., or about 56° C. (inclusive); about 56° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., about 60° C., or about 58° C. (inclusive); about 58° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., about 62° C., or about 60° C. (inclusive); about 60° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., about 64° C., or about 62° C. (inclusive); about 62° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., about 66° C., or about 64° C. (inclusive); about 64° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., about 68° C., or about 66° C. (inclusive); about 66° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., about 70° C., or about 68° C. (inclusive); about 68° C. to about 80° C., about 78° C., about 76° C., about 74° C., about 72° C., or about 70° C. (inclusive); about 70° C. to about 80° C., about 78° C., about 76° C., about 74° C., or about 72° C. (inclusive); about 72° C. to about 80° C., about 78° C., about 76° C., or about 74° C. (inclusive); about 74° C. to about 80° C., about 78° C., or about 76° C. (inclusive); about 76° C. to about 80° C. or about 78° C. (inclusive); or about 78° C. to about 80° C. (inclusive), In some embodiments, the inhibitory nucleic acid can be formulated in a nanoparticle (e.g., a nanoparticle including one or more synthetic polymers, e.g., Patil et al.,Pharmaceutical Nanotechnol.367:195-203, 2009; Yang et al.,ACS Appl. Mater. Interfaces, doi:10.1021/acsami.6b16556, 2017; Perepelyuk et al.,Mol. Ther. Nucleic Acids6:259-268, 2017). In some embodiments, the nanoparticle can be a mucoadhesive particle (e.g., nanoparticles having a positively-charged exterior surface) (Andersen et al.,Methods Mol. Biol.555:77-86, 2009). In some embodiments, the nanoparticle can have a neutrally-charged exterior surface. In some embodiments, the inhibitory nucleic acid can be formulated, e.g., as a liposome (Buyens et al.,J. Control Release158(3): 362-370, 2012; Scarabel et al.,Expert Opin. Drug Deliv.17:1-14, 2017), a micelle (e.g., a mixed micelle) (Tangsangasaksri et al.,BioMacromolecules17:246-255, 2016; Wu et al.,Nanotechnology, doi: 10.1088/1361-6528/aa6519, 2017), a microemulsion (WO 11/004395), a nanoemulsion, or a solid lipid nanoparticle (Sahay et al.,Nature Biotechnol.31:653-658, 2013; and Lin et al.,Nanomedicine9(1):105-120, 2014). Additional exemplary structural features of inhibitory nucleic acids and formulations of inhibitory nucleic acids are described in US 2016/0090598. In some embodiments, a pharmaceutical composition can include a sterile saline solution and one or more inhibitory nucleic acid (e.g., any of the inhibitory nucleic acids described herein). In some examples, a pharmaceutical composition consists of a sterile saline solution and one or more inhibitory nucleic acid (e.g., any of the inhibitory nucleic acids described herein). In certain embodiments, the sterile saline is a pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition can include one or more inhibitory nucleic acid (e.g., any of the inhibitory nucleic acids described herein) and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more inhibitory nucleic acid (e.g., any of the inhibitory nucleic acids described herein) and sterile water. In certain embodiments, a pharmaceutical composition includes one or more inhibitory nucleic acid (e.g., any of the inhibitory nucleic acids described herein) and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more inhibitory nucleic acids (e.g., any of the inhibitory nucleic acids described herein) and sterile phosphate-buffered saline (PBS). In some examples, the sterile saline is a pharmaceutical grade PBS. In certain embodiments, one or more inhibitory nucleic acids (e.g., any of the inhibitory nucleic acids described herein) may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. Pharmaceutical compositions including one or more inhibitory nucleic acids encompass any pharmaceutically acceptable salts, esters, or salts of such esters. Non-limiting examples of pharmaceutical compositions include pharmaceutically acceptable salts of inhibitory nucleic acids. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. Also provided herein are prodrugs that can include additional nucleosides at one or both ends of an inhibitory nucleic acid which are cleaved by endogenous nucleases within the body, to form the active inhibitory nucleic acid. Lipid moieties can be used to formulate an inhibitory nucleic acid. In certain such methods, the inhibitory nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, inhibitory nucleic acid complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of an inhibitory nucleic acid to a particular cell or tissue in a mammal. In some examples, a lipid moiety is selected to increase distribution of an inhibitory nucleic acid to fat tissue in a mammal. In certain embodiments, a lipid moiety is selected to increase distribution of an inhibitory nucleic acid to muscle tissue. In certain embodiments, pharmaceutical compositions provided herein comprise one or more inhibitory nucleic acid and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In some examples, a pharmaceutical composition provided herein includes liposomes and emulsions. Liposomes and emulsions can be used to formulate hydrophobic compounds. In some examples, certain organic solvents such as dimethylsulfoxide are used. In some examples, a pharmaceutical composition provided herein includes one or more tissue-specific delivery molecules designed to deliver one or more inhibitory nucleic acids to specific tissues or cell types in a mammal. For example, a pharmaceutical composition can include liposomes coated with a tissue-specific antibody. In some embodiments, a pharmaceutical composition provided herein can include a co-solvent system. Examples of such co-solvent systems include benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. As can be appreciated, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. In some examples, a pharmaceutical composition can be formulated for oral administration. In some examples, pharmaceutical compositions are formulated for buccal administration. In some examples, a pharmaceutical composition is formulated for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In some of these embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In some examples, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In some examples, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, and the like. Some pharmaceutical compositions for injection are formulated in unit dosage form, e.g., in ampoules or in multi-dose containers. Some pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Small Molecules In some embodiments, the JAK inhibitor is a small molecule. In some embodiments, the JAK inhibitory agent is a pan-JAK inhibitor (e.g., 3-O-methylthespesilactam (Li et al.,Biochem. Pharmacol.86(10):1411-8, 2013)). In some embodiments, the JAK inhibitor is a JAK1 and JAK2 inhibitor. In some embodiments, the JAK1 and JAK2 inhibitor is ruxolitinib (Jakafi®, Jakavi®, INCB018424) (Harrison et al.,N. Engl. J. Med.366:787-798, 2012; Pieri et al.,Am. J. Hematol.92(2):187-195, 2017; Mackay-Wiggan et al.,JCI Insight1(15):e89790, 2016; Rudolph et al.,Leukemia30(10):2119-2123, 2016; Furqan et al.,Biomark Res.1(1):5, 2013), baricitinib (INCB028050, LY3009104) (Gras,Drugs Today(Barc) 52(10):543-550, 2016; Smolen et al.,Ann. Rheum. Dis.76(4):694-700, 2016; Kubo et al.,Expert. Rev. Clin. Immunol.12(9):911-919, 2016; Fridman et al.,J. Immunol.84(9):5298-5307, 2010), AZD1480 (Guschin et al.,EMBO J.14:1421-1429, 1995; Ioannidis et al.,J Med. Chem.54: 262-276, 2011; Moisan et al.,Nat. Cell Biol.17(1):57-67, 2015; Qin et al.,J Neurosci.36(18):5144059, 2016; Jiang et al.,Biochem. Biophys. Res. Commun.458(4):908-912, 2015; Verstovsek et al.,Leuk. Res.39(2):157-163, 2015; Plimack et al.,Oncologist18(7): 819-820, 2013; Yan et al.,Oncotarget4(3):433-445, 2013), filgotinib (GLPG0634, G146034) (Vermeire et al.,Lancet389(10066):266-275, 2017; Menet et al.,J. Med. Chem.57(22):9323-9342, 2014; Van Rompaey et al.,J. Immunol.191(7):3568-3577, 2013; Namour et al.,Clin. Pharmacokinet.54(8):859-874, 2015), momelotinib (GS-0387, CYT387) (Pardanani et al.,Leukemia23: 1441-1445, 2009; Gupta et al.,Haematologica102(1):94-102, 2017; Hu et al.,Mol. Pharm.13(2):689-697, 2016; Abubaker et al.,BMC Cancer14: 317, 2014; Durmus et al.,Pharmacol. Res.76:9-16, 2013; Pardanani et al.,Leukemia27(6): 1322-1327, 2013; Monaghan et al.,Leukemia25(12):1891-1899, 2011; Tyner et al.,Blood115(25):5232-5240, 2010). In some embodiments, the JAK inhibitory agent is a JAK1 inhibitor (e.g., GSK2586184 (Kahl et al.,Lupus25(13): 1420-1430, 2016; Ludbrook et al.,Br. J Dermatol.174(5):985-995, 2016; van Vollenhoven et al.,Lupus24(6): 648-649, 2015), oclacitinib (PF03394197, Apoquel®) (Gonzales et al.,J Vet. Pharmacol. Ther.37(4):317-324, 2014; Collard et al.,J Vet. Pharmacol. Ther.37(3):279-285, 2014; Cosgrove et al.,Vet. Dermatol.24(6):587-597, 2013), upadacitinib (ABT494) (Kremer et al.,Arthritis Rheumatol.68(12):2867-2877, 2016; Mohamed et al.,Clin. Pharmaco.55(12): 1547-1558, 2016), GLG0778 (O'Shea et al.,Ann. Rev. Med.66(1):311-28, 2015; Schwartz et al.,Nat. Rev. Rheum.12: 25-36, 2016), INCB039110 (Mascarenhas et al.,Haematologica102(2):327-335, 2017; Bissonnette et al.,J. Dermatolog. Treat.27(4):332-338, 2016; Rosenthal et al.,Exp. Opin. Pharmacother.15(9):1265-1276, 2014), PF04965842 (Gadina et al.,Curr. Opin. Rheumatol.26(2):237-243, 2014; Degryset et al.,J. Hematol. Oncol.8:91, 2015); SAR-20347 (Works et al.,J Immunol.193(7):3278-3287, 2014)). In some embodiments, the JAK inhibitory agent is a JAK2 inhibitor (e.g., CEP-33779 (Dugan et al.,J Med. Chem.55(11):5243-5254, 2012; Seavey et al.,Mol. Cancer Ther.11(4):984-993, 2012; Stump et al.,Arthritis Res. Ther.13(2):R68, 2011), fedratinib (TG101348, SAR302503) (Pardanani et al.,J. Clin. Oncol.29:789-796, 2011; Jamieson et al.,J. Transl. Med.13:294, 2015; Zhang et al.,Oncotarget6(16):14329-14343, 2015; Wernig et al.,Blood105:4508-4515, 2008); lestaurtinib (CEP-701) (Hexnet et al.,Blood111:5663-5671, 2008; Santos et al.,Blood115: 1131-1136, 2010; Smith et al.,Blood103: 3669-3676, 2004; Hexner et al.,Leuk. Lymphoma.56(9):2543, 2015; Geyer et al.,Hematology17(Suppl1):5129-132, 2012; Diaz et al.,PLoS One6(4):e18856, 2011; Minturn et al.,Cancer Chemother. Pharmacol.68(4):1057-1065, 2011), AC-430 (O'Shea et al.,Immunity36(4):542-550, 2012; Patterson et al.,Clin. Exp. Immunol.176:1-10, 2014), pacritinib (SB1518) (Deeg et al.,J. Clin. Oncol.29: Abstract 6515, 2011; Verstovsek et al.,J. Hematol. Oncol.9(1):137, 2016; Chow et al.,Onco Targets. Ther.9:2655-2665, 2016; Komrokji et al.,Blood125(17):2649-2655, 2015; Jayaraman et al.,Drug Metab. Lett.9(1):28-47, 2015), BMS-911543 (Mace et al.,Oncotarget6(42):44509-44522, 2015; Wan et al.,ACS Med. Chem. Lett.6(8):850-855, 2015; Purandare et al.,Leukemia26(2):280-288, 2012), XL019 (Verstovsek et al.,Leuk. Res.38(3):316-322, 2014; Forsyth et al.,Bioorg. Med. Chem. Lett.22(24):7653-7658, 2012), INCB039110 (Mascarenhas et al.,Haematologica102(2):327-335, 2017; Bissonnette et al.,J. Dermatol. Treat.27(4):332-338, 2016), Gandotinib® (LY-2784544) (Ma et al.,Blood Cancer J.3:e109, 2013; Verstovsek et al.,Blood122: 665, 2013; Mitchell et al.,Org. Process Res. Dev.16(1):70-81. 2012); R723 (Shide et al.,Blood117(25): 6866-6875, 2011)); Z3 (Sayyah et al.,Mol. Cancer. Ther.7(8):2308-2318, 2008)) or a variant thereof. In some embodiments, the JAK inhibitory agent is a JAK3 inhibitor (e.g., decernotinib (VX-509) (Elwood et al.,J. Pharmacol. Exp. Ther.2017; Genovese et al.,Ann Rheum Dis.75(11):1979-1983, 2016; Gadina et al.,Arthritis Rheumatol.68(1):31-34, 2016; Farmer et al.,J. Med. Chem.58(18):7195-7216, 2015; Fleischmann et al.,Arthritis Rheumatol.67(2):334-343, 2015; Mahajan et al.,J. Pharmacol.353(2):405-414, 2015), R348 or a variant thereof (Velotta et al.,Transplantation87(5):653-659, 2009; Deuse et al.,Transplantation85(6):885-892, 2008)). In some embodiments, the small molecule is R256 or a variant thereof (Ashino et al.,J. Allergy Clin. Immunol.133(4):1162-1174, 2014). In some embodiments, the small molecule is R333 or a variant thereof. In some embodiments, the small molecule is INCB047986 or a variant thereof (Norman,Exp. Opin. Investig. Drugs23(8):1067-1077, 2014). In some embodiments, the small molecule is INCB16562 or a variant thereof (Koppikar et al.,Blood115(4):2919-2927, 2010; Li et al.,Neoplasia12(1):28-38, 2010). In some embodiments, the small molecule is NVP-BSK805 or a variant thereof (Ringel et al.,Acta Haematol.132(1):75-86, 2014; Baffert et al.,Mol. Cancer. Ther.9(7):1945-1955, 2010). In some embodiments, the small molecule is peficitinib (ASP015K, JNJ-54781532) or a variant thereof (Genovese et al.,Arthritis Rheumatol.,2017; Ito et al.,J. Pharmacol. Sci.133(1):25-33, 2017; Cao et al. (2016)Clin. Pharmacol. Drug Dev.5(6):435-449, 2016; Takeuchi et al.,Ann. Rheum. Dis.75(6):1057-1064, 2016). In some embodiments, the small molecule is tofacitinib (Xeljanz®, Jakvinus®, CP-690, 500) or a variant thereof (Ghoreschi et al.,J. Immunol.186(7):4234-4243, 2011; Yoshida et al.,Biochem. Biophys. Res. Commun418(2):234-240, 2012; Calama et al.,Pulm. Pharmacol. Ther. S1094-5539(16):30060-30068, 2017; Cutolo et al.,J. Inflamm. Res.6:129-137, 2013). In some embodiments, the small molecule is cucurbitacin I (JSI-124) or a variant thereof (Oi et al.,Int. J. Oncol.49(6):2275-2284, 2016; Qi et al.,Am. J. Chin. Med.43(2):337-347, 2015; Seo et al.,Food Chem. Toxicol.64:217-224, 2014). In some embodiments, the small molecule is CHZ868 or a variant thereof (Wu et al.,Cancer Cell28(1):29-41, 2015; Meyer et al.,Cancer Cell28(1):15-28, 2015). In some embodiments, the small molecule is a TYK2 inhibitor (e.g., Masse et al.,J. Immunol.194(1):67, 2015; Menet,Pharm. Pat. Anal.3(4):449-466, 2014; Liang et al.,Euro. J. Med. Chem.67: 175-187, 2013; Jang et al.,Bioorg. Med. Chem. Lett.25(18):3947-3952, 2015); U.S. Pat. Nos. 9,296,725 and 9,309,240; US 2013/0231340; and US 2016/0251376). In some embodiments, the TYK2 inhibitor is Ndi-031301 (Akahane et al.,Blood128:1596, 2016); BMS-986165 (Gillooly et al., 2016 ACR/ARHP Annual Meeting, Abstract 11L, 2016); SAR-20347 (Works et al.,J. Immunol.193(7):3278-3287, 2014); tyrphostin A1 (Ishizaki et al.,Int. Immunol.26(5):257-267, 2014); a triazolopyridine (US 2013/0143915); or a variant thereof. Additional examples of JAK inhibitors that are small molecules are described in, e.g., Furomoto et al.,BioDrugs27(5):431-438, 2013; O'Shea et al.,Ann. Rheum. Dis.72(2):ii111-ii-115, 2013; Sonbol et al.,Ther. Adv. Hematol.4(1):15-35, 2013; and Tanaka et al. (2015)J. Biochem.158(3): 173-179, 2015. In some embodiments, the JAK inhibitor is a pan-JAK inhibitor. As used herein, the term “pan-JAK inhibitor” is an agent that has an IC50of about 500 nM to 4 μM (e.g., about 500 nM to about 2 μM) for each of human JAK1, human JAK2, and human JAK3 isoforms, when the IC50is determined for each of wildtype human JAK1, wildtype human JAK2, and wildtype human JAK3 using similar assay conditions (e.g., the same assay conditions). In some embodiments, a pan-JAK inhibitor can be an agent that has an IC50for wildtype human JAK1, wildtype human JAK2, and wildtype human JAK3 that are within ±10% of each other, when each of the IC50values is assays under similar assay conditions (e.g., the same assay, e.g., the human wildtype JAK1, wildtype human JAK2, and wildtype human JAK3 assay described in Kim et al., 1Med. Chem.58(18):7596-5602, 2015). In some embodiments, the pan-JAK inhibitor is tofacitinib (Xeljanz®, Jakvinus®, tasocitinib, CP-690550; Yokoyama et al.,J. Clin. Immunol.33(3):586-594, 2013; and Thoma et al., 1Med. Chem.54(1):284-288, 2011); cerdulatinib (PRT2070; Coffey et al. (2014)J. Pharmacol. Exp. Ther.351(3):538-548, 2014; and Ma et al.,Oncotarget6(41):43881-43896, 2015); Pyridone 6 (P6; Nakagawa et al.,J. Immunol.187(9): 4611-4620, 2011; and Pedranzini et al.,Cancer Res.66(19):9714-9721, 2006); PF-06263276 (Jones et al. “Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin”J. Med. Chem.,2017, 60 (2), pp 767-786); JAK inhibitor 1 (CAS 457081-03-07; JAKi; Wang et al.,Antimicrob. Agents Chemother.60(5):2834-48, 2016; Bordonaro et al.,PLoS One9:e115068, 2014; and Osorio et al.,PLoS Pathogens10(6):e1004165, 2014); or baricitinib (Olumiant; LY3009104; INCB-28050; and Hsu and Armstrong,J. Immunol. Res. Article ID 283617, 2014). In some embodiments, the JAK inhibitor is a selective JAK1/JAK3 inhibitor. As used herein, the term “selective JAK1/JAK3 inhibitor” means an agent that has an IC50for wildtype human JAK1 and wildtype human JAK3, that are each at least 5-fold (e.g., at least 10-fold or at least 20-fold) lower than the IC50for wildtype human JAK2, when the IC50is determined for each of wildtype human JAK1, wildtype human JAK2, and wildtype human JAK3 using similar assay conditions (e.g., the same assay, e.g., the human wildtype JAK1, wildtype human JAK2, and wildtype human JAK3 assay described in Kim et al.,J. Med. Chem.58(18):7596-5602, 2015). In some embodiments, the JAK inhibitor is a selective JAK1 inhibitor. As used herein, the term “selective JAK1 inhibitor” means an agent that has an IC50for wildtype human JAK1 that is at least 10-fold (e.g., at least 20-fold) lower than each of the IC50for wildtype human JAK2 and the IC50for wildtype human JAK3 when measured using similar assay conditions (e.g., the same assay, e.g., the human wildtype JAK1, wildtype human JAK2, and wildtype human JAK3 assay described in Kim et al.,J. Med. Chem.58(18):7596-5602, 2015). In some embodiments, the JAK1 inhibitor is (31S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide as disclosed in international patent application PCT/US2014/062145, incorporated by reference herein in its entirety. In some embodiments, the JAK inhibitor is a selective JAK3 inhibitor. As used herein, the term “selective JAK3 inhibitor” means an agent that has an IC50for wildtype human JAK3 that is at least 10-fold (e.g., at least 20-fold) lower than each of the IC50for wildtype human JAK2 and the IC50for wildtype human JAK1 when measured using similar assay conditions (e.g., the same assay, e.g., the human wildtype JAK1, wildtype human JAK2, and wildtype human JAK3 assay described in Kim et al.,J. Med. Chem.58(18):7596-5602, 2015). In some embodiments, the JAK inhibitor is a JAK1 and JAK 3 inhibitor (e.g., a selective JAK1/JAK3 inhibitor). In some embodiments, the selective JAK1/JAK3 inhibitor is ZM 39923 (Brown et al.,Bioorg. Med. Chem. Lett.10(6):575-579, 2000; and Lai et al.,Chem. Biol.15(9):969-978, 2008); or peficitinib (ASP015K; JNJ-54781532; Ito et al.,J. Pharmacol. Sci.133(1):25-33, 2017; Cao et al.,Clin. Pharmacol. Drug Dev.5(6):435-449, 2016; Takeuchi et al.,Ann. Rheum. Dis.75(6):1057-1064, 2016); and Papp et al.,Br. J. Dermatol.173(3):767-776, 2015). In some embodiments, the JAK inhibitor is PF-06700841. In some embodiments, the JAK inhibitor is PF-06651600. In some embodiments, the inhibitor is one of the following: Common NameBrand nameCompanyTofacitinibXeljanzPfizerpeficitinibJNJ(ASP015K orJNJ-54781532)TD-1473Theravance BiopharmaPF-06263276PfizerCerdulatinib(PRT-062070)upadacitinibAbbvie(ABT494)BaricitinibOlumiantIncyte & Eli Lilly(INCB028050,LY3009104)FilgotinibTBDGilead, Galapagos NV(GLPG0634)Jakafi ®, Jakavi ®ruxolitinib(INCB018424)AZD1480AstraZenecamomelotinib(GS-0387)GSK2586184GSKoclacitinibApoquel ®Pfizer(PF03394197)oclacitinibAbbvie(PF03394197)GLG0778GLG0778fedratinib(TG101348,SAR302503)lestaurtinib(CEP-701)AC-430pacritinib(SB1518)XL019gandotinib ®(LY-2784544)BMS-911543BMSNVP-BSK805INCB16562Decernotinib(VX509)cucurbitacin I(JSI-124)R333Ndi-031301SAR-20347tyrphostin A1triazolopyridineBMS-986165TOP-1288Topivert Pharma LtdPF-06700841PfizerPF-06651600PfizerInhibitoryNucleic Acids In some embodiments, the kinase inhibitor is TOP-1288 from TopiVert Pharma Ltd., which is described in “The Pharmacological Profile of TOP1288, a Narrow Spectrum Kinase Inhibitor (NSKI) in Clinical Development as an Anti-Inflammatory Treatment for Ulcerative Colitis” Foster, Martyn et al. Gastroenterology, Volume 152, Issue 5, S766. Endoscopes, Ingestible Devices, and Reservoirs As discussed herein, in some embodiments, a method of treating a disease of the gastrointestinal tract comprises administering to the subject a pharmaceutical formulation wherein the pharmaceutical formulation is delivered proximate to one or more sites of disease by one of various methods. For example, the pharmaceutical formulation may be delivered via a medical device such as an endoscope, ingestible device, or reservoir; the pharmaceutical formulation may be a solid dosage form, a liquid dosage form, a suppository or an enema for rectal administration with different types of release such as sustained or delayed release. In one embodiment, the pharmaceutical formulation is delivered proximate to one or more sites of disease by an endoscope, ingestible device, or reservoir containing the pharmaceutical formulation. The GI tract can be imaged using endoscopes, or more recently, by ingestible devices that are swallowed. Direct visualization of the GI mucosa is useful to detect subtle mucosal alterations, as in inflammatory bowel diseases, as well as any flat or sessile lesions. As discussed herein, in some embodiments, the method of treating a disease of the gastrointestinal tract comprises administering to the subject a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is delivered proximate to one or more sites of disease by one of various methods. For example, the pharmaceutical formulation may be delivered via a medical device such as an endoscope, ingestible device, or reservoir; the pharmaceutical formulation may be a solid dosage form, a liquid dosage form, a suppository or an enema for rectal administration with different types of release such as sustained or delayed release. In one embodiment, the pharmaceutical formulation is delivered proximate to one or more sites of disease by an endoscope, ingestible device, or reservoir containing the pharmaceutical formulation. The technology behind standard colonoscopy consists of a long, semi-rigid insertion tube with a steerable tip (stiff if compared to the colon), which is pushed by the physician from the outside. However, invasiveness, patient discomfort, fear of pain, and—more often than not—the need for conscious sedation limit the take-up of screening colonoscopy. Diagnosis and treatment in the GI tract are dominated by the use of flexible endoscopes. A few large companies, namely Olympus Medical Systems Co. (Tokyo, Japan), Pentax Medical Co. (Montvale, NJ, USA), Fujinon, Inc. (Wayne, NJ, USA) and Karl Storz GmbH & Co. KG (Tuttlingen, Germany), cover the majority of the market in flexible GI endoscopy. Endoscopes may comprise a catheter. As an example, the catheter may be a spray catheter. As an example, a spray catheter may be used to deliver dyes for diagnostic purposes. As an example, a spray catheter may be used to deliver a therapeutic agent at the site of disease in the GI tract. For example, the Olypmus PW-205V is a ready-to-use spray catheter that enables efficient spraying for maximal differentiation of tissue structures during endoscopy, but may also be used to deliver drugs diseased tissue. In a review of robotic endoscopic capsules, Journal of Micro-Bio Robotics 11.1-4 (2016): 1-18, Ciuti et al. state that progress in micro-electromechanical systems (MEMS) technologies have led to the development of new endoscopic capsules with enhanced diagnostic capabilities, in addition to traditional visualization of mucosa (embedding, e.g. pressure, pH, blood detection and temperature sensors). Endoscopic capsules, however, do not have the capability of accurately locating a site autonomously. They require doctor oversight over a period of hours in order to manually determine the location. Autonomous ingestible devices are advantageous in that regard. Ingestible devices are also advantageous over spray catheters in that they are less invasive, thereby allowing for regular dosing more frequently than spray catheters. Another advantage of ingestible devices is the greater ease with which they can access, relative to a catheter, certain sections of the GI tract such as the ascending colon, the cecum, and all portions of the small intestine. Methods and Mechanisms for Localization In addition to, or as an alternative, to directly visualizing the GI tract, one or more different mechanisms can be used to determine the location of an ingestible device within the GI tract. Various implementations may be used for localization of ingestible devices within the GI tract. For example, certain implementations can include one or more electromagnetic sensor coils, magnetic fields, electromagnetic waves, electric potential values, ultrasound positioning systems, gamma scintigraphy techniques or other radio-tracker technology have been described by others. Alternatively, imaging can be used to localize, for example, using anatomical landmarks or more complex algorithms for 3D reconstruction based on multiple images. Other technologies rely on radio frequency, which relies on sensors placed externally on the body to receive the strength of signals emitted by the capsule. Ingestible devices may also be localized based on reflected light in the medium surrounding the device; pH; temperature; time following ingestion; and/or acoustic signals. The disclosure provides an ingestible device, as well as related systems and methods that provide for determining the position of the ingestible device within the GI tract of a subject with very high accuracy. In some embodiments, the ingestible device can autonomously determine its position within the GI tract of the subject. Typically, the ingestible device includes one or more processing devices, and one more machine readable hardware storage devices. In some embodiments, the one or more machine readable hardware storage devices store instructions that are executable by the one or more processing devices to determine the location of the ingestible device in a portion of a GI tract of the subject. In certain embodiments, the one or more machine readable hardware storage devices store instructions that are executable by the one or more processing devices to transmit data to an external device (e.g., a base station external to the subject, such as a base station carried on an article worn by the subject) capable of implementing the data to determine the location of the device within the GI tract of the subject. In some embodiments, the location of the ingestible device within the GI tract of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In some embodiments, the location of the ingestible device within the GI tract of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In such embodiments, the portion of the GI tract of the subject can include, for example, the esophagus, the stomach, duodenum, the jejunum, and/or the terminal ileum, cecum and colon. An exemplary and non-limiting embodiment is provided below in Example 13. In certain embodiments, the location of the ingestible device within the esophagus of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. An exemplary and non-limiting embodiment is provided below in Example 13. In some embodiments, the location of the ingestible device within the stomach of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. An exemplary and non-limiting embodiment is provided below in Example 13. In certain embodiments, the location of the ingestible device within the duodenum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. An exemplary and non-limiting embodiment is provided below in Example 13. In some embodiments, the location of the ingestible device within the jejunum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. An exemplary and non-limiting embodiment is provided below in Example 13. In certain embodiments, the location of the ingestible device within the terminal ileum, cecum and colon of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In some embodiments, the location of the ingestible device within the cecum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. An exemplary and non-limiting embodiment is provided below in Example 13. In such embodiments, the portion of the portion of the GI tract of the subject can include, for example, the esophagus, the stomach, duodenum, the jejunum, and/or the terminal ileum, cecum and colon. In certain embodiments, the location of the ingestible device within the esophagus of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In some embodiments, the location of the ingestible device within the stomach of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In certain embodiments, the location of the ingestible device within the duodenum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In some embodiments, the location of the ingestible device within the jejunum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In certain embodiments, the location of the ingestible device within the terminal ileum, cecum and colon of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. In some embodiments, the location of the ingestible device within the cecum of the subject can be determined to an accuracy of at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, 100%. As used herein, the term “reflectance” refers to a value derived from light emitted by the device, reflected back to the device, and received by a detector in or on the device. For example, in some embodiments this refers to light emitted by the device, wherein a portion of the light is reflected by a surface external to the device, and the light is received by a detector located in or on the device. As used herein, the term “illumination” refers to any electromagnetic emission. In some embodiments, an illumination may be within the range of Infrared Light (IR), the visible spectrum and ultraviolet light (UV), and an illumination may have a majority of its power centered at a particular wavelength in the range of 100 nm to 1000 nm. In some embodiments, it may be advantageous to use an illumination with a majority of its power limited to one of the infrared (750 nm-1000 nm), red (600 nm-750 nm), green (495 nm-600 nm), blue (400 nm-495 nm), or ultraviolet (100 nm-400 nm) spectrums. In some embodiments a plurality of illuminations with different wavelengths may be used. For illustrative purposes, the embodiments described herein may refer to the use of green or blue spectrums of light. However, it is understood that these embodiments may use any suitable light having a wavelength that is substantially or approximately within the green or blue spectra defined above, and the localization systems and methods described herein may use any suitable spectra of light. Referring now toFIG.1, shown therein is a view of an example embodiment of an ingestible device100, which may be used to identify a location within a gastrointestinal (GI) tract. In some embodiments, ingestible device100may be configured to autonomously determine whether it is located in the stomach, a particular portion of the small intestine such as a duodenum, jejunum, or ileum, or the large intestine by utilizing sensors operating with different wavelengths of light. Additionally, ingestible device100may be configured to autonomously determine whether it is located within certain portions of the small intestine or large intestine, such as the duodenum, the jejunum, the cecum, or the colon. Ingestible device100may have a housing102shaped similar to a pill or capsule. The housing102of ingestible device100may have a first end portion104, and a second end portion106. The first end portion104may include a first wall portion108, and second end portion106may include a second wall portion110. In some embodiments, first end portion104and second end portion106of ingestible device100may be manufactured separately, and may be affixed together by a connecting portion112. In some embodiments, ingestible device100may include an optically transparent window114. Optically transparent window114may be transparent to various types of illumination in the visible spectrum, infrared spectrum, or ultraviolet light spectrum, and ingestible device100may have various sensors and illuminators located within the housing102, and behind the transparent window114. This may allow ingestible device100to be configured to transmit illumination at different wavelengths through transparent window114to an environment external to housing102of ingestible device100, and to detect a reflectance from a portion of the illumination that is reflected back through transparent window114from the environment external to housing102. Ingestible device100may then use the detected level of reflectance in order to determine a location of ingestible device100within a GI tract. In some embodiments, optically transparent window114may be of any shape and size, and may wrap around the circumference of ingestible device100. In this case, ingestible device100may have multiple sets of sensors and illuminators positioned at different locations azimuthally behind window114. In some embodiments, ingestible device100may optionally include an opening116in the second wall portion110. In some embodiments, the second wall portion110may be configured to rotate around the longitudinal axis of ingestible device100(e.g., by means of a suitable motor or other actuator housed within ingestible device100). This may allow ingestible device100to obtain a fluid sample from the GI tract, or release a substance into the GI tract, through opening116. FIG.2shows an exploded view of ingestible device100. In some embodiments, ingestible device100may optionally include a rotation assembly118. Optional rotation assembly118may include a motor118-1driven by a microcontroller (e.g., a microcontroller coupled to printed circuit board120), a rotation position sensing ring118-2, and a storage sub-unit118-3configured to fit snugly within the second end portion104. In some embodiments, rotation assembly118may cause second end portion104, and opening116, to rotate relative to the storage sub-unit118-3. In some embodiments, there may be cavities on the side of storage sub-unit118-3that function as storage chambers. When the opening116is aligned with a cavity on the side of the storage sub-unit118-3, the cavity on the side of the storage sub-unit118-3may be exposed to the environment external to the housing102of ingestible device100. In some embodiments, the storage sub-unit118-3may be loaded with a medicament or other substance prior to the ingestible device100being administered to a subject. In this case, the medicament or other substance may be released from the ingestible device100by aligning opening116with the cavity within storage sub-unit118-3. In some embodiments, the storage sub-unit118-3may be configured to hold a fluid sample obtained from the GI tract. For example, ingestible device100may be configured to align opening116with the cavity within storage sub-unit118-3, thus allowing a fluid sample from the GI tract to enter the cavity within storage sub-unit118-3. Afterwards, ingestible device100may be configured to seal the fluid sample within storage sub-unit118-3by further rotating the second end portion106relative to storage sub-unit118-3. In some embodiments, storage sub-unit118-3may also contain a hydrophilic sponge, which may enable ingestible device100to better draw certain types of fluid samples into ingestible device100. In some embodiments, ingestible device100may be configured to either obtain a sample from within the GI tract, or to release a substance into the GI tract, in response to determining that ingestible device100has reached a predetermined location within the GI tract. For example, ingestible device100may be configured to obtain a fluid sample from the GI tract in response to determining that the ingestible device has entered the jejunum portion of the small intestine (e.g., as determined by process900discussed in relation toFIG.9). Other ingestible devices capable of obtaining samples or releasing substances are discussed in commonly-assigned PCT Application No. PCT/CA2013/000133 filed Feb. 15, 2013, commonly-assigned U.S. Provisional Application No. 62/385,553, and commonly-assigned U.S. Provisional Application No. 62/376,688, which each are hereby incorporated by reference herein in their entirety. It is understood that any suitable method of obtaining samples or releasing substances may be incorporated into some of the embodiments of the ingestible devices disclosed herein, and that the systems and methods for determining a location of an ingestible device may be incorporated into any suitable type of ingestible device. Ingestible device100may include a printed circuit board (PCB)120, and a battery128configured to power PCB120. PCB120may include a programmable microcontroller, and control and memory circuitry for holding and executing firmware or software for coordinating the operation of ingestible device100, and the various components of ingestible device100. For example, PCB120may include memory circuitry for storing data, such as data sets of measurements collected by sensing sub-unit126, or instructions to be executed by control circuitry to implement a localization process, such as, for example, one or more of the processes, discussed herein, including those discussed below in connection with one or more of the associated flow charts. PCB120may include a detector122and an illuminator124, which together form sensing sub-unit126. In some embodiments, control circuitry within PCB120may include processing units, communication circuitry, or any other suitable type of circuitry for operating ingestible device100. For illustrative purposes, only a single detector122and a single illuminator124forming a single sensing sub-unit126are shown. However, it is understood that in some embodiments there may be multiple sensing sub-units, each with a separate illuminator and detector, within ingestible device100. For example, there may be several sensing sub-units spaced azimuthally around the circumference of the PCB120, which may enable ingestible device100to transmit illumination and detect reflectances or ambient light in all directions around the circumference of the device. In some embodiments, sensing sub-unit126may be configured to generate an illumination using illuminator124, which is directed through the window114in a radial direction away from ingestible device100. This illumination may reflect off of the environment external to ingestible device100, and the reflected light coming back into ingestible device100through window114may be detected as a reflectance by detector122. In some embodiments, window114may be of any suitable shape and size. For example, window114may extend around a full circumference of ingestible device100. In some embodiments there may be a plurality of sensing sub-units (e.g., similar to sensing sub-unit126) located at different positions behind the window. For example, three sensing sub-units may be positioned behind the window at the same longitudinal location, but spaced 120 degrees apart azimuthally. This may enable ingestible device100to transmit illuminations in all directions radially around ingestible device100, and to measure each of the corresponding reflectances. In some embodiments, illuminator124may be capable of producing illumination at a variety of different wavelengths in the ultraviolet, infrared, or visible spectrum. For example, illuminator124may be implemented by using Red-Green-Blue Light-Emitting diode packages (RGB-LED). These types of RGB-LED packages are able to transmit red, blue, or green illumination, or combinations of red, blue, or green illumination. Similarly, detector122may be configured to sense reflected light of the same wavelengths as the illumination produced by illuminator124. For example, if illuminator124is configured to produce red, blue, or green illumination, detector122may be configured to detect different reflectances produced by red, blue, or green illumination (e.g., through the use of an appropriately configured photodiode). These detected reflectances may be stored by ingestible device100(e.g., within memory circuitry of PCB120), and may then be used by ingestible device100in determining a location of ingestible device100within the GI tract (e.g., through the use of process500(FIG.5), process600(FIG.6), or process900(FIG.9)). It is understood that ingestible device100is intended to be illustrative, and not limiting. It will be understood that modifications to the general shape and structure of the various devices and mechanisms described in relation toFIG.1andFIG.2may be made without significantly changing the functions and operations of the devices and mechanisms. For example, ingestible device100may have a housing formed from a single piece of molded plastic, rather than being divided into a first end portion104and a second end portion106. As an alternate example, the location of window114within ingestible device100may be moved to some other location, such as the center of ingestible device100, or to one of the ends of ingestible device100. Moreover, the systems and methods discussed in relation toFIGS.1-10may be implemented on any suitable type of ingestible device, provided that the ingestible device is capable of detecting reflectances or levels of illumination in some capacity. For example, in some embodiments ingestible device100may be modified to replace detector122with an image sensor, and the ingestible device may be configured to measure relative levels of red, blue, or green light by decomposing a recorded image into its individual spectral components. Other examples of ingestible devices with localization capabilities, which may be utilized in order to implement the systems and methods discussed in relation toFIG.1-11, are discussed in co-owned PCT Application No. PCT/US2015/052500 filed on Sep. 25, 2015, which is hereby incorporated by reference herein in its entirety. Furthermore, it should be noted that the features and limitations described in any one embodiment may be applied to any other embodiment herein, and the descriptions and examples relating to one embodiment may be combined with any other embodiment in a suitable manner. FIG.3is a diagram of an ingestible device during an example transit through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. Ingestible device300may include any portion of any other ingestible device discussed in this disclosure (e.g., ingestible device100(FIG.1)), and may be any suitable type of ingestible device with localization capabilities. For example, ingestible device300may be one embodiment of ingestible device100without the optional opening116(FIG.1) or optional rotation assembly118(FIG.2)). In some embodiments, ingestible device300may be ingested by a subject, and as ingestible device300traverses the GI tract, ingestible device300may be configured to determine its location within the GI tract. For example, the movement of ingestible device300and the amount of light detected by ingestible device300(e.g., via detector122(FIG.2)) may vary substantially depending on the location of ingestible device300within the GI tract, and ingestible device300may be configured to use this information to determine a location of ingestible device300within the GI tract. For instance, ingestible device300may detect ambient light from the surrounding environment, or reflectances based on illumination generated by ingestible device300(e.g., generated by illuminator124(FIG.1)), and use this information to determine a location of ingestible device300through processes, such as described herein. The current location of ingestible device300, and the time that ingestible device300detected each transition between the various portions of the GI tract, may then be stored by ingestible device300(e.g., in memory circuitry of PCB120(FIG.2)), and may be used for any suitable purpose. Shortly after ingestible device300is ingested, ingestible device will traverse the esophagus302, which may connect the subject's mouth to a stomach306. In some embodiments, ingestible device300may be configured to determine that it has entered the esophagus portion GI tract by measuring the amount and type of light (e.g., via detector122(FIG.2)) in the environment surrounding the ingestible device300. For instance, ingestible device300may detect higher levels of light in the visible spectrum (e.g., via detector122(FIG.2)) while outside the subject's body, as compared to the levels of light detected while within the GI tract. In some embodiments, ingestible device300may have previously stored data (e.g., on memory circuitry of PCB120(FIG.2)) indicating a typical level of light detected when outside of the body, and the ingestible device300may be configured to determine that entry to the body has occurred when a detected level of light (e.g., detected via detector122(FIG.2)) has been reduced beyond a threshold level (e.g., at least a 20-30% reduction) for a sufficient period of time (e.g., 5.0 seconds). In some embodiments, ingestible device300may be configured to detect a transition from esophagus302to stomach306by passing through sphincter304. In some embodiments, ingestible device300may be configured to determine whether it has entered stomach306based at least in part on a plurality of parameters, such as but not limited to the use of light or temperature measurements (e.g., via detector122(FIG.2) or via a thermometer within ingestible device300), pH measurements (e.g., via a pH meter within ingestible device300), time measurements (e.g., as detected through the use of clock circuitry included within PCB120(FIG.2)), or any other suitable information. For instance, ingestible device300may be configured to determine that ingestible device300has entered stomach306after detecting that a measured temperature of ingestible device300exceeds 31 degrees Celsius. Additionally, or alternately, ingestible device300may be configured to automatically determine it has entered stomach306after one minute (or another pre-set time duration parameter, 80 seconds, 90 seconds, etc.) has elapsed from the time that ingestible device300was ingested, or one minute (or another pre-set time duration parameter, 80 seconds, 90 seconds, etc.) from the time that ingestible device300detected that it has entered the GI tract. Stomach306is a relatively large, open, and cavernous organ, and therefore ingestible device300may have a relatively large range of motion. By comparison, the motion of ingestible device300is relatively restricted within the tube-like structure of the duodenum310, the jejunum314, and the ileum (not shown), all of which collectively form the small intestine. Additionally, the interior of stomach306has distinct optical properties from duodenum310and jejunum314, which may enable ingestible device300to detect a transition from stomach306to duodenum310through the appropriate use of measured reflectances (e.g., through the use of reflectances measured by detector122(FIG.2)), as used in conjunction with process600(FIG.6)). In some embodiments, ingestible device300may be configured to detect a pyloric transition from stomach306to duodenum310through the pylorus308. For instance, in some embodiments, ingestible device300may be configured to periodically generate illumination in the green and blue wavelengths (e.g., via illuminator124(FIG.2)), and measure the resulting reflectances (e.g., via detector122(FIG.2)). Ingestible device300may be configured to then use a ratio of the detected green reflectance to the detected blue reflectance to determine whether ingestible device300is located within the stomach306, or duodenum310(e.g., via process600(FIG.6)). In turn, this may enable ingestible device300to detect a pyloric transition from stomach306to duodenum310, an example of which is discussed in relation toFIG.6. Similarly, in some embodiments, ingestible device300may be configured to detect a reverse pyloric transition from duodenum310to stomach306. Ingestible device300will typically transition naturally from stomach306to duodenum310, and onward to jejunum314and the remainder of the GI tract. However, similar to other ingested substances, ingestible device300may occasionally transition from duodenum310back to stomach306as a result of motion of the subject, or due to the natural behavior of the organs with the GI tract. To accommodate this possibility, ingestible device300may be configured to continue to periodically generate illumination in the green and blue wavelengths (e.g., via illuminator124(FIG.2)), and measure the resulting reflectances (e.g., via detector122(FIG.2)) to detect whether or not ingestible device300has returned to stomach306. An exemplary detection process is described in additional detail in relation toFIG.6. After entering duodenum310, ingestible device300may be configured to detect a transition to the jejunum314through the duodenojejunal flexure312. For example, ingestible device300may be configured to use reflectances to detect peristaltic waves within the jejunum314, caused by the contraction of the smooth muscle tissue lining the walls of the jejunum314. In particular, ingestible device300may be configured to begin periodically transmitting illumination (and measuring the resulting reflectances (e.g., via detector122and illuminator124of sensing sub-unit126(FIG.2)) at a sufficiently high frequency in order to detect muscle contractions within the jejunum314. Ingestible device300may then determine that it has entered the jejunum314in response to having detected either a first muscle contraction, or a predetermined number of muscle contractions (e.g., after having detected three muscle contractions in sequence). The interaction of ingestible device300with the walls of jejunum314is also discussed in relation toFIG.4, and an example of this detection process is described in additional detail in relation toFIG.9. FIG.4is a diagram of an ingestible device during an example transit through a jejunum, in accordance with some embodiments of the disclosure. Diagrams410,420,430, and440depict ingestible device400as it traverses through a jejunum (e.g., jejunum314), and how ingestible device400interacts with peristaltic waves formed by walls406A and406B (collectively, walls406) of the jejunum. In some implementations, ingestible device400may include any portion of any other ingestible device discussed in this disclosure (e.g., ingestible device100(FIG.1) or ingestible device300(FIG.3)), and may be any suitable type of ingestible device with localization capabilities. For example, ingestible device400may be substantially similar to the ingestible device300(FIG.3) or ingestible device100(FIG.1), with window404being the same as window114(FIG.1), and sensing sub-unit402being the same as sensing sub-unit126(FIG.2). Diagram410depicts ingestible device400within the jejunum, when the walls406of the jejunum are relaxed. In some embodiments, the confined tube-like structure of the jejunum naturally causes ingestible device400to be oriented longitudinally along the length of the jejunum, with window404facing walls406. In this orientation, ingestible device400may use sensing sub-unit402to generate illumination (e.g., via illuminator124(FIG.2)) oriented towards walls406, and to detect the resulting reflectances (e.g., via detector122(FIG.2)) from the portion of the illumination reflected off of walls406and back through window404. In some embodiments, ingestible device400may be configured to use sensing sub-unit402to generate illumination and measure the resulting reflectance with sufficient frequency to detect peristaltic waves within the jejunum. For instance, in a healthy human subject, peristaltic waves may occur at a rate of approximately 0.1 Hz to 0.2 Hz. Therefore, the ingestible device400may be configured to generate illumination and measure the resulting reflectance at least once every 2.5 seconds (i.e., the minimum rate necessary to detect a 0.2 Hz signal), and preferably at a higher rate, such as once every 0.5 seconds, which may improve the overall reliability of the detection process due to more data points being available. It is understood that the ingestible device400need not gather measurements at precise intervals, and in some embodiments the ingestible device400may be adapted to analyze data gathered at more irregular intervals, provided that there are still a sufficient number of appropriately spaced data points to detect 0.1 Hz to 0.2 Hz signals. Diagram420depicts ingestible device400within the jejunum, when the walls406of the jejunum begin to contract and form a peristaltic wave. Diagram420depicts contracting portion408A of wall406A and contracting portion408B of wall406B (collectively, contracting portion408of wall406) that form a peristaltic wave within the jejunum. The peristaltic wave proceeds along the length of the jejunum as different portions of wall406contract and relax, causing it to appear as if contracting portions408of wall406proceed along the length of the jejunum (i.e., as depicted by contracting portions408proceeding from left to right in diagrams410-430). While in this position, ingestible device400may detect a similar level of reflectance (e.g., through the use of illuminator124and detector122of sensing sub-unit126(FIG.2)) as detected when there is no peristaltic wave occurring (e.g., as detected when ingestible device400is in the position indicated in diagram410). Diagram430depicts ingestible device400within the jejunum, when the walls406of the jejunum continue to contract, squeezing around ingestible device400. As the peristaltic wave proceeds along the length of the jejunum, contracting portions408of wall406may squeeze tightly around ingestible device400, bringing the inner surface of wall406into contact with window404. While in this position, ingestible device400may detect a change in a reflectance detected as a result of illumination produced by sensing sub-unit402. The absolute value of the change in the measured reflectance may depend on several factors, such as the optical properties of the window404, the spectral components of the illumination, and the optical properties of the walls406. However, ingestible device400may be configured to store a data set with the reflectance values over time, and search for periodic changes in the data set consistent with the frequency of the peristaltic waves (e.g., by analyzing the data set in the frequency domain, and searching for peaks between 0.1 Hz to 0.2 Hz). This may enable ingestible device400to detect muscle contractions due to peristaltic waves without foreknowledge of the exact changes in reflectance signal amplitude that may occur as a result of detecting the muscle contractions of the peristaltic wave. An example procedure for detecting muscle contractions is discussed further in relation toFIG.9, and an example of a reflectance data set gathered while ingestible device400is located within the jejunum is discussed in relation toFIG.10. Diagram440depicts ingestible device400within the jejunum, when the peristaltic wave has moved past ingestible device400. Diagram440depicts contracting portions408that form the peristaltic wave within the jejunum having moved past the end of ingestible device400. The peristaltic wave proceeds along the length of the jejunum as different portions of wall406contract and relax, causing it to appear as if contracting portions408of wall406proceed along the length of the jejunum (i.e., as depicted by contracting portions408proceeding from left to right in diagrams410-430). While in this position, ingestible device400may detect a similar level of reflectance (e.g., through the use of illuminator124and detector122of sensing sub-unit126(FIG.2)) as detected when there is no peristaltic wave occurring (e.g., as detected when ingestible device400is in the position indicated in diagram410, or diagram420). Depending on the species of the subject, peristaltic waves may occur with relatively predictable regularity. After the peristaltic wave has passed over ingestible device400(e.g., as depicted in diagram440), the walls406of the jejunum may relax again (e.g., as depicted in diagram410), until the next peristaltic wave begins to form. In some embodiments, ingestible device400may be configured to continue to gather reflectance value data while it is within the GI tract, and may store a data set with the reflectance values over time. This may allow ingestible device400to detect each of the muscle contractions as the peristaltic wave passes over ingestible device400(e.g., as depicted in diagram430), and may enable ingestible device400to both count the number of muscle contractions that occur, and to determine that a current location of the ingestible device400is within the jejunum. For example, ingestible device400may be configured to monitor for possible muscle contractions while is inside either the stomach or the duodenum, and may determine that ingestible device400has moved to the jejunum in response to detecting a muscle contraction consistent with a peristaltic wave. FIG.5is a flowchart illustrating some aspects of a localization process used by the ingestible device. AlthoughFIG.5may be described in connection with the ingestible device100for illustrative purposes, this is not intended to be limiting, and either portions or the entirety of the localization procedure500described inFIG.5may be applied to any device discussed in this application (e.g., the ingestible devices100,300, and400), and any of the ingestible devices may be used to perform one or more parts of the process described inFIG.5. Furthermore, the features ofFIG.5may be combined with any other systems, methods or processes described in this application. For example, portions of the process inFIG.5may be integrated into or combined with the pyloric transition detection procedure described byFIG.6, or the jejunum detection process described byFIG.9. At502, the ingestible device (e.g., ingestible device100,300, or400) gathers measurements (e.g., through detector122(FIG.2)) of ambient light. For example, ingestible device100may be configured to periodically measure (e.g., through detector122(FIG.2)) the level of ambient light in the environment surrounding ingestible device100. In some embodiments, the type of ambient light being measured may depend on the configuration of detector122within ingestible device100. For example, if detector122is configured to measure red, green, and blue wavelengths of light, ingestible device100may be configured to measure the ambient amount of red, green, and blue light from the surrounding environment. In some embodiments, the amount of ambient light measured by ingestible device100will be larger in the area external to the body (e.g., a well-lit room where ingestible device100is being administered to a subject) and in the oral cavity of the subject, as compared to the ambient level of light measured by ingestible device100when inside of an esophagus, stomach, or other portion of the GI tract (e.g., esophagus302, stomach306, duodenum310, or jejunum314(FIG.3)). At504, the ingestible device (e.g., ingestible device100,300, or400) determines (e.g., via control circuitry within PCB120(FIG.2)) whether the ingestible device has detected entry into the GI tract. For example, ingestible device100may be configured to determine when the most recent measurement of ambient light (e.g., the measurement gathered at502) indicates that the ingestible device has entered the GI tract. For instance, the first time that ingestible device100gatherers a measurement of ambient light at502, ingestible device100may store that measurement (e.g., via storage circuitry within PCB120(FIG.2)) as a typical level of ambient light external to the body. Ingestible device100may be configured to then compare the most recent measurement of ambient light to the typical level of ambient light external to the body (e.g., via control circuitry within PCB120(FIG.2)), and determine that ingestible device100has entered the GI tract when the most recent measurement of ambient light is substantially smaller than the typical level of ambient light external to the body. For example, ingestible device100may be configured to detect that it has entered the GI tract in response to determining that the most recent measurement of ambient light is less than or equal to 20% of the typical level of ambient light external to the body. If ingestible device100determines that it has detected entry into the GI tract (e.g., that ingestible device100has entered at least the esophagus302(FIG.3)), process500proceeds to506. Alternately, if ingestible device100determines that it has not detected entry into the GI tract (e.g., as a result of the most recent measurement being similar to the typical level of ambient light external to the body), process500proceeds back to502where the ingestible device100gathers further measurements. For instance, ingestible device100may be configured to wait a predetermined amount of time (e.g., five seconds, ten seconds, etc.), and then gather another measurement of the level of ambient light from the environment surrounding ingestible device100. At506, the ingestible device (e.g., ingestible device100,300, or400) waits for a transition from the esophagus to the stomach (e.g., from esophagus302to stomach306(FIG.3)). For example, ingestible device100may be configured to determine that it has entered the stomach (e.g., stomach306(FIG.3)) after waiting a predetermined period of time after having entered the GI tract. For instance, a typical esophageal transit time in a human patient may be on the order of 15-30 seconds. In this case, after having detected that ingestible device100has entered the GI tract at504(i.e., after detecting that ingestible device100has reached at least esophagus302(FIG.3)), ingestible device100may be configured to wait one minute, or a similar amount of time longer than the typical esophageal transmit time (e.g., ninety-seconds), before automatically determining that ingestible device100has entered at least the stomach (e.g., stomach306(FIG.3)). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may also determine it has entered the stomach based on measurements of pH or temperature. For example, ingestible device100may be configured to determine that it has entered the stomach if a temperature of ingestible device has increased to at least 31 degrees Celsius (i.e., consistent with the temperature inside the stomach), or if a measured pH of the environment surrounding ingestible device100is sufficiently acidic (i.e., consistent with the acidic nature of gastric juices that may be found inside the stomach). At508, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating the ingestible device has entered the stomach (e.g., stomach306(FIG.3)). For example, after having waited a sufficient amount of time at506, ingestible device100may store data (e.g., within storage circuitry of PCB120(FIG.2)) indicative of ingestible device100having entered at least the stomach. Once ingestible device100reaches at least the stomach, process500proceeds to510where ingestible device100may be configured to gather data to detect entry into the duodenum (e.g., duodenum310(FIG.3)). In some embodiments, process500may also simultaneously proceed from508to520, where ingestible device100may be configured to gather data in order to detect muscle contractions and detect entry into the jejunum (e.g., jejunum314(FIG.3)). In some embodiments, ingestible device100may be configured to simultaneously monitor for entry into the duodenum at516-518, as well as detect for entry into the jejunum at520-524. This may allow ingestible device100to determine when it has entered the jejunum (e.g., as a result of detecting muscle contractions), even when it fails to first detect entry into the duodenum (e.g., as a result of very quick transit times of the ingestible device through the duodenum). At510, the ingestible device (e.g., ingestible device100,300, or400) gathers measurements of green and blue reflectance levels (e.g., through the use of illuminator124and detector122of sensing sub-unit126(FIG.2)) while in the stomach (e.g., stomach306(FIG.3)). For example, ingestible device100may be configured to periodically gather measurements of green and blue reflectance levels while in the stomach. For instance, ingestible device100may be configured to transmit a green illumination and a blue illumination (e.g., via illuminator124(FIG.2)) every five to fifteen seconds, and measure the resulting reflectance (e.g., via detector122(FIG.2)). Every time that ingestible device100gathers a new set of measurements, the measurements may be added to a stored data set (e.g., stored within memory circuitry of PCB120(FIG.2)). The ingestible device100may then use this data set to determine whether or not ingestible device100is still within a stomach (e.g., stomach306(FIG.3)), or a duodenum (e.g., duodenum310(FIG.3)). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may be configured to detect a first reflectance based on generating an illumination of a first wavelength in approximately the green spectrum of light (between 495-600 nm), and detecting a second reflectance based on generating an illumination of the second wavelength in approximately the blue spectrum of light (between 400-495 nm). In some embodiments, the ingestible device may ensure that the illumination in the green spectrum and the illumination in the blue spectrum have wavelengths separated by at least 50 nm. This may enable ingestible device100to sufficiently distinguish between the two wavelengths when detecting the reflectances (e.g., via detector122(FIG.2)). It is understood that the separation of 50 nm is intended to be illustrative, and not limiting, and depending on the accuracy of the detectors within ingestible device100, smaller separations may be possible to be used. At512, the ingestible device (e.g., ingestible device100,300, or400) determines (e.g., using control circuitry within PCB120(FIG.2)) whether the ingestible device has detected a transition from the stomach (e.g., stomach306(FIG.3)) to a duodenum (e.g., duodenum310(FIG.3)) based on a ratio of green and blue (G/B) reflectance levels. For example, ingestible device100may obtain (e.g., from memory circuitry of PCB120(FIG.2)) a data set containing historical data for the respective ratio of the green reflectance to the blue reflectance as measured at a respective time. Generally speaking, a duodenum (e.g., duodenum310(FIG.3)) of a human subject reflects a higher ratio of green light to blue light, as compared to the ratio of green light to blue light that is reflected by a stomach (e.g., stomach306(FIG.3)). Based on this, ingestible device100may be configured to take a first set of ratios from the data set, representing the result of recent measurements, and compare them to a second set of ratios from the data set, representing the results of past measurements. When the ingestible device100determines that the mean value of the first set of ratios is substantially larger than the mean value of the second set of ratios (i.e., that the ratio of reflected green light to reflected blue light has increased), the ingestible device100may determine that it has entered the duodenum (e.g., duodenum310(FIG.3)) from the stomach (e.g., stomach306(FIG.3)). If the ingestible device100detects a transition from the stomach (e.g., stomach306(FIG.3)) to a duodenum (e.g., duodenum310(FIG.3)), process500proceeds to514, where ingestible device100stores data indicating that the ingestible device100has entered the duodenum (e.g., duodenum310(FIG.3)). Alternatively, if the ingestible device determines that the ingestible device has not transitioned from the stomach (e.g., stomach306(FIG.3)) to the duodenum (e.g., duodenum310(FIG.3)), process500proceeds back to510to gather more measurements of green and blue reflectance levels while still in the stomach (e.g., stomach306(FIG.3)). An example procedure for using measurements of green and blue reflectances to monitor for transitions between the stomach and the duodenum is discussed in greater detail in relation toFIG.6. In some embodiments, the first time that ingestible device100detects a transition from the stomach (e.g., stomach306(FIG.3)) to the duodenum (e.g., duodenum310(FIG.3)), ingestible device100may be configured to take a mean of the second set of data, (e.g., the set of data previously recorded while in stomach306(FIG.3)) and store this as a typical ratio of green light to blue light detected within the stomach (e.g., stomach306(FIG.3)) (e.g., within memory circuitry of PCB120(FIG.2)). This stored information may later be used by ingestible device100to determine when ingestible device100re-enters the stomach (e.g., stomach306(FIG.3)) from the duodenum (e.g., duodenum310(FIG.3)) as a result of a reverse pyloric transition. At514, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating that the ingestible device has entered the duodenum (e.g., duodenum310(FIG.3)). For example, ingestible device100may store a flag within local memory (e.g., memory circuitry of PCB120) indicating that the ingestible device100is currently in the duodenum. In some embodiments, the ingestible device100may also store a timestamp indicating the time when ingestible device100entered the duodenum. Once ingestible device100reaches the duodenum, process500proceeds to520where ingestible device100may be configured to gather data in order to detect muscle contractions and detect entry into the jejunum (e.g., jejunum314(FIG.3)). Process500also proceeds from514to516, where ingestible device100may be configured to gather data additional data in order to detect re-entry into the stomach (e.g., stomach306(FIG.3)) from the duodenum (e.g., duodenum310(FIG.3)). At516, the ingestible device (e.g., ingestible device100,300, or400) gathers measurements (e.g., via sensing sub-unit126(FIG.2)) of green and blue reflectance levels while in the duodenum (e.g., duodenum310(FIG.3)). For example, ingestible device100may be configured to periodically gather measurements (e.g., via sensing sub-unit126(FIG.2)) of green and blue reflectance levels while in the duodenum, similar to the measurements made at510while in the stomach. For instance, ingestible device100may be configured to transmit a green illumination and a blue illumination (e.g., via illuminator124(FIG.2)) every five to fifteen seconds, and measure the resulting reflectance (e.g., via detector122(FIG.2)). Every time that ingestible device100gathers a new set of measurements, the measurements may be added to a stored data set (e.g., stored within memory circuitry of PCB120(FIG.2)). The ingestible device100may then use this data set to determine whether or not ingestible device100is still within the duodenum (e.g., duodenum310(FIG.3)), or if the ingestible device100has transitioned back into the stomach (e.g., stomach306(FIG.3)). At518, the ingestible device (e.g., ingestible device100,300, or400) determines a transition from the duodenum (e.g., duodenum310(FIG.3)) to the stomach (e.g., stomach306(FIG.3)) based on a ratio of the measured green reflectance levels to the measured blue reflectance levels. In some embodiments, ingestible device100may compare the ratio of the measured green reflectance levels to the measured blue reflectance levels recently gathered by ingestible device100(e.g., measurements gathered at516), and determine whether or not the ratio of the measured green reflectance levels to the measured blue reflectance levels is similar to the average ratio of the measured green reflectance levels to the measured blue reflectance levels seen in the stomach (e.g., stomach306(FIG.3)). For instance, ingestible device100may retrieve data (e.g., from memory circuitry of PCB120(FIG.2)) indicative of the average ratio of the measured green reflectance levels to the measured blue reflectance levels seen in the stomach, and determine that ingestible device100has transitioned back to the stomach if the recently measured ratio of the measured green reflectance levels to the measured blue reflectance levels is sufficiently similar to the average level in the stomach (e.g., within 20% of the average ratio of the measured green reflectance levels to the measured blue reflectance levels seen in the stomach, or within any other suitable threshold level). If the ingestible device detects a transition from the duodenum (e.g., duodenum310(FIG.3)) to the stomach (e.g., stomach306(FIG.3)), process500proceeds to508to store data indicating the ingestible device has entered the stomach (e.g., stomach306(FIG.3)), and continues to monitor for further transitions. Alternatively, if the ingestible device does not detect a transition from the duodenum (e.g., duodenum310(FIG.3)) to the stomach (e.g., stomach306(FIG.3)), process500proceeds to516to gather additional measurements of green and blue reflectance levels while in the duodenum (e.g., duodenum310(FIG.3)), which may be used to continuously monitor for possible transitions back into the stomach. An example procedure for using measurements of green and blue reflectances to monitor for transitions between the stomach and the duodenum is discussed in greater detail in relation toFIG.6. At520, the ingestible device (e.g., ingestible device100,300, or400) gathers periodic measurements of the reflectance levels (e.g., via sensing sub-unit126(FIG.2)) while in the duodenum (e.g., duodenum310(FIG.3)). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may gather similar periodic measurements while in the stomach as well. In some embodiments, these periodic measurements may enable ingestible device100to detect muscle contractions (e.g., muscle contractions due to a peristaltic wave as discussed in relation toFIG.4), which may be indicative of entry into a jejunum (e.g., jejunum314(FIG.3)). Ingestible device100may be configured to gather periodic measurements using any suitable wavelength of illumination (e.g., by generating illumination using illuminator124, and detecting the resulting reflectance using detector122(FIG.2)), or combinations of wavelengths of illumination. For example, in some embodiments, ingestible device100may be configured to generate red, green, and blue illumination, store separate data sets indicative of red, green, and blue illumination, and analyze each of the data sets separately to search for frequency components in the recorded data indicative of detected muscle contractions. In some embodiments, the measurements gathered by ingestible device100at520may be sufficiently fast as to detect peristaltic waves in a subject. For instance, in a healthy human subject, peristaltic waves may occur at a rate of approximately 0.1 Hz to 0.2 Hz. Therefore, the ingestible device400may be configured to generate illumination and measure the resulting reflectance at least once every 2.5 seconds (i.e., the minimum rate necessary to detect a 0.2 Hz signal), and preferably at a higher rate, such as once every 0.5 seconds or faster, and store values indicative of the resulting reflectances in a data set (e.g., within memory circuitry of PCB120(FIG.2)). After gathering additional data (e.g., after gathering one new data point, or a predetermined number of new data points), process500proceeds to522, where ingestible device100determines whether or not a muscle contraction has been detected. At522, the ingestible device (e.g., ingestible device100,300, or400) determines (e.g., via control circuitry within PCB120(FIG.0.2)) whether the ingestible device detects a muscle contraction based on the measurements of reflectance levels (e.g., as gathered by sensing sub-unit126(FIG.2)). For example, ingestible device100may obtain a fixed amount of data stored as a result of measurements made at520(e.g., retrieve the past minute of data from memory circuitry within PCB120(FIG.2)). Ingestible device100may then convert the obtained data into the frequency domain, and search for peaks in a frequency range that would be consistent with peristaltic waves. For example, in a healthy human subject, peristaltic waves may occur at a rate of approximately 0.1 Hz to 0.2 Hz, and an ingestible device100may be configured to search for peaks in the frequency domain representation of the data between 0.1 Hz and 0.2 Hz above a threshold value. If the ingestible device100detects a contraction based on the reflectance levels (e.g., based on detecting peaks in the frequency domain representation of the data between 0.1 Hz and 0.2 Hz), process500proceeds to524to store data indicating that the device has entered the jejunum. Alternatively, if the ingestible device100does not detect a muscle contraction, process500proceeds to520to gather periodic measurements of the reflectance levels while in the duodenum (e.g., duodenum310(FIG.3)). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may store data (e.g., within memory circuitry of PCB120(FIG.2)) indicating that a muscle contraction was detected, and process500will not proceed from522to524until a sufficient number of muscle contractions have been detected. At524, the ingestible device (e.g., ingestible device100,300, or400) stores data (e.g., within memory circuitry of PCB120(FIG.2)) indicating that the device has entered the jejunum (e.g., jejunum314(FIG.3)). For example, in response to detecting that muscle contraction has occurred at522, ingestible device100may determine that it has entered the jejunum314, and is no longer inside of the duodenum (e.g., duodenum310(FIG.3)) or the stomach (e.g., stomach306(FIG.3)). In some embodiments, the ingestible device100may continue to measure muscle contractions while in the jejunum, and may store data indicative of the frequency, number, or strength of the muscle contractions over time (e.g., within memory circuitry of PCB120(FIG.2)). In some embodiments, the ingestible device100may also be configured to monitor for one or more transitions. Such transitions can include a transition from the jejunum to the ileum, an ileoceacal transition from the ileum to the cecum, a transition from the cecum to the colon, or detect exit from the body (e.g., by measuring reflectances, temperature, or levels of ambient light). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may also determine that it has entered the jejunum (e.g., jejunum314(FIG.3)) after a pre-determined amount of time has passed after having detected entry into the duodenum (e.g., duodenum310(FIG.3)). For example, barring a reverse pyloric transition from the duodenum (e.g., duodenum310(FIG.3)) back to the stomach (e.g., stomach306(FIG.3)), the typical transit time for an ingestible device to reach the jejunum from the duodenum in a healthy human subject is less than three minutes. In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may therefore be configured to automatically determine that it has entered the jejunum after spending at least three minutes within the duodenum. This determination may be made separately from the determination made based on measured muscle contractions (e.g., the determination made at522), and in some embodiments, ingestible device100may determine that it has entered the jejunum in response to either detecting muscle contractions, or after three minutes has elapsed from having entered the duodenum (e.g., as determined by storing data at514indicative of the time that ingestible device entered the duodenum). For illustrative purposes,512-518of process500describe the ingestible device (e.g., ingestible device100,300, or400) measuring green reflectances and blue reflectances, calculating a ratio of the two reflectances, and using this information to determine when the ingestible device has transitioned between the duodenum and stomach. However, in some embodiments, other wavelengths of light may be used other than green and blue, provided that the wavelengths of light chosen have different reflective properties within the stomach and the duodenum (e.g., as a result of different reflection coefficients of the stomach tissue and the tissue of the duodenum). It will be understood that the steps and descriptions of the flowcharts of this disclosure, includingFIG.5, are merely illustrative. Any of the steps and descriptions of the flowcharts, includingFIG.5, may be modified, omitted, rearranged, and performed in alternate orders or in parallel, two or more of the steps may be combined, or any additional steps may be added, without departing from the scope of the present disclosure. For example, the ingestible device100may calculate the mean and the standard deviation of multiple data sets in parallel in order to speed up the overall computation time. As another example, ingestible device100may gather data periodic measurements and detect possible muscle contractions (e.g., at520-522) while simultaneously gathering green and blue reflectance levels to determine transitions to and from the stomach and duodenum (e.g., at510-518). Furthermore, it should be noted that the steps and descriptions ofFIG.5may be combined with any other system, device, or method described in this application, including processes600(FIG.6) and900(FIG.9), and any of the ingestible devices or systems discussed in this application (e.g., ingestible devices100,300, or400) could be used to perform one or more of the steps inFIG.5. FIG.6is a flowchart illustrating some aspects of a process for detecting transitions from a stomach to a duodenum and from a duodenum back to a stomach, which may be used when determining a location of an ingestible device as it transits through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. In some embodiments, process600may begin when an ingestible device first detects that it has entered the stomach, and will continue as long as the ingestible device determines that it is within the stomach or the duodenum. In some embodiments, process600may only be terminated when an ingestible device determines that it has entered the jejunum, or otherwise progressed past the duodenum and the stomach. AlthoughFIG.6may be described in connection with the ingestible device100for illustrative purposes, this is not intended to be limiting, and either portions or the entirety of the duodenum detection process600described inFIG.6may be applied to any device discussed in this application (e.g., the ingestible devices100,300, or400), and any of the ingestible devices may be used to perform one or more parts of the process described inFIG.6. Furthermore, the features ofFIG.6may be combined with any other systems, methods or processes described in this application. For example, portions of the process described by the process inFIG.6may be integrated into process500discussed in relation toFIG.5. At602, the ingestible device (e.g., ingestible device100,300, or400) retrieves a data set (e.g., from memory circuitry within PCB120(FIG.2)) with ratios of the measured green reflectance levels to the measured blue reflectance levels over time. For example, ingestible device100may retrieve a data set from PCB120containing recently recorded ratios of the measured green reflectance levels to the measured blue reflectance levels (e.g., as recorded at510or516of process500(FIG.5)). In some embodiments, the retrieved data set may include the ratios of the measured green reflectance levels to the measured blue reflectance levels over time. Example plots of data sets of ratios of the measured green reflectance levels to the measured blue reflectance levels are discussed further in relation toFIG.7andFIG.8. At604, the ingestible device (e.g., ingestible device100,300, or400) includes a new measurement (e.g., as made with sensing sub-unit126(FIG.2)) of a ratio of the measured green reflectance level to the measured blue reflectance level in the data set. For example, ingestible device100may be configured to occasionally record new data by transmitting green and blue illumination (e.g., via illuminator124(FIG.2)), detecting the amount of reflectance received due to the green and blue illumination (e.g., via detector122(FIG.2)), and storing data indicative of the amount of the received reflectance (e.g., in memory circuitry of PCB120(FIG.2)). The ingestible device100may be configured to record new data every five to fifteen seconds, or at any other convenient interval of time. For illustrative purposes, ingestible device100is described as storing and retrieving the ratio of the measured green reflectance levels to the measured blue reflectance levels (e.g., if the amount of detected green reflectance was identical to the amount of detected blue reflectance at a given time, the ratio of the green and blue reflectances would be “1.0” at that given time); however, it is understood that the green reflectance data and the blue reflectance data may be stored separately within the memory of ingestible device100(e.g., stored as two separate data sets within memory circuitry of PCB120(FIG.2)). At606, the ingestible device (e.g., ingestible device100,300, or400) retrieves a first subset of recent data by applying a first sliding window filter to the data set. For example, ingestible device100may use a sliding window filter to obtain a predetermined amount of the most recent data within the data set, which may include any new values of the ratio of the measured green reflectance level to the measured blue reflectance level obtained at604. For instance, the ingestible device may be configured to select between ten and forty data points from the data set, or ingestible device100may be configured to select a predetermined range of data values between fifteen seconds of data and five minutes of data. In some embodiments, other ranges of data may be selected, depending on how frequently measurements are recorded, and the particular application at hand. For instance, any suitable amount of data may be selected in the sliding window, provided that it is sufficient to detect statistically significant differences between the data selected in a second sliding window (e.g., the second subset of data selected at614). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may also be configured to remove outliers from the data set, or to smooth out unwanted noise in the data set. For example, ingestible device100may select the first subset of data, or any other subset of data, by first obtaining a raw set of values by applying a window filter to the data set (e.g., selecting a particular range of data to be included). Ingestible device100may then be configured to identify outliers in the raw set of values; for instance, by identifying data points that are over three standard deviations away from the mean value of the raw set of values, or any other suitable threshold. Ingestible device100may then determine the subset of data by removing outliers from the raw set of values. This may enable ingestible device100to avoid spurious information when determining whether or not it is located within the stomach or the duodenum. At608, the ingestible device (e.g., ingestible device100,300, or400) determines whether the most recently detected location was the duodenum (e.g., duodenum310(FIG.3)). In some embodiments, ingestible device100may store a data flag (e.g., within memory circuitry of PCB120(FIG.2)) indicating the most recent portion of the GI tract that the ingestible device100detected itself to be within. For instance, every time ingestible device100detects entry to the stomach (e.g., detects entry into stomach306(FIG.3) as a result of the decision made at610), a flag is stored in memory indicating the ingestible device100is in the stomach (e.g., as part of storing data at612). If ingestible device100subsequently detects entry into the duodenum (e.g., detects entry into duodenum310(FIG.3) as a result of a decision made at624), another different flag is stored in memory indicating that the ingestible device100is in the duodenum (e.g., as part of storing data at624). In this case, ingestible device100may retrieve the most recently stored flag at608, and determine whether or not the flag indicates that the ingestible device100was most recently within the duodenum. If ingestible device100detects that it was most recently in the duodenum, process600proceeds to610where the ingestible device compares the recent measurements of the ratios of the measured green reflectance levels to the measured blue reflectance levels (e.g., measurements that include the recent measurement made at606) to the typical ratios measured within the stomach, and uses this information to determine whether a reverse pyloric transition from the duodenum back to the stomach has occurred. Alternately, if ingestible device100detects that it was not most recently in the duodenum (e.g., because it was in the stomach instead), process600proceeds to614where the ingestible device compares the recent measurements of the ratios of the measured green reflectance levels to the measured blue reflectance levels (e.g., measurements that include the recent measurement made at606) to past measurements, and uses this information to determine whether a pyloric transition from the stomach to the duodenum has occurred. Process600proceeds from608to610when the ingestible device determined that it was most recently in the duodenum. At610, the ingestible device (e.g., ingestible device100,300, or400) determines (e.g., via control circuitry within PCB120(FIG.2)) whether the current G/B signal is similar to a recorded average G/B signal in the stomach. For example, ingestible device100may be configured to have previously stored data (e.g., within memory circuitry of PCB120(FIG.2)) indicative of the average ratio of the measured green reflectance levels to the measured blue reflectance levels measured in the stomach. Ingestible device100may then retrieve this stored data indicative of the average ratio of the measured green reflectance levels to the measured blue reflectance levels in the stomach, and compare this against the recent measurements in order to determine whether or not ingestible device100has returned back to the stomach from the duodenum. For instance, ingestible device100may determine if the mean value of the first subset of recent data (i.e., the average value of the recently measured ratios of the measured green reflectance levels to the measured blue reflectance levels) is less than the average ratio of the measured green reflectance levels to the measured blue reflectance levels within the stomach, or less that the average ratio measured within the stomach plus a predetermined number times the standard deviation of the ratios measured within the stomach. For instance, if the average ratio of the measured green reflectance levels to the measured blue reflectance levels in the stomach was “1,” with a standard deviation of “0.2,” ingestible device100may determine whether or not the mean value of the first subset of data is less than “1.0+k*0.2,” where “k” is a number between zero and five. It is understood that, in some embodiments, the ingestible device100may be configured to use a different threshold level to determine whether or not the mean value of the first subset of recent data is sufficiently similar to the average ratio of the measured green reflectance levels to the measured blue reflectance levels within the stomach. In response to determining that the recent ratio of the measured green reflectance levels to the measured blue reflectance levels is similar to the average ratio of measured green and blue reflectance levels seen in the stomach, process600proceeds to612where ingestible device100stores data indicating that it has re-entered the stomach from the duodenum. Alternately, in response to determining that the recent ratio of measured green and blue reflectance levels is sufficiently different from the average ratio of measured green and blue reflectance levels seen in the stomach, ingestible device100proceeds directly to604, and continues to obtain new data on an ongoing basis. At612, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating a reverse pyloric transition from the duodenum to the stomach was detected. For example ingestible device100may store a data flag (e.g., within memory circuitry of PCB120(FIG.2)) indicating that the ingestible device100most recently detected itself to be within the stomach portion of the GI tract (e.g., stomach306(FIG.3)). In some embodiments, ingestible device100may also store data (e.g., within memory circuitry of PCB120(FIG.2)) indicating a time that ingestible device100detected the reverse pyloric transition from the duodenum to the stomach. This information may be used by ingestible device100at608, and as a result process600may proceed from608to614, rather than proceeding from618to610. After ingestible device100stores the data indicating a reverse pyloric transition from the duodenum to the stomach was detected, process600proceeds to604where ingestible device100continues to gather additional measurements, and continues to monitor for further transitions between the stomach and the duodenum. Process600proceeds from608to614when the ingestible device determined that it was not most recently in the duodenum (e.g., as a result of having most recently been in the stomach instead). At614, the ingestible device (e.g., ingestible device100,300, or400) retrieves a second subset of previous data by applying a second sliding window filter to the data set. For example, ingestible device100may use a sliding window filter to obtain a predetermined amount of older data from a past time range, which may be separated from recent time range used to select the first subset of data gathered at606by a predetermined period of time. In some embodiments, any suitable amount of data may be selected by the first and second window filters, and the first and second window filters may be separated by any appropriate predetermined amount of time. For example, in some embodiments, the first window filter and the second window filter may each be configured to select a predetermined range of data values from the data set, the predetermined range being between fifteen seconds of data and five minutes of data. In some embodiments, the recent measurements and the past measurements may then be separated by a predetermined period of time that is between one to five times the predetermined range of data values. For instance, ingestible device100may select the first subset of data and the second subset of data to each be one minute of data selected from the dataset (i.e., selected to have a predetermined range of one minute), and the first subset of data and the second subset of data are selected from recorded measurements that are at least two minutes apart (i.e., the predetermined period of time is two minutes, which is twice the range used to select the subsets of data using the window filters). As another example, ingestible device100may select the first subset of data and the second subset of data to each be five minutes of data selected from the dataset (i.e., selected to have a predetermined range of five minutes), and the first subset of data and the second subset of data are selected from recorded measurements that are at least 10 minutes apart (i.e., the predetermined period of time is two minutes, which is twice the range used to select the subsets of data using the window filters). In some embodiments, if ingestible device100recently transitioned to the stomach from the duodenum (e.g., as determined by checking for recent data stored within ingestible device100at612), ingestible device100may select the second subset of data at614from a time frame when ingestible device100is known to be within the stomach. In some embodiments, ingestible device100may alternately select a previously recorded average and standard deviation for ratios of green reflectances and blue reflectances within the stomach (e.g., an average and standard deviation typical of data recorded within the stomach, as previously recorded within memory circuitry of PCB120at620) in place of the second subset of data. In this case, ingestible device100may simply use the previously recorded average and previously recorded standard deviation when making a determination at616, rather than expending resources to calculate the mean and standard deviation of the second subset. At616, the ingestible device (e.g., ingestible device100,300, or400) determines whether the difference between the mean of the second subset and the mean of the first subset is greater than a predetermined multiple of the standard deviation of the first subset. For example, ingestible device100may compute a difference between a mean of the first subset of recent data and a mean of a second subset of past data, and determine whether this difference is greater than three times the standard deviation of the second subset of past data. In some embodiments, it is understood that any convenient threshold level may be used other than three times the standard deviation, such as any value between one and five times the standard deviation. Also, in some embodiments, the ingestible device may instead set the threshold level based on the standard deviation of the second subset instead of the first subset. In response to determining that the difference between the mean of the first subset and the mean of the second subset is greater than a predetermined multiple of the standard deviation of the second subset, process600proceeds to618. Otherwise, process600proceeds back to604, where the ingestible device604continues to gather new data to be used in monitoring for transitions between the stomach (e.g., stomach306(FIG.3)) and the duodenum (e.g., duodenum310(FIG.3)). At618, the ingestible device (e.g., ingestible device100,300, or400) determines (e.g., via control circuitry within PCB120(FIG.2)) whether the determination made at616is the first time that the difference between the mean of the first subset of recent data and the mean of the second subset of past data is calculated to be greater than the standard deviation of the second subset. If the ingestible device determines that this is the first time that the difference between the mean of the first subset and the mean of the second subset is calculated to be greater than the standard deviation of the second subset, process600proceeds to620to store the mean of the second subset of past data as an average G/B signal in the stomach. Alternatively, if the ingestible device determines that the immediately preceding determination made at616is not the first time that the difference between the mean of the first subset of recent data and the mean of the second subset of past data is calculated to be greater than the standard deviation of the second subset, process600proceeds directly to622. At620, the ingestible device (e.g., ingestible device100,300, or400) stores the mean of the second subset as an average G/B signal in the stomach. For example, ingestible device100may be configured to store the mean of the second subset of past data (e.g., store within memory circuitry of PCB120(FIG.2)) as the average ratio of the measured green reflectance levels to the measured blue reflectance levels measured in the stomach. In some embodiments, ingestible device100may also store the standard deviation of the second subset of past data as a typical standard deviation of the ratios of the measured green reflectance levels to the measured blue reflectance levels detected within the stomach. This stored information may be used by the ingestible device later on (e.g., at610) to compare against future data, which may enable the ingestible device to detect reverse pyloric transitions from the duodenum (e.g., duodenum310(FIG.3)) back to the stomach (e.g., stomach306(FIG.3)), and may generally be used in place of other experimental data gathered from the stomach (e.g., in place of the second subset of data at616). After storing the mean of the second subset as an average G/B signal in the stomach, process600proceeds to622. At622, the ingestible device (e.g., ingestible device100,300, or400) determines whether a difference of the mean of the first subset of recent data to the mean of the second subset of past data is greater than a predetermined threshold, “M”. In some embodiments, the predetermined threshold, “M,” will be sufficiently large to ensure that the mean of the first subset is substantially larger than the mean of the second subset, and may enable ingestible device100to ensure that it detected an actual transition to the duodenum. This may be particularly advantageous when the determination made at616is potentially unreliable due to the standard deviation of the second subset of past data being abnormally small. For example, a typical value of the predetermined threshold “M,” may be on the order of 0.1 to 0.5. If ingestible device100determines that the difference of the mean of the first subset of recent data to the second subset of past data is greater than a predetermined threshold, process600proceeds to624to store data indicating that a pyloric transition from the stomach to the duodenum (e.g., from stomach306to duodenum310(FIG.3)) was detected. Alternatively, if the ingestible device determines that the ratio of the mean of the first subset to the second subset is less than or equal to the predetermined threshold, “M” (i.e., determines that a transition to the duodenum has not occurred), process600proceeds directly to604where ingestible device100continues to make new measurements and monitor for possible transitions between the stomach and the duodenum. In some embodiments, instead of using a difference of the mean of the first subset of recent data to the mean of the second subset of past data, the ingestible device (e.g., ingestible device100,300, or400) determines whether the ratio of the mean of the first subset of recent data to the mean of the second subset of past data is greater than a predetermined threshold, “M”. In some embodiments, the predetermined threshold, “M,” will be sufficiently large to ensure that the mean of the first subset is substantially larger than the mean of the second subset, and may enable ingestible device100to ensure that it detected an actual transition to the duodenum. This may be particularly advantageous when the determination made at616is potentially unreliable due to the standard deviation of the second subset of past data being abnormally small. For example, a typical value of the predetermined threshold “M,” may be on the order of 1.2 to 2.0. It is understood any convenient type of threshold or calculation may be used to determine whether or not the first subset of data and the second subset of data are both statistically distinct from one another, and also substantially different from one another in terms of overall average value. At624, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating a pyloric transition from the stomach to the duodenum was detected. For example ingestible device100may store a data flag (e.g., within memory circuitry of PCB120(FIG.2)) indicating that the ingestible device100most recently detected itself to be within the duodenum portion of the GI tract (e.g., duodenum310(FIG.3)). In some embodiments, ingestible device100may also store data (e.g., within memory circuitry of PCB120(FIG.2)) indicating a time that ingestible device100detected the pyloric transition from the stomach to the duodenum. This information may be used by ingestible device100at608, and as a result process600may proceed from608to610, rather than proceeding from618to614. After ingestible device100stores the data indicating a pyloric transition from the stomach to the duodenum was detected, process600proceeds to604where ingestible device100continues to gather additional measurements, and continues to monitor for further transitions between the stomach and the duodenum. It will be understood that the steps and descriptions of the flowcharts of this disclosure, includingFIG.6, are merely illustrative. Any of the steps and descriptions of the flowcharts, includingFIG.6, may be modified, omitted, rearranged, and performed in alternate orders or in parallel, two or more of the steps may be combined, or any additional steps may be added, without departing from the scope of the present disclosure. For example, the ingestible device100may calculate the mean and the standard deviation of multiple data sets in parallel in order to speed up the overall computation time. Furthermore, it should be noted that the steps and descriptions ofFIG.6may be combined with any other system, device, or method described in this application, and any of the ingestible devices or systems discussed in this application could be used to perform one or more of the steps inFIG.6. For example, portions of process600may be incorporated into508-516of process500(FIG.5), and may be part of a more general process for determining a location of the ingestible device. As another example, the ratio of detected blue and green light (e.g., as measured and added to the data set at604) may continue even outside of the stomach or duodenum, and similar information may be recorded by the ingestible device throughout its transit in the GI tract. Example plots of data sets of ratios of measured green and blue reflectance levels, which may be gathered throughout the GI tract, are discussed further in relation toFIG.7andFIG.8below. FIG.7is a plot illustrating data collected during an example operation of an ingestible device (e.g., ingestible device100,300, or400), which may be used when determining a location of an ingestible device as it transits through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. AlthoughFIG.7may be described in connection with ingestible device100for illustrative purposes, this is not intended to be limiting, and plot700and data set702may be typical of data gathered by any device discussed in this application. Plot700depicts the ratios of the measured green reflectance levels to the measured blue reflectance levels over time. For example, ingestible device100may have computed the value for each point in the data set702by transmitting green and blue illumination at a given time (e.g., via illuminator124(FIG.2)), measuring the resulting green and blue reflectances (e.g., via detector122(FIG.2)), calculating the ratio of the resulting reflectances, and storing the ratio in the data set along with a timestamp indicating the time that the reflectances were gathered. At704, shortly after ingestible device100begins operation, ingestible device100determines that it has reached at least the stomach (e.g., as a result of making a determination similar to the determination discussed in relation to506in process500(FIG.5)). Ingestible device100continues to gather additional measurements of green and blue reflectance levels, and at706ingestible device100determines that a pyloric transition has occurred from the stomach to the duodenum (e.g., as a result of making a determination similar to the determinations discussed in relation to616-624of process600(FIG.6)). Notably, the values in data set702around706jump up precipitously, which is indicative of the higher ratios of measured green reflectance levels to measured blue reflectance levels typical of the duodenum. The remainder of the data set702depicts the ratios of the measured green reflectance levels to the measured blue reflectance levels throughout the remainder of the GI tract. At708, ingestible device100has reached the jejunum (e.g., as determined through measurements of muscle contractions, as discussed in relation toFIG.9), and by710, ingestible device100has reached the cecum. It is understood that, in some embodiments, the overall character and appearance of data set702changes within the small intestine (i.e., the duodenum, jejunum, and ileum) versus the cecum. Within the jejunum and ileum, there may typically be a wide variation in the ratios of the measured green reflectance levels to the measured blue reflectance levels, resulting in relatively noisy data with a high standard deviation. By comparison, within the cecum ingestible device100may measure a relatively stable ratio of the measured green reflectance levels to the measured blue reflectance levels. In some embodiments, ingestible device100may be configured to determine transitions from the small intestine to the cecum based on these differences. For example, ingestible device100may compare recent windows of data to past windows of data, and detect a transition to the cecum in response to determining that the standard deviation of the ratios in the recent window of data is substantially less than the standard deviation of the ratios in the past window of data. FIG.8is another plot illustrating data collected during an example operation of an ingestible device, which may be used when determining a location of an ingestible device as it transits through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. Similar toFIG.7,FIG.8may be described in connection with the ingestible device100for illustrative purposes. However, this is not intended to be limiting, and plot800and data set802may be typical of data gathered by any device discussed in this application. At804, shortly after ingestible device100begins operation, ingestible device100determines that it has reached at least the stomach (e.g., as a result of making a determination similar to the determination discussed in relation to506in process500(FIG.5)). Ingestible device100continues to gather additional measurements of green and blue reflectance levels (e.g., via sensing sub-unit126(FIG.2)), and at806ingestible device100determines that a pyloric transition has occurred from the stomach to the duodenum (e.g., as a result of making a determination similar to the determinations discussed in relation to616-624of process600(FIG.6)). Notably, the values in data set802around806jump up precipitously, which is indicative of the higher ratios of measured green reflectance levels to measured blue reflectance levels typical of the duodenum, before falling shortly thereafter. As a result of the reduced values in data set802, ingestible device100determines that a reverse pyloric transition has occurred from the duodenum back to the stomach at808(e.g., as a result of making a determination similar to the determinations discussed in relation to610-612of process600(FIG.6)). At810, as a result of the values in data set802increasing again, ingestible device100determines that another pyloric transition has occurred from the stomach to the duodenum, and shortly thereafter ingestible device100proceeds onwards to the jejunum, ileum, and cecum. The remainder of the data set802depicts the ratios of the measured green reflectance levels to the measured blue reflectance levels throughout the remainder of the GI tract. Notably, at812, ingestible device reaches the transition point between the ileum and the cecum. As discussed above in relation toFIG.7, the transition to the cecum is marked by a reduced standard deviation in the ratios of measured green reflectances and measured blue reflectances over time, and ingestible device100may be configured to detect a transition to the cecum based on determining that the standard deviation of a recent set of measurements is substantially smaller than the standard deviation of past measurements taken from the jejunum or ileum. FIG.9is a flowchart of illustrative steps for detecting a transition from a duodenum to a jejunum, which may be used when determining a location of an ingestible device as it transits through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. AlthoughFIG.9may be described in connection with the ingestible device100for illustrative purposes, this is not intended to be limiting, and either portions or the entirety of process900described inFIG.9may be applied to any device discussed in this application (e.g., the ingestible devices100,300, and400), and any of these ingestible devices may be used to perform one or more parts of the process described inFIG.9. Furthermore, the features ofFIG.9may be combined with any other systems, methods or processes described in this application. For example, portions of the process described by the process inFIG.9may be integrated into the localization process described byFIG.5(e.g., as part of520-524of process500(FIG.5)). In some embodiments, an ingestible device100may perform process900while in the duodenum, or in response to detecting entry to the duodenum. In other embodiments, an ingestible device100may perform process900while in the stomach, or in response to detecting entry into the GI tract. It is also understood that process900may be performed in parallel with any other process described in this disclosure (e.g., process600(FIG.6)), which may enable ingestible device100to detect entry into various portions of the GI tract, without necessarily detecting entry into a preceding portion of the GI tract. For illustrative purposes,FIG.9may be discussed in terms of ingestible device100generating and making determinations based on a single set of reflectance levels generated at a single wavelength by a single sensing sub-unit (e.g., sensing sub-unit126(FIG.2)). However, it is understood that ingestible device100may generate multiple wavelengths of illumination from multiple different sensing sub-units positioned around the circumference of ingestible device (e.g., multiple sensing sub-units positioned at different locations behind window114of ingestible device100(FIG.1), and each of the resulting reflectances may be stored as a separate data set. Moreover, each of these sets of reflectance levels may be used to detect muscle contractions by running multiple versions of process900, each one of which processes data for a different set of reflectances corresponding to data sets obtained from measurements of different wavelengths or measurements made by different sensing sub-units. At902, the ingestible device (e.g., ingestible device100,300, or400) retrieves a set of reflectance levels. For example, ingestible device100may retrieve a data set of previously recorded reflectance levels from memory (e.g., from memory circuitry of PCB120(FIG.2)). Each of the reflectance levels may correspond to reflectances previously detected by ingestible device100(e.g., via detector122(FIG.2)) from illumination generated by ingestible device100(e.g., via illuminator124(FIG.2)), and may represent a value indicative of an amount of light detected in a given reflectance. However, it is understood that any suitable frequency of light may be used, such as light in the infrared, visible, or ultraviolet spectrums. In some embodiments, the reflectance levels may correspond to reflectances previously detected by ingestible device100at periodic intervals. At904, the ingestible device (e.g., ingestible device100,300, or400) includes new measurements of reflectance levels in the data set. For example, ingestible device100may be configured to detect a new reflectance (e.g., transmit illumination and detect the resulting reflectance using sensing sub-unit126(FIG.2)) at regular intervals, or with sufficient speed as to detect peristaltic waves. For example, ingestible device100may be configured to generate illumination and measure the resulting reflectance once every three seconds (i.e., the minimum rate necessary to detect a 0.17 Hz signal), and preferably at a higher rate, as fast at 0.1 second or even faster. It is understood that the periodic interval between measurements may be adapted as needed based on the species of the subject, and the expected frequency of the peristaltic waves to be measured. Every time ingestible device100makes a new reflectance level measurement at904, the new data is included to the data set (e.g., a data set stored within memory circuitry of PCB120(FIG.2)). At906, the ingestible device (e.g., ingestible device100,300, or400) obtains a first subset of recent data by applying a sliding window filter to the data set. For example, ingestible device100may retrieve a one-minute worth of data from the data set. If the data set includes values for reflectances measured every second, this would be approximately 60 data points worth of data. Any suitable type of window size may be used, provided that the size of the window is sufficiently large to detect peristaltic waves (e.g., fluctuations on the order of 0.1 Hz to 0.2 Hz for healthy human subjects). In some embodiments, ingestible device100may also clean the data, for example, by removing outliers from the first subset of data obtained through the use of the sliding window filter. At908, the ingestible device (e.g., ingestible device100,300, or400) obtains a second subset of recent data by interpolating the first subset of recent data. For example, ingestible device100may interpolate the first subset of data in order to generate a second subset of data with a sufficient number of data points (e.g., data points spaced every 0.5 seconds or greater). In some embodiments, this may enable ingestible device100to also replace any outlier data points that may have been removed as part of applying the window filter at906. At910, the ingestible device (e.g., ingestible device100,300, or400) calculates a normalized frequency spectrum from the second subset of data. For example, ingestible device100may be configured to perform a fast Fourier transform to convert the second subset of data from a time domain representation into a frequency domain representation. It is understood that depending on the application being used, and the nature of the subset of data, any number of suitable procedures (e.g., Fourier transform procedures) may be used to determine a frequency spectrum for the second subset of data. For example, the sampling frequency and size of the second subset of data may be known in advance, and ingestible device100may be configured to have pre-stored values of a normalized discreet Fourier transform (DFT) matrix, or the rows of the DFT matrix corresponding to the 0.1 Hz to 0.2 Hz frequency components of interest, within memory (e.g., memory circuitry of PCB120(FIG.2)). In this case, the ingestible device may use matrix multiplication between the DFT matrix and the data set to generate an appropriate frequency spectrum. An example data set and corresponding frequency spectrum that may be obtained by the ingestible device is discussed in greater detail in relation toFIG.10. At912, the ingestible device (e.g., ingestible device100,300, or400) determines whether at least a portion of the normalized frequency spectrum is between 0.1 Hz and 0.2 Hz above a threshold value of 0.5 Hz. Peristaltic waves in a healthy human subject occur at a rate between 0.1 Hz and 0.2 Hz, and an ingestible device experiencing peristaltic waves (e.g., ingestible device400detecting contractions in walls406of the jejunum (FIG.4)) may detect sinusoidal variations in the amplitude of detected reflectances levels that follow a similar 0.1 Hz to 0.2 Hz frequency. If the ingestible device determines that a portion of the normalized frequency spectrum between 0.1 Hz and 0.2 Hz is above a threshold value of 0.5, this measurement may be consistent with peristaltic waves in a healthy human subject, and process900proceeds to914where ingestible device100stores data indicating a muscle contraction was detected. Alternatively, if the ingestible device determines that no portion of the normalized frequency spectrum between 0.1 Hz and 0.2 Hz above a threshold value of 0.5, process900proceeds directly to904to make new measurements and to continue to monitor for new muscle contractions. It is understood that a threshold value other than 0.5 may be used, and that the exact threshold may depend on the sampling frequency and type of frequency spectrum used by ingestible device100. At914, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating a muscle contraction was detected. For example, ingestible device100may store data in memory (e.g., memory circuitry of PCB120(FIG.2)) indicating that a muscle contraction was detected, and indicating the time that the muscle contraction was detected. In some embodiments, ingestible device100may also monitor the total number of muscle contractions detected, or the number of muscle contractions detected in a given time frame. In some embodiments, detecting a particular number of muscle contractions may be consistent with ingestible device100being within the jejunum (e.g., jejunum314(FIG.3)) of a healthy human subject. After detecting a muscle contraction, process900proceeds to916. At916, the ingestible device (e.g., ingestible device100,300, or400) determines whether a total number of muscle contractions exceeds a predetermined threshold number. For example, ingestible device100may retrieve the total number of muscle contractions detected from memory (e.g., from memory circuitry of PCB120(FIG.2)), and compare the total number to a threshold value. In some embodiments, the threshold value may be one, or any number larger than one. The larger the threshold value, the more muscle contractions need to be detected before ingestible device100stores data indicating that it has entered the jejunum. In practice, setting the threshold value as three or higher may prevent the ingestible device from detecting false positives (e.g., due to natural movement of the GI tract organs, or due to movement of the subject). If the total number of contractions exceeds the predetermined threshold number, process900proceeds to918to store data indicating detection of a transition from the duodenum to the jejunum. Alternatively, if the total number of contractions does not exceed a predetermined threshold number, process900proceeds to904to include new measurements of reflectance levels in the data set. An example plot of the muscle contractions detected over time is discussed in greater detail in relation toFIG.11. At918, the ingestible device (e.g., ingestible device100,300, or400) stores data indicating detection of a transition from the duodenum to the jejunum. For example, ingestible device100may store data in memory (e.g., from memory circuitry of PCB120(FIG.2)) indicating that the jejunum has been reached. In some embodiments, if ingestible device100is configured to perform all or part of process900while in the stomach, ingestible device100may store data at918indicating detection of a transition from the stomach directly to the jejunum (e.g., as a result of transitioning too quickly through the duodenum for the pyloric transition to be detected using process600(FIG.6)). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may be configured to obtain a fluid sample from the environment external to a housing of the ingestible device in response to identifying a change in the location of the ingestible device. For example, ingestible device100may be configured to obtain a fluid sample from the environment external to the housing of ingestible device100(e.g., through the use of optional opening116and optional rotating assembly118(FIG.2)) in response to determining that the ingestible device is located within the jejunum (e.g., jejunum314(FIG.3)). In some embodiments, ingestible device100may also be equipped with appropriate diagnostics to detect certain medical conditions based on the retrieved fluid sample, such as small intestinal bacterial overgrowth (SIBO). In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may be configured to deliver a dispensable substance that is pre-stored within the ingestible device from the ingestible device into the gastrointestinal tract in response to identifying the change in the location of the ingestible device. For example, ingestible device100may have a dispensable substance pre-stored within the ingestible device100(e.g., within a storage chamber or cavity on optional storage sub-unit118-3(FIG.2)), and ingestible device100may be configured to dispense the substance into the gastrointestinal tract (e.g., through the use of optional opening116and optional rotating assembly118(FIG.2)) when the ingestible device100detects that the ingestible device100is located within the jejunum (e.g., jejunum314(FIG.3)). In some embodiments, this may enable ingestible device100to deliver substances (e.g., therapeutics and medicaments) at targeted locations within the GI tract. In some embodiments, the ingestible device (e.g., ingestible device100,300, or400) may be configured to perform an action based on the total number of detected muscle contractions. For example, ingestible device100may be configured to retrieve data indicative of the total number of muscle contractions (e.g., from memory circuitry of PCB120(FIG.2)), and compare that to an expected number muscle contractions in a healthy individual. In response, the ingestible device may either dispense a substance into the gastrointestinal tract (e.g., through the use of optional opening116and optional rotating assembly118(FIG.2)), or may obtain a fluid sample from the environment external to the housing of ingestible device100(e.g., through the use of optional opening116and optional rotating assembly118(FIG.2)). For instance, ingestible device100may be configured to obtain a sample in response to determining that a number of detected muscle contractions is abnormal, and differs greatly from the expected number. As another example, ingestible device100may be configured to deliver a substance into the GI tract (such as a medicament), in response to determining that the detected muscle contractions are consistent with a functioning GI tract in a healthy individual. It will be understood that the steps and descriptions of the flowcharts of this disclosure, includingFIG.9, are merely illustrative. Any of the steps and descriptions of the flowcharts, includingFIG.9, may be modified, omitted, rearranged, performed in alternate orders or in parallel, two or more of the steps may be combined, or any additional steps may be added, without departing from the scope of the present disclosure. For example, the ingestible device100may calculate the mean and the standard deviation of multiple data sets in parallel (e.g., multiple data sets, each one corresponding to a different wavelength of reflectance or different sensing sub-unit used to detect the reflectance) in order to speed up the overall computation time. Furthermore, it should be noted that the steps and descriptions ofFIG.9may be combined with any other system, device, or method described in this application, and any of the ingestible devices or systems discussed in this application could be used to perform one or more of the steps inFIG.9. FIG.10is a plot illustrating data collected during an example operation of an ingestible device, which may be used when detecting a transition from a duodenum to a jejunum, in accordance with some embodiments of the disclosure. Diagram1000depicts a time domain plot1002of a data set of reflectance levels measured by an ingestible device (e.g., the second subset of data discussed in relation to908ofFIG.9). In some embodiments, ingestible device100may be configured to gather data points at semi-regular intervals approximately 0.5 seconds apart. By comparison, diagram1050depicts a frequency domain plot1004of the same data set of reflectance levels measured by an ingestible device (e.g., as a result of ingestible device100calculating a frequency spectrum at910ofFIG.9). In some embodiments, ingestible device100may be configured to calculate the frequency spectrum through any convenient means. In diagram1050, the range of frequencies1006between 0.1 Hz and 0.2 Hz may be the range of frequencies that ingestible device100searches in order to detect muscle contractions. As shown in diagram1050, there is a strong peak in the frequency domain plot1004around 0.14 Hz, which is consistent with the frequency of peristaltic motion in a healthy human individual. In this case, an ingestible device100analyzing frequency domain plot1004may be configured to determine that the data is consistent with a detected muscle contraction (e.g., using a process similar to912of process900(FIG.9)), and may store data (e.g., in memory circuitry of PCB120(FIG.2)) indicating that a muscle contraction has been detected. Because the muscle contraction was detected from the one-minute window of data ending at 118 minutes, ingestible device100may also store data indicating that the muscle contraction was detected at the 118-minute mark (i.e., which may indicate that the ingestible device100was turned on and ingested by the subject 118 minutes ago). FIG.11is a plot illustrating muscle contractions detected by an ingestible device over time, which may be used when determining a location of an ingestible device as it transits through a gastrointestinal (GI) tract, in accordance with some embodiments of the disclosure. In some embodiments, ingestible device100may be configured to detect muscle contractions, and store data indicative of when each muscle contraction is detected (e.g., as part of914of process900(FIG.9)). Plot1100depicts the detected muscle contractions1106over time, with each muscle contraction being represented by a vertical line reaching from “0” to “1” on the y-axis. At1102, around the 10-minute mark, ingestible device100first enters the duodenum (e.g., as determined by ingestible device100performing process600(FIG.6)). Shortly thereafter, at1108, ingestible device100begins to detect several muscle contractions1106in quick succession, which may be indicative of the strong peristaltic waves that form in the jejunum (e.g., jejunum314(FIG.3)). Later, around1110, ingestible device100continues to detect intermittent muscle contractions, which may be consistent with an ingestible device100within the ileum. Finally at1104, ingestible device100transitions out of the small intestine, and into the cecum. Notably, ingestible device100detects more frequent muscle contractions in the jejunum portion of the small intestine as compared to the ileum portion of the small intestine, and ingestible device100does not measure any muscle contractions after having exited the small intestine. In some embodiments, ingestible device100may incorporate this information into a localization process. For example, ingestible device100may be configured to detect a transition from a jejunum to an ileum in response to determining that a frequency of detected muscle contractions (e.g., the number of muscle contractions measured in a given 10-minute window) has fallen below a threshold number. As another example, ingestible device100may be configured to detect a transition from an ileum to a cecum in response to determining that no muscle contractions have been detected for a threshold period of time. It is understood that these examples are intended to be illustrative, and not limiting, and that measurements of muscle contractions may be combined with any of the other processes, systems, or methods discussed in this disclosure. FIG.12is a flowchart1200for certain embodiments for determining a transition of the device from the jejunum to the ileum. It is to be noted that, in general, the jejunum is redder and more vascular than the ileum. Moreover, generally, in comparison to the ileum, the jejunum has a thicker intestine wall with more messentary fat. These differences between the jejunum and the ileum are expected to result in differences in optical responses in the jejunum relative to the ileum. Optionally, one or more optical signals may be used to investigate the differences in optical responses. For example, the process can include monitoring a change in optical response in reflected red light, blue light, green light, ratio of red light to green light, ratio of red light to blue light, and/or ratio of green light to blue light. In some embodiments, reflected red light is detected in the process. Flowchart1200represents a single sliding window process. In step1210, the jejenum reference signal is determined based on optical reflection. Typically, this signal is as the average signal (e.g., reflected red light) over a period of time since the device was determined to enter the jejenum. The period of time can be, for example, from five minutes to 40 minutes (e.g., from 10 minutes to 30 minutes, from 15 minutes to 25 minutes). In step1220, the detected signal (e.g., reflected red light) just after the period of time used in step1210is normalized to the reference signal determined in step1210. In step1230, the signal (e.g., reflected red light) is detected. In step1240, the mean signal detected based on the single sliding window is compared to a signal threshold. The signal threshold in step1240is generally a fraction of the reference signal of the jejenum reference signal determined in step1210. For example, the signal threshold can be from 60% to 90% (e.g., from 70% to 80%) of the jejenum reference signal. If the mean signal exceeds the signal threshold, then the process determines that the device has entered the ileum at step1250. If the mean signal does not exceed the signal threshold, then the process returns to step1230. FIG.13is a flowchart1200for certain embodiments for determining a transition of the device from the jejunum to the ileum using a two sliding window process. In step1310, the jejenum reference signal is determined based on optical reflection. Typically, this signal is as the average signal (e.g., reflected red light) over a period of time since the device was determined to enter the jejenum. The period of time can be, for example, from five minutes to 40 minutes (e.g., from 10 minutes to 30 minutes, from 15 minutes to 25 minutes). In step1320, the detected signal (e.g., reflected red light) just after the period of time used in step1310is normalized to the reference signal determined in step1310. In step1330, the signal (e.g., reflected red light) is detected. In step1340, the mean difference in the signal detected based on the two sliding windows is compared to a signal threshold. The signal threshold in step1340is based on whether the mean difference in the detected signal exceeds a multiple (e.g., from 1.5 times to five times, from two times to four times) of the detected signal of the first window. If signal threshold is exceeded, then the process determines that the device has entered the ileum at step1350. If the signal threshold is not exceeded, then the process returns to step1330. FIG.14is a flowchart1400for a process for certain embodiments for determining a transition of the device from the ileum to the cecum. In general, the process involves detecting changes in the reflected optical signal (e.g., red light, blue light, green light, ratio of red light to green light, ratio of red light to blue light, and/or ratio of green light to blue light). In some embodiments, the process includes detecting changes in the ratio of reflected red light to reflected green light, and also detecting changes in the ratio of reflected green light to reflected blue light. Generally, in the process1400, the sliding window analysis (first and second windows) discussed with respect to process600is continued. Step1410includes setting a first threshold in a detected signal, e.g., ratio of detected red light to detected green light, and setting a second threshold for the coefficient of variation for a detected signal, e.g., the coefficient of variation for the ratio of detected green light to detected blue light. The first threshold can be set to a fraction (e.g., from 0.5 to 0.9, from 0.6 to 0.8) of the average signal (e.g., ratio of detected red light to detected green light) in the first window, or a fraction (e.g., from 0.4 to 0.8, from 0.5 to 0.7) of the mean difference between the detected signal (e.g., ratio of detected red light to detected green light) in the two windows. The second threshold can be set to 0.1 (e.g., 0.05, 0.02). Step1420includes detecting the signals in the first and second windows that are to be used for comparing to the first and second thresholds. Step1430includes comparing the detected signals to the first and second thresholds. If the corresponding value is not below the first threshold or the corresponding value is not below the second threshold, then it is determined that the device has not left the ileum and entered the cecum, and the process returns to step1420. If the corresponding value is below the first threshold and the corresponding value is below the second threshold, then it is determined that the device has left the ileum and entered the cecum, and the proceeds to step1440. Step1450includes determining whether it is the first time that that the device was determined to leave the ileum and enter the cecum. If it is the first time that the device was determined to leave the ileum and enter the cecum, then the process proceeds to step1460. If it is not the first time that the device has left the ileum and entered the cecum, then the process proceeds to step1470. Step1460includes setting a reference signal. In this step the optical signal (e.g., ratio of detected red light to detected green light) as a reference signal. Step1470includes determining whether the device may have left the cecum and returned to the ileum. The device is determined to have left the cecum and returned to the ileum if the corresponding detected signal (e.g., ratio of detected red light to detected green light) is statistically comparable to the reference signal (determined in step1460) and the coefficient of variation for the corresponding detected signal (e.g., ratio of detected green light to detected blue light) exceeds the second threshold. If it is determined that the device may have left the cecum and returned to the ileum, the process proceeds to step1480. Step1480includes continuing to detect the relevant optical signals for a period of time (e.g., at least one minute, from five minutes to 15 minutes). Step1490includes determining whether the signals determined in step1480indicate (using the methodology discussed in step1470) that the device re-entered the ileum. If the signals indicate that the device re-entered the ileum, the process proceeds to step1420. If the signals indicate that the device is in the cecum, the process proceeds to step1492. Step1492includes continuing to monitor the relevant optical signals for a period of time (e.g., at least 30 minutes, at least one hour, at least two hours). Step1494includes determining whether the signals determined in step1492indicate (using the methodology discussed in step1470) that the device re-entered the ileum. If the signals indicate that the device re-entered the ileum, the process proceeds to step1420. If the signals indicate that the device is in the cecum, the process proceeds to step1496. At step1496, the process determines that the device is in the cecum. FIG.15is a flowchart1500for a process for certain embodiments for determining a transition of the device from the cecum to the colon. In general, the process involves detecting changes in the reflected optical signal (e.g., red light, blue light, green light, ratio of red light to green light, ratio of red light to blue light, and/or ratio of green light to blue light). In some embodiments, the process includes detecting changes in the ratio of reflected red light to reflected green light, and also detecting changes in the ratio of reflected blue light. Generally, in the process1500, the sliding window analysis (first and second windows) discussed with respect to process1400is continued. In step1510, optical signals (e.g., the ratio of reflected red signal to reflected green signal, and reflected blue signal) are collected for a period of time (e.g., at least one minute, at least five minutes, at least 10 minutes) while the device is in the cecum (e.g., during step1480). The average values for the recorded optical signals (e.g., the ratio of reflected red signal to reflected green signal, and reflected blue signal) establish the cecum reference signals. In step1520, the optical signals are detected after it has been determined that the device entered the cecum (e.g., at step1440). The optical signals are normalized to the cecum reference signals. Step1530involves determining whether the device has entered the colon. This includes determining whether any of three different criteria are satisfied. The first criterion is satisfied if the mean difference in the ratio of a detected optical signal (e.g., ratio of detected red signal to the detected green) is a multiple greater than one (e.g., 2×, 3×, 4×) the standard deviation of the corresponding signal (e.g., ratio of detected red signal to the detected green) in the second window. The second criterion is satisfied if the mean of a detected optical signal (e.g., a ratio of detected red light to detected green light) exceeds a given value (e.g., exceeds one). The third criterion is satisfied if the coefficient of variation of an optical signal (e.g., detected blue light) in the first window exceeds a given value (e.g., exceeds 0.2). If any of the three criteria are satisfied, then the process proceeds to step1540. Otherwise, none of the three criteria are satisfied, the process returns to step1520. For illustrative purposes the disclosure focuses primarily on a number of different example embodiments of an ingestible device, and example embodiments of methods for determining a location of an ingestible device within a GI tract. However, the possible ingestible devices that may be constructed are not limited to these embodiments, and variations in the shape and design may be made without significantly changing the functions and operations of the device. Similarly, the possible procedures for determining a location of the ingestible device within the GI tract are not limited to the specific procedures and embodiments discussed (e.g., process500(FIG.5), process600(FIG.6), process900(FIG.9), process1200(FIG.12), process1300(FIG.13), process1400(FIG.14) and process1500(FIG.15)). Also, the applications of the ingestible devices described herein are not limited merely to gathering data, sampling and testing portions of the gastrointestinal tract, or delivering medicament. For example, in some embodiments the ingestible device may be adapted to include a number of chemical, electrical, or optical diagnostics for diagnosing a number of diseases. Similarly, a number of different sensors for measuring bodily phenomenon or other physiological qualities may be included on the ingestible device. For example, the ingestible device may be adapted to measure elevated levels of certain chemical compounds or impurities in the gastrointestinal tract, or the combination of localization, sampling, and appropriate diagnostic and assay techniques incorporated into a sampling chamber may be particularly well suited to determine the presence of small intestinal bacterial overgrowth (SIBO). At least some of the elements of the various embodiments of the ingestible device described herein that are implemented via software (e.g., software executed by control circuitry within PCB120(FIG.2)) may be written in a high-level procedural language such as object oriented programming, a scripting language or both. Accordingly, the program code may be written in C, C++ or any other suitable programming language and may comprise modules or classes, as is known to those skilled in object oriented programming. Alternatively, or in addition, at least some of the elements of the embodiments of the ingestible device described herein that are implemented via software may be written in assembly language, machine language or firmware as needed. In either case, the language may be a compiled or an interpreted language. At least some of the program code used to implement the ingestible device can be stored on a storage media or on a computer readable medium that is readable by a general or special purpose programmable computing device having a processor, an operating system and the associated hardware and software that is necessary to implement the functionality of at least one of the embodiments described herein. The program code, when read by the computing device, configures the computing device to operate in a new, specific and predefined manner in order to perform at least one of the methods described herein. Furthermore, at least some of the programs associated with the systems, devices, and methods of the example embodiments described herein are capable of being distributed in a computer program product comprising a computer readable medium that bears computer usable instructions for one or more processors. The medium may be provided in various forms, including non-transitory forms such as, but not limited to, one or more diskettes, compact disks, tapes, chips, and magnetic and electronic storage. In some embodiments, the medium may be transitory in nature such as, but not limited to, wire-line transmissions, satellite transmissions, internet transmissions (e.g. downloads), media, digital and analog signals, and the like. The computer useable instructions may also be in various formats, including compiled and non-compiled code. The techniques described above can be implemented using software for execution on a computer. For instance, the software forms procedures in one or more computer programs that execute on one or more programmed or programmable computer systems (which may be of various architectures such as distributed, client/server, or grid) each including at least one processor, at least one data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device or port, and at least one output device or port. The software may be provided on a storage medium, such as a CD-ROM, readable by a general or special purpose programmable computer or delivered (encoded in a propagated signal) over a communication medium of a network to the computer where it is executed. All of the functions may be performed on a special purpose computer, or using special-purpose hardware, such as coprocessors. The software may be implemented in a distributed manner in which different parts of the computation specified by the software are performed by different computers. Each such computer program is preferably stored on or downloaded to a storage media or device (e.g., solid state memory or media, or magnetic or optical media) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer system to perform the procedures described herein. The inventive system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer system to operate in a specific and predefined manner to perform the functions described herein. Methods and Mechanisms of Delivery FIG.16provides an example mock-up diagram illustrating aspects of a structure of an ingestible device1600for delivering a dispensable substance, such as a formulation of a therapeutic agent described herein, according to some embodiments described herein. In some embodiments, the ingestible device1600may generally be in the shape of a capsule, a pill or any swallowable form that may be orally consumed by an individual. In this way, the ingestible device1600may be ingested by a patient and may be prescribed by healthcare practitioners and patients. The ingestible device1600includes a housing1601that may take a shape similar to a capsule, a pill, and/or the like, which may include two ends1602a-b. The housing1601may be designed to withstand the chemical and mechanical environment of the GI tract (e.g., effects of muscle contractile forces and concentrated hydrochloric acid in the stomach). A broad range of materials that may be used for the housing1601. Examples of these materials include, but are not limited to, thermoplastics, fluoropolymers, elastomers, stainless steel and glass complying with ISO 10993 and USP Class VI specifications for biocompatibility; and any other suitable materials and combinations thereof. In some embodiment, the wall of the housing1601may have a thickness of 0.5 mm-1 mm, which is sufficient to sustain an internal explosion (e.g., caused by hydrogen ignition or over pressure inside the housing). The housing1601may or may not have a pH-sensitive enteric coating to detect or otherwise be sensitive to a pH level of the environment external to the ingestible device. As discussed elsewhere in the application in more detail, the ingestible device1600may additionally or alternatively include one more sensors, e.g., temperature sensor, optical sense. The housing1601may be formed by coupling two enclosure portions together. The ingestible device1600may include an electronic component within the housing1600. The electronic component may be placed proximally to an end1602bof the housing, and includes a printed circuit board (PCB), a battery, an optical sensing unit, and/or the like. The ingestible device1600further includes a gas generating cell1603that is configured to generate gas and thus cause an internal pressure within the housing1601. In some embodiments, the gas generating cell may include or be connected to a separate channel or valve of the ingestible device such that gas may be release through the channel or valve to create a motion to alter the position of the ingestible device within the GI tract. Such gas release can also be used to position the ingestible device relative to the intestinal lining. In another embodiment, gas may be released through the separate channel or valve to alter the surface orientation of the intestinal tissue prior to delivery of the dispensable substance. A traveling plunger1604may be placed on top of the gas generating cell1603within the housing1601. The traveling plunger1604is a membrane that separates the gas generating cell1603and a storage reservoir that stores the dispensable substance1605. In some embodiments, the traveling plunger1604may be a movable piston. In some embodiments, the traveling plunger1604may instead be a flexible membrane such as but not limited to a diaphragm. In some embodiments, the traveling plunger1604, which may have the form of a flexible diaphragm, may be placed along an axial direction of the housing1601, instead of being placed on top of the gas generating cell1603. The traveling plunger or the membrane1604may move (when the membrane1604is a piston) or deform (when the membrane1604is a diaphragm) towards a direction of the end1602aof the housing, when the gas generating cell1603generates gas to create an internal pressure that pushes the membrane1604. In this way, the membrane or traveling plunger1604may push the dispensable substance1605out of the housing via a dispensing outlet1607. The housing1601may include a storage reservoir storing one or more dispensable substances1605adjacent to the traveling plunger1604. The dispensable substance1605may be a therapeutic or medical agent that may take a form of a powder, a compressed powder, a fluid, a semi-liquid gel, or any other dispensable or deliverable form. The delivery of the dispensable substance1605may take a form such as but not limited to bolus, semi-bolus, continuous, burst drug delivery, and/or the like. In some embodiments, a single bolus is delivered proximate to the disease location. In some embodiments, more than one bolus is released at one location or more than one location. In some embodiments the release of more than one bolus is triggered according to a pre-programmed algorithm. In some embodiments the release profile is continuous. In some embodiments the release profile is time-based. In some embodiments the release profile is location-based. In some embodiments, the amount delivered is based on the severity and/or extent of the disease in the following manner. In some embodiments, the bolus is delivered in one or more of the following locations: stomach; duodenum; proximal jejunum; ileum; cecum; ascending colon; transverse colon; descending colon. In some embodiments, the JAK inhibitor is ustekinumab. In some embodiments, the JAK inhibitor is briakinumab. In some embodiments, the JAK inhibitor is guselkumab. In some embodiments, the JAK inhibitor is tildrakizumab. In some embodiments, the JAK inhibitor is brazikumab. In some embodiments, the JAK inhibitor is ustekinumab. In some embodiments the dispensable substance is a small molecule therapeutic that is released in the cecum and/or other parts of the large intestine. Small molecules that are administered by typical oral routes are primarily absorbed in the small intestine, with much lower absorption taking place in the large intestine (outside of the rectum). Accordingly, an ingestible device that is capable of releasing a small molecule selectively in the large intestine (e.g., the cecum) with resulting low systemic levels (even when high doses are used) is attractive for subjects with inflammatory bowel disease in the large intestine. In some embodiments, the storage reservoir may include multiple chambers, and each chamber stores a different dispensable substance. For example, the different dispensable substances can be released at the same time via the dispensing outlet1607. Alternatively, the multiple chambers may take a form of different layers within the storage reservoir such that the different dispensable substance from each chamber is delivered sequentially in an order. In one example, each of the multiple chambers is controlled by a separate traveling plunger, which may be propelled by gas generation. The electronic component may control the gas generating cell1603to generate gas to propel a specific traveling plunger, e.g., via a separate gas generation chamber, etc., to delivery the respective substance. In some embodiments, the content of the multiple chambers may be mixed or combined prior to release, for example, to activate the drug. The ingestible device1600may include a dispensing outlet1607at one end1602aof the housing1601to direct the dispensable substance105out of the housing. The dispensing outlet1607may include an exit valve, a slit or a hole, a jet injection nozzle with a syringe, and/or the like. When the traveling plunger1604moves towards the end1602aof the housing1601, an internal pressure within the storage reservoir may increase and push the dispensing outlet to be open to let the dispensable substance1605be released out of the housing1601. In an embodiment, a pressure relief device1606may be placed within the housing1601, e.g., at the end1602aof the housing1601. In some embodiments, the housing1601may include small holes (e.g., with a diameter smaller than 2 mm), e.g., on the side of the housing1601, or at the end1602ato facilitate loading the dispensable substance into the storage reservoir. In some embodiments, a feedback control circuit (e.g., a feedback resistor, etc.) may be added to send feedback from the gas generating cell1603to the electronic component such that when the internal pressure reaches a threshold level, the electronic component may control the gas generating cell1603to turn off gas generation, or to activate other safety mechanism (e.g., feedback-controlled release valve, etc.). For example, an internal pressure sensor may be used to measure the internal pressure within the ingestible device and generate feedback to the feedback control circuit. FIG.17provides an example diagram illustrating aspects of a mechanism for a gas generating cell1603configured to generate a gas to dispense a substance, according to some embodiments described herein. As shown inFIG.17, the gas generating cell1603generates a gas1611which can propel the dispensable substance1605out of the dispensing outlet1607. A variable resistor1608may be connected to a circuit with the gas generating cell1603such that the variable resistor1608may be used to control an intensity and/or an amount of gas1611(e.g., hydrogen) generated by the cell1603. Specifically, the gas generating cell1603may be a battery form factor cell that is capable of generating hydrogen when a resistor is applied. In this way, as the gas generating cell1603only needs the use of a resistor only without any active power requirements, the gas generating cell1603may be integrated into an ingestible device such as a capsule with limited energy/power available. For example, the gas generating cell1603may be compatible with a capsule at a size of 26 mm×13 mm or smaller. In some embodiments, based on the elution rate of gas from the cell, and an internal volume of the ingestible device, it may take time to generate sufficient gas1611to deliver the substance1605, and the time required may be 30 seconds or longer. For example, the time to generate a volume of hydrogen equivalent to5004of fluid would be approximately 5 minutes. A longer period of time may be needed based upon non-ideal conditions within the ingestible device, such as friction, etc. Thus, given that the production of gas (e.g., hydrogen) may take time, gas generation may need to start prior to the ingestible device arriving at the site of delivery to build pressure up within the device. The ingestible device may then need to know when it is approaching the site of delivery. For example, the device may start producing gas on an “entry transition,” which is determined by temperature, so as to produce enough gas to be close to the pressure high enough to deliver the dispensable substance. The ingestible device may then only start producing gas again when it arrives at the site of delivery, which will cause the internal pressure within the ingestible device to reach a level required by the dispensing outlet to release the dispensable substance. Also, for regio-specific delivery, the ingestible device may estimate the time it takes to build up enough pressure to deliver the dispensable substance before the ingestible device arrives at a specific location, to activate gas generation. For example, for systemic delivery, when an internal volume of the ingestible device is around 5004, a gas generation time of 2 hours, an initial pressure of approximately 300 pound per square inch absolute (psia) may be generated, with higher and lower pressures possible. The generated pressure may drop when air enters the storage reservoir which was previously occupied by the dispensable substance during the dispensing process. For systemic drug delivery, a force with a generated pressure of approximately 100 to 360 pound per square inch (psi) may be required for dermal penetration, e.g., to penetrate the mucosa or epithelial layer. The pressure may also vary depending on the nozzle design at the dispensing outlet, fluid viscosity, and surrounding tissue proximity and properties. The gas1611that may be generated for a continuous delivery of drug (e.g., 1 cc H2in 4 hours, 16 breaths per minute at 0.5 L tidal volume) may equate to 1 cc hydrogen in approximately 2000 L of exhaled air, or approximately 0.5 ppm H2, which is below physiologic values of exhaled hydrogen. Reducing this time to 10 minutes equates to approximately 13 ppm hydrogen. Thus, due to the length of intestine that may be covered during this time period, the ingestible device may possess a higher localized value than physiologic. FIGS.18and19, disclosed in U.S. Provisional Application No. 62/385,553, incorporated by reference herein in its entirety, illustrates an example of an ingestible device for localized delivery of pharmaceutical compositions disclosed herein, in accordance with particular implementations. The ingestible device1600includes a piston or drive element1634to push for drug delivery, in accordance with particular implementations described herein. The ingestible device1600may have one or more batteries1631placed at one end1602aof a housing1601to provide power for the ingestible device1600. A printed circuit board (PCB)1632may be placed adjacent to a battery or other power source1631, and a gas generating cell1603may be mounted on or above the PCB1632. The gas generating cell1603may be sealed from the bottom chamber (e.g., space including1631and1632) of the ingestible device1600. A movable piston1634may be placed adjacent to the gas generating cell1603. In this way, gas generation from the gas generating cell1603may propel a piston1634to move towards another end1602bof the housing1601such that the dispensable substance in a reservoir compartment1635can be pushed out of the housing through a dispensing outlet1607, e.g., the movement is shown at1636, with the piston1634at a position after dispensing the substance. The dispensing outlet1607may comprise a plug. The reservoir compartment1635can store the dispensable substance (e.g., drug substance), or alternatively the reservoir compartment can house a storage reservoir1661which comprises the dispensable substance. The reservoir compartment1635or storage reservoir1661may have a volume of approximately 600 μL or even more dispensable substance, which may be dispensed in a single bolus, or gradually over a period of time. The battery cells1631may have a height of 1.65 mm each, and one to three batteries may be used. The height of the piston may be reduced with custom molded part for around 1.5 mm to save space. If the gas generating cell1603is integrated with the piston1634, the overall height of the PCB, batteries and gas generating cell in total can be reduced to around 5 mm, thus providing more space for drug storage. For example, for an ingestible device of 7.8 mm in length (e.g., from end1602ato the other end1602b), a reservoir compartment1635or a storage reservoir1661of approximately 600 μL may be used for drug delivery. For another example, for an ingestible device of 17.5 mm in length, a reservoir compartment1635or a storage reservoir1661of approximately 1300 μL may be used for drug release. In some implementations, at the reservoir1635or1661for storing a therapeutically effective amount of the JAK inhibitor forms at least a portion of the device housing1601. The therapeutically effective amount of the JAK inhibitor can be stored in the reservoir1635or1661at a particular pressure, for example, determined to be higher than a pressure inside the GI tract so that once the reservoir1635or1661is in fluid communication with the GI tract, the JAK inhibitor is automatically released. In certain implementations, the reservoir compartment1635includes a plurality of chambers, and each of the plurality of the chambers stores a different dispensable substance or a different storage reservoir1661. In certain embodiments, the storage reservoir1661is a compressible component or has compressible side walls. In particular embodiments, the compressible component can be composed, at least in part, or coated (e.g., internally) with polyvinyl chloride (PVC), silicone, DEHP (di-2-ethylhexyl phthalate), Tyvek, polyester film, polyolefin, polyethylene, polyurethane, or other materials that inhibit the JAK inhibitor from sticking to the reservoir and provide a sterile reservoir environment for the JAK inhibitor. The storage reservoir1661can be hermetically sealed. The reservoir compartment1635or storage reservoir1661can be configured to store JAK inhibitor in quantities in the range of 0.01 mL-2 mL, such as 0.05 mL-2 mL, such as 0.05 mL-2 mL, such as 0.6 mL-2 mL. In some embodiments, the storage reservoir1661is attachable to the device housing1601, for example, in the reservoir compartment. Accordingly, the storage reservoir1635can be loaded with the JAK inhibitor prior to being positioned in and/or coupled to the ingestible device housing1601. The ingestible device housing1601includes one or more openings configured as a loading port to load the dispensable substance into the reservoir compartment. In another embodiment, the ingestible device housing1601includes one or more openings configured as a vent. As noted above, in some embodiments, a storage reservoir (optionally, containing a JAK inhibitor, such as a therapeutically effective amount of JAK inhibitor) is attachable to an ingestible device. In general, in such embodiments the storage reservoir and ingestible device can be designed in any appropriate fashion so that the storage reservoir can attach to the ingestible device when desired. Examples of designs include a storage reservoir that fits entirely within the ingestible device (e.g., in the ingestible device so that the storage reservoir is sealed within the device at the time the device is ingested by a subject), a storage reservoir that fits partially within the ingestible device, and a storage reservoir that is carried by the housing of the device. In some embodiments, the storage reservoir snap fits with the ingestible device. In certain embodiments, the storage reservoir is friction fit with the ingestible device. In some embodiments, the storage reservoir is held together with the ingestible device via a biasing mechanism, such as one or more springs, one or more latches, one or more hooks, one or more magnets, and/or electromagnetic radiation. In certain embodiments, the storage reservoir can be a piercable member. In some embodiments, the ingestible device has a sleeve into which the storage reservoir securely fits. In some embodiments, the storage reservoir is disposed in/on a slidable track/groove so that it can move onto a piercing needle when delivery of the therapeutic agent is desired. In certain embodiments, the storage reservoir is made of a soft plastic coating, which is contacted with a needle at any orientation to deliver the therapeutic agent when desired. Generally, the storage reservoir can be made of one or more appropriate materials, such as, for example, one or more plastics and/or one or more metals or alloys. Exemplary materials include silicone, polyvinyl chloride, polycarbonate and stainless steel. Optionally, the design may be such that the storage reservoir carries some or all of the electrical componentry to be used by the ingestible device. Although the foregoing discussion relates to one storage reservoir, it is to be understood that an ingestible device can be designed to carry any desired number (e.g., two, three, four, five) storage reservoirs. Different storage reservoirs can have the same or different designs. In some embodiments, the ingestible device (when fully assembled and packaged) satisfies the regulatory requirements for marketing a medical device in one or more jurisdictions selected from the United States of America, the European Union or any member state thereof, Japan, China, Brazil, Canada, Mexico, Colombia, Argentina, Chile, Peru, Russia, the UK, Switzerland, Norway, Turkey, Israel, any member state of the Gulf Cooperative Council, South Africa, India, Australia, New Zealand, South Korea, Singapore, Thailand, the Philippines, Malaysia, Viet Nam, Indonesia, Taiwan and Hong Kong. In certain embodiments, the ingestible device housing1601includes one or more actuation systems (e.g., gas generating cell1603) for pumping the JAK inhibitor from the reservoir1635. In some embodiments, the actuation system can include a mechanical, electrical, electromechanical, hydraulic, and/or fluid actuation system. For example, a chemical actuation means may use chemical reaction of mixing one or more reagents to generate a sufficient volume of gas to propel the piston or drive element1634for drug release. The actuation system can be integrated into the reservoir compartment1635or can be an auxiliary system acting on or outside of the reservoir compartment1635. For example, the actuation system can include pumping system for pushing/pulling the JAK inhibitor out of the reservoir compartment1635or the actuation system can be configured to cause the reservoir compartment1635to change structurally so that the volume inside of the reservoir compartment1635changes, thereby dispensing the JAK inhibitor from the reservoir compartment1635. The actuation system can include an energy storage component such as a battery or a capacitor for powering the actuation system. The actuation system can be actuated via gas pressure or a system storing potential energy, such as energy from an elastic reservoir component being expanded during loading of the reservoir and after being positioned in the ingestible device housing1601being subsequently released from the expanded state when the ingestible device housing is at the location for release within the GI tract. In certain embodiments, the reservoir compartment1635can include a membrane portion, whereby the JAK inhibitor is dispensed from the reservoir compartment1635or storage reservoir1661via osmotic pressure. In particular embodiments the storage reservoir1661is in a form of a bellow that is configured to be compressed via a pressure from the gas generating cell. The JAK inhibitor may be loaded into the bellow, which may be compressed by gas generation from the gas generating cell or other actuation means to dispense the dispensable substance through the dispensing outlet1607and out of the housing1601. In some embodiments, the ingestible device includes a capillary plate placed between the gas generating cell and the first end of the housing, and a wax seal between the gas generating cell and the reservoir, wherein the wax seal is configured to melt and the dispensable substance is pushed through the capillary plate by a pressure from the gas generating cell. The shape of the bellow may aid in controlled delivery. The reservoir compartment1635includes a dispensing outlet, such as a valve or dome slit1662extending out of an end of the housing1601, in accordance with particular implementations. Thus when the bellow is being compressed, the dispensable substance may be propelled out of the bellow through the valve or the dome slit. In certain embodiments, the reservoir compartment1635includes one or more valves (e.g. a valve in the dispensing outlet1607) that are configured to move or open to fluidly couple the reservoir compartment1635to the GI tract. In certain embodiments, a housing wall of the housing1601can form a portion of the reservoir compartment1635. In certain embodiments, the housing walls of the reservoir serve as a gasket. One or more of the one or more valves are positioned in the housing wall of the device housing1601, in accordance with particular implementations. One or more conduits may extend from the reservoir1635to the one or more valves, in certain implementations. In certain embodiments, a housing wall of the housing1601can be formed of a material that is configured to dissolve, for example, in response to contact at the disease site. In certain embodiments, a housing wall of the housing1601can be configured to dissolve in response to a chemical reaction or an electrical signal. The one or more valves and/or the signals for causing the housing wall of the housing1601to dissolve or dissipate can be controlled by one or more processors or controllers positioned on PCB1632in the device housing1601. The controller is communicably coupled to one or more sensors or detectors configured to determine when the device housing1601is proximate to a disease site. The sensors or detectors comprise a plurality of electrodes comprising a coating, in certain implementations. Releasing of the JAK inhibitor from the reservoir compartment1635is triggered by an electric signal from the electrodes resulting from the interaction of the coating with the one or more sites of disease site. The one or more sensors can include a chemical sensor, an electrical sensor, an optical sensor, an electromagnetic sensor, a light sensor, and/or a radiofrequency sensor. In particular embodiments, the device housing1601can include one or more pumps configured to pump the therapeutically effective amount of the JAK inhibitor from the reservoir compartment1635. The pump is communicably coupled to the one or more controllers. The controller is configured to activate the pump in response to detection by the one or more detectors of the disease site and activation of the valves to allow the reservoir1635to be in fluid communication with the GI tract. The pump can include a fluid actuated pump, an electrical pump, or a mechanical pump. In certain embodiments, the device housing1601comprises one or more anchor systems for anchoring the device housing1601or a portion thereof at a particular location in the GI tract adjacent the disease site. In some embodiments, a storage reservoir comprises an anchor system, and the storage reservoir comprising a releasable substance is anchored to the GI tract. The anchor system can be activated by the controller in response to detection by the one or more detectors of the disease site. In certain implementations, the anchor system includes legs or spikes configured to extend from the housing wall(s) of the device housing1601. The spikes can be configured to retract and/or can be configured to dissolve over time. An example of an attachable device that becomes fixed to the interior surface of the GI tract is described in PCT Patent Application PCT/US2015/012209, “Gastrointestinal Sensor Implantation System”, filed Jan. 21, 2015, which is hereby incorporated by reference herein in its entirety. FIG.20provides an example structural diagram having a flexible diaphragm1665that may deform towards the dispensing outlet1607when the gas generating cell1603generates gas. The dispensable substance may then be propelled by the deformed diaphragm out of the housing through the dispensing outlet1607. The dispensing outlet1607shown atFIG.20is in the form of a ring valve, however, any outlet design can be applied. In some embodiments, an ingestible device can have an umbrella-shaped exit valve structure as a dispensing outlet of the ingestible device. Optionally, an ingestible device can have a flexible diaphragm to deform for drug delivery, and/or an integrated piston and gas generating cell such that the gas generating cell is movable with the piston to push for drug delivery. In certain embodiments, an ingestible device can be anchored within the intestine by extending hooks from the ingestible device after it has entered the region of interest. For example, when the ingestible device determines it has arrived at a location within the GI tract, the hooks can be actuated to extend outside of the ingestible device to catch in the intestinal wall and hold the ingestible device in the respective location. In some embodiments, the hook can pierce into the intestinal wall to hold the ingestible device100in place. The hooks can be hollow. A hollow hook can be used to anchor the ingestible device and/or to dispense a substance from the dispensable substance, e.g., into the intestinal wall. In some embodiments an ingestible device includes an intestinal gripper to grip a portion of the intestinal wall for delivering the dispensable substance. Such a gripper can include two or more arms configured to out of the device and close to grip a portion of the intestinal wall. An injecting needle can be used with the anchoring arms to inject dispensable substance into the intestinal wall after a portion of the intestinal wall is gripped. In some embodiments, when the gas generating cell generates gas to propel the piston to move towards the nozzle such that the dispensable substance can be pushed under the pressure to break a burst disc to be injected via the nozzle. In some embodiments, an ingestible device has a jet delivery mechanism with enhanced usable volume of dispensable substance. For example, the nozzle may be placed at the center of the ingestible device, and gas channels may be placed longitudinally along the wall of the ingestible device to transport gas from the gas generating cell to propel the piston, which is placed at an end of the ingestible device. In some embodiments, the ingestible device can use osmotic pressure to adhere a suction device of the ingestible device to the intestinal wall. For example, the ingestible device may have an osmotic mechanism that has a chamber storing salt crystals. The chamber can include a mesh placed in proximate to a burst valve at one end of the chamber, and a reverse osmosis (RO) membrane placed in proximate to a valve on the other end of the chamber. A suction device, e.g., two or more suction fingers, is placed outside of the chamber with an open outlet exposed to luminal fluid in the GI tract. When the osmotic mechanism is inactivated, e.g., the valve is closed so that no luminal fluid is drawn into the osmotic chamber. When the osmotic mechanism is activated by opening the valve, luminal fluid enters the ingestible device through an outlet of the suction device and enters the osmotic chamber through the valve. The salt in the chamber is then dissolved into the fluid. The RO membrane prevents any fluid to flow in the reverse direction, e.g., from inside the chamber to the valve. The fluid continues to flow until all the salt contained in the chamber is dissolved or until intestinal tissue is drawn into the suction device. As luminal fluid keeps flowing into the chamber, the solution of the luminal fluid with dissolved salt in the chamber may reduce osmotic pressure such that the suction force at may also be reduced. In this way, suction of the intestinal tissue may stall before the tissue is in contact with the valve to avoid damage to the intestinal tissue. An ingestible device employing an osmotic mechanism can also include a suction device as illustrated. The suction device can be two or more suction fingers347a-bdisposed proximate to the outlet. The outlet can be connected to a storage reservoir storing the dispensable substance (e.g., therapeutic agent). The storage reservoir can contact a piston (similar to104inFIG.16), which can be propelled by pressure generated from the osmotic pump to move towards the outlet. The osmotic pump can be similar to the osmotic mechanism described in the preceding paragraph. A breakaway section can be placed in proximate to the other end (opposite to the end where the outlet107is disposed) of the ingestible device. In some embodiments, tumbling suction by an ingestible device is used. Such an ingestible device does not require any electronics or other actuation elements. Such an ingestible device may constantly, intermittently, or periodically tumble when travelling through the intestine. When the ingestible device tumbles to a position that the outlet is in direct contact with the intestinal wall, a suction process similar to that described in the preceding paragraph may occur. Additional structural elements such as fins, flutes or the like may be added to the outer wall of the ingestible device100to promote the tumbling motion. In certain embodiments, the reservoir is an anchorable reservoir, which is a reservoir comprising one or more anchor systems for anchoring the reservoir at a particular location in the GI tract adjacent the disease site. In certain embodiments, the anchor system includes legs or spikes or other securing means such as a piercing element, a gripping element, a magnetic-flux-guiding element, or an adhesive material, configured to extend from the anchorable reservoir of the device housing. The spikes can be configured to retract and/or can be configured to dissolve over time. In some embodiments, the anchorable reservoir is suitable for localizing, positioning and/or anchoring. In some embodiments, the anchorable reservoir is suitable for localizing, and positioning and/or anchoring by an endoscope. In some embodiments, the anchorable reservoir is connected to the endoscope. In some embodiments, the anchorable reservoir is connected to the endoscope in a manner suitable for oral administration. In some embodiments, the anchorable reservoir is connected to the endoscope in a manner suitable for rectal administration. Accordingly, provided herein in some embodiments is an anchorable reservoir is connected to an endoscope wherein the anchorable reservoir comprises a therapeutically effective amount of the JAK inhibitor. In some embodiments the endoscope is fitted with a spray catheter. Exemplary embodiments of anchorable reservoirs are as follows. In more particular examples of the following exemplary embodiments the reservoir is connected to an endoscope. In one embodiment, the anchorable reservoir comprises an implant capsule for insertion into a body canal to apply radiation treatment to a selected portion of the body canal. The reservoir includes a body member defining at least one therapeutic treatment material receiving chamber and at least one resilient arm member associated with the body member for removably engaging the body canal when the device is positioned therein. In one embodiment the anchorable reservoir has multiple suction ports and permits multiple folds of tissue to be captured in the suction ports with a single positioning of the device and attached together by a tissue securement mechanism such as a suture, staple or other form of tissue bonding. The suction ports may be arranged in a variety of configurations on the reservoir to best suit the desired resulting tissue orientation. In some embodiments an anchorable reservoir comprises a tract stimulator and/or monitor IMD comprising a housing enclosing electrical stimulation and/or monitoring circuitry and a power source and an elongated flexible member extending from the housing to an active fixation mechanism adapted to be fixed into the GI tract wall is disclosed. After fixation is effected, the elongated flexible member bends into a preformed shape that presses the housing against the mucosa so that forces that would tend to dislodge the fixation mechanism are minimized. The IMD is fitted into an esophageal catheter lumen with the fixation mechanism aimed toward the catheter distal end opening whereby the bend in the flexible member is straightened. The catheter body is inserted through the esophagus into the GI tract cavity to direct the catheter distal end to the site of implantation and fix the fixation mechanism to the GI tract wall. The IMD is ejected from the lumen, and the flexible member assumes its bent configuration and lodges the hermetically sealed housing against the mucosa. A first stimulation/sense electrode is preferably an exposed conductive portion of the housing that is aligned with the bend of the flexible member so that it is pressed against the mucosa. A second stimulation/sense electrode is located at the fixation site. In some embodiments a reservoir for sensing one or more parameters of a patient is anchored to a tissue at a specific site and is released from a device, using a single actuator operated during a single motion. As an example, a delivery device may anchor the capsule to the tissue site and release the reservoir from the delivery device during a single motion of the actuator. In some embodiments a device is provided comprising: a reservoir configured to contain a fluid, the reservoir having at least one outlet through which the fluid may exit the reservoir; a fluid contained within the reservoir; a primary material contained within the reservoir and having a controllable effective concentration in the fluid; and at least one electromagnetically responsive control element located in the reservoir or in a wall of the reservoir and adapted for modifying the distribution of the primary material between a first active form carried in the fluid and a second form within the reservoir in response to an incident electromagnetic control signal, the effective concentration being the concentration of the first active form in the fluid, whereby fluid exiting the reservoir carries the primary material in the first active form at the effective concentration. In some embodiments systems and methods are provided for implementing or deploying medical or veterinary devices or reservoirs (a) operable for anchoring at least partly within a digestive tract, (b) small enough to pass through the tract per vias naturales and including a wireless-control component, (c) having one or more protrusions positionable adjacent to a mucous membrane, (d) configured to facilitate redundant modes of anchoring, (e) facilitating a “primary” material supply deployable within a stomach for an extended and/or controllable period, (f) anchored by one or more adaptable extender modules supported by a subject's head or neck, and/or (g) configured to facilitate supporting at least a sensor within a subject's body lumen for up to a day or more. In certain embodiments, the reservoir is attachable to an ingestible device. In certain embodiments, the ingestible device comprises a housing and the reservoir is attachable to the housing. In certain embodiments, the attachable reservoir is also an anchorable reservoir, such as an anchorable reservoir comprising one or more anchor systems for anchoring the reservoir at a particular location in the GI tract as disclosed hereinabove. Accordingly, in certain embodiments, provided herein is a JAK inhibitor for use in a method of treating a disease of the gastrointestinal tract as disclosed herein, wherein the JAK inhibitor is contained in a reservoir suitable for attachment to a device housing, and wherein the method comprises attaching the reservoir to the device housing to form the ingestible device, prior to orally administering the ingestible device to the subject. In certain embodiments, provided herein is an attachable reservoir containing a JAK inhibitor for use in a method of treating a disease of the gastrointestinal tract, wherein the method comprises attaching the reservoir to a device housing to form an ingestible device and orally administering the ingestible device to a subject, wherein the JAK inhibitor is released by device at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease. In certain embodiments, provided herein is an attachable reservoir containing a JAK inhibitor, wherein the reservoir is attachable to a device housing to form an ingestible device that is suitable for oral administration to a subject and that is capable of releasing the JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease. In particular implementation the ingestible device includes cameras (e.g., video cameras) that affords inspection of the entire GI tract without discomfort or the need for sedation, thus avoiding many of the potential risks of conventional endoscopy. Video imaging can be used to help determine one or more characteristics of the GI tract, including the location of disease (e.g., presence or location of inflamed tissue and/or lesions associated with inflammatory bowel disease). In some embodiments, the ingestible device101may comprise a camera for generating video imaging data of the GI tract which can be used to determine, among other things, the location of the device. Examples of video imaging capsules include Medtronic's PillCam™, Olympus' Endocapsule®, and IntroMedic's MicroCam™. For a review of imaging capsules, see Basar et al. “Ingestible Wireless Capsule Technology: A Review of Development and Future Indication” International Journal of Antennas and Propagation (2012); 1-14). Other imaging technologies implemented with the device101can include thermal imaging cameras, and those that employ ultrasound or Doppler principles to generate different images (see Chinese patent application CN104473611: “Capsule endoscope system having ultrasonic positioning function”. Ingestible devices can be equipped with sources for generating reflected light, including light in the Ultraviolet, Visible, Near-infrared and/or Mid-infrared spectrum, and the corresponding detectors for spectroscopy and hyperspectral imaging. Likewise, autofluorescense may be used to characterize GI tissue (e.g., subsurface vessel information), or low-dose radiation (see Check-Cap™) can be used to obtain 3D reconstructed images. Device Components An ingestible device in accordance with particular embodiments of the present invention may comprise a component made of a non-digestible material and contain the JAK inhibitor. In some embodiments, the material is plastic. It is envisaged that the device is single-use. The device is loaded with a drug prior to the time of administration. In some embodiments, it may be preferred that there is provided a medicinal product comprising the device pre-filled with the drug. Anchoring Components Several systems may actively actuate and control the capsule position and orientation in different sections of the GI tract. Examples include leg-like or anchor-like mechanisms that can be deployed by an ingestible device to resist peristaltic forces in narrowed sections of the GI tract, such as the intestine, and anchor the device to a location. Other systems employ magnetic shields of different shapes that can interact with external magnetic fields to move the device. These mechanisms may be particularly useful in areas outside of the small intestine, like the cecum and large intestine. An anchoring mechanism may be a mechanical mechanism. For example, a device may be a capsule comprising a plurality of legs configured to steer the capsule. The number of legs in the capsule may be, for example, two, four, six, eight, ten or twelve. The aperture between the legs of the device may be up to about 35 mm; about 30 to about 35 mm; about 35 to about 75 mm; or about 70 to about 75 mm. The contact area of each leg may be varied to reduce impact on the tissue. One or more motors in the capsule may each actuate a set of legs independently from the other. The motors may be battery-powered motors. An anchoring mechanism may be a non-mechanical mechanism. For example, a device may be a capsule comprising a permanent magnet located inside the capsule. The capsule may be anchored at the desired location of the GI tract by an external magnetic field. An anchoring mechanism may comprise a non-mechanical mechanism and a mechanical mechanism. For example, a device may be a capsule comprising one or more legs, one or more of which are coated with an adhesive material. Locomotion Components Ingestible devices can be active or passive, depending on whether they have controlled or non-controlled locomotion. Passive (non-controlled) locomotion is more commonly used among ingestible devices given the challenges of implementing a locomotion module. Active (controlled) locomotion is more common in endoscopic ingestible capsules. For example, a capsule may comprise a miniaturized locomotion system (internal locomotion). Internal locomotion mechanisms may employ independent miniaturized propellers actuated by DC brushed motors, or the use of water jets. As an example, a mechanism may comprise flagellar or flap-based swimming mechanisms. As an example, a mechanism may comprise cyclic compression/extension shape-memory alloy (SMA) spring actuators and anchoring systems based on directional micro-needles. As an example, a mechanism may comprise six SMA actuated units, each provided with two SMA actuators for enabling bidirectional motion. As an example, a mechanism may comprise a motor adapted to electrically stimulating the GI muscles to generate a temporary restriction in the bowel. As an example, a capsule may comprise a magnet and motion of the capsule is caused by an external magnetic field. For example, a locomotion system may comprise an ingestible capsule and an external magnetic field source. For example, the system may comprise an ingestible capsule and magnetic guidance equipment such as, for example, magnetic resonance imaging and computer tomography, coupled to a dedicated control interface. In some embodiments drug release mechanisms may also be triggered by an external condition, such as temperature, pH, movement, acoustics, or combinations thereof. Use of an Endoscope or an Ingestible Device in Biopsy and Surgery Sampling Ingestible devices may comprise a mechanism adapted to permit the collection of tissue samples. In some examples, this is achieved using electro-mechanical solutions to collect and store the sample inside an ingestible device. As an example, a biopsy mechanism may include a rotational tissue cutting razor fixed to a torsional spring or the use of microgrippers to fold and collect small biopsies. As an example, Over-the-scope clips (OTSC®) may be used to perform endoscopic surgery and/or biopsy. As an example of the methods disclosed herein, the method may comprise releasing a JAK inhibitor and collecting a sample inside the device. As an example, the method may comprise releasing a JAK inhibitor and collecting a sample inside the device in a single procedure. FIG.21illustrates an example ingestible device2100with multiple openings in the housing. The ingestible device2100has an outer housing with a first end2102A, a second end2102B, and a wall2104extending longitudinally from the first end2102A to the second end2102B. Ingestible device2100has a first opening2106in the housing, which is connected to a second opening2108in the housing. The first opening2106of the ingestible device2100is oriented substantially perpendicular to the second opening2108, and the connection between the first opening2106and the second opening2108forms a curved chamber2110within the ingestible device2100. The overall shape of the ingestible device2100, or any of the other ingestible devices discussed in this disclosure, may be similar to an elongated pill or capsule. In some embodiments, a portion of the curved chamber2110may be used as a sampling chamber, which may hold samples obtained from the GI tract. In some embodiments the curved chamber2110is subdivided into sub-chambers, each of which may be separated by a series of one or more valves or interlocks. In some embodiments, the first opening2106, the second opening2108, or the curved chamber2110include one or more of a hydrophilic or hydrophobic material, a sponge, a valve, or an air permeable membrane. The use of a hydrophilic material or sponge may allow samples to be retained within the curved chamber2110, and may reduce the amount of pressure needed for fluid to enter through the first opening2106and dislodge air or gas in the curved chamber2110. Examples of hydrophilic materials that may be incorporated into the ingestible device2100include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, and the like. Similarly, materials that have undergone various types of treatments, such as plasma treatments, may have suitable hydrophilic properties, and may be incorporated into the investible device2100. Sponges may be made of any suitable material or combination of materials, such as fibers of cotton, rayon, glass, polyester, polyethylene, polyurethane, and the like. Sponges generally may be made from commercially available materials, such as those produced by Porex®. As discussed in more detail below, in some embodiments, the sponges may be treated in order to change their absorbency or to help preserve samples. In some embodiments, the sponges may be cut or abraded to change their absorbency or other physical properties. Hydrophobic materials located near the second opening2108may repel liquids, discouraging liquid samples from entering or exiting the curved chamber2110through the second opening2108. This may serve a similar function as an air permeable membrane. Examples of hydrophobic materials which may be incorporated into the ingestible device2100include polycarbonate, acrylics, fluorocarbons, styrenes, certain forms of vinyl, stainless steel, silicone, and the like. The various materials listed above are provided as examples, and are not limiting. In practice, any type of suitable hydrophilic, hydrophobic, or sample preserving material may be used in the ingestible device2100. In some embodiments, an ingestible device includes a moveable valve as a diaphragm valve, which uses a mechanical actuator to move a flexible diaphragm in order to seal or unseal an aperture in a second portion of an inlet region, which may effectively block or unblock the inlet region. However, it will be understood that, in some embodiments, the moveable valve may be a different type of valve. For example, in some embodiments the moveable valve may be replaced by a pumping mechanism. As another example, in some embodiments the moveable valve is replaced with an osmotic valve A sampling chamber of an ingestible device can have an exit port to allow air or gas to exit the sampling chamber, while preventing at least a portion of the sample obtained by the ingestible device from exiting the sampling chamber. For example, the exit port may include a gas-permeable membrane. An ingestible device can include one-way valve as part of its exit port. An ingestible device can include an outlet port connected to the volume within housing of the ingestible device. The outlet port may provide a path for the gas to exit the ingestible device and be released into the environment surrounding the ingestible device. This may prevent pressure from building up within the housing of the ingestible device. In some embodiments, an ingestible device does not include an outlet port, and the gas stays inside the volume of the ingestible device. In some embodiments, the outlet port may contain a gas permeable membrane, a one-way valve, a hydrophobic channel, or some other mechanism to avoid unwanted material, (e.g., fluids and solid particulates from within the GI tract), from entering the ingestible device through the outlet port. In some embodiments, the ingestible device may include a sensor within or proximate to the sampling chamber. For example, this sensor may be used to detect various properties of a sample contained within the sampling chamber, or this sensor may be used to detect the results of an assay technique applied to the sample contained within the sampling chamber. In some embodiments, a hydrophilic sponge is located within the sampling chamber, and the hydrophilic sponge may be configured to absorb the sample as the sample enters the sampling chamber. In some embodiments, the hydrophilic sponge fills a substantial portion of the sampling chamber, and holds the sample for an extended period of time. This may be particularly advantageous if the sample is collected from the ingestible device after the ingestible device exits the body. In some embodiments, the hydrophilic sponge is placed on only certain surfaces or fills only certain portions of the sampling chamber. For example, it may be possible to line certain walls (or all walls) of the sampling chamber with a hydrophilic sponge to assist in drawing in the sample, while leaving some (or none) of the walls of the sampling chamber uncovered. Leaving walls uncovered may allow the use of diagnostics or assay techniques that require a relatively un-obscured optical path. In some embodiments, the ingestible device may include a sealed vacuum chamber connected to the exit port, or connected directly or indirectly to the sampling chamber. In some embodiments a pin valve may be used as a moveable valve (e.g., as moveable valve of ingestible device). In certain embodiments, a rotary valve may be used as a moveable valve (e.g., as moveable valve of ingestible device). In some embodiments, a flexible diaphragm, or diaphragm valve, may be used as a moveable valve (e.g., as moveable valve of ingestible device). In certain embodiments, a mechanism is near the diaphragm or in direct contact with the diaphragm. The spring mechanism may apply pressure to the diaphragm to oppose the pressure applied by the mechanical actuator, which may cause the flexible diaphragm to be moved into an open position when the mechanical actuator is not applying pressure to the flexible diaphragm. Additionally, this may ensure that the diaphragm valve remains open when the mechanical actuator is not applying pressure across the flexible diaphragm. In some embodiments, moving the mechanical actuator from a closed position to an open position causes a volume of the inlet region within the ingestible device to increase. This may cause the pressure within the inlet region to be reduced, generating suction to draw a sample into the inlet region. Similarly, moving the mechanical actuator from an open position to a closed position may cause the volume of the inlet region to be reduced. This may cause the pressure within the inlet region to be increased, pushing the sample out of the inlet region. Depending on the design of the inlet region, the mechanical actuator, and the moveable valve, this may push the sample into the sampling chamber rather than pushing the sample back through the opening in the ingestible device. FIG.22depicts a cross-sectional view of a portion of the interior of ingestible device3000. As shown inFIG.22, the interior of ingestible device3000includes a valve system3100and a sampling system3200. Valve system3100is depicted as having a portion that is flush with the opening3018so that valve system3100prevents fluid exterior to ingestible device2000from entering sampling system3200. However, as described in more detail below with reference toFIGS.22-27, valve system3100can change position so that valve system3100allows fluid exterior to ingestible device3000to enter sampling system3200. FIGS.23and27illustrate valve system3100in more detail. As shown inFIG.23, valve system3100includes an actuation mechanism3110, a trigger3120, and a gate3130. InFIGS.23and7, a leg3132of gate3130is flush against, and parallel with, housing wall3016so that gate leg3132covers opening3018to prevent fluid exterior to ingestible device3000(e.g., fluid in the GI tract) from entering the interior of ingestible device3000. A protrusion3134of gate3130engages a lip3122of trigger3120. A peg3124of trigger3120engages a wax pot3112of actuation mechanism3110. Referring toFIG.27, a biasing mechanism3140includes a compression spring3142that applies an upward force on gate3130. Biasing mechanism3140also includes a torsion spring3144that applies a force on trigger3120in the counter-clockwise direction. InFIGS.23and27, the force applied by torsion spring3144is counter-acted by the solid wax in pot3112, and the force applied by compression spring3142is counter-acted by lip3122. FIG.24AandFIG.24Bshow an embodiment of the manner in which actuation mechanism3110actuates movement of trigger3120. Similar toFIGS.23and27,FIG.24Ashows a configuration in which peg3124applies a force against solid wax pot3112due to torsion spring3144, and in which the solid nature of wax pot3112resists the force applied by peg3124. A control unit3150is in signal communication with valve system3100. During use of ingestible device3000, a control unit3150receives a signal, indicating that the position of valve system3100should change, e.g., so that ingestible device3000can take a sample of a fluid in the GI tract. Control unit3150sends a signal that causes a heating system3114of actuation system3100to heat the wax in pot3112so that the wax melts. As shown inFIG.24B, the melted wax is not able to resist the force applied by peg3124so that, under the force of torsion spring3144, trigger3120moves in a counter-clockwise fashion. FIGS.25A and25Billustrate the interaction of trigger3120and gate3130before and after actuation. As shown inFIG.25A, when wax pot3112is solid (corresponding to the configuration shown inFIG.24A), protrusion3134engages lip3122, which prevents the force of compression spring3142from moving gate3130upward. As shown inFIG.25B, when the wax in pot3112melts (FIG.24B), trigger3120moves counter-clockwise, and lip3122disengages from protrusion3134. This allows the force of compression spring3142to move gate3130upward. As seen by comparingFIG.25AtoFIG.25B, the upward movement of gate3130results in an upward movement of an opening3136in gate leg3132. FIGS.26A and26Billustrate the impact of the upward movement of opening3136on the ability of ingestible device3000to obtain a sample. As shown inFIG.26A, when the wax in pot3112is solid (FIGS.24A and25A), opening3136in is not aligned with opening3018in wall3016of ingestible device3000. Instead, gate leg3132covers opening3018and blocks fluid from entering the interior of ingestible device3000. As shown inFIG.26B, when the wax in pot3112is melted and trigger3120and gate3130have moved (FIGS.24B and42B), opening3136in gate3130is aligned with opening3018in wall3016. In this configuration, fluid that is exterior to ingestible device3000(e.g., in the GI tract) can enter the interior of ingestible device3000via openings3018and3036. FIG.27illustrates a more detailed view of ingestible device3000including valve system3100and sampling system3200. While the foregoing description is made with regard to a valve system having one open position and one closed position (e.g., a two-stage valve system), the disclosure is not limited in this sense. Rather, the concepts described above with regard to a two stage valve system can be implemented with a valve system have more than two stages (e.g., three stages, four stages, five stages, etc.). As noted above in addition to a valve system, an ingestible device includes a sampling system.FIG.28illustrates a partial cross sectional view of ingestible device3000with sampling system3200and certain components of valve system3100. Sampling system3200includes a series of sponges configured to absorb fluid from an opening, move the fluid to a location within the housing, and prepare the fluid for testing. Preparation for testing may include filtering the fluid and combining the fluid with a chemical assay. The assay may be configured to dye cells in the filtered sample. The series of sponges includes a wicking sponge3210, a transfer sponge3220, a volume sponge3230, and an assay sponge3240. Sampling system3200also includes a membrane3270located between assay sponge3240and a vent3280for gases to leave sampling system3200. A cell filter3250is located between distal end3214of wicking sponge3210and a first end3222of transfer sponge3220. Membrane3270is configured to allow one or more gases to leave sampling system3200via an opening3280, while maintaining liquid in sampling system3200. FIG.29is a highly schematic illustration of an ingestible device4000that contains multiple different systems that cooperate for obtaining a sample and analyzing a sample, e.g., within the GI tract of a subject. Ingestible device4000includes a power system4100(e.g., one or more batteries), configured to power an electronics system4200(e.g., including a control system, optionally in signal communication with an external base station), a valve system4300, a sampling system4400, and an analytic system4500. Exemplary analytical systems include assay systems, such as, for example, optical systems containing one or more sources of radiation and/or one more detectors. Some or all of the sponges of the above-described sampling systems may contain one or more preservatives (see discussion above). Typically, the assay sponge and/or the volume sponge3230and/or the transfer sponge contain one or more preservatives. Typically, the preservative(s) are selected based on the analyte of interest, e.g., an analyte (such as a protein biomarker) for a GI disorder. Communication Systems An ingestible device may be equipped with a communication system adapted to transmit and/or receive data, including imaging and/or localization data. As an example, a communication system may employ radiofrequency transmission. Ingestible devices using radiofrequency communication are attractive because of their efficient transmission through the layers of the skin. This is especially true for low frequency transmission (UHF-433 ISM and lower, including the Medical Device Radio Communication Service band (MDRS) band 402-406 MHz). In another embodiment, acoustics are used for communications, including the transmission of data. For example, an ingestible capsule may be able to transmit information by applying one or more base voltages to an electromechanical transducer or piezoelectric (e.g., PZT, PVDF, etc.) device to cause the piezoelectric device to ring at particular frequencies, resulting in an acoustic transmission. A multi-sensor array for receiving the acoustic transmission may include a plurality of acoustic transducers that receive the acoustic transmission from a movable device such as an ingestible capsule as described in U.S. patent application Ser. No. 11/851,214 filed Sep. 6, 2007, incorporated by reference herein in its entirety. As an example, a communication system may employ human body communication technology. Human body communication technology uses the human body as a conductive medium, which generally requires a large number of sensor electrodes on the skin. As an example, a communication system may integrate a data storage system. Environmental Sensors In some embodiments the device may comprise environmental sensors to measure pH, temperature, transit times, or combinations thereof. Other examples of environmental sensors include, but are not limited to a capacitance sensor, an impedance sensor, a heart rate sensor, acoustic sensor such as a microphone or hydrophone, image sensor, and/or a movement sensor. In one embodiment, the ingestible device comprises a plurality of different environmental sensors for generating different kinds of environmental data. In order to avoid the problem of capsule retention, a thorough past medical and surgical history should be undertaken. In addition, several other steps have been proposed, including performing investigations such as barium follow-through. In cases where it is suspected that there is a high risk of retention, the patient is given a patency capsule a few days before swallowing an ingestible device. Any dissolvable non-endoscopic capsule may be used to determine the patency of the GI tract. The patency capsule is usually the same size as the ingestible device and can be made of cellophane. In some embodiments, the patency capsule contains a mixture of barium and lactose, which allows visualization by x-ray. The patency capsule may also include a radiotag or other label, which allows for it to be detected by radio-scanner externally. The patency capsule may comprise wax plugs, which allow for intestinal fluid to enter and dissolve the content, thereby dividing the capsule into small particles. Accordingly, in some embodiments, the methods herein comprise (a) identifying a subject having a disease of the gastrointestinal tract and (b) evaluating the subject for suitability to treatment. In some embodiments, the methods herein comprise evaluating for suitability to treatment a subject identified as having a disease of the gastrointestinal tract. In some embodiments, evaluating the subject for suitability to treatment comprises determining the patency of the subject's GI tract. In some embodiments, an ingestible device comprises a tissue anchoring mechanism for anchoring the ingestible device to a subject's tissue. For example, an ingestible device could be administered to a subject and once it reaches the desired location, the tissue attachment mechanism can be activated or deployed such that the ingestible device, or a portion thereof, is anchored to the desired location. In some embodiments, the tissue anchoring mechanism is reversible such that after initial anchoring, the tissue attachment device is retracted, dissolved, detached, inactivated or otherwise rendered incapable of anchoring the ingestible device to the subject's tissue. In some embodiments the attachment mechanism is placed endoscopically. In some embodiments, a tissue anchoring mechanism comprises an osmotically-driven sucker. In some embodiments, the osmotically-driven sucker comprises a first valve on the near side of the osmotically-driven sucker (e.g., near the subject's tissue) and a second one-way valve that is opened by osmotic pressure on the far side of the osmotically-driven sucker, and an internal osmotic pump system comprising salt crystals and semi-permeable membranes positioned between the two valves. In such embodiments, osmotic pressure is used to adhere the ingestible device to the subject's tissue without generating a vacuum within the ingestible capsule. After the osmotic system is activated by opening the first valve, fluid is drawn in through the sucker and expelled through the second burst valve. Fluid continues to flow until all the salt contained in the sucker is dissolved or until tissue is drawn into the sucker. As liminal fluid is drawn through the osmotic pump system, solutes build up between the tissue and the first valve, reducing osmotic pressure. In some embodiments, the solute buildup stalls the pump before the tissue contacts the valve, preventing tissue damage. In some embodiments, a burst valve is used on the far side of the osmotically-driven sucker rather than a one-way valve, such that luminal fluid eventually clears the saline chamber and the osmotic flow reverses, actively pushing the subject's tissue out of the sucker. In some embodiments, the ingestible device may be anchored to the interior surface of tissues forming the GI tract of a subject. In one embodiment, the ingestible device comprises a connector for anchoring the device to the interior surface of the GI tract. The connector may be operable to ingestible device to the interior surface of the GI tract using an adhesive, negative pressure and/or fastener. In some embodiments a device comprises a tract stimulator and/or monitor IMD comprising a housing enclosing electrical stimulation and/or monitoring circuitry and a power source and an elongated flexible member extending from the housing to an active fixation mechanism adapted to be fixed into the GI tract wall is disclosed. After fixation is effected, the elongated flexible member bends into a preformed shape that presses the housing against the mucosa so that forces that would tend to dislodge the fixation mechanism are minimized. The IMD is fitted into an esophageal catheter lumen with the fixation mechanism aimed toward the catheter distal end opening whereby the bend in the flexible member is straightened. The catheter body is inserted through the esophagus into the GI tract cavity to direct the catheter distal end to the site of implantation and fix the fixation mechanism to the GI tract wall. The IMD is ejected from the lumen, and the flexible member assumes its bent configuration and lodges the hermetically sealed housing against the mucosa. A first stimulation/sense electrode is preferably an exposed conductive portion of the housing that is aligned with the bend of the flexible member so that it is pressed against the mucosa. A second stimulation/sense electrode is located at the fixation site. In some embodiments a device includes a fixation mechanism to anchor the device to tissue within a body lumen, and a mechanism to permit selective de-anchoring of the device from the tissue anchoring site without the need for endoscopic or surgical intervention. An electromagnetic device may be provided to mechanically actuate the de-anchoring mechanism. Alternatively, a fuse link may be electrically blown to de-anchor the device. As a further alternative, a rapidly degradable bonding agent may be exposed to a degradation agent to de-anchor the device from a bonding surface within the body lumen. In some embodiments a device is as disclosed in patent publication WO2015112575A1, incorporated by reference herein in its entirety. The patent publication is directed to a gastrointestinal sensor implantation system. In some embodiments an orally-administrable capsule comprises a tissue capture device or reservoir removably coupled to the orally-administrable capsule, where the tissue capture device including a plurality of fasteners for anchoring the tissue capture device to gastrointestinal tissue within a body In some embodiments, the ingestible device contains an electric energy emitting means, a radio signal transmitting means, a medicament storage means and a remote actuatable medicament releasing means. The capsule signals a remote receiver as it progresses through the alimentary tract in a previously mapped route and upon reaching a specified site is remotely triggered to release a dosage of medicament. Accordingly, in some embodiments, releasing the JAK inhibitor is triggered by a remote electromagnetic signal. In some embodiments, the ingestible device includes a housing introducible into a body cavity and of a material insoluble in the body cavity fluids, but formed with an opening covered by a material which is soluble in body cavity fluids. A diaphragm divides the interior of the housing into a medication chamber including the opening, and a control chamber. An electrolytic cell in the control chamber generates a gas when electrical current is passed therethrough to deliver medication from the medication chamber through the opening into the body cavity at a rate controlled by the electrical current. Accordingly, in some embodiments, releasing the JAK inhibitor is triggered by generation in the composition of a gas in an amount sufficient to expel the JAK inhibitor. In some embodiments, the ingestible device includes an oral drug delivery device having a housing with walls of water permeable material and having at least two chambers separated by a displaceable membrane. The first chamber receives drug and has an orifice through which the drug is expelled under pressure. The second chamber contains at least one of two spaced apart electrodes forming part of an electric circuit which is closed by the ingress of an aqueous ionic solution into the second chamber. When current flows through the circuit, gas is generated and acts on the displaceable membrane to compress the first chamber and expel the active ingredient through the orifice for progressive delivery to the gastrointestinal tract. In some embodiments, the ingestible device includes an ingestible device for delivering a substance to a chosen location in the GI tract of a mammal includes a receiver of electromagnetic radiation for powering an openable part of the device to an opened position for dispensing of the substance. The receiver includes a coiled wire that couples the energy field, the wire having an air or ferrite core. In a further embodiment the invention includes an apparatus for generating the electromagnetic radiation, the apparatus including one or more pairs of field coils supported in a housing. The device optionally includes a latch defined by a heating resistor and a fusible restraint. The device may also include a flexible member that may serve one or both the functions of activating a transmitter circuit to indicate dispensing of the substance; and restraining of a piston used for expelling the substance. In some embodiments, the ingestible device includes an ingestible device for delivering a substance to a chosen location in the GI tract of a mammal includes a receiver of electromagnetic radiation for powering an openable part of the device to an opened position for dispensing of the substance. The receiver includes a coiled wire that couples the energy field, the wire having an air or ferrite core. In a further embodiment the invention includes an apparatus for generating the electromagnetic radiation, the apparatus including one or more pairs of field coils supported in a housing. The device optionally includes a latch defined by a heating resistor and a fusible restraint. The device may also include a flexible member that may serve one or both the functions of activating a transmitter circuit to indicate dispensing of the substance; and restraining of a piston used for expelling the substance. In some embodiments, the ingestible device is a device a swallowable capsule. A sensing module is disposed in the capsule. A bioactive substance dispenser is disposed in the capsule. A memory and logic component is disposed in the capsule and in communication with the sensing module and the dispenser. In some embodiments, localized administration is implemented via an electronic probe which is introduced into the intestinal tract of a living organism and which operates autonomously therein, adapted to deliver one or more therapy agents. In one embodiment, the method includes loading the probe with one or more therapy agents, and selectively releasing the agents from the probe at a desired location of the intestinal tract in order to provide increased efficacy over traditional oral ingestion or intravenous introduction of the agent(s). In some embodiments, the ingestible device includes electronic control means for dispensing the drug substantially to the diseased tissue sites of the GI tract, according to a pre-determined drug release profile obtained prior to administration from the specific mammal. Accordingly, in some embodiments, releasing the JAK inhibitor is triggered by an electromagnetic signal generated within the device. The releasing may occur according to a pre-determined drug release profile. In some embodiments, the ingestible device can include at least one guide tube, one or more tissue penetrating members positioned in the guide tube, a delivery member, an actuating mechanism and a release element. The release element degrades upon exposure to various conditions in the intestine so as to release and actuate the actuating mechanism. Embodiments of the invention are particularly useful for the delivery of drugs which are poorly absorbed, tolerated and/or degraded within the GI tract. In some embodiments, the ingestible device includes an electronic pill comprising at least one reservoir with a solid powder or granulate medicament or formulation, a discharge opening and an actuator responsive to control circuitry for displacing medicine from the reservoir to the discharge opening. The medicament or formulation comprises a dispersion of one or more active ingredients—e.g., solids in powder or granulate form—in an inert carrier matrix. Optionally, the active ingredients are dispersed using intestinal moisture absorbed into the pill via a semi-permeable wall section. In some embodiments, the ingestible device includes a sensor comprising a plurality of electrodes having a miniature size and a lower power consumption and a coating exterior to the electrodes, wherein the coating interacts with a target condition thereby producing a change in an electrical property of the electrodes, wherein the change is transduced into an electrical signal by the electrodes. Accordingly, in some embodiments, releasing the JAK inhibitor is triggered by an electric signal by the electrodes resulting from the interaction of the coating with the one or more sites of disease. Further provided herein is a system for medication delivery comprising such sensor and a pill. In some embodiments, the ingestible device includes an electronic pill comprising a plurality of reservoirs, each of the reservoirs comprising a discharge opening covered by a removable cover. The pill comprises at least one actuator responsive to control circuitry for removing the cover from the discharge opening. The actuator can for example be a spring loaded piston breaking a foil cover when dispensing the medicament. Alternatively, the cover can be a rotatable disk or cylinder with an opening which can be brought in line with the discharge opening of a reservoir under the action of the actuator. In some embodiments, the ingestible device includes an electronically and remotely controlled pill or medicament delivery system. The pill includes a housing; a reservoir for storing a medicament; an electronically controlled release valve or hatch for dispensing one or more medicaments stored in the reservoir while traversing the gastrointestinal tract; control and timing circuitry for opening and closing the valve; and a battery. The control and timing circuitry opens and closes the valve throughout a dispensing time period in accordance with a preset dispensing timing pattern which is programmed within the control and timing circuitry. RF communication circuitry receives control signals for remotely overriding the preset dispensing timing pattern, reprogramming the control and timing circuitry or terminating the dispensing of the medicament within the body. The pill includes an RFID tag for tracking, identification, inventory and other purposes. In some embodiments, the ingestible device includes an electronic capsule which has a discrete drive element comprising: a housing, electronics for making the electronic capsule operable, a pumping mechanism for dosing and displacing a substance, a power source for powering the electronic capsule and enabling the electronics and the pumping mechanism to operate, and a locking mechanism; and a discrete payload element comprising: a housing, a reservoir for storing the substance, one or more openings in the housing for releasing the substance from the reservoir and a locking mechanism for engaging the drive element locking mechanism. Engagement of the drive element locking mechanism with the payload element locking mechanism secures the drive element to the payload element, thereby making the electronic capsule operable and specific. In some embodiments, the ingestible device may be a mucoadhesive device configured for release of an active agent. In some embodiments, the ingestible device includes an apparatus that includes an ingestible medical treatment device, which is configured to initially assume a contracted state having a volume of less than 4 cm3. The device includes a gastric anchor, which initially assumes a contracted size, and which is configured to, upon coming in contact with a liquid, expand sufficiently to prevent passage of the anchor through a round opening having a diameter of between 1 cm and 3 cm. The device also includes a duodenal unit, which is configured to pass through the opening, and which is coupled to the gastric anchor such that the duodenal unit is held between 1 cm and 20 cm from the gastric anchor. In some embodiments, the ingestible device includes a medical robotic system and method of operating such comprises taking intraoperative external image data of a patient anatomy, and using that image data to generate a modeling adjustment for a control system of the medical robotic system (e.g., updating anatomic model and/or refining instrument registration), and/or adjust a procedure control aspect (e.g., regulating substance or therapy delivery, improving targeting, and/or tracking performance). In one embodiment the ingestible device may also include one or more environmental sensors. Environmental sensor may be used to generate environmental data for the environment external to device in the gastrointestinal (GI) tract of the subject. In some embodiments, environmental data is generated at or near the location within the GI tract of the subject where a drug is delivered. Examples of environmental sensor include, but are not limited to a capacitance sensor, a temperature sensor, an impedance sensor, a pH sensor, a heart rate sensor, acoustic sensor, image sensor (e.g., a hydrophone), and/or a movement sensor (e.g., an accelerometer). In one embodiment, the ingestible device comprises a plurality of different environmental sensors for generating different kinds of environmental data. In one embodiment, the image sensor is a video camera suitable for obtaining images in vivo of the tissues forming the GI tract of the subject. In one embodiment, the environmental data is used to help determine one or more characteristics of the GI tract, including the location of disease (e.g., presence or location of inflamed tissue and/or lesions associated with inflammatory bowel disease). In some embodiments, the ingestible device may comprise a camera for generating video imaging data of the GI tract which can be used to determine, among other things, the location of the device. In another embodiment, the ingestible device described herein may be localized using a gamma scintigraphy technique or other radio-tracker technology as employed by Phaeton Research's Enterion™ capsule (See Teng, Renli, and Juan Maya. “Absolute bioavailability and regional absorption of ticagrelor in healthy volunteers.” Journal of Drug Assessment 3.1 (2014): 43-50), or monitoring the magnetic field strength of permanent magnet in the ingestible device (see T. D. Than, et al., “A review of localization systems for robotic endoscopic capsules,” IEEE Trans. Biomed. Eng., vol. 59, no. 9, pp. 2387-2399, September 2012). In one embodiment, drug delivery is triggered when it encounters the site of disease in the GI tract. In one embodiment, the one or more environmental sensors measure pH, temperature, transit times, or combinations thereof. In some embodiments, releasing the JAK inhibitor is dependent on the pH at or in the vicinity of the location. In some embodiments the pH in the jejunum is from 6.1 to 7.2, such as 6.6. In some embodiments the pH in the mid small bowel is from 7.0 to 7.8, such as 7.4. In some embodiments the pH in the ileum is from 7.0 to 8.0, such as 7.5. In some embodiments the pH in the right colon is from 5.7 to 7.0, such as 6.4. In some embodiments the pH in the mid colon is from 5.7 to 7.4, such as 6.6. In some embodiments the pH in the left colon is from 6.3 to 7.7, such as 7.0. In some embodiments, the gastric pH in fasting subjects is from about 1.1 to 2.1, such as from 1.4 to 2.1, such as from 1.1 to 1.6, such as from 1.4 to 1.6. In some embodiments, the gastric pH in fed subjects is from 3.9 to 7.0, such as from 3.9 to 6.7, such as from 3.9 to 6.4, such as from 3.9 to 5.8, such as from 3.9 to 5.5, such as from 3.9 to 5.4, such as from 4.3 to 7.0, such as from 4.3 to 6.7, such as from 4.3 to 6.4, such as from 4.3 to 5.8, such as from 4.3 to 5.5, such as from 4.3 to 5.4. In some embodiments, the pH in the duodenum is from 5.8 to 6.8, such as from 6.0 to 6.8, such as from 6.1 to 6.8, such as from 6.2 to 6.8, such as from 5.8 to 6.7, such as from 6.0 to 6.7, such as from 6.1 to 6.7, such as from 6.2 to 6.7, such as from 5.8 to 6.6, such as from 6.0 to 6.6, such as from 6.1 to 6.6, such as from 6.2 to 6.6, such as from 5.8 to 6.5, such as from 6.0 to 6.5, such as from 6.1 to 6.5, such as from 6.2 to 6.5. In some embodiments, releasing the JAK inhibitor is not dependent on the pH at or in the vicinity of the location. In some embodiments, releasing the JAK inhibitor is triggered by degradation of a release component located in the capsule. In some embodiments, the JAK inhibitor is not triggered by degradation of a release component located in the capsule. In some embodiments, wherein releasing the JAK inhibitor is not dependent on enzymatic activity at or in the vicinity of the location. In some embodiments, releasing the JAK inhibitor is not dependent on bacterial activity at or in the vicinity of the location. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;a reservoir located within the housing and containing the JAK inhibitor,wherein a first end of the reservoir is attached to the first end of the housing;a mechanism for releasing the JAK inhibitor from the reservoir;and;an exit valve configured to allow the JAK inhibitor to be released out of the housing from the reservoir. In some embodiments, the ingestible device further comprises:an electronic component located within the housing; anda gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas. In some embodiments, the ingestible device further comprises:a safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;an exit valve located at the first end of the housing,wherein the exit valve is configured to allow the dispensable substance to be released out of the first end of the housing from the reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing,a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;an injection device located at the first end of the housing,wherein the jet injection device is configured to inject the dispensable substance out of the housing from the reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an optical sensing unit located on a side of the housing,wherein the optical sensing unit is configured to detect a reflectance from an environment external to the housing;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas in response to identifying a location of the ingestible device based on the reflectance;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;a membrane in contact with the gas generating cell and configured to move or deform into the reservoir by a pressure generated by the gas generating cell; anda dispensing outlet placed at the first end of the housing,wherein the dispensing outlet is configured to deliver the dispensable substance out of the housing from the reservoir. In one embodiment, drug delivery is triggered when it encounters the site of disease in the GI tract. In one embodiment, the one or more environmental sensors measure pH, temperature, transit times, or combinations thereof. In some embodiments, releasing the JAK inhibitor is dependent on the pH at or in the vicinity of the location. In some embodiments the pH in the jejunum is from 6.1 to 7.2, such as 6.6. In some embodiments the pH in the mid small bowel is from 7.0 to 7.8, such as 7.4. In some embodiments the pH in the ileum is from 7.0 to 8.0, such as 7.5. In some embodiments the pH in the right colon is from 5.7 to 7.0, such as 6.4. In some embodiments the pH in the mid colon is from 5.7 to 7.4, such as 6.6. In some embodiments the pH in the left colon is from 6.3 to 7.7, such as 7.0. In some embodiments, the gastric pH in fasting subjects is from about 1.1 to 2.1, such as from 1.4 to 2.1, such as from 1.1 to 1.6, such as from 1.4 to 1.6. In some embodiments, the gastric pH in fed subjects is from 3.9 to 7.0, such as from 3.9 to 6.7, such as from 3.9 to 6.4, such as from 3.9 to 5.8, such as from 3.9 to 5.5, such as from 3.9 to 5.4, such as from 4.3 to 7.0, such as from 4.3 to 6.7, such as from 4.3 to 6.4, such as from 4.3 to 5.8, such as from 4.3 to 5.5, such as from 4.3 to 5.4. In some embodiments, the pH in the duodenum is from 5.8 to 6.8, such as from 6.0 to 6.8, such as from 6.1 to 6.8, such as from 6.2 to 6.8, such as from 5.8 to 6.7, such as from 6.0 to 6.7, such as from 6.1 to 6.7, such as from 6.2 to 6.7, such as from 5.8 to 6.6, such as from 6.0 to 6.6, such as from 6.1 to 6.6, such as from 6.2 to 6.6, such as from 5.8 to 6.5, such as from 6.0 to 6.5, such as from 6.1 to 6.5, such as from 6.2 to 6.5. In some embodiments, releasing the JAK inhibitor is not dependent on the pH at or in the vicinity of the location. In some embodiments, releasing the JAK inhibitor is triggered by degradation of a release component located in the capsule. In some embodiments, the JAK inhibitor is not triggered by degradation of a release component located in the capsule. In some embodiments, wherein releasing the JAK inhibitor is not dependent on enzymatic activity at or in the vicinity of the location. In some embodiments, releasing the JAK inhibitor is not dependent on bacterial activity at or in the vicinity of the location. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;a reservoir located within the housing and containing the JAK inhibitor,wherein a first end of the reservoir is attached to the first end of the housing;a mechanism for releasing the JAK inhibitor from the reservoir;and;an exit valve configured to allow the JAK inhibitor to be released out of the housing from the reservoir. In some embodiments, the ingestible device further comprises:an electronic component located within the housing; anda gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas. In some embodiments, the ingestible device further comprises:a safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;an exit valve located at the first end of the housing,wherein the exit valve is configured to allow the dispensable substance to be released out of the first end of the housing from the reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing,a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;an injection device located at the first end of the housing,wherein the jet injection device is configured to inject the dispensable substance out of the housing from the reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing. In some embodiments, the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an optical sensing unit located on a side of the housing,wherein the optical sensing unit is configured to detect a reflectance from an environment external to the housing;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas in response to identifying a location of the ingestible device based on the reflectance;a reservoir located within the housing,wherein the reservoir stores a dispensable substance and a first end of the reservoir is attached to the first end of the housing;a membrane in contact with the gas generating cell and configured to move or deform into the reservoir by a pressure generated by the gas generating cell; anda dispensing outlet placed at the first end of the housing,wherein the dispensing outlet is configured to deliver the dispensable substance out of the housing from the reservoir. In some embodiments, the pharmaceutical composition is an ingestible device as disclosed in U.S. Patent Application Ser. No. 62/385,553, incorporated by reference herein in its entirety. In some embodiments, the pharmaceutical composition is an ingestible device as disclosed in the following applications, each of which is incorporated by reference herein in its entirety:U.S. Ser. Nos. 14/460,893; 15/514,413; 62/376,688; 62/385,344; 62/478,955; 62/434,188; 62/434,320; 62/431,297; 62/434,797; 62/480,187; 62/502,383; and 62/540,873. In some embodiments, the pharmaceutical composition is an ingestible device comprising a localization mechanism as disclosed in international patent application PCT/US2015/052500, incorporated by reference herein in its entirety. In some embodiments, the pharmaceutical composition is not a dart-like dosage form. In some embodiments of any ingestible device disclosed herein comprising a JAK inhibitor, the JAK inhibitor is present in a therapeutically effective amount. In case of conflict between the present specification and any subject matter incorporated by reference herein, the present specification, including definitions, will control. Devices and Methods for Detection of Analytes in GI Tract Detection of certain analytes in the GI tract may be useful in the identification of the nature and severity of the disease, in accurately locating the site(s) of disease, and in assessing patient response to a therapeutic agent. The appropriate therapeutic agent may accordingly be released at the correct locations(s), dosage, or timing for the disease. As discussed further herein, analytes may include biomarkers associated with a disease or associated with patient response and/or therapeutic agents previously administered to treat the disease. In some embodiments, the disclosure provides an ingestible device for detecting an analyte in a sample, the ingestible device comprising a sampling chamber that is configured to hold a composition comprising: (1) a plurality of donor particles, each of the plurality of donor particles comprising a photosensitizer and having coupled thereto a first antigen-binding agent that binds to the analyte, wherein the photosensitizer, in its excited state, is capable of generating singlet oxygen; and (2) a plurality of acceptor particles, each of the plurality of acceptor particles comprising a chemiluminescent compound and having coupled thereto a second antigen-binding agent that binds to the analyte, wherein the chemiluminescent compound is capable of reacting with singlet oxygen to emit luminescence. In some embodiments, the first and the second analyte-binding agents are antigen-binding agents (e.g., antibodies). In some embodiments, the first and the second antigen-binding agents bind to the same epitope of the analyte (e.g., a protein). In some embodiments, the first and the second antigen-binding agents bind to separate epitopes of the analyte (e.g., a protein) that spatially overlap. In some embodiments, the first and the second antigen-binding agents bind to the separate epitopes of the analyte (e.g., a protein) that do not spatially overlap. In some embodiments, this disclosure provides an ingestible device for detecting an analyte in a sample, the ingestible device comprising a sampling chamber that is configured to hold an absorbable material (e.g., an absorbable pad or sponge) having absorbed therein a composition comprising: (1) a plurality of donor particles, each of the plurality of donor particles comprising a photosensitizer and having coupled thereto a first antigen-binding agent that binds to the analyte, wherein the photosensitizer, in its excited state, is capable of generating singlet oxygen; and (2) a plurality of acceptor particles, each of the plurality of acceptor particles comprising a chemiluminescent compound and having coupled thereto a second antigen-binding agent that binds to the analyte, wherein the chemiluminescent compound is capable of reacting with singlet oxygen to emit luminescence. In some embodiments, the first and the second analyte-binding agents are antigen-binding agents (e.g., antibodies). In some embodiments, the first and the second antigen-binding agents bind to the same epitope of the analyte (e.g., a protein). In some embodiments, the first and the second antigen-binding agents bind to separate epitopes of the analyte (e.g., a protein) that spatially overlap. In some embodiments, the first and the second antigen-binding agents bind to the separate epitopes of the analyte (e.g., a protein) that do not spatially overlap. In certain embodiments, the disclosure provides a kit comprising an ingestible device as described herein. In some embodiments, the kit further comprises instructions, e.g., for detecting or quantifying an analyte in a sample. In some embodiments, the disclosure provides methods for determining an analyte in a sample. In certain embodiments, this disclosure provides a method of detecting an analyte in a fluid sample of a subject, comprising: (1) providing an ingestible device; (2) transferring the fluid sample of the subject into the sampling chamber of the ingestible device in vivo; (3) irradiating the composition held in the sampling chamber of the ingestible device with light to excite the photosensitizer; and (4) measuring total luminescence or rate of change of luminescence emitted from the composition held in the sampling chamber of the ingestible device as a function of time, thereby determining the level of the analyte in the fluid sample. In some embodiments, the method further comprises comparing the level of the analyte in the fluid sample with the level of analyte in a reference sample (e.g., a reference sample obtained from a healthy subject). In some embodiments, the level of the analyte in the sample is used to diagnose and/or monitor a disease or disorder in the subject. In some embodiments, the disclosure provides a method of detecting an analyte in a fluid sample of a subject, comprising: (1) providing an ingestible device, the device comprising a sampling chamber that is configured to hold an absorbable material (e.g., an absorbable pad or sponge) having absorbed therein a composition, as described herein; (2) transferring the fluid sample of the subject into the sampling chamber of the ingestible device in vivo; (3) fully or partially saturating the absorbable material held in the sampling chamber of the ingestible device with the fluid sample; (4) irradiating the absorbable material held in the sampling chamber of the ingestible device with light to excite the photosensitizer; and (5) measuring total luminescence or rate of change of luminescence emitted from the composition held in the sampling chamber of the ingestible device as a function of time, thereby determining the level of the analyte in the fluid sample. In some embodiments, the method further comprises comparing the level of the analyte in the fluid sample with the level of analyte in a reference sample (e.g., a reference sample obtained from a healthy subject). In some embodiments, the level of the analyte in the sample is used to diagnose and/or monitor a disease or disorder in the subject. In some embodiments, the disclosure provides a method of assessing or monitoring the need to treat a subject suffering from or at risk of overgrowth of bacterial cells in the gastrointestinal (GI) tract, comprising: (1) providing an ingestible device for detecting an analyte; (2) transferring a fluid sample from the GI tract of the subject into the sampling chamber of the ingestible device in vivo; (3) irradiating the composition held in the sampling chamber of the ingestible device with light to excite the photosensitizer; (4) measuring total luminescence or rate of change of luminescence emitted from the composition held in the sampling chamber of the ingestible device as a function of time; (5) correlating the total luminescence or the rate of change of luminescence as a function of time measured in step (4) to the amount of the analyte in the fluid sample; and (6) correlating the amount of the analyte in the fluid sample to the number of viable bacterial cells in the fluid sample. In some embodiments, a number of viable bacterial cells determined in step (6) greater than a control number of viable bacterial cells, indicates a need for treatment (e.g., with an antibiotic agent described herein). In some embodiments, the control number of viable bacterial cells is 103, 104, 105, 106, 107, 108, 109, or more. For example, in some embodiments, a number of viable bacterial cells determined in step (6) greater that about 103CFU/mL indicates a need for treatment. In some embodiments, a number of viable bacterial cells determined in step (6) greater that about 104CFU/mL indicates a need for treatment. In some embodiments, a number of the viable bacterial cells determined in step (6) greater than about 105CFU/mL indicates a need for treatment, e.g., with an antibiotic agent as described herein. In some embodiments, a number of viable bacterial cells determined in step (6) greater that about 106or more CFU/mL indicates a need for treatment. In some embodiments, the total luminescence or the rate of change of luminescence as a function of time of the sponge is measured over multiple time points for an extended period of time in step (4). For instance, in some embodiments, the total luminescence or rate of change of luminescence as a function of time of the sample is measured continuously for a period of 0-1800 minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In some embodiments, the total luminescence or the rate of change of luminescence as a function of time of said sample is measured continuously for a period of 0-330 minutes. In some embodiments, the method is performed in vivo. In some embodiments, the method includes communicating the results of the onboard assay(s) to an ex vivo receiver. In some embodiments, the total luminescence or the rate of change of luminescence as a function of time of the sponge is measured over multiple time points for an extended period of time in step (5). For instance, in some embodiments, the total luminescence or rate of change of luminescence as a function of time of the sample is measured continuously for a period of 0-1800 minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In some embodiments, the total luminescence or the rate of change of luminescence as a function of time of said sample is measured continuously for a period of 0-330 minutes. In some embodiments, the method is performed in vivo. In some embodiments, the method includes communicating the results of the onboard assay(s) to an ex vivo receiver. In some embodiments, the disclosure provides a method of assessing or monitoring the need to treat a subject suffering from or at risk of overgrowth of bacterial cells in the gastrointestinal tract, comprising: (1) providing an ingestible device for detecting an analyte, the device comprising a sampling chamber that is configured to hold an absorbable material (e.g., an absorbable pad or sponge) having absorbed therein a composition, as described herein; (2) transferring a fluid sample from the GI tract of the subject into the sampling chamber of the ingestible device in vivo; (3) fully or partially saturating the absorbable material held in the sampling chamber of the ingestible device with the fluid sample; (4) irradiating the absorbable material held in the sampling chamber of the ingestible device with light to excite the photosensitizer; (5) measuring total luminescence or rate of change of luminescence emitted from the composition held in the sampling chamber of the ingestible device as a function of time; (6) correlating the total luminescence or the rate of change of luminescence as a function of time measured in step (5) to the amount of the analyte in the fluid sample; and (7) correlating the amount of the analyte in the fluid sample to the number of viable bacterial cells in the fluid sample. In some embodiments, a number of viable bacterial cells determined in step (7) greater than a control number of viable bacterial cells indicates a need for treatment (e.g., with an antibiotic agent described herein). In some embodiments, the control number of viable bacterial cells is 103, 104, 105, 106, 107, 108, 109, or more. For example, in some embodiments, a number of viable bacterial cells determined in step (7) greater that about 103CFU/mL indicates a need for treatment. In some embodiments, a number of viable bacterial cells determined in step (7) greater that about 104CFU/mL indicates a need for treatment. In some embodiments, a number of the viable bacterial cells determined in step (7) greater than about 105CFU/mL indicates a need for treatment, e.g., with an antibiotic agent as described herein. In some embodiments, a number of viable bacterial cells determined in step (7) greater that about 106or more CFU/mL indicates a need for treatment. In some embodiments, the disclosure, provides a method of measuring the presence, absence or amount of one or more analytes from one or more samples in the gastrointestinal tract. In some embodiments the one or more analytes are measured multiple times, for example, at different time points or at different locations. In one embodiment, a single device measures one or more analytes or more time points or locations; thereby creating a “molecular map” of a physiological region. Measurements can be taken at any location in the gastrointestinal tract. For example, in one aspect, analytes from samples from one or more of the duodenum, jejunum, ileum, ascending colon, transverse colon or descending colon can be measured to create a molecular map of the small and large intestine. In one aspect, the sample is from the duodenum. In one aspect, In one aspect, the sample is from the jejunum. In one aspect, the sample is from the ileum. In one aspect, the sample is from the ascending colon. In one aspect, the sample is from the transverse colon. In one aspect, the sample is from the descending colon. In another aspect, a series of measurements can be taken over a shorter distance of the gastrointestinal tract (e.g., the ileum) to create a higher resolution molecular map. In some embodiments, previous endoscopic imaging may identify a diseased area for molecular mapping. For example, a gastroenterologist may use imaging (e.g., an endoscope equipped with a camera) to identify the presence of Crohn's Disease in the ileum and cecum of a patient, and the methods and techniques herein may be used to measure inflammation-associated analytes in this diseased area of the patient. In a related embodiment, the inflammation-associated analytes, or any analyte, may be measured every one or more days to monitor disease flare-ups, or response to therapeutics. Analytes The compositions and methods described herein can be used to detect, analyze, and/or quantitate a variety of analytes in a human subject. “Analyte” as used herein refers to a compound or composition to be detected in a sample. Exemplary analytes suitable for use herein include those described in U.S. Pat. No. 6,251,581, which is incorporated by reference herein in its entirety. Broadly speaking, an analyte can be any substance (e.g., a substance with one or more antigens) capable of being detected. An exemplary and non-limiting list of analytes includes ligands, proteins, blood clotting factors, hormones, cytokines, polysaccharides, mucopolysaccharides, microorganisms (e.g., bacteria), microbial antigens, and therapeutic agents (including fragments and metabolites thereof). For instance, the analyte may be a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic), usually antigenic or haptenic, and is a single compound or plurality of compounds which share at least one common epitopic or determinant site. The analyte can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., a human leukocyte antigen (HLA), or other cell surface antigen, or a microorganism, e.g., bacterium (e.g. a pathogenic bacterium), a fungus, protozoan, or a virus (e.g., a protein, a nucleic acid, a lipid, or a hormone). In some embodiments, the analyte can be a part of an exosome (e.g., a bacterial exosome). In some embodiments, the analyte is derived from a subject (e.g., a human subject). In some embodiments, the analyte is derived from a microorganism present in the subject. In some embodiments, the analyte is a nucleic acid (e.g., a DNA molecule or a RNA molecule), a protein (e.g., a soluble protein, a cell surface protein), or a fragment thereof, that can be detected using any of the devices and methods provided herein. The polyvalent ligand analytes will normally be poly(amino acids), i.e., a polypeptide (i.e., protein) or a peptide, polysaccharides, nucleic acids (e.g., DNA or RNA), and combinations thereof. Such combinations include components of bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes, and the like. In some embodiments, the polyepitopic ligand analytes have a molecular weight of at least about 5,000 Da, more usually at least about 10,000 Da. In the poly(amino acid) category, the poly(amino acids) of interest may generally have a molecular weight from about 5,000 Da to about 5,000,000 Da, more usually from about 20,000 Da to 1,000,000 Da; among the hormones of interest, the molecular weights will usually range from about 5,000 Da to 60,000 Da. In some embodiments, the monoepitopic ligand analytes generally have a molecular weight of from about 100 to 2,000 Da, more usually from 125 to 1,000 Da. A wide variety of proteins may be considered as to the family of proteins having similar structural features, proteins having particular biological functions, proteins related to specific microorganisms, particularly disease causing microorganisms, etc. Such proteins include, for example, immunoglobulins, cytokines, enzymes, hormones, cancer antigens, nutritional markers, tissue specific antigens, etc. In some embodiments, the analyte is a protein. In some embodiments, the analyte is a protein, e.g., an enzyme (e.g., a hemolysin, a protease, a phospholipase), a soluble protein, an exotoxin. In some embodiments, the analyte is a fragment of a protein, a peptide, or an antigen. In some embodiments, the analyte is a peptide of at least 5 amino acids (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least, 50, or at least 100 amino acids). Exemplary lengths include 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, or 100 amino acids. Exemplary classes of protein analytes include, but are not limited to: protamines, histones, albumins, globulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, cell surface receptors, membrane-anchored proteins, transmembrane proteins, secreted proteins, HLA, and unclassified proteins. In some embodiments, the analyte is an affimer (see, e.g., Tiede et al. (2017)eLife6: e24903, which is expressly incorporated herein by reference). Exemplary analytes include: Prealbumin, Albumin, α1-Lipoprotein, α1-Antitrypsin, α1-Glycoprotein, Transcortin, 4.6S-Postalbumin, α1-glycoprotein, α1x-Glycoprotein, Thyroxin-binding globulin, Inter-α-trypsin-inhibitor, Gc-globulin (Gc 1-1, Gc 2-1, Gc 2-2), Haptoglobin (Hp 1-1, Hp 2-1, Hp 2-2), Ceruloplasmin, Cholinesterase, α2-Lipoprotein(s), Myoglobin, C-Reactive Protein, α2-Macroglobulin, α2-HS-glycoprotein, Zn-α2-glycoprotein, α2-Neuramino-glycoprotein, Erythropoietin, β-lipoprotein, Transferrin, Hemopexin, Fibrinogen, Plasminogen, β2-glycoprotein I, β2-glycoprotein II, Immunoglobulin G (IgG) or γG-globulin, Immunoglobulin A (IgA) or γA-globulin, Immunoglobulin M (IgM) or γM-globulin, Immunoglobulin D (IgD) or γD-Globulin (γD), Immunoglobulin E (IgE) or γE-Globulin (γE), Free κ and λ light chains, and Complement factors: C′1, (C′1q, C′1r, C′1s, C′2, C′3 (β1A, α2D), C′4, C'S, C′6, C′7, C′8, C′9. Additional examples of analytes include tumor necrosis factor-α (TNFα), interleukin-12 (IL-12), IL-23, IL-6, α2β1 integrin, α1β1 integrin, α4β7 integrin, integrin α4β1 (VLA-4), E-selectin, ICAM-1, α5β1 integrin, α4β1 integrin, VLA-4, α2β1 integrin, α5β3 integrin, α5β5 integrin, α1β33 integrin, MAdCAM-1, SMAD7, JAK1, JAK2, JAK3, TYK-2, CHST15, IL-1, IL-1α, IL-1β, IL-18, IL-36α, IL-36β, IL-36γ, IL-38, IL-33, IL-13, CD40L, CD40, CD3γ, CD3δ, CD3ε, CD3ζ, TCR, TCRα, TCRβ, TCRδ, TCRγ, CD14, CD20, CD25, IL-2, IL-2 β chain, IL-2 γ chain, CD28, CD80, CD86, CD49, MMP1, CD89, IgA, CXCL10, CCL11, an ELR chemokine, CCR2, CCR9, CXCR3, CCR3, CCR5, CCL2, CCL8, CCL16, CCL25, CXCR1m CXCR2m CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and CXCL8, and a nucleic acid (e.g., mRNA) encoding any of the same. In some embodiments, the analyte is a blood clotting factor. Exemplary blood clotting factors include, but are not limited to: International designationNameIFibrinogenIIProthrombinIIaThrombinIIITissue thromboplastinV and VIProaccelerin, acceleratorglobulinVIIProconvertinVIIIAntihemophilic globulin(AHG)IXChristmas factorplasma thromboplastincomponent (PTC)XStuart-Prower factor,autoprothrombin IIIXIPlasma thromboplastinantecedent (PTA)XIIHagemann factorXIIIFibrin-stabilizing factor In some embodiments, the analyte is a hormone. Exemplary hormones include, but are not limited to: Peptide and Protein Hormones, Parathyroid hormone, (parathromone), Thyrocalcitonin, Insulin, Glucagon, Relaxin, Erythropoietin, Melanotropin (melancyte-stimulating hormone; intermedin), Somatotropin (growth hormone), Corticotropin (adrenocorticotropic hormone), Thyrotropin, Follicle-stimulating hormone, Luteinizing hormone (interstitial cell-stimulating hormone), Luteomammotropic hormone (luteotropin, prolactin), Gonadotropin (chorionic gonadotropin), Secretin, Gastrin, Angiotensin I and II, Bradykinin, and Human placental lactogen, thyroxine, cortisol, triiodothyronine, testosterone, estradiol, estrone, progestrone, luteinizing hormone-releasing hormone (LHRH), and immunosuppressants such as cyclosporin, FK506, mycophenolic acid, and so forth. In some embodiments, the analyte is a peptide hormone (e.g., a peptide hormone from the neurohypophysis). Exemplary peptide hormones from the neurohypophysis include, but are not limited to: Oxytocin, Vasopressin, and releasing factors (RF) (e.g., corticotropin releasing factor (CRF), luteinizing hormone releasing factor (LRF), thyrotropin releasing factor (TRF), Somatotropin-RF, growth hormone releasing factor (GRF), follicle stimulating hormone-releasing factor (FSH-RF), prolactin inhibiting factor (PIF), and melanocyte stimulating hormone inhibiting factor (MIF)). In some embodiments, the analyte is a cytokine or a chemokine. Exemplary cytokines include, but are not limited to: interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), epidermal growth factor (EGF), tumor necrosis factor (TNF, e.g., TNF-α or TNF-β), and nerve growth factor (NGF). In some embodiments, the analyte is a cancer antigen. Exemplary cancer antigens include, but are not limited to: prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), α-fetoprotein, Acid phosphatase, CA19.9, and CA125. In some embodiments, the analyte is a tissue-specific antigen. Exemplary tissue specific antigens include, but are not limited to: alkaline phosphatase, myoglobin, CPK-MB, calcitonin, and myelin basic protein. In some embodiments, the analyte is a mucopolysaccharide or a polysaccharide. In some embodiments, the analyte is a microorganism, or a molecule derived from or produced by a microorganism (e.g., a bacteria, a virus, prion, or a protozoan). For example, in some embodiments, the analyte is a molecule (e.g., an protein or a nucleic acid) that is specific for a particular microbial genus, species, or strain (e.g., a specific bacterial genus, species, or strain). In some embodiments, the microorganism is pathogenic (i.e., causes disease). In some embodiments, the microorganism is non-pathogenic (e.g., a commensal microorganism). Exemplary microorganisms include, but are not limited to: CorynebacteriaCorynebacterium diphtheriaPneumococciDiplococcus pneumoniaeStreptococciStreptococcus pyrogenesStreptococcus salivarusStaphylococciStaphylococcus aureusStaphylococcus albusNeisseriaNeisseria meningitidisNeisseria gonorrheaEnterobacteriaciaeEscherichia coliAerobacter aerogenesThe coliformKlebsiella pneumoniaebacteriaSalmonella typhosaSalmonella choleraesuisTheSalmonellaeSalmonella typhimuriumShigella dysenteriaShigella schmitziiShigella arabinotardaTheShigellaeShigella flexneriShigella boydiiShigella sonneiOther entericbacilliProteus vulgarisProteus mirabilisProteusspeciesProteus morganiPseudomonas aeruginosaAlcaligenes faecalisVibrio choleraeHemophilus-BordetellagroupRhizopus oryzaeHemophilus influenza,H. ducryiRhizopus arrhizuaPhycomycetesHemophilus hemophilusRhizopus nigricansHemophilus aegypticusSporotrichum schenkiiHemophilus parainfluenzaFlonsecaea pedrosoiBordetella pertussisFonsecacea compactPasteurellaeFonsecacea dermatidisPasteurella pestisCladosporium carrioniiPasteurella tulareusisPhialophora verrucosaBrucellaeAspergillus nidulansBrucella melltensisMadurella mycetomiBrucella abortusMadurella griseaBrucella suisAllescheria boydiiAerobic Spore-formingBacilliPhialophora jeanselmeiBacillus anthracisMicrosporum gypseumBacillus subtilisTrichophyton mentagrophytesBacillus megateriumKeratinomyces ajelloiBacillus cereusMicrosporum canisAnaerobic Spore-formingBacilliTrichophyton rubrumClostridium botulinumMicrosporum adouiniClostridium tetaniVirusesClostridium perfringensAdenovirusesClostridium novyiHerpes VirusesClostridium septicumHerpes simplexClostridium histoyticumVaricella (Chicken pox)Clostridium tertiumHerpes Zoster (Shingles)Clostridium bifermentansVirus BClostridium sporogenesCytomegalovirusMycobacteriaPox VirusesMycobacterium tuberculosishominisVariola (smallpox)Mycobacterium bovisVacciniaMycobacterium aviumPoxvirus bovisMycobacterium lepraeParavacciniaMycobacterium paratuberculosisMolluscum contagiosumActinomycetes(fungus-ike bacteria)PicornavirusesActinomyces IsaeliPoliovirusActinomyces bovisCoxsackievirusActinomyces naeslundiiEchovirusesNocardia asteroidesRhinovirusesNocardia brasiliensisMyxovirusesThe SpirochetesInfluenza(A, B, and C)Treponema pallidumParainfluenza (1-4)Treponema pertenueMumps VirusSpirillum minusStreptobacillus monoiliformisNewcastle Disease VirusTreponema carateumMeasles VirusBorrelia recurrentisRinderpest VirusLeptospira icterohemorrhagiaeCanine Distemper VirusLeptospira canicolaRespiratory Syncytial VirusTrypanasomesRubella VirusMycoplasmasArbovirusesMycoplasma pneumoniaeOther pathogensEastern Equine Encephalitis VirusListeria monocytogenesWestern Equine EncephalitisVirusErysipeothrix rhusiopathiaeSindbis VirusStreptobacillus moniliformisChikugunya VirusDonvania granulomatisSemliki Forest VirusEntamoeba histolyticaMayora VirusPlasmodium falciparumSt. Louis EncephalitisPlasmodium japonicumCalifornia Encephalitis VirusBartonella bacilliformisColorado Tick Fever VirusRickettsia(bacteria-like parasites)Yellow Fever VirusRickettsia prowazekiiDengue VirusRickettsia mooseriReovirusesRickettsia rickettsiiReovirus Types 1-3Rickettsia conoriRetrovirusesRickettsia australisHuman ImmunodeficiencyRickettsia sibiricusViruses I and II (HTLV)Rickettsia akariHuman T-cell LymphotrophicRickettsia tsutsugamushiVirus I & II (HIV)Rickettsia burnettiHepatitisRickettsia quintanaHepatitis A VirusChlamydia(unclassifiable parasitesHepatitis B Virusbacterial/viral)Hepatitis C VirusChlamydiaagents (naming uncertain)Tumor VirusesChlamydia trachomatisFungiRauscher Leukemia VirusCryptococcus neoformansGross VirusBlastomyces dermatidisMaloney Leukemia VirusHistoplasma capsulatumCoccidioides immitisHuman Papilloma VirusParacoccidioides brasliensisCandida albicansAspergillus fumigatusMucor corymbifer(Absidia corymbifera) In some embodiments, the analyte is a bacterium. Exemplary bacteria include, but are not limited to:Escherichia coli(orE. coli),Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Yersinia pestis, Yersinia enterocolitica, Francisella tularensis, Brucella species, Clostridium perfringens, Burkholderia mallei, Burkholderia pseudomallei, Staphylococcus species, Mycobacteriumspecies, Group AStreptococcus, Group BStreptococcus, Streptococcus pneumoniae, Helicobacter pylori, Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia, Rickettsia akari, Rickettsia prowazekii, Rickettsia canada, Bacillus subtilis, Bacillus subtilis niger, Bacillus thuringiensis, Coxiella burnetti, Faecalibacterium prausnitzii(also known asBacteroidespraussnitzii),Roseburia hominis, Eubacterium rectale, Dialister invisus, Ruminococcus albus, Ruminococcus callidus, andRuminococcus bromii. Additional exemplary bacteria include bacteria of the phyla Firmicutes (e.g.,Clostridiumclusters XIVa and IV), bacteria of the phyla Bacteroidetes (e.g.,Bacteroides fragilisorBacteroides vulgatus), and bacteria of the phyla Actinobacteria (e.g., Coriobacteriaceae spp. orBifidobacterium adolescentis). Bacteria of theClostridiumcluster XIVa includes species belonging to, for example, theClostridium, Ruminococcus, Lachnospira, Roseburia, Eubacterium, Coprococcus, Dorea, andButyrivibriogenera. Bacteria of theClostridiumcluster IV includes species belonging to, for example, theClostridium, Ruminococcus, EubacteriumandAnaerofilumgenera. In some embodiments, the analyte isCandida, e.g.,Candida albicans. In some embodiments, the analyte is a byproduct from a bacterium or other microorganism, e.g., helminth ova, enterotoxin (Clostridium difficiletoxin A; TcdA) or cytotoxin (Clostridium difficiletoxin B; TcdB). In some embodiments, the bacterium is a pathogenic bacterium. Non-limiting examples of pathogenic bacteria belong to the generaBacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, andYersinia. Non-limiting examples of specific pathogenic bacterial species include a strain ofBacillusanthraces, a strain of a strain ofBordetella pertussis, a strain of a strain ofBorrelia burgdorferi, a strain of a strain ofBrucella abortus, a strain of a strain ofBrucella canis, a strain of a strain ofBrucella melitensis, a strain of a strain ofBrucella suis, a strain of a strain ofCampylobacter jejuni, a strain ofChlamydia pneumoniae, a strain ofChlamydia trachomatis, a strain ofChlamydophila psittaci, a strain ofClostridium botulinum, a strain ofClostridium difficile, a strain ofClostridium perfringens, a strain ofClostridium tetani, a strain ofCorynebacterium diphtheria, a strain ofEnterobacter sakazakii, a strain ofEnterococcus faecalis, a strain ofEnterococcus faecium, a strain ofEscherichia coli(e.g.,E. coliO157 H7), a strain ofFrancisella tularensis, a strain ofHaemophilusinfluenza, a strain ofHelicobacter pylori, a strain ofLegionella pneumophila, a strain ofLeptospira interrogans, a strain ofListeria monocytogenes, a strain ofMycobacterium leprae, a strain ofMycobacterium tuberculosis, a strain ofMycobacterium ulcerans, a strain ofMycoplasmapneumonia, a strain ofNeisseria gonorrhoeae, a strain ofNeisseria meningitides, a strain ofPseudomonas aeruginosa, a strain ofRickettsia rickettsia, a strain ofSalmonella typhiandSalmonella typhimurium, a strain ofShigella sonnei, a strain ofStaphylococcus aureus, a strain ofStaphylococcus epidermidis, a strain ofStaphylococcus saprophyticus, a strain ofStreptococcus agalactiae, a strain ofStreptococcus pneumonia, a strain ofStreptococcus pyogenes, a strain ofTreponema pallidum, a strain ofVibrio cholera, a strain ofYersinia enterocolitica, and, a strain ofYersinia pestis. In some embodiments, the bacterium is a commensal bacterium (e.g., a probiotic). In some embodiments, the bacterium has been previously administered to a subject, e.g., as a live biotherapeutic agent. Exemplary commensal bacteria include, but are not limited to,Faecalibacterium prausnitzii(also referred to asBacteroidespraussnitzii),Roseburia hominis, Eubacterium rectale, Dialister invisus, Ruminococcus albus, Ruminococcus gnavus, Ruminococcus torques, Ruminococcus callidus, andRuminococcus bromii. In some embodiments, the analyte is a virus. In some embodiments, the virus is a pathogenic virus. Non-limiting examples of pathogenic viruses belong to the families Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. In some embodiments, the analyte is a fungus. In some embodiments, the fungi is a pathogenic fungus. Non-limiting examples of pathogenic fungi belong to the generaAsperfillus, Canidia, Cryptococcus, Histoplasma, Pneumocystis, andStachybotrys. Non-limiting examples of specific pathogenic fungi species include a strain ofAspergillus clavatus, Aspergillus fumigatus, Aspergillus flavus, Canidia albicans, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis firovecii, Pneumocystis carinii, andStachybotrys chartarum. In some embodiments, the analyte is a protozoan. In some embodiments, the analyte is a pathogenic protozoan. Non-limiting examples of pathogenic protozoa belong to the generaAcanthamoeba, Balamuthia, Cryptosporidium, Dientamoeba, Endolimax, Entamoeba, Giardia, Iodamoeba, Leishmania, Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas, andTrypanosoma. Non-limiting examples of specific pathogenic protozoa species include a strain ofAcanthamoebaspp.,Balamuthia mandrillaris, Cryptosporidium canis, Cryptosporidium fells, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Dientamoeba fragilis, Endolimax nana, Entamoeba dispar, Entamoeba hartmanni, Entamoeba histolytica, Entamoeba coli, Entamoeba moshkovskii, Giardia lamblia, Iodamoeba butschlii, Leishmania aethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Naegleria fowleri, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Sappinia diploidea, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, andTrypanosoma cruzi. In some embodiments, the analyte is secreted by or expressed on the cell surface of a microorganism (e.g., a bacterium, a colonic bacterium, a viable bacterium, a dead bacterium, a parasite (e.g.,Giardia lamblia, Cryptosporidium, Cystoisosporiasis belli, andBalantidium coli), a virus (e.g., a herpes virus, a cytomegalovirus, a herpes simplex virus, an Epstein-Barr virus, a human papilloma virus, a rotavirus, a human herpesvirus-8; Goodgame (1999) Curr. Gastroenterol. Rep. 1(4): 292-300). In some embodiments, the analyte is secreted by or expressed on the cell surface of a Gram-negative bacterium (e.g.,E. coli, Helicobacter pylori). In some embodiments, the analyte is secreted by or expressed on the cell surface (e.g., a bacterial surface epitope) of a Gram-positive bacterium (e.g.,Staphylococcus aureus, Clostridium botulinum, Clostridium difficile). In some embodiments, the analyte is a molecule expressed on the surface of a bacterial cell (e.g., a bacterial cell surface protein). In some embodiments, the analyte is a bacterial toxin (e.g., TcdA and/or TcdB fromClostridium difficile). In some embodiments, the analyte is CFA/I fimbriae, flagella, lipopolysaccharide (LPS), lipoteichoic acid, or a peptidoglycan. Non-limiting examples of bacterium that may express an analyte that can be detected using any of the devices and methods described herein include:Bacillusanthraces,Bacillus cereus, Clostridium botulinum, Clostridium difficile, Escherichia coli, Yersinia pestis, Yersinia enterocolitica, Francisella tularensis, Brucella species, Clostridium perfringens, Burkholderia mallei, Burkholderia pseudomallei, Helicobacter pylori, Staphylococcus species, Mycobacteriumspecies, Group AStreptococcus, Group BStreptococcus, Streptococcus pneumoniae, Francisella tularensis, Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia, Rickettsia akari, Rickettsia prowazekii, Rickettsia canada, Bacillus subtilis, Bacillus subtilis niger, Bacillus thuringiensis, Coxiella bumetti, Candida albicans, Bacteroides fragilis, Leptospira interrogans, Listeria monocytogenes, Pasteurella multocida, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneria, Shigella sonnei, Vibrio cholera, andVibrio parahaemolyticus. In some embodiments, the analyte is a byproduct from a bacterium or another microorganism, e.g., helminth ova, enterotoxin (Clostridium difficiletoxin A; TcdA), cytotoxin (Clostridium difficiletoxin B; TcdB), ammonia. In some embodiments, the analyte is an antigen from a microorganism (e.g., a bacteria, virus, prion, fungus, protozoan or a parasite). In some embodiments, the analytes include drugs, metabolites, pesticides, pollutants, and the like. Included among drugs of interest are the alkaloids. Among the alkaloids are morphine alkaloids, which includes morphine, codeine, heroin, dextromethorphan, their derivatives and metabolites; cocaine alkaloids, which include cocaine and benzyl ecgonine, their derivatives and metabolites; ergot alkaloids, which include the diethylamide of lysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinoline alkaloids, which include quinine and quinidine; diterpene alkaloids, their derivatives and metabolites. In some embodiments, the analyte is a steroid selected from the estrogens, androgens, andreocortical steroids, bile acids, cardiotonic glycosides and aglycones, which includes digoxin and digoxigenin, saponins and sapogenins, their derivatives and metabolites. Also included are the steroid mimetic substances, such as diethylstilbestrol. In some embodiments, the analyte is a bile acid. In some embodiments, the presence, absence, and/or a specific level of one or more bile acids in the GI tract of a subject is indicative of a condition or disease state (e.g., a GI disorder and/or a non-GI disorder (e.g., a systemic disorder). For example, in some embodiments, the compositions and methods described herein may be used to detect and/or quantify a bile acid in the GI tract of the subject to diagnose a condition such as bile acid malabsorption (also known as bile acid diarrhea). In some embodiments, the analyte is a metabolite in the serotonin, tryptophan and/or kynurenine pathways, including but not limited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilic acid, and combinations thereof 5-HT is a molecule that plays a role in the regulation of gastrointestinal motility, secretion, and sensation. Imbalances in the levels of 5-HT are associated with several diseases including inflammatory bowel syndrome (IBS), autism, gastric ulcer formation, non-cardiac chest pain, and functional dyspepsia (see, e.g., Faure et al. (2010)Gastroenterology139(1): 249-58 and Muller et al. (2016)Neuroscience321: 24-41, and International Publication No. WO 2014/188377, each of which are incorporated herein by reference). Conversion of metabolites within the serotonin, tryptophan and/or kynurenine pathways affects the levels of 5-HT in a subject. Therefore, measuring the levels of one or more of the metabolites in this pathway may be used for the diagnosis, management and treatment of a disease or disorder associated with 5-HT imbalance including but not limited to IBS, autism, carcinoid syndrome, depression, hypertension, Alzheimer's disease, constipation, migraine, and serotonin syndrome. One or more analytes in the serotonin, tryptophan and/or kynurenine pathways can be detected and/or quantitated using, for example, methods and analyte-binding agents that bind to these metabolites including, e.g., antibodies, known in the art (see, e.g., International Publication No. WO2014/188377, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the analyte is a lactam having from 5 to 6 annular members selected from barbituates, e.g., phenobarbital and secobarbital, diphenylhydantonin, primidone, ethosuximide, and metabolites thereof. In some embodiments, the analyte is an aminoalkylbenzene, with alkyl of from 2 to 3 carbon atoms, selected from the amphetamines; catecholamines, which includes ephedrine, L-dopa, epinephrine; narceine; papaverine; and metabolites thereof. In some embodiments, the analyte is a benzheterocyclic selected from oxazepam, chlorpromazine, tegretol, their derivatives and metabolites, the heterocyclic rings being azepines, diazepines and phenothiazines. In some embodiments, the analyte is a purine selected from theophylline, caffeine, their metabolites and derivatives. In some embodiments, the analyte is marijuana, cannabinol or tetrahydrocannabinol. In some embodiments, the analyte is a vitamin such as vitamin A, vitamin B, e.g. vitamin B12, vitamin C, vitamin D, vitamin E and vitamin K, folic acid, thiamine. In some embodiments, the analyte is selected from prostaglandins, which differ by the degree and sites of hydroxylation and unsaturation. In some embodiments, the analyte is a tricyclic antidepressant selected from imipramine, dismethylimipramine, amitriptyline, nortriptyline, protriptyline, trimipramine, chlomipramine, doxepine, and desmethyldoxepin. In some embodiments, the analyte is selected from anti-neoplastics, including methotrexate. In some embodiments, the analyte is an antibiotic as described herein, including, but not limited to, penicillin, chloromycetin, actinomycetin, tetracycline, terramycin, and metabolites and derivatives. In some embodiments, the analyte is a nucleoside and nucleotide selected from ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine with their appropriate sugar and phosphate substituents. In some embodiments, the analyte is selected from methadone, meprobamate, serotonin, meperidine, lidocaine, procainamide, acetylprocainamide, propranolol, griseofulvin, valproic acid, butyrophenones, antihistamines, chloramphenicol, anticholinergic drugs, such as atropine, their metabolites and derivatives. In some embodiments, the analyte is a metabolite related to a diseased state. Such metabolites include, but are not limited to spermine, galactose, phenylpyruvic acid, and porphyrin Type 1. In some embodiments, the analyte is an aminoglycoside, such as gentamicin, kanamicin, tobramycin, or amikacin. In some embodiments, the analyte is a pesticide. Among pesticides of interest are polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenated sulfenamides, their metabolites and derivatives. In some embodiments, the analyte has a molecular weight of about 500 Da to about 1,000,000 Da (e.g., about 500 to about 500,000 Da, about 1,000 to about 100,000 Da). In some embodiments, the analyte is a receptor, with a molecular weight ranging from 10,000 to 2×108Da, more usually from 10,000 to 106Da. For immunoglobulins, IgA, IgG, IgE and IgM, the molecular weights will generally vary from about 160,000 Da to about 106Da. Enzymes will normally range in molecular weight from about 10,000 Da to about 1,000,000 Da. Natural receptors vary widely, generally having a molecular weight of at least about 25,000 Da and may be 106or higher Da, including such materials as avidin, DNA, RNA, thyroxine binding globulin, thyroxine binding prealbumin, transcortin, etc. In some embodiments, the term “analyte” further includes polynucleotide analytes such as those polynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA, DNA-RNA duplexes, etc. The term analyte also includes polynucleotide-binding agents, such as, for example, restriction enzymes, trascription factors, transcription activators, transcription repressors, nucleases, polymerases, histones, DNA repair enzymes, intercalating gagents, chemotherapeutic agents, and the like. In some embodiments, the analyte may be a molecule found directly in a sample such as a body fluid from a host. The sample can be examined directly or may be pretreated to render the analyte more readily detectible. Furthermore, the analyte of interest may be determined by detecting an agent probative of the analyte of interest (i.e., an analyte-binding agent), such as a specific binding pair member complementary to the analyte of interest, whose presence will be detected only when the analyte of interest is present in a sample. Thus, the agent probative of the analyte becomes the analyte that is detected in an assay. In some embodiments, the analyte a nucleic acid (e.g., a bacterial DNA molecule or a bacterial RNA molecule (e.g., a bacterial tRNA, a transfer-messenger RNA (tmRNA)). See, e.g., Sjostrom et al. (2015) Scientific Reports 5: 15329; Ghosal (2017) Microbial Pathogenesis 104: 161-163; Shen et al. (2012) Cell Host Microbe. 12(4): 509-520. In some embodiments, the analyte is a component of an outer membrane vesicle (OMV) (e.g., an OmpU protein, Elluri et al. (2014) PloS One 9: e106731). See, e.g., Kulp and Kuehn (2010) Annual Review of microbiology 64: 163-184; Berleman and Auer (2013) Environmental microbiology 15: 347-354; Wai et al. (1995) Microbiology and immunology 39: 451-456; Lindmark et al. (2009) BMC microbiology 9: 220; Sjostrom et al. (2015) Scientific Reports 5: 15329. In some embodiments, the analyte is G-CSF, which can stimulate the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. In some embodiments, the analyte is an enzyme such as glutathione 5-transferase. For example, the ingestible device can include P28GST, a 28 kDa helminth protein fromSchistosomawith potent immunogenic and antioxidant properties. P28GST prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils and can be recombinantly produced (e.g., inS. cerevisiae). See, for example, U.S. Pat. No. 9,593,313, Driss et at,Mucosal Immunology,2016 9, 322-335; and Capron et al.,Gastroenterology,146(5): S-638. In some embodiments, the analyte is a metabolite in the serotonin, tryptophan and/or kynurenine pathways, including but not limited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilic acid, and combinations thereof. In some embodiments, analytes are therapeutic agents or drugs. In some embodiments, analytes are biomarkers. The therapeutic agents disclosed herein are can also be analytes. Examples of biomarkers are provided herein. In some embodiments, analytes are therapeutic agents, fragments thereof, and metabolites thereof (e.g., antibiotics). In some embodiments, the analytes are antibodies. In some embodiments, the analytes are antibiotics. Additional exemplary analytes (e.g., antibodies and antibiotics) are provided below. a. Antibodies In some embodiments, the analyte or the analyte-binding agent is an antibody. An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv) and domain antibodies), and fusion proteins including an antibody portion, and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site. The term antibody includes antibody fragments (e.g., antigen-binding fragments) such as an Fv fragment, a Fab fragment, a F(ab′)2 fragment, and a Fab′ fragment. Additional examples of antigen-binding fragments include an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM). An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) that contain hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al, 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches. As known in the art, a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. A “derivative” refers to any polypeptide (e.g., an antibody) having a substantially identical amino acid sequence to the naturally occurring polypeptide, in which one or more amino acids have been modified at side groups of the amino acids (e.g., an biotinylated protein or antibody). The term “derivative” shall also include any polypeptide (e.g., an antibody) which has one or more amino acids deleted from, added to, or substituted from the natural polypeptide sequence, but which retains a substantial amino acid sequence homology to the natural sequence. A substantial sequence homology is any homology greater than 50 percent. In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, a multivalent antibody, or a fragment thereof. In some embodiments, an antibody can be a scFv-Fc (Sokolowska-Wedzina et al.,Mol. Cancer Res.15(8):1040-1050, 2017), a VHH domain (Li et al.,Immunol. Lett.188:89-95, 2017), a VNAR domain (Hasler et al.,Mol. Immunol.75:28-37, 2016), a (scFv)2, a minibody (Kim et al.,PLoS One10(1):e113442, 2014), or a BiTE. In some embodiments, an antibody can be a DVD-Ig (Wu et al.,Nat. Biotechnol.25(11):1290-1297, 2007; WO 08/024188; WO 07/024715), and a dual-affinity re-targeting antibody (DART) (Tsai et al.,Mol. Ther. Oncolytics3:15024, 2016), a triomab (Chelius et al.,MAbs2(3):309-319, 2010), kih IgG with a common LC (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a crossmab (Regula et al.,EMBO Mol. Med.9(7):985, 2017), an ortho-Fab IgG (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a 2-in-1-IgG (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), IgG-scFv (Cheal et al.,Mol. Cancer Ther.13(7):1803-1812, 2014), scFv2-Fc (Natsume et al.,J. Biochem.140(3):359-368, 2006), a bi-nanobody (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), tanden antibody (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a DART-Fc (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a scFv-HSA-scFv (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), DNL-Fab3 (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), DAF (two-in-one or four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair antibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab, kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody (e.g., antibodies derived fromCamelus bactriamus, Calelus dromaderius, orLama paccos) (U.S. Pat. No. 5,759,808; Stijlemans et al.,J. Biol. Chem.279:1256-1261, 2004; Dumoulin et al.,Nature424:783-788, 2003; and Pleschberger et al.,Bioconjugate Chem.14:440-448, 2003), nanobody-HSA, a diabody (e.g., Poljak,Structure2(12):1121-1123, 1994; Hudson et al.,J. Immunol. Methods23(1-2):177-189, 1999), a TandAb (Reusch et al.,mAbs6(3):727-738, 2014), scDiabody (Cuesta et al.,Trends in Biotechnol.28(7):355-362, 2010), scDiabody-CH3 (Sanz et al.,Trends in Immunol.25(2):85-91, 2004), Diabody-CH3 (Guo et al.), Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody (Huston et al.,Human Antibodies10(3-4):127-142, 2001; Wheeler et al.,Mol. Ther.8(3):355-366, 2003; Stocks,Drug Discov. Today9(22):960-966, 2004), dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HSA, tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2. In some embodiments, an antibody can be an IgNAR, a bispecific antibody (Milstein and Cuello,Nature305:537-539, 1983; Suresh et al.,Methods in Enzymology121:210, 1986; WO 96/27011; Brennan et al.,Science229:81, 1985; Shalaby et al.,J. Exp. Med.175:217-225, 1992; Kolstelny et al.,J. Immunol.148(5):1547-1553, 1992; Hollinger et al.,Proc. Natl. Acad. Sci. U.S.A.90:6444-6448, 1993; Gruber et al.,J. Immunol.152:5368, 1994; Tuft et al.,J. Immunol.147:60, 1991), a bispecific diabody, a triabody (Schoonooghe et al.,BMC Biotechnol.9:70, 2009), a tetrabody, scFv-Fc knobs-into-holes, a scFv-Fc-scFv, a (Fab′scFv)2, a V-IgG, a IvG-V, a dual V domain IgG, a heavy chain immunoglobulin or a camelid (Holt et al.,Trends Biotechnol.21(11):484-490, 2003), an intrabody, a monoclonal antibody (e.g., a human or humanized monoclonal antibody), a heteroconjugate antibody (e.g., U.S. Pat. No. 4,676,980), a linear antibody (Zapata et al.,Protein Eng.8(10:1057-1062, 1995), a trispecific antibody (Tuft et al.,J. Immunol.147:60, 1991), a Fabs-in-Tandem immunoglobulin (WO 15/103072), or a humanized camelid antibody. In some embodiments, the antibody binds specifically to a metabolite in the serotonin, tryptophan and/or kynurenine pathways, including but not limited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilic acid. Exemplary antibodies that bind to metabolites in these pathways are disclosed, for example, in International Publication No. WO2014/188377, the entire contents of which are incorporated herein by reference. In some embodiments, the antibody is specific for a particular genus, species, or strain of a microorganism, and may therefore be used for the detection, analysis and/or quantitation of the microorganism using the detection methods described below. In some embodiments, the antibody specifically binds to a surface-specific biomolecule (e.g., a pilus subunit or a flagella protein) present in a particular genus, species or strain of microorganism, and does not cross-react with other microorganisms. In some embodiments, these antibodies may be used in the methods described herein to diagnose a subject with a particular infection or disease, or to monitor an infection (e.g., during or after treatment). In some embodiments, the antibody specifically binds to an antigen present in a particular genera, species or strain of a microorganism. Exemplary antigens, the corresponding microorganism that can be detected, and the disease caused by the microorganism (in parentheticals) include: outer membrane protein A OmpA (Acinetobacter baumannii, Acinetobacterinfections)); HIV p24 antigen, HIV Eenvelope proteins (Gp120, Gp41, Gp160) (HIV (Human immunodeficiency virus), AIDS (Acquired immunodeficiency syndrome)); galactose-inhibitable adherence protein GIAP, 29 kDa antigen Eh29, GaVGaINAc lectin, protein CRT, 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica, Amoebiasis); protective Antigen PA, edema factor EF, lethal factor LF, the S-layer homology proteins SLH (Bacillus anthracis, Anthrax); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junin virus, Argentine hemorrhagic fever); 41 kDa allergen Asp v13, allergen Asp f3, major conidial surface protein rodlet A, protease Pep1p, GPI-anchored protein Gel1p, GPI-anchored protein Crf1p (Aspergillusgenus, Aspergillosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, flagellar filament 41 kDa core protein Fla, basic membrane protein A precursor BmpA (Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vIsE (Borreliagenus,Borreliainfection); OmpA-like transmembrane domain-containing protein Omp31, immunogenic 39-kDa protein M5 P39, 25 kDa outer-membrane immunogenic protein precursor Omp25, outer membrane protein MotY Omp16, conserved outer membrane protein D15, malate dehydrogenase Mdh, component of the Type-IV secretion system (T4SS) VirJ, lipoprotein of unknown function BAB1-0187 (Brucellagenus, Brucellosis); major outer membrane protein PorA, flagellin FIaA, surface antigen CjaA, fibronectin binding protein CadF, aspartate/glutamate-binding ABC transporter protein Peb1A, protein FspA1, protein FspA2 (Campylobactergenus, Campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl proteinases SAP1-10, glycophosphatidylinositol (GPI)-linked cell wall protein, adhesin Als3p, cell surface hydrophobicity protein CSH (usuallyCandida albicansand otherCandidaspecies, Candidiasis); envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL) (Varicella zoster virus (VZV), Chickenpox); major outer membrane protein MOMP, probable outer membrane protein PMPC, outer membrane complex protein B OmcB (Chlamydia trachomatis, Chlamydia); major outer membrane protein MOMP, outer membrane protein 2 Omp2, (Chlamydophila pneumoniae, Chlamydophila pneumoniaeinfection); outer membrane protein U Porin ompU, (Vibrio cholerae, Cholera); surface layer proteins SLPs, Cell Wall Protein CwpV, flagellar protein FliC, flagellar protein FliD (Clostridium difficile, Clostridium difficileinfection); acidic ribosomal protein P2 CpP2, mucin antigens Muc1, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherence protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptides gp40, surface-associated glycopeptides gp15, oocyst wall protein AB, profilin PRF, apyrase (Cryptosporidiumgenus, Cryptosporidiosis); membrane protein pp15, capsid-proximal tegument protein pp150 (Cytomegalovirus, Cytomegalovirus infection); prion protein (vCJD prion, Variant Creutzfeldt-Jakob disease (vCJD, nvCJD)); cyst wall proteins CWP1, CWP2, CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen (Giardia intestinalis, Giardiasis); minor pilin-associated subunit pilC, major pilin subunit and variants pilE, pilS (Neisseria gonorrhoeae, Gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein K17 OmpK17 (Klebsiella granulomatis, Granuloma inguinale (Donovanosis)); fibronectin-binding protein Sfb (Streptococcus pyogenes, Group A streptococcal infection); outer membrane protein P6 (Haemophilus influenzae, Haemophilus influenzaeinfection); integral membrane proteins, aggregation-prone proteins, O-antigen, toxin-antigens Stx2B, toxin-antigen Stx1B, adhesion-antigen fragment Int28, protein EspA, protein EspB, Intimin, protein Tir, protein IntC300, protein Eae (Escherichia coliO157:H7, O111 and O104:H4, Hemolytic-uremic syndrome (HUS)); hepatitis A surface antigen HBAg (Hepatitis A Virus, Hepatitis A); hepatitis B surface antigen HBsAg (Hepatitis B Virus, Hepatitis B); envelope glycoprotein E1 gp32 gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C, (Hepatitis C Virus, Hepatitis C); type IV pilin PilE, outer membrane protein MIP, major outer membrane protein MompS (Legionella pneumophila, Legionellosis (Legionnaires' disease, Pontiac fever)); minor pilin-associated subunit pilC, major pilin subunit and variants pilE, pilS (Neisseria meningitidis, Meningococcal disease); adhesin P1, adhesion P30 (Mycoplasma pneumoniae, Mycoplasmapneumonia); F1 capsule antigen, outer membrane protease Pla, (Yersinia pestis, Plague); surface adhesin PsaA, cell wall surface anchored protein psrP (Streptococcus pneumoniae, Pneumococcal infection); flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein NmpC, outer membrane protein FadL, transport protein SadA (Salmonellagenus,Salmonellosis); collagen adhesin Cna, fibronectin-binding protein A FnbA, secretory antigen SssA (Staphylococcusgenus, Staphylococcal food poisoning); collagen adhesin Can (Staphylococcusgenus, Staphylococcal infection); fibronectin-binding protein A FbpA (Ag85A), fibronectin-binding protein D FbpD, fibronectin-binding protein C FbpCl, heat-shock protein HSP65, protein PST-S (Mycobacterium tuberculosis, Tuberculosis); and outer membrane protein FobA, outer membrane protein FobB, type IV pili glycosylation protein, outer membrane protein tolC, protein TolQ (Francisella tularensis, Tularemia). Additional exemplary microorganisms and corresponding antigens are disclosed, e.g., in U.S. Publication No. 2015/0118264, the entire contents of which are expressly incorporated herein by reference. In some embodiments, a plurality of antibodies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more antibodies) are used as analyte-binding agents in any of the methods described herein (e.g., to detect the presence of one or more analytes in a sample). In some embodiments, the plurality of antibodies bind to the same analyte (e.g., an antigen). In some embodiments, the plurality of antibodies bind to the same epitope present on the analyte (e.g., an antigen). In some embodiments, the plurality of antibodies bind to different epitopes present on the same analyte. In some embodiments, the plurality of antibodies bind to overlapping epitopes present on the same analyte. In some embodiments, the plurality of antibodies bind to non-overlapping epitopes present on the same analyte. b. Antibiotics In some embodiments, the analyte or analyte-binding agent is an antibiotic. An “antibiotic” or “antibiotic agent” refers to a substance that has the capacity to inhibit or slow down the growth of, or to destroy bacteria and/or other microorganisms. In some embodiments, the antibiotic agent is a bacteriostatic antibiotic agent. In some embodiments, the antibiotic is a bacteriolytic antibiotic agent. Exemplary antibiotic agents are set forth in the U.S. Patent Publication US 2006/0269485, which is hereby incorporated by reference herein in its entirety. In some embodiments, the antibiotic agent is selected from the classes consisting of beta-lactam antibiotics, aminoglycosides, ansa-type antibiotics, anthraquinones, antibiotic azoles, antibiotic glycopeptides, macrolides, antibiotic nucleosides, antibiotic peptides, antibiotic polyenes, antibiotic polyethers, quinolones, antibiotic steroids, sulfonamides, tetracycline, dicarboxylic acids, antibiotic metals, oxidizing agents, substances that release free radicals and/or active oxygen, cationic antimicrobial agents, quaternary ammonium compounds, biguanides, triguanides, bisbiguanides and analogs and polymers thereof and naturally occurring antibiotic compounds. In some embodiments, the antibiotic is rifaximin. Beta-lactam antibiotics include, but are not limited to, 2-(3-alanyl)clavam, 2-hydroxymethylclavam, 8-epi-thienamycin, acetyl-thienamycin, amoxicillin, amoxicillin sodium, amoxicillin trihydrate, amoxicillin-potassium clavulanate combination, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin-sulbactam, apalcillin, aspoxicillin, azidocillin, azlocillin, aztreonam, bacampicillin, biapenem, carbenicillin, carbenicillin disodium, carfecillin, carindacillin, carpetimycin, cefacetril, cefaclor, cefadroxil, cefalexin, cefaloridine, cefalotin, cefamandole, cefamandole, cefapirin, cefatrizine, cefatrizine propylene glycol, cefazedone, cefazolin, cefbuperazone, cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren, cefditoren pivoxil, cefepime, cefetamet, cefetamet pivoxil, cefixime, cefinenoxime, cefinetazole, cefminox, cefminox, cefmolexin, cefodizime, cefonicid, cefoperazone, ceforanide, cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran, cefpiramide, cefpirome, cefpodoxime, cefpodoxime proxetil, cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime, cefteram, cefteram pivoxil, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuroxime axetil, cephalosporin, cephamycin, chitinovorin, ciclacillin, clavulanic acid, clometocillin, cloxacillin, cycloserine, deoxy pluracidomycin, dicloxacillin, dihydro pluracidomycin, epicillin, epithienamycin, ertapenem, faropenem, flomoxef, flucloxacillin, hetacillin, imipenem, lenampicillin, loracarbef, mecillinam, meropenem, metampicillin, meticillin, mezlocillin, moxalactam, nafcillin, northienamycin, oxacillin, panipenem, penamecillin, penicillin, phenethicillin, piperacillin, tazobactam, pivampicillin, pivcefalexin, pivmecillinam, pivmecillinam hydrochloride, pluracidomycin, propicillin, sarmoxicillin, sulbactam, sulbenicillin, talampicillin, temocillin, terconazole, thienamycin, ticarcillin and analogs, salts and derivatives thereof. Aminoglycosides include, but are not limited to, 1,2′-N-DL-isoseryl-3′,4′-dideoxykanamycin B, 1,2′-N-DL-isoseryl-kanamycin B, 1,2′-N—[(S)-4-amino-2-hydroxybutyryl]-3′,4′-dideoxykanamycin B, 1,2′-N—[(S)-4-amino-2-hydroxybutyryl]-kanamycin B, 1-N-(2-Aminobutanesulfonyl) kanamycin A, 1-N-(2-aminoethanesulfonyl)3′,4′-dideoxyribostamycin, 1-N-(2-Aminoethanesulfonyl)3′-deoxyribostamycin, 1-N-(2-aminoethanesulfonyl)3′4′-dideoxykanamycin B, 1-N-(2-aminoethanesulfonyl)kanamycin A, 1-N-(2-aminoethanesulfonyl)kanamycin B, 1-N-(2-aminoethanesulfonyl)ribostamycin, 1-N-(2-aminopropanesulfonyl)3′-deoxykanamycin B, 1-N-(2-aminopropanesulfonyl)3′4′-dideoxykanamycin B, 1-N-(2-aminopropanesulfonyl)kanamycin A, 1-N-(2-aminopropanesulfonyl)kanamycin B, 1-N-(L-4-amino-2-hydroxy-butyryl)2′,3′-dideoxy-2′-fluorokanamycin A, 1-N-(L-4-amino-2-hydroxy-propionyl)2′,3′-dideoxy-2′-fluorokanamycin A, 1-N-DL-3′,4′-dideoxy-isoserylkanamycin B, 1-N-DL-isoserylkanamycin, 1-N-DL-isoserylkanamycin B, 1-N-[L-(−)-(alpha-hydroxy-gamma-aminobutyryl]-XK-62-2,2′,3′-dideoxy-2′-fluorokanamycin A,2-hydroxygentamycin A3,2-hydroxygentamycin B, 2-hydroxygentamycin B1, 2-hydroxygentamycin JI-20A, 2-hydroxygentamycin JI-20B, 3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy kanamycin A, 3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy kanamycin B, 3″-N-methyl-4″-C-methyl-3′,4′-dodeoxy-6′-methyl kanamycin B, 3′,4′-Dideoxy-3′-eno-ribostamycin,3′,4′-dideoxyneamine,3′,4′-dideoxyribostamycin, 3′-deoxy-6′-N-methyl-kanamycin B,3′-deoxyneamine,3′-deoxyribostamycin, 3′-oxysaccharocin,3,3′-nepotrehalosadiamine, 3-demethoxy-2″-N-formimidoylistamycin B disulfate tetrahydrate, 3-demethoxyistamycin B,3-O-demethyl-2-N-formimidoylistamycin B, 3-O-demethylistamycin B,3-trehalosamine,4″,6″-dideoxydibekacin, 4-N-glycyl-KA-6606VI, 5″-Amino-3′,4′,5″-trideoxy-butirosin A, 6″-deoxydibekacin,6′-epifortimicin A, 6-deoxy-neomycin (structure 6-deoxy-neomycin B),6-deoxy-neomycin B, 6-deoxy-neomycin C, 6-deoxy-paromomycin, acmimycin, AHB-3′,4′-dideoxyribostamycin, AHB-3′-deoxykanamycin B, AHB-3′-deoxyneamine, AHB-3′-deoxyribostamycin, AHB-4″-6″-dideoxydibekacin, AHB-6″-deoxydibekacin, AHB-dideoxyneamine, AHB-kanamycin B, AHB-methyl-3′-deoxykanamycin B, amikacin, amikacin sulfate, apramycin, arbekacin, astromicin, astromicin sulfate, bekanamycin, bluensomycin, boholmycin, butirosin, butirosin B, catenulin, coumamidine gamma1, coumamidine gamma2,D,L-1-N-(alpha-hydroxy-beta-aminopropionyl)-XK-62-2, dactimicin, de-O-methyl-4-N-glycyl-KA-6606VI, de-O-methyl-KA-66061, de-O-methyl-KA-7038I, destomycin A, destomycin B, di-N6′,O3-demethylistamycin A, dibekacin, dibekacin sulfate, dihydrostreptomycin, dihydrostreptomycin sulfate, epi-formamidoylglycidylfortimicin B, epihygromycin, formimidoyl-istamycin A, formimidoyl-istamycin B, fortimicin B, fortimicin C, fortimicin D, fortimicin KE, fortimicin KF, fortimicin KG, fortimicin KG1 (stereoisomer KG1/KG2), fortimicin KG2 (stereoisomer KG1/KG2), fortimicin KG3, framycetin, framycetin sulphate, gentamicin, gentamycin sulfate, globeomycin, hybrimycin A1, hybrimycin A2, hybrimycin B1, hybrimycin B2, hybrimycin C1, hybrimycin C2, hydroxystreptomycin, hygromycin, hygromycin B, isepamicin, isepamicin sulfate, istamycin, kanamycin, kanamycin sulphate, kasugamycin, lividomycin, marcomycin, micronomicin, micronomicin sulfate, mutamicin, myomycin, N-demethyl-7-O-demethylcelesticetin, demethylcelesticetin, methanesulfonic acid derivative of istamycin, nebramycin, nebramycin, neomycin, netilmicin, oligostatin, paromomycin, quintomycin, ribostamycin, saccharocin, seldomycin, sisomicin, sorbistin, spectinomycin, streptomycin, tobramycin, trehalosmaine, trestatin, validamycin, verdamycin, xylostasin, zygomycin and analogs, salts and derivatives thereof. Ansa-type antibiotics include, but are not limited to, 21-hydroxy-25-demethyl-25-methylth ioprotostreptovaricin, 3-methylth iorifamycin, ansamitocin, atropisostreptovaricin, awamycin, halomicin, maytansine, naphthomycin, rifabutin, rifamide, rifampicin, rifamycin, rifapentine, rifaximin (e.g., Xifaxan®), rubradirin, streptovaricin, tolypomycin and analogs, salts and derivatives thereof. Antibiotic anthraquinones include, but are not limited to, auramycin, cinerubin, ditrisarubicin, ditrisarubicin C, figaroic acid fragilomycin, minomycin, rabelomycin, rudolfomycin, sulfurmycin and analogs, salts and derivatives thereof. Antibiotic azoles include, but are not limited to, azanidazole, bifonazole, butoconazol, chlormidazole, chlormidazole hydrochloride, cloconazole, cloconazole monohydrochloride, clotrimazol, dimetridazole, econazole, econazole nitrate, enilconazole, fenticonazole, fenticonazole nitrate, fezatione, fluconazole, flutrimazole, isoconazole, isoconazole nitrate, itraconazole, ketoconazole, lanoconazole, metronidazole, metronidazole benzoate, miconazole, miconazole nitrate, neticonazole, nimorazole, niridazole, omoconazol, ornidazole, oxiconazole, oxiconazole nitrate, propenidazole, secnidazol, sertaconazole, sertaconazole nitrate, sulconazole, sulconazole nitrate, tinidazole, tioconazole, voriconazol and analogs, salts and derivatives thereof. Antibiotic glycopeptides include, but are not limited to, acanthomycin, actaplanin, avoparcin, balhimycin, bleomycin B (copper bleomycin), chloroorienticin, chloropolysporin, demethylvancomycin, enduracidin, galacardin, guanidylfungin, hachimycin, demethylvancomycin, N-nonanoyl-teicoplanin, phleomycin, platomycin, ristocetin, staphylocidin, talisomycin, teicoplanin, vancomycin, victomycin, xylocandin, zorbamycin and analogs, salts and derivatives thereof. Macrolides include, but are not limited to, acetylleucomycin, acetylkitasamycin, angolamycin, azithromycin, bafilomycin, brefeldin, carbomycin, chalcomycin, cirramycin, clarithromycin, concanamycin, deisovaleryl-niddamycin, demycinosyl-mycinamycin, Di-O-methyltiacumicidin, dirithromycin, erythromycin, erythromycin estolate, erythromycin ethyl succinate, erythromycin lactobionate, erythromycin stearate, flurithromycin, focusin, foromacidin, haterumalide, haterumalide, josamycin, josamycin ropionate, juvenimycin, juvenimycin, kitasamycin, ketotiacumicin, lankavacidin, lankavamycin, leucomycin, machecin, maridomycin, megalomicin, methylleucomycin, methymycin, midecamycin, miocamycin, mycaminosyltylactone, mycinomycin, neutramycin, niddamycin, nonactin, oleandomycin, phenylacetyideltamycin, pamamycin, picromycin, rokitamycin, rosaramicin, roxithromycin, sedecamycin, shincomycin, spiramycin, swalpamycin, tacrolimus, telithromycin, tiacumicin, tilmicosin, treponemycin, troleandomycin, tylosin, venturicidin and analogs, salts and derivatives thereof. Antibiotic nucleosides include, but are not limited to, amicetin, angustmycin, azathymidine, blasticidin S, epiroprim, flucytosine, gougerotin, mildiomycin, nikkomycin, nucleocidin, oxanosine, oxanosine, puromycin, pyrazomycin, showdomycin, sinefungin, sparsogenin, spicamycin, tunicamycin, uracil polyoxin, vengicide and analogs, salts and derivatives thereof. Antibiotic peptides include, but are not limited to, actinomycin, aculeacin, alazopeptin, amfomycin, amythiamycin, antifungal fromZalerion arboricola, antrimycin, apid, apidaecin, aspartocin, auromomycin, bacileucin, bacillomycin, bacillopeptin, bacitracin, bagacidin, beminamycin, beta-alanyl-L-tyrosine, bottromycin, capreomycin, caspofungine, cepacidine, cerexin, cilofungin, circulin, colistin, cyclodepsipeptide, cytophagin, dactinomycin, daptomycin, decapeptide, desoxymulundocandin, echanomycin, echinocandin B, echinomycin, ecomycin, enniatin, etamycin, fabatin, ferrimycin, ferrimycin, ficellomycin, fluoronocathiacin, fusaricidin, gardimycin, gatavalin, globopeptin, glyphomycin, gramicidin, herbicolin, iomycin, iturin, iyomycin, izupeptin, janiemycin, janthinocin, jolipeptin, katanosin, killertoxin, lipopeptide antibiotic, lipopeptide fromZalerionsp., lysobactin, lysozyme, macromomycin, magainin, melittin, mersacidin, mikamycin, mureidomycin, mycoplanecin, mycosubtilin, neopeptifluorin, neoviridogrisein, netropsin, nisin, nocathiacin, nocathiacin 6-deoxyglycoside, nosiheptide, octapeptin, pacidamycin, pentadecapeptide, peptifluorin, permetin, phytoactin, phytostreptin, planothiocin, plusbacin, polcillin, polymyxin antibiotic complex, polymyxin B, polymyxin B1, polymyxin F, preneocarzinostatin, quinomycin, quinupristin-dalfopristin, safracin, salmycin, salmycin, salmycin, sandramycin, saramycetin, siomycin, sperabillin, sporamycin, aStreptomycescompound, subtilin, teicoplanin aglycone, telomycin, thermothiocin, thiopeptin, thiostrepton, tridecaptin, tsushimycin, tuberactinomycin, tuberactinomycin, tyrothricin, valinomycin, viomycin, virginiamycin, zervacin and analogs, salts and derivatives thereof. In some embodiments, the antibiotic peptide is a naturally-occurring peptide that possesses an antibacterial and/or an antifungal activity. Such peptide can be obtained from an herbal or a vertebrate source. Polyenes include, but are not limited to, amphotericin, amphotericin, aureofungin, ayfactin, azalomycin, blasticidin, candicidin, candicidin methyl ester, candimycin, candimycin methyl ester, chinopricin, filipin, flavofungin, fradicin, hamycin, hydropricin, levorin, lucensomycin, lucknomycin, mediocidin, mediocidin methyl ester, mepartricin, methylamphotericin, natamycin, niphimycin, nystatin, nystatin methyl ester, oxypricin, partricin, pentamycin, perimycin, pimaricin, primycin, proticin, rimocidin, sistomycosin, sorangicin, trichomycin and analogs, salts and derivatives thereof. Polyethers include, but are not limited to, 20-deoxy-epi-narasin, 20-deoxysalinomycin, carriomycin, dianemycin, dihydrolonomycin, etheromycin, ionomycin, iso-lasalocid, lasalocid, lenoremycin, lonomycin, lysocellin, monensin, narasin, oxolonomycin, a polycyclic ether antibiotic, salinomycin and analogs, salts and derivatives thereof. Quinolones include, but are not limited to, an alkyl-methylendioxy-4(1H)-oxocinnoline-3-carboxylic acid, alatrofloxacin, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, danofloxacin, dermofongin A, enoxacin, enrofloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, lomefloxacin, hydrochloride, miloxacin, moxifloxacin, nadifloxacin, nalidixic acid, nifuroquine, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pazufloxacine, pefloxacin, pefloxacin mesylate, pipemidic acid, piromidic acid, premafloxacin, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin and analogs, salts and derivatives thereof. Antibiotic steroids include, but are not limited to, aminosterol, ascosteroside, cladosporide A, dihydrofusidic acid, dehydro-dihydrofusidic acid, dehydrofusidic acid, fusidic acid, squalamine and analogs, salts and derivatives thereof. Sulfonamides include, but are not limited to, chloramine, dapsone, mafenide, phthalylsulfathiazole, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfadiazine, sulfadiazine silver, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaguanidine, sulfalene, sulfamazone, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxol, sulfanilamide, sulfaperine, sulfaphenazol, sulfapyridine, sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea, sulfatolamide, sulfatriazin, sulfisomidine, sulfisoxazole, sulfisoxazole acetyl, sulfacarbamide and analogs, salts and derivatives thereof. Tetracyclines include, but are not limited to, dihydrosteffimycin, demethyltetracycline, aclacinomycin, akrobomycin, baumycin, bromotetracycline, cetocyclin, chlortetracycline, clomocycline, daunorubicin, demeclocycline, doxorubicin, doxorubicin hydrochloride, doxycycline, lymecyclin, marcellomycin, meclocycline, meclocycline sulfosalicylate, methacycline, minocycline, minocycline hydrochloride, musettamycin, oxytetracycline, rhodirubin, rolitetracycline, rubomycin, serirubicin, steffimycin, tetracycline and analogs, salts and derivatives thereof. Dicarboxylic acids, having between about 6 and about 14 carbon atoms in their carbon atom skeleton are particularly useful in the treatment of disorders of the skin and mucosal membranes that involve microbial. Suitable dicarboxylic acid moieties include, but are not limited to, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, 1,11-undecanedioic acid, 1,12-dodecanedioic acid, 1,13-tridecanedioic acid and 1,14-tetradecanedioic acid. Thus, in one or more embodiments of the present disclosure, dicarboxylic acids, having between about 6 and about 14 carbon atoms in their carbon atom skeleton, as well as their salts and derivatives (e.g., esters, amides, mercapto-derivatives, anhydrides), are useful immunomodulators in the treatment of disorders of the skin and mucosal membranes that involve inflammation. Azelaic acid and its salts and derivatives are preferred. It has antibacterial effects on both aerobic and anaerobic organisms, particularlyPropionibacterium acnesandStaphylococcus epidermidis, normalizes keratinization, and has a cytotoxic effect on malignant or hyperactive melanocytes. In a preferred embodiment, the dicarboxylic acid is azelaic acid in a concentration greater than 10%. Preferably, the concentration of azelaic acid is between about 10% and about 25%. In such concentrates, azelaic acid is suitable for the treatment of a variety of skin disorders, such as acne, rosacea and hyperpigmentation. In some embodiments, the antibiotic agent is an antibiotic metal. A number of metals ions have been shown to possess antibiotic activity, including silver, copper, zinc, mercury, tin, lead, bismutin, cadmium, chromium and ions thereof. It has been theorized that these antibiotic metal ions exert their effects by disrupting respiration and electron transport systems upon absorption into bacterial or fungal cells. Anti-microbial metal ions of silver, copper, zinc, and gold, in particular, are considered safe for in vivo use. Anti-microbial silver and silver ions are particularly useful due to the fact that they are not substantially absorbed into the body. Thus, in one or more embodiment, the antibiotic metal consists of an elemental metal, selected from the group consisting of silver, copper, zinc, mercury, tin, lead, bismutin, cadmium, chromium and gold, which is suspended in the composition as particles, microparticles, nanoparticles or colloidal particles. The antibiotic metal can further be intercalated in a chelating substrate. In further embodiments, the antibiotic metal is ionic. The ionic antibiotic metal can be presented as an inorganic or organic salt (coupled with a counterion), an organometallic complex or an intercalate. Non-binding examples of counter inorganic and organic ions are sulfadiazine, acetate, benzoate, carbonate, iodate, iodide, lactate, laurate, nitrate, oxide, and palmitate, a negatively charged protein. In preferred embodiments, the antibiotic metal salt is a silver salt, such as silver acetate, silver benzoate, silver carbonate, silver iodate, silver iodide, silver lactate, silver laurate, silver nitrate, silver oxide, silver palmitate, silver protein, and silver sulfadiazine. In one or more embodiments, the antibiotic metal or metal ion is embedded into a substrate, such as a polymer, or a mineral (such as zeolite, clay and silica). In one or more embodiments, the antibiotic agent includes strong oxidants and free radical liberating compounds, such as oxygen, hydrogen peroxide, benzoyl peroxide, elemental halogen species, as well as oxygenated halogen species, bleaching agents (e.g., sodium, calcium or magnesium hypochloride and the like), perchlorite species, iodine, iodate, and benzoyl peroxide. Organic oxidizing agents, such as quinones, are also included. Such agents possess a potent broad-spectrum activity. In one or more embodiments, the antibiotic agent is a cationic antimicrobial agent. The outermost surface of bacterial cells universally carries a net negative charge, making them sensitive to cationic substances. Examples of cationic antibiotic agents include: quaternary ammonium compounds (QAC's)—QAC's are surfactants, generally containing one quaternary nitrogen associated with at least one major hydrophobic moiety; alkyltrimethyl ammonium bromides are mixtures of where the alkyl group is between 8 and 18 carbons long, such as cetrimide (tetradecyltrimethylammonium bromide); benzalkonium chloride, which is a mixture of n-alkyldimethylbenzyl ammonium chloride where the alkyl groups (the hydrophobic moiety) can be of variable length; dialkylmethyl ammonium halides; dialkylbenzyl ammonium halides; and QAC dimmers, which bear bi-polar positive charges in conjunction with interstitial hydrophobic regions. In one or more embodiments, the cationic antimicrobial agent is a polymer. Cationic antimicrobial polymers include, for example, guanide polymers, biguanide polymers, or polymers having side chains containing biguanide moieties or other cationic functional groups, such as benzalkonium groups or quarternium groups (e.g., quaternary amine groups). It is understood that the term “polymer” as used herein includes any organic material including three or more repeating units, and includes oligomers, polymers, copolymers, block copolymers, terpolymers, etc. The polymer backbone may be, for example a polyethylene, polypropylene or polysilane polymer. In one or more embodiments, the cationic antimicrobial polymer is a polymeric biguanide compound. When applied to a substrate, such a polymer is known to form a barrier film that can engage and disrupt a microorganism. An exemplary polymeric biguanide compound is polyhexamethylene biguanide (PHMB) salts. Other exemplary biguanide polymers include, but are not limited to poly(hexamethylenebiguanide), poly(hexamethylenebiguanide) hydrochloride, poly(hexamethylenebiguanide) gluconate, poly(hexamethylenebiguanide) stearate, or a derivative thereof. In one or more embodiments, the antimicrobial material is substantially water-insoluble. In some embodiments, the antibiotic agent is selected from the group of biguanides, triguanides, bisbiguanides and analogs thereof. Guanides, biguanides, biguanidines and triguanides are unsaturated nitrogen containing molecules that readily obtain one or more positive charges, which make them effective antimicrobial agents. The basic structures a guanide, a biguanide, a biguanidine and a triguanide are provided below. In some embodiments, the guanide, biguanide, biguanidine or triguanide, provide bi-polar configurations of cationic and hydrophobic domains within a single molecule. Examples of guanides, biguanides, biguanidines and triguanides that are currently been used as antibacterial agents include chlorhexidine and chlorohexidine salts, analogs and derivatives, such as chlorhexidine acetate, chlorhexidine gluconate and chlorhexidine hydrochloride, picloxydine, alexidine and polihexanide. Other examples of guanides, biguanides, biguanidines and triguanides that can conceivably be used according to the present disclosure are chlorproguanil hydrochloride, proguanil hydrochloride (currently used as antimalarial agents), mefformin hydrochloride, phenformin and buformin hydrochloride (currently used as antidiabetic agents). Yet, in one or more embodiments, the antibiotic is a non-classified antibiotic agent, including, without limitation, aabomycin, acetomycin, acetoxycycloheximide, acetylnanaomycin, anActinoplanessp. compound, actinopyrone, aflastatin, albacarcin, albacarcin, albofungin, albofungin, alisamycin, alpha-R,S-methoxycarbonylbenzylmonate, altromycin, amicetin, amycin, amycin demanoyl compound, amycine, amycomycin, anandimycin, anisomycin, anthramycin, anti-syphilis immune substance, anti-tuberculosis immune substance, an antibiotic fromEscherichia coli, an antibiotic fromStreptomycesrefuineus, anticapsin, antimycin, aplasmomycin, aranorosin, aranorosinol, arugomycin, ascofuranone, ascomycin, ascosin,Aspergillus flavusantibiotic, asukamycin, aurantinin, an Aureolic acid antibiotic substance, aurodox, avilamycin, azidamfenicol, azidimycin, bacillaene, aBacillus larvaeantibiotic, bactobolin, benanomycin, benzanthrin, benzylmonate, bicozamycin, bravomicin, brodimoprim, butalactin, calcimycin, calvatic acid, candiplanecin, carumonam, carzinophilin, celesticetin, cepacin, cerulenin, cervinomycin, chartreusin, chloramphenicol, chloramphenicol palmitate, chloramphenicol succinate sodium, chlorflavonin, chlorobiocin, chlorocarcin, chromomycin, ciclopirox, ciclopirox olamine, citreamicin, cladosporin, clazamycin, clecarmycin, clindamycin, coliformin, collinomycin, copiamycin, corallopyronin, corynecandin, coumermycin, culpin, cuprimyxin, cyclamidomycin, cycloheximide, dactylomycin, danomycin, danubomycin, delaminomycin, demethoxyrapamycin, demethylscytophycin, dermadin, desdamethine, dexylosyl-benanomycin, pseudoaglycone, dihydromocimycin, dihydronancimycin, diumycin, dnacin, dorrigocin, dynemycin, dynemycin triacetate, ecteinascidin, efrotomycin, endomycin, ensanchomycin, equisetin, ericamycin, esperamicin, ethylmonate, everninomicin, feldamycin, flambamycin, flavensomycin, florfenicol, fluvomycin, fosfomycin, fosfonochlorin, fredericamycin, frenolicin, fumagillin, fumifungin, funginon, fusacandin, fusafungin, gelbecidine, glidobactin, grahamimycin, granaticin, griseofulvin, griseoviridin, grisonomycin, hayumicin, hayumicin, hazymicin, hedamycin, heneicomycin, heptelicid acid, holomycin, humidin, isohematinic acid, karnatakin, kazusamycin, kristenin, L-dihydrophenylalanine, a L-isoleucyl-L-2-amino-4-(4′-amino-2′,5′-cyclohexadienyl) derivative, lanomycin, leinamycin, leptomycin, libanomycin, lincomycin, lomofungin, lysolipin, magnesidin, manumycin, melanomycin, methoxycarbonylmethylmonate, methoxycarbonylethylmonate, methoxycarbonylphenylmonate, methyl pseudomonate, methylmonate, microcin, mitomalcin, mocimycin, moenomycin, monoacetyl cladosporin, monomethyl cladosporin, mupirocin, mupirocin calcium, mycobacidin, myriocin, myxopyronin, pseudoaglycone, nanaomycin, nancimycin, nargenicin, neocarcinostatin, neoenactin, neothramycin, nifurtoinol, nocardicin, nogalamycin, novobiocin, octylmonate, olivomycin, orthosomycin, oudemansin, oxirapentyn, oxoglaucine methiodide, pactacin, pactamycin, papulacandin, paulomycin, phaeoramularia fungicide, phenelfamycin, phenyl, cerulenin, phenylmonate, pholipomycin, pirlimycin, pleuromutilin, a polylactone derivative, polynitroxin, polyoxin, porfiromycin, pradimicin, prenomycin, prop-2-enylmonate, protomycin,Pseudomonasantibiotic, pseudomonic acid, purpuromycin, pyrinodemin, pyrrolnitrin, pyrrolomycin, amino, chloro pentenedioic acid, rapamycin, rebeccamycin, resistomycin, reuterin, reveromycin, rhizocticin, roridin, rubiflavin, naphthyridinomycin, saframycin, saphenamycin, sarkomycin, sarkomycin, sclopularin, selenomycin, siccanin, spartanamicin, spectinomycin, spongistatin, stravidin, streptolydigin,Streptomyces arenaeantibiotic complex, streptonigrin, streptothricins, streptovitacin, streptozotocine, a strobilurin derivative, stubomycin, sulfamethoxazol-trimethoprim, sakamycin, tejeramycin, terpentecin, tetrocarcin, thermorubin, thermozymocidin, thiamphenicol, thioaurin, thiolutin, thiomarinol, thiomarinol, tirandamycin, tolytoxin, trichodermin, trienomycin, trimethoprim, trioxacarcin, tyrissamycin, umbrinomycin, unphenelfamycin, urauchimycin, usnic acid, uredolysin, variotin, vermisporin, verrucarin and analogs, salts and derivatives thereof. In one or more embodiments, the antibiotic agent is a naturally occurring antibiotic compound. As used herein, the term “naturally-occurring antibiotic agent” includes all antibiotics that are obtained, derived or extracted from plant or vertebrate sources. Non-limiting examples of families of naturally-occurring antibiotic agents include phenol, resorcinol, antibiotic aminoglycosides, anamycin, quinines, anthraquinones, antibiotic glycopeptides, azoles, macrolides, avilamycin, agropyrene, cnicin, aucubin antibioticsaponin fractions, berberine (isoquinoline alkaloid), arctiopicrin (sesquiterpene lactone), lupulone, humulone (bitter acids), allicin, hyperforin, echinacoside, coniosetin, tetramic acid, imanine and novoimanine. Ciclopirox and ciclopiroxolamine possess fungicidal, fungistatic and sporicidal activity. They are active against a broad spectrum of dermatophytes, yeasts, moulds and other fungi, such as Trichophytons species,Microsporumspecies,Epidermophytonspecies and yeasts (Candida albicans, Candida glabrata, othercandidaspecies andCryptococcus neoformans). SomeAspergillusspecies are sensitive to ciclopirox as are somePenicillium. Likewise, ciclopirox is effective against many Gram-positive and Gram-negative bacteria (e.g.,Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, StaphylococcusandStreptococcusspecies), as well asMycoplasmaspecies,Trichomonas vaginalisandActinomyces. Plant oils and extracts which contain antibiotic agents are also useful. Non-limiting examples of plants that contain agents include thyme,Perilla, lavender, tea tree,Terfezia clayeryi, Micromonospora, Putterlickia verrucosa, Putterlickla pyracantha, Putterlickla retrospinosa, Maytenus ilicifolia, Maytenus evonymoides, Maytenus aquifolia, Faenia interjecta, Cordyceps sinensis, couchgrass, holy thistle, plantain, burdock, hops,echinacea, buchu, chaparral, myrrh, red clover and yellow dock, garlic, and St. John's wort. Mixtures of the antibiotic agents as described herein may also be employed. Combination Detection: Any combination of the analytes disclosed herein can be detected using any of the methods described herein. In particular, any combination disclosed herein can be detected using any of the methods described herein. A “photosensitizer” as used herein refers to a sensitizer for generation of singlet oxygen usually by excitation with light. Exemplary photosensitizers suitable for use include those described in U.S. Pat. Nos. 6,251,581, 5,516,636, 8,907,081, 6,545,012, 6,331,530, 8,247,180, 5,763,602, 5,705,622, 5,516,636, 7,217,531, and U.S. Patent Publication No. 2007/0059316, all of which are herein expressly incorporated by reference in their entireties. The photosensitizer can be photoactivatable (e.g., dyes and aromatic compounds) or chemiactivated (e.g., enzymes and metal salts). When excited by light the photosensitizer is usually a compound comprised of covalently bonded atoms, usually with multiple conjugated double or triple bonds. The compound should absorb light in the wavelength range of 200-1100 nm, usually 300-1000 nm, e.g., 450-950 nm, with an extinction coefficient at its absorbance maximum greater than 500 M−1cm−1, e.g., at least 5000 M−1cm−1, or at least 50,000 M−1cm−1at the excitation wavelength. The lifetime of an excited state produced following absorption of light in the absence of oxygen will usually be at least 100 nsec, e.g., at least 1 μsec. In general, the lifetime must be sufficiently long to permit energy transfer to oxygen, which will normally be present at concentrations in the range of 10−5to 10313M depending on the medium. The sensitizer excited state will usually have a different spin quantum number (S) than its ground state and will usually be a triplet (S=1) when, as is usually the case, the ground state is a singlet (S═O). In some embodiments, the sensitizer will have a high intersystem crossing yield. That is, photoexcitation of a sensitizer will produce the long lived state (usually triplet) with an efficiency of at least 10%, at least 40%, e.g., greater than 80%. The photosensitizer will usually be at most weakly fluorescent under the assay conditions (quantum yield usually less that 0.5, or less that 0.1). Photosensitizers that are to be excited by light will be relatively photostable and will not react efficiently with singlet oxygen. Several structural features are present in most useful sensitizers. Most sensitizers have at least one and frequently three or more conjugated double or triple bonds held in a rigid, frequently aromatic structure. They will frequently contain at least one group that accelerates intersystem crossing such as a carbonyl or imine group or a heavy atom selected from rows 3-6 of the periodic table, especially iodine or bromine, or they may have extended aromatic structures. Typical sensitizers include acetone, benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such as hematoporphyrin, phthalocyanines, chlorophylls, rose bengal, buckminsterfullerene, etc., and derivatives of these compounds having substituents of 1 to 50 atoms for rendering such compounds more lipophilic or more hydrophilic and/or as attaching groups for attachment. Examples of other photosensitizers that may be utilized are those that have the above properties and are enumerated in N. J. Turro, “Molecular Photochemistry,” page 132, W. A. Benjamin Inc., N.Y. 1965. In some embodiments, the photosensitizers are relatively non-polar to assure dissolution into a lipophilic member when the photosensitizer is incorporated in an oil droplet, liposome, latex particle, etc. In some embodiments, the photosensitizers suitable for use herein include other substances and compositions that can produce singlet oxygen with or without activation by an external light source. Thus, for example, molybdate (MoO4=) salts and chloroperoxidase and myeloperoxidase plus bromide or chloride ion (Kanofsky,J. Biol. Chem. (1983) 259 5596) have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Either of these compositions can, for example, be included in particles and used in the assay method wherein hydrogen peroxide is included as an ancillary reagebly, chloroperoxidase is bound to a surface and molybdate is incorporated in the aqueous phase of a liposome. Also included within the scope of the invention as photosensitizers are compounds that are not true sensitizers but which on excitation by heat, light, or chemical activation will release a molecule of singlet oxygen. The best known members of this class of compounds includes the endoperoxides such as 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen. A “chemiluminescent compound” as used herein refers to a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate that can decompose with the simultaneous or subsequent emission of light within the wavelength range of 250 to 1200 nm. Exemplary chemiluminescent compounds suitable for use include those described in U.S. Pat. Nos. 6,251,581 and 7,709,273, and Patent Cooperation Treaty (PCT) International Application Publication No. WO1999/042838. Exemplary chemiluminescent compound includes the following: EmissionChemiluminescerHalf-LifeMaxThioxene + Diphenyl anthracence:0.6seconds430 nmThioxene + Umbelliferone derivative0.6seconds500 nmThioxene + Europium chelate0.6seconds615 nmThioxene + Samarium Chelate0.6seconds648 nmThioxene + terbium Chelate0.6seconds540 nmN-Phenyl Oxazine + Umbelliferone30seconds500 nmderivativeN-Phenyl Oxazine + Europium chelate30seconds613 nmN-phenyl Oxazine + Samarium Chelate30seconds648 nmN-phenyl Oxazine + terbium Chelate30seconds540 nmDioxene + Umbelliferone derivative300seconds500 nmDioxene + Europium chelate300seconds613 nmDioxene + Samarium Chelate300seconds648 nmN-phenyl Oxazine + terbium Chelate300seconds540 nm All of the above mentioned applications are hereby expressly incorporated by reference herein in their entireties. Emission will usually occur without the presence of an energy acceptor or catalyst to cause decomposition and light emission. In some embodiments, the intermediate decomposes spontaneously without heating or addition of ancillary reagents following its formation. However, addition of a reagent after formation of the intermediate or the use of elevated temperature to accelerate decomposition will be required for some chemiluminescent compounds. The chemiluminescent compounds are usually electron rich compounds that react with singlet oxygen, frequently with formation of dioxetanes or dioxetanones. Exemplary of such compounds are enol ethers, enamines, 9-alkylidenexanthans, 9-alkylidene-N-alkylacridans, aryl vinyl ethers, dioxenes, arylimidazoles and lucigenin. Other chemiluminescent compounds give intermediates upon reaction with singlet oxygen, which subsequently react with another reagent with light emission. Exemplary compounds are hydrazides such as luminol and oxalate esters. The chemiluminescent compounds of interest will generally emit at wavelengths above 300 nanometers and usually above 400 nm. Compounds that alone or together with a fluorescent molecule emit light at wavelengths beyond the region where serum components absorb light will be of particular use. The fluorescence of serum drops off rapidly above 500 nm and becomes relatively unimportant above 550 nm. Therefore, when the analyte is in serum, chemiluminescent compounds that emit light above 550 nm, e.g., above 600 nm may be suitable for use. In order to avoid autosensitization of the chemiluminescent compound, in some embodiments, the chemiluminescent compounds do not absorb light used to excite the photosensitizer. In some embodiments, the sensitizer is excited with light wavelengths longer than 500 nm, it will therefore be desirable that light absorption by the chemiluminescent compound be very low above 500 nm. Where long wave length emission from the chemiluminescent compound is desired, a long wavelength emitter such as a pyrene, bound to the chemiluminescent compound can be used. Alternatively, a fluorescent molecule can be included in the medium containing the chemiluminescent compound. In some embodiments, fluorescent molecules will be excited by the activated chemiluminescent compound and emit at a wavelength longer than the emission wavelength of the chemiluminescent compound, usually greater that 550 nm. It is usually also desirable that the fluorescent molecules do not absorb at the wavelengths of light used to activate the photosensitizer. Examples of useful dyes include rhodamine, ethidium, dansyl, Eu(fod)3, Eu(TTA)3, Ru(bpy)3(wherein bpy=2,2′-dipyridyl, etc. In general these dyes act as acceptors in energy transfer processes and in some embodiments, have high fluorescent quantum yields and do not react rapidly with singlet oxygen. They can be incorporated into particles simultaneously with the incorporation of the chemiluminescent compound into the particles. In some embodiments, the disclosure provides diffractive optics detection technology that can be used with, for example, ingestible device technology. In certain embodiments, an ingestible device includes the diffractive optics technology (e.g., diffractive optics detection system). In certain embodiments, the disclosure provides diffractive optics technology (e.g., diffractive optics detection systems) that are used outside the body of subject. As an example, an ingestible device can be used to obtain one more samples in the body (e.g., in the gastrointestinal tract) of a subject, and the diffractive optics technology can be used to analyze the sample(s). Such analysis can be performed in vivo (e.g., when the ingestible device contains the diffractive optics). Diffraction is a phenomenon that occurs due to the wave nature of light. When light hits an edge or passes through a small aperture, it is scattered in different directions. But light waves can interfere to add (constructively) and subtract (destructively) from each other, so that if light hits a non-random pattern of obstacles, the subsequent constructive and destructive interference will result in a clear and distinct diffraction pattern. A specific example is that of a diffraction grating, which is of uniformly spaced lines, typically prepared by ruling straight, parallel grooves on a surface. Light incident on such a surface produces a pattern of evenly spaced spots of high light intensity. This is called Bragg scattering, and the distance between spots (or ‘Bragg scattering peaks’) is a unique function of the diffraction pattern and the wavelength of the light source. Diffraction gratings, like focusing optics, can be operated in both transmission and reflection modes. In general, the light used in the diffractive optics can be of any appropriate wavelength. Exemplary wavelengths include visible light, infrared red (IR) and ultraviolet (UV). Optionally, the light can be monochromatic or polychromatic. The light can be coherent or incoherent. The light can be collimated or non-collimated. In some embodiments, the light is coherent and collimated. Generally, any appropriate light source may be used, such as, for example, a laser (e.g., a laser diode) or a light emitting diode. In some embodiments, the light source is a laser diode operating at 670 nm wavelength, e.g., at 3 mWatts power. Optionally, an operating wavelength of a laser diode can be 780 nm, e.g., when larger grating periods are used. In certain embodiments, the light source is a laser, such as, for example, a He—Ne laser, a Nd:YVO4 laser, or an argon-ion laser. In some embodiments, the light source is a low power, continuous waver laser. The diffracted light can be detected using any appropriate light detector(s). Examples of light detectors include photodetectors, such as, for example, position sensitive photodiodes, photomultiplier tubes (PMTs), photodiodes (PDs), avalanche photodiodes (APDs), charged-coupled device (CCD) arrays, and CMOS detectors. In some embodiments, the diffracted light is detected via one or more individual photodiodes. In general, the diffraction grating is made of a material that is transparent in the wavelength of the radiation used to illuminate the sensor. Any appropriate material may be used for the diffraction grating substrate, such as glass or a polymer. Exemplary polymers include polystyrene polymers (PSEs), cyclo-olefin polymers (COPs), polycarbonate polymers, polymethyl methacrylates, and methyl methacrylate styrene copolymers. Exemplary COPs include Zeonex (e.g., Zeonex E48R, Zeonex F52R). The light may be incident on the diffraction grating any appropriate angle. In some embodiments, the light is incident on the diffraction grating with an angle of incidence of from 30° to 80° (e.g., from 40° to 80°, from 50° to 70°, from 55° to 65°, 60°). Optionally, the system is configured so that that diffractive grating and light source can move relative to each other In general, the light detector can be positioned with respect to the diffractive grating so that the diffraction grating can be illuminated at a desired angle of incidence and/or so that diffracted light can be detected at a desired angle and/or so that diffracted light of a desired order can be detected. The period P of the diffraction grating can be selected as desired. In some embodiments, the period P is from 0.5 microns to 50 microns (e.g., from one micron to 15 microns, from one micron to five microns). In some embodiments, the grating is a repeating patter of 1.5 micron and 4.5 micron lines with a period of 15 microns. The height h of the diffraction grating can be selected as desired. In certain embodiments, the height h is from one nanometer to about 1000 nanometers (e.g., from about five nanometers to about 250 nanometers, from five nanometers to 100 nanometers). In general, the diffractive optics can be prepared using any appropriate method, such as, for example, surface ablation, photolithograph (e.g., UV photolithography), laser etching, electron beam etching, nano-imprint molding, or microcontact printing. Optionally, the diffractive optics system can include one or more additional optical elements, such as, for example, one or more mirrors, filters and/or lenses. Such optical elements can, for example, be arranged between the light source and the diffractive grating and/or between the diffractive grating and the detector. In some of the embodiments of the devices described herein, a primary binding partner specifically binds to a secondary binding partner through non-covalent interactions (e.g., electrostatic, van der Waals, hydrophobic effect). In some embodiments, a primary binding partner specifically binds to a secondary binding partner via a covalent bond (e.g., a polar covalent bond or a non-polar covalent bond). In some embodiments of any of the devices described herein, the primary and the secondary binding partner can be interchanged. For example, the primary binding partner can be biotin, or a derivative thereof, and the secondary binding partner is avidin, or a derivative thereof. In other examples, the primary binding partner can be avidin, or a derivative thereof, and the secondary binding partner is biotin. In some embodiments, the binding of the primary and the secondary binding partner is essentially irreversible. In some embodiments, the binding of the primary and the secondary binding partner is reversible. In some embodiments, the primary binding partner is CaptAvidin™ biotin-binding protein and the secondary binding partner is biotin, or vice versa. In some embodiments, the primary binding partner is DSB-X™ biotin and the secondary binding partner is avidin, or vice versa. In some embodiments, the primary binding partner is desthiobiotin and the secondary binding partner is avidin, or vice versa (Hirsch et al.,Anal Biochem.308(2):343-357, 2002). In some embodiments, the primary binding partner is glutathione (GSH) or a derivative thereof, and the secondary binding partner is glutathione-S-transferase (GST). In some embodiments, the primary binding partner can bind to a target analyte that is a nucleic acid (e.g., a DNA molecule, a RNA molecule). In some embodiments, the primary binding partner comprises a portion of a nucleic acid that is complementary to the nucleic acid sequence of the target analyte. In some embodiments of any of the devices described herein, the device can include a label that binds to the target analyte and does not prevent binding of the target analyte to the primary binding partner. In some embodiments, the label can amplify the diffraction signal of the target analyte. In some embodiments, the label is from about 1 nm to 200 nm (e.g., about 50 nm to about 200 nm). In some embodiments, the label (e.g., any of the labels described herein) includes one or more antibodies (e.g., any of the antibodies and/or antibody fragments described herein). In some embodiments, the label is a nanoparticle (e.g., a gold nanoparticle) that includes the primary binding partner that has a nucleic acid sequence that is complementary to the target analyte, and is covalently linked to the nanoparticle. One or more additional steps can be performed in any of the methods described herein. In some embodiments, the one or more additional steps are performed: prior to the binding of the primary binding partner to the secondary binding partner, after the binding of the primary binding partner to the secondary binding partner, prior to the binding of the primary binding partner to the target analyte, or after the binding of the primary binding partner to the target analyte. In some embodiments of any of the methods described herein, the determining step (during which the primary binding partner binds to the target analyte is detected) can occur in at least 15 seconds. In some embodiments, the binding of the primary binding partner to the target analyte can occur during a period of time of, for example, five at least seconds. In some embodiments, the one or more additional steps can include: a blocking of the sensors step, at least one wash step, a capturing step, and/or a filtering step. In some embodiments, the blocking step can include blocking a sensor within the ingestible device with a solution comprising at least 1% bovine serum albumin (BSA) in a buffered solution (e.g., phosphate buffered saline (PBS), Tris buffered saline (TBS)). In some embodiments, the at least one wash step can include washing with a buffered solution (e.g., phosphate buffered saline (PBS), Tris buffered saline (TBS)). In general, blocking is performed during capsule manufacture, rather than in vivo. In some embodiments, the capturing step includes enriching the target analyte. In some embodiments, the capturing step includes physically separating the target analyte from the remaining sample using a filter, a pore, or a magnetic bead. In some embodiments, the target analyte is captured by size exclusion. In some embodiments, the disclosure provides methods of obtaining, culturing, and/or detecting target cells and/or target analytes in vivo within the gastrointestinal (GI) tract or reproductive tract of a subject. Associated devices are also disclosed. The methods and devices described provide a number of advantages for obtaining and/or analyzing fluid samples from a subject. In some embodiments, diluting the fluid sample increases the dynamic range of analyte detection and/or reduces background signals or interference within the sample. For example, interference may be caused by the presence of non-target analytes or non-specific binding of a dye or label within the sample. In some embodiments, culturing the sample increases the concentration of target cells and/or target analytes produced by the target cells thereby facilitating their detection and/or characterization. In certain embodiments, the methods and devices a described herein may be used to obtain information regarding bacteria populations in the GI tract of a subject. This has a number of advantages and is less invasive than surgical procedures such as intubation or endoscopy to obtain fluid samples from the GI tract. The use of an ingestible device as described herein also allows for fluid samples to be obtained and data to be generated on bacterial populations from specific regions of the GI tract. In some embodiments, the methods and devices described herein may be used to generate data such as by analyzing the fluid sample, dilutions thereof or cultured samples for one or more target cells and/or target analytes. The data may include, but is not limited to, the types of bacteria present in the fluid sample or the concentration of bacteria in specific regions of the GI tract. Such data may be used to determine whether a subject has an infection, such as Small Intestinal Bacterial Overgrowth (SIBO), or to characterize bacterial populations within the GI tract for diagnostic or other purposes. Thus, in some embodiments, analytes disclosed herein are indicative of disorders of the gastrointestinal tract associated with anomalous bacterial populations. For example, in one aspect, the data may include, but is not limited to, the concentration of bacteria in a specific region of the GI tract that is one or more of the duodenum, jejunum, ileum, ascending colon, transverse colon or descending colon. In one aspect, the specific region of the GI tract is the duodenum. In one aspect, the specific region of the GI tract is the jejunum. In one aspect, the specific region of the GI tract is the ileum. In one aspect, the specific region of the GI tract is the ascending colon. In one aspect, the specific region of the GI tract is the transverse colon. In one aspect, the specific region of the GI tract is the descending colon. In a related embodiment, the data may be generated every one or more days to monitor disease flare-ups, or response to the therapeutic agents disclosed herein. Data may be generated after the device has exited the subject, or the data may be generated in vivo and stored on the device and recovered ex vivo. Alternatively, the data can be transmitted wirelessly from the device while the device is passing through the GI tract of the subject or in place within the reproductive tract of the subject. In some embodiments, a method comprises: providing a device comprising one or more dilution chambers and dilution fluid; transferring all or part of a fluid sample obtained from the GI tract or reproductive tract of the subject into the one or more dilution chambers in vivo; and combining the fluid sample and the dilution fluid to produce one or more diluted samples in the one or more dilution chambers. In certain embodiments, a method comprises: providing an ingestible device comprising one or more dilution chambers; transferring all or part of a fluid sample obtained from the GI tract into the one or more dilution chambers comprising sterile media; culturing the sample in vivo within the one or more dilution chambers to produce one or more cultured samples; and detecting bacteria in the one or more cultured samples. In some embodiments, a method comprises: providing a device comprising one or more dilution chambers; transferring all or part of a fluid sample obtained from the GI tract or reproductive tract into the one or more dilution chambers; combining all or part of the fluid sample with a dilution fluid in the one or more dilution chambers; and detecting the target analyte in the one or more diluted samples. In certain embodiments, a device comprises: one or more dilution chambers for diluting a fluid sample obtained from the GI tract or reproductive tract; and dilution fluid for diluting the sample within the one or more dilution chambers. In some embodiments, the device comprises: one or more dilution chambers for culturing a fluid sample obtained from the GI tract; sterile media for culturing the sample within the one or more dilution chambers; and a detection system for detecting bacteria. In certain embodiments, a device comprises: one or more dilution chambers for culturing a fluid sample obtained from the GI tract; sterile media for culturing the sample within the one or more dilution chambers; and a detection system for detecting bacteria. Also provided is the use of a device as described herein for diluting one or more samples obtained from the GI tract or reproductive tract of a subject. In one embodiment, there is provided the use of an ingestible device as described herein for detecting target cells and/or target analytes in vivo within the gastrointestinal (GI) tract of a subject. Further provided is a system comprising a device as described herein and a base station. In one embodiment, the device transmits data to the base station, such as data indicative of the concentration and/or types of bacteria in the GI tract of the subject. In one embodiment, the device receives operating parameters from the base station. Some embodiments described herein provide an ingestible device for obtaining one or more samples from the GI tract or reproductive tract of a subject and diluting and/or culturing all or part of the one or more samples. The ingestible device includes a cylindrical rotatable element having a port on the wall of the cylindrical rotatable element. The ingestible device further includes a shell element wrapping around the cylindrical rotatable element to form a first dilution chamber between the cylindrical rotatable element and the shell element. The shell element has an aperture that exposes a portion of the wall of the cylindrical rotatable element to an exterior of the ingestible device. In certain embodiments, the medical device comprises one or more dilution chambers for receiving a fluid sample from the GI tract or reproductive tract of a subject or a dilution thereof. In some embodiments, one or more dilutions of the fluid sample are cultured in one or more dilution chambers. In certain embodiments, the dilution chambers each define a known volume, optionally the same volume or different volumes. In some embodiments, the dilution chambers define a fluid volume ranging from about 10 μL to about 1 mL. The dilution chambers may define a fluid volume less than or equal to about 500 μL, less than or equal to about 250 μL, less than or equal to about 100 μL, or less than or equal to about 50 pt. In certain embodiments, the dilution chambers define a fluid volume of greater than or equal to about 10 μL, greater than or equal to about 20 μL, greater than or equal to about 30 μL, or greater than or equal to about 50 μL. In some embodiments, the dilution chambers define a fluid volume between about 10 μL and 500 μL, between about 20 μL and 250 μL, between about 30 μL and 100 μL or about 50 μL. In some embodiments, dilution fluid in the device is combined with all or part of the fluid sample, or dilution thereof, to produce one or more dilutions. In certain embodiments, the dilution fluid is sterile media suitable for culturing one or more target cells within the dilution chambers. In certain embodiments, the one or more dilution chambers may be filled with the dilution fluid prior to a patient ingesting the ingestible device. In some embodiments, the dilution fluid may be added into the one or more dilution chambers in vivo from a reservoir of the ingestible device. Sampling and dilution of the GI fluid sample may take place in vivo. For example, an actuator of the ingestible device may pump the dilution fluid from the reservoir into a dilution chamber when it is determined that the ingestible device is located at a predetermined location within the GI tract. In some embodiments, the dilution chambers each contain a volume of sterile media suitable for culturing a fluid sample from the GI tract or reproductive tract. In certain embodiments, the dilution chambers are at least 95%, at least 97%, at least 98%, or at least 99% full of sterile media. In some embodiments, the dilution chambers each contain oxygen to facilitate aerobic bacteria growth. In certain embodiments, a non-dilution chamber comprises oxygen and is added to one or more of the dilution chambers to facilitate aerobic bacteria growth. In some embodiments, the culturing may take place in vivo immediately after the GI fluid sample has been diluted. Or alternatively, the culturing may take place ex vivo, e.g., when the ingestible device has been evacuated and recovered such that the dilution chamber containing the diluted GI fluid sample may be extracted and the culturing may be performed in a laboratory. The recovery of the ingestible device may be performed in a similar manner as embodiments described in U.S. Provisional Application No. 62/434,188, filed on Dec. 14, 2016, which is herein expressly incorporated by reference in its entirety. As used herein “culturing” refers to maintaining target cells in an environment that allows a population of one or more target cells to increase in number through cell division. For example, in some embodiments, “culturing” may include combining the cells with media in an dilution chamber at a temperature that permits cell growth, optionally a temperature found in vivo within the GI tract or reproductive tract of a subject. In certain embodiments, the cells are cultured at a temperature between about 35° C. and 42° C. As used herein “dilution fluid” refers to a fluid within the device for diluting a fluid sample from the GI tract or reproductive tract. In some embodiments, the dilution fluid is an aqueous solution. In certain embodiments, the dilution fluid comprises one or more agents that promote or inhibit the growth of an organism, such as a fungus or bacteria. In some embodiments, the dilution fluid comprises one or more agents that facilitate the detection of a target analyte, such as dyes or binding agents for target analytes. In some embodiments, the dilution fluid is a sterile media. As used herein, “sterile media” refers to media that does not contain any viable bacteria or other cells that would grow and increase in number through cell division. Media may be rendered sterile by various techniques known in the art such as, but not limited to, autoclaving and/or preparing the media using asceptic techniques. In certain embodiments, the media is a liquid media. Examples of media suitable for culturing bacteria include nutrient broth, Lysogeny Broth (LB) (also known as Luria Broth), Wilkins chalgren, and Tryptic Soy Broth (TSB), Other growth or culture media known in the art may also be used in the methods and devices described herein. In some embodiments, the media has a carbon source, such as glucose or glycerol, a nitrogen source such as ammonium salts or nitrates or amino acids, as well as salts and/or trace elements and vitamins required for microbial growth. In certain embodiments, the media is suitable for maintaining eukaryotic cells. In some embodiments, the media comprises one or more agents that promote or inhibit the growth of bacteria, optionally agents that promote or inhibit the growth of specific types of bacteria. In certain embodiments, the media is a selective media. As used herein, “selective media” refers to a media that allows certain types of target cells to grow and inhibits the growth of other organisms. Accordingly, the growth of cells in a selective media indicates the presence of certain types of cells within the cultured sample. For example, in some embodiments, the media is selective for gram-positive or gram-negative bacteria. In certain embodiments, the media contains crystal violet and bile salts (such as found in MacConkey agar) that inhibit the growth of gram-positive organisms and allows for the selection and isolation of gram-negative bacteria. In some embodiments, the media contains a high concentration of salt (NaCl) (such as found in Mannitol salt agar) and is selective for Gram-positive bacteria. In some embodiments, the media selectively kills eukaryotic cells or only grows prokaryotic cells, for example, using a media comprising Triton™ X-100. In certain embodiments, the media selectively kills prokaryotic cells (or alternatively only grows eukaryotic cells), for example, using a media that comprises antibiotics. In some embodiments, the media is an indicator media. As used herein, “indicator media” refers to a media that contains specific nutrients or indicators (such as, but not limited to neutral red, phenol red, eosin y, or methylene blue) that produce a detectable signal when a certain type of cells are cultured in the indicator media. In some embodiments, the disclosure provides a composition comprising a dye and optionally a reagent for selective lysis of eukaryotic cells. In certain embodiments, the composition comprises both a dye and a reagent for selective lysis of eukaryotic cells. In some embodiments, the composition further comprises one or more reagents independently selected from the group consisting of: a second reagent for selective lysis of eukaryotic cells (e.g., Triton X-100), an electrolyte (e.g., MgCl2), an anti-fungi reagent (e.g., amphotericin-B), and an antibiotic. In some embodiments, the composition comprises water and is in the form of an aqueous solution. In some embodiments, the composition is a solid or semi-solid. In some embodiments, the compositions described here are suitable for use in a kit or device for detecting or quantifying viable bacterial cells in a sample. In some embodiments, such a device is an ingestible device for detecting or quantifying viable bacterial cells in vivo (e.g., in the GI tract). In some embodiments, viable bacterial cells in a sample are detected or quantified in the presence of one or more antibiotics to determine antibiotic resistance of the bacteria in the sample. In some embodiments, anomalous bacterial populations in a sample may be detected or quantified, for example through the use of one a composition comprising a dye as disclosed herein, to determine whether a subject has an infection, such as Small Intestinal Bacterial Overgrowth (SIBO), or to characterize bacterial populations within the GI tract for diagnostic or other purposes. In some embodiments, a method comprises: (a) contacting the sample with a composition as described herein; and (b) measuring total fluorescence or rate of change of fluorescence as a function of time of said sample, thereby detecting viable bacterial cells in said sample. In some embodiments, a control as described herein may be employed in the method. In some embodiments, the total fluorescence or the rate of change of fluorescence as a function of time of the sample is measured over multiple time points for an extended period of time in step (b), thereby detecting viable bacterial cells in said sample. In some embodiments, the method further comprises correlating the total fluorescence or the rate of change of fluorescence as a function of time determined in step (b) to the number of viable bacterial cells in the sample. In some embodiments, the rate of change of fluorescence as a function of time of the sample measured over multiple time points is determined and compared to the rate of change of fluorescence as a function of time of a control measured over the same time points to determine the number of viable bacterial cells in the sample. In some embodiments, the method does not require ex vivo plating or culturing. In some embodiments, the method does not require aspiration. In some embodiments, the method is performed in vivo (e.g., in an ingestible device in vivo). In some embodiments, the method comprises communicating the results of the onboard assay(s) to an ex vivo receiver. In certain embodiments, a kit comprises a composition as described herein and instructions, e.g., for detecting or quantifying viable bacterial cells in a sample. In some embodiments, a device comprises a composition as described herein, e.g., for detecting or quantifying viable bacterial cells in a sample. The detection of live cells, as opposed to the detection of bacterial components (such as endotoxins) which can be present in the sample environment and lead to conflicting results, is the gold standard of viable plate counting and represents one of the advantages of the compositions and methods described herein. The systems employ methods, compositions and detection systems found to accurately and reliably correlate fluorescence to total bacteria count (TBC) in an autonomous, ingestible device, or other similarly-sized device. The compositions include novel combinations of dyes, buffers and detergents that allow for the selective staining of viable bacterial cells in samples that comprise non-bacterial cells and other components that otherwise make detecting or quantifying live bacterial cells challenging. In some embodiments, the systems allow for bacteria to be quantified in near real-time and the results to be shared telemetrically outside of the device. In certain embodiments, the disclosure provides a method of assessing or monitoring the need to treat a subject suffering from or at risk of overgrowth of bacterial cells in the gastrointestinal tract, which comprises: (a) obtaining a sample from the gastrointestinal tract of said subject; (b) contacting the sample with a composition as described herein; (c) measuring total fluorescence or rate of change of fluorescence as a function of time of said sample; and (d) correlating the total fluorescence or the rate of change of fluorescence as a function of time measured in step (c) to the number of viable bacterial cells in the sample, wherein the number of the viable bacterial cells determined in step (e) greater than about 105 CFU/mL indicates a need for treatment, e.g., with an antibiotic agent as described herein. In some embodiments, a control as described herein may be employed in the method. In some embodiments, the total fluorescence or the rate of change of fluorescence as a function of time of the sample is measured over multiple time points for an extended period of time in step (c). In some embodiments, the rate of change of fluorescence as a function of time of the sample measured over multiple time points is determined and compared to the rate of change of fluorescence as a function of time of a control measured over the same time points to determine the number of viable bacterial cells in the sample. In some embodiments, the method does not require ex vivo plating or culturing. In some embodiments, the method does not require aspiration. In some embodiments, the method is performed in vivo (e.g., in an ingestible device in vivo). In some embodiments, the method comprises communicating the results of the onboard assay(s) to an ex vivo receiver. In some embodiments, the method may be further used to monitor the subject after the treatment (e.g., with an antibiotic). In some embodiments, the method may be used to assess the efficacy of the treatment. For example, efficacious treatment may be indicated by the decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. Efficacy of the treatment may be evaluated by the rate of decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. In some embodiments, the method may be used to detect infection with antibiotic-resistant strains of bacteria in a subject. For instance, such infection may be indicated where the number of viable bacterial cells in a sample from the GI tract of the subject does not substantially decrease after antibiotic treatment. In some embodiments, the disclosure provides an absorbable material, (e.g., absorbable sponge), having absorbed therein a composition as described herein. In some embodiments, the absorbable sponge is Ahlstrom Grade 6613H (Lot 150191) or Porex PSU-567, having absorbed therein a composition as described herein. In some embodiments, the absorbable sponge may be prepared by injecting into the absorbable sponge an aqueous solution comprising a composition as described herein, and optionally further comprising a step of drying the resulting absorbable sponge. In certain embodiments, the disclosure provides a method for detecting the presence of viable bacterial cells in a sample, which comprises: (a) fully or partially saturating an absorbable sponge as described herein, or an absorbable sponge prepared as described herein, with the sample; and (b) measuring total fluorescence or rate of change of fluorescence as a function of time of the fully or partially saturated sponge prepared in step (a), thereby detecting viable bacterial cells. In some embodiments, a control as described herein may be employed in the method. In some embodiments, the total fluorescence or the rate of change of fluorescence as a function of time of the fully or partially saturated sponge is measured over multiple time points for an extended period of time in step (b), thereby detecting viable bacterial cells in said sample. In some embodiments, the method further comprises correlating the total fluorescence or the rate of change of fluorescence as a function of time measured in step (b) to the number of viable bacterial cells in the sample. In some embodiments, the rate of change of fluorescence as a function of time of the fully or partially saturated sponge measured over multiple time points is determined and compared to the rate of change of fluorescence as a function of time of a control measured over the same time points to determine the number of viable bacterial cells in the sample. In some embodiments, the method does not require ex vivo plating or culturing. In some embodiments, the method does not require aspiration. In some embodiments, the method is performed in vivo (e.g., in an ingestible device in vivo). In some embodiments, the method comprises communicating the results of the onboard assay(s) to an ex vivo receiver. In one aspect, provided herein is a kit comprising an absorbable sponge as described herein and instructions, e.g., for detecting or quantifying viable bacterial cells in a sample. In another aspect, provided herein is a device comprising an absorbable sponge as described herein, e.g., for detecting or quantifying viable bacterial cells in a sample. In certain embodiments, the disclosure provides a method of assessing or monitoring the need to treat a subject suffering from or at risk of overgrowth of bacterial cells in the gastrointestinal tract, which comprises: (a) obtaining a sample from the gastrointestinal tract of said subject; (b) fully or partially saturating an absorbable sponge described herein, or an absorbable sponge prepared as described herein, with the sample; (c) measuring total fluorescence or rate of change of fluorescence as a function of time of the fully or partially saturated sponge prepared in step (b); (d) correlating the total fluorescence or the rate of change of fluorescence as a function of time measured in step (c) to the number of viable bacterial cells in the sample, wherein the number of the viable bacterial cells as determined in step (e) greater than about 105CFU/mL indicates a need for treatment, e.g., with an antibiotic agent as described herein. In some embodiments, a control as described herein may be employed in the method. In some embodiments, the total fluorescence or the rate of change of fluorescence as a function of time of the fully or partially saturated sponge is measured over multiple time points for an extended period of time in step (c). In some embodiments, the rate of change of fluorescence as a function of time of the fully or partially saturated sponge measured over multiple time points is determined and compared to the rate of change of fluorescence as a function of time of a control measured over the same time points to determine the number of viable bacterial cells in the sample. In some embodiments, the method does not require ex vivo plating or culturing. In some embodiments, the method does not require aspiration. In some embodiments, the method is performed in vivo (e.g., in an ingestible device in vivo). In some embodiments, the method comprises communicating the results of the onboard assay(s) to an ex vivo receiver. In some embodiments, the method may be further used to monitor the subject after the treatment (e.g., with an antibiotic). In some embodiments, the method may be used to assess the efficacy of the treatment. For example, efficacious treatment may be indicated by the decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. Efficacy of the treatment may be evaluated by the rate of decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. In some embodiments, the method may be used to detect infection with antibiotic-resistant strains of bacteria in a subject. For instance, such infection may be indicated where the number of viable bacterial cells in a sample from the GI tract of the subject does not substantially decrease after antibiotic treatment In certain embodiments, the disclosure provides and ingestible device comprising a housing; a first opening in the wall of the housing; a second opening in the first end of the housing; and a chamber connecting the first opening and the second opening, wherein at least a portion of the chamber forms a sampling chamber within the ingestible device. In some embodiments, the sampling chamber is configured to hold an absorbable sponge described herein. In some embodiments, the sampling chamber is configured to hold a sample obtained from a gastrointestinal (GI) tract of a body. In some embodiments, the ingestible device is individually calibrated (for example, by comparing to a positive or negative control as described herein), wherein the fluorescent properties of the absorbable sponge held in the sampling chamber of the device are determined prior to the introduction of the sample. The ingestible device as described herein is useful for detecting or quantifying viable bacterial cells in vivo. In some embodiments, provided herein is a method for detecting or quantifying viable bacterial cells in a GI tract sample in vivo using an ingestible device as described herein. In some embodiments, provided herein is a method of assessing or monitoring the need to treat a subject suffering from or at risk of overgrowth of bacterial cells in the GI tract in vivo using an ingestible device as described herein. In some embodiments, provided herein is a method of altering the treatment regimen of a subject suffering from or at risk of overgrowth of bacterial cells in the GI tract in vivo using an ingestible device as described herein. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the duodenum. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the jejunum. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the ileum. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the ascending colon. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the transverse colon. In one aspect, the subject is a subject suffering from or at risk of overgrowth of bacterial cells in the descending colon. In some embodiments, the method may be further used to monitor the subject after the treatment (e.g., with an antibiotic). In some embodiments, the method may be used to assess the efficacy of the treatment. For example, efficacious treatment may be indicated by the decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. Efficacy of the treatment may be evaluated by the rate of decrease of the number of viable bacterial cells in a sample from the GI tract of the subject post-treatment. In some embodiments, the method may be used to detect infection with antibiotic-resistant strains of bacteria in a subject. For instance, such infection may be indicated where the number of viable bacterial cells in a sample from the GI tract of the subject does not substantially decrease after antibiotic treatment. In some embodiments, the method is performed autonomously and does not require instructions, triggers or other inputs from outside the body after the device has been ingested. “Eukaryotic” as recited herein relates to any type of eukaryotic organism excluding fungi, such as animals, in particular animals containing blood, and comprises invertebrate animals such as crustaceans and vertebrates. Vertebrates comprise both cold-blooded (fish, reptiles, amphibians) and warm blooded animal (birds and mammals). Mammals comprise in particular primates and more particularly humans “Selective lysis” as used herein is obtained in a sample when the percentage of bacterial cells in that sample that remain intact is significantly higher (e.g. 2, 5, 10, 20, 50, 100, 250, 500, or 1,000 times more) than the percentage of the eukaryotic cells in that sample that remain intact, upon treatment of or contact with a composition or device as described herein. In some embodiments, the dye suitable for use herein is a dye that is capable of being internalized by a viable cell, binding to or reacting with a target component of the viable cell, and having fluorescence properties that are measurably altered when the dye is bound to or reacted with the target component of the viable cell. In some embodiments, the dye herein is actively internalized by penetrating viable cells through a process other than passible diffusion across cell membranes. Such internalization includes, but is not limited to, internalization through cell receptors on cell surfaces or through channels in cell membranes. In some embodiments, the target component of a viable cell to which the dye is bound to or reacted with is selected from the group consisting of: nucleic acids, actin, tubulin, enzymes, nucleotide-binding proteins, ion-transport proteins, mitochondria, cytoplasmic components, and membrane components. In some embodiments, the dye suitable for use herein is a fluorogenic dye that is capable of being internalized and metabolized by a viable cell, and wherein said dye fluoresces when metabolized by the viable cell. In some embodiments, the dye is a chemiluminescent dye that is capable of being internalized and metabolized by a viable cell, and wherein said dye becomes chemiluminescent when metabolized by the viable cell. In some embodiments, the composition comprises a dye that fluoresces when bond to nucleic acids. Examples of such dyes include, but are not limited to, acridine orange (U.S. Pat. No. 4,190,328); calcein-AM (U.S. Pat. No. 5,314,805); DAPI; Hoechst 33342; Hoechst 33258; PicoGreen™; SYTO® 16; SYBR® Green I; Texas Red®; Redmond Red™; Bodipy® Dyes; Oregon Green™; ethidium bromide; and propidium iodide. In some embodiments, the composition comprises a lipophilic dye that fluoresces when metabolized by a cell. In some embodiments, the dye fluoresces when reduced by a cell or a cell component. Examples of dyes that fluoresce when reduced include, but are not limited to, resazurin; C12-resazurin; 7-hydroxy-9H-(1,3 dichloro-9,9-dimethylacridin-2-ol)N-oxide; 6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine; and tetrazolium salts. In some embodiment, the dye fluoresces when oxidized by a cell or a cell component. Examples of such dyes include, but are not limited to, dihydrocalcein AM; dihydrorhodamine 123; dihydroethidium; 2,3,4,5,6-pentafluorotetramethyldihydrorosamine; and 3′-(p-aminophenyl) fluorescein. In some embodiments, the composition comprises a dye that becomes chemiluminescent when oxidized by a cell or a cell component, such as luminol. In some embodiments, the composition comprises a dye that fluoresces when de-acetylated and/or oxidized by a cell or a cell component. Examples of such dyes include, but are not limited to, dihydrorhodamines; dihydrofluoresceins; 2′,7′-dichlorodihydrofluorescein diacetate; 5-(and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate; and chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester. In some embodiments, the composition comprises a dye that fluoresces when reacted with a peptidase. Examples of such dyes include, but are not limited to, (CBZ-Ala-Ala-Ala-Ala)2-R110 elastase 2; (CBZ-Ala-Ala-Asp)2-R110 granzyme B; and 7-amino-4-methylcoumarin, N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide. In some embodiments, the composition comprises a dye selected from the group consisting of resazurin, FDA, Calcein AM, and SYTO® 9. In some embodiments, the dye is FDA or SYTO® 9. SYTO® 9, when used alone, labels the nucleic acid of bacteria cells. The excitation/emission wavelengths for SYTO® 9 is 480/500 nm, with the background remaining non-fluorescent. See, e.g., J. Appl. Bacteriol. 72, 410 (1992); Lett. Appl. Microbiol. 13, 58 (1991); Curr. Microbiol. 4, 321 (1980); J. Microbiol. Methods 13, 87 (1991); and Microbiol. Rev. 51, 365 (1987); and J. Med. Microbiol. 39, 147 (1993). FDA is a non-polar, non-fluorescent compound that can cross the membranes of mammalian and bacterial cells. The acetyl esterases (present only within viable cells) hydrolyze the FDA into the fluorescent compound fluorescein. Fluorescein is a fluorescent polar compound that is retained within these cells. Living cells can be visualized in a photospectrometer when assayed with an excitation wavelength of 494 nm and an emission wavelength of 518 nm. See, e.g., Brunius, G. (1980).Technical aspects of the use of3′, 6′—Diacetyl fluorescein for vital fluorescent staining of bacteria. Current Microbiol. 4: 321-323; Jones, K. H. and Senft, J. A. (1985).An improved method to determine cell viability by simultaneous staining with fluorescein diacetate—propidium iodide. J. Histochem. Cytochem. 33: 77-79; Ross, R. D., Joneckis, C. C., Ordonez, J. V., Sisk, A. M., Wu, R. K., Hamburger, A. W., and Nora, R. E. (1989).Estimation of cell survival by flow cytometric quantification of fluorescein diacetate/propidium iodide viable cell number. Cancer Research. 49: 3776-3782. Calcein-AM, which is an acetoxylmethyl ester of calcein, is highly lipophilic and cell permeable. Calcein-AM in itself is not fluorescent, but the calcein generated by esterase in a viable cell emits a green fluorescence with an excitation wavelength of 490 nm and an emission of 515 nm. Therefore, Calcein-AM can only stain viable cells. See, e.g., Kimura, K., et al.,Neurosci. Lett.,208, 53 (1998); Shimokawa, I., et al.,J. Geronto.,51a, b49 (1998); Yoshida, S., et al.,Clin. Nephrol.,49, 273 (1998); and Tominaga, H., et al.,Anal. Commun.,36, 47 (1999). Resazuirn (also known as Alamar Blue) is a blue compound that can be reduced to pink resorufin which is fluorescent. This dye is mainly used in viability assays for mammalian cells. C12—resazurin has better cell permeability than resazurin. When lipohilic C12—resazurin crosses the cell membranes, it is subsequently reduced by living cells to make a red fluorescent resorufin. The adsorption/emission of C12—resazurin is 563/587 nm. See, e.g., Appl Environ Microbiol 56, 3785 (1990); J Dairy Res 57, 239 (1990); J Neurosci Methods 70, 195 (1996); J Immunol Methods 210, 25 (1997); J Immunol Methods 213, 157 (1998); Antimicrob Agents Chemother 41, 1004 (1997). In some embodiments, the composition optionally further comprises a reagent for selective lysis of eukaryotic cells. In some embodiments, the composition comprises a dye as described herein and a reagent for selective lysis of eukaryotic cells. In some embodiments, the reagent for selective lysis of eukaryotic cells is a detergent, such as a non-ionic or an ionic detergent. Examples of the reagent for selective lysis of eukaryotic cells include, but are not limited to, alkylglycosides, Brij 35 (C12E23 Polyoxyethyleneglycol dodecyl ether), Brij 58 (C16E20 Polyoxyethyleneglycol dodecyl ether), Genapol, glucanids such as MEGA-8, -9, -10, octylglucoside, Pluronic F127, Triton X-100 (C14H22O(C2H4O)n), Triton X-114 (C24H42O6), Tween 20 (Polysorbate 20) and Tween 80 (Polysorbate 80), Nonidet P40, deoxycholate, reduced Triton X-100 and/or Igepal CA 630. In some embodiments, the composition comprises a dye as described herein and deoxycholate (e.g., sodium deoxycholate) as a reagent for selective lysis of eukaryotic cells. In some embodiments, the composition comprises deoxycholate at a concentration selected from 0.0001% to 1 wt %. In some embodiments, the composition comprises deoxycholate at a concentration of 0.005 wt %. In some embodiments, the composition may comprise more than one reagent for selective lysis of eukaryotic cells. In some embodiments, the composition may comprise two different reagents for selective lysis of eukaryotic cells. In some instances, when more than one selective lysis reagents are used, more effective and/or complete selective lysis of eukaryotic cells in a sample may be achieved. For example, the composition may comprise deoxycholate (e.g., sodium deoxycholate) and Triton X-100 as two different reagents for selective lysis of eukaryotic cells. In some embodiments, the composition comprises deoxycholate (e.g., sodium deoxycholate) at a concentration selected from 0.0001% to 1 wt % (e.g., 0.005 wt %) and Triton X-100 at a concentration selected from 0.1 to 0.05 wt %. In some embodiments, after a sample (e.g., a biological sample) is treated or contacted with a composition comprising a dye and one or more reagents for selective lysis of eukaryotic cells as described herein, the eukaryotic cells (e.g., animal cells) in the sample are selectively lysed whereby a substantial percentage (e.g., more than 20%, 40%, 60%, 80%, 90% or even more that 95%) of the bacterial cells in the same sample remains intact or alive. In some embodiments, the composition does not comprise a reagent for selective lysis of eukaryotic cells, and such a composition is useful for detecting or quantifying viable bacterial cells in a sample (e.g., an environmental sample such as a water sample) that does not contain any eukaryotic cells. In some embodiments, the composition further comprises an electrolyte, such as a divalent electrolyte (e.g., MgCl2). In some embodiments, the composition comprises MgCl2at a concentration selected from 0.1 mM to 100 mM (e.g., a concentration selected from 0.5 mM to 50 mM). In some embodiments, the composition further comprises water and is in a form of an aqueous solution. In some embodiments, the composition has a pH selected from 5-8 (e.g., a pH selected from 6-7.8, such as pH being 6.0). In some embodiments, the composition is a solid or a semi-solid. In some embodiments, the composition further comprises an anti-fungal agent. Suitable anti-fungal agents for use herein include, but are not limited to, fungicidal and fungistatic agents including terbinafine, itraconazole, micronazole nitrate, thiapendazole, tolnaftate, clotrimazole and griseofulvin. In some embodiments, the anti-fungal agent is a polyene anti-fungal agent, such as amphotericin-B, nystatin, and pimaricin. In some embodiments, the composition does not contain any anti-fungal agent. In some embodiments, the composition contains broad spectrum antibiotics but not any anti-fungal agent. Such compositions that do not contain anti-fungal agents but contain broad spectrum antibiotics may be useful in detecting or quantifying fungi (e.g., yeast) in a sample. In some embodiments, the composition does not contain any anti-fungal agent, any antibiotics or any anti-mammalian agent. Such compositions that do not selectively lyse mammalian cells may be useful in detecting or quantifying mammalian cells (e.g., cells from the GI tract) in a sample since many dyes have a higher affinity for mammalian as compared to bacteria or fungi cells. In some embodiments, the composition contains broad spectrum antibiotics and one or more anti-fungal agents. Such compositions that contain anti-fungal agents and broad spectrum antibiotics may be useful in detecting or quantifying mammalian cells (e.g., cells from the GI tract) in a sample. The detection or quantification of mammalian cells may be useful for determining cell turnover in a subject. High cell turnover is sometimes associated with a GI injury (e.g., lesion), the presence of a tumor(s), or radiation-induced colitis or radiation enteropathy. In some embodiments, the composition further comprises an antibiotic agent as described herein. Such a composition may be useful in detecting or quantifying antibiotic-resistant strains of bacteria in a sample. In certain embodiments, the composition comprises Triton X-100, deoxycholate, resazurin, and MgCl2. In some embodiments, the composition comprises Triton X-100, deoxycholate, resazurin, amphotericin-B and MgCl2. In some embodiments, the composition comprises 0.1 wt % or 0.05 wt % Triton X-100; 0.005 wt % deoxycholate; 10 mM resazurin; 2.5 mg/L amphotericin-B and 50 mM MgCl2. In some embodiments, the composition has a pH of 6.0. In certain embodiments, the compositions are suitable for use in a kit or device, e.g., for detecting or quantifying viable bacterial cells in a sample. In some embodiments, such a device is an ingestible device for detecting or quantifying viable bacterial cells in vivo (e.g., in the GI tract). FIG.62illustrates a nonlimiting example of a system for collecting, communicating and/or analyzing data about a subject, using an ingestible device as disclosed herein. For example, an ingestible device may be configured to communicate with an external base station. As an example, an ingestible device can have a communications unit that communicates with an external base station which itself has a communications unit.FIG.62illustrates exemplary implementation of such an ingestible device. As shown inFIG.62, a subject ingests an ingestible device as disclosed herein. Certain data about the subject (e.g., based on a collected sample) and/or the location of the ingestible device in the GI tract of the subject is collected or otherwise available and provided to a mobile device, which then forwards the data via the internet and a server/data store to a physician's office computer. The information collected by the ingestible device is communicated to a receiver, such as, for example, a watch or other object worn by the subject. The information is then communicated from the receiver to the mobile device which then forwards the data via the internet and a server/data store to a physician's office computer. The physician is then able to analyze some or all of the data about the subject to provide recommendations, such as, for example, delivery a therapeutic agent. WhileFIG.62shows a particular approach to collecting and transferring data about a subject, the disclosure is not limited. As an example, one or more of the receiver, mobile device, internet, and/or server/data store can be excluded from the data communication channel. For example, a mobile device can be used as the receiver of the device data, e.g., by using a dongle. In such embodiments, the item worn by the subject need not be part of the communication chain. As another example, one or more of the items in the data communication channel can be replaced with an alternative item. For example, rather than be provided to a physician's office computer, data may be provided to a service provider network, such as a hospital network, an HMO network, or the like. In some embodiments, subject data may be collected and/or stored in one location (e.g., a server/data store) while device data may be collected and/or stored in a different location (e.g., a different server/data store). Locations of Treatment In some embodiments, the JAK inhibitor is delivered at a location in the large intestine of the subject. In some embodiments, the location is in the proximal portion of the large intestine. In some embodiments, the location is in the distal portion of the large intestine. In some embodiments, the JAK inhibitor is delivered at a location in the ascending colon of the subject. In some embodiments, the location is in the proximal portion of the ascending colon. In some embodiments, the location is in the distal portion of the ascending colon. In some embodiments, the JAK inhibitor is delivered at a location in the cecum of the subject. In some embodiments, the location is in the proximal portion of the cecum. In some embodiments, the location is in the distal portion of the cecum. In some embodiments, the JAK inhibitor is delivered at a location in the sigmoid colon of the subject. In some embodiments, the location is in the proximal portion of the sigmoid colon. In some embodiments, the location is in the distal portion of the sigmoid colon. In some embodiments, the JAK inhibitor is delivered at a location in the transverse colon of the subject. In some embodiments, the location is in the proximal portion of the transverse colon. In some embodiments, the location is in the distal portion of the transverse colon. In some embodiments, the JAK inhibitor is delivered at a location in the descending colon of the subject. In some embodiments, the location is in the proximal portion of the descending colon. In some embodiments, the location is in the distal portion of the descending colon. In some embodiments, the JAK inhibitor is delivered at a location in the small intestine of the subject. In some embodiments, the location is in the proximal portion of the small intestine. In some embodiments, the location is in the distal portion of the small intestine. In some embodiments, the JAK inhibitor is delivered at a location in the duodenum of the subject. In some embodiments, the location is in the proximal portion of the duodenum. In some embodiments, the location is in the distal portion of the duodenum. In some embodiments, the JAK inhibitor is delivered at a location in the jejunum of the subject. In some embodiments, the location is in the proximal portion of the jejunum. In some embodiments, the location is in the distal portion of the jejunum. In some embodiments, the JAK inhibitor is delivered at a location in the duodenum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the duodenum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the duodenum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the duodenum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the duodenum and a second site of disease is in the stomach and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal duodenum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal duodenum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the duodenum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal duodenum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the duodenum and a second site of disease is in the stomach and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the jejunum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the jejunum and a second site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the jejunum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the jejunum and a second site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the jejunum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the jejunum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the jejunum and a second site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the ileum of the subject. In some embodiments, the location is in the proximal portion of the ileum. In some embodiments, the location is in the distal portion of the ileum. In some embodiments, the JAK inhibitor is delivered at a location in the ileum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and/or ascending colon, and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the proximal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and/or ascending colon, and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the ileum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the ileum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the ileum and a second site of disease is in the cecum and/or ascending colon, and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the cecum of the subject and is not delivered at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the cecum of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a site of disease is in the cecum and/or ascending colon, and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, the JAK inhibitor is delivered at a location in the distal portion of the ileum or the proximal portion of the ascending colon of the subject and is not delivered at other locations in the gastrointestinal tract, wherein a first site of disease is in the cecum and a second site of disease is in the ascending colon, and no site of disease is present at other locations in the gastrointestinal tract. In some embodiments, a site of disease is in the colon and the JAK inhibitor is released in the colon, such as in the cecum. In some embodiments, a site of disease is in the ascending colon and the JAK inhibitor is released in the ascending colon, such as in the cecum. In some embodiments, a site of disease is in the ileum and the JAK inhibitor is released in the ileum. In some embodiments the subject is diagnosed with ileal Crohn's disease and the JAK inhibitor is released in the ileum. In some embodiments the subject is diagnosed with ileal colonic Crohn's disease and the JAK inhibitor is released in both the ileum and the colon. In some more particular embodiments, the JAK inhibitor is released in both the ileum and the colon from the same ingestible device. In some more particular embodiments, the JAK inhibitor is released in the ileum from a first ingestible device and in the colon from a second ingestible device, wherein the first ingestible device and the second ingestible device are ingested at substantially the same time or at different times. In some embodiments the subject is diagnosed with colitis throughout the colon and the JAK inhibitor is released (a) in the cecum, (b) in the cecum and in the transverse colon, and/or release (c) in the descending colon. In some embodiments the subject is diagnosed with right sided colitis and the JAK inhibitor is released in the transverse colon or in the descending colon. In some embodiments the subject is diagnosed with rectosigmoidal colitis and the JAK inhibitor is released in the descending colon. In some embodiments, the location at which the JAK inhibitor is delivered is proximate to a site of disease. The site of disease may be, for example, an injury, inflamed tissue, or one or more lesions. In some embodiments, the location at which the JAK inhibitor is delivered is proximate to one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 150 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 125 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 100 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 50 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 40 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 30 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 20 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 10 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 5 cm or less from the one or more sites of disease. In some embodiments, the JAK inhibitor is delivered 2 cm or less from the one or more sites of disease. In some embodiments, the method further comprises using an ingestible device to deliver the JAK inhibitor and using localization methods disclosed herein (e.g., such as discussed in Example 13 below) to determine the location of the ingestible device within the GI tract (e.g., relative to the site of disease). In some embodiments, the method further comprises using an ingestible device to deliver the JAK inhibitor and determining the period of time since the ingestible device was ingested to determine the location of the ingestible device within the GI tract (e.g., relative to the site of disease). In some embodiments, the method further comprises identifying the one or more sites of disease by a method comprising imaging of the gastrointestinal tract. In some embodiments, imaging of the gastrointestinal tract comprises video imaging. In some embodiments, imaging of the gastrointestinal tract comprises thermal imaging. In some embodiments, imaging of the gastrointestinal tract comprises ultrasound imaging. In some embodiments, imaging of the gastrointestinal tract comprises Doppler imaging. In some embodiments the method does not comprise releasing more than 20% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method does not comprise releasing more than 10% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method does not comprise releasing more than 5% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method does not comprise releasing more than 4% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method does not comprise releasing more than 3% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method does not comprise releasing more than 2% of the JAK inhibitor at a location that is not proximate to a site of disease. In some embodiments the method comprises releasing at least 80% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments the method comprise releasing at least 90% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments the method comprises releasing at least 95% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments the method comprises releasing at least 96% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments the method comprises releasing at least 97% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments the method comprises releasing at least 98% of the JAK inhibitor at a location proximate to a site of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 150 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 125 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 100 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 50 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 40 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 30 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 20 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 10 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 5 cm or less from the one or more sites of disease. In some embodiments, the at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the JAK inhibitor is delivered 2 cm or less from the one or more sites of disease. In some embodiments, the method further comprises using an ingestible device to deliver the JAK inhibitor and using localization methods disclosed herein (e.g., such as discussed in Example 13 below) to determine the location of the ingestible device within the GI tract (e.g., relative to the site of disease). In some embodiments, the method further comprises using an ingestible device to deliver the JAK inhibitor and determining the period of time since the ingestible device was ingested to determine the location of the ingestible device within the GI tract (e.g., relative to the site of disease). In some embodiments, the amount of JAK inhibitor that is delivered is a Human Equivalent Dose. In some embodiments the method comprises releasing the JAK inhibitor at a location that is proximate to a site of disease, wherein the JAK inhibitor and, if applicable, any carriers, excipients or stabilizers admixed with the JAK inhibitor, are substantially unchanged, at the time of release of the JAK inhibitor at the location, relatively to the time of administration of the composition to the subject. In some embodiments the method comprises releasing the JAK inhibitor at a location that is proximate to a site of disease, wherein the JAK inhibitor and, if applicable, any carriers, excipients or stabilizers admixed with the JAK inhibitor, are substantially unchanged by any physiological process (such as, but not limited to, degradation in the stomach), at the time of release of the JAK inhibitor at the location, relatively to the time of administration of the composition to the subject. In some embodiments, the JAK inhibitor is delivered to the location by mucosal contact. In some embodiments, a method of treatment disclosed herein includes determining the level of JAK inhibitor at a site of disease or a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease. In some examples, a method of treatment as described herein can include determining the level of JAK inhibitor at a site of disease or a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease within a time period of about 10 minutes to about 10 hours following administration of the device. In some examples, a method of treatment disclosed herein includes determining the level of the JAK inhibitor at a site of disease or a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease at a time point following administration of the device that is elevated as compared to a level of the JAK inhibitor at the same site of disease or location at substantially the same time point in a subject following systemic administration of an equal amount of the JAK inhibitor. In some examples where the JAK inhibitor is an antibody or an antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding antibody fragments described herein) are administered to a subject using any of the compositions or devices described herein, the antibody or antigen-binding antibody fragment can penetrate the GI tissue of the subject. As used herein, “GI tissue” refers to tissue in the gastrointestinal (GI) tract, such as tissue in one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. In one particular embodiment, GI tissue refers to tissue in the proximal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon. In one particular embodiment, GI tissue refers to tissue in the distal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon. The GI tissue may be, for example, GI tissue proximate to one or more sites of disease. Accordingly, in some embodiments the antibody or antigen-binding antibody fragment can penetrate the dudodenum tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the jejunum tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the ileum tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the cecum tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the ascending colon tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the transverse colon tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the descending colon tissue proximate to one or more sites of disease. In some embodiments the antibody or antigen-binding antibody fragment can penetrate the sigmoid colon tissue proximate to one or more sites of disease. For example, an antibody or antigen-binding fragment thereof (e.g., a F(ab′)2, a Fv, or a scFv) can penetrate one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa. In some embodiments, any of the devices or compositions described herein can release a recombinant antibody (e.g., a humanized or fully human antibody, e.g., human or humanized IgG1, human or humanized IgG2, human or humanized IgG3, human or humanized IgG4, human or humanized IgA1, human or humanized IgA2, human or humanized IgD, human or humanized IgE, or human or humanized IgM), which is degraded into an antigen-binding antibody fragment (e.g., a Fab, a Fv, or a F(ab′)2), which in turn is able to penetrate GI tissue (e.g., one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa) of the subject. In some embodiments, the device releases an antigen-binding antibody fragment (e.g., any of the antigen-binding antibody fragments described herein). In some examples, administration of an antibody or an antigen-binding fragment thereof using any of the compositions or devices described herein results in penetration (e.g., a detectable level of penetration) of GI tissue (e.g., one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa) within a time period of about 10 minutes to about 10 hours, about 10 minutes to about 9 hours, about 10 minutes to about 8 hours, about 10 minutes to about 7 hours, about 10 minutes to about 6 hours, about 10 minutes to about 5 hours, about 10 minutes to about 4.5 hours, about 10 minutes to about 4 hours, about 10 minutes to about 3.5 hours, about 10 minutes to about 3 hours, about 10 minutes to about 2.5 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1.5 hours, about 10 minutes to about 1 hour, about 10 minutes to about 55 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 45 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 10 hours, about 15 minutes to about 9 hours, about 15 minutes to about 8 hours, about 15 minutes to about 7 hours, about 15 minutes to about 6 hours, about 15 minutes to about 5 hours, about 15 minutes to about 4.5 hours, about 15 minutes to about 4 hours, about 15 minutes to about 3.5 hours, about 15 minutes to about 3 hours, about 15 minutes to about 2.5 hours, about 15 minutes to about 2 hours, about 15 minutes to about 1.5 hours, about 15 minutes to about 1 hour, about 15 minutes to about 55 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 10 hours, about 20 minutes to about 9 hours, about 20 minutes to about 8 hours, about 20 minutes to about 7 hours, about 20 minutes to about 6 hours, about 20 minutes to about 5 hours, about 20 minutes to about 4.5 hours, about 20 minutes to about 4 hours, about 20 minutes to about 3.5 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2.5 hours, about 20 minutes to about 2 hours, about 20 minutes to about 1.5 hours, about 20 minutes to about 1 hour, about 20 minutes to about 55 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 45 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 25 minutes, about 25 minutes to about 10 hours, about 25 minutes to about 9 hours, about 25 minutes to about 8 hours, about 25 minutes to about 7 hours, about 25 minutes to about 6 hours, about 25 minutes to about 5 hours, about 25 minutes to about 4.5 hours, about 25 minutes to about 4 hours, about 25 minutes to about 3.5 hours, about 25 minutes to about 3 hours, about 25 minutes to about 2.5 hours, about 25 minutes to about 2 hours, about 25 minutes to about 1.5 hours, about 25 minutes to about 1 hour, about 25 minutes to about 55 minutes, about 25 minutes to about 50 minutes, about 25 minutes to about 45 minutes, about 25 minutes to about 40 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 30 minutes, about 30 minutes to about 10 hours, about 30 minutes to about 9 hours, about 30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30 minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3.5 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2.5 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1.5 hours, about 30 minutes to about 1 hour, about 30 minutes to about 55 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 45 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 35 minutes to about 10 hours, about 35 minutes to about 9 hours, about 35 minutes to about 8 hours, about 35 minutes to about 7 hours, about 35 minutes to about 6 hours, about 35 minutes to about 5 hours, about 35 minutes to about 4.5 hours, about 35 minutes to about 4 hours, about 35 minutes to about 3.5 hours, about 35 minutes to about 3 hours, about 35 minutes to about 2.5 hours, about 35 minutes to about 2 hours, about 35 minutes to about 1.5 hours, about 35 minutes to about 1 hour, about 35 minutes to about 55 minutes, about 35 minutes to about 50 minutes, about 35 minutes to about 45 minutes, about 35 minutes to about 40 minutes, about 40 minutes to about 10 hours, about 40 minutes to about 9 hours, about 40 minutes to about 8 hours, about 40 minutes to about 7 hours, about 40 minutes to about 6 hours, about 40 minutes to about 5 hours, about 40 minutes to about 4.5 hours, about 40 minutes to about 4 hours, about 40 minutes to about 3.5 hours, about 40 minutes to about 3 hours, about 40 minutes to about 2.5 hours, about 40 minutes to about 2 hours, about 40 minutes to about 1.5 hours, about 40 minutes to about 1 hour, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 40 minutes to about 45 minutes, about 45 minutes to about 10 hours, about 45 minutes to about 9 hours, about 45 minutes to about 8 hours, about 45 minutes to about 7 hours, about 45 minutes to about 6 hours, about 45 minutes to about 5 hours, about 45 minutes to about 4.5 hours, about 45 minutes to about 4 hours, about 45 minutes to about 3.5 hours, about 45 minutes to about 3 hours, about 45 minutes to about 2.5 hours, about 45 minutes to about 2 hours, about 45 minutes to about 1.5 hours, about 45 minutes to about 1 hour, about 45 minutes to about 55 minutes, about 45 minutes to about 50 minutes, about 50 minutes to about 10 hours, about 50 minutes to about 9 hours, about 50 minutes to about 8 hours, about 50 minutes to about 7 hours, about 50 minutes to about 6 hours, about 50 minutes to about 5 hours, about 50 minutes to about 4.5 hours, about 50 minutes to about 4 hours, about 50 minutes to about 3.5 hours, about 50 minutes to about 3 hours, about 50 minutes to about 2.5 hours, about 50 minutes to about 2 hours, about 50 minutes to about 1.5 hours, about 50 minutes to about 1 hour, about 50 minutes to about 55 minutes, about 55 minutes to about 10 hours, about 55 minutes to about 9 hours, about 55 minutes to about 8 hours, about 55 minutes to about 7 hours, about 55 minutes to about 6 hours, about 55 minutes to about 5 hours, about 55 minutes to about 4.5 hours, about 55 minutes to about 4 hours, about 55 minutes to about 3.5 hours, about 55 minutes to about 3 hours, about 55 minutes to about 2.5 hours, about 55 minutes to about 2 hours, about 55 minutes to about 1.5 hours, about 55 minutes to about 1 hour, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 1.5 hours to about 10 hours, about 1.5 hours to about 9 hours, about 1.5 hours to about 8 hours, about 1.5 hours to about 7 hours, about 1.5 hours to about 6 hours, about 1.5 hours to about 5 hours, about 1.5 hours to about 4.5 hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3.5 hours, about 1.5 hours to about 3 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 10 hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours to about 4.5 hours, about 2 hours to about 4 hours, about 2 hours to about 3.5 hours, about 2 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2.5 hours to about 10 hours, about 2.5 hours to about 9 hours, about 2.5 hours to about 8 hours, about 2.5 hours to about 7 hours, about 2.5 hours to about 6 hours, about 2.5 hours to about 5 hours, about 2.5 hours to about 4.5 hours, about 2.5 hours to about 4 hours, about 2.5 hours to about 3.5 hours, about 2.5 hours to about 3 hours, about 3 hours to about 10 hours, about 3 hours to about 9 hours, about 3 hours to about 8 hours, about 3 hours to about 7 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, about 3 hours to about 4.5 hours, about 3 hours to about 4 hours, about 3 hours to about 3.5 hours, about 3.5 hours to about 10 hours, about 3.5 hours to about 9 hours, about 3.5 hours to about 8 hours, about 3.5 hours to about 7 hours, about 3.5 hours to about 6 hours, about 3.5 hours to about 5 hours, about 3.5 hours to about 4.5 hours, about 3.5 hours to about 4 hours, about 4 hours to about 10 hours, about 4 hours to about 9 hours, about 4 hours to about 8 hours, about 4 hours to about 7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 4 hours to about 4.5 hours, about 4.5 hours to about 10 hours, about 4.5 hours to about 9 hours, about 4.5 hours to about 8 hours, about 4.5 hours to about 7 hours, about 4.5 hours to about 6 hours, about 4.5 hours to about 5 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours, about 5 hours to about 8 hours, about 5 hours to about 7 hours, about 5 hours to about 6 hours, about 6 hours to about 10 hours, about 6 hours to about 9 hours, about 6 hours to about 8 hours, about 6 hours to about 7 hours, about 7 hours to about 10 hours, about 7 hours to about 9 hours, about 7 hours to about 8 hours, about 8 hours to about 10 hours, about 8 hours to about 9 hours, or about 9 hours to about 10 hours. Penetration of GI tissue by an antibody or an antigen-binding antibody fragment can be detected by administering a labeled antibody or labeled antigen-binding antibody fragment, and performing imaging on the subject (e.g., ultrasound, computed tomography, or magnetic resonance imaging). For example, the label can be a radioisotope, a heavy metal, a fluorophore, or a luminescent agent (e.g., any suitable radioisotopes, heavy metals, fluorophores, or luminescent agents used for imaging known in the art). While not wishing to be bound to a particular theory, the inventors contemplate that at or near the site of release a concentration gradient of the JAK inhibitor is generated in the mucosa, and that administration of an JAK inhibitor using a device as described herein advantageously results in a “reverse” concentration gradient when compared to the concentration gradient resulting from systemic administration. In such “reverse” concentration gradient, the drug concentration is highest from superficial to deep with respect to the mucosal surface. Systemic administration instead typically results in concentrations of the drug being highest from deep to superficial. A “reverse” concentration gradient as described above aligns more favorably with the pathophysiology of IBD. In some embodiments, administration of an antibody or an antigen-binding antibody fragment can provide for treatment (e.g., a reduction in the number, severity, and/or duration of one or more symptoms of any of the disorders described herein in a subject) for a time period of between about 1 hour to about 30 days, about 1 hour to about 28 days, about 1 hour to about 26 days, about 1 hour to about 24 days, about 1 hour to about 22 days, about 1 hour to about 20 days, about 1 hour to about 18 days, about 1 hour to about 16 days, about 1 hour to about 14 days, about 1 hour to about 12 days, about 1 hour to about 10 days, about 1 hour to about 8 days, about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 3 hours, about 3 hours to about 30 days, about 3 hours to about 28 days, about 3 hours to about 26 days, about 3 hours to about 24 days, about 3 hours to about 22 days, about 3 hours to about 20 days, about 3 hours to about 18 days, about 3 hours to about 16 days, about 3 hours to about 14 days, about 3 hours to about 12 days, about 3 hours to about 10 days, about 3 hours to about 8 days, about 3 hours to about 6 days, about 3 hours to about 5 days, about 3 hours to about 4 days, about 3 hours to about 3 days, about 3 hours to about 2 days, about 3 hours to about 1 day, about 3 hours to about 12 hours, about 3 hours to about 6 hours, about 6 hours to about 30 days, about 6 hours to about 28 days, about 6 hours to about 26 days, about 6 hours to about 24 days, about 6 hours to about 22 days, about 6 hours to about 20 days, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 6 hours to about 12 hours, about 12 hours to about 30 days, about 12 hours to about 28 days, about 12 hours to about 26 days, about 12 hours to about 24 days, about 12 hours to about 22 days, about 12 hours to about 20 days, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 30 days, about 1 day to about 28 days, about 1 day to about 26 days, about 1 day to about 24 days, about 1 day to about 22 days, about 1 day to about 20 days, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 30 days, about 2 days to about 28 days, about 2 days to about 26 days, about 2 days to about 24 days, about 2 days to about 22 days, about 2 days to about 20 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 30 days, about 3 days to about 28 days, about 3 days to about 26 days, about 3 days to about 24 days, about 3 days to about 22 days, about 3 days to about 20 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 30 days, about 4 days to about 28 days, about 4 days to about 26 days, about 4 days to about 24 days, about 4 days to about 22 days, about 4 days to about 20 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 30 days, about 5 days to about 28 days, about 5 days to about 26 days, about 5 days to about 24 days, about 5 days to about 22 days, about 5 days to about 20 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 6 days, about 6 days to about 30 days, about 6 days to about 28 days, about 6 days to about 26 days, about 6 days to about 24 days, about 6 days to about 22 days, about 6 days to about 20 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 8 days to about 30 days, about 8 days to about 28 days, about 8 days to about 26 days, about 8 days to about 24 days, about 8 days to about 22 days, about 8 days to about 20 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 10 days to about 30 days, about 10 days to about 28 days, about 10 days to about 26 days, about 10 days to about 24 days, about 10 days to about 22 days, about 10 days to about 20 days, about 10 days to about 18 days, about 10 days to about 16 days, about 10 days to about 14 days, about 10 days to about 12 days, about 12 days to about 30 days, about 12 days to about 28 days, about 12 days to about 26 days, about 12 days to about 24 days, about 12 days to about 22 days, about 12 days to about 20 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 30 days, about 14 days to about 28 days, about 14 days to about 26 days, about 14 days to about 24 days, about 14 days to about 22 days, about 14 days to about 20 days, about 14 days to about 18 days, about 14 days to about 16 days, about 16 days to about 30 days, about 16 days to about 28 days, about 16 days to about 26 days, about 16 days to about 24 days, about 16 days to about 22 days, about 16 days to about 20 days, about 16 days to about 18 days, about 18 days to about 30 days, about 18 days to about 28 days, about 18 days to about 26 days, about 18 days to about 24 days, about 18 days to about 22 days, about 18 days to about 20 days, about 20 days to about 30 days, about 20 days to about 28 days, about 20 days to about 26 days, about 20 days to about 24 days, about 20 days to about 22 days, about 22 days to about 30 days, about 22 days to about 28 days, about 22 days to about 26 days, about 22 days to about 24 days, about 24 days to about 30 days, about 24 days to about 28 days, about 24 days to about 26 days, about 26 days to about 30 days, about 26 days to about 28 days, or about 28 days to about 30 days in a subject following first administration of an antibody or antigen-binding antibody fragment using any of the compositions or devices described herein. Non-limiting examples of symptoms of a disease described herein are described below. For example, treatment can result in a decrease (e.g., about 1% to about 99% decrease, about 1% to about 95% decrease, about 1% to about 90% decrease, about 1% to about 85% decrease, about 1% to about 80% decrease, about 1% to about 75% decrease, about 1% to about 70% decrease, about 1% to about 65% decrease, about 1% to about 60% decrease, about 1% to about 55% decrease, about 1% to about 50% decrease, about 1% to about 45% decrease, about 1% to about 40% decrease, about 1% to about 35% decrease, about 1% to about 30% decrease, about 1% to about 25% decrease, about 1% to about 20% decrease, about 1% to about 15% decrease, about 1% to about 10% decrease, about 1% to about 5% decrease, about 5% to about 99% decrease, about 5% to about 95% decrease, about 5% to about 90% decrease, about 5% to about 85% decrease, about 5% to about 80% decrease, about 5% to about 75% decrease, about 5% to about 70% decrease, about 5% to about 65% decrease, about 5% to about 60% decrease, about 5% to about 55% decrease, about 5% to about 50% decrease, about 5% to about 45% decrease, about 5% to about 40% decrease, about 5% to about 35% decrease, about 5% to about 30% decrease, about 5% to about 25% decrease, about 5% to about 20% decrease, about 5% to about 15% decrease, about 5% to about 10% decrease, about 10% to about 99% decrease, about 10% to about 95% decrease, about 10% to about 90% decrease, about 10% to about 85% decrease, about 10% to about 80% decrease, about 10% to about 75% decrease, about 10% to about 70% decrease, about 10% to about 65% decrease, about 10% to about 60% decrease, about 10% to about 55% decrease, about 10% to about 50% decrease, about 10% to about 45% decrease, about 10% to about 40% decrease, about 10% to about 35% decrease, about 10% to about 30% decrease, about 10% to about 25% decrease, about 10% to about 20% decrease, about 10% to about 15% decrease, about 15% to about 99% decrease, about 15% to about 95% decrease, about 15% to about 90% decrease, about 15% to about 85% decrease, about 15% to about 80% decrease, about 15% to about 75% decrease, about 15% to about 70% decrease, about 15% to about 65% decrease, about 15% to about 60% decrease, about 15% to about 55% decrease, about 15% to about 50% decrease, about 15% to about 45% decrease, about 15% to about 40% decrease, about 15% to about 35% decrease, about 15% to about 30% decrease, about 15% to about 25% decrease, about 15% to about 20% decrease, about 20% to about 99% decrease, about 20% to about 95% decrease, about 20% to about 90% decrease, about 20% to about 85% decrease, about 20% to about 80% decrease, about 20% to about 75% decrease, about 20% to about 70% decrease, about 20% to about 65% decrease, about 20% to about 60% decrease, about 20% to about 55% decrease, about 20% to about 50% decrease, about 20% to about 45% decrease, about 20% to about 40% decrease, about 20% to about 35% decrease, about 20% to about 30% decrease, about 20% to about 25% decrease, about 25% to about 99% decrease, about 25% to about 95% decrease, about 25% to about 90% decrease, about 25% to about 85% decrease, about 25% to about 80% decrease, about 25% to about 75% decrease, about 25% to about 70% decrease, about 25% to about 65% decrease, about 25% to about 60% decrease, about 25% to about 55% decrease, about 25% to about 50% decrease, about 25% to about 45% decrease, about 25% to about 40% decrease, about 25% to about 35% decrease, about 25% to about 30% decrease, about 30% to about 99% decrease, about 30% to about 95% decrease, about 30% to about 90% decrease, about 30% to about 85% decrease, about 30% to about 80% decrease, about 30% to about 75% decrease, about 30% to about 70% decrease, about 30% to about 65% decrease, about 30% to about 60% decrease, about 30% to about 55% decrease, about 30% to about 50% decrease, about 30% to about 45% decrease, about 30% to about 40% decrease, about 30% to about 35% decrease, about 35% to about 99% decrease, about 35% to about 95% decrease, about 35% to about 90% decrease, about 35% to about 85% decrease, about 35% to about 80% decrease, about 35% to about 75% decrease, about 35% to about 70% decrease, about 35% to about 65% decrease, about 35% to about 60% decrease, about 35% to about 55% decrease, about 35% to about 50% decrease, about 35% to about 45% decrease, about 35% to about 40% decrease, about 40% to about 99% decrease, about 40% to about 95% decrease, about 40% to about 90% decrease, about 40% to about 85% decrease, about 40% to about 80% decrease, about 40% to about 75% decrease, about 40% to about 70% decrease, about 40% to about 65% decrease, about 40% to about 60% decrease, about 40% to about 55% decrease, about 40% to about 50% decrease, about 40% to about 45% decrease, about 45% to about 99% decrease, about 45% to about 95% decrease, about 45% to about 90% decrease, about 45% to about 85% decrease, about 45% to about 80% decrease, about 45% to about 75% decrease, about 45% to about 70% decrease, about 45% to about 65% decrease, about 45% to about 60% decrease, about 45% to about 55% decrease, about 45% to about 50% decrease, about 50% to about 99% decrease, about 50% to about 95% decrease, about 50% to about 90% decrease, about 50% to about 85% decrease, about 50% to about 80% decrease, about 50% to about 75% decrease, about 50% to about 70% decrease, about 50% to about 65% decrease, about 50% to about 60% decrease, about 50% to about 55% decrease, about 55% to about 99% decrease, about 55% to about 95% decrease, about 55% to about 90% decrease, about 55% to about 85% decrease, about 55% to about 80% decrease, about 55% to about 75% decrease, about 55% to about 70% decrease, about 55% to about 65% decrease, about 55% to about 60% decrease, about 60% to about 99% decrease, about 60% to about 95% decrease, about 60% to about 90% decrease, about 60% to about 85% decrease, about 60% to about 80% decrease, about 60% to about 75% decrease, about 60% to about 70% decrease, about 60% to about 65% decrease, about 65% to about 99% decrease, about 65% to about 95% decrease, about 65% to about 90% decrease, about 65% to about 85% decrease, about 65% to about 80% decrease, about 65% to about 75% decrease, about 65% to about 70% decrease, about 70% to about 99% decrease, about 70% to about 95% decrease, about 70% to about 90% decrease, about 70% to about 85% decrease, about 70% to about 80% decrease, about 70% to about 75% decrease, about 75% to about 99% decrease, about 75% to about 95% decrease, about 75% to about 90% decrease, about 75% to about 85% decrease, about 75% to about 80% decrease, about 80% to about 99% decrease, about 80% to about 95% decrease, about 80% to about 90% decrease, about 80% to about 85% decrease, about 85% to about 99% decrease, about 85% to about 95% decrease, about 85% to about 90% decrease, about 90% to about 99% decrease, about 90% to about 95% decrease, or about 95% to about 99% decrease) in one or more (e.g., two, three, four, five, six, seven, eight, or nine) of: the level of interferon-γ in GI tissue, the level of IL-1β in GI tissue, the level of IL-6 in GI tissue, the level of IL-22 in GI tissue, the level of IL-17A in the GI tissue, the level of TNFα in GI tissue, the level of IL-2 in GI tissue, and endoscopy score in a subject (e.g., as compared to the level in the subject prior to treatment or compared to a subject or population of subjects having a similar disease but receiving a placebo or a different treatment) (e.g., for a time period of between about 1 hour to about 30 days (e.g., or any of the subranges herein) following the first administration of an antibody or antigen-binding antibody fragment using any of the compositions or devices described herein. Exemplary methods for determining the endoscopy score are described herein and other methods for determining the endoscopy score are known in the art. Exemplary methods for determining the levels of interferon-γ, IL-1β, IL-6, IL-22, IL-17A, TNFα, and IL-2 are described herein. Additional methods for determining the levels of these cytokines are known in the art. In some examples, treatment can result in an increase (e.g., about 1% to about 500% increase, about 1% to about 400% increase, about 1% to about 300% increase, about 1% to about 200% increase, about 1% to about 150% increase, about 1% to about 100% increase, about 1% to about 90% increase, about 1% to about 80% increase, about 1% to about 70% increase, about 1% to about 60% increase, about 1% to about 50% increase, about 1% to about 40% increase, about 1% to about 30% increase, about 1% to about 20% increase, about 1% to about 10% increase, a 10% to about 500% increase, about 10% to about 400% increase, about 10% to about 300% increase, about 10% to about 200% increase, about 10% to about 150% increase, about 10% to about 100% increase, about 10% to about 90% increase, about 10% to about 80% increase, about 10% to about 70% increase, about 10% to about 60% increase, about 10% to about 50% increase, about 10% to about 40% increase, about 10% to about 30% increase, about 10% to about 20% increase, about 20% to about 500% increase, about 20% to about 400% increase, about 20% to about 300% increase, about 20% to about 200% increase, about 20% to about 150% increase, about 20% to about 100% increase, about 20% to about 90% increase, about 20% to about 80% increase, about 20% to about 70% increase, about 20% to about 60% increase, about 20% to about 50% increase, about 20% to about 40% increase, about 20% to about 30% increase, about 30% to about 500% increase, about 30% to about 400% increase, about 30% to about 300% increase, about 30% to about 200% increase, about 30% to about 150% increase, about 30% to about 100% increase, about 30% to about 90% increase, about 30% to about 80% increase, about 30% to about 70% increase, about 30% to about 60% increase, about 30% to about 50% increase, about 30% to about 40% increase, about 40% to about 500% increase, about 40% to about 400% increase, about 40% to about 300% increase, about 40% to about 200% increase, about 40% to about 150% increase, about 40% to about 100% increase, about 40% to about 90% increase, about 40% to about 80% increase, about 40% to about 70% increase, about 40% to about 60% increase, about 40% to about 50% increase, about 50% to about 500% increase, about 50% to about 400% increase, about 50% to about 300% increase, about 50% to about 200% increase, about 50% to about 150% increase, about 50% to about 100% increase, about 50% to about 90% increase, about 50% to about 80% increase, about 50% to about 70% increase, about 50% to about 60% increase, about 60% to about 500% increase, about 60% to about 400% increase, about 60% to about 300% increase, about 60% to about 200% increase, about 60% to about 150% increase, about 60% to about 100% increase, about 60% to about 90% increase, about 60% to about 80% increase, about 60% to about 70% increase, about 70% to about 500% increase, about 70% to about 400% increase, about 70% to about 300% increase, about 70% to about 200% increase, about 70% to about 150% increase, about 70% to about 100% increase, about 70% to about 90% increase, about 70% to about 80% increase, about 80% to about 500% increase, about 80% to about 400% increase, about 80% to about 300% increase, about 80% to about 200% increase, about 80% to about 150% increase, about 80% to about 100% increase, about 80% to about 90% increase, about 90% to about 500% increase, about 90% to about 400% increase, about 90% to about 300% increase, about 90% to about 200% increase, about 90% to about 150% increase, about 90% to about 100% increase, about 100% to about 500% increase, about 100% to about 400% increase, about 100% to about 300% increase, about 100% to about 200% increase, about 100% to about 150% increase, about 150% to about 500% increase, about 150% to about 400% increase, about 150% to about 300% increase, about 150% to about 200% increase, about 200% to about 500% increase, about 200% to about 400% increase, about 200% to about 300% increase, about 300% to about 500% increase, about 300% to about 400% increase, or about 400% to about 500% increase) in one or both of stool consistency score and weight of a subject (e.g., as compared to the level in the subject prior to treatment or compared to a subject or population of subjects having a similar disease but receiving a placebo or a different treatment) (e.g., for a time period of between about 1 hour to about 30 days (e.g., or any of the subranges herein) following the first administration of an antibody or antigen-binding antibody fragment using any of the compositions or devices described herein. Exemplary methods for determining stool consistency score are described herein. Additional methods for determining a stool consistency score are known in the art. In some examples, administration of an antibody or an antigen-binding antibody fragment using any of the devices or compositions described herein can result in a ratio of GI tissue concentration of the antibody or the antigen-binding antibody fragment to the blood, serum, or plasma concentration of the antibody or the antigen-binding antibody fragment of, e.g., about 2.8 to about 6.0, about 2.8 to about 5.8, about 2.8 to about 5.6, about 2.8 to about 5.4, about 2.8 to about 5.2, about 2.8 to about 5.0, about 2.8 to about 4.8, about 2.8 to about 4.6, about 2.8 to about 4.4, about 2.8 to about 4.2, about 2.8 to about 4.0, about 2.8 to about 3.8, about 2.8 to about 3.6, about 2.8 to about 3.4, about 2.8 to about 3.2, about 2.8 to about 3.0, about 3.0 to about 6.0, about 3.0 to about 5.8, about 3.0 to about 5.6, about 3.0 to about 5.4, about 3.0 to about 5.2, about 3.0 to about 5.0, about 3.0 to about 4.8, about 3.0 to about 4.6, about 3.0 to about 4.4, about 3.0 to about 4.2, about 3.0 to about 4.0, about 3.0 to about 3.8, about 3.0 to about 3.6, about 3.0 to about 3.4, about 3.0 to about 3.2, about 3.2 to about 6.0, about 3.2 to about 5.8, about 3.2 to about 5.6, about 3.2 to about 5.4, about 3.2 to about 5.2, about 3.2 to about 5.0, about 3.2 to about 4.8, about 3.2 to about 4.6, about 3.2 to about 4.4, about 3.2 to about 4.2, about 3.2 to about 4.0, about 3.2 to about 3.8, about 3.2 to about 3.6, about 3.2 to about 3.4, about 3.4 to about 6.0, about 3.4 to about 5.8, about 3.4 to about 5.6, about 3.4 to about 5.4, about 3.4 to about 5.2, about 3.4 to about 5.0, about 3.4 to about 4.8, about 3.4 to about 4.6, about 3.4 to about 4.4, about 3.4 to about 4.2, about 3.4 to about 4.0, about 3.4 to about 3.8, about 3.4 to about 3.6, about 3.6 to about 6.0, about 3.6 to about 5.8, about 3.6 to about 5.6, about 3.6 to about 5.4, about 3.6 to about 5.2, about 3.6 to about 5.0, about 3.6 to about 4.8, about 3.6 to about 4.6, about 3.6 to about 4.4, about 3.6 to about 4.2, about 3.6 to about 4.0, about 3.6 to about 3.8, about 3.8 to about 6.0, about 3.8 to about 5.8, about 3.8 to about 5.6, about 3.8 to about 5.4, about 3.8 to about 5.2, about 3.8 to about 5.0, about 3.8 to about 4.8, about 3.8 to about 4.6, about 3.8 to about 4.4, about 3.8 to about 4.2, about 3.8 to about 4.0, about 4.0 to about 6.0, about 4.0 to about 5.8, about 4.0 to about 5.6, about 4.0 to about 5.4, about 4.0 to about 5.2, about 4.0 to about 5.0, about 4.0 to about 4.8, about 4.0 to about 4.6, about 4.0 to about 4.4, about 4.0 to about 4.2, about 4.2 to about 6.0, about 4.2 to about 5.8, about 4.2 to about 5.6, about 4.2 to about 5.4, about 4.2 to about 5.2, about 4.2 to about 5.0, about 4.2 to about 4.8, about 4.2 to about 4.6, about 4.2 to about 4.4, about 4.4 to about 6.0, about 4.4 to about 5.8, about 4.4 to about 5.6, about 4.4 to about 5.4, about 4.4 to about 5.2, about 4.4 to about 5.0, about 4.4 to about 4.8, about 4.4 to about 4.6, about 4.6 to about 6.0, about 4.6 to about 5.8, about 4.6 to about 5.6, about 4.6 to about 5.4, about 4.6 to about 5.2, about 4.6 to about 5.0, about 4.6 to about 4.8, about 4.8 to about 6.0, about 4.8 to about 5.8, about 4.8 to about 5.6, about 4.8 to about 5.4, about 4.8 to about 5.2, about 4.8 to about 5.0, about 5.0 to about 6.0, about 5.0 to about 5.8, about 5.0 to about 5.6, about 5.0 to about 5.4, about 5.0 to about 5.2, about 5.2 to about 6.0, about 5.2 to about 5.8, about 5.2 to about 5.6, about 5.2 to about 5.4, about 5.4 to about 6.0, about 5.4 to about 5.8, about 5.4 to about 5.6, about 5.6 to about 6.0, about 5.6 to about 5.8, or about 5.8 to about 6.0. Accordingly, in some embodiments, a method of treatment disclosed herein can include determining the ratio of the level of the JAK inhibitor in the GI tissue to the level of the JAK inhibitor in the blood, serum, or plasma of a subject at substantially the same time point following administration of the device is about 2.8 to about 6.0. Exemplary methods for measuring the concentration of an antibody or an antigen-binding antibody fragment in the plasma or the GI tissue of a subject are described herein. Additional methods for measuring the concentration of an antibody or an antigen-binding antibody fragment in the plasma or the GI tissue of a subject are known in the art. Accordingly, in some embodiments, a method of treatment disclosed herein includes determining the level of the JAK inhibitor in the GI tissue (e.g., one or more of any of the exemplary GI tissues described herein). In some embodiments, a method of treatment disclosed herein can include determining the level of JAK inhibitor in one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa. In some embodiments, a method of treatment disclosed herein includes determining that the level of the JAK inhibitor in the GI tissue (e.g., one or more of any of the exemplary types of GI tissues described herein) at a time point following administration of the device is higher than the level of JAK inhibitor in the GI tissue at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor. In some embodiments, a method of treatment disclosed herein can include determining that the level of the JAK inhibitor in one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa at a time point following administration of the device is higher than the level of the JAK inhibitor in one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor. In some embodiments, a method of treatment disclosed herein includes determining the level of JAK inhibitor in the feces of the subject. In some embodiments, a method of treatment disclosed herein includes determining the level of JAK inhibitor in the GI tissue, e.g., in one or more (e.g., two, three, or four) of the lumen/superficial mucosa, the lamina propria, the submucosa, and the tunica muscularis/serosa within a time period of about 10 minutes to about 10 hours following administration of the device. In some embodiments, a method of treatment as disclosed herein comprises determining the level of the JAK inhibitor at the location of disease following administration of the device. In some embodiments, a method of treatment as disclosed herein comprises determining that the level of JAK inhibitor at the location of disease at a time point following administration of the device is higher than the level of the JAK inhibitor at the same location of disease at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor. In some embodiments, a method of treatment as disclosed herein comprises determining that the level of JAK inhibitor in plasma in a subject at a time point following administration of the device is lower than the level of the JAK inhibitor in plasma in a subject at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor. In some embodiments, a method of treatment as disclosed herein comprises determining the level of the JAK inhibitor in the tissue of the subject within a time period of about 10 minutes to 10 hours following administration of the device. Some examples of any of the methods described herein can, e.g., result in a selective suppression of a local inflammatory response (e.g., an inflammatory response in local GI tissue), while maintaining the systemic immune response (e.g., blood). The GI tissue may be, for example, GI tissue proximate to one or more sites of disease. FAs used herein, “GI content” refers to the content of the gastrointestinal (GI) tract, such as the content of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum, more particularly of the proximal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, or of the distal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon. Accordingly, in some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the dudodenum tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the jejunum tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the ileum tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the cecum tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the ascending colon tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the transverse colon tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the descending colon tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some embodiments, the methods described herein can result in a selective suppression of the inflammatory response in the sigmoid colon tissue proximate to one or more sites of disease, while maintaining the systemic immune response. In some examples, the methods described herein can result in a 1% increase to 500% increase (e.g., a 1% increase to 450% increase, a 1% increase to 400% increase, a 1% increase to 350% increase, a 1% increase to 300% increase, a 1% increase to 250% increase, a 1% increase to 200% increase, a 1% increase to 190% increase, a 1% increase to 180% increase, a 1% increase to 170% increase, a 1% increase to 160% increase, a 1% increase to 150% increase, a 1% increase to 140% increase, a 1% increase to 130% increase, a 1% increase to 120% increase, a 1% increase to 110% increase, a 1% increase to 100% increase, a 1% increase to 90% increase, a 1% increase to 80% increase, a 1% increase to 70% increase, a 1% increase to 60% increase, a 1% increase to 50% increase, a 1% increase to 40% increase, a 1% increase to 30% increase, a 1% increase to 25% increase, a 1% increase to 20% increase, a 1% increase to 15% increase, a 1% increase to 10% increase, a 1% increase to 5% increase, a 5% increase to 500% increase, a 5% increase to 450% increase, a 5% increase to 400% increase, a 5% increase to 350% increase, a 5% increase to 300% increase, a 5% increase to 250% increase, a 5% increase to 200% increase, a 5% increase to 190% increase, a 5% increase to 180% increase, a 5% increase to 170% increase, a 5% increase to 160% increase, a 5% increase to 150% increase, a 5% increase to 140% increase, a 5% increase to 130% increase, a 5% increase to 120% increase, a 5% increase to 110% increase, a 5% increase to 100% increase, a 5% increase to 90% increase, a 5% increase to 80% increase, a 5% increase to 70% increase, a 5% increase to 60% increase, a 5% increase to 50% increase, a 5% increase to 40% increase, a 5% increase to 30% increase, a 5% increase to 25% increase, a 5% increase to 20% increase, a 5% increase to 15% increase, a 5% increase to 10% increase, a 10% increase to 500% increase, a 10% increase to 450% increase, a 10% increase to 400% increase, a 10% increase to 350% increase, a 10% increase to 300% increase, a 10% increase to 250% increase, a 10% increase to 200% increase, a 10% increase to 190% increase, a 10% increase to 180% increase, a 10% increase to 170% increase, a 10% increase to 160% increase, a 10% increase to 150% increase, a 10% increase to 140% increase, a 10% increase to 130% increase, a 10% increase to 120% increase, a 10% increase to 110% increase, a 10% increase to 100% increase, a 10% increase to 90% increase, a 10% increase to 80% increase, a 10% increase to 70% increase, a 10% increase to 60% increase, a 10% increase to 50% increase, a 10% increase to 40% increase, a 10% increase to 30% increase, a 10% increase to 25% increase, a 10% increase to 20% increase, a 10% increase to 15% increase, a 15% increase to 500% increase, a 15% increase to 450% increase, a 15% increase to 400% increase, a 15% increase to 350% increase, a 15% increase to 300% increase, a 15% increase to 250% increase, a 15% increase to 200% increase, a 15% increase to 190% increase, a 15% increase to 180% increase, a 15% increase to 170% increase, a 15% increase to 160% increase, a 15% increase to 150% increase, a 15% increase to 140% increase, a 15% increase to 130% increase, a 15% increase to 120% increase, a 15% increase to 110% increase, a 15% increase to 100% increase, a 15% increase to 90% increase, a 15% increase to 80% increase, a 15% increase to 70% increase, a 15% increase to 60% increase, a 15% increase to 50% increase, a 15% increase to 40% increase, a 15% increase to 30% increase, a 15% increase to 25% increase, a 15% increase to 20% increase, a 20% increase to 500% increase, a 20% increase to 450% increase, a 20% increase to 400% increase, a 20% increase to 350% increase, a 20% increase to 300% increase, a 20% increase to 250% increase, a 20% increase to 200% increase, a 20% increase to 190% increase, a 20% increase to 180% increase, a 20% increase to 170% increase, a 20% increase to 160% increase, a 20% increase to 150% increase, a 20% increase to 140% increase, a 20% increase to 130% increase, a 20% increase to 120% increase, a 20% increase to 110% increase, a 20% increase to 100% increase, a 20% increase to 90% increase, a 20% increase to 80% increase, a 20% increase to 70% increase, a 20% increase to 60% increase, a 20% increase to 50% increase, a 20% increase to 40% increase, a 20% increase to 30% increase, a 20% increase to 25% increase, a 25% increase to 500% increase, a 25% increase to 450% increase, a 25% increase to 400% increase, a 25% increase to 350% increase, a 25% increase to 300% increase, a 25% increase to 250% increase, a 25% increase to 200% increase, a 25% increase to 190% increase, a 25% increase to 180% increase, a 25% increase to 170% increase, a 25% increase to 160% increase, a 25% increase to 150% increase, a 25% increase to 140% increase, a 25% increase to 130% increase, a 25% increase to 120% increase, a 25% increase to 110% increase, a 25% increase to 100% increase, a 25% increase to 90% increase, a 25% increase to 80% increase, a 25% increase to 70% increase, a 25% increase to 60% increase, a 25% increase to 50% increase, a 25% increase to 40% increase, a 25% increase to 30% increase, a 30% increase to 500% increase, a 30% increase to 450% increase, a 30% increase to 400% increase, a 30% increase to 350% increase, a 30% increase to 300% increase, a 30% increase to 250% increase, a 30% increase to 200% increase, a 30% increase to 190% increase, a 30% increase to 180% increase, a 30% increase to 170% increase, a 30% increase to 160% increase, a 30% increase to 150% increase, a 30% increase to 140% increase, a 30% increase to 130% increase, a 30% increase to 120% increase, a 30% increase to 110% increase, a 30% increase to 100% increase, a 30% increase to 90% increase, a 30% increase to 80% increase, a 30% increase to 70% increase, a 30% increase to 60% increase, a 30% increase to 50% increase, a 30% increase to 40% increase, a 40% increase to 500% increase, a 40% increase to 450% increase, a 40% increase to 400% increase, a 40% increase to 350% increase, a 40% increase to 300% increase, a 40% increase to 250% increase, a 40% increase to 200% increase, a 40% increase to 190% increase, a 40% increase to 180% increase, a 40% increase to 170% increase, a 40% increase to 160% increase, a 40% increase to 150% increase, a 40% increase to 140% increase, a 40% increase to 130% increase, a 40% increase to 120% increase, a 40% increase to 110% increase, a 40% increase to 100% increase, a 40% increase to 90% increase, a 40% increase to 80% increase, a 40% increase to 70% increase, a 40% increase to 60% increase, a 40% increase to 50% increase, a 50% increase to 500% increase, a 50% increase to 450% increase, a 50% increase to 400% increase, a 50% increase to 350% increase, a 50% increase to 300% increase, a 50% increase to 250% increase, a 50% increase to 200% increase, a 50% increase to 190% increase, a 50% increase to 180% increase, a 50% increase to 170% increase, a 50% increase to 160% increase, a 50% increase to 150% increase, a 50% increase to 140% increase, a 50% increase to 130% increase, a 50% increase to 120% increase, a 50% increase to 110% increase, a 50% increase to 100% increase, a 50% increase to 90% increase, a 50% increase to 80% increase, a 50% increase to 70% increase, a 50% increase to 60% increase, a 60% increase to 500% increase, a 60% increase to 450% increase, a 60% increase to 400% increase, a 60% increase to 350% increase, a 60% increase to 300% increase, a 60% increase to 250% increase, a 60% increase to 200% increase, a 60% increase to 190% increase, a 60% increase to 180% increase, a 60% increase to 170% increase, a 60% increase to 160% increase, a 60% increase to 150% increase, a 60% increase to 140% increase, a 60% increase to 130% increase, a 60% increase to 120% increase, a 60% increase to 110% increase, a 60% increase to 100% increase, a 60% increase to 90% increase, a 60% increase to 80% increase, a 60% increase to 70% increase, a 70% increase to 500% increase, a 70% increase to 450% increase, a 70% increase to 400% increase, a 70% increase to 350% increase, a 70% increase to 300% increase, a 70% increase to 250% increase, a 70% increase to 200% increase, a 70% increase to 190% increase, a 70% increase to 180% increase, a 70% increase to 170% increase, a 70% increase to 160% increase, a 70% increase to 150% increase, a 70% increase to 140% increase, a 70% increase to 130% increase, a 70% increase to 120% increase, a 70% increase to 110% increase, a 70% increase to 100% increase, a 70% increase to 90% increase, a 70% increase to 80% increase, a 80% increase to 500% increase, a 80% increase to 450% increase, a 80% increase to 400% increase, a 80% increase to 350% increase, a 80% increase to 300% increase, a 80% increase to 250% increase, a 80% increase to 200% increase, a 80% increase to 190% increase, a 80% increase to 180% increase, a 80% increase to 170% increase, a 80% increase to 160% increase, a 80% increase to 150% increase, a 80% increase to 140% increase, a 80% increase to 130% increase, a 80% increase to 120% increase, a 80% increase to 110% increase, a 80% increase to 100% increase, a 80% increase to 90% increase, a 90% increase to 500% increase, a 90% increase to 450% increase, a 90% increase to 400% increase, a 90% increase to 350% increase, a 90% increase to 300% increase, a 90% increase to 250% increase, a 90% increase to 200% increase, a 90% increase to 190% increase, a 90% increase to 180% increase, a 90% increase to 170% increase, a 90% increase to 160% increase, a 90% increase to 150% increase, a 90% increase to 140% increase, a 90% increase to 130% increase, a 90% increase to 120% increase, a 90% increase to 110% increase, a 90% increase to 100% increase, a 100% increase to 500% increase, a 100% increase to 450% increase, a 100% increase to 400% increase, a 100% increase to 350% increase, a 100% increase to 300% increase, a 100% increase to 250% increase, a 100% increase to 200% increase, a 100% increase to 190% increase, a 100% increase to 180% increase, a 100% increase to 170% increase, a 100% increase to 160% increase, a 100% increase to 150% increase, a 100% increase to 140% increase, a 100% increase to 130% increase, a 100% increase to 120% increase, a 100% increase to 110% increase, a 110% increase to 500% increase, a 110% increase to 450% increase, a 110% increase to 400% increase, a 110% increase to 350% increase, a 110% increase to 300% increase, a 110% increase to 250% increase, a 110% increase to 200% increase, a 110% increase to 190% increase, a 110% increase to 180% increase, a 110% increase to 170% increase, a 110% increase to 160% increase, a 110% increase to 150% increase, a 110% increase to 140% increase, a 110% increase to 130% increase, a 110% increase to 120% increase, a 120% increase to 500% increase, a 120% increase to 450% increase, a 120% increase to 400% increase, a 120% increase to 350% increase, a 120% increase to 300% increase, a 120% increase to 250% increase, a 120% increase to 200% increase, a 120% increase to 190% increase, a 120% increase to 180% increase, a 120% increase to 170% increase, a 120% increase to 160% increase, a 120% increase to 150% increase, a 120% increase to 140% increase, a 120% increase to 130% increase, a 130% increase to 500% increase, a 130% increase to 450% increase, a 130% increase to 400% increase, a 130% increase to 350% increase, a 130% increase to 300% increase, a 130% increase to 250% increase, a 130% increase to 200% increase, a 130% increase to 190% increase, a 130% increase to 180% increase, a 130% increase to 170% increase, a 130% increase to 160% increase, a 130% increase to 150% increase, a 130% increase to 140% increase, a 140% increase to 500% increase, a 140% increase to 450% increase, a 140% increase to 400% increase, a 140% increase to 350% increase, a 140% increase to 300% increase, a 140% increase to 250% increase, a 140% increase to 200% increase, a 140% increase to 190% increase, a 140% increase to 180% increase, a 140% increase to 170% increase, a 140% increase to 160% increase, a 140% increase to 150% increase, a 150% increase to 500% increase, a 150% increase to 450% increase, a 150% increase to 400% increase, a 150% increase to 350% increase, a 150% increase to 300% increase, a 150% increase to 250% increase, a 150% increase to 200% increase, a 150% increase to 190% increase, a 150% increase to 180% increase, a 150% increase to 170% increase, a 150% increase to 160% increase, a 160% increase to 500% increase, a 160% increase to 450% increase, a 160% increase to 400% increase, a 160% increase to 350% increase, a 160% increase to 300% increase, a 160% increase to 250% increase, a 160% increase to 200% increase, a 160% increase to 190% increase, a 160% increase to 180% increase, a 160% increase to 170% increase, a 170% increase to 500% increase, a 170% increase to 450% increase, a 170% increase to 400% increase, a 170% increase to 350% increase, a 170% increase to 300% increase, a 170% increase to 250% increase, a 170% increase to 200% increase, a 170% increase to 190% increase, a 170% increase to 180% increase, a 180% increase to 500% increase, a 180% increase to 450% increase, a 180% increase to 400% increase, a 180% increase to 350% increase, a 180% increase to 300% increase, a 180% increase to 250% increase, a 180% increase to 200% increase, a 180% increase to 190% increase, a 190% increase to 500% increase, a 190% increase to 450% increase, a 190% increase to 400% increase, a 190% increase to 350% increase, a 190% increase to 300% increase, a 190% increase to 250% increase, a 190% increase to 200% increase, a 200% increase to 500% increase, a 200% increase to 450% increase, a 200% increase to 400% increase, a 200% increase to 350% increase, a 200% increase to 300% increase, a 200% increase to 250% increase, a 250% increase to 500% increase, a 250% increase to 450% increase, a 250% increase to 400% increase, a 250% increase to 350% increase, a 250% increase to 300% increase, a 300% increase to 500% increase, a 300% increase to 450% increase, a 300% increase to 400% increase, a 300% increase to 350% increase, a 350% increase to 500% increase, a 350% increase to 450% increase, a 350% increase to 400% increase, a 400% increase to 500% increase, a 400% increase to 450% increase, or a 450% increase to 500% increase) in one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of: the plasma, serum, or blood level of IL-6; the plasma, serum, or blood level of IL-2; the plasma, serum, or blood level of IL-1β; the plasma, serum, or blood level of TNFα; the plasma, serum, or blood level of IL-17A; the plasma, serum, or blood level of IL-22; the plasma, serum, or blood level of interferon-γ; the level of blood Th memory cells (CD44+CD45RB−CD4+cells); and the level of α4β7 expression in blood cells; e.g., each as compared to the corresponding level in a subject systemically administered the same dose of the same JAK inhibitor. Methods for determining the plasma, serum, or blood level of IL-6; the plasma, serum, or blood level of IL-2; the plasma, serum, or blood level of IL-1β; the plasma, serum, or blood level of TNFα; the plasma, serum, or blood level of IL-17A; the plasma, serum, or blood level of IL-22; the plasma, serum, or blood level of interferon-γ; the level of blood Th memory cells (CD44+CD45RB−CD4+cells); and the level of α4β7 expression in blood cells are known in the art. In some examples of any of the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of: the level of interferon-γ in GI tissue or GI content; the level of IL-1β in GI tissue or GI content; the level of IL-6 in GI tissue or GI content; the level of IL-22 in GI tissue or GI content; the level of IL-17A in GI tissue or GI content; the level of TNFα in GI tissue or GI content; and the level of IL-2 in GI tissue or GI content, e.g., as compared to the corresponding level in a subject not administered a treatment, or not administered a JAK inhibitor locally as disclosed herein. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the duodenum tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the ileum tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the jejunum tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the cecum tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the ascending colon tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the transverse colon tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the decending colon tissue proximate to one or more sites of disease. Accordingly, in some embodiments, the methods described herein can result, e.g., in a 1% to 99% decrease (or any of the subranges of this range described herein) in one or more (e.g., two, three, four, five, six, or seven) of the level of interferon-γ; the level of IL-1β; the level of IL-6; the level of IL-22; the level of IL-17A; the level of TNFα; and the level of IL-2, in the sigmoid colon tissue proximate to one or more sites of disease. In some embodiments, the JAK inhibitor is delivered to the location by a process that does not comprise systemic transport of the JAK inhibitor. In some embodiments, the amount of the JAK inhibitor that is administered is from about 1 mg to about 500 mg. In some embodiments, the amount of the JAK inhibitor that is administered is from about 1 mg to about 100 mg. In some embodiments, the amount of the JAK inhibitor that is administered is from about 10 mg to about 300 mg. In some embodiments, the amount of the JAK inhibitor that is administered is from about 5 mg to about 50 mg. In some embodiments, the amount of the JAK inhibitor that is administered is from about 10 mg to about 50 mg. In some embodiments, the amount of the JAK inhibitor that is administered is from about 5 mg to about 40 mg. In some embodiments, the amount of the JAK inhibitor that is administered is less than an amount that is effective when the JAK inhibitor is delivered systemically. In some embodiments, the amount of the JAK inhibitor that is administered is an induction dose. In some embodiments, such induction dose is effective to induce remission of the TNF and cytokine storm and healing of acute inflammation and lesions. In some embodiments, the induction dose is administered once a day. In some embodiments, the induction dose is administered once every three days. In some embodiments, the induction dose is administered once a week. In some embodiments, the induction dose is administered once a day, once every three days, or once a week, over a period of about 6-8 weeks. In some embodiments, the method comprises administering (i) an amount of the JAK inhibitor that is an induction dose, and (ii) an amount of the JAK inhibitor that is a maintenance dose, in this order. In some embodiments, step (ii) is repeated one or more times. In some embodiments, the induction dose is equal to the maintenance dose. In some embodiments, the induction dose is greater than the maintenance dose. In some embodiments, the induction dose is five times greater than the maintenance dose. In some embodiments, the induction dose is two times greater than the maintenance dose. In some embodiments, the induction dose is the same as or higher than an induction dose administered systemically for treatment of the same disorder to a subject. In more particular embodiments, the induction dose is the same as or higher than an induction dose administered systemically for treatment of the same disorder to a subject, and the maintenance dose is lower than the maintenance dose administered systemically for treatment of the same disorder to a subject. In some embodiments, the induction dose is the same as or higher than an induction dose administered systemically for treatment of the same disorder to a subject, and the maintenance dose is higher than the maintenance dose administered systemically for treatment of the same disorder to a subject. In some embodiments an induction dose of JAK inhibitor and a maintenance dose of JAK inhibitor are each administered to the subject by administering a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor, wherein the pharmaceutical composition is a device. In some embodiments an induction dose of JAK inhibitor is administered to the subject in a different manner from the maintenance dose. As an example, the induction dose may be administered systemically. In some embodiments, the induction dose may be administered other than orally. As an example, the induction dose may be administered rectally. As an example, the induction dose may be administered intravenously. As an example, the induction dose may be administered subcutaneously. In some embodiments, the induction dose may be administered by spray catheter. In some embodiments, the concentration of the JAK inhibitor delivered at the location in the gastrointestinal tract is 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, 2000% greater than the concentration of JAK inhibitor in plasma. In some embodiments, the method provides a concentration of the JAK inhibitor at a location that is a site of disease or proximate to a site of disease that is 2-100 times greater than at a location that is not a site of disease or proximate to a site of disease. In some embodiments, the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract as a single bolus. In some embodiments, the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract as more than one bolus. In some embodiments, the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract in a continuous manner. In some embodiments, the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract over a time period of 20 or more minutes. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 10 μg/ml. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 3 μg/ml. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 1 μg/ml. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 0.3 μg/ml. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 0.1 μg/ml. In some embodiments, the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 0.01 μg/ml. In some embodiments, the values of the concentration of the JAK inhibitor in the plasma of the subject provided herein refer to Ctrough, that is, the lowest value of the concentration prior to administration of the next dose. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 10 μg/ml. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 3 μg/ml. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 1 μg/ml. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 0.3 μg/ml. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 0.1 μg/ml. In some embodiments, the method provides a concentration Cmaxof the JAK inhibitor in the plasma of the subject that is less than 0.01 μg/ml. In some embodiments, the method does not comprise delivering a JAK inhibitor rectally to the subject. In some embodiments, the method does not comprise delivering a JAK inhibitor via an enema to the subject. In some embodiments, the method does not comprise delivering a JAK inhibitor via suppository to the subject. In some embodiments, the method does not comprise delivering a JAK inhibitor via instillation to the rectum of a subject. In some embodiments, the methods disclosed herein comprise producing a therapeutically effective degradation product of the JAK inhibitor in the gastrointestinal tract. In some embodiments, the degradation product is a therapeutic antibody fragment. In some embodiments, a therapeutically effective amount of the degradation product is produced. In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, a multivalent antibody, or a fragment thereof. In some embodiments, an antibody can be a scFv-Fc (Sokolowska-Wedzina et al.,Mol. Cancer Res.15(8):1040-1050, 2017), a VHH domain (Li et al.,Immunol. Lett.188:89-95, 2017), a VNAR domain (Hasler et al.,Mol. Immunol.75:28-37, 2016), a (scFv)2, a minibody (Kim et al.,PLoS One10(1):e113442, 2014), or a BiTE. In some embodiments, an antibody can be a DVD-Ig (Wu et al.,Nat. Biotechnol.25(11):1290-1297, 2007; WO 08/024188; WO 07/024715), and a dual-affinity re-targeting antibody (DART) (Tsai et al.,Mol. Ther. Oncolytics3:15024, 2016), a triomab (Chelius et al.,MAbs2(3):309-319, 2010), kih IgG with a common LC (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a crossmab (Regula et al.,EMBO Mol. Med.9(7):985, 2017), an ortho-Fab IgG (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a 2-in-1-IgG (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), IgG-scFv (Cheal et al.,Mol. Cancer Ther.13(7):1803-1812, 2014), scFv2-Fc (Natsume et al.,J. Biochem.140(3):359-368, 2006), a bi-nanobody (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), tanden antibody (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a DART-Fc (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), a scFv-HSA-scFv (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), DNL-Fab3 (Kontermann et al.,Drug Discovery Today20(7):838-847, 2015), DAF (two-in-one or four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair antibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab, kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody (e.g., antibodies derived fromCamelus bactriamus, Calelus dromaderius, orLama paccos) (U.S. Pat. No. 5,759,808; Stijlemans et al.,J. Biol. Chem.279:1256-1261, 2004; Dumoulin et al.,Nature424:783-788, 2003; and Pleschberger et al.,Bioconjugate Chem.14:440-448, 2003), nanobody-HSA, a diabody (e.g., Poljak,Structure2(12):1121-1123, 1994; Hudson et al.,J. Immunol. Methods23(1-2):177-189, 1999), a TandAb (Reusch et al.,mAbs6(3):727-738, 2014), scDiabody (Cuesta et al.,Trends in Biotechnol.28(7):355-362, 2010), scDiabody-CH3 (Sanz et al.,Trends in Immunol.25(2):85-91, 2004), Diabody-CH3 (Guo et al. Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody (Huston et al.,Human Antibodies10(3-4):127-142, 2001; Wheeler et al.,Mol. Ther.8(3):355-366, 2003; Stocks,Drug Discov. Today9(22):960-966, 2004), dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HSA, tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2. Non-limiting examples of an antigen-binding fragment of an antibody include an Fv fragment, a Fab fragment, a F(ab′)2 fragment, and a Fab′ fragment. Additional examples of an antigen-binding fragment of an antibody is an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM). In some embodiments, an antibody can be an IgNAR, a bispecific antibody (Milstein and Cuello,Nature305:537-539, 1983; Suresh et al.,Methods in Enzymology121:210, 1986; WO 96/27011; Brennan et al.,Science229:81, 1985; Shalaby et al.,J. Exp. Med.175:217-225, 1992; Kolstelny et al.,J. Immunol.148(5):1547-1553, 1992; Hollinger et al.,Proc. Natl. Acad. Sci. U.S.A.90:6444-6448, 1993; Gruber et al.,J. Immunol.152:5368, 1994; Tuft et al.,J. Immunol.147:60, 1991), a bispecific diabody, a triabody (Schoonooghe et al.,BMC Biotechnol.9:70, 2009), a tetrabody, scFv-Fc knobs-into-holes, a scFv-Fc-scFv, a (Fab′scFv)2, a V-IgG, a IvG-V, a dual V domain IgG, a heavy chain immunoglobulin or a camelid (Holt et al.,Trends Biotechnol.21(11):484-490, 2003), an intrabody, a monoclonal antibody (e.g., a human or humanized monoclonal antibody), a heteroconjugate antibody (e.g., U.S. Pat. No. 4,676,980), a linear antibody (Zapata et al.,Protein Eng.8(10:1057-1062, 1995), a trispecific antibody (Tuft et al.,J. Immunol.147:60, 1991), a Fabs-in-Tandem immunoglobulin (WO 15/103072), or a humanized camelid antibody. In some embodiments, the methods comprising administering the JAK inhibitor in the manner disclosed herein disclosed herein result in a reduced immunosuppressive properties relative to methods of administration of the JAK inhibitor systemically. In some embodiments, the methods comprising administering the JAK inhibitor in the manner disclosed herein disclosed herein result in reduced immunogenicity relative to methods of administration of the JAK inhibitor systemically. Methods for Treating Colitis in Subjects in Immune-Oncology Therapy In some embodiments, provided herein is a method for treating colitis as disclosed herein in a subject, comprising releasing a JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor, wherein the colitis is associated with treatment of the subject with one or more immuno-oncology agents. In some embodiments, the pharmaceutical composition is an ingestible device. In some embodiments, the pharmaceutical composition is an ingestible device and the method comprises administering orally to the subject the pharmaceutical composition. In some embodiments, at least one of the one or more immuno-oncology agents is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a chemotherapeutic immunomodulator. In some embodiments, the chemotherapeutic immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets an immune checkpoint protein or decreases an activity of an immune checkpoint protein selected from the group of CTLA-4, PD-1, PD-L1, PD-1-PD-L1, PD-1-PD-L2, interleukin 2 (IL 2), indoleamine 2,3-dioxygenase (IDO), IL 10, transforming growth factor-β (TGFβ), T cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), Galectin 9-TIM3, Phosphatidylserine-TIM3, lymphocyte activation gene 3 protein (LAG3), MHC class II-LAG3, 4 1BB-4 1BB ligand, OX40-OX40 ligand, GITR, GITR ligand-GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA, HVEM-CD160, HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80-PDL-1, PDL2-CD80, CD244, CD48-CD244, CD244, ICOS, ICOS-ICOS ligand, B7 H3, B7 H4, VISTA, TMIGD2, HHLA2-TMIGD2, Butyrophilins, including BTNL2, Siglec family, TIGIT and PVR family members, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICA and MICB, CD244, CD28, CD86-CD28, CD86-CTLA, CD80-CD28, CD39, CD73 Adenosine-CD39-CD73, CXCR4-CXCL12, Phosphatidylserine, TIM3, Phosphatidylserine-TIM3, SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, and CD155. In some examples, the immune checkpoint inhibitor is selected from the group consisting of: Urelumab, PF 05082566, MEDI6469, TRX518, Varlilumab, CP 870893, Pembrolizumab (PD1), Nivolumab (PD1), Atezolizumab (formerly MPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab (PD-L1), PDR001 (PD1), BMS 986016, MGA271, Lirilumab, IPH2201, Emactuzumab, INCB024360, Galunisertib, Ulocuplumab, BKT140, Bavituximab, CC 90002, Bevacizumab, and MNRP1685A, and MGA271. In some examples, the immune checkpoint inhibitor targets or decreases an activity of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the antibody is ipilimumab or tremelimumab. In some examples, the immune checkpoint inhibitor targets PD1 or PD-L1. In some examples, the immune checkpoint inhibitor is selected from nivolumab, lambroizumab, and BMS-936559. In some embodiments, at least one of the one or more immuno-oncology agents is a T-cell capable of expressing a chimeric antigen receptor (CAR). In some embodiments, at least one of the one or more immuno-oncology agents is a PI-3-kinase inhibitor. In some embodiments, the treatment of the subject with one or more immuno-oncology agents further comprises treatment of the subject with an immunosuppressant. In some embodiments, provided herein is a method for reducing the development of colitis in a subject administered an immuno-oncology agent, comprising releasing a JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor. In some embodiments, the pharmaceutical composition is an ingestible device. In some embodiments, the pharmaceutical composition is an ingestible device and the method comprises administering orally to the subject the pharmaceutical composition. In some embodiments of these methods, a subject is administered at least one dose of an immuno-oncology agent prior to administering a pharmaceutical composition comprising any of the devices described herein as described herein to the subject. In some embodiments of these methods, a subject is first administered any of the devices as described herein, prior to administration of the first dose of the immuno-oncology agent. In some embodiments of these methods, the immuno-oncology agent is administered at substantially the same time as the device described herein. Also provided herein are methods of treating a subject having a cancer that include: administering a first dose of an immuno-oncology agent to the subject; monitoring one or more biomarkers, markers, or symptoms of colitis (e.g., any of the biomarkers, markers, or symptoms of colitis described herein or known in the art); identifying a subject having a level of a biomarker or marker, or having a symptom of colitis; and releasing a JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of disease, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor. In some embodiments, the pharmaceutical composition is an ingestible device. In some embodiments, the pharmaceutical composition is an ingestible device and the method comprises administering orally to the subject the pharmaceutical composition. Also provided herein are methods of reducing the severity of colitis in a subject having a cancer and administered an immuno-oncology agent that include administering to the subject any of the devices described herein. In some embodiments, provided herein is a method for treating colitis in a subject comprising:determining that the subject has colitis associated with treatment of the subject with one or more immuno-oncology agents; andreleasing a JAK inhibitor at a location in the gastrointestinal tract of the subject that is proximate to one or more sites of colitis, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor. In some embodiments, the pharmaceutical composition is an ingestible device. In some embodiments, the pharmaceutical composition is an ingestible device and the method comprises administering orally to the subject the pharmaceutical composition. In some embodiments, provided herein is a method for treating colitis in a subject comprising:determining that the subject has colitis associated with treatment of the subject with one or more immuno-oncology agents; andadministering to the subject an ingestible device comprising any of the JAK inhibitors described herein, to treat the colitis. In some embodiments, provided herein is a method for treating colitis,comprising releasing a JAK inhibitor at a location in the gastrointestinal tract of a subject who has been determined to have colitis associated with treatment of the subject with one or more immuno-oncology agents, wherein the location is proximate to one or more sites of colitis, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor. In some embodiments, the pharmaceutical composition is an ingestible device. In some embodiments, the pharmaceutical composition is an ingestible device and the method comprises administering orally to the subject the pharmaceutical composition. In some embodiments, provided herein is a method for treating colitis, comprising administering an ingestible device comprising any of the JAK inhibitors described herein to a subject who has been determined to have colitis associated with treatment of the subject with one or more immuno-oncology agents. In some embodiments, provided herein is an ingestible device comprising any of the JAK inhibitors described herein for treating colitis associated with treatment of a subject with one or more immuno-oncology agents. Monitoring Progress of Disease In some embodiments, the methods provided herein comprise monitoring the progress of the disease. In some embodiments, monitoring the progress of the disease comprises measuring the levels of IBD serological markers. In some embodiments, monitoring the progress of the disease comprises determining mucosal healing at the location of release. In some embodiments, monitoring the progress of the disease comprises determining the Crohn's Disease Activity Index (CDAI) over a period of about 6-8 weeks, or over a period of about 52 weeks, following administration of the JAK inhibitor. In some embodiments, monitoring the progress of the disease comprises determining the Harvey-Bradshaw Index (HBI) following administration of the JAK inhibitor. Possible markers may include the following: anti-glycan antibodies: anti-Saccharomices cerevisiae(ASCA); anti-laminaribioside (ALCA); anti-chitobioside (ACCA); anti-mannobioside (AMCA); anti-laminarin (anti-L); anti-chitin (anti-C) antibodies: anti-outer membrane porin C (anti-OmpC), anti-Cbir1 flagellin; anti-12 antibody; autoantibodies targeting the exocrine pancreas (PAB); perinuclear anti-neutrophil antibody (pANCA). In some embodiments, monitoring the progress of the disease comprises measuring JAK inhibitor levels in serum over a period of about 1-14 weeks, such as about 6-8 weeks following administration of the JAK inhibitor, including at the 6-8 week time point. In some embodiments, monitoring the progress of the disease comprises measuring JAK inhibitor levels in serum over a period of about 52 weeks following administration of the JAK inhibitor, including at the 52 week time point. Patients Condition, Diagnosis and Treatment In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises one or more of the following:a) identifying a subject having a disease of the gastrointestinal tract, for example by endoscopy or colonoscopy;b) determination of the severity of the disease, for example with reference to the Mayo Clinic Score, the Crohn's Disease Activity Index (CDAI), the Harvey-Bradshaw Index (HBI), or a combination of the above;c) determination of the location of the disease, for example as determined by the presence of lesions indicative of the disease;d) evaluating the subject for suitability to treatment, for example by determining the patency of the subject's GI tract, for example if the indication is small intestinal diseases, pancolitis, Crohn's disease, or if the patients has strictures or fistulae;e) administration of an induction dose or of a maintenance dose of a drug, such as the JAK inhibitor or such as another drug that is effective in the treatment of IBD conditions;f) monitoring the progress of the disease, for example with reference to the Mayo Clinic Score, the Crohn's Disease Activity Index (CDAI), the Harvey-Bradshaw Index (HBI), the PRO, PRO2 or PRO3 tools, or a combination of the above; and/org) optionally repeating steps e) and f) one or more times, for example over a period of about 1-14 weeks, such as about 6-8 weeks following administration of the JAK inhibitor, including at the 6-8 week time point, or over a period of about 52 weeks following administration of the JAK inhibitor, including at the 52 week time point. As used herein, an induction dose is a dose of drug that may be administered, for example, at the beginning of a course of treatment, and that is higher than the maintenance dose administered during treatment. An induction dose may also be administered during treatment, for example if the condition of the patients becomes worse. As used herein, a maintenance dose is a dose of drug that is provided on a repetitive basis, for example at regular dosing intervals. In some embodiments the JAK inhibitor is released from an ingestible device. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises c) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises d) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises e) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises g) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and b) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and c) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and d) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and e) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises a) and g) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) and c) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) and d) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) and e) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) and f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises b) and g) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises c) and d) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises c) and e) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises c) and f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises c) and g) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises d) and e) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises d) and f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises d) and g) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises e) and f) hereinabove. In some embodiments herein, the method of treating a disease of the gastrointestinal tract that comprises releasing a JAK inhibitor at a location in the gastrointestinal tract that is proximate to one or more sites of disease comprises g) hereinabove. In some embodiments, one or more steps a) to e) herein comprise endoscopy of the gastrointestinal tract. In some embodiments, one or more steps a) to e) herein comprise colonoscopy of the gastrointestinal tract. In some embodiments, one or more steps a) to e) herein is performed one or more times. In some embodiments, such one or more of such one or more steps a) to e) is performed after releasing the JAK inhibitor at the location in the gastrointestinal tract that is proximate to one or more sites of disease. In some embodiments, the method comprises administering one or more maintenance doses following administration of the induction dose in step e). In some embodiments an induction dose of JAK inhibitor and a maintenance dose of JAK inhibitor are each administered to the subject by administering a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor. In some embodiments an induction dose of JAK inhibitor is administered to the subject in a different manner from the maintenance dose. As an example, the maintenance dose may be administered systemically, while the maintenance dose is administered locally using a device. In one embodiment, a maintenance dose is administered systemically, and an induction dose is administered using a device every 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 35, 40, or 45 days. In another embodiment, a maintenance dose is administered systemically, and an induction dose is administered when a disease flares up is detected or suspected. In some embodiments, the induction dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In some embodiments, the maintenance dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In some embodiments, the induction dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In some embodiments, the maintenance dose is a dose of the JAK inhibitor delivered systemically, such as orally with a tablet or capsule, or subcutaneously, or intravenously. In some embodiments, the induction dose is a dose of the JAK inhibitor delivered systemically, such as orally with a tablet or capsule, or subcutaneously, or intravenously. In some embodiments, the maintenance dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In some embodiments, the induction dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In some embodiments, the maintenance dose is a dose of a second agent as disclosed herein delivered systemically, such as orally with a tablet or capsule, or subcutaneously, or intravenously. In some embodiments, the induction dose is a dose of a second agent as disclosed herein delivered systemically, such as orally with a tablet or capsule, or subcutaneously, or intravenously. In some embodiments, the maintenance dose is a dose of the JAK inhibitor administered in an ingestible device as disclosed herein. In one embodiment of the methods provided herein, the patient is not previously treated with a JAK inhibitor. In one embodiment, the gastrointestinal inflammatory disorder is an inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis and the response is selected from clinical response, mucosal healing and remission. In certain embodiments, remission in the patient is determined to be induced when the Mayo Clinic Score <2 and no individual subscore >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopy subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, patients who experience mucosal healing are determined to have an endoscopy subscore of 0. In certain embodiments, clinical response is determined to have occurred when the patient experiences a 3-point decrease and 30% reduction from baseline in MCS and >1-point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1. In some embodiments, the method comprises identifying the disease site substantially at the same time as releasing the JAK inhibitor. In some embodiments, the method comprises monitoring the progress of the disease. In some embodiments, monitoring the progress of the disease comprises measuring the weight of the subject over a period of about 1-14 weeks, such as about 6-8 weeks following administration of the JAK inhibitor, including at the 6-8 week time point, or over a period of about 52 weeks following administration of the JAK inhibitor, including at the 52 week time point. In some embodiments, monitoring the progress of the disease comprises measuring the food intake of the subject; measuring the level of blood in the feces of the subject; measuring the level of abdominal pain of the subject; and/or a combination of the above, for example over a period of about 1-14 weeks, such as about 6-8 weeks following administration of the JAK inhibitor, including at the 6-8 week time point, or over a period of about 52 weeks following administration of the JAK inhibitor, including at the 52 week time point. In some embodiments, the method comprises administering a JAK inhibitor with a spray catheter. For example, administering a JAK inhibitor with a spray catheter may be performed in step (e) hereinabove. In some embodiments, the method does not comprise administering a JAK inhibitor with a spray catheter. In some embodiments, data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given JAK inhibitor. The effectiveness and dosing of any JAK inhibitor can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more disease symptoms in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases). In some embodiments, the subject is further administered an additional therapeutic agent (e.g., any of the additional therapeutic agents described herein). The additional therapeutic agent can be administered to the subject at substantially the same time as the JAK inhibitor or pharmaceutical composition comprising it is administered and/or at one or more other time points. In some embodiments, the additional therapeutic agent is formulated together with the JAK inhibitor (e.g., using any of the examples of formulations described herein). In some embodiments, the subject is administered a dose of the JAK inhibitor at least once a month (e.g., at least twice a month, at least three times a month, at least four times a month, at least once a week, at least twice a week, three times a week, once a day, or twice a day). The JAK inhibitor may be administered to a subject chronically. Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, more than five years, more than 10 years, more than 15 years, more than 20 years, more than 25 years, more than 30 years, more than 35 years, more than 40 years, more than 45 years, or longer. Alternatively, or in addition, chronic treatments may be administered. Chronic treatments can involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month. For example, chronic treatment can include administration (e.g., intravenous administration) about every two weeks (e.g., between about every 10 to 18 days). A suitable dose may be the amount that is the lowest dose effective to produce a desired therapeutic effect. Such an effective dose will generally depend upon the factors described herein. If desired, an effective daily dose of JAK inhibitor can be administered as two, three, four, five, or six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some examples, administration of an JAK inhibitor using any of the compositions or devices described herein can result in the onset of treatment (e.g., a reduction in the number, severity, or duration of one or more symptoms and/or markers of any of the diseases described herein) or drug-target engagement in a subject within a time period of about 10 minutes to about 10 hours, about 10 minutes to about 9 hours, about 10 minutes to about 8 hours, about 10 minutes to about 7 hours, about 10 minutes to about 6 hours, about 10 minutes to about 5 hours, about 10 minutes to about 4.5 hours, about 10 minutes to about 4 hours, about 10 minutes to about 3.5 hours, about 10 minutes to about 3 hours, about 10 minutes to about 2.5 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1.5 hours, about 10 minutes to about 1 hour, about 10 minutes to about 55 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 45 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 10 hours, about 15 minutes to about 9 hours, about 15 minutes to about 8 hours, about 15 minutes to about 7 hours, about 15 minutes to about 6 hours, about 15 minutes to about 5 hours, about 15 minutes to about 4.5 hours, about 15 minutes to about 4 hours, about 15 minutes to about 3.5 hours, about 15 minutes to about 3 hours, about 15 minutes to about 2.5 hours, about 15 minutes to about 2 hours, about 15 minutes to about 1.5 hours, about 15 minutes to about 1 hour, about 15 minutes to about 55 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 10 hours, about 20 minutes to about 9 hours, about 20 minutes to about 8 hours, about 20 minutes to about 7 hours, about 20 minutes to about 6 hours, about 20 minutes to about 5 hours, about 20 minutes to about 4.5 hours, about 20 minutes to about 4 hours, about 20 minutes to about 3.5 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2.5 hours, about 20 minutes to about 2 hours, about 20 minutes to about 1.5 hours, about 20 minutes to about 1 hour, about 20 minutes to about 55 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 45 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 25 minutes, about 25 minutes to about 10 hours, about 25 minutes to about 9 hours, about 25 minutes to about 8 hours, about 25 minutes to about 7 hours, about 25 minutes to about 6 hours, about 25 minutes to about 5 hours, about 25 minutes to about 4.5 hours, about 25 minutes to about 4 hours, about 25 minutes to about 3.5 hours, about 25 minutes to about 3 hours, about 25 minutes to about 2.5 hours, about 25 minutes to about 2 hours, about 25 minutes to about 1.5 hours, about 25 minutes to about 1 hour, about 25 minutes to about 55 minutes, about 25 minutes to about 50 minutes, about 25 minutes to about 45 minutes, about 25 minutes to about 40 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 30 minutes, about 30 minutes to about 10 hours, about 30 minutes to about 9 hours, about 30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30 minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3.5 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2.5 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1.5 hours, about 30 minutes to about 1 hour, about 30 minutes to about 55 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 45 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 35 minutes to about 10 hours, about 35 minutes to about 9 hours, about 35 minutes to about 8 hours, about 35 minutes to about 7 hours, about 35 minutes to about 6 hours, about 35 minutes to about 5 hours, about 35 minutes to about 4.5 hours, about 35 minutes to about 4 hours, about 35 minutes to about 3.5 hours, about 35 minutes to about 3 hours, about 35 minutes to about 2.5 hours, about 35 minutes to about 2 hours, about 35 minutes to about 1.5 hours, about 35 minutes to about 1 hour, about 35 minutes to about 55 minutes, about 35 minutes to about 50 minutes, about 35 minutes to about 45 minutes, about 35 minutes to about 40 minutes, about 40 minutes to about 10 hours, about 40 minutes to about 9 hours, about 40 minutes to about 8 hours, about 40 minutes to about 7 hours, about 40 minutes to about 6 hours, about 40 minutes to about 5 hours, about 40 minutes to about 4.5 hours, about 40 minutes to about 4 hours, about 40 minutes to about 3.5 hours, about 40 minutes to about 3 hours, about 40 minutes to about 2.5 hours, about 40 minutes to about 2 hours, about 40 minutes to about 1.5 hours, about 40 minutes to about 1 hour, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 40 minutes to about 45 minutes, about 45 minutes to about 10 hours, about 45 minutes to about 9 hours, about 45 minutes to about 8 hours, about 45 minutes to about 7 hours, about 45 minutes to about 6 hours, about 45 minutes to about 5 hours, about 45 minutes to about 4.5 hours, about 45 minutes to about 4 hours, about 45 minutes to about 3.5 hours, about 45 minutes to about 3 hours, about 45 minutes to about 2.5 hours, about 45 minutes to about 2 hours, about 45 minutes to about 1.5 hours, about 45 minutes to about 1 hour, about 45 minutes to about 55 minutes, about 45 minutes to about 50 minutes, about 50 minutes to about 10 hours, about 50 minutes to about 9 hours, about 50 minutes to about 8 hours, about 50 minutes to about 7 hours, about 50 minutes to about 6 hours, about 50 minutes to about 5 hours, about 50 minutes to about 4.5 hours, about 50 minutes to about 4 hours, about 50 minutes to about 3.5 hours, about 50 minutes to about 3 hours, about 50 minutes to about 2.5 hours, about 50 minutes to about 2 hours, about 50 minutes to about 1.5 hours, about 50 minutes to about 1 hour, about 50 minutes to about 55 minutes, about 55 minutes to about 10 hours, about 55 minutes to about 9 hours, about 55 minutes to about 8 hours, about 55 minutes to about 7 hours, about 55 minutes to about 6 hours, about 55 minutes to about 5 hours, about 55 minutes to about 4.5 hours, about 55 minutes to about 4 hours, about 55 minutes to about 3.5 hours, about 55 minutes to about 3 hours, about 55 minutes to about 2.5 hours, about 55 minutes to about 2 hours, about 55 minutes to about 1.5 hours, about 55 minutes to about 1 hour, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 1.5 hours to about 10 hours, about 1.5 hours to about 9 hours, about 1.5 hours to about 8 hours, about 1.5 hours to about 7 hours, about 1.5 hours to about 6 hours, about 1.5 hours to about 5 hours, about 1.5 hours to about 4.5 hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3.5 hours, about 1.5 hours to about 3 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 10 hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours to about 4.5 hours, about 2 hours to about 4 hours, about 2 hours to about 3.5 hours, about 2 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2.5 hours to about 10 hours, about 2.5 hours to about 9 hours, about 2.5 hours to about 8 hours, about 2.5 hours to about 7 hours, about 2.5 hours to about 6 hours, about 2.5 hours to about 5 hours, about 2.5 hours to about 4.5 hours, about 2.5 hours to about 4 hours, about 2.5 hours to about 3.5 hours, about 2.5 hours to about 3 hours, about 3 hours to about 10 hours, about 3 hours to about 9 hours, about 3 hours to about 8 hours, about 3 hours to about 7 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, about 3 hours to about 4.5 hours, about 3 hours to about 4 hours, about 3 hours to about 3.5 hours, about 3.5 hours to about 10 hours, about 3.5 hours to about 9 hours, about 3.5 hours to about 8 hours, about 3.5 hours to about 7 hours, about 3.5 hours to about 6 hours, about 3.5 hours to about 5 hours, about 3.5 hours to about 4.5 hours, about 3.5 hours to about 4 hours, about 4 hours to about 10 hours, about 4 hours to about 9 hours, about 4 hours to about 8 hours, about 4 hours to about 7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 4 hours to about 4.5 hours, about 4.5 hours to about 10 hours, about 4.5 hours to about 9 hours, about 4.5 hours to about 8 hours, about 4.5 hours to about 7 hours, about 4.5 hours to about 6 hours, about 4.5 hours to about 5 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours, about 5 hours to about 8 hours, about 5 hours to about 7 hours, about 5 hours to about 6 hours, about 6 hours to about 10 hours, about 6 hours to about 9 hours, about 6 hours to about 8 hours, about 6 hours to about 7 hours, about 7 hours to about 10 hours, about 7 hours to about 9 hours, about 7 hours to about 8 hours, about 8 hours to about 10 hours, about 8 hours to about 9 hours, or about 9 hours to about 10 hours of administration of a dose of an JAK inhibitor using any of the devices or compositions described herein. Drug-target engagement may be determined, for example, as disclosed in Simon G M, Niphakis M J, Cravat B F,Nature chemical biology.2013; 9(4):200-205, incorporated by reference herein in its entirety. In some embodiments, administration of an JAK inhibitor using any of the devices or compositions described herein can provide for treatment (e.g., a reduction in the number, severity, and/or duration of one or more symptoms and/or markers of any of the disorders described herein in a subject) for a time period of between about 1 hour to about 30 days, about 1 hour to about 28 days, about 1 hour to about 26 days, about 1 hour to about 24 days, about 1 hour to about 22 days, about 1 hour to about 20 days, about 1 hour to about 18 days, about 1 hour to about 16 days, about 1 hour to about 14 days, about 1 hour to about 12 days, about 1 hour to about 10 days, about 1 hour to about 8 days, about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 3 hours, about 3 hours to about 30 days, about 3 hours to about 28 days, about 3 hours to about 26 days, about 3 hours to about 24 days, about 3 hours to about 22 days, about 3 hours to about 20 days, about 3 hours to about 18 days, about 3 hours to about 16 days, about 3 hours to about 14 days, about 3 hours to about 12 days, about 3 hours to about 10 days, about 3 hours to about 8 days, about 3 hours to about 6 days, about 3 hours to about 5 days, about 3 hours to about 4 days, about 3 hours to about 3 days, about 3 hours to about 2 days, about 3 hours to about 1 day, about 3 hours to about 12 hours, about 3 hours to about 6 hours, about 6 hours to about 30 days, about 6 hours to about 28 days, about 6 hours to about 26 days, about 6 hours to about 24 days, about 6 hours to about 22 days, about 6 hours to about 20 days, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 6 hours to about 12 hours, about 12 hours to about 30 days, about 12 hours to about 28 days, about 12 hours to about 26 days, about 12 hours to about 24 days, about 12 hours to about 22 days, about 12 hours to about 20 days, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 30 days, about 1 day to about 28 days, about 1 day to about 26 days, about 1 day to about 24 days, about 1 day to about 22 days, about 1 day to about 20 days, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 30 days, about 2 days to about 28 days, about 2 days to about 26 days, about 2 days to about 24 days, about 2 days to about 22 days, about 2 days to about 20 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 30 days, about 3 days to about 28 days, about 3 days to about 26 days, about 3 days to about 24 days, about 3 days to about 22 days, about 3 days to about 20 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 30 days, about 4 days to about 28 days, about 4 days to about 26 days, about 4 days to about 24 days, about 4 days to about 22 days, about 4 days to about 20 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 30 days, about 5 days to about 28 days, about 5 days to about 26 days, about 5 days to about 24 days, about 5 days to about 22 days, about 5 days to about 20 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 6 days, about 6 days to about 30 days, about 6 days to about 28 days, about 6 days to about 26 days, about 6 days to about 24 days, about 6 days to about 22 days, about 6 days to about 20 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 8 days to about 30 days, about 8 days to about 28 days, about 8 days to about 26 days, about 8 days to about 24 days, about 8 days to about 22 days, about 8 days to about 20 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 10 days to about 30 days, about 10 days to about 28 days, about 10 days to about 26 days, about 10 days to about 24 days, about 10 days to about 22 days, about 10 days to about 20 days, about 10 days to about 18 days, about 10 days to about 16 days, about 10 days to about 14 days, about 10 days to about 12 days, about 12 days to about 30 days, about 12 days to about 28 days, about 12 days to about 26 days, about 12 days to about 24 days, about 12 days to about 22 days, about 12 days to about 20 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 30 days, about 14 days to about 28 days, about 14 days to about 26 days, about 14 days to about 24 days, about 14 days to about 22 days, about 14 days to about 20 days, about 14 days to about 18 days, about 14 days to about 16 days, about 16 days to about 30 days, about 16 days to about 28 days, about 16 days to about 26 days, about 16 days to about 24 days, about 16 days to about 22 days, about 16 days to about 20 days, about 16 days to about 18 days, about 18 days to about 30 days, about 18 days to about 28 days, about 18 days to about 26 days, about 18 days to about 24 days, about 18 days to about 22 days, about 18 days to about 20 days, about 20 days to about 30 days, about 20 days to about 28 days, about 20 days to about 26 days, about 20 days to about 24 days, about 20 days to about 22 days, about 22 days to about 30 days, about 22 days to about 28 days, about 22 days to about 26 days, about 22 days to about 24 days, about 24 days to about 30 days, about 24 days to about 28 days, about 24 days to about 26 days, about 26 days to about 30 days, about 26 days to about 28 days, or about 28 days to about 30 days in a subject following first administration of an JAK inhibitor using any of the compositions or devices described herein. Non-limiting examples of symptoms and/or markers of a disease described herein are described below. For example, treatment can result in a decrease (e.g., about 1% to about 99% decrease, about 1% to about 95% decrease, about 1% to about 90% decrease, about 1% to about 85% decrease, about 1% to about 80% decrease, about 1% to about 75% decrease, about 1% to about 70% decrease, about 1% to about 65% decrease, about 1% to about 60% decrease, about 1% to about 55% decrease, about 1% to about 50% decrease, about 1% to about 45% decrease, about 1% to about 40% decrease, about 1% to about 35% decrease, about 1% to about 30% decrease, about 1% to about 25% decrease, about 1% to about 20% decrease, about 1% to about 15% decrease, about 1% to about 10% decrease, about 1% to about 5% decrease, about 5% to about 99% decrease, about 5% to about 95% decrease, about 5% to about 90% decrease, about 5% to about 85% decrease, about 5% to about 80% decrease, about 5% to about 75% decrease, about 5% to about 70% decrease, about 5% to about 65% decrease, about 5% to about 60% decrease, about 5% to about 55% decrease, about 5% to about 50% decrease, about 5% to about 45% decrease, about 5% to about 40% decrease, about 5% to about 35% decrease, about 5% to about 30% decrease, about 5% to about 25% decrease, about 5% to about 20% decrease, about 5% to about 15% decrease, about 5% to about 10% decrease, about 10% to about 99% decrease, about 10% to about 95% decrease, about 10% to about 90% decrease, about 10% to about 85% decrease, about 10% to about 80% decrease, about 10% to about 75% decrease, about 10% to about 70% decrease, about 10% to about 65% decrease, about 10% to about 60% decrease, about 10% to about 55% decrease, about 10% to about 50% decrease, about 10% to about 45% decrease, about 10% to about 40% decrease, about 10% to about 35% decrease, about 10% to about 30% decrease, about 10% to about 25% decrease, about 10% to about 20% decrease, about 10% to about 15% decrease, about 15% to about 99% decrease, about 15% to about 95% decrease, about 15% to about 90% decrease, about 15% to about 85% decrease, about 15% to about 80% decrease, about 15% to about 75% decrease, about 15% to about 70% decrease, about 15% to about 65% decrease, about 15% to about 60% decrease, about 15% to about 55% decrease, about 15% to about 50% decrease, about 15% to about 45% decrease, about 15% to about 40% decrease, about 15% to about 35% decrease, about 15% to about 30% decrease, about 15% to about 25% decrease, about 15% to about 20% decrease, about 20% to about 99% decrease, about 20% to about 95% decrease, about 20% to about 90% decrease, about 20% to about 85% decrease, about 20% to about 80% decrease, about 20% to about 75% decrease, about 20% to about 70% decrease, about 20% to about 65% decrease, about 20% to about 60% decrease, about 20% to about 55% decrease, about 20% to about 50% decrease, about 20% to about 45% decrease, about 20% to about 40% decrease, about 20% to about 35% decrease, about 20% to about 30% decrease, about 20% to about 25% decrease, about 25% to about 99% decrease, about 25% to about 95% decrease, about 25% to about 90% decrease, about 25% to about 85% decrease, about 25% to about 80% decrease, about 25% to about 75% decrease, about 25% to about 70% decrease, about 25% to about 65% decrease, about 25% to about 60% decrease, about 25% to about 55% decrease, about 25% to about 50% decrease, about 25% to about 45% decrease, about 25% to about 40% decrease, about 25% to about 35% decrease, about 25% to about 30% decrease, about 30% to about 99% decrease, about 30% to about 95% decrease, about 30% to about 90% decrease, about 30% to about 85% decrease, about 30% to about 80% decrease, about 30% to about 75% decrease, about 30% to about 70% decrease, about 30% to about 65% decrease, about 30% to about 60% decrease, about 30% to about 55% decrease, about 30% to about 50% decrease, about 30% to about 45% decrease, about 30% to about 40% decrease, about 30% to about 35% decrease, about 35% to about 99% decrease, about 35% to about 95% decrease, about 35% to about 90% decrease, about 35% to about 85% decrease, about 35% to about 80% decrease, about 35% to about 75% decrease, about 35% to about 70% decrease, about 35% to about 65% decrease, about 35% to about 60% decrease, about 35% to about 55% decrease, about 35% to about 50% decrease, about 35% to about 45% decrease, about 35% to about 40% decrease, about 40% to about 99% decrease, about 40% to about 95% decrease, about 40% to about 90% decrease, about 40% to about 85% decrease, about 40% to about 80% decrease, about 40% to about 75% decrease, about 40% to about 70% decrease, about 40% to about 65% decrease, about 40% to about 60% decrease, about 40% to about 55% decrease, about 40% to about 50% decrease, about 40% to about 45% decrease, about 45% to about 99% decrease, about 45% to about 95% decrease, about 45% to about 90% decrease, about 45% to about 85% decrease, about 45% to about 80% decrease, about 45% to about 75% decrease, about 45% to about 70% decrease, about 45% to about 65% decrease, about 45% to about 60% decrease, about 45% to about 55% decrease, about 45% to about 50% decrease, about 50% to about 99% decrease, about 50% to about 95% decrease, about 50% to about 90% decrease, about 50% to about 85% decrease, about 50% to about 80% decrease, about 50% to about 75% decrease, about 50% to about 70% decrease, about 50% to about 65% decrease, about 50% to about 60% decrease, about 50% to about 55% decrease, about 55% to about 99% decrease, about 55% to about 95% decrease, about 55% to about 90% decrease, about 55% to about 85% decrease, about 55% to about 80% decrease, about 55% to about 75% decrease, about 55% to about 70% decrease, about 55% to about 65% decrease, about 55% to about 60% decrease, about 60% to about 99% decrease, about 60% to about 95% decrease, about 60% to about 90% decrease, about 60% to about 85% decrease, about 60% to about 80% decrease, about 60% to about 75% decrease, about 60% to about 70% decrease, about 60% to about 65% decrease, about 65% to about 99% decrease, about 65% to about 95% decrease, about 65% to about 90% decrease, about 65% to about 85% decrease, about 65% to about 80% decrease, about 65% to about 75% decrease, about 65% to about 70% decrease, about 70% to about 99% decrease, about 70% to about 95% decrease, about 70% to about 90% decrease, about 70% to about 85% decrease, about 70% to about 80% decrease, about 70% to about 75% decrease, about 75% to about 99% decrease, about 75% to about 95% decrease, about 75% to about 90% decrease, about 75% to about 85% decrease, about 75% to about 80% decrease, about 80% to about 99% decrease, about 80% to about 95% decrease, about 80% to about 90% decrease, about 80% to about 85% decrease, about 85% to about 99% decrease, about 85% to about 95% decrease, about 85% to about 90% decrease, about 90% to about 99% decrease, about 90% to about 95% decrease, or about 95% to about 99% decrease) in one or more (e.g., two, three, four, five, six, seven, eight, or nine) of: the level of interferon-γ in GI tissue, the level of IL-1β in GI tissue, the level of IL-6 in GI tissue, the level of IL-22 in GI tissue, the level of IL-17A in the GI tissue, the level of TNFα in GI tissue, the level of IL-2 in GI tissue, and endoscopy score in a subject (e.g., as compared to the level in the subject prior to treatment or compared to a subject or population of subjects having a similar disease but receiving a placebo or a different treatment) (e.g., for a time period of between about 1 hour to about 30 days (e.g., or any of the subranges herein) following the first administration of an JAK inhibitor using any of the compositions or devices described herein. As used herein, “GI tissue” refers to tissue in the gastrointestinal (GI) tract, such as tissue in one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum, more particularly in the proximal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, or in the distal portion of one or more of duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and sigmoid colon. The GI tissue may be, for example, GI tissue proximate to one or more sites of disease. Exemplary methods for determining the endoscopy score are described herein and other methods for determining the endoscopy score are known in the art. Exemplary methods for determining the levels of interferon-γ, IL-1β, IL-6, IL-22, IL-17A, TNFα, and IL-2 are described herein. Additional methods for determining the levels of these cytokines are known in the art. In some examples, treatment can result in an increase (e.g., about 1% to about 500% increase, about 1% to about 400% increase, about 1% to about 300% increase, about 1% to about 200% increase, about 1% to about 150% increase, about 1% to about 100% increase, about 1% to about 90% increase, about 1% to about 80% increase, about 1% to about 70% increase, about 1% to about 60% increase, about 1% to about 50% increase, about 1% to about 40% increase, about 1% to about 30% increase, about 1% to about 20% increase, about 1% to about 10% increase, a 10% to about 500% increase, about 10% to about 400% increase, about 10% to about 300% increase, about 10% to about 200% increase, about 10% to about 150% increase, about 10% to about 100% increase, about 10% to about 90% increase, about 10% to about 80% increase, about 10% to about 70% increase, about 10% to about 60% increase, about 10% to about 50% increase, about 10% to about 40% increase, about 10% to about 30% increase, about 10% to about 20% increase, about 20% to about 500% increase, about 20% to about 400% increase, about 20% to about 300% increase, about 20% to about 200% increase, about 20% to about 150% increase, about 20% to about 100% increase, about 20% to about 90% increase, about 20% to about 80% increase, about 20% to about 70% increase, about 20% to about 60% increase, about 20% to about 50% increase, about 20% to about 40% increase, about 20% to about 30% increase, about 30% to about 500% increase, about 30% to about 400% increase, about 30% to about 300% increase, about 30% to about 200% increase, about 30% to about 150% increase, about 30% to about 100% increase, about 30% to about 90% increase, about 30% to about 80% increase, about 30% to about 70% increase, about 30% to about 60% increase, about 30% to about 50% increase, about 30% to about 40% increase, about 40% to about 500% increase, about 40% to about 400% increase, about 40% to about 300% increase, about 40% to about 200% increase, about 40% to about 150% increase, about 40% to about 100% increase, about 40% to about 90% increase, about 40% to about 80% increase, about 40% to about 70% increase, about 40% to about 60% increase, about 40% to about 50% increase, about 50% to about 500% increase, about 50% to about 400% increase, about 50% to about 300% increase, about 50% to about 200% increase, about 50% to about 150% increase, about 50% to about 100% increase, about 50% to about 90% increase, about 50% to about 80% increase, about 50% to about 70% increase, about 50% to about 60% increase, about 60% to about 500% increase, about 60% to about 400% increase, about 60% to about 300% increase, about 60% to about 200% increase, about 60% to about 150% increase, about 60% to about 100% increase, about 60% to about 90% increase, about 60% to about 80% increase, about 60% to about 70% increase, about 70% to about 500% increase, about 70% to about 400% increase, about 70% to about 300% increase, about 70% to about 200% increase, about 70% to about 150% increase, about 70% to about 100% increase, about 70% to about 90% increase, about 70% to about 80% increase, about 80% to about 500% increase, about 80% to about 400% increase, about 80% to about 300% increase, about 80% to about 200% increase, about 80% to about 150% increase, about 80% to about 100% increase, about 80% to about 90% increase, about 90% to about 500% increase, about 90% to about 400% increase, about 90% to about 300% increase, about 90% to about 200% increase, about 90% to about 150% increase, about 90% to about 100% increase, about 100% to about 500% increase, about 100% to about 400% increase, about 100% to about 300% increase, about 100% to about 200% increase, about 100% to about 150% increase, about 150% to about 500% increase, about 150% to about 400% increase, about 150% to about 300% increase, about 150% to about 200% increase, about 200% to about 500% increase, about 200% to about 400% increase, about 200% to about 300% increase, about 300% to about 500% increase, about 300% to about 400% increase, or about 400% to about 500% increase) in one or both of stool consistency score and weight of a subject (e.g., as compared to the level in the subject prior to treatment or compared to a subject or population of subjects having a similar disease but receiving a placebo or a different treatment) (e.g., for a time period of between about 1 hour to about 30 days (e.g., or any of the subranges herein) following the first administration of an JAK inhibitor using any of the compositions or devices described herein. Exemplary methods for determining stool consistency score are described herein. Additional methods for determining a stool consistency score are known in the art. Accordingly, in some embodiments, a method of treatment disclosed herein includes determining the level of a marker at the location of disease in a subject (e.g., either before and/or after administration of the device). In some embodiments, the marker is a biomarker and the method of treatment disclosed herein comprises determining that the level of a biomarker at the location of disease is a subject following administration of the device is decreased as compared to the level of the biomarker at the same location of disease in a subject either before administration or at the same time point following systemic administration of an equal amount of the JAK inhibitor. In some examples, the level of the biomarker at the same location of disease following administration of the device is 1% decreased to 99% decreased as compared to the level of the biomarker at the same location of disease in a subject either before administration or at the same time point following systemic administration of an equal amount of the JAK inhibitor. In some embodiments, the level of the marker is one or more of: the level of interferon-γ in GI tissue, the level of IL-17A in the GI tissue, the level of TNFα in the GI tissue, the level of IL-2 in the GI tissue, and the endoscopy score in a subject. In some embodiments, the method of treatment disclosed herein includes determining that the level of a marker at a time point following administration of a device is lower than the level of the marker at a time point following administration of the device is lower than the level of the marker in a subject prior to administration of the device or in a subject at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor. In some examples, the level of the marker following administration of the device is 1% decreased to 99% decreased as compared to the level of the marker in a subject prior to administration of the device or in a subject at the same time point following systemic administration of an equal amount of the JAK inhibitor. In some examples, a method of treatment disclosed herein includes determining the level of the biomarker at the location of disease in a subject within a time period of about 10 minutes to 10 hours following administration of the device. In some embodiments, a method of treatment described herein includes: (i) determining the ratio RBof the level L1Bof a biomarker at the location of disease at a first time point following administration of the device and the level L2Bof the biomarker at the same location of disease in a subject at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor; (ii) determining the ratio of RDof the level of L1Dof the JAK inhibitor at the same location and the substantially the same time point as in (i) and the level L2Dof the JAK inhibitor at the same location of disease in a subject at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor; and (iii) determining the ratio of RB/RD. In some embodiments, a method of treatment disclosed herein can include: (i) determining the ratio RBof the level L1Bof a biomarker at the location of disease at a time point following administration of the device and the level L2Bof the biomarker at the same location of disease in a subject at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor; (ii) determining the ratio RDof the level L1Dof the JAK inhibitor at the same location and at substantially the time point as in (i) and the level L2Dof the JAK inhibitor in a subject at the same location of disease at substantially the same time point following systemic administration of an equal amount of the JAK inhibitor; and (iii) determining the product RB×RD. In some embodiments, a method of treatment disclosed herein can include determining that the level of a marker in a subject at a time point following administration of the device is elevated as compared to a level of the marker in a subject prior to administration of the device or a level at substantially the same time point in a subject following systemic administration of an equal amount of the JAK inhibitor. In some examples, the level of the marker at a time point following administration of the device is 1% increased or 400% increased as compared to the level of the marker in a subject prior to administration of the device or a level at substantially the same time point in a subject following systemic administration of an equal amount of the JAK inhibitor. In some examples, the level of the marker is one or more of subject weight and stool consistency (e.g., stool consistency score). In some examples, a method of treatment disclosed herein includes determining the level of the marker in a subject within a period of about 10 minutes to about 10 hours following administration of the device. In some embodiments, a method of treatment disclosed herein can include determining the level of a marker in a subject's blood, serum or plasma. An illustrative list of examples of biomarkers for GI disorders includes interferon-γ, IL-1β, IL-6, IL-22, IL-17A, TNFα, IL-2, memory cells (CD44+CD45RB−CD4+cells); α4β37; VEGF; ICAM; VCAM; SAA; Calprotectin; lactoferrin; FGF2; TGFb; ANG-1; ANG-2; PLGF; Biologics (Infliximab; Humira; Stelara; Vedolizumab; Simponi; Jak inhibitors; Others); EGF; IL12/23p40; GMCSF; A4 B7; AeB7; CRP; SAA; ICAM; VCAM; AREG; EREG; HB-EGF; HRG; BTC; TGFα; SCF; TWEAK; MMP-9; MMP-6; Ceacam CD66; IL10; ADA; Madcam-1; CD166 (AL CAM); FGF2; FGF7; FGF9; FGF19; ANCA Antineutrophil cytoplasmic antibody; ASCAA Anti-Saccharomyces CerevisiaeAntibody IgA; ASCAG Anti-Saccharomyces CerevisiaeAntibody IgG; CBir1 Anti-Clostridiumcluster XIVa flagellin CBir1 antibody; A4-Fla2 Anti-Clostridiumcluster XIVa flagellin 2 antibody; FlaX Anti-Clostridiumcluster XIVa flagellin X antibody; OmpC Anti-Escherichia coliOuter Membrane Protein C; ANCA Perinuclear AntiNeutrophil Cytoplasmic Antibody; AREG Amphiregulin Protein; BTC Betacellulin Protein; EGF Epidermal Growth Factor EREG Epiregulin Protein; HBEGF Heparin Binding Epidermal Growth Factors; HGF Hepatocyte Growth Factor; HRG Neuregulin-1; TGFA Transforming Growth Factor alpha; CRP C-Reactive Protein; SAA Serum Amyloid A; ICAM-1 Intercellular Adhesion Molecule 1; VCAM-1 Vascular Cell Adhesion Molecule 1; fibroblasts underlying the intestinal epithelium; and HGF. In some embodiments, a marker is an IBD biomarker, such as, for example: anti-glycan; anti-Saccharomices cerevisiae(ASCA); anti-laminaribioside (ALCA); anti-chitobioside (ACCA); anti-mannobioside (AMCA); anti-laminarin (anti-L); anti-chitin (anti-C) antibodies: anti-outer membrane porin C (anti-OmpC), anti-Cbir1 flagellin; anti-12 antibody; autoantibodies targeting the exocrine pancreas (PAB); and perinuclear anti-neutrophil antibody (pANCA); and calprotectin. In some embodiments, a biomarker is associated with membrane repair, fibrosis, angiogenesis. In certain embodiments, a biomarker is an inflammatory biomarker, an anti-inflammatory biomarker, an MMP biomarker, an immune marker, or a TNF pathway biomarker. In some embodiments, a biomarker is gut specific. For tissue samples, HER2 can be used as a biomarker relating to cytotoxic T cells. Additionally, other cytokine levels can be used as biomarkers in tissue (e.g., phospho STAT 1, STAT 3 and STAT 5), in plasma (e.g., VEGF, VCAM, ICAM, IL-6), or both. In some embodiments, the biomarkers include one or more immunoglobulins, such as, for example, immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE) and/or immunoglobulin A (IgA). In some embodiments, IgM is a biomarker of infection and/or inflammation. In some embodiments, IgD is a biomarker of autoimmune disease. In some embodiments, IgG is a biomarker of Alzheimer's disease and/or for cancer. In some embodiments, IgE is a biomarker of asthma and/or allergen immunotherapy. In some embodiments, IgA is a biomarker of kidney disease. In some embodiments, the biomarker is High Sensitivity C-reactive Protein (hsCRP); 7 α-hydroxy-4-cholesten-3-one (7C4); Anti-Endomysial IgA (EMA IgA); Anti-Human Tissue Transglutaminase IgA (tTG IgA); Total Serum IgA by Nephelometry; Fecal Calprotectin; or Fecal Gastrointestinal Pathogens. In some embodiments, the biomarker isa) an anti-gliadin IgA antibody, an anti-gliadin IgG antibody, an anti-tissue transglutaminase (tTG) antibody, an anti-endomysial antibody;b)i) a serological marker that is ASCA-A, ASCA-G, ANCA, pANCA, anti-OmpC antibody, anti-CBir1 antibody, anti-FlaX antibody, or anti-A4-Fla2 antibody;b)ii) an inflammation marker that is VEGF, ICAM, VCAM, SAA, or CRP;b)iii) the genotype of the genetic markers ATG16L1, ECM1, NKX2-3, or STAT3;c) a bacterial antigen antibody marker;d) a mast cell marker;e) an inflammatory cell marker;f) a bile acid malabsorption (BAM) marker;g) a kynurenine marker;orh) a serotonin marker. In some embodiments, the bacterial antigen antibody marker is selected from the group consisting of an anti-Fla1 antibody, anti-Fla2 antibody, anti-FlaA antibody, anti-FliC antibody, anti-FliC2 antibody, anti-FliC3 antibody, anti-YBaN1 antibody, anti-ECFliC antibody, anti-Ec0FliC antibody, anti-SeFljB antibody, anti-CjFlaA antibody, anti-CjFlaB antibody, anti-SfFliC antibody, anti-CjCgtA antibody, anti-Cjdmh antibody, anti-CjGT-A antibody, anti-EcYidX antibody, anti-EcEra antibody, anti-EcFrvX antibody, anti-EcGabT antibody, anti-EcYedK antibody, anti-EcYbaN antibody, anti-EcYhgN antibody, anti-RtMaga antibody, anti-RbCpaF antibody, anti-RgPilD antibody, anti-LaFrc antibody, anti-LaEno antibody, anti-LjEFTu antibody, anti-BfOmpa antibody, anti-PrOmpA antibody, anti-Cp10bA antibody, anti-CpSpA antibody, anti-EfSant antibody, anti-LmOsp antibody, anti-SfET-2 antibody, anti-Cpatox antibody, anti-Cpbtox antibody, anti-EcSta2 antibody, anti-Ec0Stx2A antibody, anti-CjcdtB/C antibody, anti-CdtcdA/B antibody, and combinations thereof. In some embodiments, the mast cell marker is selected from the group consisting of beta-tryptase, histamine, prostaglandin E2 (PGE2), and combinations thereof. In some embodiments, the inflammatory marker is selected from the group consisting of CRP, ICAM, VCAM, SAA, GRO.alpha., and combinations thereof. In some embodiments, the bile acid malabsorption marker is selected from the group consisting of 7α-hydroxy-4-cholesten-3-one, FGF19, and a combination thereof. In some embodiments, the kynurenine marker is selected from the group consisting of kynurenine (K), kynurenic acid (KyA), anthranilic acid (AA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), quinolinic acid (QA), tryptophan, 5-hydroxytryptophan (5-HTP), and combinations thereof. In some embodiments, the serotonin marker is selected from the group consisting of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), serotonin-O-sulfate, serotonin-O-phosphate, and combinations thereof. In some embodiments, the biomarker is a biomarker as disclosed in U.S. Pat. No. 9,739,786, incorporated by reference herein in its entirety. The following markers can be expressed by mesenchymal stem cells (MSC): CD105, CD73, CD90, CD13, CD29, CD44, CD10, Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3, SISD2, Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1 (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more of such markers), and lack expression of one or more of CD45, CD34, CD14, CD19, and HLA-DR (e.g., lack expression of two or more, three or more, four or more, or five or more such markers). In some embodiments, MSC can express CD105, CD73, and CD90. In some embodiments, MSC can express CD105, CD73, CD90, CD13, CD29, CD44, and CD10. In some embodiments, MSC can express CD105, CD73, and CD90 and one or more stemness markers such as Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3. SISD2, Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1. In some embodiments, MSC can express CD105, CD73, CD90, CD13, CD29, CD44, and CD10 and one or more stemness markers such as Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3. SISD2, Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1. See, e.g., Lv, et al.,Stem Cells,2014, 32:1408-1419. Intestinal stem cells (ISC) can be positive for one or more markers such as Musashi-1 (Msi-1), Ascl2, Bmi-1, Doublecortin and Ca2+/calmodulin-dependent kinase-like 1 (DCAMKL1), and Leucin-rich repeat-containing G-protein-coupled receptor 5 (Lgrs). See, e.g., Mohamed, et al., Cytotechnology, 2015 67(2): 177-189. Any of the foregoing biomarkers can be used as a biomarker for one or more of other conditions as appropriate. In some embodiments of the methods herein, the methods comprise determining the time period of onset of treatment following administration of the device. Combination Therapy: The JAK inhibitors disclosed herein may be optionally be used with additional agents in the treatment of the diseases disclosed herein. Nonlimiting examples of such agents for treating or preventing inflammatory bowel disease in such adjunct therapy (e.g., Crohn's disease, ulcerative colitis) include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); non-steroidal antiinflammatory drugs (NSAIDs); ganciclovir; tacrolimus; lucocorticoids such as Cortisol or aldosterone; anti-inflammatory agents such as a cyclooxygenase inhibitor; a 5-lipoxygenase inhibitor; or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporine; 6-mercaptopurine; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL®, methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies or antagonists including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor (TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists; anti-LFA-1 antibodies, including anti-CD 1 la and anti-CD 18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990); streptokinase; transforming growth factor-beta (TGF-beta); streptodomase; RNA or DNA from the host; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al, Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF or BR3 antibodies or immunoadhesins and zTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol, 23: 113-5 (2002) and see also definition below); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand. (e.g., Durie et al, Science, 261: 1328-30 (1993); Mohan et al, J. Immunol, 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Non-limiting examples of adjunct agents also include the following: budenoside; epidermal growth factor; aminosalicylates; metronidazole; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; TNF antagonists; IL-4, IL-1β, IL-13 and/or TGFβ cytokines or agonists thereof (e.g., agonist antibodies); IL-11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-I antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TPlO; T Cell Sciences, Inc.); slow-release mesalazine; antagonists of platelet activating factor (PAF); ciprofloxacin; and lignocaine. Examples of agents for UC are sulfasalazine and related salicylate-containing drugs for mild cases and corticosteroid drugs in severe cases. Topical administration of either salicylates or corticosteroids is sometimes effective, particularly when the disease is limited to the distal bowel, and is associated with decreased side effects compared with systemic use. Supportive measures such as administration of iron and antidiarrheal agents are sometimes indicated. Azathioprine, 6-mercaptopurine and methotrexate are sometimes also prescribed for use in refractory corticosteroid-dependent cases. In other embodiments, a JAK inhibitor as described herein can be administered with one or more of: a CHST15 inhibitor, a IL-6 receptor inhibitor, a TNF inhibitor, an integrin inhibitor, an IL-12/IL-23 inhibitor, a SMAD7 inhibitor, a IL-13 inhibitor, an IL-1 receptor inhibitor, a TLR agonist, an immunosuppressant, a live biotherapeutic such as a stem cell, IL-10 or an IL-10 agonist, copaxone, a CD40 inhibitor, an S1P-inhibitor, or a chemokine/chemokine receptor inhibitor. In other embodiments, a JAK inhibitor as described herein can be administered with a vitamin C infusion, one or more corticosteroids, and optionally thiamine. Examples of particular combinations include the following. Unless otherwise specified, the first component (component (1)) is administered in an ingestible device, while the second component (component (2)) is administered either in an ingestible device, which may be the same or different ingestible device as the first component, or by another form of administration.(1) JAK inhibitor; (2) TNF inhibitor.(1) JAK inhibitor; (2) TNF inhibitor in an ingestible device.(1) JAK inhibitor; (2) TNF inhibitor intravenously or subcutaneously.(1) TNF inhibitor; (2) JAK inhibitor(1) TNF inhibitor; (2) JAK inhibitor in an ingestible device.(1) TNF inhibitor; (2) JAK inhibitor orally.(1) Neoregulin-4; (2) JAK inhibitor.(1) Neoregulin-4; (2) JAK inhibitor in an ingestible device.(1) Neoregulin-4; (2) JAK inhibitor intravenously or subcutaneously. In some embodiments, the methods disclosed herein comprise administering (i) the JAK inhibitor as disclosed herein, and (ii) a second agent orally, intravenously or subcutaneously, wherein the second agent in (ii) is the same JAK inhibitor in (i); a different JAK inhibitor; or an agent having a different biological target from the JAK inhibitor. In some embodiments, the methods disclosed herein comprise administering (i) the JAK inhibitor in the manner disclosed herein, and (ii) a second agent orally, intravenously or subcutaneously, wherein the second agent in (ii) is an agent suitable for treating an inflammatory bowel disease. In some embodiments, the JAK inhibitor is administered prior to the second agent. In some embodiments, the JAK inhibitor is administered after the second agent. In some embodiments, the JAK inhibitor and the second agent are administered substantially at the same time. In some embodiments, the JAK inhibitor is delivered prior to the second agent. In some embodiments, the JAK inhibitor is delivered after the second agent. In some embodiments, the JAK inhibitor and the second agent are delivered substantially at the same time. In some embodiments, the second agent is an agent suitable for the treatment of a disease of the gastrointestinal tract. In some embodiments, the second agent is an agent suitable for the treatment of an inflammatory bowel disease. In some embodiments, the second agent is administered intravenously. In some embodiments, the second agent is administered subcutaneously. In some embodiments, the second agent is methotrexate. In some embodiments, delivery of the JAK inhibitor to the location, such as delivery to the location by mucosal contact, results in systemic immunogenicity levels at or below systemic immunogenicity levels resulting from administration of the JAK inhibitor systemically. In some embodiments comprising administering the JAK inhibitor in the manner disclosed herein and a second agent systemically, delivery of the JAK inhibitor to the location, such as delivery to the location by mucosal contact, results in systemic immunogenicity levels at or below systemic immunogenicity levels resulting from administration of the JAK inhibitor systemically and the second agent systemically. In some embodiments, the method comprises administering the JAK inhibitor in the manner disclosed herein and a second agent, wherein the amount of the second agent is less than the amount of the second agent when the JAK inhibitor and the second agent are both administered systemically. In some aspects of these embodiments, the second agent is a JAK inhibitor. In some embodiments, the method comprises administering the JAK inhibitor in the manner disclosed herein and does not comprise administering a second agent. Example 1—Preclinical Murine Colitis Model Experimental Induction of Colitis Colitis is experimentally induced to mice via the dextran sulfate sodium (DSS)-induced colitis model. This model is widely used because of its simplicity and many similarities with human ulcerative colitis. Briefly, mice are subjected to DSS via cecal catheterization, which is thought to be directly toxic to colonic epithelial cells of the basal crypts, for several days until colitis is induced. Groups Mice are allocated to one of seven cohorts, depending on the agent that is administered:1. Control (no agent)2. Adalimumab (2.5 mg/kg)3. Adalimumab (5 mg/kg)4. Adalimumab (10 mg/kg) The control or agent is applied to a damaged mucosal surface of the bowel via administration through a cecal catheter at the dose levels described above. Additionally, for each cohort, the animals are separated into two groups. One group receives a single dose of the control or agent on day 10 or 12. The other group receives daily (or similar) dosing of the control or agent. Analysis For each animal, efficacy is determined (e.g., by endoscopy, histology, etc.), and cytotoxic T-cell levels are determined in blood, feces, and tissue (tissue levels are determined after animal sacrifice). For tissue samples, levels HER2 are additionally determined, and the level of cytotoxic T cells is normalized to the level of HER2. Additionally, other cytokine levels are determined in tissue (e.g., phospho STAT 1, STAT 3 and STAT 5), in plasma (e.g., VEGF, VCAM, ICAM, IL-6), or both. Pharmacokinetics are determined both systemically (e.g., in the plasma) and locally (e.g., in colon tissue). For systemic pharmacokinetic analysis, blood and/or feces is collected from the animals at one or more timepoints after administration (e.g., plasma samples are collected at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and/or 8 hours after administration). Local/colon tissue samples are collected once after animal sacrifice. Example 2A—Development of Preclinical Porcine Colitis Model Experimental Induction of Colitis Female swine weighing approximately 35 to 45 kg at study start are fasted at least 24 hours prior to intra-rectal administration of trinitrobenzene sulfonic acid (TNBS). Animals are lightly anesthetized during the dosing and endoscopy procedure. An enema to clean the colon is used, if necessary. One animal is administered 40 ml of 100% EtOH mixed with 5 grams of TNBS diluted in 10 ml of water via an enema using a ball-tipped catheter. The enema is deposited in the proximal portion of the descending colon just past the bend of the transverse colon. The TNBS is retained at the dose site for 12 minutes by use of two Foley catheters with 60-ml balloons placed in the mid-section of the descending colon below the dose site. A second animal is similarly treated, but with a solution containing 10 grams of TNBS. An Endoscope is employed to positively identify the dose site in both animals prior to TNBS administration. Dosing and endoscopy are performed by a veterinary surgeon Seven (7) days after TNBS administration, after light anesthesia, the dose site and mucosal tissues above and below the dose site are evaluated by the veterinary surgeon using an endoscope. Pinch Biopsies are obtained necessary, as determined by the surgeon. Based on the endoscopy findings, the animals may be euthanized for tissue collection on that day, or may proceed on study pending the results of subsequent endoscopy exams for 1 to 4 more days. Macroscopic and microscopic alterations of colonic architecture, possible necrosis, thickening of the colon, and substantial histologic changes are observed at the proper TNBS dose. Clinical signs (e.g., ill health, behavioral changes, etc.) are recorded at least daily during acclimation and throughout the study. Additional pen-side observations are conducted twice daily (once-daily on weekends). Body weight is measured for both animals Days 1 and 7 (and on the day of euthanasia if after Day 7). On the day of necropsy, the animals are euthanized via injection of a veterinarian-approved euthanasia solution. Immediately after euthanasia in order to avoid autolytic changes, colon tissues are collected, opened, rinsed with saline, and a detailed macroscopic examination of the colon is performed to identify macroscopic finings related to TNBS-damage. Photos are taken. Tissue samples are taken from the proximal, mid, and distal transverse colon; the dose site; the distal colon; the rectum; and the anal canal. Samples are placed into NBF and evaluated by a board certified veterinary pathologist. Example 2B—Pharmacokinetic/Pharmacodynamic and Bioavailability of Adalimumab after Topical Application Groups Sixteen (16) swine (approximately 35 to 45 kg at study start) are allocated to one of five groups:1. Vehicle Control: (3.2 mL saline); intra-rectal; (n=2)2. Treated Control: Adalimumab (40 mg in 3.2 mL saline); subcutaneous; (n=2)3. Adalimumab (low): Adalimumab (40 mg in 3.2 mL saline); intra-rectal; (n=4)4. Adalimumab (med): Adalimumab (80 mg in 3.2 mL saline); intra-rectal; (n=4)5. Adalimumab (high): Adalimumab (160 mg in 3.2 mL saline); intra-rectal;(n=4) On Day 0, the test article is applied to a damaged mucosal surface of the bowel via intra-rectal administration or subcutaneous injection by a veterinary surgeon at the dose levels and volume described above. Clinical Observations and Body Weight Clinical observations are conducted at least once daily. Clinical signs (e.g., ill health, behavioral changes, etc.) are recorded on all appropriate animals at least daily prior to the initiation of experiment and throughout the study until termination. Additional clinical observations may be performed if deemed necessary. Animals whose health condition warrants further evaluation are examined by a Clinical Veterinarian. Body weight is measured for all animals Days −6, 0, and after the last blood collections. Samples Blood: Blood is collected (cephalic, jugular, and/or catheter) into EDTA tubes during acclimation on Day −7, just prior to dose on Day 0, and 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours post-dose. The EDTA samples are split into two aliquots and one is centrifuged for pharmacokinetic plasma and either analyzed immediately, or stored frozen (−80° C.) for later pharmacokinetic analyses. The remaining sample of whole blood is used for pharmacodynamic analyses. Feces: Feces is collected Day −7, 0 and 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 hours post-dose, and either analyzed immediately, or flash-frozen on liquid nitrogen and stored frozen at −70° C. pending later analysis of drug levels and inflammatory cytokines. Tissue: Immediately after euthanasia in order to avoid autolytic changes, colon tissues are collected, opened, rinsed with saline, and a detailed macroscopic examination of the colon is performed to identify macroscopic finings related to TNBS-damage. Triplicate samples of normal and damaged tissues are either analyzed immediately, or are flash-frozen on liquid nitrogen and stored frozen at −70° C. pending later analysis of drug concentration, inflammatory cytokines and histology. Samples are analyzed for adalimumab levels (local mucosal tissue levels and systemic circulation levels), and for levels of inflammatory cytokines including TNF-alpha. Terminal Procedures Animals are euthanized as per the schedule in Table AA, where one animal each of Vehicle and Treated Control groups is euthanized at 6 and 48 hours post-dose, and one animal of each the adalimumab groups are euthanized at 6, 12, 24 and 48 hours post-dose. Animals are discarded after the last blood collection unless retained for a subsequent study. TABLE AASampleDaysHourssizeDoseRoute−7−6−5−4−3−2−100.512468122448GeneralFast•Food/Waterad libidumoral••••••••••••••••Observationsclinical observations••••••••••body weight••••Treatments (groups)TNBS (all animals)intra rectal•1. Vehicle controln = 21.6 mL salineintra rectal•(vehicle)euthanizedn = 1n = 12. Treated controln = 240 mg insub-cutaneous1.6 mL salineeuthanizedn = 1n = 13. Adalimumab (low)n = 440 mg inintra rectal•1.6 mL salineeuthanizedn = 1n = 1n = 1n = 14. Adalimumab (med)n = 480 mg inintra rectal•1.6 mL salineeuthanizedn = 1n = 1n = 1n = 15. Adalimumab (high)n = 4160 mg inintra rectal•1.6 mL salineeuthanizedn = 1n = 1n = 1n = 1Adalimumab (required)1200SamplesBloodcephalic, jugular or•••••••••••catheterFecalrectal•••••••••••Tissuenecropsy••••• Example 2C—Pharmacokinetic/Pharmacodynamic and Bioavailability of Adalimumab after Topical Application Groups DSS-induced colitis Yorkshire-Cross Farm Swine (approximately 5-10 kg at study start) are allocated to one of five groups:1. Vehicle Control: (saline); intra-rectal;2. Treated Control: Adalimumab (13 mg in saline); subcutaneous;3. Adalimumab: Adalimumab (13 mg in saline); intra-rectal; At t=0, the test article is applied to a damaged mucosal surface of the bowel via intra-rectal administration or subcutaneous injection by a veterinary surgeon at the dose levels and volume described above. Clinical Observations Clinical signs (e.g., ill health, behavioral changes, etc.) are recorded on all appropriate animals at least daily prior to the initiation of experiment and throughout the study until termination. Additional clinical observations may be performed if deemed necessary. Animals whose health condition warrants further evaluation are examined by a Clinical Veterinarian. Samples Blood: Blood is collected (cephalic, jugular, and/or catheter) into EDTA tubes during acclimation on Day −7, just prior to dose on Day 0, and 12 hours post-dose. The EDTA samples are split into two aliquots and one is centrifuged for pharmacokinetic plasma and either analyzed immediately, or stored frozen (−80° C.) for later pharmacokinetic analyses. The remaining sample of whole blood is used for pharmacodynamic analyses. Feces: Feces is collected Day −7, 0 and 12 hours post-dose, and either analyzed immediately, or flash-frozen on liquid nitrogen and stored frozen at −70° C. pending later analysis of drug levels and inflammatory cytokines. Tissue: Immediately after euthanasia (12 hours after dosing) in order to avoid autolytic changes, colon tissues are collected, opened, rinsed with saline, and a detailed macroscopic examination of the colon is performed to identify macroscopic finings related to DSS-damage. Triplicate samples of normal and damaged tissues are either analyzed immediately, or are flash-frozen on liquid nitrogen and stored frozen at −70° C. pending later analysis of drug concentration, inflammatory cytokines and histology. Samples are analyzed for adalimumab levels (local mucosal tissue levels and systemic circulation levels), and for levels of inflammatory cytokines including TNF-alpha. Terminal Procedures Animals are euthanized at 12 hours post-dose. Example 3. Comparison of Systemic Versus Intracecal Delivery of an Anti-Il-12 Antibody The objective of this study was to compare the efficacy of an IL-12 inhibitor (anti-IL-12 p40; anti-p40 mAb; BioXCell (Cat #: BE0051)), when dosed systemically versus intracecally, to the treat dextran sulfate sodium salt (DSS)-induced colitis in male C57Bl/6 mice. Materials and Methods Mice Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing 20-24 g, were obtained from Charles River Laboratories. The mice were randomized into thirteen groups of twelve animals and two groups of eight animals, and housed in groups of 6-8 per cage, and acclimatized for at least three days prior to entering the study. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour, with an automatic timer for a light/dark cycle of 12 hours on/off, and fed with Labdiet 5053 sterile rodent chow, with water administered ad libitum. Cecal Cannulation Animals were placed under isoflurane anesthesia, with the cecum exposed via a midline incision in the abdomen. A small point incision was made in the distal cecum where 1-2 cm of the cannula was inserted. The incision was closed with a purse string suture using 5-0 silk. An incision was then made in the left abdominal wall through which the distal end of the cannula was inserted and pushed subcutaneously to the dorsal aspect of the back. The site was then washed copiously with warmed saline prior to closing the abdominal wall. A small incision was also made in the skin of the back between the shoulder blades, exposing the tip of the cannula. The cannula was secured in place using suture, wound clips, and tissue glue. All animals received 1 mL of warm sterile saline (subcutaneous injection) and were monitored closely until recovery before returning to their cage. All animals received 0.6 mg/kg BID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg every day for the first 5 days post surgery. Induction of Colitis Colitis was induced in male C57Bl/6 mice by exposure to 3% DSS drinking water (MP Biomedicals #0260110) from Day 0 to Day 5. Fresh DSS/water solutions were made again on Day 3 and any of the remaining original DSS solution will be discarded. Assessment of Colitis All animals were weighed daily and visually assessed for the presence of diarrhea and/or bloody stool at the time of dosing. The mice underwent two video endoscopies, one on day 10 and one on day 14, to assess colitis severity. Images were captured from each animal at the most severe region of disease identified during the endoscopy, and assessed using the rubric demonstrated in Table 1.1. Additionally, stool consistency was scored during the endoscopy using this rubric (Table 1.2) (0=Normal, well-formed pellet, 1=Loose stool, soft, staying in shape, 2=Loose stool, abnormal form with excess moisture, 3=Watery or diarrhea, 4=Bloody diarrhea). At necropsy, intestinal contents, peripheral blood, and tissue, and cecum/colon contents were collected for analysis. TABLE 1.1Endoscopy ScoringScoreDescription of Endoscopy Score0Normal1Loss of vascularity2Loss of vascularity and friability3Friability and erosions4Ulcerations and bleeding TABLE 1.2Stool Consistency ScoreScoreDescription of Stool Consistency0Normal, well-formed pellet1Loose stool, soft, staying in shape2Loose stool, abnormal form with excess moisture3Watery or diarrhea4Bloody diarrhea Treatment of Colitis Mice were treated with anti-IL-12 p40 during the acute phase of colitis due to its efficacy in the treatment of DSS-induced colitis. The test article was dosed at a volume of 0.1 mL/20 g from days 0 to 14. Anti-IL-12 p40 was administered intraperitoneally at a dose of 10 mg/kg every 3 days, and intracecally at a dose of 10 mg/kg, either every 3 days or every day. There was also a lower dose of 1 mg/kg given every day intracecally. The control groups were not administered drugs, and the vehicles (sterile PBS) were administered the placebo drug intraperitoneally and intracecally every day. These drugs were given from days 5-14, which is 9 days of administration. A more detailed explanation of dosing and groups can be seen in Table 1.3. TABLE 1.3Groups of AnimalsGroup# ofCecalTreat-DoseDosing#AnimalsDSSCannulament(mg/kg)RouteSchedule18 males—NO————28 males—YES————312 males3%NOVehicle—POQDDSSday 0-14(day0-5)412 males3%YESVehicle—ICQDDSSday 0-14(day0-5)512 males3%NOAnti-p4010IPQ3DSS0, 3, 6,(day9, 120-5)612 males3%YESAnti-p4010ICQ3DSS0, 3, 6,(day9, 120-5)712 males3%YESAnti-p4010ICQDDSSday 0-14(day0-5)812 males3%YESAnti-p401ICQDDSSday 0-14(day0-5) Sample Collection Intestinal contents, peripheral blood, and tissue were collected at sacrifice on day 14, as follows: at the end of each study period, mice were euthanized by CO2inhalation immediately following endoscopy on day 14. The blood was collected via cardiac puncture into K2EDTA-coated tubes and centrifuged at 4000×g for 10 minutes. The blood cell pellet was retained and snapped frozen. The resulting plasma was then split into two separate cryotubes, with 100 μL in one tube and the remainder in the second. Plasma and cell pellet were also collected, flash frozen, and stored at −80 degrees Celsius. The cecum and colon were removed from each animal and contents were collected, weighed, and snap frozen in separate cryovials. The colon was excised, rinsed, measured, weighed, and then trimmed to 6 cm in length and divided into 5 pieces. The most proximal 1 cm of colon was snapped frozen for subsequent bioanalysis of test article levels. Of the remaining 5 cm of colon, the most distal and proximal 1.5-cm sections was placed in formalin for 24 hours then transferred to 70% ethanol for subsequent histological evaluation. The middle 2-cm portion was bisected longitudinally and placed into two separate cryotubes, weighed, and snap frozen in liquid nitrogen. Results The data inFIG.30show that the DSS mice that were intracecally administered an anti-IL-12 p40 (IgG2A) antibody had decreased weight loss as compared to DSS mice that were intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.31show that the plasma concentration of the anti-IL-12 p40 antibody was decreased in DSS mice that were intracecally administered the anti-IL-12 p40 antibody as compared to DSS mice that were intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.32show that the cecum and colon concentration of the anti-IL-12 p40 antibody is increased in DSS mice that were intracecally administered the anti-IL-12 p40 antibody as compared to the DSS mice that were intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIGS.33and34show that the anti-IL-12 p40 antibody is able to penetrate colon tissues (the lumen superficial, lamina propria, submucosa, and tunica muscularis/serosa) in DSS mice intracecally administered the anti-IL-12 p40 antibody, while the anti-IL-12 p40 antibody did not detectably penetrate the colon tissues of DSS mice intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.35also show that the ratio of the concentration of anti-IL-12 p40 antibody in colon tissue to the concentration of the anti-IL-12 p40 antibody in plasma is increased in DSS mice intracecally administered the anti-IL-12 p40 antibody as compared to the ratio in DSS mice intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.36show that the concentration of IL-1β in colon tissue is decreased in DSS mice intracecally administered the anti-IL-12 p40 antibody as compared to the concentration of IL-1β in colon tissue in DSS mice intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.37show that the concentration of IL-6 in colon tissue is decreased in DSS mice intracecally administered the anti-IL-12 p40 antibody as compared to the concentration of IL-6 in colon tissue in DSS mice intraperitoneally administered the anti-IL-12 p40 antibody. The data inFIG.38show that the concentration of IL-17A in colon tissue is decreased in DSS mice intracecally administered the anti-IL-12 p40 antibody as compared to the concentration of IL-17A in colon tissue in DSS mice intraperitoneally administered the anti-IL-12 p40 antibody. No significant differences in clinical observations or gastrointestinal-specific adverse effects, including stool consistency and/or bloody stool, were observed due to cannulation or intra-cecal treatments when compared with vehicle. No toxicity resulting from the treatments was reported. A significant reduction in body weight-loss (AUC) was found in groups treated with anti-IL-12 p40 antibody (10 mg/kg and 1 mg/kg, QD) via intra-cecal delivery when compared with vehicle control and intraperitoneal delivery (10 mg/kg, Q3D). The immunohistochemistry staining in anti-IL-12 p40 antibody (10 mg/kg, QD) treatment groups showed penetration of the antibody in all layers of colon tissue, including lumen mucosa, lamina propria, submucosa, tunica muscularis, via intra-cecal delivery. The distribution of anti-IL-12 p40 antibody was found in all segments of the colon, however, higher levels were detected in the proximal region. A significantly higher mean concentration of anti-IL-12 p40 antibody was found in the gastrointestinal contents and colon tissues when delivered via intra-cecal administration (Anti-p40: 10 mg/kg and 1 mg/kg, QD) compared with intraperitoneal administration (anti-p40: 10 mg/kg, Q3D). The blood level of anti-IL-12 p40 antibody was significantly higher when delivered via intraperitoneal administration (Q3D) as compared to intra-cecal administration (Q3D & QD). The concentrations of inflammatory cytokines, including IL-1β, IL-6, and IL-17, were significantly reduced by anti-IL-12 p40 antibody (10 mg/kg, QD) treatment when delivered via intra-cecal administration as compared to vehicle controls. In sum, these data show that the compositions and devices provided herein can suppress the local immune response in the intestine, while having less of a suppressive effect on the systemic immune response of an animal. These data also suggest that the presently claimed compositions and devices will provide for treatment of colitis and other pro-inflammatory disorders of the intestine. Example 4. Comparison of Systemic Versus Intracecal Delivery of an Anti-Integrin α4β7 Antibody The objective of this study was to compare the efficacy of an integrin inhibitor (anti-integrin α4β7; anti-LPAM1; DATK-32 mAb; BioXCell (Cat #: BE0034)) when dosed systemically versus intracecally for treating dextran sulfate sodium salt (DSS)-induced colitis in male C57Bl/6 mice. Materials and Methods Mice Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing 20-24 g, were obtained from Charles River Laboratories. The mice were randomized into thirteen groups of twelve animals and two groups of eight animals, and housed in groups of 6-8 per cage, and acclimatized for at least three days prior to entering the study. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour, with an automatic timer for a light/dark cycle of 12 hours on/off, and fed with Labdiet 5053 sterile rodent chow, with water administered ad libitum. Cecal Cannulation The animals were placed under isoflurane anesthesia, with the cecum exposed via a midline incision in the abdomen. A small point incision was made in the distal cecum where 1-2 cm of the cannula was inserted. The incision was closed with a purse string suture using 5-0 silk. An incision was then made in the left abdominal wall through which the distal end of the cannula was inserted and pushed subcutaneously to the dorsal aspect of the back. The site was then washed copiously with warmed saline prior to closing the abdominal wall. A small incision was also made in the skin of the back between the shoulder blades, exposing the tip of the cannula. The cannula was secured in place using suture, wound clips, and tissue glue. All animals received 1 mL of warm sterile saline (subcutaneous injection) and were monitored closely until recovery before returning to their cage. All animals received 0.6 mg/kg BID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg every day for the first 5 days post-surgery. Induction of Colitis Colitis was induced in male C57Bl/6 mice by exposure to 3% DSS drinking water (MP Biomedicals #0260110) from day 0 to day 5. Fresh DSS/water solutions were made again on day 3 and any of the remaining original DSS solution will be discarded. Assessment of Colitis All animals were weighed daily and visually assessed for the presence of diarrhea and/or bloody stool at the time of dosing. Mice underwent two video endoscopies, one on day 10 and one on day 14, to assess colitis severity. Images were captured from each animal at the most severe region of disease identified during the endoscopy, and assessed using the rubric demonstrated in Table 2.1. Additionally, stool consistency was scored during the endoscopy using this rubric (Table 2.2) (0=Normal, well-formed pellet, 1=Loose stool, soft, staying in shape, 2=Loose stool, abnormal form with excess moisture, 3=Watery or diarrhea, 4=Bloody diarrhea). At necropsy, intestinal contents, peripheral blood and tissue, and cecum/colon contents were collected for analysis. TABLE 2.1Endoscopy ScoreScoreDescription of Endoscopy Score0Normal1Loss of vascularity2Loss of vascularity and friability3Friability and erosions4Ulcerations and bleeding TABLE 2.2Stool Consistency ScoreScoreDescription of Stool Consistency0Normal, well-formed pellet1Loose stool, soft, staying in shape2Loose stool, abnormal form with excess moisture3Watery or diarrhea4Bloody diarrhea Treatment of Colitis Mice were treated with DATK32 during the acute phase of colitis due to its efficacy in the treatment of DSS-induced colitis. The test article was dosed at a volume of 0.1 mL/20 g from days 0 to 14. DATK32 was administered intraperitoneally at a dose of 25 mg/kg every 3 days, and intracecally at a dose of 25 mg/kg, either every 3 days or every day. There was also a lower dose of 5 mg/kg given every day intracecally. The control groups were not administered drugs, and the vehicle (sterile PBS) was administered as the placebo drug intraperitoneally and intracecally every day. These drugs were given from days 5-14, which is 9 days of administration. A more detailed explanation of dosing and groups can be seen in Table 2.3. TABLE 2.3Groups of MiceGroup# ofCecalTreat-DoseDosing#AnimalsDSSCannulament(mg/kg)RouteSchedule18 males—NO————28 males—YES————312 males3%NOVehicle—POQDDSSday 0-14(day0-5)412 males3%YESVehicle—ICQDDSSday 0-14(day0-5)912 males3%NODATK3225IPQ3DSS0, 3, 6,(day9, 120-5)1012 males3%YESDATK3225ICQ3DSS0, 3, 6,(day9, 120-5)1112 males3%YESDATK3225ICQDDSSday 0-14(day0-5)1212 males3%YESDATK325ICQDDSSday 0-14(day0-5) Sample Collection Intestinal contents, peripheral blood, and tissue were collected at sacrifice on day 14, as follows: at the end of each study period, mice were euthanized by CO2inhalation immediately following endoscopy on day 14. The blood was collected via cardiac puncture into K2EDTA-coated tubes and centrifuged at 4000×g for 10 minutes. The blood cell pellet was retained and snapped frozen. The resulting plasma was then split into two separate cryotubes, with 100 μL in one tube and the remainder in the second. Plasma and the cell pellet were also collected, flash frozen, and stored at −80 degrees Celsius. An ELISA was used to determine the level of rat IgG2A. The cecum and colon were removed from each animal and contents were collected, weighed, and snap frozen in separate cryovials. The colon was excised, rinsed, measured, weighed, and then trimmed to 6 cm in length and divided into 5 pieces. The most proximal 1 cm of colon was snapped frozen for subsequent bioanalysis of anti-DATK32 levels. Of the remaining 5 cm of colon, the most distal and proximal 1.5-cm sections was placed in formalin for 24 hours then transferred to 70% ethanol for subsequent histological evaluation. The middle 2-cm portion was bisected longitudinally and placed into two separate cryotubes, weighed, and snap frozen in liquid nitrogen. There was an additional collection of 100 μL of whole blood from all animals and processed for FACS analysis of α4 and β7 expression on T-helper memory cells. Tissue and blood were immediately placed in FACS buffer (lx PBS containing 2.5% fetal calf serum) and analyzed using the following antibody panel (Table 2.4). TABLE 2.4Fluorophore Labelled Antibodies Used in FACS AnalysisAntibody TargetFlurochromePurposeCD4APC-Vio770Defines T-Helper CellsCD44VioBlueMemory/NaiveDiscriminationCD45RBFITCMemory/NaiveDiscriminationα4APCDefines T-helper memorysubset of interestβ7PEDefines T-helper memorysubset of interestCD16/32—Fc Block Results The data inFIG.39show decreased weight loss in DSS mice intracecally administered DATK antibody as compared to DSS mice that were intraperitoneally administered the DATK antibody. The data inFIG.40show that DSS mice intracecally administered DATK antibody have a decreased plasma concentration of DATK antibody as compared to DSS mice that were intraperitoneally administered DATK antibody. The data inFIGS.41and42show that DSS mice intracecally administered DATK antibody have an increased concentration of DATK antibody in the cecum and colon content as compared to DSS mice intraperitoneally administered DATK antibody. The data inFIGS.43and44show that DSS mice intracecally administered DATK antibody have an increased concentration of DATK antibody in colon tissue as compared to DSS mice intraperitoneally administered DATK antibody. The data inFIGS.45and46show an increased level of penetration of DATK antibody into colon tissue in DSS mice intracecally administered the DATK antibody as compared to an intracecal vehicle control (PBS). The data inFIG.47show that DSS mice intracecally administered DATK antibody have an increased ratio of the concentration of DATK antibody in colon tissue to the plasma concentration of the DATK antibody, as compared to the same ratio in DSS mice intraperitoneally administered the DATK antibody. The data inFIG.48show that DSS mice intracecally administered the DATK antibody have an increased percentage of blood Th memory cells as compared to DSS mice intraperitoneally administered the DATK antibody. No significant differences in clinical observations or gastrointestinal-specific adverse effects, including stool consistency and/or bloody stool, were observed due to cannulation or intra-cecal treatments when compared with vehicle. No toxicity resulting from the treatments was reported. A significant reduction in body weight-loss was also found with DATK32 (5 mg/kg, QD) treatment (IC) when compared to vehicle control at the endpoint (day 14). The immunohistochemistry staining in DATK32 (25 mg/kg, QD) treatment groups showed penetration of DATK32 in all layers of colon tissue, including lumen mucosa, lamina propria, submucosa, tunica muscularis, via intra-cecal delivery. The distribution of DATK32 was found in all segments of the colon, however, higher levels were detected in the proximal region. A significantly higher mean concentration of DATK32 was found in gastrointestinal contents and colon tissues when delivered via intra-cecal administration (DATK32: 25 mg/kg and 5 mg/kg, QD) as compared to intraperitoneal administration (DATK32: 25 mg/kg, Q3D). The blood level of DATK32 was significantly higher when delivered via intraperitoneal administration (Q3D) as compared to intra-cecal administration (Q3D & QD). The pharmacokinetics of DATK32 (25 mg/kg, QD) showed significantly higher mean concentrations of DATK32 when delivered via intra-cecal administration at 1, 2, and 4 h post-dose in the gastrointestinal contents, and 1, 2, 4 and 24 h in colon tissue as compared with the mean concentrations of DATK32 following intraperitoneal administration. The mean number of gut-homing T cells (Th memory cells) was significantly higher in the blood of groups treated with DATK32 via intra-cecal administration (QD 25 mg/kg and QD 5 mg/kg) as compared to the groups treated with DATK32 via intraperitoneal administration (Q3D 25 mg/kg). The mean number of Th memory cells was significantly lower in the Peyer's Patches of groups treated with DATK32 via intra-cecal administration (QD 25 mg/kg and 5 mg/kg) as compared to the groups treated with DATK32 via intraperitoneal administration (Q3D 25 mg/kg). The mean number of Th memory cells in mesenteric lymph nodes (MLN) was significantly lower in groups treated with DATK32 via intra-cecal administration (QD and Q3D 25 mg/kg and QD 5 mg/kg) as compared to the groups treated with DATK32 via intraperitoneal administration (Q3D 25 mg/kg). In sum, these data show that the compositions and devices provided herein can suppress the local immune response in the intestine, while having less of a suppressive effect on the systemic immune response of an animal. These data also show that the release of DATK-32 antibody in the colon can result in a suppression of leukocyte recruitment and may provide for the treatment of colitis and other pro-inflammatory diseases of the intestine. Example 5. An Assessment of Datk32 Bio-Distribution Following Intracecal Administration in Male C57Bl/6 Mice The objective of this study is to assess DATK32 bio-distribution when dosed intracecally in male C57Bl/6 mice. A minimum of 10 days prior to the start of the experiment a cohort of animals will undergo surgical implantation of a cecal cannula. A sufficient number of animals will undergo implantation to allow for 24 cannulated animals to be enrolled in the main study (e.g., 31 animals). Animals were dosed with vehicle or test article via intracecal injection (IC) on Day 0 as indicated in Table 3. Animals from all groups were sacrificed for terminal sample collection three hours following test article administration. Materials and Methods Mice Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing 20-24 g, were obtained from Charles River Laboratories. The mice were randomized into two groups of twelve animals, and housed in groups of 12 per cage, and acclimatized for at least three days prior to entering the study. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour, with an automatic timer for a light/dark cycle of 12 hours on/off, and fed with Labdiet 5053 sterile rodent chow, with water administered ad libitum. Cecal Cannulation The animals were placed under isoflurane anesthesia, with the cecum exposed via a midline incision in the abdomen. A small point incision was made in the distal cecum where 1-2 cm of the cannula was inserted. The incision was closed with a purse string suture using 5-0 silk. An incision was then made in the left abdominal wall through which the distal end of the cannula was inserted and pushed subcutaneously to the dorsal aspect of the back. The site was then washed copiously with warmed saline prior to closing the abdominal wall. A small incision was also made in the skin of the back between the shoulder blades, exposing the tip of the cannula. The cannula was secured in place using suture, wound clips, and tissue glue. All animals received 1 mL of warm sterile saline (subcutaneous injection) and were monitored closely until recovery before returning to their cage. All animals received 0.6 mg/kg BID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg every day for the first 5 days post-surgery. Dosing Animals were dosed IC at a volume of 0.075 mL/animal on Days 0 as indicated in Table 3. Sacrifice All animals were euthanized by CO2inhalation three hours after dosing on Day 0. Sample Collection Terminal blood was collected and prepared for plasma using K2EDTA as the anti-coagulant. The plasma will be split into two cryotubes, with 50 μL in one tube (PK analysis) and the remainder in another (other). Both samples were flash-frozen in liquid nitrogen. Plasma was stored at −80° C. for downstream analysis. Mesenteric lymph nodes (mLN) were collected, weighed, and flash-frozen in liquid nitrogen. Mesenteric lymph nodes were stored at −80° C. for downstream analysis. The small intestine was excised and rinsed, and the most distal 1 cm of ilium was dissected, weighed, and flash-frozen in liquid nitrogen. The samples were stored at −80° C. for downstream analysis. The cecum and colon were removed from each animal and contents collected, weighed, and snap frozen in separate cryovials. The samples were stored at −80° C. for downstream analysis. The colon was rinsed, and the most proximal 1 cm of colon was weighed and flash-frozen in liquid nitrogen. The snap frozen tissues were stored at −80° C. TABLE 3Study DesignTerminalNoCollectionsGroupAnimalsTreatmentRouteScheduleDay 0112VehicleICDay 0 **Blood (plasma) Small(PBS)intestine mLN212DATK32Colon(625 μg)*Colon ContentsCecum Contents*Per mouse. TA was administered in 0.075 mL/animal. DATK32 was delivered in sterile PBS.** Animals were dosed on Day 0 and collections were performed 3 hours later. Results The data inFIGS.63A-Fshow no significant differences in clinical observations. No gastrointestinal-specific or adverse effects were found in the group administered DATK32 via intra-cecal administration as compared to the group administered a vehicle control. No toxicity resulting from the treatments was reported. The level of DATK32 in the group intra-cecally administered DATK32 was significantly higher in cecum and colon content, and colon tissue compared to the group administered a vehicle control at 3 h post-dose. A small amount of DATK32 was also detected in plasma, small intestine, and mesenteric lymph node in the group intra-cecally administered DATK32. Example 6. Pharmacokinectics/Pharmacodynamics and Bioavailability of Adalimumab when Applied to a Tnbs-Damaged Mucosal Surface (Induced Colitis) in Swine The purpose of this non-Good Laboratory Practice (GLP) study was to explore the PK/PD, and bioavailability of adalimumab when applied to a TNBS-damaged mucosal surface (induced colitis) in Yorkshire-Cross farm swine, and to determine an appropriate dose and frequency for studies where a drug will be delivered by the ingestible device system. The ingestible device system will be capable of delivering a TNF inhibitor (adalimumab) topically and locally to damaged mucosa in human patients with inflammatory bowel disease (IBD). The TNBS-induced colitis model was validated when a single administration on Day 1 of 40 mL of 100% ethanol (EtOH) mixed with 5 grams of TNBS diluted in 10 mL of water via an enema using a rubber catheter resulted in the intended reproducible induction of damaged mucosal surface (induced colitis) in Yorkshire-Cross farm swine. This study investigated whether topical delivery of adalimumab would result in increased local mucosal tissue levels with limited drug reaching systemic circulation, as compared to subcutaneous administration; whether local mucosal tissue levels of drug would be greater in damaged tissues when compared to normal tissues; whether increasing the dose of drug would result in increased mucosal tissue levels in local and distal TNBS-damaged tissues; and whether topical delivery of adalimumab would result in reductions in inflammatory cytokines such as TNF-α in damaged tissues, feces, and possibly blood. All animals were subjected to intra-rectal administration of trinitrobenzene sulfonic acid (TNBS) to induce chronic colitis on day −2. All animals were fasted prior to colitis induction. Bedding was removed and replaced with rubber mats on day −3 to prevent ingestion of straw bedding material. The dose was 40 mL of 100% EtOH mixed with 5 grams of TNBS diluted in 10 mL of water, then instilled into the colon intra-rectally using a flexible gavage tube by a veterinary surgeon (deposited in a 10-cm portion of the distal colon and proximal rectum, and retained for 12 minutes by use of two Foley catheters with 60-mL balloons). Approximately 3 days after induction, macroscopic and microscopic alterations of colonic architecture were apparent: some necrosis, thickening of the colon, and substantial histologic changes were observed (FIGS.49and50). The study employed 15 female swine (approximately 35 to 45 kg at study start) allocated to one of five groups. Group 1 employed three animals that were the treated controls. Each animal in Group 1 was administered adalimumab by subcutaneous injection at 40 mg in 0.8 mL saline. Groups 2, 3, 4, and 5 employed 3 animals in each group. Animals in these groups were administered intra-rectal adalimumab at 40 mg in 0.8 mL saline. The test drug (adalimumab) was administered to all groups on study day 1. The intra-rectal administrations (Groups 2-5) were applied to damaged mucosal surface of the bowel vial intra-rectal administration by a veterinary surgeon. Blood (EDTA) was collected from all animals (cephalic, jugular, or catheter) on day −3 (n=15), −1 (n=15), and 6 (n=15), 12 (n=12), 24 (n=9), and 48 (n=6) hours post-dose (87 bleeds total). The EDTA samples were split into two aliquots, and one was centrifuged for PK plasma, and stored frozen (−80° C.) for PK analyses and reporting. Fecal samples were collected for the same time-points (87 fecal collections). Fecal samples were flash-frozen in liquid nitrogen and then stored at −80° C. for analysis of drug levels and inflammatory cytokines. Groups 2, 3, 4, and 5 were euthanized and subjected to gross necropsy and tissue collection 6, 12, 24, and 48 hours post-dose, respectively. Group 1 was similarly euthanized and necropsied 48 hours post-dose. The animals were euthanized via injection of a veterinarian-approved euthanasia solution as per the schedule. Immediately after euthanasia in order to avoid autolytic changes, colon tissues were collected, opened, rinsed with saline, and a detailed macroscopic examination of the colon were performed to identify macroscopic findings related to TNBS-damage. Tissue samples were taken from the proximal, mid, and distal transverse colon; the dose site; and the distal colon. Each tissue sample was divided into two approximate halves; one tissue section was placed into 10% neutral buffered formalin (NBF) and evaluated by a Board certified veterinary pathologist, and the remaining tissue section was flash frozen in liquid nitrogen and stored frozen at −80° C. Clinical signs (ill health, behavioral changes, etc.) were recorded daily beginning on day −3. Additional pen-side observations were conducted once or twice daily. Animals observed to be in ill health were examined by a veterinarian. Body weight was measured for all animals on day −3, and prior to scheduled euthanasia. Table 4.1, depicted below, shows the study design. Materials and Methods Test Article Adalimumab (EXEMPTIA™) is a Tumour Necrosis Factor (TNF) inhibitor. A single dose was pre-filled in a syringe (40 mg in a volume of 0.8 mL). TABLE 4.1Study Design TableDaysHoursSample sizeDoseRoute−3−2−110.512468122448GeneralFast•Food/Waterad libidumoral••••••••••••Observationsclinical observations••••••body weight••••Treatments (groups)TNBS (all animals)intra rectal•1. Treated controln = 340 mg insub-cutaneous•0.8 mL salineeuthanizedn = 32. Adalimumabn = 340 mg inintra rectal•0.8 mL salineeuthanizedn = 33. Adalimumabn = 340 mg inintra rectal•0.8 mL salineeuthanizedn = 34. Adalimumabn = 340 mg inintra rectal•0.8 mL salineeuthanizedn = 35. Adalimumabn = 340 mg inintra rectal•0.8 mL salineeuthanizedn = 3Adalimumab (required)600SamplesPBMCscephalic, jugular or•••••catheterSerumcephalic, jugular or•••••••catheterFecalrectal•••••••Tissuenecropsy••••AnalysisHistopathology1 location4 locationsinflammed45180H&Enormal45180H&EBloodadalimumab57pbl15151296TNFα87pbl151515151296Fecesadalimumab57pbl15151296TNFα87pbl151515151296TissueInflammedadalimumab45180pbl3336TNFα45180pbl3336HER245180pbl3336Normaladalimumab45180pbl3336TNFα45180pbl3336HER245180pbl3336 Results While subcutaneously administered adalimumab was detected at all times points tested in plasma, topically administered adalimumab was barely detectable in plasma (FIGS.51and52). Both topical delivery and subcutaneous delivery of adalimumab resulted in reduced levels of TNF-α in colon tissue of TNBS-induced colitis animals, yet topical delivery of adalimumab was able to achieve a greater reduction in TNF-α levels (FIGS.53and54). Either subcutaneous or intra-rectal administration of adalimumab was well tolerated and did not result in death, morbidity, adverse clinical observations, or body weight changes. A decreased level of total TNBS-related inflammatory response was observed by adalimumab treatment via intra-rectal administration when applied to the damaged mucosal surface of the bowel when compared to subcutaneous delivery. A significantly higher concentration of adalimumab was measured in blood following subcutaneous delivery as compared to the blood concentration following intra-rectal administration. Intra-rectal administration of adalimumab decreased the total and normalized TNFα concentration over time (6˜48 h) and was more effective at reducing TNFα at the endpoint (48 h) as compared to groups administered adalimumab subcutaneously. In sum, these data show that the compositions and devices provided herein can suppress the local immune response in the intestine, while having less of a suppressive effect on the systemic immune response of an animal. For example, these data show that intracecal administration of adalimumab using a device as described herein can provide for local delivery of adalimumab to the site of disease, without suppressing the systemic immune response. These data also show that local administration of adalimumab using a device as described herein can result in a significant reduction of the levels of TNFα in diseases animals. Example 7. Comparison of Systemic Versus Intracecal Delivery of Cyclosporine A The objective of this study was to compare the efficacy of an immunosuppressant agent (cyclosporine A; CsA) when dosed systemically versus intracecally to treat dextran sulfate sodium salt (DSS)-induced colitis in male C57Bl/6 mice. Experimental Design A minimum of 10 days prior to the start of the experiment a cohort of animals underwent surgical implantation of a cecal cannula. A sufficient number of animals underwent implantation to allow for 44 cannulated animals to be enrolled in the main study (e.g., 76 animals). Colitis was induced in 60 male C5Bl/6 mice by exposure to 3% DSS-treated drinking water from day 0 to day 5. Two groups of eight additional animals (cannulated and non-cannulated) served as no-disease controls (Groups 1 and 2). Animals were dosed with cyclosporine A via intraperitoneal injection (IP), oral gavage (PO), or intracecal injection (IC) from day 0 to 14 as indicated in Table 5.1. All animals were weighed daily and assessed visually for the presence of diarrhea and/or bloody stool at the time of dosing. Mice underwent video endoscopy on days 10 and 14 to assess colitis severity. Images were captured from each animal at the most severe region of disease identified during endoscopy. Additionally, stool consistency was scored during endoscopy using the parameters defined in Table 5.2. Following endoscopy on day 14, animals from all groups were sacrificed and underwent terminal sample collection. Specifically, animals in all treatment groups dosed on day 14 were sacrificed at a pre-dosing time point, or 1, 2, and 4 hours after dosing (n=3/group/time point). Terminal blood was collected via cardiac puncture and prepared for plasma using K2EDTA as the anti-coagulant. The blood cell pellet was retained and snap frozen while the resulting plasma was split into two separate cryotubes, with 100 μL in one tube and the remainder in the second. Additionally, the cecum and colon were removed from all animals; the contents were collected, weighed, and snap frozen in separate cyrovials. The colon was then rinsed, measured, weighed, and then trimmed to 6 cm in length and divided into five pieces. The most proximal 1 cm of colon was snap frozen for subsequent bioanalysis of cyclosporine A levels. Of the remaining 5 cm of colon, the most distal and proximal 1.5-cm sections were each placed in formalin for 24 hours, then transferred to 70% ethanol for subsequent histological evaluation. The middle 2-cm portion was bisected longitudinally and placed into two separate cryotubes, weighed, and snap frozen in liquid nitrogen. All plasma and frozen colon tissue were stored at −80° C. for selected end point analysis. For all control animals in Groups 1-4, there was an additional collection of 100 μL of whole blood from all animals which was then processed for FACS analysis of α4 and β7 expression on Tx memory cells. The details of the study are shown in Table 5.1. TABLE 5.1Study DesignGroup1234131415NumberNumber of881212121212AnimalsCecalNOYESNOYESNOYESYESCannulaDSSN/AN/A3% DSS on Day 0 to Day 5TreatmentnonenonevehiclevehicleCsACsACsADoseN/AN/AN/AN/A10103(mg/kg)RouteN/AN/AN/AN/APOICICDosingN/AN/AQD:QD:QD:QD:QD:ScheduleDayDayDayDayDay0 to 140 to 140 to 140 to 140 to 14EndoscopyDays 10 and 14Schedule*EndpointsEndoscopy, Colon weight/ length, stool scoreDay 14Terminal Collection (all groups): Cecal contents,colon contents, plasma, and colon tissueFACS analysis collection of Groups 1-4:Whole blood for the following FACS panel: CD4,CD44, CD45RB, α4, β7, CD16/32PKN = 3/ time pointsSacrificeAt pre-dose and 1, 2, and 4 hours post-dosing(Day 14)*Animals were dosed once (QD) on Day 14 and plasma collected (K2EDTA) at pre-dosing, 1, 2, and 4 hours post-dosing from n = 3/group/time point. Each collection was terminal. Experimental Procedures Cecal Cannulation Animals were placed under isoflurance anesthesia, and the cecum exposed via a mid-line incision in the abdomen. A small point incision was made in the distal cecum through which 1-2 cm of the cannula was inserted. The incision was closed with a purse-string suture using 5-0 silk. An incision was made in the left abdominal wall through which the distal end of the cannula was inserted and pushed subcutaneously to the dorsal aspect of the back. The site was washed copiously with warmed saline prior to closing the abdominal wall. A small incision was made in the skin of the back between the shoulder blades, exposing the tip of the cannula. The cannula was secured in place using suture, wound clips, and tissue glue. All animals received 1 mL of warm sterile saline (subcutaneous injection) and were monitored closely until fully recovered before returning to the cage. All animals received buprenorphine at 0.6 mg/kg BID for the first 3 days, and Baytril® at 10 mg/kg QD for the first 5 days following surgery. Disease Induction Colitis was induced on day 0 via addition of 3% DSS (MP Biomedicals, Cat #0260110) to the drinking water. Fresh DSS/water solutions were made on day 3 and any of the remaining original DSS solution was discarded. Dosing Animals were dosed by oral gavage (PO), intraperitoneal injection (IP), or intracecal injection (IC) at a volume of 0.1 mL/20 g on days 0 to 14 as indicated in Table 5.1. Body Weight and Survival Animals were observed daily (weight, morbidity, survival, presence of diarrhea, and/or bloody stool) in order to assess possible differences among treatment groups and/or possible toxicity resulting from the treatments. Animals Found Dead or Moribund Animals were monitored on a daily basis and those exhibiting weight loss greater than 30% were euthanized, and samples were not collected from these animals. Endoscopy Each mouse underwent video endoscopy on days 10 and 14 using a small animal endoscope (Karl Storz Endoskope, Germany) under isoflurane anesthesia. During each endoscopic procedure still images as well as video were recorded to evaluate the extent of colitis and the response to treatment. Additionally, we attempted to capture an image from each animal at the most severe region of disease identified during endoscopy. Colitis severity was scored using a 0-4 scale (0=normal; 1=loss of vascularity; 2=loss of vascularity and friability; 3=friability and erosions; 4=ulcerations and bleeding). Additionally, stool consistency was scored during endoscopy using the parameters defined in Table 5.2. TABLE 5.2Stool ConsistencyScoreDescription0Normal, well-formed pellet1Loose stool, soft, staying in shape2Loose stool, abnormal form with excess moisture3Watery or diarrhea4Bloody diarrhea Tissue/Blood for FACS Tissue and blood were immediately placed in FACS buffer (lx phosphate-buffered saline (PBS) containing 2.5% fetal calf serum (FCS)) and analyzed using the antibody panel in Table 5.3. TABLE 5.3FACS Antibody PanelAntibody TargetFluorochromePurposeCD4APC-Vio770Defines THcellsCD44VioBlueMemory/NaïvediscriminationCD45RBFITCMemory/Naïvediscriminationα4APCDefines TH-memory subset ofinterestβ7PEDefines TH-memory subset ofinterestCD16/32—Fc block Results The data inFIG.55show a decrease in weight loss is observed in DSS mice intracecally administered cyclosporine A as compared to DSS mice orally administered cyclosporine A. The data inFIG.56show a decrease in plasma concentration of cyclosporine A in DSS mice intracecally administered cyclosporine A as compared to DSS mice orally administered cyclosporine A. The data inFIGS.57-59show an increased concentration of cyclosporine A in the colon tissue of DSS mice intracecally administered cyclosporine A as compared to the concentration of cyclosporine A in the colon tissue of DSS mice orally administered cyclosporine A. The data inFIG.60show that DSS mice intracecally administered cyclosporine A have an increased concentration of IL-2 in colon tissue as compared to DSS mice orally administered cyclosporine A. The data inFIG.61show that DSS mice intracecally administered cyclosporine A have a decreased concentration of IL-6 in colon tissue as compared to DSS mice orally administered cyclosporine A. In sum, these data show that the compositions and devices provided herein can suppress the local immune response in the intestine, while having less of a suppressive effect on the systemic immune response of an animal. For example, these data demonstrate that the present compositions and devices can be used to release cyclosporine A to the intestine and that this results in a selective immune suppression in the colon, while having less of an effect on the immune system outside of the intestine. These data also suggest that the present compositions and devices will provide for the treatment of colitis and other pro-inflammatory disorders of the intestine. Example 8. Bellows Testing: Drug Stability Bench Test Experiments were run to evaluate the effects that bellows material would have on the function of a drug used as the dispensable substance. The experiments also evaluated the effects on drug function due to shelf life in the bellows. The adalimumab was loaded into simulated device jigs containing either tapered silicone bellows or smooth PVC bellows and allowed to incubate for 4, 24, or 336 hours at room temperature while protected from light.FIG.64illustrates the tapered silicone bellows, andFIG.65illustrates the tapered silicone bellows in the simulated device jig.FIG.66illustrates the smooth PVC bellows, andFIG.67illustrates the smooth PVC in the simulated device jig. The drug was subsequently extracted using the respective dispensing systems and tested by a competitive inhibition assay. The test method has been developed from the literature (Velayudhan et al., “Demonstration of functional similarity of proposed biosimilar ABP501 to adalimumab”BioDrugs30:339-351 (2016) and Barbeauet et al., “Application Note: Screening for inhibitors of TNFα/s TNFR1 Binding using AlphaScreen™ Technology”. PerkinElmer Technical Note ASC-016. (2002)), as well as pre-testing development work using control drug and experiments using the provided AlphaLISA test kits.FIG.68demonstrates the principle of the competition assay performed in the experiment. The bellows were loaded as follows: aseptically wiped the dispensing port of the simulated ingestible device jig with 70% ethanol; allowed to air dry for one minute; used an adalimumab delivery syringe to load each set of bellows with 200 μL of drug; took a photo of the loaded device; gently rotated the device such that the drug is allowed to come in contact with all bellows surfaces; protected the bellows from light; and incubate at room temperature for the predetermined time period to allow full contact of the drug with all bellows' surfaces. The drug was extracted as follows: after completion of the incubation period; the device jig was inverted such that the dispensing port was positioned over a sterile collection microfuge tube and petri dish below; five cubic centimeters of air was drawn into an appropriate syringe; the lure lock was attached to the device jig; the syringe was used to gently apply positive pressure to the bellow with air such that the drug was recovered in the collection microfuge tube; where possible, a video of drug dispensing was taken; samples were collected from each bellows type; a control drug sample was collected by directly dispensing 200 μL of drug from the commercial dispensing syringe into a sterile microfuge tube; the control drug-free sample was collected by directly dispensing 200 μL of PBS using a sterile pipette into a sterile microfuge tube; the collected drug was protected from light; and the drug was diluted over the following dilution range (250, 125, 25, 2.5, 0.25, 0.025, 0.0125, 0.0025 μg) in sterile PBS to determine the IC50range of the drug. To determine any effects storage conditions may have on drug efficacy in the device, the drug (stored either in the syringe, silicon bellows, PVC bellows) was stored at room temperature while protected from light for 24 hours and 72 hours. Samples were then extracted and the steps in the preceding paragraph were repeated. The AlphaLISA (LOCI™) test method was used. Human TNFα standard dilution ranges were prepared as described in Table 6. TABLE 6[human TNFα]Vol. ofVol. ofin standard curvehuman TNFαdiluent(g/mL in(pg/mL inTube(μL)(μL)*5 μL)5 μL)A10 μL of901E−07100 000reconstitutedhuman TNFαB60 μL of tube A1403E−0830 000C60 μL of tube B1201E−0810 000D60 μL of tube C1403E−093 000E60 μL of tube D1201E−091 000F60 μL of tube E1403E−10300G60 μL of tube F1201E−10100H60 μL of tube G1403E−1130I60 μL of tube H1201E−1110J60 μL of tube I1403E−123K60 μL of tube J1201E−121L60 μL of tube K1403E−130.3M**(background)010000N**(background)010000O**(background)010000P**(background)010000 The test was performed as follows: the above standard dilution ranges were in a separate 96-well plate; to ensure consistent mixing, samples were mixed up and down gently with a pipette five times; a 384-well test plate was prepared according to the test layout diagram depicted Table 7; five microliters of 10,000 pg/mL TNFα standard from the previously made dilution plate was added to each corresponding concentration as shown in Table 6; five microliters of recovered drug (directly from the commercial syringe (A), from the silicone bellows (B Si), from the PVC bellows (B PVC), or from the PBS control (C) was added into the corresponding wells described in Table 5; the test plate was incubated for one hour at room temperature while protected from light; 10 microliters of acceptor beads were added to each previously accessed well; the wells were incubated for 30 minutes at room temperature while protected from light; 10 μL of biotinylated antibody was added to each previously accessed well; the wells were incubated for 15 minutes at room temperature, while protected from light; the room lights were darkened and 25 microliters of streptavidin (SA) donor beads were added to each previously accessed well; the wells were incubated for 30 minutes at room temperature while protected from light; the plate was read in Alpha Mode; and the results were recorded. Upon addition of reagent(s) in the various steps, each well was pipetted up and down three times to achieve good mixing. TABLE 7123456789101112ASTD2STD102502502502502502502502502501.00E+0510AAAAAB SiB SiB SiB SiBCSTD3STD11125125125125125125125125125300003AAAAAB SiB SiB SiB SiDESTD4STD12252525252525252525100001AAAAAB SiB SiB SiB SiFGSTD5STD132.52.52.52.52.52.52.52.52.530000.333AAAAAB SiB SiB SiB SiHISTD6Blank0.250.250.250.250.250.250.250.250.2510000AAAAAB SiB SiB SiB SiJKSTD7Blank0.0250.0250.0250.0250.0250.0250.0250.0250.0253000AAAAAB SiB SiB SiB SiLMSTD8Blank0.0130.0130.0130.0130.0130.0130.0130.0130.0131000AAAAAB SiB SiB SiB SiNOSTD9Blank0.0030.0030.0030.0030.0030.0030.0030.0030.003300AAAAAB SiB SiB SiB SiP1314151617181920212223A250250250250250250250250250250250B SiB PVCB PVCB PVCB PVCB PVCCCCCCBC125125125125125125125125125125125B SiB PVCB PVCB PVCB PVCB PVCCCCCCDE2525252525252525252525B SiB PVCB PVCB PVCB PVCB PVCCCCCCFG2.52.52.52.52.52.52.52.52.52.52.5B SiB PVCB PVCB PVCB PVCB PVCCCCCCHI0.250.250.250.250.250.250.250.250.250.250.25B SiB PVCB PVCB PVCB PVCB PVCCCCCCJK0.0250.0250.0250.0250.0250.0250.0250.0250.0250.0250.025B SiB PVCB PVCB PVCB PVCB PVCCCCCCLM0.0130.0130.0130.0130.0130.0130.0130.0130.0130.0130.013B SiB PVCB PVCB PVCB PVCB PVCCCCCCNO0.0030.0030.0030.0030.0030.0030.0030.0030.0030.0030.003B SiB PVCB PVCB PVCB PVCB PVCCCCCCP The data are shown inFIGS.69-71. The data demonstrate that the bellows do not negatively impact the drug function after shelf lives of 4 hours, 24 hours, or 336 hours. The IC50values of the drug dispensed from the bellows were comparable to the IC50values of the standard dispensation method (Table 6). A slight right shift was noted in the bellows curves after 24 hours (FIG.70), but this shift was well within the error bars of the curves. Tables 8-11 represent data ofFIGS.69-71, respectively. Of note, when comparing mean (n=5) RFU data between test articles over the concentration ranges significant differences (p<0.05) were discerned. However, these significant differences did not favor either test article over time, suggesting that they were not related to the performance of the material in response to the drug (FIGS.69-71). TABLE 8NeedleSiliconePVCcontrol (A)Bellows (B)Bellows (C)4Hours0.01740.01690.017224Hours0.01800.01800.0180336Hours0.01440.01590.0163 TABLE 9Statistics (Student's T-test, 2 tailed, non-pair-wise, for significance p < 0.05)DrugNeedle control (A)Needle control (A)Silicone(micrograms)vs. Silicone (B)vs. PVCvs. PVC0.00010.9110.008*0.2680.00250.1380.3900.8220.01250.1220.1180.7710.0250.1430.4650.020*0.250.5910.9840.3502.50.2430.1240.1691250.8670.6880.1822500.6810.1840.108*p < 0.5 data set TABLE 10Statistics (Student's T-test, 2 tailed, non-pair-wise, for significance p < 0.05)DrugNeedle control (A)Needle control (A)Silicone(micrograms)vs. Silicone (B)vs. PVCvs. PVC0.00010.1320.038*0.2920.00250.003*0.0760.5750.01250.1610.022*0.7830.0250.0580.0780.5380.250.9740.3840.1982.50.7140.0800.017*1250.8730.7310.2692500.7980.9560.903*p < 0.5 data set TABLE 11Statistics (Student's T-test, 2 tailed, non-pair-wise, for significance p < 0.05)DrugNeedle control (A)Needle control (A)Silicone(micrograms)vs. Silicone (B)vs. PVCvs. PVC0.00010.8584490.036847*0.026444*0.00250.0873790.2803020.046767*0.01250.4692820.0572320.1171940.0250.02758*0.0782340.3734190.250.4115480.2589280.4004982.50.3689590.1565740.006719*1250.9486490.2467020.4637352500.4850460.1289930.705543*p < 0.5 data set Example 9. A Comparison Study of Systemic Vs Intracecal Delivery of SMAD7 Bio-Distribution in DSS-Induced Colitis in Male C57Bl/6 Mice The objective of this study was to compare the efficacy of novel test articles, e.g., fluorescent SMAD7 antisense oligonucleotides (SMAD7 AS), when dosed systemically versus intracecally in the treatment of DSS-induced colitis, in male C57Bl/6 mice. Experimental Design A minimum of 10 days prior to the start of the experiment a cohort of animals underwent surgical implantation of a cecal cannula. A sufficient number of animals underwent implantation to allow for 12 cannulated animals to be enrolled in the main study (i.e., 16 animals). Colitis was induced in 12 male C57Bl/6 mice (Groups 4-5) by exposure to 3% DSS-treated drinking water from Day 0 to Day 5. Three groups of six additional animals per group (n=6 cannulated; n=12 non-cannulated; Groups 1-3) served as no-disease controls (Groups 1-3). All animals were weighed daily and assessed visually for the presence of diarrhea and/or bloody stool during this time. Animals were dosed with test-article via oral gavage (PO) or intracecal injection (IC) once on Day 9 as indicated in Table 12. The animals in Group 0 were not dosed. The animals in Groups 2 and 4 were dosed PO with SMAD7 antisense. The animals in Groups 3 and 5 were dosed IC with SMAD7 antisense. All animals were euthanized by CO2inhalation 12 hours after dosing, on Day 10. Terminal blood was collected into two K2EDTA tubes and processed for plasma. Both plasma and pellet samples were snap-frozen in liquid nitrogen and stored at −80° C. Cecum contents were removed and the contents were split into two aliquots. Both aliquots were weighed and snap frozen in separate cryovials in liquid nitrogen. The cecum was excised and bisected longitudinally; each piece is separately weighed and flash-frozen in liquid nitrogen. The colon contents were removed and the contents were split into two aliquots. Both aliquots were weighed and snap frozen in separate cryovials in liquid nitrogen. The colon was then rinsed, and the most proximal 2 cm of colon was collected. This 2-cm portion was bisected longitudinally; each piece was separately weighed and flash-frozen in liquid nitrogen. Snap-frozen blood pellet, cecum/colon contents, and tissue samples were used for downstream fluoremetry or RP-HPLC. The details of the study design are shown in Table 12. TABLE 12Study designTerminalNoCecalColitisCollectionsGroupAnimalsCannulaInductionTreatmentRouteScheduleDay 1016NO————Whole blood,26NOFluorescentlyPOQDplasma, cecal36YESlabeledICDay 9**contents, colon46NO3% DSSSMAD7POcontents, cecal56YESDays 0-5antisenseICtissue, colon50 μg*tissue*Per mouse. TA is administered in 0.075 mL/animal.**Animals are dosed on Day 9 and collections are performed 12 hours later. Materials and Methods Mice Normal male C57Bl/6 mice between the ages of 6-8 weeks old, weighing 20-24 g, were obtained from Charles River Laboratories. The mice were randomized into five groups of six mice each, and housed in groups of 8-15 per cage, and acclimatized for at least three days prior to entering the study. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour, with an automatic timer for a light/dark cycle of 12 hours on/off, and fed with Labdiet 5053 sterile rodent chow, with water administered ad libitum. Cecal Cannulation The animals were placed under isoflurane anesthesia, with the cecum exposed via a midline incision in the abdomen. A small point incision was made in the distal cecum, where 1-2 cm of the cannula was inserted. The incision was closed with a purse string suture using 5-0 silk. An incision was then made in the left abdominal wall through which the distal end of the cannula was inserted and pushed subcutaneously to the dorsal aspect of the back. The site was then washed copiously with warmed saline prior to closing the abdominal wall. A small incision was also made in the skin of the back between the shoulder blades, exposing the tip of the cannula. The cannula was secured in place using suture, wound clips, and tissue glue. All animals were administered 1 mL of warm sterile saline (subcutaneous injection) and were monitored closely until recovery before returning to their cage. All animals were administered 0.6 mg/kg BID buprenorphine for the first 3 days, and Baytril® at 10 mg/Kg every day for the first 5 days post-surgery. Disease Induction Colitis was induced on Day 0 via addition of 3% DSS (MP Biomedicals, Cat #0260110) to the drinking water. Fresh DSS/water solutions was provided on Day 3 and any of the remaining original DSS solution is discarded. Body Weight and Survival Animals were observed daily (weight, morbidity, survival, presence of diarrhea and/or bloody stool) in order to assess possible differences among treatment groups and/or possible toxicity resulting from the treatments. Animals Found Dead or Moribund Animals were monitored on a daily basis. Animals exhibiting weight loss greater than 30% were euthanized, and samples were not collected from these animals. Dosing Animals were dosed with test-article via oral gavage (PO) or intracecal injection (IC) once on Day 9 as indicated in Table 12. Animals in Group 0 were not dosed. Animals in Groups 2 and 4 were dosed PO with SMAD7 antisense. Animals in Groups 3 and 5 were dosed IC with SMAD7 antisense. Sacrifice All animals were euthanized by CO2inhalation 12 hours after dosing, on Day 10. Sample Collection Intestinal contents, peripheral blood and tissue were collected at sacrifice on Day 10, as follows: Blood/Plasma Terminal blood was collected into two K2EDTA tubes and processed for plasma. The approximate volume of each blood sample was recorded prior to centrifugation. Both plasma and pellet samples were snap-frozen in liquid nitrogen and stored at −80° C. The first pellet sample (sample 1) was used for fluoremetry. The second pellet sample (sample 2) was used for RP-HPLC. Cecum Contents Cecum contents was removed and contents were split into two aliquots. Both aliquots were weighed and snap frozen in separate cryovials in liquid nitrogen. The first sample (sample 1) was used for fluorometry. The second sample (sample 2) was used for RP-HPLC. Cecum The cecum was excised and bisected longitudinally; each piece was separately weighed and snap-frozen. The first sample (sample 1) was used for fluoremetry. The second sample (sample 2) was used for RP-HPLC. Colon Contents Colon contents were removed and contents were split into two aliquots. Both aliquots were weighed and snap frozen in separate cryovials in liquid nitrogen. The first sample (sample 1) was used for fluorometry. The second sample (sample 2) was used for RP-HPLC. Colon The colon was rinsed, and the most proximal 2 cm of colon was collected and bisected longitudinally. Each piece was separately weighed and flash-frozen in liquid nitrogen. The first sample (sample 1) was used for fluorometry. The second sample (sample 2) was used for RP-HPLC. SMAD7 Antisense Bioanalysis Samples flash-frozen for fluoremetry were homogenized in 0.5 mL buffer RLT+ (Qiagen). Homogenate was centrifuged (4000×g; 10 minutes), and supernatant was collected. Forty microliters of the sample was diluted 1:6 in 200 μL of bicarbonate solution and 100 μL of diluted supernatant was analyzed on a fluorescent plate reader (485 excitation; 535 emission) in duplicate. Prior to the above, assay development was performed as follows. Samples (as indicated in Sample Collection) were harvested from a naïve animal and flash-frozen. Samples were then homogenized in 0.5 mL buffer RLT+, homogenate was centrifuged (4000×g; 10 minutes) and supernatant was collected and diluted 1:6 with bicarbonate solution (i.e., 0.5 mL supernatant was added to 2.5 mL of PBS). An aliquot (0.200 mL (90 μL for each duplicate) of each diluted sample was pipetted into 15 (14 dilution of FAM-AS-SAMD7+ blank control) Eppendorf tubes. One tube was set-aside to be used as a blank sample. Ten microliters of fluorescently-labeled SMAD7 antisense was then spiked into all other sample to achieve final concentrations of 50 μg/mL, 16.67 μg/mL, 5.56 μg/mL, 1.85 μg/mL, 0.62 μg/mL, 0.21 μg/mL, 0.069 μg/mL, 0.023 μg/mL, 7.6 μg/mL, 2.5 μg/mL, 0.847 μg/mL, 0.282 ng/mL, 0.094 μg/mL, and 0.024 μg/mL respectively. The fluorescently-labeled SMAD7 antisense was prepared and serially diluted such that the volume added to each organ homogenate sample was the same for each of the above concentrations. These samples were analyzed on a fluorescent plate reader (485 excitation; 535 emission) in duplicate. Processing for RP-HPLC Samples flash-frozen for RP-HPLC were homogenized in buffer RLT+ (Qiagen). Homogenate was centrifuged (4000×g; 10 minutes), and supernatant was used to perform RP-HPLC analysis. Results The data inFIGS.73and74show that significantly more SMAD7 anstisense oligonucleotide was present in cecum tissue and colon tissue for mice with or without DSS treatment that were intra-cecally administered the SMAD7 antisense oligonucleotide as compared to mice with or without DSS treatment that were orally administered the SMAD7 antisense oligonucleotide. The data inFIG.75show that there is about the same level of SMAD7 antisense oligonucleotide in the cecum contents of mice with or without DSS treatment that were orally or intra-cecally administered the SMAD7 antisense oligonucleotide. No SMAD7 antisense oligonucleotide was found in the plasma or white blood cell pellet of SMAD7 antisense oligonucleotide treated mice. Example 10. Comparison of the Tissue, Plasma, and GI Content Pharmacokinetics of Tacrolimus Through Oral Vs. Intra-Cecal Ingestible Device Delivery in Yorkshire-Cross Farm Swine The primary objective of this study was to compare the tissue, plasma, rectal sample, and GI content pharmacokinetics of tacrolimus through oral versus intra-cecal ingestible device delivery in normal Yorkshire-Cross farm swine. This study compares the effects of administration of: a single intra-cecal administration of an ingestible device containing 0.8 mL sterile vehicle solution (80% alcohol, 20% castor oil (HCO-60)); a single oral dose of tacrolimus at 4 mg/0.8 mL (in sterile vehicle solution); and a single intra-cecal administration of an ingestible device containing either 1 mg/0.8 mL (in sterile vehicle solution), 2 mg/0.8 mL (in sterile vehicle solution), or 4 mg/0.8 mL (in sterile vehicle solution). This study employed five groups of three female swine weighing approximately 45 to 50 kg at study start. Swine were randomly placed into animal rooms/pens as they are transferred from the delivery vehicle without regard to group. Group numbers were assigned to the rooms in order of room number. No further randomization procedure was employed. The study design is provided in Table 13. TABLE 13Study Design TableDaysHoursGroupPre-DosePost-doseGeneralsizeDoseRoute−11 −10 −5 −1 10.5 1 2 3 4 6 12Fastad libidumFood/WaterObservationsclinical observationsDay −10~−5 &body weight*Day 1Treatments (Groups)1. Vehicle controln = 30.8 mL (20%ICn = 3Surgical placement ofHCO-60, 80%IC port**EtOH)Euthanized(1 IngestibleDevice)2. Tacrolimus (PO)n = 34 mg in 0.8 mLOraln = 3Surgical placement of0.08 mg/kgIC port**(solution)Euthanized3. Tacrolimus (IC)n = 31 mg in 0.8 mLICn = 3Surgical placement of0.02 mg/kgIC port**(1 IngestibleEuthanizedDevice)4. Tacrolimus (IC)n = 32 mg in 0.8 mLICn = 3Surgical placement of0.04 mg/kgIC port**(1 IngestibleEuthanizedDevice)5. Tacrolimus (IC)n = 34 mg in 0.8 mLICn = 3Surgical placement of0.08 mg/kgIC port**(1 IngestibleEuthanizedDevice)Tacrolimus (required)20 mgSamples*****Plasmacephalic,Rectal contentsjugular orTissue***×5catheterLuminal contents**×5rectalnecropsynecropsyAnalysis (AgriluxTotalCharles River)SamplesPlasma[Tacrolimus]105151515151515 15Rectal contents[Tacrolimus]6015 15 15 15Tissue (intact)***[Tacrolimus]105105Luminal contents[Tacrolimus]7575Tissue after removingluminal content[Tacrolimus]7575Notes:*Animal weight was ~45-50 kg for drug doses proposed.**Surgical placement of IC port in all animals to control.***Tissue samples [drug] (five GI section cecum (CAC); proximal colon (PCN); transverse colon (TCN); distal colon (DCN); rectum (RTM), plus mesenteric lymph nodes and Peyer's Patch).****Luminal contents (cecum (CAC); proximal colon (PCN); transverse colon (TCN); distal colon (DCN); rectum (RTM)). Animals in Group 1 received an ingestible device containing 0.8 mL of vehicle solution (80% alcohol, 20% HCO-60). Animals in Group 2 received orally 4 mL liquid formulation of tacrolimus at 4 mg/0.8 mL per animal (Prograf: 5 mg/mL). Animals in Group 3 received intra-cecally an ingestible device containing tacrolimus at 1 mg in 0.8 mL per ingestible device. Animals in Group 4 received intra-cecally an ingestible device containing tacrolimus at 2 mg in 0.8 mL per ingestible device. Animals in Group 5 received intra-cecally an ingestible device containing tacrolimus at 4 mg in 0.8 mL per ingestible device. To control for potential confounding effects of the surgery, all groups fast on Day −11 at least 24 hr before being subjected to anesthesia followed by surgical placements of a cecal port by a veterinary surgeon at Day −10. All animals were fasted for at least 12 hr prior to dosing on Day 1. Animals were dosed via either intra-cecal dosing (IC) or oral dosing (PO) at Day 1 (between 6-8 p.m.). All animals resumed feeding at approximately 4 hours after dose (11-12 p.m. after dosing). Animals in Group 1 (Vehicle Control) were administered a single intra-cecal ingestible device containing 0.8 mL Vehicle solution (80% alcohol, 20% castor oil (HCO-60) on Day 1. On Day −10 the animals were anesthetized, and a veterinary surgeon surgically placed an intra-cecal port in each animal. On Day 1, each animal was placed into a sling then a single intra-cecal ingestible device containing 0.8 mL vehicle solution (80% alcohol, 20% castor oil (HCO-60)) is introduced by the veterinary surgeon into the cecum via the cecal port in each animal. Following ingestible device placement, the animals were removed from the slings and placed back into their pens with water. All animals resumed feeding at approximately 4 hours after dose. Samples of rectal contents were collected for pharmacokinetic analyses from each animal at each of 1, 3, 6, and 12 hours post-ingestible device placement using a fecal swab (rectal swab). A total of 60 samples were collected. Approximately 200˜400 mg of rectal content were collected, if available, with a fecal swab (Copan Diagnostics Nylon Flocked Dry Swabs, 502CS01). The fecal swab was pre-weighed and weighed after collection in the collection tube (Sterile Tube and Cap No Media, PFPM913S), and the sample weight was recorded. The fecal swab was broken via the breakpoint, and was stored in the collection tube, and immediately frozen at −70° C. Whole blood (2 mL) was collected into K2EDTA coated tubes for pharmacokinetics at each time-point of pre-dose and 1, 2, 3, 4, 6 and 12 hours post-dose. Immediately following euthanasia, tissue was collected. A total of 105 samples were collected. For tissue necropsy, small intestine fluid and cecal fluid were collected separately from all the animals into two separate square plastic bottles, and stored at −20° C. The length and diameter of the cecum and the colon was measured from one animal in each group and recorded for reference. Tissues were collected for pharmacokinetic analyses and include mesenteric lymph nodes, a Peyer's Patch, and five gastrointestinal sections, including cecum, proximal colon, transverse colon, distal colon, and rectum. All samples were weighed, and the tissue sample weights were recorded. In each of the five gastrointestinal sections, tissue samples were collected in three different areas where the mucosal surface was visible and not covered by luminal content by using an 8.0-mm punch biopsy tool. Around 3 grams of the total punched sample were collected into a pre-weighed 15-mL conical tube, and the tissue weight was recorded. Three mesenteric lymph nodes were collected from different areas and weighed. At least one Peyer's Patch was collected and weighed. Tissues were snap-frozen in liquid nitrogen and stored frozen at approximately −70° C. or below (total of 105 samples). Luminal contents were collected for pharmacokinetic analyses from the surface of the tissue from each of five gastrointestinal sections: cecum, proximal colon, transverse colon, distal colon, and rectum (total of 75). The contents were collected in pre-weighed 15-mL conical tubes and the sample weights were recorded. Samples were snap-frozen in liquid nitrogen stored frozen at approximately −70° C. or below. After removing the luminal content, another set of tissue samples from 3 different areas were collected via an 8.0-mm punch biopsy in each section of the five tissue gastrointestinal sections described above. Around 3 grams of the total punched sample were collected into a pre-weighed 15-mL conical tube, and the tissue weight was recorded (total of 75). Tissues were snap-frozen in liquid nitrogen and stored frozen at approximately −70° C. or below. A 30-cm length of jejunum (separated into two 15 cm lengths), and the remaining distal and transverse colon tissue sample (after tissue and luminal content were collected for PK) were collected in one animal in each group of treatment, snap-frozen in liquid nitrogen and stored frozen at approximately −70° C. or below. All samples for pharmacokinetic analyses were stored on dry ice before analyses. Group 2 animals were administered a single oral dose of tacrolimus at 1 mg/0.8 mL (in the vehicle solution) on Day 1. Plasma, rectal content sample, tissue collection, GI content collection and related procedures/storage/shipments was the same as those employed in Group 1. Group 3 animals were administered a single intra-cecal ingestible device containing tacrolimus at 0.5 mg/0.8 mL (in the vehicle solution) on Day 1 by a veterinary surgeon. Plasma, rectal content sample, tissue collection, GI content collection and related procedures/storage/shipments was the same as those employed in Group 1. All samples were analyzed for tacrolimus. Group 4 animals were administered a single intra-cecal ingestible device of tacrolimus at 2 mg/0.8 mL (in sterile vehicle solution) on Day 1 by a veterinary surgeon. Plasma, rectal content sample, tissue collection, GI content collection and related procedures/storage/shipments were the same as those employed in Group 1. All samples were analyzed for tacrolimus. Group 5 animals are administered a single intra-cecal ingestible device containing tacrolimus at 4 mg/0.8 mL (in the vehicle solution) on Day 1 by a veterinary surgeon. Plasma, rectal content sample, tissue collection, GI content collection and related procedures/storage/shipments were the same as those employed in Group 1. All samples were analyzed for tacrolimus. Detailed clinical observations were conducted daily from Day −10 to −5, and on Day 1. Additional pen-side observations were conducted at least once each day. The animals remained under constant clinical observation for the entire 12 hours from dose until euthanasia. Body weights were collected on Day −10, Day −5, and pre-dose on Day 1. Animals were euthanized via injection of a veterinarian-approved euthanasia. Test Article and Formulation 1. Vehicle solution, 20 mL Description: 80% alcohol, 20% PEG-60 castor oil Physical characteristics: clear liquid solution. 2. Prograf (tacrolimus injection), 10 ampules Description: A sterile solution containing the equivalent of 5 mg anhydrous tacrolimus in 1 mL. Tacrolimus is macrolide immunosuppressant and the active ingredient of Prograf. 0.8 mL of Prograf (5 mg/mL) was administrated through oral gavage per animal in group 2. Prograf (5 mg/mL) was diluted 2× folds (2.5 mg/mL) and 4× folds (1.25 mg/mL) by using vehicle solution. 0.8 mL of each concentration, 1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL of Prograf, was injected into a DSS ingestible device for group 3, 4, and 5. Formulation: Each mL contained polyoxyl 60 hydrogenated castor oil (HCO-60), 200 mg, and dehydrated alcohol, USP, 80.0% v/v. Physical characteristics: clear liquid solution. 3. DDS ingestible device containing Tacrolimus Description: Three (3) DDS ingestible devices containing vehicle solution for Group 1, three (3) DSS ingestible devices containing 1 mg tacrolimus for Group 3, three (3) DDS ingestible devices containing 2 mg tacrolimus for Group 4, and three (3) DDS ingestible devices containing 4 mg tacrolimus for Group 5. Acclimation Animals were acclimated prior to study initiation for at least 7 days. Animals in obvious poor health were not placed on study. Concurrent Medication Other than veterinary-approved anesthetics and medications used during surgery to install the ileocecal ports, or for vehicle or test article administration, and analgesia and antibiotics post-surgery, no further medications were employed. Feed All swine were fasted at least 24 hours before being anesthetized and properly medicated for surgery or overnight before dosing. Otherwise, animals were fed ad-libitum. Tap water was pressure-reduced and passed through a particulate filter, then a carbon filter prior to supply to an automatic watering system. Water was supplied ad libitum. There were no known contaminants in the feed or water that would be expected to interfere with this study. Results The data inFIG.76show that the mean concentration of tacrolimus in the cecum tissue and the proximate colon tissue were higher in swine that were inta-cecally administered tacrolimus as compared to swine that were orally administered tacrolimus. These data suggest that intra-cecal administration of tacrolimus is able to locally deliver tacrolimus to the tissues in the GI tract of a mammal, while not decreasing the systemic immune system of a mammal. Example 11. Comparison of the Tissue, Plasma, and GI Content Pharmacokinetics of Adalimumab Through Sc Vs. Intra-Cecal Ingestible Device Delivery in Yorkshire-Cross Farm Swine in Dss-Induced Colitis The purpose of this non-Good Laboratory Practice (GLP) study is to explore the PK/PD and bioavailability of adalimumab when applied to DSS-induced colitis in Yorkshire-cross farm swine. All animals are randomized into groups of three. Animals are dosed once with adalimumab via subcutaneous (SC), perirectal (PR), or intracecal (IC) administration. The concentration of adalimumab and TNFα is measured in plasma at 1, 2, 3, 4, 6, and 12 hours post-dose. The concentration of adalimumab is measured in rectal contents at 1, 3, 6, and 12 hours post-dose and in luminal content at 12 hours post-dose. Concentration of adalimumab and TNFα, HER2, and total protein is measured in gastrointestinal tissue, e.g., cecum sample (CAC), proximal colon sample (PCN), transverse colon sample (TCN), distal colon sample (DCNi) inflamed, distal colon non-inflamed sample (DCNn), and rectum sample (RTM), at 12 hours post-dose. Example 12. Human Clinical Trial of Treatment of Ulcerative Colitis Using Adalimumab As a proof of concept, the patient population of this study is patients that (1) have moderate to severe ulcerative colitis, regardless of extent, and (2) have had an insufficient response to a previous treatment, e.g., a conventional therapy (e.g., 5-ASA, corticosteroid, and/or immunosuppressant) or a FDA-approved treatment. In this placebo-controlled eight-week study, patients are randomized. All patient undergo a colonoscopy at the start of the study (baseline) and at week 8. Patients enrolled in the study are assessed for clinical status of disease by stool frequency, rectal bleeding, abdominal pain, physician's global assessment, and biomarker levels such as fecal calprotectin and hsCRP. The primary endpoint is a shift in endoscopy scores from Baseline to Week 8. Secondary and exploratory endpoints include safety and tolerability, change in rectal bleeding score, change in abdominal pain score, change in stool frequency, change in partial Mayo score, change in Mayo score, proportion of subjects achieving endoscopy remission, proportion of subjects achieving clinical remission, change in histology score, change in biomarkers of disease such as fecal calprotectin and hsCRP, level of adalimumab in the blood/tissue/stool, change in cytokine levels (e.g., TNFα, IL-6) in the blood and tissue. FIG.72describes an exemplary process of what would occur in clinical practice, and when, where, and how the ingestible device will be used. Briefly, a patient displays symptoms of ulcerative colitis, including but not limited to: diarrhea, bloody stool, abdominal pain, high c-reactive protein (CRP), and/or high fecal calprotectin. A patient may or may not have undergone a colonoscopy with diagnosis of ulcerative colitis at this time. The patient's primary care physician refers the patient. The patient undergoes a colonoscopy with a biopsy, CT scan, and/or MRI. Based on this testing, the patient is diagnosed with ulcerative colitis. Most patients are diagnosed with ulcerative colitis by colonoscopy with biopsy. The severity based on clinical symptoms and endoscopic appearance, and the extent, based on the area of involvement on colonoscopy with or without CT/MRI is documented. Treatment is determined based on diagnosis, severity and extent. For example, treatment for a patient that is diagnosed with ulcerative colitis is an ingestible device programmed to release a single bolus of a therapeutic agent, e.g., 40 mg adalimumab, in the cecum or proximal to the cecum. Prior to administration of the treatment, the patient is fasted overnight and is allowed to drink clear fluids. Four hours after swallowing the ingestible device, the patient can resume a normal diet. An ingestible device is swallowed at the same time each day. The ingestible device is not recovered. In some embodiments, there may be two different ingestible devices: one including an induction dose (first 8 to 12 weeks) and a different ingestible device including a different dose or a different dosing interval. In some examples, the ingestible device can include a mapping tool, which can be used after 8 to 12 weeks of induction therapy, to assess the response status (e.g., based on one or more of the following: drug level, drug antibody level, biomarker level, and mucosal healing status). Depending on the response status determined by the mapping tool, a subject may continue to receive an induction regimen or maintenance regimen of adalimumab. In different clinical studies, the patients may be diagnosed with Crohn's disease and the ingestible devices (including adalimumab) can be programmed to release adalimumab in the cecum, or in both the cecum and transverse colon. In different clinical studies, the patients may be diagnosed with illeocolonic Crohn's disease and the ingestible devices (including adalimumab) can be programmed to release adalimumab in the late jejunum or in the jejunum and transverse colon. Example 13 An ingestible medical device according to the disclosure (“TLC1”) was tested on 20 subjects to investigate its localization ability. TLC1 was a biocompatible polycarbonate ingestible device that contained a power supply, electronics and software. An onboard software algorithm used time, temperature and reflected light spectral data to determine the location of the ingestible device as it traveled the GI tract. The ingestible device is 0.51×1.22 inches which is larger than a vitamin pill which is 0.4×0.85 inches. The subjects fasted overnight before participating in the study. Computerized tomography (“CT”) were used as a basis for determining the accuracy of the localization data collected with TLC1. One of the 20 subjects did not follow the fasting rule. CT data was lacking for another one of the 20 subjects. Thus, these two subjects were excluded from further analysis. TLC1 sampled RGB data (radially transmitted) every 15 seconds for the first 14 hours after it entered the subject's stomach, and then samples every five minutes after that until battery dies. TLC1 did not start to record optical data until it reached the subject's stomach. Thus, there was no RGB-based data for the mouth-esophagus transition for any of the subjects. In addition, a PillCam® SB (Given Imaging) device was tested on 57 subjects. The subjects fasted overnight before joining the study. PillCam videos were recorded within each subject. The sampling frequency of PillCam is velocity dependent. The faster PillCam travels, the faster it would sample data. Each video is about seven to eight hours long, starting from when the ingestible device was administrated into the subject's mouth. RGB optical data were recorded in a table. A physician provided notes on where stomach-duodenum transition and ileum-cecum transition occurred in each video. Computerized tomography (“CT”) was used as a basis for determining the accuracy of the localization data collected with PillCam. Esophagus-Stomach Transition For TLC1, it was assumed that this transition occurred one minute after the patient ingested the device. For PillCam, the algorithm was as follows:1. Start mouth-esophagus transition detection after ingestible device is activated/administrated2. Check whether Green <102.3 and Blue <94.6a. If yes, mark as mouth-esophagus transitionb. If no, continue to scan the data3. After detecting mouth-esophagus transition, continue to monitor Green and Blue signals for another 30 seconds, in case of location reversala. If either Green >110.1 or Blue >105.5, mark it as mouth-esophagus location reversalb. Reset the mouth-esophagus flag and loop through step 2 and 3 until the confirmed mouth-esophagus transition detected4. Add one minute to the confirmed mouth-esophagus transition and mark it as esophagus-stomach transition For one of the PillCam subjects, there was not a clear cut difference between the esophagus and stomach, so this subject was excluded from future analysis of stomach localization. Among the 56 valid subjects, 54 of them have correct esophagus-stomach transition localization. The total agreement is 54/56=96%. Each of the two failed cases had prolonged esophageal of greater than one minute. Thus, adding one minute to mouth-esophagus transition was not enough to cover the transition in esophagus for these two subjects. Stomach-Duodenum For both TLC1 and PillCam, a sliding window analysis was used. The algorithm used a dumbbell shape two-sliding-window approach with a two-minute gap between the front (first) and back (second) windows. The two-minute gap was designed, at least in part, to skip the rapid transition from stomach to small intestine and capture the small intestine signal after ingestible device settles down in small intestine. The algorithm was as follows:1. Start to check for stomach-duodenum transition after ingestible device enters stomach2. Setup the two windows (front and back)a. Time length of each window: 3 minutes for TLC1; 30 seconds for PillCamb. Time gap between two windows: 2 minutes for both devicesc. Window sliding step size: 0.5 minute for both devices3. Compare signals in the two sliding windowsa. If difference in mean is higher than 3 times the standard deviation of Green/Blue signal in the back windowi. If this is the first time ever, record the mean and standard deviation of signals in the back window as stomach referenceii. If mean signal in the front window is higher than stomach reference signal by a certain threshold (0.3 for TLC1 and 0.18 for PillCam), mark this as a possible stomach-duodenum transitionb. If a possible pyloric transition is detected, continue to scan for another 10 minutes in case of false positive flagi. If within this 10 minutes, location reversal is detected, the previous pyloric transition flag is a false positive flag. Clear the flag and continue to checkii. If no location reversal has been identified within 10 minutes following the possible pyloric transition flag, mark it as a confirmed pyloric transitionc. Continue monitoring Green/Blue data for another 2 hours after the confirmed pyloric transition, in case of location reversali. If a location reversal is identified, flag the timestamp when reversal happened and then repeat steps a-c to look for the next pyloric transitionii. If the ingestible device has not gone back to stomach 2 hours after previously confirmed pyloric transition, stops location reversal monitoring and assume the ingestible device would stay in intestinal area For TLC1, one of the 18 subjects had too few samples (<3 minutes) taken in the stomach due to the delayed esophagus-stomach transition identification by previously developed localization algorithm. Thus, this subject was excluded from the stomach-duodenum transition algorithm test. For the rest of the TLC1 subjects, CT images confirmed that the detected pyloric transitions for all the subjects were located somewhere between stomach and jejunum. Two out of the 17 subjects showed that the ingestible device went back to stomach after first the first stomach-duodenum transition. The total agreement between the TLC1 algorithm detection and CT scans was 17/17=100%. For one of the PillCam subjects, the ingestible device stayed in the subject's stomach all the time before the video ended. For another two of the PillCam subjects, too few samples were taken in the stomach to run the localization algorithm. These three PillCam subjects were excluded from the stomach-duodenum transition localization algorithm performance test. The performance summary of pyloric transition localization algorithm for PillCam was as follows:1. Good cases (48 subjects):a. For 25 subjects, our detection matches exactly with the physician's notesb. For 19 subjects, the difference between the two detections is less than five minutesc. For four subjects, the difference between the two detections is less than 10 minutes (The full transition could take up to 10 minutes before the G/B signal settled)2. Failed cases (6 subjects):a. Four subjects had high standard deviation of Green/Blue signal in the stomachb. One subject had bile in the stomach, which greatly affected Green/Blue in stomachc. One subject had no Green/Blue change at pyloric transition The total agreement for the PillCam stomach-duodenum transition localization algorithm detection and physician's notes was 48/54=89%. Duodenum-Jejenum Transition For TLC1, it was assumed that the device left the duodenum and entered the jejenum three minutes after it was determined that the device entered the duodenum. Of the 17 subjects noted above with respect to the TLC1 investigation of the stomach-duodenum transition, 16 of the subjects mentioned had CT images that confirmed that the duodenum-jejenum transition was located somewhere between stomach and jejunum. One of the 17 subjects had a prolonged transit time in duodenum. The total agreement between algorithm detection and CT scans was 16/17=94%. For PillCam, the duodenum-jejenum transition was not determined. Jejenum-Ileum Transition It is to be noted that the jejunum is redder and more vascular than ileum, and that the jejenum has a thicker intestine wall with more mesentery fat. These differences can cause various optical responses between jejunum and ileum, particularly for the reflected red light signal. For both TLC1 and PillCam, two different approaches were explored to track the change of red signal at the jejunum-ileum transition. The first approach was a single-sliding-window analysis, where the window is 10 minutes long, and the mean signal was compared with a threshold value while the window was moving along. The second approach was a two-sliding-window analysis, where each window was 10 minutes long with a 20 minute spacing between the two windows. The algorithm for the jejunum-ileum transition localization was as follows:1. Obtain 20 minutes of Red signal after the duodenum jejenum transition, average the data and record it as the jejunum reference signal2. Start to check the jejunum-ileum transition 20 minutes after the device enters the jejunuma. Normalize the newly received data by the jejunum reference signalb. Two approaches:i. Single-sliding-window analysisSet the transition flag if the mean of reflected red signal is less than 0.8ii. Two-sliding-window analysis:Set the transition flag if the mean difference in reflected red is higher than 2× the standard deviation of the reflected red signal in the front window For TLC1, 16 of the 18 subjects had CT images that confirmed that the detected jejunum-ileum transition fell between jejunum and cecum. The total agreement between algorithm and CT scans was 16/18=89%. This was true for both the single-sliding-window and double-sliding-window approaches, and the same two subjects failed in both approaches. The performance summary of the jejunum-ileum transition detection for PillCam is listed below:1. Single-sliding-window analysis:a. 11 cases having jejunum-ileum transition detected somewhere between jejunum and cecumb. 24 cases having jejunum-ileum transition detected after cecumc. 19 cases having no jejunum-ileum transition detectedd. Total agreement: 11/54=20%2. Two-sliding-window analysis:a. 30 cases having jejunum-ileum transition detected somewhere between jejunum and cecumb. 24 cases having jejunum-ileum transition detected after cecumc. Total agreement: 30/54=56% Ileum-Cecum Transition Data demonstrated that, for TLC1, mean signal of reflected red/green provided the most statistical difference before and after the ileum-cecum transition. Data also demonstrated that, for TLC1, the coefficient of variation of reflected green/blue provided the most statistical contrast at ileum-cecum transition. The analysis based on PillCam videos showed very similar statistical trends to those results obtained with TLC1 device. Thus, the algorithm utilized changes in mean value of reflected red/green and the coefficient of variation of reflected green/blue. The algorithm was as follows:1. Start to monitor ileum-cecum transition after the ingestible device enters the stomach2. Setup the two windows (front (first) and back (second))a. Use a five-minute time length for each windowb. Use a 10-minute gap between the two windowsc. Use a one-minute window sliding step size3. Compare signals in the two sliding windowsa. Set ileum-cecum transition flag ifi. Reflected red/green has a significant change or is lower than a thresholdii. Coefficient of variation of reflected green/blue is lower than a thresholdb. If this is the first ileum-cecum transition detected, record average reflected red/green signal in small intestine as small intestine reference signalc. Mark location reversal (i.e. ingestible device returns to terminal ileum) ifi. Reflected red/green is statistically comparable with small intestine reference signalii. Coefficient of variation of reflected green/blue is higher than a thresholdd. If a possible ileum-cecum transition is detected, continue to scan for another 10 minutes for TLC1 (15 minutes for PillCam) in case of false positive flagi. If within this time frame (10 minutes for TLC1, 15 minutes for PillCam), location reversal is detected, the previous ileum-cecum transition flag is a false positive flag. Clear the flag and continue to checkii. If no location reversal has been identified within this time frame (10 minutes for TLC1, 15 minutes for PillCam) following the possible ileum-cecum transition flag, mark it as a confirmed ileum-cecum transitione. Continue monitoring data for another 2 hours after the confirmed ileum-cecum transition, in case of location reversali. If a location reversal is identified, flag the timestamp when reversal happened and then repeat steps a-d to look for the next ileum-cecum transitionii. If the ingestible device has not gone back to small intestine 2 hours after previously confirmed ileum-cecum transition, stop location reversal monitoring and assume the ingestible device would stay in large intestinal area The flag setting and location reversal criteria particularly designed for TLC1 device were as follows:1. Set ileum-cecum transition flag ifa. The average reflected red/Green in the front window is less than 0.7 or mean difference between the two windows is higher than 0.6b. And the coefficient of variation of reflected green/blue is less than 0.022. Define as location reversal ifa. The average reflected red/green in the front window is higher than small intestine reference signalb. And the coefficient of variation of reflected green/blue is higher than 0.086 For TLC1, 16 of the 18 subjects had CT images that confirmed that the detected ileum-cecum transition fell between terminal ileum and colon. The total agreement between algorithm and CT scans was 16/18=89%. Regarding those two subject where the ileum-cecum transition localization algorithm failed, for one subject the ileum-cecum transition was detected while TLC1 was still in the subject's terminal ileum, and for the other subject the ileum-cecum transition was detected when the device was in the colon. Among the 57 available PillCam endoscopy videos, for three subjects the endoscopy video ended before PillCam reached cecum, and another two subjects had only very limited video data (less than five minutes) in the large intestine. These five subjects were excluded from ileum-cecum transition localization algorithm performance test. The performance summary of ileum-cecum transition detection for PillCam is listed below:1. Good cases (39 subjects):a. For 31 subjects, the difference between the PillCam detection and the physician's notes was less than five minutesb. For 3 subjects, the difference between the PillCam detection and the physician's notes was less than 10 minutesc. For 5 subjects, the difference between the PillCam detection and the physician's notes was less than 20 minutes (the full transition can take up to 20 minutes before the signal settles)2. Marginal/bad cases (13 subjects):a. Marginal cases (9 subjects)i. The PillCam ileum-cecum transition detection appeared in the terminal ileum or colon, but the difference between the two detections was within one hourb. Failed cases (4 subjects)i. Reasons of failure:1. The signal already stabilized in the terminal ileum2. The signal was highly variable from the entrance to exit3. There was no statistically significant change in reflected red/green at ileum-cecum transition The total agreement between ileocecal transition localization algorithm detection and the physician's notes is 39/52=75% if considering good cases only. Total agreement including possibly acceptable cases is 48/52=92.3% Cecum-Colon Transition Data demonstrated that, for TLC1, mean signal of reflected red/green provided the most statistical difference before and after the cecum-colon transition. Data also demonstrated that, for TLC1, the coefficient of variation of reflected bluee provided the most statistical contrast at cecum-colon transition. The same signals were used for PillCam. The cecum-colon transition localization algorithm was as follows:1. Obtain 10 minutes of reflected red/green and reflected blue signals after ileum-cecum transition, average the data and record it as the cecum reference signals2. Start to check cecum-colon transition after ingestible device enters cecum (The cecum-colon transition algorithm is dependent on the ileum-cecum transition flag)a. Normalize the newly received data by the cecum reference signalsb. Two-sliding-window analysis:i. Use two adjacent 10 minute windowsii. Set the transition flag if any of the following criteria were metThe mean difference in reflected red/green was more than 4× the standard deviation of reflected red/green in the back (second) windowThe mean of reflected red/green in the front (first) window was higher than 1.03The coefficient of variation of reflected blue signal in the front (first) window was greater than 0.23 The threshold values above were chosen based on a statistical analysis of data taken by TLC1. For TLC1, 15 of the 18 subjects had the cecum-colon transition detected somewhere between cecum and colon. One of the subjects had the cecum-colon transition detected while TLC1 was still in cecum. The other two subjects had both wrong ileum-cecum transition detection and wrong cecum-colon transition detection. The total agreement between algorithm and CT scans was 15/18=83%. For PillCam, for three subjects the endoscopy video ended before PillCam reached cecum, and for another two subjects there was very limited video data (less than five minutes) in the large intestine. These five subjects were excluded from cecum-colon transition localization algorithm performance test. The performance summary of cecum-colon transition detection for PillCam is listed below:1. 27 cases had the cecum-colon transition detected somewhere between the cecum and the colon2. one case had the cecum-colon transition detected in the ileum3. 24 cases had no cecum-colon transition localized The total agreement: 27/52=52%. The following table summarizes the localization accuracy results. TransitionTLC1PillCamStomach-Duodenum100%(17/17)89% (48/54)Duodenum-Jejenum94%(16/17)N/AIleum-Cecum89%(16/18)75% (39/52)Ileum-terminal100%(18/18)92% (48/52)ileum/cecum/colon Exemplary Embodiments: The following exemplary embodiments 1)-94) are provided herein:1) A method of treating a disease of the gastro-intestinal tract in a subject, comprising:delivering a JAK inhibitor at a location in the gastrointestinal tract of the subject, wherein the method comprises administering orally to the subject a pharmaceutical composition comprising a therapeutically effective amount of the JAK inhibitor.2) The method of exemplary embodiment 1, wherein the disease of the GI tract is an inflammatory bowel disease.3) The method of exemplary embodiment 1, wherein the disease of the GI tract is ulcerative colitis.4) The method of exemplary embodiment 1, wherein the disease of the GI tract is Crohn's disease.5) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the large intestine of the subject.6) The method of exemplary embodiment 5, wherein the location is in the proximal portion of the large intestine.7) The method of exemplary embodiment 5, wherein the location is in the distal portion of the large intestine.8) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the ascending colon of the subject.9) The method of exemplary embodiment 8, wherein the location is in the proximal portion of the ascending colon.10) The method of exemplary embodiment 8, wherein the location is in the distal portion of the ascending colon.11) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the cecum of the subject.12) The method of exemplary embodiment 11, wherein the location is in the proximal portion of the cecum.13) The method of exemplary embodiment 11, wherein the location is in the distal portion of the cecum.14) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the sigmoid colon of the subject.15) The method of exemplary embodiment 14, wherein the location is in the proximal portion of the sigmoid colon.16) The method of exemplary embodiment 14, wherein the location is in the distal portion of the sigmoid colon.17) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the transverse colon of the subject.18) The method of exemplary embodiment 17, wherein the location is in the proximal portion of the transverse colon.19) The method of exemplary embodiment 17, wherein the location is in the distal portion of the transverse colon.20) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the descending colon of the subject.21) The method of exemplary embodiment 20, wherein the location is in the proximal portion of the descending colon.22) The method of exemplary embodiment 20, wherein the location is in the distal portion of the descending colon.23) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the small intestine of the subject.24) The method of exemplary embodiment 23, wherein the location is in the proximal portion of the small intestine.25) The method of exemplary embodiment 23, wherein the location is in the distal portion of the small intestine.26) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the duodenum of the subject.27) The method of exemplary embodiment 26, wherein the location is in the proximal portion of the duodenum.28) The method of exemplary embodiment 26, wherein the location is in the distal portion of the duodenum.29) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the jejunum of the subject.30) The method of exemplary embodiment 29, wherein the location is in the proximal portion of the jejunum.31) The method of exemplary embodiment 29, wherein the location is in the distal portion of the jejunum.32) The method of any one of exemplary embodiments 1, 2, or 3, 4, wherein the JAK inhibitor is delivered at a location in the ileum of the subject.33) The method of exemplary embodiment 32, wherein the location is in the proximal portion of the ileum.34) The method of exemplary embodiment 32, wherein the location is in the distal portion of the ileum.35) The method of any one of the preceding exemplary embodiments, wherein the location is proximate to one or more sites of disease.36) The method of exemplary embodiment 35, further comprising identifying the one or more sites of disease by a method comprising imaging of the gastrointestinal tract.37) The method of any one of the preceding exemplary embodiments, wherein the JAK inhibitor is delivered to the location by mucosal contact.38) The method of any one of the preceding exemplary embodiments, wherein the JAK inhibitor is delivered to the location by a process that does not comprise systemic transport of the JAK inhibitor.39) The method of any one of the preceding exemplary embodiments, wherein the amount of the JAK inhibitor that is administered is from about 1 mg to about 300 mg.40) The method of exemplary embodiment 39, wherein the amount of the JAK inhibitor that is administered is from about 1 mg to about 100 mg.41) The method of exemplary embodiment 40, wherein the amount of the JAK inhibitor that is administered is from about 5 mg to about 40 mg.42) The method of any one of exemplary embodiments 1 to 41, wherein the amount of the JAK inhibitor is less than an amount that is effective when the JAK inhibitor is administered systemically.43) The method of any one of the preceding exemplary embodiments, comprising administering (i) an amount of the JAK inhibitor that is an induction dose.44) The method of exemplary embodiment 43, further comprising (ii) administering an amount of the JAK inhibitor that is a maintenance dose following the administration of the induction dose.45) The method of exemplary embodiment 43 or 44, wherein the induction dose is administered once a day.46) The method of exemplary embodiment 43 or 44, wherein the induction dose is administered once every three days.47) The method of exemplary embodiment 43 or 44, wherein the induction dose is administered once a week.48) The method of exemplary embodiment 44, wherein step (ii) is repeated one or more times.49) The method of exemplary embodiment 44, wherein the induction dose is equal to the maintenance dose.50) The method of exemplary embodiment 44, wherein the induction dose is greater than the maintenance dose.51) The method of exemplary embodiment 44, wherein the induction dose is 5 greater than the maintenance dose.52) The method of exemplary embodiment 44, wherein the induction dose is 2 greater than the maintenance dose.53) The method of any one of the preceding exemplary embodiments, wherein the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract as a single bolus.54) The method of any one of exemplary embodiments 1 to 52, wherein the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract as more than one bolus.55) The method of any one of exemplary embodiments 1 to 52, wherein the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract in a continuous manner.56) The method of exemplary embodiment 55, wherein the method comprises delivering the JAK inhibitor at the location in the gastrointestinal tract over a time period of 20 or more minutes.57) The method of any one of the preceding exemplary embodiments, wherein the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 3 μg/ml.58) The method of exemplary embodiment 57, wherein the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 0.3 μg/ml.59) The method of exemplary embodiment 58, wherein the method provides a concentration of the JAK inhibitor in the plasma of the subject that is less than 0.01 μg/ml.60) The method of any one of exemplary embodiments 1 to 59, wherein the method does not comprise delivering a JAK inhibitor rectally to the subject.61) The method of any one of exemplary embodiments 1 to 59, wherein the method does not comprise delivering a JAK inhibitor via an enema to the subject.62) The method of any one of exemplary embodiments 1 to 59, wherein the method does not comprise delivering a JAK inhibitor via suppository to the subject.63) The method of any one of exemplary embodiments 1 to 59, wherein the method does not comprise delivering a JAK inhibitor via instillation to the rectum of the subject.64) The method of exemplary embodiment 63, wherein the JAK inhibitor is a JAK1 inhibitor.65) The method of exemplary embodiment 63, wherein the JAK inhibitor is selected from tofacitinib (Xeljanz®), filgotinib (GLPG0634, Galapagos NV); TD-1473 (Theravance Biopharma, Inc.); ruxolitinib; and generic equivalents thereof.66) The method of any one of the preceding exemplary embodiments, wherein the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;a storage reservoir located within the housing and containing the JAK inhibitor,a first end of the storage reservoir is connected to the first end of the housing;a mechanism for releasing the JAK inhibitor from the storage reservoir;and;an exit valve configured to allow the JAK inhibitor to be released out of the housing from the storage reservoir.67) The method of exemplary embodiment 66, wherein the ingestible device further comprises:an electronic component located within the housing; anda gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas.68) The method of exemplary embodiment 66 or 67, wherein the ingestible device further comprises:a safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level.69) The method of exemplary embodiment 66, wherein the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a storage reservoir located within the housing,wherein the storage reservoir stores a dispensable substance and a first end of the storage reservoir is connected to the first end of the housing;an exit valve located at the first end of the housing,wherein the exit valve is configured to allow the dispensable substance to be released out of the first end of the housing from the storage reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing when the internal pressure exceeds a threshold level.70) The method of exemplary embodiment 66, wherein the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an electronic component located within the housing,a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas;a storage reservoir located within the housing,wherein the storage reservoir stores a dispensable substance and a first end of the storage reservoir is connected to the first end of the housing;an injection device located at the first end of the housing,wherein the jet injection device is configured to inject the dispensable substance out of the housing from the storage reservoir; anda safety device placed within or attached to the housing,wherein the safety device is configured to relieve an internal pressure within the housing.71) The method of exemplary embodiment 66, wherein the pharmaceutical composition is an ingestible device, comprising:a housing defined by a first end, a second end substantially opposite from the first end, and a wall extending longitudinally from the first end to the second end;an optical sensing unit located on a side of the housing,wherein the optical sensing unit is configured to detect a reflectance from an environment external to the housing;an electronic component located within the housing;a gas generating cell located within the housing and adjacent to the electronic component,wherein the electronic component is configured to activate the gas generating cell to generate gas in response to identifying a location of the ingestible device based on the reflectance;a storage reservoir located within the housing,wherein the storage reservoir stores a dispensable substance and a first end of the storage reservoir is connected to the first end of the housing;a membrane in contact with the gas generating cell and configured to move or deform into the storage reservoir by a pressure generated by the gas generating cell; anda dispensing outlet placed at the first end of the housing,wherein the dispensing outlet is configured to deliver the dispensable substance out of the housing from the storage reservoir.72) The method of any one of exemplary embodiments 1-71, wherein the pharmaceutical composition is an ingestible device as disclosed in U.S. Patent Application Ser. No. 62/385,553, incorporated by reference herein in its entirety.73) The method of any one of exemplary embodiments 1-71, wherein the pharmaceutical composition is an ingestible device comprising a localization mechanism as disclosed in international patent application PCT/US2015/052500, incorporated by reference herein in its entirety.74) The method of any one of exemplary embodiments 1-73, wherein the pharmaceutical composition is not a dart-like dosage form.75) A method of treating a disease of the large intestine of a subject, comprising:delivering of a JAK inhibitor at a location in the proximal portion of the large intestine of the subject,wherein the method comprises administering endoscopically to the subject a therapeutically effective amount of the JAK inhibitor.76) The method of exemplary embodiment 75, wherein the disease of the large intestine is an inflammatory bowel disease.77) The method of exemplary embodiment 75, wherein the disease of the large intestine is ulcerative colitis.78) The method of exemplary embodiment 75, wherein the disease the large intestine is Crohn's disease.79) The method of any one of exemplary embodiments 75 to 78, wherein the JAK inhibitor is delivered at a location in the proximal portion of the ascending colon.80) The method of any one of exemplary embodiments 75 to 78, wherein the JAK inhibitor is delivered at a location in the proximal portion of the cecum.81) The method of any one of exemplary embodiments 75 to 78, wherein the JAK inhibitor is delivered at a location in the proximal portion of the sigmoid colon.82) The method of any one of exemplary embodiments 75 to 78, wherein the JAK inhibitor is delivered at a location in the proximal portion of the transverse colon.83) The method of any one of exemplary embodiments 75 to 78, wherein the JAK inhibitor is delivered at a location in the proximal portion of the descending colon.84) The method of any one of the preceding exemplary embodiments, further comprising administering a second agent orally, intravenously or subcutaneously, wherein the second agent is the same JAK inhibitor as in exemplary embodiment 1 or 75; a different JAK inhibitor; or an agent having a different biological target from JAK.85) The method of any one of the preceding exemplary embodiments, further comprising administering a second agent orally, intravenously or subcutaneously, wherein the second agent is an agent suitable for treating an inflammatory bowel disease.86) The method of exemplary embodiment 84 or 85, wherein the JAK inhibitor is administered prior to the second agent.87) The method of exemplary embodiment 84 or 85, wherein the JAK inhibitor is administered after the second agent.88) The method of exemplary embodiment 84 or 85, wherein the JAK inhibitor and the second agent are administered substantially at the same time.89) The method of any one of exemplary embodiments 84 to 88, wherein the second agent is administered intravenously.90) The method of any one of exemplary embodiments 84 to 88, wherein the second agent is administered subcutaneously.91) The method of any one of exemplary embodiments 84 to 90, wherein the amount of the second agent is less than the amount of the second agent when the JAK inhibitor and the second agent are both administered systemically.92) The method of exemplary embodiment 91, wherein the second agent is an immunosuppressant.93) In some aspects of these embodiments, the second agent is methotrexate.94) The method of any one of exemplary embodiments 1 to 83, wherein the method does not comprise Other Embodiments The various embodiments of systems, processes and apparatuses have been described herein by way of example only. It is contemplated that the features and limitations described in any one embodiment may be applied to any other embodiment herein, and flowcharts or examples relating to one embodiment may be combined with any other embodiment in a suitable manner, done in different orders, or done in parallel. It should be noted, the systems and/or methods described above may be applied to, or used in accordance with, other systems and/or methods. Various modifications and variations may be made to these example embodiments without departing from the spirit and scope of the embodiments, and the appended listing of embodiments should be given the broadest interpretation consistent with the description as a whole. | 865,884 |
11857670 | DETAILED DESCRIPTION Definitions “Bicontinuous Morphology” refers to at least two regions, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. Thus, a bicontinuous morphology will have two continuous pathways or two sets of continuous pathways extending from one surface of the material to the other surface. “Metabolically Active” means cellular or biotherapeutic agents that produce therapeutic metabolites in a biologically relevant environment. “Nanoporous” means very small pores extending through a surface, and measured in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Nanoscale” and “nanophase” means measurements in increments in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Rigid” means stiff and not bending. “Through-porous membrane” means a semipermeable membrane, also termed a selectively permeable membrane, a partially permeable membrane or a differentially permeable membrane, is a type of biological membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized “facilitated diffusion” along with other types of passive transport and active transport. NOMENCLATURE 10First Nanoporous Region 20Second Nanoporous Region 100Canister 102First Major Surface 104Second Major Surface 106Side Surface 108Port 108aExtended Silicone Tube 110Internal Barrier 112Interior Chamber 212Internal Channel 300Canister 308Port 400Canister 408Port 412Internal Channel 500Nanoporous and Patterned Surface 600Nanoporous and Patterned Surface 700Nanoporous and Patterned Surface 800Nanoporous and Patterned Surface 900Nanoporous and Patterned Surface 1000Nanoporous and Patterned Surface 1100Nanoporous and Patterned Surface 1400Canister 1402Outer Metallic Canister 1404Inner Polymeric Pouch 1406Port 1500Canister 1502First Major Surface 1504Second Major Surface 1506Side Surface 1508First Sealed Chamber 1510Second Sealed Chamber 1512Third Sealed Chamber 1514Fourth Sealed Chamber 1516Fifth Sealed Chamber 1518Sixth Sealed Chamber A platform for cellular and biotherapeutic agent delivery in a mammalian host, primarily humans, utilizing an implantable metal canister is described herein. Such a cell and biotherapeutics delivery canister provides a suitable environment for the cells and biotherapeutics to survive and function (e.g. produce and secrete therapeutic bioactive factors) without adversely affecting the mammalian host recipient or impaired by normal immunoprotective response. The cell and biotherapeutics delivery canister construct is made of medical-grade metal(s). In its simplest configuration, it is a metallic canister composed of two identical cup-shaped halves to create a hollow chamber. The medical-grade metals are modified to contain a nanoscale through-porous and bicontinuous membrane morphology. The internal void chamber of the envisioned canister is accessed through an incorporated silicon septum or attached infusion tube built into the canister, also sealed with a silicon septum for needle injection. The metals material is processed in a way that to create a porous, membrane-like structure. This modification takes place at the nanoscale level. The nanoscale pore size is sized and exacted to control bioactive factor exchange and diffusion. Specifically, a tailored nanoscale, through-porous feature with bicontinuous morphology within the canister superstructure allows for highly controlled therapeutic factor diffusion, both in and out of the delivery canister. The pore sizes range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The incorporated and tailored membrane also precludes certain unwanted biomaterials from penetrating the implanted delivery canister and contacting its therapeutic agent contents. These include immunogenic factors (e.g. immune cells or immune agents). The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. Nanophase biofunctional surfacing of implanted devices has been shown to match the recognition ability of biological systems, especially microvascularization. As such, topographic patterns can be executed on the canister delivery surface to match proteins at the nanometer scale and cells at the micrometer scale. The pore size feature of the canister superstructure metal material will facilitate desired vascular tissue incorporation of the canister to aid in the survival and/or function of its contents. The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The availability of vascular supply to the encapsulated therapeutic agents relates to their survival and function. A constant supply of oxygen, nutrients, and waste removal are required for nourishing the implanted cells and maintaining an optimal environment within the delivery canister for their longevity. In the case of therapeutic cells, a well-vascularized environment prevents death or damage through the effects of ischemia or hypoxia. The nanoscale texturing of the external surface of the delivery canister will encourage growth of vascularized tissue into the porous construct of the device. At the same time, scar tissue formation, triggered by a foreign body response, is mitigated. The desired ingrown vascular tissues also stabilize the implant canister, preventing its translocation into other body regions or tissues. Nanoscale texturing of the internal void surface of the canister device will encourage distribution of therapeutic contents within the canister to the outermost regions of the delivery canister. This will encourage more immediate access to the vascularized tissues resident to the outer surface of the canister device, resulting in a large surface to volume ratio associated with nanophase materials construction. The implanted delivery canister, having as well a nanoporous external surface, can be coated with an approved antibiotic compound to minimize infections. The porous surface texture of the delivery canister can be tailored at the nanoscale level for specific retention and release of such pharmaceutical agents. Representative antibiotics alone and in combination, include but are not limited to ampicillin, tetracycline, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, vancomycin, gentamicin, streptomycin, erythromycin, penicillin, amoxicillin, sulfonamides and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved antifibrotic drugs to inhibit the formation of unwanted fibrous tissues. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Antifibrotic agents alone and in combination may include but are not limited to paclitaxel, everolimus, tacrolimus, rapamycin, and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved biological or pharmaceutical agents (e.g. growth factors) to stimulate tissue in-growth and angiogenesis. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Such biological or non-biological agents, alone or in combination, stimulate tissue incorporation and angiogenesis include but are not limited to PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), FGF-1 (fibroblast growth factor), endoglin, ephrin, plasminogen activators, angiogenin and derivatives and analogues thereof. Functionality of the envisioned cell and biotherapeutics delivery canister is also based on proper dimensions of the delivery canister as they relate to particular cells, biotherapeutic volumes and clinical indications. Overall canister thickness, individual canister wall thickness, contour, shape, and the ability to load and recharge are important to sustaining a therapeutic number of cells for their ongoing survival and effect. The delivery canister shape must be anatomically convenient and non-protruding while meeting patient comfort and aesthetic considerations. In addition to size and shape, the metal material can be finely finished to avoid sharp traumatic edges. There may also be a clinical need to ultimately remove the delivery canister. Another embodiment of the cell and biotherapeutics delivery canister includes multiple divided, internal chambers within the canister. These can either be connected or separate depending on septal or cell infusion tube access to the crafted, internal chambers. This embodiment would for example, facilitate the delivery of multiple biotherapeutic compounds where phased delivery is critical to therapeutic endpoints. This phased delivery would be dependent upon size of these internal chambers and location of sized nanoscale through-porosity. Specifically important to cell survival is the ability to control cell distribution within the canister as cell clustering can impact their survival. Cell quantities can be positioned and controlled within the chamber utilizing internally crafted structures such as interconnected channels. In all embodiments, the cells are loaded using needled injection through either a septum or self-sealing infusion tube that is designed for needle injection. In one embodiment, the septum is made of a self-sealing medical grade silicone. The septum is positioned to one side of the delivery canister, thereby facilitating manual palpation when implanted in subcutaneous tissues. Deeper tissue placement will utilize a defined tube length and a self-sealing tube with an access port that facilitates needle injection. This self-sealing tube can be brought to the exterior of the patient's body in conjunction with surgical placement and standard wound closure. The method for loading cells first requires fixating the cell and biotherapeutics delivery canister in the mammalian host body. As such, the nanoporous through-porous membrane of the delivery canister is infiltrated with vascular and connective tissues. Once the cell and biotherapeutics delivery canister is encapsulated in a vascularized collagen matrix, delivering a cell and/or biotherapeutic agent is executed via needle injection, either through the built-in silicon septum or through the self-sealing access tube, which is connected to the delivery canister. Throughout the disclosure, the terms cell and biotherapeutic infusion and cell and biotherapeutic transplantation are used interchangeably. A transplanted cell and biotherapeutics delivery canister for containing therapeutic cells and biotherapeutics in vivo, in a mammalian host, is provided. The envisioned implanted cell and biotherapeutics delivery canister comprises a nanoporous canister that can be configured to create a hollow void chamber that is accessed for cell and biotherapeutic loading by way of syringe injection. Metallurgists have specifically graded metals for clinical applications with enhanced characteristics that make them highly compatible within living tissues. Included in this list are medical-grade metals. Examples of such biomedical grade metals and alloys include stainless steel based alloys, cobalt-chromium based alloys, alloys and nickel-titanium based alloys. More recently platinum containing alloys have been perfected for intravascular applications. The porous canister is formed of a biocompatible medical grade metal material that elicits only a mild inflammatory response in the body. The nanophase porous exterior portion of a through-porous membrane stimulates microvascular vessels to enter the cell and biotherapeutics delivery canister and promotes a vascularized collagen matrix to envelop the device, while curtailing a significant inflammation of tissues surrounding the delivery canister. The pore size and density of the nanoscale porous canister through-porous membrane encourages the growth and maintenance of these healthy vessels, which relate to the survival and targeted function of the therapeutic agents (e.g. molecular factor diffusion) contained within the delivery canister. The required size of the porous canister depends on the optimal surface area-to-volume ratios for holding metabolically active agents in vivo and for ensuring their long-term survival within the vascularized void chamber(s). The number of chambers in the implanted delivery canister is determined by the volume and/or number of cells and/or biotherapeutics that are to be transplanted. The total volume of the cell and biotherapeutics delivery canister can be adjusted by increasing or decreasing the number of chambers and the optimum surface area-to-volume ratio of each individual chamber. The length, width and height of the chambers are also defined and manufactured to meet total therapeutic volume requirements. Method of Using The cell and biotherapeutics delivery canister disclosed can be implanted using standard surgical techniques. Applied surgical implantation can occur at the following anatomical locations: subcutaneous, intraperitoneal including the omentum, intramuscular, intravascular, intraocular, intracerebral or other appropriate sites including the digestive tract, spinal cord area or any other organ as required to elicit a therapeutic factor from implanted cells or biotherapeutic agents. The loading procedure is a two-step process comprising a cell and biotherapeutics delivery canister being implanted and then followed by agent transplantation. After an in vivo incubation period during which the implanted cell and biotherapeutics delivery canister is infiltrated with a vascularized collagen matrix, the agent infusion step is then executed. The desired incubation period is generally thirty days to allow for angiogenesis and collagen infiltration of the porous canister. The incubation period may vary, depending on the extent of desired neovascularization and tissue formation. For example, the device may vascularize at different rates depending on the cell and biotherapeutics delivery canister material, dimensions, or coatings (e.g. antibiotic/antifibrotic coatings, growth factors, vascularizing agents etc.). There may be different vascularization rates pending locations in different body cavities and tissues. A clinically prepared expert can determine the appropriate incubation period while applying imaging tools that can help measure the extent of connective tissue deposition around and through the walls of the porous implanted canister. For the metabolically active agent step, the implantation site is generally identified and needle accessed following (subcutaneous) palpation or a small surgical incision for deep tissue access. Specifically, the clinician will identify the septum built into the surface of the delivery canister. The cell and/or biotherapeutic agent is then delivered via needle injection through the incorporated silicon septum or self-sealing infusion tubing (e.g. polyethylene tubing) or any other suitable material to deliver the therapeutic agents into porous chamber of cell and biotherapeutics delivery canister during the agent infusion step. The number of septum or infusion tubes in the delivery system may correspond to the number of porous chambers. Deep tissue placement and access will likely involve image-guided technology commonly used in other medical device implant procedures. As a metallic device, medically accepted imaging is readily enhanced. It is also envisioned that the delivery canister is placed along with the therapeutic agent using a single step and thus implanted together. The potential need exists for a biodegradable polymer coating for short-term containment of the encapsulated cell and biotherapeutic agent during such a singular implant procedure to control leakage loss of the molecular factors within the porous canister. Construction A void and porous canister may be created, for example, by joining (e.g. welding) the top and bottom halves of the canister along an edge. This would result in the canister being a single void chamber for holding the desired therapeutic agent. Its overall dimensions will generally be defined by the volume requirements and targeted anatomical location. In most embodiments the preferred delivery canister will measure 2-10 cm in length, 2-8 cm in width and have a height of 0.5 mm-5 mm. Different iterations of this device include similarly parallel halves with patterned gross textures. These geometric patterns, such as wave patterns, circular divots or indents, ridges, grooves and other roughened or contoured surfaces would aid in increasing the desired maximum surface area for enhanced cellular and biotherapeutic factor exchange and vascularization. As an implantable medical device, the cell and biotherapeutics delivery canister is sterilized using standard techniques prior to implantation. These include ethylene oxide, gamma radiation, cold plasma or dry heat autoclaving. The type of sterilization method used is dependent on the canister material. The cell and biotherapeutics delivery canister may be packaged in a self-seal package or any other sterilizable package along with a sterility indicator strip. The disclosed delivery canisters can be used for transplantation of any cells, or a combination of cells, any biotherapeutic agent or combination of agents into a mammalian host body for providing therapeutic factors to the mammalian host for the treatment of a disease condition. Allogeneic, xenogeneic or syngeneic donor cells, patient-derived cells, including stem cells, cord blood cells and embryonic stem cells are appropriate for such transplantation. Living tissue derived and active factors include but are not limited to proteins, peptides, genes, antibodies hormones, growth factors and neurotransmitters. FIG.1shows an implantable canister100defining a first major surface102, second major surface104and a side surface106. A port108extends from the side surface106, allowing access to the interior chamber112for infusion or flushing.FIG.1Ashows the canister100prior to being sealed shut, including an interior chamber112formed by combining both the upper shell110and lower shell114.FIG.1Bshows the canister100with one the upper shell110cut away, showing the interior chamber112. In all embodiments, the major surfaces can be fitted and/or joined and/or welded along an edge of the joined top and bottom halves, creating a single delivery canister. FIG.1Ccorresponds to the area shown on the first major surface inFIG.1Aand illustrates a microscopic view of the nanoscale through-porous membrane structure10of the canister100.FIG.2illustrates a microscopic view of the nanoscale through-porous metallic membrane material of the canister100,300400,1400,1500having a uniform or homogeneous nanoporous structure.FIG.2Ashows similarly illustrates a microscopic view of the metallic material of the canister100,300,400,1400,1500that is processed to possess an interconnected bicontinuous morphology which containing two distinct porous regions10,20, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. FIG.3shows a cut away view of a canister300configured to have an internal curved, raised pattern formed by an internal barrier212to physically stabilize a biotherapeutic agent (not shown) encased within.FIG.4similarly is a cut away view of an embodiment of a canister400having a squared shape with a plurality of raised internal barriers412to physically stabilize a biotherapeutic agent (not shown) held by the canister400.FIGS.3and4are shown for purposes of illustration only and are not intended to be limiting as variations of shape and internal barrier configuration within a canister are infinite. FIGS.5-11illustrate exterior nanoporous surface treatment patterns500,600,700,800,900,1400,1500that could be formed into the exterior and/or interior surface (unnumbered) of the canister100,300,400,1400,1500. The purpose of the different exterior and/or interior nanoporous surface treatment configurations is to increase the surface area of the canister100,300,400,1400,1500to allow improved inflow and outflow which positively affects the output of cellular and/or biotherapeutic agents contained in the canister and diffused from the canister.FIGS.5to11are shown for purposes of illustration only and are not intended to be limiting as surface treatment variations of canister surface are infinite. FIG.12illustrates the canister100being loaded through its port108with a biotherapeutic agent.FIG.13similarly illustrates a canister100having an extended tube108abetween the canister100and the exterior of a patient's body to allow loading of biotherapeutic agent (not shown) following a deep body (e.g., abdominal) implantation. FIG.14is a view of a metallic nanoporous outer protective canister1400with an inner polymeric pouch1404constructed of a semi-permeable material and containing biotherapeutic agent (not shown). The outer protective canister1400is constructed of a nanoscale or microscale through-porous membrane to facilitate cellular or biotherapeutic agent diffusion. The polymeric pouch1404is provided fluid communication by means of extended silicone tube1406. In another embodiment, as shown inFIG.15, the delivery canister1500is composed of multiple individual and independent chambers1508,1510,1512,1514,1516,1518. Each individual and independent chamber is accessed through its own port108. Each independent chamber can have the same or varied nanophase through-porous membrane structure10,20for controlled diffusion of various cellular or biotherapeutic agents, thus allowing sequenced delivery. Referring now toFIGS.16and17, implanting the canister100, or any other canister disclosed herein, within the mammal, includes depositing a metabolically active agent into the implantable canister. To that end, an injection device1602may be included and configured to deliver the metabolically active agent into the canister. In one configuration, the injection device1602includes a push button, squeeze handle, or similar actuator1604to deliver a substantially uniform distribution of the metabolically active agent into the implantable canister100. In one configuration, the canister100includes a membrane1606, which may be a tri-leaf valve or other valve structure in which the injection device1602penetrates to deliver the metabolically agent into the canister. For example, the membrane1606may be positioned on the surface of the canister or spaced a distance away from the canister within one or more injection device anchoring elements1608. In one configuration, the anchoring element1608defines a beveled edge to facilitate engagement to the injection device1602and may further be conical in shape. In one configuration, the anchoring element1608includes threads configured to engage a corresponding threaded portion on the injection device1602to threadably engage the injection device1602to the anchoring element1608. In the configuration shown inFIG.17, a second anchoring element1608is included on an opposite side of the canister. In an exemplary configuration, the injection device1602includes an injection tube1610extending outward therefrom, the injection tube1610being in communication with as source of metabolically active agent disposed within the injection device1602. The injection tube1610may be a straight tube or may define a helical or cork-screw shape to penetrate the member1606. Once the injection tube is engaged to the anchoring element1608, the injection tube1610may penetrate the membrane1606to inject the metabolically active agent. Following the injection, the injection tube1610may be withdrawn and the membrane1606may reseal itself to retain the contents within the canister. In other configurations, the injection device1602may snap fit, or otherwise lock with the anchoring element1608of the canister. For example, the injection device1602may include a plurality of arms that engage the canister and injection the contents therein. In other configurations, the injection tube1610may break off from the injection device1602and degrade over time inside the body. In still other configurations, the cannister100and/or the anchoring element1608may include a radiopaque or echogenic marker such that it can be visible under fluoroscopy or other imaging techniques to locate a port in the cannister100. Other embodiments of the cell and biotherapeutic delivery canister will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. | 25,600 |
11857671 | DETAILED DESCRIPTION FIG.1shows a very basic structure of a prior art subcutaneous medical implant device. Implant disc2consists of a top4, a bottom6and an outside wall8. It also has an opening10that is used for drug delivery. The size of opening10and the number of openings may vary. Line2-2will be used in the remaining Figures to illustrate various internal structures of the prior art implants and of the improved implants disclosed in this application. However, please understand that these Figures are not intended to cover all of applicant's improved implant structures. In addition, for example, the Figures are not representative of the number of layers of materials in an implant. Also, although the matrix materials are shown in regular shapes, they need not have such a regular shape—e.g., the channel may have a curved or irregular shape, and it have different heights/widths (such as lower/narrower near the opening and expanded/broader thereafter, or vice versa). In that regard, the preferred 3-D printing process is believed to provide, inter alia, the capability and flexibility to design different matrix channel shapes, sizes, designs, et cetera. If non-3-D printing processes (such as extrusion) are used to make the matrix, the channels and barriers are likely to be more arbitrarily configured. Nevertheless, non-3-D processes (such as hot-melt casting, extrusion and shrink wrap) may be used in the formation of some (or all) of the improved implant. FIG.1shows a generally cylindrical implant device. However, the shape of the implant in this embodiment (and in all other embodiments) may be modified to whatever shape is desirable. In other words, a particular exterior shape of the implant is not critical to the improved implant of this application. In looking at the Figures, it should be borne in mind that the structures are not drawn to scale. Instead, they are drawn in a manner to illustrate the general subject matter of this application. Thus, the relative sizes/shapes/dimensions of the coating, matrix materials, matrix channels, matrix barrier materials, drug materials, anticoagulant channels/materials and the like are not intended to be realistic. FIG.2shows the very basic structure of the prior art implant along line2-2ofFIG.1. More specifically, an impermeable coating12generally surrounds matrix14. In that regard, the coating must be impermeable in terms of (a) prohibiting the flow of drug materials and (b) having a relatively high breaking strength. Opening10extends all of the way through implant2. As a result, edges of the coating and matrix create sidewalls16to the opening. Although the opening in this and all other embodiments is shown to extend entirely through the implant, this is not always necessary. Moreover, it should be understood that there may be one or more openings that extend fully or only partially through the implant. Circle18inFIG.2will be used in illustrate the applicant's embodiments disclosed below inFIGS.3to7. Circle18is intended to create a somewhat microscopic view of a portion of the improved implant so as to help explain some of the structures, functions and purposes of the subject matter of this application. More specifically, in one possible situation, the matrix is surrounded by an impervious coating. The matrix is comprised of, inter alia, at least one (1) non-randomly located biodegradable barrier material, (2) non-biodegradable material and (2) drug material. In addition, the matrix and coating have at least one opening for drug delivery. Likewise, the matrix should have one or, preferably, more channels for drug delivery. Further, it also is contemplated that the drug material may or may not be mixed with the barrier material. Further design options are discussed below. FIG.3illustrates some of the novel aspects of this application. Circle18of this Figure shows a representative close-up view of one section of the implant device2for the first embodiment. Again, it should be understood that the size, shape, location and structure of the channel(s) in the matrix may be configured in many different ways to ensure the desired drug delivery mechanism. Thus, the present invention is intended to provide great flexibility in drug delivery, especially when 3-D printing processes are used to make some or all of the matrix layers. More specifically, the implant2(partially shown) inFIG.3has an opening10(partially shown), an opening sidewall16(partially shown), an impervious coating12(partially shown) and a matrix14(partially shown). In that regard, matrix14contains several elements. For example, the matrix14in this embodiment includes a non-biodegradable matrix portion19having channels20containing at least two different materials. The different materials in the channels of this embodiment are drug material22and biodegradable barrier24. The biodegradable barrier material24for this embodiment (and at least some other embodiments) may be the same as or different from other biodegradable materials in the matrix. Furthermore, it is expressly contemplated that the barriers may be made of different biodegradable materials and may be of different thicknesses or other dimensions. Thus, for example, different biodegradable materials and thicknesses may be utilized to provide enhanced drug release timing options. As shown inFIG.3, barriers24can be placed in various locations within the drug containing channels20. For example, one or more biodegradable barriers24can be created at or near opening sidewall16to moderate the initial drug burst phenomena. Barriers24also may be placed in other locations in channel20to create mini-chambers for drug materials. As explained above, these biodegradable barriers are structures used to regulate the time and amount of drug release. It is expressly contemplated (but not required) that the barriers be staggered in the various channels so that the initial burst of a mini-chamber in one channel is somewhat or largely cancelled out by the drug delivery from the mini-chambers of other channels. This staggering approach may be used from the beginning to the end of the drug delivery. In addition, in a preferred embodiment, the non-randomly located biodegradable barriers may be created at the end of every channel at the opening sidewall. This will avoid any premature release of drug material prior to implanting. Likewise, the drug delivery may be regulated by the use of different thicknesses of the barriers. Alternatively, or in addition, the barriers may be made of different biodegradable materials so that drug delivery may be regulated in that way as well. Finally, another approach is regulate drug delivery is to incorporate some drug material into the barriers (especially in barriers located at the opening sidewalls). As previously discussed, drug material22may be one or more different types of drugs. Thus, for example, one or more types of drug material may be used in a first group of mini-channels and other types of drug material may be used in later mini-chambers or in different channels. Alternatively, the %'s of drug materials may be varied in particular mini-chambers/channels. The ability to flexibly employ various drugs and various drug levels in different mini-chambers/channels is believed to be enhanced by 3-D printing processes. In a second embodiment, the matrix is surrounded by an impervious coating and the matrix is comprised of, inter alia, at least one (1) non-randomly located biodegradable barrier material, (2) coating material used as a non-biodegradable material and (3) drug material. In addition, the matrix and coating have at least one opening for drug delivery. Once again, the drug material may or may not be mixed with the barrier material. Further design options are discussed elsewhere in this application. FIG.4illustrates some of the other novel aspects of this application. Circle18of this Figure shows a representative close-up view of one section of the implant device2for the second embodiment. More specifically, the implant2(partially shown) has an opening110(partially shown), an opening sidewall116(partially shown), an impervious coating112(partially shown) and a matrix114(partially shown). Once again, matrix114contains several elements. For example, the matrix114in this embodiment includes a non-biodegradable matrix portion112′ made from the same impervious materials as coating112. In addition, the non-biodegradable matrix material112′ has channels120containing different materials. The different materials in this embodiment are drug material122and non-randomly located biodegradable barriers124. As shown inFIG.4, barriers124can be placed in various locations within the drug containing channels120. For example, one or more biodegradable barriers124can be created at or near opening sidewall116to moderate the initial drug burst phenomena. Barriers124also may be placed in other locations in channel120to create mini-chambers for drug materials. As previously discussed, drug material122may be one or more different types of drugs. In addition to the above concepts, the use of only biodegradable materials in the matrix may be beneficial in the delivery of the drug material because it may lessen the % of drug materials that are remain in the implant device when (a) the drug delivery is substantially completed and/or (b) the implant is removed. For example, the capillary action effect in terms of drug delivery may decrease as the distance from the opening(s) increase. This may inhibit the delivery of all drug materials in the implant to the patient. Thus, in a third embodiment, the matrix surrounded by an impervious coating and the matrix is comprised of, inter alia, of (1) at least two different biodegradable materials and (2) at least one drug material. The two biodegradable materials typically have different rates of biodegradability so as to regulate/control drug delivery. In addition, the matrix and coating have at least one opening for drug delivery. As indicated previously, a drug material may or may not be mixed with the barrier material. Further design options are discussed elsewhere in this application. For example, one option is for one or more drug materials to be mixed with a biodegradable material in a matrix barrier and/or in the biodegradable material of the matrix. In addition, another option is to form the barriers from different and/or multiple biodegradable materials. This is yet another way in which drug delivery may be regulated by non-randomly located biodegradable materials. FIG.5illustrates some of the other novel aspects of this application. Circle18of this Figure shows a representative close-up view of one section of the implant device2for the third embodiment. More specifically, the implant2(partially shown) has an opening210(partially shown), an opening sidewall216(partially shown), an impervious coating212(partially shown) and a matrix214(partially shown). In that regard, matrix214contains several elements. For example, the matrix in this embodiment includes at least two different biodegradable materials218and224. The matrix also has channels220containing different materials. The different materials in this embodiment are drug material222and biodegradable barrier224. As shown inFIG.5, non-randomly located barriers224may be placed in various locations within the drug containing channels220. For example, one or more biodegradable barriers224can be created at or near opening sidewall216to moderate the initial drug burst phenomena. Barriers224also may be placed in other locations in channel220to create mini-chambers for drug materials. As previously discussed, drug material222may be one or more different types of drugs. In a fourth embodiment, the matrix does not have an impervious coating. Instead, the coating also is biodegradable. In that situation, the matrix is comprised of, inter alia, of (1) at least two different biodegradable materials and (2) at least one drug material. The two biodegradable materials in the matrix have different rates of biodegradability so as to regulate/control drug delivery. Furthermore, because the coating is biodegradable, the coating preferably should have a much lower/slower rate of biodegradability than the biodegradable materials in the matrix so that the drug delivery is maintained only through the one or more original openings in the coating. As indicated previously, the drug material may or may not be mixed with the barrier material. In addition, the matrix and coating have at least one opening for drug delivery. FIG.6illustrates some of the other novel aspects of this application. Circle18of this Figure shows a representative close-up view of one section of the implant device2for the fourth embodiment. More specifically, the implant2(partially shown) has an opening310(partially shown), an opening sidewall316(partially shown), a biodegradable or semi-biodegradable coating312(partially shown) and a matrix314(partially shown). In that regard, matrix214contains several elements. For example, the matrix314in this embodiment includes a biodegradable matrix portion318that has channels320containing different materials. The different materials in this embodiment are drug material322and biodegradable barrier324. As shown inFIG.6, barriers324can be placed in various locations within the drug containing channels320. For example, one or more biodegradable barriers324can be created at or near opening sidewall316to moderate the initial drug burst phenomena. Barriers324also may be placed in other locations in channel320to create mini-chambers for drug materials. As previously discussed, drug material322may be one or more different types of drugs. In a fifth embodiment, the previous four embodiments are modified so as to also incorporate the use of anticoagulant materials to avoid and/or limit blood clotting when the device is implanted. The anticoagulant materials may be applied to various parts of the implant. For example, the anticoagulant material may be, inter alia, (i) applied to various areas of the coating such as on top of the coating or as a part of the exterior of the coating, (ii) applied to one or more surfaces of the opening(s) and/or (iii) mixed with the matrix materials. The fifth embodiment is illustrated inFIG.7. There, anticoagulant material is applied topically to various locations (such as locations428) on coating412. Alternatively, anticoagulant material can be topically applied to surfaces (such as opening sidewall surface416) of opening410. And/Or, the anticoagulant material may be mixed with drug material422, matrix material418and/or barriers324within matrix414. In a sixth embodiment (not shown in a Figure), anticoagulant material may be incorporated within a portion of the coating. In one approach, the anticoagulant material may incorporated into or on top of the coating by 3-D printing methods (via, for example, very small channels opening on the surface of the coating) or by non-3-D printing methods (via, for example, a separate biodegradable material located on the outside surface of the coating). In a seventh embodiment (also not shown in a Figure), the matrix is formed as a mixture of materials—i.e., without defined channels. Although a 3-D printing process may be used, this matrix structure also may be obtained by a non-3-D printing process. In that seventh embodiment situation, it is envisioned that the materials (e.g., the composition and % mixtures) will vary throughout the matrix in order to reduce the “initial burst,” to maintain a more level of drug delivery (or, alternatively, to adjust the rate of drug so that at certain desired times drug material is delivered in a higher or lower %) and/or to provide anticoagulant material. Thus, this is another way in which the use of different matrix material compositions may be formed (e.g., by extrusion, partial material removal and subsequent liquid deposition) so as to create so-called non-randomly located biodegradable materials/barriers having different compositions which are intended to regulate the delivery of drug materials. A coating material(s) may be subsequently applied to the matrix (via, e.g., shrink wrap) and, thereafter, one or more openings may be created in the implant. In another approach, the type of biodegradable material may vary with, in one approach, a slower dissolving rate biodegradable material being close to the opening and with different biodegradable material having faster dissolving rates farther from the opening. Thus, an initial level of drug delivery may be established and then a higher rate of drug delivery is established during a subsequent drug delivery period(s). In addition to or as an alternative, a lower % of drug material may be located closer to the implant opening to avoid/lessen the initial drug burst. Thus, the present invention contemplates that the % of the drug material may be varied (e.g., increased and/or decreased) as the distance increases from the opening. Moreover, in addition to or as yet another alternative, the anticoagulant material may be located in the matrix mixture just in the area nearer to the opening or that material may be included, for example, in lower, higher or the same dosages elsewhere in the biodegradable matrix. In that regard, it may be desirable to have anticoagulant material delivered at a time relatively close to the removal of the implant. Furthermore, the invention is intended to provide an improved implant where the matrix barrier materials and drug materials are varied—in terms of locations materials and %. The exact choice of biodegradable materials and the % concentration at different locations may be adjusted depending, for example, upon the drug material(s) to be delivered to the patient. As indicated above, the present invention covers the situation where the 3-D printing method is used to create all or just a portion of the implant device—e.g., at least only 3 or more layers of the matrix. However, the invention also contemplates the situation where one or more layers of the matrix and/or coating are created by other methods. Further, the present invention also envisions processes that deposit layers having the same or different thicknesses. The seventh embodiment also may be used with distinct walls and/or distinct channels as shown in other embodiments. In other words, modifications and/or variations may be readily made to all embodiments without departing from the spirit or scope of my inventions. Finally, in the situation where more than one drug material is desired, this invention also envisions the use of one or more openings to deliver these different drugs either separately, serially or together in terms of times and locations. Some of the potential advantages resulting from the use of the above non-randomly located biodegradable barriers and/or anticoagulant materials include at least the following:1. The use of non-randomly located biodegradable barrier structures may permit a higher % of drug materials in the implant to be delivered to the patient; and2. The use of non-randomly located biodegradable barrier structures may permit a more “flat” or “steady” level of drug delivery; and3. Blood clotting may be reduced by incorporating anticoagulant material in or on the implant; and4. Removal of the implant may be easier if anticoagulant materials are used; and5. The timing and level of drug delivery may be adjusted by the use of the biodegradable barrier structures and/or other biodegradable matrix materials having different compositions and dimensions; and6. The use of non-randomly located biodegradable barrier structures may enhance the timed delivery of two or more drugs. These embodiments and potential advantages are intended to merely be examples. As may be readily appreciated by those of ordinary skill in the manufacture and design of medical implant art, the present inventions can be practiced in ways other than as specifically disclosed herein. Thus, while the inventions have been described generally and with respect to certain preferred embodiments, it is to be understood that the foregoing and other modifications and variations may be made without departing from the scope or spirit of my inventions. | 20,338 |
11857672 | DETAILED DESCRIPTION Definitions “Bicontinuous Morphology” refers to at least two regions, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. Thus, a bicontinuous morphology will have two continuous pathways or two sets of continuous pathways extending from one surface of the material to the other surface. “Metabolically Active” means cellular or biotherapeutic agents that produce therapeutic metabolites in a biologically relevant environment. “Nanoporous” means very small pores extending through a surface, and measured in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Nanoscale” and “nanophase” means measurements in increments in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Rigid” means stiff and not bending. “Through-porous membrane” means a semipermeable membrane, also termed a selectively permeable membrane, a partially permeable membrane or a differentially permeable membrane, is a type of biological membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized “facilitated diffusion” along with other types of passive transport and active transport. NOMENCLATURE 10First Nanoporous Region20Second Nanoporous Region100Canister102First Major Surface104Second Major Surface106Spacer Ring108Port108aExtended Silicone Tube110Internal Barrier112Interior Chamber212Internal Channel300Canister308Port400Canister408Port412Internal Channel500Nanoporous and Patterned Surface600Nanoporous and Patterned Surface700Nanoporous and Patterned Surface800Nanoporous and Patterned Surface900Nanoporous and Patterned Surface1000Nanoporous and Patterned Surface1100Nanoporous and Patterned Surface1400Canister1402Outer Metallic Canister1404Inner Polymeric Pouch1406Port1500Canister1502First Major Surface1504Second Major Surface1506Side Surface1508First Sealed Chamber1510Second Sealed Chamber1512Third Sealed Chamber1514Fourth Sealed Chamber1516Fifth Sealed Chamber1518Sixth Sealed Chamber A platform for cellular and biotherapeutic agent delivery in a mammalian host, primarily humans, utilizing an implantable metal canister is described herein. Such a cell and biotherapeutics delivery canister provides a suitable environment for the cells and biotherapeutics to survive and function (e.g. produce and secrete therapeutic bioactive factors) without adversely affecting the mammalian host recipient or impaired by normal immunoprotective response. The cell and biotherapeutics delivery canister construct is made of medical-grade metal(s). In its simplest configuration, it is a metallic canister composed of two identical cup-shaped halves to create a hollow chamber. The medical-grade metals are modified to contain a nanoscale through-porous and bicontinuous membrane morphology. The internal void chamber of the envisioned canister is accessed through an incorporated silicon septum or attached infusion tube built into the canister, also sealed with a silicon septum for needle injection. The metals material is processed in a way that to create a porous, membrane-like structure. This modification takes place at the nanoscale level. The nanoscale pore size is sized and exacted to control bioactive factor exchange and diffusion. Specifically, a tailored nanoscale, through-porous feature with bicontinuous morphology within the canister superstructure allows for highly controlled therapeutic factor diffusion, both in and out of the delivery canister. The pore sizes range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The incorporated and tailored membrane also precludes certain unwanted biomaterials from penetrating the implanted delivery canister and contacting its therapeutic agent contents. These include immunogenic factors (e.g. immune cells or immune agents). The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. Nanophase biofunctional surfacing of implanted devices has been shown to match the recognition ability of biological systems, especially microvascularization. As such, topographic patterns can be executed on the canister delivery surface to match proteins at the nanometer scale and cells at the micrometer scale. The pore size feature of the canister superstructure metal material will facilitate desired vascular tissue incorporation of the canister to aid in the survival and/or function of its contents. The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The availability of vascular supply to the encapsulated therapeutic agents relates to their survival and function. A constant supply of oxygen, nutrients, and waste removal are required for nourishing the implanted cells and maintaining an optimal environment within the delivery canister for their longevity. In the case of therapeutic cells, a well-vascularized environment prevents death or damage through the effects of ischemia or hypoxia. The nanoscale texturing of the external surface of the delivery canister will encourage growth of vascularized tissue into the porous construct of the device. At the same time, scar tissue formation, triggered by a foreign body response, is mitigated. The desired ingrown vascular tissues also stabilize the implant canister, preventing its translocation into other body regions or tissues. Nanoscale texturing of the internal void surface of the canister device will encourage distribution of therapeutic contents within the canister to the outermost regions of the delivery canister. This will encourage more immediate access to the vascularized tissues resident to the outer surface of the canister device, resulting in a large surface to volume ratio associated with nanophase materials construction. The implanted delivery canister, having as well a nanoporous external surface, can be coated with an approved antibiotic compound to minimize infections. The porous surface texture of the delivery canister can be tailored at the nanoscale level for specific retention and release of such pharmaceutical agents. Representative antibiotics alone and in combination, include but are not limited to ampicillin, tetracycline, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, vancomycin, gentamicin, streptomycin, erythromycin, penicillin, amoxicillin, sulfonamides and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved antifibrotic drugs to inhibit the formation of unwanted fibrous tissues. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Antifibrotic agents alone and in combination may include but are not limited to paclitaxel, everolimus, tacrolimus, rapamycin, and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved biological or pharmaceutical agents (e.g. growth factors) to stimulate tissue in-growth and angiogenesis. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Such biological or non-biological agents, alone or in combination, stimulate tissue incorporation and angiogenesis include but are not limited to PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), FGF-1 (fibroblast growth factor), endoglin, ephrin, plasminogen activators, angiogenin and derivatives and analogues thereof. Functionality of the envisioned cell and biotherapeutics delivery canister is also based on proper dimensions of the delivery canister as they relate to particular cells, biotherapeutic volumes and clinical indications. Overall canister thickness, individual canister wall thickness, contour, shape, and the ability to load and recharge are important to sustaining a therapeutic number of cells for their ongoing survival and effect. The delivery canister shape must be anatomically convenient and non-protruding while meeting patient comfort and aesthetic considerations. In addition to size and shape, the metal material can be finely finished to avoid sharp traumatic edges. There may also be a clinical need to ultimately remove the delivery canister. Another embodiment of the cell and biotherapeutics delivery canister includes multiple divided, internal chambers within the canister. These can either be connected or separate depending on septal or cell infusion tube access to the crafted, internal chambers. This embodiment would for example, facilitate the delivery of multiple biotherapeutic compounds where phased delivery is critical to therapeutic endpoints. This phased delivery would be dependent upon size of these internal chambers and location of sized nanoscale through-porosity. Specifically important to cell survival is the ability to control cell distribution within the canister as cell clustering can impact their survival. Cell quantities can be positioned and controlled within the chamber utilizing internally crafted structures such as interconnected channels. In all embodiments, the cells are loaded using needled injection through either a septum or self-sealing infusion tube that is designed for needle injection. In one embodiment, the septum is made of a self-sealing medical grade silicone. The septum is positioned to one side of the delivery canister, thereby facilitating manual palpation when implanted in subcutaneous tissues. Deeper tissue placement will utilize a defined tube length and a self-sealing tube with an access port that facilitates needle injection. This self-sealing tube can be brought to the exterior of the patient's body in conjunction with surgical placement and standard wound closure. The method for loading cells first requires fixating the cell and biotherapeutics delivery canister in the mammalian host body. As such, the nanoporous through-porous membrane of the delivery canister is infiltrated with vascular and connective tissues. Once the cell and biotherapeutics delivery canister is encapsulated in a vascularized collagen matrix, delivering a cell and/or biotherapeutic agent is executed via needle injection, either through the built-in silicon septum or through the self-sealing access tube, which is connected to the delivery canister. Throughout the disclosure, the terms cell and biotherapeutic infusion and cell and biotherapeutic transplantation are used interchangeably. A transplanted cell and biotherapeutics delivery canister for containing therapeutic cells and biotherapeutics in vivo, in a mammalian host, is provided. The envisioned implanted cell and biotherapeutics delivery canister comprises a nanoporous canister that can be configured to create a hollow void chamber that is accessed for cell and biotherapeutic loading by way of syringe injection. Metallurgists have specifically graded metals for clinical applications with enhanced characteristics that make them highly compatible within living tissues. Included in this list are medical-grade metals. Examples of such biomedical grade metals and alloys include stainless steel based alloys, cobalt-chromium based alloys, alloys and nickel-titanium based alloys. More recently platinum containing alloys have been perfected for intravascular applications. The porous canister is formed of a biocompatible medical grade metal material that elicits only a mild inflammatory response in the body. The nanophase porous exterior portion of a through-porous membrane stimulates microvascular vessels to enter the cell and biotherapeutics delivery canister and promotes a vascularized collagen matrix to envelop the device, while curtailing a significant inflammation of tissues surrounding the delivery canister. The pore size and density of the nanoscale porous canister through-porous membrane encourages the growth and maintenance of these healthy vessels, which relate to the survival and targeted function of the therapeutic agents (e.g. molecular factor diffusion) contained within the delivery canister. The required size of the porous canister depends on the optimal surface area-to-volume ratios for holding metabolically active agents in vivo and for ensuring their long-term survival within the vascularized void chamber(s). The number of chambers in the implanted delivery canister is determined by the volume and/or number of cells and/or biotherapeutics that are to be transplanted. The total volume of the cell and biotherapeutics delivery canister can be adjusted by increasing or decreasing the number of chambers and the optimum surface area-to-volume ratio of each individual chamber. The length, width and height of the chambers are also defined and manufactured to meet total therapeutic volume requirements. Method of Using The cell and biotherapeutics delivery canister disclosed can be implanted using standard surgical techniques. Applied surgical implantation can occur at the following anatomical locations: subcutaneous, intraperitoneal including the omentum, intramuscular, intravascular, intraocular, intracerebral or other appropriate sites including the digestive tract, spinal cord area or any other organ as required to elicit a therapeutic factor from implanted cells or biotherapeutic agents. The loading procedure is a two-step process comprising a cell and biotherapeutics delivery canister being implanted and then followed by agent transplantation. After an in vivo incubation period during which the implanted cell and biotherapeutics delivery canister is infiltrated with a vascularized collagen matrix, the agent infusion step is then executed. The desired incubation period is generally thirty days to allow for angiogenesis and collagen infiltration of the porous canister. The incubation period may vary, depending on the extent of desired neovascularization and tissue formation. For example, the device may vascularize at different rates depending on the cell and biotherapeutics delivery canister material, dimensions, or coatings (e.g. antibiotic/antifibrotic coatings, growth factors, vascularizing agents etc.). There may be different vascularization rates pending locations in different body cavities and tissues. A clinically prepared expert can determine the appropriate incubation period while applying imaging tools that can help measure the extent of connective tissue deposition around and through the walls of the porous implanted canister. For the metabolically active agent step, the implantation site is generally identified and needle accessed following (subcutaneous) palpation or a small surgical incision for deep tissue access. Specifically, the clinician will identify the septum built into the surface of the delivery canister. The cell and/or biotherapeutic agent is then delivered via needle injection through the incorporated silicon septum or self-sealing infusion tubing (e.g. polyethylene tubing) or any other suitable material to deliver the therapeutic agents into porous chamber of cell and biotherapeutics delivery canister during the agent infusion step. The number of septum or infusion tubes in the delivery system may correspond to the number of porous chambers. Deep tissue placement and access will likely involve image-guided technology commonly used in other medical device implant procedures. As a metallic device, medically accepted imaging is readily enhanced. It is also envisioned that the delivery canister is placed along with the therapeutic agent using a single step and thus implanted together. The potential need exists for a biodegradable polymer coating for short-term containment of the encapsulated cell and biotherapeutic agent during such a singular implant procedure to control leakage loss of the molecular factors within the porous canister. Construction A void and porous canister may be created, for example, by joining (e.g. welding) the top and bottom halves of the canister along an edge. This would result in the canister being a single void chamber for holding the desired therapeutic agent. Its overall dimensions will generally be defined by the volume requirements and targeted anatomical location. In most embodiments the preferred delivery canister will measure 2-10 cm in length, 2-8 cm in width and have a height of 0.5 mm-5 mm. Different iterations of this device include similarly parallel halves with patterned gross textures. These geometric patterns, such as wave patterns, circular divots or indents, ridges, grooves and other roughened or contoured surfaces would aid in increasing the desired maximum surface area for enhanced cellular and biotherapeutic factor exchange and vascularization. As an implantable medical device, the cell and biotherapeutics delivery canister is sterilized using standard techniques prior to implantation. These include ethylene oxide, gamma radiation, cold plasma or dry heat autoclaving. The type of sterilization method used is dependent on the canister material. The cell and biotherapeutics delivery canister may be packaged in a self-seal package or any other sterilizable package along with a sterility indicator strip. The disclosed delivery canisters can be used for transplantation of any cells, or a combination of cells, any biotherapeutic agent or combination of agents into a mammalian host body for providing therapeutic factors to the mammalian host for the treatment of a disease condition. Allogeneic, xenogeneic or syngeneic donor cells, patient-derived cells, including stem cells, cord blood cells and embryonic stem cells are appropriate for such transplantation. Living tissue derived and active factors include but are not limited to proteins, peptides, genes, antibodies hormones, growth factors and neurotransmitters. FIG.1shows an implantable canister100defining a first major surface102, second major surface104and a spacer ring106. A port108defined by the first major surface102or second major surface104allows access to the interior chamber112for infusion or flushing.FIG.1Ashows the canister100prior to being sealed shut, including an interior chamber112formed by combining both the upper shell110and lower shell114.FIG.1Bshows the canister100with one the upper shell110cut away, showing the interior chamber112. In all embodiments, the major surfaces can be fitted and/or joined and/or welded along an edge of the joined top and bottom halves, creating a single delivery canister. As shown inFIG.1B, the canister includes a plurality of suture retention tabs111configured to provide access to one or more sutures to mount the canister within a target area. Moreover, tubes113may also be include coupled to the canister100to load bioactive compounds within the canister100. For example, one or tubes113may be in fluid communication with one or the other of the first major surface102or the second major surface104. Moreover, a non-metal, polymeric membrane material may be stretched over the spacer ring106. FIG.1Ccorresponds to the area shown on the first major surface inFIG.1Aand illustrates a microscopic view of the nanoscale through-porous membrane structure10of the canister100.FIG.2illustrates a microscopic view of the nanoscale through-porous metallic membrane material of the canister100,300400,1400,1500having a uniform or homogeneous nanoporous structure.FIG.2Ashows similarly illustrates a microscopic view of the metallic material of the canister100,300,400,1400,1500that is processed to possess an interconnected bicontinuous morphology which containing two distinct porous regions10,20, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. FIG.3shows a cut away view of a canister300configured to have an internal curved, raised pattern formed by an internal barrier212to physically stabilize a biotherapeutic agent (not shown) encased within.FIG.4similarly is a cut away view of an embodiment of a canister400having a squared shape with a plurality of raised internal barriers412to physically stabilize a biotherapeutic agent (not shown) held by the canister400.FIGS.3and4are shown for purposes of illustration only and are not intended to be limiting as variations of shape and internal barrier configuration within a canister are infinite. FIGS.5-11illustrate exterior nanoporous surface treatment patterns500,600,700,800,900,1400,1500that could be formed into the exterior and/or interior surface (unnumbered) of the canister100,300,400,1400,1500. The purpose of the different exterior and/or interior nanoporous surface treatment configurations is to increase the surface area of the canister100,300,400,1400,1500to allow improved inflow and outflow which positively affects the output of cellular and/or biotherapeutic agents contained in the canister and diffused from the canister.FIGS.5to11are shown for purposes of illustration only and are not intended to be limiting as surface treatment variations of canister surface are infinite. FIG.12illustrates the canister100being loaded through its port108with a biotherapeutic agent.FIG.13similarly illustrates a canister100having an extended tube108abetween the canister100and the exterior of a patient's body to allow loading of biotherapeutic agent (not shown) following a deep body (e.g., abdominal) implantation. FIG.14is a view of a metallic nanoporous outer protective canister1400with an inner polymeric pouch1404constructed of a semi-permeable material and containing biotherapeutic agent (not shown). The outer protective canister1400is constructed of a nanoscale or microscale through-porous membrane to facilitate cellular or biotherapeutic agent diffusion. The polymeric pouch1404is provided fluid communication by means of extended silicone tube1406. In another embodiment, as shown inFIG.15, the delivery canister1500is composed of multiple individual and independent chambers1508,1510,1512,1514,1516,1518. Each individual and independent chamber is accessed through its own port108. Each independent chamber can have the same or varied nanophase through-porous membrane structure10,20for controlled diffusion of various cellular or biotherapeutic agents, thus allowing sequenced delivery. Referring now toFIGS.16and17, implanting the canister100, or any other canister disclosed herein, within the mammal, includes depositing a metabolically active agent into the implantable canister. To that end, an injection device1602may be included and configured to deliver the metabolically active agent into the canister. In one configuration, the injection device1602includes a push button, squeeze handle, or similar actuator1604to deliver a substantially uniform distribution of the metabolically active agent into the implantable canister100. In one configuration, the canister100includes a membrane1606, which may be a tri-leaf valve or other valve structure in which the injection device1602penetrates to deliver the metabolically agent into the canister. For example, the membrane1606may be positioned on the surface of the canister or spaced a distance away from the canister within one or more injection device anchoring elements1608. In one configuration, the anchoring element1608defines a beveled edge to facilitate engagement to the injection device1602and may further be conical in shape. In one configuration, the anchoring element1608includes threads configured to engage a corresponding threaded portion on the injection device1602to threadably engage the injection device1602to the anchoring element1608. In the configuration shown inFIG.17, a second anchoring element1608is included on an opposite side of the canister. In an exemplary configuration, the injection device1602includes an injection tube1610extending outward therefrom, the injection tube1610being in communication with as source of metabolically active agent disposed within the injection device1602. The injection tube1610may be a straight tube or may define a helical or cork-screw shape to penetrate the member1606. Once the injection tube is engaged to the anchoring element1608, the injection tube1610may penetrate the membrane1606to inject the metabolically active agent. Following the injection, the injection tube1610may be withdrawn and the membrane1606may reseal itself to retain the contents within the canister. In other configurations, the injection device1602may snap fit, or otherwise lock with the anchoring element1608of the canister. For example, the injection device1602may include a plurality of arms that engage the canister and injection the contents therein. In other configurations, the injection tube1610may break off from the injection device1602and degrade over time inside the body. In still other configurations, the canister100and/or the anchoring element1608may include a radiopaque or echogenic marker such that it can be visible under fluoroscopy or other imaging techniques to locate a port in the canister100. Referring now toFIGS.18-19, in which another embodiment of an implantable medical device1800is shown. The medical device includes a first portion1802having a nanoscale through-porous structure and a second portion1804opposite the first portion1802having a nanoscale through porous structure. In one configuration the combination of the first portion1802with the second portion1804produces a bicontinuous morphology, as discussed in more detail above. In the configuration shown inFIG.18, the first portion1802and the second portion1804are metallic and rigid planar discs that are the same size, but in other configurations the first portion1802and the second portion1804may be differently sized, any shape, and may be flexible. Continuing to refer toFIG.18, the first portion1802and the second portion1804are engaged to an implantable spacer ring1806sized and configured to retain the first portion1802and the second portion1804. In the configuration shown inFIG.18, the spacer ring1806is ring shaped to accommodate the first and second portions1802,1804. In other configurations, the spacer ring1806may be any shape to accommodate the first portion1802and the second portion1804. The spacer ring1806is sized and configured to be implantable with the body of a human or animal patient. That is, the spacer ring1806may be composed of corrosion resistant and biocompatible materials. The spacer ring1806may further define or otherwise includes a plurality of suture holes1808disposed about a perimeter or circumference of the spacer ring1806. The suture holes1808are through holes such that the spacer ring1806may be sutured within the patient. In the configuration shown inFIG.18, the spacer ring1806further defines a first bezel1810or a recessed area sized to receive the first portion1802and a second bezel (not shown) or recessed area on the opposite side of the spacer ring1806sized to receive the second portion1804. When seated within the respective bezels, the first portion1802and the second portion1804are substantially flush within the spacer ring1806. Moreover, the spacer ring1806defines an aperture1812within its center such that when the first portion and the second portions1802,1804are engaged to the spacer ring1806, they are in fluid communication with each other through the aperture1812. Continuing to refer toFIG.18, the spacer ring1806includes a port1814disposed within its circumference. In one configuration the port1814is disposed within a bulbous portion of the spacer ring1806. The port1814is further engageable with a fluid delivery tube1816extending from the port1814, which is in fluid communication with a source of fluid, for example, bioactive compounds. The fluid delivery tube1816may be flexible and may affixed to the port1814. In an exemplary configuration, the first portion1802and the second portion1804are loaded with fluid injection into the fluid delivery tube1816through the port1814to saturate each of the first portion1802and the second portion1804with fluid. Other embodiments of the cell and biotherapeutic delivery canister will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. | 29,084 |
11857673 | DETAILED DESCRIPTION Definitions “Bicontinuous Morphology” refers to at least two regions, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. Thus, a bicontinuous morphology will have two continuous pathways or two sets of continuous pathways extending from one surface of the material to the other surface. “Metabolically Active” means cellular or biotherapeutic agents that produce therapeutic metabolites in a biologically relevant environment. “Nanoporous” means very small pores extending through a surface, and measured in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Nanoscale” and “nanophase” means measurements in increments in increments of a millionth of a meter or as 10−9of a meter (abbreviated “nm”). “Rigid” means stiff and not bending. “Through-porous membrane” means a semipermeable membrane, also termed a selectively permeable membrane, a partially permeable membrane or a differentially permeable membrane, is a type of biological membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized “facilitated diffusion” along with other types of passive transport and active transport. “Integrated Nanophase Morphologies” refers to at least two regions, each constructed from the same metal but with substantially different compositions which differs from the other and each of which forms a continuous pathway from one surface on an article to another surface of an article. Thus, an integrated or joined nanophase morphology will have two continuous pathways or sets of continuous pathways extending across both and from one surface of the material to the other surface. A platform for cellular and biotherapeutic agent delivery in a mammalian host, primarily humans, utilizing an implantable metal canister is described herein. Such a cell and biotherapeutics delivery canister provides a suitable environment for the cells and biotherapeutics to survive and function (e.g. produce and secrete therapeutic bioactive factors) without adversely affecting the mammalian host recipient or impaired by normal immunoprotective response. The cell and biotherapeutics delivery canister construct is made of medical-grade metal(s). In its simplest configuration, it is a metallic canister composed of two identical cup-shaped halves to create a hollow chamber. The medical-grade metals are modified to contain a nanoscale through-porous and bicontinuous membrane morphology. The internal void chamber of the envisioned canister is accessed through an incorporated silicon septum or attached infusion tube built into the canister, also sealed with a silicon septum for needle injection. The metals material is processed in a way that to create a porous, membrane-like structure. This modification takes place at the nanoscale level. The nanoscale pore size is sized and exacted to control bioactive factor exchange and diffusion. Specifically, a tailored nanoscale, through-porous feature with bicontinuous morphology within the canister superstructure allows for highly controlled therapeutic factor diffusion, both in and out of the delivery canister. The pore sizes range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The incorporated and tailored membrane also precludes certain unwanted biomaterials from penetrating the implanted delivery canister and contacting its therapeutic agent contents. These include immunogenic factors (e.g. immune cells or immune agents). The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. Nanophase biofunctional surfacing of implanted devices has been shown to match the recognition ability of biological systems, especially microvascularization. As such, topographic patterns can be executed on the canister delivery surface to match proteins at the nanometer scale and cells at the micrometer scale. The pore size feature of the canister superstructure metal material will facilitate desired vascular tissue incorporation of the canister to aid in the survival and/or function of its contents. The pore sizes may range from approximately 20 nm to 5000 nm with a wall thickness of 5 to 250 microns. The wall thickness can be varied to provide a balance between efficient diffusion and structural integrity of the implanted device. The availability of vascular supply to the encapsulated therapeutic agents relates to their survival and function. A constant supply of oxygen, nutrients, and waste removal are required for nourishing the implanted cells and maintaining an optimal environment within the delivery canister for their longevity. In the case of therapeutic cells, a well-vascularized environment prevents death or damage through the effects of ischemia or hypoxia. The nanoscale texturing of the external surface of the delivery canister will encourage growth of vascularized tissue into the porous construct of the device. At the same time, scar tissue formation, triggered by a foreign body response, is mitigated. The desired ingrown vascular tissues also stabilize the implant canister, preventing its translocation into other body regions or tissues. Nanoscale texturing of the internal void surface of the canister device will encourage distribution of therapeutic contents within the canister to the outermost regions of the delivery canister. This will encourage more immediate access to the vascularized tissues resident to the outer surface of the canister device, resulting in a large surface to volume ratio associated with nanophase materials construction. The implanted delivery canister, having as well a nanoporous external surface, can be coated with an approved antibiotic compound to minimize infections. The porous surface texture of the delivery canister can be tailored at the nano scale level for specific retention and release of such pharmaceutical agents. Representative antibiotics alone and in combination, include but are not limited to ampicillin, tetracycline, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, vancomycin, gentamicin, streptomycin, erythromycin, penicillin, amoxicillin, sulfonamides and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved antifibrotic drugs to inhibit the formation of unwanted fibrous tissues. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Antifibrotic agents alone and in combination may include but are not limited to paclitaxel, everolimus, tacrolimus, rapamycin, and derivatives and analogues thereof. The implanted cell and biotherapeutics delivery canister, having a nanoporous external surface, can be coated with approved biological or pharmaceutical agents (e.g. growth factors) to stimulate tissue in-growth and angiogenesis. The porous surface texture of the delivery canister can be tailored at the nanoscale level for retention and release of such pharmaceutical agents. Such biological or non-biological agents, alone or in combination, stimulate tissue incorporation and angiogenesis include but are not limited to PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), FGF-1 (fibroblast growth factor), endoglin, ephrin, plasminogen activators, angiogenin and derivatives and analogues thereof. Functionality of the envisioned cell and biotherapeutics delivery canister is also based on proper dimensions of the delivery canister as they relate to particular cells, biotherapeutic volumes and clinical indications. Overall canister thickness, individual canister wall thickness, contour, shape, and the ability to load and recharge are important to sustaining a therapeutic number of cells for their ongoing survival and effect. The delivery canister shape must be anatomically convenient and non-protruding while meeting patient comfort and aesthetic considerations. In addition to size and shape, the metal material can be finely finished to avoid sharp traumatic edges. There may also be a clinical need to ultimately remove the delivery canister. Another embodiment of the cell and biotherapeutics delivery canister includes multiple divided, internal chambers within the canister. These can either be connected or separate depending on septal or cell infusion tube access to the crafted, internal chambers. This embodiment would for example, facilitate the delivery of multiple biotherapeutic compounds where phased delivery is critical to therapeutic endpoints. This phased delivery would be dependent upon size of these internal chambers and location of sized nanoscale through-porosity. Specifically important to cell survival is the ability to control cell distribution within the canister as cell clustering can impact their survival. Cell quantities can be positioned and controlled within the chamber utilizing internally crafted structures such as interconnected channels. In all embodiments, the cells are loaded using needled injection through either a septum or self-sealing infusion tube that is designed for needle injection. In one embodiment, the septum is made of a self-sealing medical grade silicone. The septum is positioned to one side of the delivery canister, thereby facilitating manual palpation when implanted in subcutaneous tissues. Deeper tissue placement will utilize a defined tube length and a self-sealing tube with an access port that facilitates needle injection. This self-sealing tube can be brought to the exterior of the patient's body in conjunction with surgical placement and standard wound closure. The method for loading cells first requires fixating the cell and biotherapeutics delivery canister in the mammalian host body. As such, the nanoporous through-porous membrane of the delivery canister is infiltrated with vascular and connective tissues. Once the cell and biotherapeutics delivery canister is encapsulated in a vascularized collagen matrix, delivering a cell and/or biotherapeutic agent is executed via needle injection, either through the built-in silicon septum or through the self-sealing access tube, which is connected to the delivery canister. Throughout the disclosure, the terms cell and biotherapeutic infusion and cell and biotherapeutic transplantation are used interchangeably. A transplanted cell and biotherapeutics delivery canister for containing therapeutic cells and biotherapeutics in vivo, in a mammalian host, is provided. The envisioned implanted cell and biotherapeutics delivery canister comprises a nanoporous canister that can be configured to create a hollow void chamber that is accessed for cell and biotherapeutic loading by way of syringe injection. Metallurgists have specifically graded metals for clinical applications with enhanced characteristics that make them highly compatible within living tissues. Included in this list are medical-grade metals. Examples of such biomedical grade metals and alloys include stainless steel-based alloys, cobalt-chromium based alloys, alloys and nickel-titanium based alloys. More recently platinum containing alloys have been perfected for intravascular applications. The porous canister is formed of a biocompatible medical grade metal material that elicits only a mild inflammatory response in the body. The nanophase porous exterior portion of a through-porous membrane stimulates microvascular vessels to enter the cell and biotherapeutics delivery canister and promotes a vascularized collagen matrix to envelop the device, while curtailing a significant inflammation of tissues surrounding the delivery canister. The pore size and density of the nanoscale porous canister through-porous membrane encourages the growth and maintenance of these healthy vessels, which relate to the survival and targeted function of the therapeutic agents (e.g. molecular factor diffusion) contained within the delivery canister. The required size of the porous canister depends on the optimal surface area-to-volume ratios for holding metabolically active agents in vivo and for ensuring their long-term survival within the vascularized void chamber(s). The number of chambers in the implanted delivery canister is determined by the volume and/or number of cells and/or biotherapeutics that are to be transplanted. The total volume of the cell and biotherapeutics delivery canister can be adjusted by increasing or decreasing the number of chambers and the optimum surface area-to-volume ratio of each individual chamber. The length, width and height of the chambers are also defined and manufactured to meet total therapeutic volume requirements. Method of Using The cell and biotherapeutics delivery canister disclosed can be implanted using standard surgical techniques. Applied surgical implantation can occur at the following anatomical locations: subcutaneous, intraperitoneal including the omentum, intramuscular, intravascular, intraocular, intracerebral or other appropriate sites including the digestive tract, spinal cord area or any other organ as required to elicit a therapeutic factor from implanted cells or biotherapeutic agents. The loading procedure is a two-step process comprising a cell and biotherapeutics delivery canister being implanted and then followed by agent transplantation. After an in vivo incubation period during which the implanted cell and biotherapeutics delivery canister is infiltrated with a vascularized collagen matrix, the agent infusion step is then executed. The desired incubation period is generally thirty days to allow for angiogenesis and collagen infiltration of the porous canister. The incubation period may vary, depending on the extent of desired neovascularization and tissue formation. For example, the device may vascularize at different rates depending on the cell and biotherapeutics delivery canister material, dimensions, or coatings (e.g., antibiotic/antifibrotic coatings, growth factors, vascularizing agents etc.). There may be different vascularization rates pending locations in different body cavities and tissues. A clinically prepared expert can determine the appropriate incubation period while applying imaging tools that can help measure the extent of connective tissue deposition around and through the walls of the porous implanted canister. For the metabolically active agent step, the implantation site is generally identified and needle accessed following (subcutaneous) palpation or a small surgical incision for deep tissue access. Specifically, the clinician will identify the septum built into the surface of the delivery canister. The cell and/or biotherapeutic agent is then delivered via needle injection through the incorporated silicon septum or self-sealing infusion tubing (e.g., polyethylene tubing) or any other suitable material to deliver the therapeutic agents into porous chamber of cell and biotherapeutics delivery canister during the agent infusion step. The number of septum or infusion tubes in the delivery system may correspond to the number of porous chambers. Deep tissue placement and access will likely involve image-guided technology commonly used in other medical device implant procedures. As a metallic device, medically accepted imaging is readily enhanced. It is also envisioned that the delivery canister is placed along with the therapeutic agent using a single step and thus implanted together. The potential need exists for a biodegradable polymer coating for short-term containment of the encapsulated cell and biotherapeutic agent during such a singular implant procedure to control leakage loss of the molecular factors within the porous canister. A void and porous canister may be created, for example, by joining (e.g. welding) the top and bottom halves of the canister along an edge. This would result in the canister being a single void chamber for holding the desired therapeutic agent. Its overall dimensions will generally be defined by the volume requirements and targeted anatomical location. In most embodiments the preferred delivery canister will measure 2-10 cm in length, 2-8 cm in width and have a height of 0.5 mm-5 mm. Different iterations of this device include similarly parallel halves with patterned gross textures. These geometric patterns, such as wave patterns, circular divots or indents, ridges, grooves and other roughened or contoured surfaces would aid in increasing the desired maximum surface area for enhanced cellular and biotherapeutic factor exchange and vascularization. As an implantable medical device, the cell and biotherapeutics delivery canister is sterilized using standard techniques prior to implantation. These include ethylene oxide, gamma radiation, cold plasma or dry heat autoclaving. The type of sterilization method used is dependent on the canister material. The cell and biotherapeutics delivery canister may be packaged in a self-seal package or any other sterilizable package along with a sterility indicator strip. The disclosed delivery canisters can be used for transplantation of any cells, or a combination of cells, any biotherapeutic agent or combination of agents into a mammalian host body for providing therapeutic factors to the mammalian host for the treatment of a disease condition. Allogeneic, xenogeneic or syngeneic donor cells, patient-derived cells, including stem cells, cord blood cells and embryonic stem cells are appropriate for such transplantation. Living tissue derived and active factors include but are not limited to proteins, peptides, genes, antibodies hormones, growth factors and neurotransmitters. FIG.1shows an implantable canister100defining a first major surface102, second major surface104and a spacer ring106. A port108defined by the first major surface102or second major surface104allows access to the interior chamber112for infusion or flushing.FIG.1Ashows the canister100prior to being sealed shut, including an interior chamber112formed by combining both the upper shell110and lower shell114.FIG.1Bshows the canister100with one the upper shell110cut away, showing the interior chamber112. In all embodiments, the major surfaces can be fitted and/or joined and/or welded along an edge of the joined top and bottom halves, creating a single delivery canister. As shown inFIG.1B, the cannister includes a plurality of suture retention tabs111configured to provide access to one or more sutures to mount the cannister within a target area. Moreover, tubes113may also be include coupled to the cannister100to load bioactive compounds within the cannister100. For example, one or tubes113may be in fluid communication with one or the other of the first major surface102or the second major surface104. Moreover, a non-metal, polymeric membrane material may be stretched over the spacer ring106. FIG.1Ccorresponds to the area shown on the first major surface inFIG.1Aand illustrates a microscopic view of the nanoscale through-porous membrane structure10of the canister100.FIG.2illustrates a microscopic view of the nanoscale through-porous metallic membrane material of the canister100,300400,1400,1500having a uniform or homogeneous nanoporous structure.FIG.2Ashows similarly illustrates a microscopic view of the metallic material of the canister100,300,400,1400,1500that is processed to possess an interconnected bicontinuous morphology which containing two distinct porous regions10,20, each of substantially uniform composition which differs from the other and each of which forms a continuous pathway from one surface of an article to another surface of an article. The above nanoscale through-porous membrane structure10can include other configurations. For example,FIG.2Billustrates a microscopic view of a material that includes a nanoporous, bi-continuous morphology “A” that is layered or integrated with a tubular nanoporous morphology “B” such that the material includes two distinct porous regions of different composition and which form a continuous fluid pathway through the structure. Turning now toFIG.2C, a top or bottom face of the tubular nanoporous structure “B” is presented to show openings, tubular pores “C,” in the structure into which, through, or from fluid may flow. The pores “C” can be loaded with biodegradable polymers and a wide range of drugs, such as insulin, cytokines, or growth factors. The structure “B” provides excellent control over small molecule diffusion and inhibits large molecule release. As shown, the structure “B” provides some pores “C” with a porosity of 80 nm inner diameter and a 100 nm outer diameter, while others have a smaller or larger pore diameter. In this way, it is possible to filter more than one proteins/nanoparticle of selected sizes. However, the structure “B” can include pores all having substantially the same diameter. FIG.2Dis a cross section ofFIG.2Cshowing the tubular pores “B” from the side and illustrating the comparative length to diameter ratio. While the tubular pores “C” are shown to be substantially straight, they can also be curved or serpentine as required by the shape of the substrate to which they are affixed. The nanoporous structure10is metallic, and more particularly fabricated from medical grade metals such as titanium and/or 316L stainless steel. However other metal oxide chemistry is suitable. The metallic tubular pores “C” are very rigid and close enough together (generally 10 nm apart) that fluid with select drugs as described above flows through the tubes and not around or between them. For a nanoporous titanium or 316L stainless steel structure, excellent diffusion of insulin analogue is diffused while completely inhibiting IgG analogue release. For a nanoporous structure10having pores in the range of 20 nm to 5000 nm, immune cells such as macrophages and T-cells are restricted by their size from infiltrating through the membrane and prevented from gaining contact with the living tissue contained within. In one configuration the nanopores or no larger than 800 nm in diameter. The nanoporous structure10can be provided with a surface that is created with a subtractive process such as an acid wash, or an additive process like 3D printing. In one embodiment the textured surface is nano-sericeous, at the nanoscale to change the surface energy. Nano-serious topography can be considered as nanofeatures that lie on the surface and/or down or along the direction of a porous substrate. They can reach any depth of the nanoporous interior, whether tubular or bicontinuous or any other nano-porous matrix (e.g., serpentine). The nanofeatures do not affect the through-porous flow rates within the 20 nm to microns sized pores of the underlying substrate. The nanofeatures are 1-5 nm in height and can take many shapes such as oval bumps, straight or staggered ridges, cones, clusters, all with varying widths, from 1-200 nm. However, the process to create nano-sericeousness material does not substantively add any material. The underlying through-porous substrate and nano-sericeous topography are of the same, homogenous metal material. The nano-sericeous topography provided significant benefits in biological applications. For example, when incorporated, these specialized topographical features can impact biological interactions with an implanted device. Cell or protein binding for example, can be stimulated to attract or be repulsed based in part on surface texture/charge. Specifically, initial protein adsorption dictates cell adhesion and growth. Since proteins possess a charge and energy, changing the surface energy of a material will alter the adsorption and 3-dimensional conformation of proteins (or bioactivity) which will in turn change cell adhesion and growth. A nano-sericeous topography can stimulate vascularization and induce epithelial cells to enter a larger diameter nanoporous matrix thus establishing neovascularity closer to encapsulated living tissue. Additionally, nano-sericeous topography allows for depositing nanoparticles on the surfaces of medical implants. For example, deposited Ag nanoparticles help reduce infection. As described above, the nanoporous structure10can be considered a “stand-alone” material that can be integral with a capsule. However, the nanoporous structure can also be applied to any surface of any object, such as a medical device including the capsules described herein. For example, similar to what has been described above, a medical device can include a substrate having a nanophase tubular structure “B.” The nanophase tubular structure can have a thickness between 8 microns and 200 microns and the nanophase tubular structure can include multiple nanotubes made of a material including one of titanium and 316L stainless steel. In one embodiment the nanophase tubular structure includes nanotubes having a diameter of 80 nm that are integrated with a metallic capsule as described herein, wherein the metallic capsule is apertured to permit fluid passage into the metallic capsule. Referring now toFIG.3, a cut away view of a canister300configured to have an internal curved, raised pattern formed by an internal barrier212to physically stabilize a biotherapeutic agent (not shown) encased within.FIG.4similarly is a cut away view of an embodiment of a canister400having a squared shape with a plurality of raised internal barriers412to physically stabilize a biotherapeutic agent (not shown) held by the canister400.FIGS.3and4are shown for purposes of illustration only and are not intended to be limiting as variations of shape and internal barrier configuration within a canister are infinite. FIGS.5-11illustrate exterior nanoporous surface treatment patterns500,600,700,800,900,1400,1500that could be formed into the exterior and/or interior surface (unnumbered) of the canister100,300,400,1400,1500. The purpose of the different exterior and/or interior nanoporous surface treatment configurations is to increase the surface area of the canister100,300,400,1400,1500to allow improved inflow and outflow which positively affects the output of cellular and/or biotherapeutic agents contained in the canister and diffused from the canister.FIGS.5to11are shown for purposes of illustration only and are not intended to be limiting as surface treatment variations of canister surface are infinite. FIG.12illustrates the canister100being loaded through its port108with a biotherapeutic agent.FIG.13similarly illustrates a canister100having an extended tube108abetween the canister100and the exterior of a patient's body to allow loading of biotherapeutic agent (not shown) following a deep body (e.g., abdominal) implantation. FIG.14is a view of a metallic nanoporous outer protective canister1400with an inner polymeric pouch1404constructed of a semi-permeable material and containing biotherapeutic agent (not shown). The outer protective canister1400is constructed of a nanoscale or microscale through-porous membrane to facilitate cellular or biotherapeutic agent diffusion. The polymeric pouch1404is provided fluid communication by means of extended silicone tube1406. In another embodiment, as shown inFIG.15, the delivery canister1500is composed of multiple individual and independent chambers1508,1510,1512,1514,1516,1518. Each individual and independent chamber is accessed through its own port108. Each independent chamber can have the same or varied nanophase through-porous membrane structure10,20for controlled diffusion of various cellular or biotherapeutic agents, thus allowing sequenced delivery. Referring now toFIGS.16and17, implanting the canister100, or any other canister disclosed herein, within the mammal, includes depositing a metabolically active agent into the implantable canister. To that end, an injection device1602may be included and configured to deliver the metabolically active agent into the canister. In one configuration, the injection device1602includes a push button, squeeze handle, or similar actuator1604to deliver a substantially uniform distribution of the metabolically active agent into the implantable canister100. In one configuration, the canister100includes a membrane1606, which may be a tri-leaf valve or other valve structure in which the injection device1602penetrates to deliver the metabolically agent into the canister. For example, the membrane1606may be positioned on the surface of the canister or spaced a distance away from the canister within one or more injection device anchoring elements1608. In one configuration, the anchoring element1608defines a beveled edge to facilitate engagement to the injection device1602and may further be conical in shape. In one configuration, the anchoring element1608includes threads configured to engage a corresponding threaded portion on the injection device1602to threadably engage the injection device1602to the anchoring element1608. In the configuration shown inFIG.17, a second anchoring element1608is included on an opposite side of the canister. In an exemplary configuration, the injection device1602includes an injection tube1610extending outward therefrom, the injection tube1610being in communication with as source of metabolically active agent disposed within the injection device1602. The injection tube1610may be a straight tube or may define a helical or cork-screw shape to penetrate the member1606. Once the injection tube is engaged to the anchoring element1608, the injection tube1610may penetrate the membrane1606to inject the metabolically active agent. Following the injection, the injection tube1610may be withdrawn and the membrane1606may reseal itself to retain the contents within the canister. In other configurations, the injection device1602may snap fit, or otherwise lock with the anchoring element1608of the canister. For example, the injection device1602may include a plurality of arms that engage the canister and injection the contents therein. In other configurations, the injection tube1610may break off from the injection device1602and degrade over time inside the body. In still other configurations, the cannister100and/or the anchoring element1608may include a radiopaque or echogenic marker such that it can be visible under fluoroscopy or other imaging techniques to locate a port in the cannister100. Referring now toFIGS.18-19, in which another embodiment of an implantable medical device1800is shown. The medical device includes a first portion1802having a nanoscale through-porous structure and a second portion1804opposite the first portion1802having a nanoscale through porous structure. In one configuration the combination of the first portion1802with the second portion1804produces a bicontinuous morphology, as discussed in more detail above. In the configuration shown inFIG.18, the first portion1802and the second portion1804are metallic and rigid planar discs that are the same size, but in other configurations the first portion1802and the second portion1804may be differently sized, any shape, and may be flexible. Continuing to refer toFIG.18, the first portion1802and the second portion1804are engaged to an implantable spacer ring1806sized and configured to retain the first portion1802and the second portion1804. In the configuration shown inFIG.18, the spacer ring1806is ring shaped to accommodate the first and second portions1802,1804. In other configurations, the spacer ring1806may be any shape to accommodate the first portion1802and the second portion1804. The spacer ring1806is sized and configured to be implantable with the body of a human or animal patient. That is, the spacer ring1806may be composed of corrosion resistant and biocompatible materials. The spacer ring1806may further define or otherwise includes a plurality of suture holes1808disposed about a perimeter or circumference of the spacer ring1806. The suture holes1808are through holes such that the spacer ring1806may be sutured within the patient. In the configuration shown inFIG.18, the spacer ring1806further defines a first bezel1810or a recessed area sized to receive the first portion1802and a second bezel (not shown) or recessed area on the opposite side of the spacer ring1806sized to receive the second portion1804. When seated within the respective bezels, the first portion1802and the second portion1804are substantially flush within the spacer ring1806. Moreover, the spacer ring1806defines an aperture1812within its center such that when the first portion and the second portions1802,1804are engaged to the spacer ring1806, they are in fluid communication with each other through the aperture1812. Continuing to refer toFIG.18, the spacer ring1806includes a port1814disposed within its circumference. In one configuration the port1814is disposed within a bulbous portion of the spacer ring1806. The port1814is further engageable with a fluid delivery tube1816extending from the port1814, which is in fluid communication with a source of fluid, for example, bioactive compounds. The fluid delivery tube1816may be flexible and may affixed to the port1814. In an exemplary configuration, the first portion1802and the second portion1804are loaded with fluid injection into the fluid delivery tube1816through the port1814to saturate each of the first portion1802and the second portion1804with fluid. Other embodiments of the cell and biotherapeutic delivery canister will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. | 34,165 |
11857674 | DETAILED DESCRIPTION OF CERTAIN INVENTIVE EMBODIMENTS In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the FIGURES, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. Lubricants, including personal lubricants are described herein that are useful for increasing lubricity. In some embodiments, the personal lubricant is useful for penile and/or vaginal application for increasing lubricity and for enhancing the ease and comfort of sexual intimacy, and for supplementing the body's natural lubrication. In some embodiment, the personal lubricant may be used alone or in combination with condoms or other devices to improve lubrication and comfort during sexual intimacy. In some embodiments, lubricants may be useful for treating, ameliorating, or reducing the itchiness associated with fungal, viral, or other skin conditions. In some embodiments, the personal lubricant is a hybrid-based lubricant that includes water and silicone. In some embodiments described herein are methods of making and using the lubricant formulations. Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For purposes of the present disclosure, the following terms are defined below. By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. In some embodiments, the “purity” of any given agent (for example, dimethicone or hypochlorous acid) in a composition may be specifically defined. For instance, certain compositions may include, for example, an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by analytical chemistry techniques. As used herein, the terms “function” and “functional” and the like refer to a biological, chemical, mechanical, or therapeutic function. “Hypochlorous acid”, as used herein, refers to a weak acid having the chemical formula HClO. Hypochlorous acid is also known as chloric (I) acid, chloranol, or hydroxidochlorine. Salts of hypochlorite are also referred to herein and can include sodium hypochlorite (NaClO), calcium hypochlorite (Ca(ClO)2), or potassium hypochlorite (KClO). As described herein, hypochlorous acid and hypochlorite are used as killing agents, skin cleansing agents, disinfectants, antibacterial agents, sanitizers, and/or preservatives. Hypochlorite, or acids and salts thereof, may be used in the lubricants and personal lubricants of the present invention at an amount of about 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or greater w/v %, or within a range defined by any two of the aforementioned amounts. In some embodiments, the w/v % of hypochlorite or an acid or salt thereof is about 25% w/v. In some embodiments, the hypochlorite salt or hypochlorous acid is added directly to a personal lubricant, wherein the final amount of hypochlorite is less than, greater than, or equal to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 175, 200, 300 ppm or within a range defined by any two of the aforementioned amounts. In some embodiments, the amount of hypochlorite in the lubricant is between about 50 to about 100 ppm. In some embodiments, the amount of hypochlorite in the lubricant is about 75 ppm. In some embodiments, the hypochlorite is added to the lubricant as a hypochlorite solution. In some embodiments, the hypochlorite solution is prepared from hypochlorite salt or hypochlorous acid. In some embodiments, the solution of hypochlorite is prepared by passing a sodium chloride solution through electrolysis. In some embodiments, the sodium chloride solution is a 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% or greater w/v % or within a range defined by any two of the aforementioned amounts. In some embodiments, the sodium chloride is 0.28%, and the resulting hypochlorite solution is 300 ppm. In some embodiments, the hypochlorite solution is added to the personal lubricant in an amount of about 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or greater w/v %, or within a range defined by any two of the aforementioned amounts. In some embodiments, the solution includes, for example, about 300 ppm hypochlorite is added to a personal lubricant in an amount of about 25% w/v. As used herein, silicone polymers include dimethicone, which is also known as polydimethylsiloxane (PDMS), dimethylpolysiloxane, E900, or polymerized siloxane and has the chemical formula of CH3[Si(CH3)2O]nSi(CH3)3where n is the number of repeating monomer [Si(CH3)2] units. Silicone polymers are used as an inert slip agent or lubricant. The silicone polymer may be used in the lubricant or personal lubricant in an amount of about 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, or greater w/v %, or in an amount within any two of the aforementioned values or between a range defined by these values. In some embodiments, the amount of silicone polymer is about 10% w/v. As used herein, the term “sodium magnesium silicate” refers to a silicate of sodium and magnesium and is a synthetic silicate clay, having magnesium and sodium silicate. It is used as a binder and bulking agent in cosmetics and personal care products, in part because of its ability to absorb water. It is also used in the creation of concrete. Sodium magnesium silicate is effective in slowing the decomposition of formulas, and can prevent premature darkening of the cosmetic composition and prevent premature development of a foul odor, thereby improving the shelf life of cosmetic compositions. In some embodiments, the sodium magnesium silicate is Laponite. As used herein, sodium magnesium silicate is useful as a gelling agent or rheology modifier. Sodium magnesium silicate may be used in the lubricant or personal lubricant in an amount of about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 10%, 15%, or greater w/v %, or in an amount within any two of the aforementioned values or between a range defined by these values. In some embodiments, the amount of sodium magnesium silicate is about 3.25% w/v. As used herein, the term “sodium phosphate monobasic” refers to the chemical formula of NaH2PO4, an inorganic compound of sodium with dihydrogen phosphate. Sodium phosphate monobasic is also referred to as sodium dihydrogen phosphate, sodium phosphate, monosodium phosphate, sodium biphosphate, acid sodium phosphate, monosodium orthophosphate, or primary sodium phosphate. As described herein, it may be used for adjustment of pH, as a thickening agent, or as an emulsifier. Sodium phosphate monobasic may be used in the lubricant or personal lubricant in an amount of about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 10%, 15%, or greater w/v %, or in an amount within any two of the aforementioned values. In some embodiments, the amount of sodium phosphate is about 3.25% w/v. The personal lubricants described herein may further include an additive known in the art can be included. Exemplary additives include emollients, moisturizers, humectants, pigments, dyes, pearlescent compounds, nacreous pigments, bismuth oxychloride coated mica, titanium dioxide coated mica, colorants, fragrances, biocides, preservatives, alpha hydroxy acids, antioxidants, anti-microbial agents, anti-fungal agents, antiperspirant agents, exfoliants, hormones, enzymes, medicinal compounds, vitamins, salts, electrolytes, alcohols, polyols, polypropylene glycol, polyisobutene, polyoxyethylene, behenic acid, behenyl, sugar-alcohols, absorbing agents for ultraviolet radiation, botanical extracts, surfactants, silicone oils, organic oils, waxes, alkaline or acidic or buffering agents, film formers, thickening agents, hyaluronic acid, fumed silica, hydrated silica, talc, kaolin, starch, modified starch, mica, nylon, clay, bentonite, organo-modified clays and combinations thereof. In some embodiments, the personal lubricant described herein is characterized in having an osmolality by vapor pressure of about 10, 20, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, or 100 mmol/kg, or within a range defined by any two of the aforementioned amounts. In some embodiments, the osmolality by vapor pressure is about 38 mmol/kg. In some embodiments, the osmolality by vapor pressure is about 49 mmol/kg. In some embodiments, the lubricant is characterized in having an osmolality by freezing point depression of about 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 80, 90, or 100 mOsm/kg, or within a range defined by any two of the aforementioned amounts. In some embodiments, the osmolality by freezing point depression is about 22 mOsm/kg. In some embodiments, the osmolality by freezing point depression is about 54 mOsm/kg. In some embodiments is provided a method of making the lubricant formulation. In some embodiments, the method includes providing hypochlorite. In some embodiments, the hypochlorite is provided as a hypochlorite acid or salt. In some embodiments, the hypochlorite is provided as a hypochlorite solution. In some embodiments, the method of making the lubricant includes providing hypochlorite in an amount of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 175, 200, 250, or 300 ppm or within a range defined by any two of the aforementioned amounts. In some embodiments, hypochlorite is provided in an amount of about 50 to 100 ppm. In some embodiments, the hypochlorite is provided in an amount of about 75 ppm. In some embodiments, the method of making the lubricant includes providing a hypochlorite solution. In some embodiments, the hypochlorite solution is prepared from hypochlorite acids or salts. In some embodiments, the hypochlorite salt is sodium hypochlorite. In some embodiments, the hypochlorite solution is prepared from sodium chloride. In some embodiments, the method includes running sodium chloride solution through electrolysis. In some embodiments, the sodium chloride solution is provided in an amount of about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, or 0.4% w/v. In some embodiments, the sodium chloride is 0.28%, and the resulting hypochlorite solution is about 300 ppm. In some embodiments, the method of making the lubricant includes diluting the hypochlorite solution to provide hypochlorite in an amount of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 175, 200, 250, or 300 ppm or within a range defined by any two of the aforementioned amounts. In some embodiments, hypochlorite is provided in an amount of about 50 to 100 ppm. In some embodiments, the hypochlorite is provided in an amount of about 75 ppm. In some embodiments, the method of making the lubricant further includes providing a silicone polymer. In some embodiments, the silicone polymer is dimethicone. In some embodiments, the silicone polymer is provided in an amount of about 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% w/v, or within a range defined by any two of the aforementioned amounts. In some embodiments, the silicone polymer is provided in an amount of about 10% w/v. In some embodiments, the method further includes providing an emulsifier. In some embodiments, the emulsifier is sodium phosphate. In some embodiments, the emulsifier is provided in an amount of about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, or 2.5% w/v or within a range defined by any two of the aforementioned amounts. In some embodiments, the emulsifier is provided in an amount of about 0.2% w/v. In some embodiments, the method of making the lubricant further includes providing sodium magnesium silicate, water, or buffer or combinations thereof. In some embodiments, the sodium magnesium silicate is provided in an amount of about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 10%, or 15% w/v or within a range defined by any two of the aforementioned amounts. In some embodiments, the sodium magnesium silicate is provided in an amount of about 3.25% w/v. In some embodiments, the water and/or buffer is provided in an amount to make up the balance of the lubricant, and is provided in an amount of about 20%, 30%, 40%, 45%, 50%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 61.55%, 62%, 62.5%, 63%, 65%, 70%, 75%, 80%, or 90% w/v or within a range defined by any two of the aforementioned amounts. As used herein, the term “buffer” refers to a buffering agent and is used for balancing the pH and/or osmolality of the lubricant. Examples of a buffer for use herein include, for example, salts of phosphates, borates, citrates, ascorbates, carbonates, bicarbonates, TRIS, HEPES, sodium ions, potassium ions, chloride ions, bicarbonate ions, glucose, sucrose, peptides, proteins, a combination or mixture thereof or other agents that are chemically, functionally, or physiologically equivalent or similar. The lubricant compositions provided herein have an optimum pH and viscosity, with an osmolality that is hypo-osmotic, having an osmolality by vapor pressure of about 10, 20, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, or 100 mmol/kg, or within a range defined by any two of the aforementioned amounts. In some embodiments, the osmolality by vapor pressure is about 38 mmol/kg. In some embodiments, the osmolality by vapor pressure is about 49 mmol/kg. In some embodiments, the lubricant is characterized in having an osmolality by freezing point depression of about 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 80, 90, or 100 mOsm/kg, or within a range defined by any two of the aforementioned amounts. In some embodiments, the osmolality by freezing point depression is about 22 mOsm/kg. In some embodiments, the osmolality by freezing point depression is about 54 mOsm/kg. The osmolality of the lubricant can be determined by vapor pressure osmometry or freezing point osmometry. In some embodiments is provided a method of using a lubricant. In some embodiments, the method includes providing the lubricant and applying the lubricant. In some embodiments, the lubricant is provided as a ready-to-use formulation that includes hypochlorite or acids or salts thereof, silicone polymer, and an emulsifier, and further may include sodium magnesium silicate, water, or buffer. In some embodiments, the lubricant is provided in portions, and further additions and/or mixing is required prior to use. In some embodiments, the lubricant is applied in the penile or vaginal regions. In some embodiments, the lubricant is applied on a condom or other device. In some embodiments, the lubricant is applied multiple times daily, once daily, multiple times weekly, once weekly, multiple times monthly, or once monthly, or within a time frame defined by any two of the aforementioned time frames. In some embodiments, the lubricant is applied liberally. In some embodiments, the lubricant is applied meagerly. In some embodiments, the personal lubricant as disclosed herein is useful for improving lubrication and comfort during sexual intimacy. In some embodiments, the personal lubricant described herein is a hybrid lubricant that includes both water and silicone. In some embodiments, the personal lubricant includes hypochlorite, or a salt or acid thereof, dimethicone, and an emulsifier. In some embodiments, the personal lubricant includes water and/or buffer, hypochlorous acid solution, dimethicone, sodium magnesium silicate, and sodium phosphate. In some embodiments is provided a method of using a lubricant for the cessation, amelioration, prevention, or inhibition of the development of itching from the vagina or other body parts by eliminating microbial causing infections. In some embodiments, the personal lubricant is useful for alleviating discomfort in a subject having inflammation or discomfort in the vaginal or vulvovaginal area. Symptoms can include but are not limited to irritation and/or itching of the genital area, inflammation of the vaginal or perineal area or pain. Causes can include but are not limited to disruption of the healthy microbiota, infections, yeast, bacteria or viruses. Pathogens that can cause irritation can include but are not limited toCandida, Gardnerella, gonorrhea,chlamydia, Mycoplasma, herpes,Campylobacter, orTrichomonas vaginalis. Irritation can also occur due to effects of diabetes, birth control, bad diet, tight clothing, use of antibiotics, hormonal vaginitis due to post-menopause or postpartum, or loss of estrogen. Irritants also originate from condoms, spermicides, soaps, perfumes, and lubricants. Loss of estrogen or hormonal vaginitis can also lead to dryness of tissues. In some embodiments is provided a method of using the lubricant composition to stop a fungal infection. In some embodiments, the fungal infection is caused by a yeast of theCandidagenus. In one embodiment, the yeast is of theCandida albicansspecies. In other embodiments, theCandidayeast may be of theCandida dubliniensis, Candida parapsilosis, Candida tropicalis, Candida kefyr, Candida guilliermondii, Candida inconspicua, Candida famata, Candida glabrata, Candida krusei, Candida lusitaniae, or otherCandidaspecies, or combinations thereof. In some embodiments, the lubricant composition is used to stop a viral infection. In some embodiments, the lubricant as disclosed herein is useful as a medical or surgical lubricant for use with medical instruments for insertion, penetration, or introduction of a catheter, ultra sound, or other medical device into a subject. The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. EXAMPLES Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims. Example 1 Preparation of Personal Lubricant Formulations The following example demonstrates the method of preparing the personal lubricant and various compositions or formulations thereof. A personal lubricant was prepared with the ingredients as provided in Table 1. Hypochlorite or a salt or acid thereof was added to water, dimethicone, sodium magnesium silicate, and sodium phosphate in the preparation of a personal lubricant formulation PL-10. TABLE 1Personal Lubricant 10 (PL-10) FormulationIngredientQuantityWater and/or bufferbalanceHypochlorite75ppmDimethicone10%w/vSodium Magnesium Silicate3.25%w/vSodium Phosphate0.2%w/v The personal lubricant formulation described in Table 1 was tested on various organisms associated with sexually transmitted diseases to determine the efficacy of inhibiting, eradication, or reducing the organismal population. Various microbial pathogens were tested, including human immunodeficiency virus (HIV), Herpes simplex virus (HSV), Hepatitis B virus (HBV),Chlamydia trachomatis, andNeisseria gonorrhoeae. These studies are described in detail in the following examples. Alternative Preparation Example 1 A personal lubricant having 25% of a 220 ppm hypochlorite solution was added to 10% w/v dimethicone, with 3.25% w/v sodium magnesium silicate and 0.2% sodium phosphate, with the balance of 61.55% water. The hypochlorite solution was prepared by passing 0.28% sodium chloride through electrolysis to provide a 220 ppm hypochlorite solution. Example 2 Efficacy of Personal Lubricant Against HIV-1 in Suspension The following example shows the results of the efficacy of the personal lubricant against HIV-1 in suspension. HIV-1 was evaluated in a virucidal suspension assay. The test medium was Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 15% (v/v) heat-inactivated fetal bovine serum (FBS). The medium was also supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin. The suspension containing HIV-1 was exposed to the personal lubricant formulation. At a pre-determined exposure time, an aliquot was removed, neutralized by serial dilution, and assayed for the presence of virus. The virus controls, cytotoxicity control, and neutralization control were assays in parallel. Antiviral properties of the personal lubricant were evaluated and compared at the specified concentrations and time intervals. HIV-1 strain HTLV MB was exposed to a personal lubricant formulation, PL-10, for an exposure time of either 5 minutes or 10 minutes at a temperature of 20.0° C. in the presence of a 5% FBS organic soil load. PL-10 demonstrated a greater than 99.99% reduction in viral titer following 5 and 10 minute exposure times to HIV-1, as compared to the titer of the corresponding virus control. The 50% tissue culture infective dose (TCID50)/200 μL, a measure of infectious virus titer, was less than 102.50at both 5 and 10 minutes. Table 2 summarizes the effects of exposure of the personal lubricant to a suspension of HIV-1. The cytotoxicity and neutralization control results are presented in Table 3. MT-2 (human T cell leukemia cells) were used as indicator cell cultures. TABLE 2Effects of PL-10 Against HIV-1 in SuspensionVirus ControlHIV-1 + PL-105 minute10 minute5 minute10 minuteDilutionexposureexposureexposureexposureCell Control0 0 0 00 0 0 00 0 0 00 0 0 010−2+ + + ++ + + +T T T TT T T T10−3+ + + ++ + + +0 0 0 00 0 0 010−4+ + + ++ + + +0 0 0 00 0 0 010−5+ + + ++ + + +0 0 0 00 0 0 010−6+ + + ++ + + +0 0 0 00 0 0 010−70 0 0 00 0 0 00 0 0 00 0 0 0TCID50/200 μL106.50106.50≤102.50≤102.50Percent Reduction≥99.99%≥99.99%Log Reduction≥4.00 log10≥4.00 log10(+) = positive test for the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present(T) = cytotoxicity present TABLE 3PL-10 Cytotoxicity and Neutralization Results for HIV-1Cytotoxicity ControlNeutralization ControlDilutionPL-10HIV-1 + PL-10Cell Control0 0 0 00 0 0 010−2T T T TT T T T10−30 0 0 0+ + + +10−40 0 0 0+ + + +10−50 0 0 0+ + + +10−60 0 0 0+ + + +10−70 0 0 0+ + + +TCID50/200 μL102.50*Neutralized at ≤2.50 Log10(+) = positive test for the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present(T) = cytotoxicity present*Neutralization control reported as TCID50/250 μL Example 3 Efficacy of Personal Lubricant Against HSV-2 in Suspension The following example shows the results of the efficacy of the personal lubricant against HSV-2 in suspension. HSV-2 was evaluated in a virucidal suspension assay. The test medium was minimum essential medium (MEM) supplemented with 5% (v/v) heat-inactivated FBS. The medium was also supplemented with 100 units/mL penicillin, 10 μg/mL gentamicin, and 2.5 μg/mL amphotericin B. HSV-2 was exposed to the personal lubricant formulation. At a pre-determined exposure time, an aliquot was removed, neutralized by serial dilution, and assayed for the presence of virus. The virus controls, cytotoxicity control, and neutralization control were assays in parallel. Antiviral properties of the personal lubricant were evaluated and compared at the specified concentrations and time intervals. HSV-2, ATCC VR-734, Strain G was exposed to a personal lubricant formulation, PL-10. The exposure time was of either 5 minutes or 10 minutes at a temperature of 21.0° C. in the presence of a 5% FBS organic soil load. PL-10 demonstrated a greater than 99.9997% reduction in viral titer following 5 minute exposure time and a greater than 99.998% reduction in viral titer following a 10 minute exposure time to HSV-2, as compared to the titer of the corresponding virus control. The log reductions in viral titer were greater than 5.50 log10and greater than 4.75 log10, respectively. Table 4 summarizes the effects of exposure of the personal lubricant to a suspension of HSV-2. The cytotoxicity and neutralization control results are presented in Table 5. Vero cells were used as indicator cell cultures. TABLE 4Effects of PL-10 Against HSV-2 in SuspensionVirus ControlHSV-2 + PL-105 minute10 minute5 minute10 minuteDilutionexposureexposureexposureexposureCell Control0 0 0 00 0 0 00 0 0 00 0 0 010−2+ + + ++ + + +0 0 0 00 0 0 010−3+ + + ++ + + +0 0 0 00 0 0 010−4+ + + ++ + + +0 0 0 00 0 0 010−5+ + + ++ + + +0 0 0 00 0 0 010−6+ + + ++ 0 + +0 0 0 00 0 0 010−7+ 0 + 00 0 0 00 0 0 00 0 0 010−80 0 0 00 0 0 00 0 0 00 0 0 0TCID50/100 μL107.00106.25≤101.50≤101.50Percent Reduction≥99.9997%≥99.998%Log Reduction5.50 log104.75 log10(+) = positive test tor the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present TABLE 5PL-10 Cytotoxicity and Neutralization Results for HSV-2Cytotoxicity ControlNeutralization ControlDilutionPL-10HSV-2 + PL-10Cell Control0 0 0 00 0 0 010−20 0 0 0+ + + +10−30 0 0 0+ + + +10−40 0 0 0+ + + +TCID50/100 μL≤101.50*Neutralized at ≤1.50 Log10(+) = positive test for the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present*Neutralization control reported as TCID50/100 μL Example 4 Efficacy of Personal Lubricant Against HBV in Suspension The following example shows the results of the efficacy of the personal lubricant against HBV in suspension. HBV was evaluated in a virucidal suspension assay. The test medium was Leibovitz L-15 medium supplemented with 0.1% glucose, 10 μM dexamethasone, 10 μg/mL insulin, 20 mM HEPES, 100 units/mL penicillin, and 10 μg/mL gentamicin. HBV was exposed to the personal lubricant formulation. At a pre-determined exposure time, an aliquot was removed, neutralized by serial dilution, and assayed for the presence of virus. The virus controls, cytotoxicity control, and neutralization control were assays in parallel. Antiviral properties of the personal lubricant were evaluated and compared at the specified concentrations and time intervals. Duck HBV was exposed to a personal lubricant formulation, PL-10. The exposure time was of either 5 minutes or 10 minutes at a temperature of 20.0° C. in the presence of 100% duck serum, with no additional soil load added. PL-10 demonstrated a greater than 99.999% reduction in viral titer following 5 minute exposure time and a greater than 99.998% reduction in viral titer following a 10 minute exposure time to duck HBV, as compared to the titer of the corresponding virus control. The log reductions in viral titer were greater than 5.00 log10and greater than 4.75 log10, respectively. Table 6 summarizes the effects of exposure of the personal lubricant to a suspension of HSV-2. The cytotoxicity and neutralization control results are presented in Table 7. Primary duck hepatocytes were used as indicator cell cultures. TABLE 6Effects of PL-10 Against HBV in SuspensionVirus ControlHBV + PL-105 minute10 minute5 minute10 minuteDilutionexposureexposureexposureexposureCell Control0 0 0 00 0 0 00 0 0 00 0 0 010−2+ + + ++ + + +0 0 0 00 0 0 010−3+ + + ++ + + +0 0 0 00 0 0 010−4+ + + ++ + + +0 0 0 00 0 0 010−5+ + + ++ + + +0 0 0 00 0 0 010−6+ + + ++ + + 00 0 0 00 0 0 010−70 0 0 00 0 0 00 0 0 00 0 0 0TCID50/250 μL106.50106.25≤101.50≤101.50Percent Reduction≥99.999%≥99.998%Log Reduction≥5.00 log10≥4.75 log10(+) = positive test for the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present TABLE 7PL-10 Cytotoxicity and Neutralization Results for HBVCytotoxicity ControlNeutralization ControlDilutionPL-10HBV + PL-10Cell Control0 0 0 00 0 0 010−20 0 0 0+ + + +10−30 0 0 0+ + + +10−40 0 0 0+ + + +TCID50/250 μL≤101.50*Neutralized at ≤1.50 Log10(+) = positive test for the presence of test virus(0) = no test virus recovered and/or no cytotoxicity present*Neutralization control reported as TCID50/250 μL Example 5 Efficacy of Personal Lubricant AgainstChlamydiain Suspension The following example shows the results of the efficacy of the personal lubricant againstChlamydia trachomatisin suspension. Chlamydia trachomatiswas evaluated in a chlamydial suspension assay. The test medium was MEM supplemented with 10% (v/v) heat-inactivated FBS, 2 μg/mL cycloheximide, 4.5 g/L glucose, 10 mM HEPES, 10 μg/mL gentamicin, and 2.5 μg/mL amphotericin B. A suspension ofchlamydiawas exposed to the personal lubricant formulation. At a pre-determined exposure time, an aliquot was removed, neutralized by serial dilution, and assayed for the presence ofchlamydia. Thechlamydiacontrols, cytotoxicity control, and neutralization control were assays in parallel. Antichlamydia properties of the personal lubricant were evaluated and compared at the specified concentrations and time intervals. Chlamydia trachomatis(Serotype K), ATCC VR-887, strain UW-31/Cx was exposed to a personal lubricant formulation, PL-10. The exposure time was of either 5 minutes or 10 minutes at a temperature of 20.0° C. in the presence of 5% FBS organic soil load. PL-10 demonstrated a greater than 99.999% reduction inchlamydiatiter following 5 minute exposure time and a greater than 99.998% reduction inchlamydiatiter following a 10 minute exposure time toChlamydia trachomatis(Serotype K), as compared to the titer of the correspondingChlamydiacontrol. The log reductions inchlamydiatiter were greater than 5.00 log10and greater than 4.75 log10, respectively. Table 8 summarizes the effects of exposure of the personal lubricant to a suspension ofchlamydia. The cytotoxicity and neutralization control results are presented in Table 9. McCoy indicator cell cultures were used. TABLE 8Effects of PL-10 Against Chlamydia in SuspensionChlamydia ControlChlamydia trachomatis + PL-105 minute10 minute5 minute10 minuteDilutionexposureexposureexposureexposureCell Control0 0 0 00 0 0 00 0 0 00 0 0 010−2+ + + ++ + + +0 0 0 00 0 0 010−3+ + + ++ + + +0 0 0 00 0 0 010−4+ + + ++ + + +0 0 0 00 0 0 010−5+ + + ++ + + +0 0 0 00 0 0 010−6+ + + ++ + 0 +0 0 0 00 0 0 010−70 0 0 00 0 0 00 0 0 00 0 0 0TCID50/200 μL106.50106.25≤101.50≤101.50Percent≥99.999%≥99.998%ReductionLog Reduction≥5.00 log10≥4.75 log10(+) = positive test for the presence of test chlamydia(0) = no test chlamydia recovered and/or no cytotoxicity present TABLE 9PL-10 Cytotoxicity and Neutralization Results for ChlamydiaCytotoxicity ControlNeutralization ControlDilutionPL-10Chlamydia trachomatis + PL-10Cell Control0 0 0 00 0 0 010−20 0 0 0+ + + +10−30 0 0 0+ + + +10−40 0 0 0+ + + +TCID50/100 μL≤101.50*Neutralized at ≤1.50 Log10(+) = positive test for the presence of test chlamydia(0) = no test chlamydia recovered and/or no cytotoxicity present*Neutralization control reported as TCID50/200 μL Example 6 Efficacy of Personal Lubricant AgainstNeisseria gonorrhoeaein Suspension The following example shows the results of the efficacy of the personal lubricant againstNeisseria gonorrhoeaein suspension. Neisseria gonorrhoeaewas evaluated in a time kill assay. The test was conducted in an agar plate medium of chocolate agar.Neisseria gonorrhoeae, ATCC 43069, was exposed to a personal lubricant formulation, PL-10 at exposure times of 1, 2, 5, and 10 minutes in suspension at a temperature of 21.0° C. After exposure, an aliquot of the suspension was transferred to a neutralizer and was assayed for survivors. Appropriate culture purity, neutralizer sterility, test population, and neutralization confirmation controls were performed. The neutralizer was Letheen broth with 0.07% lecithin and 0.5% Tween 80. The neutralizer sterility control shows no growth ofNeisseria gonorrhoeae. The control population ofNeisseria gonorrhoeaeshows 3.2×104colony forming units, and log reduction of 4.51 log10. The exposure ofNeisseria gonorrhoeaeto PL-10 for any of 1, 2, 5, and 10 minutes showed no survivors at any of the dilution. Table 10 summarizes the effects of exposure for PL-10 againstNeisseria gonorrhoeae. TABLE 10PL-10 AgainstNeisseria gonorrhoeaeCFU/mLin TestExposurepopulationTimecontrolCFU/mL ofLog10PercentLog10(minutes)(Log10)SurvivorsSurvivorsReductionReduction13.2 × 104<5<0.70>99.9%>3.812(4.51)<5<0.70>99.9%>3.815<5<0.70>99.9%>3.8110<5<0.70>99.9%>3.81CFU = colony forming units Table 11 summarizes the results of Examples 2-6 for the efficacy of PL-10 in reducing a variety of organismal populations. TABLE 11PL-10 Destroys Major Sexually Transmitted InfectionsMaxPopulation atPopulation atTestControl5 minutePercent10 minutePercentOrganismPopulationexposureReductionexposureReductionHIV-14.00 Log100>99.99%0>99.99%Herpes5.50 Log100>99.9997%0>99.998%simplexvirustype 2Hepatitis5.00 Log100>99.999%0>99.998%B virusChlamydia5.00 Log100>99.999%0>99.998%trachomatisNeisseria3.8 Log100>99.9%0>99.9%gonorrhoeae Example 7 Cytotoxicity of Personal Lubricant on Cell Culture The following example shows the results of the cytotoxicity of the personal lubricant on cell cultures. The personal lubricant PL-10 was added to mouse fibroblast cells and incubated for 24 hours. The cells were observed under 100× magnification and the amount of morphology was scored using a 0-4 scale, where 0=no reactivity and 4=severe reactivity. A score of 3 or greater indicates a cytotoxic effect. The personal lubricant received a score of 2, indicating that the personal lubricant is not cytotoxic on mouse fibroblast cultures. Example 8 Sensitivity Test of Personal Lubricant The following example shows the results of the sensitivity of the personal lubricant on animal models. The personal lubricant was tested on guinea pigs to determine if exposure to the product produced a delayed-type hypersensitivity skin reaction. In this example, guinea pigs were exposed to the personal lubricant and to a control substance both underneath the skin and topically. After 14 days, the animals were again exposed to the lubricant or control. The test sites on the skin were evaluated at both 24 and 48 hours post treatment. No sensitization reactions were observed, and the test group did not exhibit differences from the control group. Example 9 Vaginal Mucosal Irritation Test of Personal Lubricant The following example shows the results of the personal lubricant on vaginal mucosal irritation. The personal lubricant of Example 1, PL-10, was used on rabbits to determine the effects of the personal lubricant on vaginal tissue. The study complied with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR 1-3), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. Test procedures were reviewed and approved by PBL's Institutional Animal Care and Use Committee (IACUC) in compliance with Animal Welfare Act. Environment. New Zealand white rabbits were housed individually in stainless steel cages. Animals were maintained in a controlled environment at a nominal temperature range of 16 to 22° C., a humidity range of 50 to 20%, and a light/dark cycle of 12 hours. Animals were maintained in rooms with at least ten room air changes per hour. Diet and Feed. Animals received a Certified Laboratory Rabbit Diet approximately 165 g per day. The feed is analyzed by the supplier for nutritional components and environmental contaminants. There are no known contaminants in the feed that interfered with the conduct of this example. Water. Fresh, potable drinking water was provided ad libitum to all animals via a sipper tube. Water testing is conducted two times a year for total dissolved solids and specified microbiological content and selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. There are no known contaminants in the water that interfered with the conduct of this example. Acclimation. Animals placed on study were acclimated to the testing facility for at least 6 days prior to initiation of the study. Health observations were performed prior to the study to ensure that the animals were acceptable for study use. Assignment to Study and Disposition. Animals were examined prior to study initiation, and determined (based on clinical observations) suitable as test subjects. Test and Control Article Preparation: The personal lubricant PL-10 is applied. Physiological saline (SCI) is used as a negative control. Procedure: Six female rabbits were used in this example (three test and three control animals). Prior to the test and prior to each treatment, the animals were checked for vaginal discharge, swelling and/or other evidence of vaginal infection, irritation or injury. The animals were weighed prior to the initial dosing and at the termination of the test. Rabbits were dosed at 24±2 hour intervals every day for a minimum of five consecutive days (Days 0, 1, 2, 3 and 4). A short, soft catheter (approximately 6 cm) or blunt-tipped cannula (for example, 12 French Nelaton catheter) attached to a syringe was used for administration of the personal lubricant. The dose volume was approximately 1 mL. The tip of the catheter used for the test group animals was moistened with the personal lubricant and inserted into vagina. The tip of the catheter used for the control group animals was moistened with a control lubricant (for example, Lubrivet) and inserted into the vagina. The personal lubricant or control (at least 1 mL) was introduced no more than 6 cm into the anterior vagina. Any expelled material was gently removed with a soft tissue and rabbit returned to its cage. Clinical Observation: At 24±2 hours after the initial application and immediately prior to each treatment, the appearance of the vaginal opening and perineum was noted for signs of discharge, erythema and edema. At 24±2 hours after the last dose, all animals were euthanized. The entire urogenital tract (including the vagina and the cervix) was removed. The entire vagina was opened longitudinally, and examined for gross evidence of irritation, injury to epithelial layer of tissue and necrosis. The entire urogenital tract was placed in 10% formalin and samples further processed by approved histopathology laboratory (for example, HSRL, VA). Histopathological evaluation of tissues was performed by a Board Certified Pathologist. The vaginal tissues were evaluated for the irritant effects. Results: No evidence of mucosal irritation was found in the test animals following vaginal exposure to the personal lubricant, based on both macroscopic (evidence of irritation, epithelial cell injury, and necrosis) and microscopic (histopathological) analysis. Example 10 Acute Systemic Toxicity of Personal Lubricant The following example shows that the personal lubricant described herein does not show systemic toxicity in mice. Mice were injected intraperitoneally with the personal lubricant and observed for 3 days. None of the animals tested exhibited any biological reactivity during the test period, including no difference in body weight, no signs of dehydration, no abnormal posture or appearance of skin, eyes, fur and mucous membranes, no change in urine and fecal output, and no change in locomotor behavior. Example 11 Condom Compatibility of Personal Lubricant The following example demonstrates that the personal lubricant as described herein is compatible with a variety of condoms in terms of being compatible with standard condom testing measures, such as burst pressure, burst volume, break force, and elongation. The personal lubricant was tested with latex, polyisoprene, and polyurethane condoms. As a baseline, each condom was tested as received, with no heat or lubricant applied. A control was performed with each condom exposed to 40° C. for 1 hour, without lubricant. A positive control was performed with each condom exposed to 40° C. for 1 hour with mineral oil applied. The test was performed at 40° C. for 1 hour with the personal lubricant, PL-10. Test results indicate that the personal lubricant passed all natural latex and polyisoprene condom compatibility testing. In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. | 47,963 |
11857675 | As it is shown, there is rolled up on each side of the horizontal arm a strand of copper (C); there are also rolled up two superimposed filament layers of copper (D) on the vertical arm wherein the coils of these filaments are spaced between them (E). The FIGURE also shows that there are placed ends of a length of coiled copper within the sphere (G); said length can have different shapes as a Solomon Bar (H), Celtic Knot (I) or Bracelet (J). DETAILED DESCRIPTION Any person skilled in the art must understand all technical terms described herein. However, certain terms are defined in order to clarify the invention. Therapeutic effect.—refers to a therapeutic and/or prophylactic benefit wherein the prophylactic benefit encompass the delay or the elimination of a disease or condition like the incidence of the human papilloma virus. Treatment.—refers to the treatment of a disease or condition in a human, particularly in women and includes: the prevention (when the disease or condition is not yet suffered); the inhibition and the relief (once the woman is already infected with the disease or is suffering certain condition) which involves the detention of the development of the disease and/or condition, and the regression of the disease and/or condition relieving the suffered symptoms respectively. Approximately or about—all measurable technical features like sizes, parameters, concentrations are not neither do need to be exact, i.e., these measurable technical features are disclosed as ranges which already includes the allowable tolerance. Hence, the use of the term “approximately” or “about” provides an additional determined range regarding the numeric value to which it is being applied. Said additional range provided by the term is approximately +10%. By way of example, but not in a limitative manner, if it reads “approximately 40 cm”, the exact range which it describes and/or claims is between 36 to 44 cm. Leukocytes—also called as a white blood cell, refers to those components present in the human body that defend it against all kind of diseases. They are divided into granular and agranular leukocytes, wherein the first group comprises the neutrophils, eosinophils, and basophils, and the second group comprises monocytes and lymphocytes. Cytokines.—are proteins or glycoproteins produced by different cell types that act as regulators of immune and inflammatory responses. Cervicovaginal microbiota.—refers to a dynamic group of microorganisms which can modulate the local immune response in the cervix and it can be classified into five groups according to [CURTY, Gislaine, et. al.; “The Role of the Cervicovaginal Microbiome on the Genesis and as a Biomarker of Premalignant Cervical Intraepithelial Neoplasia and Invasive Cervical Cancer”; Int. J. Mol. Sci. (2020), 21, 222, p. 5 of 24]; i.e., includes all community state types (CSTs) stated as CSTs I, II, III, IV and V according to dominant bacteria. For instance,Lactobacillus crispatus, L. gasseri, L. iners, andL. jenseniiare the dominant species of CSTs I, II, III and V; meanwhile, the CST IV shows an increase of anaerobic species likeGardnerella, Megasphera, Atopobium, andPrevotella. A lot of environmental factors, for instance, sexual activity, the use of oral contraceptive or others, stress, etc., can change the composition of microbiota. Unless expressly stated otherwise, whenever we refer to cervicovaginal microbioma, it should be considered that it is formed by a lot of microorganisms already disclosed in the art. The invention provides a method for reducing the incidence and prevalence of the human papillomavirus in women, mainly types 16 and 18, thus preventing the development of uterine cervical cancer in women and comprises the following steps:1. Providing a uterine cervical device comprising copper between approximately 380 mm2to approximately 524 mm2, preferably approximately 418 mm2to approximately 524 mm2, distributed on a T-shaped frame having a horizontal arm having a length of between about 17.8 millimeters and about 32.2 millimeters, said T-shaped frame having a vertical post extending from the horizontal arm, the vertical post having a length of between about 25.8 millimeters and about 36.2 millimeters and a diameter of about 1.7 millimeters, the horizontal arm having portions on opposite sides of the vertical post and a sphere affixed to the vertical post; said T-shaped frame made of inert plastic like polyethylene, propylene, polyester or silicone elastomer.2. Inserting said uterine cervical device inside of the female reproductive system, particularly in the lower part of the uterine cavity and endocervix where the human papillomavirus is incubated.3. The device must be maintained in position up to approximately 5 years. In an embodiment of the invention, the inserting step 2) is performed manually and without the use of any specific device for the insertion. That is, devices such as speculum, tenaculum, uterine sound, ring clips, scissors, usually used in the art are also used in said inserting step 2). In a preferred embodiment of the invention, the uterine cervical device being used in the present method corresponds to that claimed and granted in the U.S. patent application Ser. No. 15/100,900, U.S. Pat. No. 10,702,470 which comprises: A T-shaped frame formed of inert plastic material having between 15 and 23 weight percent radiopaque material, the radiopaque material being a combination of barium sulfate and titanium oxide, said T-shaped frame having a horizontal arm having a length of between 17.8 millimeters and 32.2 millimeters, said T-shaped frame having a vertical post extending from the horizontal arm, the vertical post having a length of between 25.8 millimeters and 36.2 millimeters and a diameter of 1.7 millimeters, the horizontal arm having portions on opposite sides of the vertical post; a sphere affixed to the vertical post, said sphere being formed of inert plastic or copper, said sphere having a diameter of 3.1 millimeters; a strand of copper wound on each of the portions of the horizontal arm, said strand of copper having a diameter of between 0.25 and 0.26 millimeters and having a total area of copper of between 35.8 and 36.2 square millimeters; a pair of copper filaments wound on the vertical post, each of said pair of copper filaments having a diameter of 0.25 and 0.26 millimeters and a total area of copper of 100 square millimeters, the pair of copper filaments extending for a distance of between 20 and 25 millimeters; a length of a copper filament having an area of between 146 and 250 square millimeters wound in a shape of a Soloman bar or a Celtic knot or a bracelet, wherein said length of the copper filament being positioned distal said sphere on said T-shaped frame, said sphere positioned between the vertical post and said length of the copper filament; and a low-density polyethylene thread having a length of 20 centimeters and a diameter of between 0.20 and 0.30 millimeters, said low-density polyethylene thread being affixed to said T-shaped frame and positioned inside said length of copper filament, a total amount of copper of said strand of copper and said pair of copper filaments and said length of copper filaments being between approximately 380 mm2to approximately 524 mm2, preferably between approximately 418 and 524 square millimeters. In the most preferred embodiment of the invention, the used uterine cervical device comprises 418 mm2of copper distributed on a T-shaped frame. According to the method of the invention, the uterine cervical device may be maintained in position up to approximately 5 years, and more preferably up to approximately 3 years. In one embodiment of the invention, the method as it is claimed also comprises the change of the uterine cervical device between approximately 3 to approximately 5 years after being inserted. In other embodiment of the invention, the method disclosed above is also novel and inventive because it prevents the development of uterine cervical cancer showing a therapeutic effect because within the area of the sphere affixed to the vertical post are located the ends of a length of 99.9% pure copper winding with a length of approximately 150 to approximately 300 mm, and approximately 146 to approximately 250 mm2placed on the uterine cervical canal right opposite the folds or cavities of the cervix; i.e, the structure of the uterine cervical device of the invention allows to be placed within the uterus and the cervix of the woman where lesions caused by the human papilloma virus, mainly types 16 and 18, can develop into cancer. According to the known measurements of cavimetries in women, the uterine cervical device used in the method of the present invention, advantageously, has the necessary dimensions so that it has contact not only with the uterus but also with the cervix of said population. Direct contact with the cervix is important, because as previously stated, it is in this area where HPV lodges and can become cancer. Another embodiment of the invention is that the method disclosed above permits treatment of the uterine cervical cancer developed in women by allowing the involution of premalignant and malignant lesions through the decreasing of the local inflammatory response and the modification of the local microbiota. Other embodiment of the invention is a method modifying the cervical microbiota of the women thus reducing the inflammatory response measured with cytokines and the progression of precancerous lesions in the cervix related to the human papilloma virus including the high-risk and the low-risk viruses; mainly the HPV types 16 and 18. This method comprises inserting the uterine cervical device disclosed previously into the female reproductive system, specifically in the lower part of the uterine cavity and endocervix. Said device must be maintained in position up to approximately 5 years, preferably up to three years. In an additional aspect, one method of the invention further provides an increase in leukocytes which, consequently, causes an increase in local immunity. The increased local immunity provides protection for other sexually transmitted infections in women aschlamydiainfection or gonococcal infection. Said method permitting an increment on the leukocytes comprises inserting the uterine cervical device disclosed previously into the female reproductive system, specifically in the lower part of the uterine cavity and endocervix. Said device must be maintained in position up to approximately 5 years, preferably up to three years. Experimental Studies The uterine cervical device disclosed in the invention will be mainly tested in patients already infected with the human papilloma virus, mainly types 16 and 18, by inserting said device into the lower part of the uterine cavity and endocervix, to demonstrate the reduction in the incidence and prevalence of the human papillomavirus in women, mainly types 16 and 18, thus preventing the development of uterine cervical cancer in women. Further, said device will be also tested to demonstrate that by inserting it into patients already suffering from uterine cervical cancer can be effectively treated such that involution of premalignant and malignant lesions can be expected thus leading to an improvement of the patient's life. In another aspect, said device will be tested in patients already infected with the human papilloma virus, mainly types 16 and 18, to determine and evaluate the viral load and the inflammatory load within the uterine and cervix of the woman by measuring the cytokines, in particular cytokines CD4 and CC4, in three different moments. The first measurement will be made before inserting the uterine cervical device; the second one will be done six months after the uterine cervical device was inserted; and the third one will be performed twelve months after the uterine cervical was inserted. Variations in these time intervals may occur as should be evident for a skilled person. Additionally, the device will be tested in patients already infected with the human papilloma virus, mainly types 16 and 18, when introducing the uterine cervical device to demonstrate the women's microbiome was modified after the device was introduced and placed inside their uterus and cervix. In a further aspect, the therapeutic effect of the device will be tested in women not suffering from a VPH infection or a uterine cervical cancer to demonstrate the preventing effect of the method. Finally, the embodiments of the invention which have been described do not attempt to limit the scope of the invention; rather simply illustrate some of the variations which are found comprised within the spirit of the invention and the scope of the same. As will be obvious to a person skilled in the art, the variations or amendments which do not depart from the spirit of the invention are found to lie within the scope thereof. | 12,991 |
11857676 | DETAILED DESCRIPTION The present disclosure relates to compositions and methods for treating oral pain. The compositions disclosed herein may comprise a dissolvable carrier containing at least one active ingredient. In some embodiments, the active ingredients comprise guaiacol, eugenol, glycerin, menthol oil, peppermint oil, oregano oil, and cardamom oil. The methods disclosed herein comprise treating oral pain in a subject using the compositions disclosed herein, wherein such compositions are configured as a paste that can be molded on or about a surface exhibiting oral pain in a subject's oral cavity. Such compositions are resistant to the moist environment of the subject's oral cavity and provide rapid, effective pain relief in the subject. The term “active ingredient” as used herein means an ingredient that may be administered to a subject to treat or prevent a disease, condition or symptom that would adversely affect the subject. Active ingredients may include, but are not limited to, anti-inflammatory agents, anti-microbial agents, and other agents known to those of skill in the art. Non-limiting examples of active ingredients include guaiacol, eugenol, glycerin, menthol oil, peppermint oil, oregano oil, and cardamom oil. The term “carrier” as used herein means a physiologically acceptable carrier of one or more active ingredients for oral application to a subject. As a preferred embodiment, the carrier is a dissolvable paste comprising xylitol and aloe. In some embodiments, the carrier is configured to adhere to a surface in a subject's oral cavity and dissolve overtime. Alternatively, the carrier is a gelfoam formulation, for example Surgifoam® available from Ethicon. The carrier can also be a surgical sponge, for example the absorbable gelatin sponge Gelita-Spon® available from Gelita Medical. The term “dissolvable” as used herein means substances that, upon administration to a subject, dissolve over time upon exposure to saliva and other mouth fluids. The term “effective amount” means an amount of an active ingredient that is sufficient to at least reduce or relieve the condition, symptom or disease being treated in a subject, but sufficiently low to avoid any adverse side effects in the subject. The effective amount of the active ingredient may vary with the type and/or severity of the disease, symptom or condition, the age and physical condition of the subject being treated, the duration of treatment, the nature of concurrent therapy, the specific form (i.e., salt) of the pharmaceutically active agent employed, and the particular carrier in which the active ingredient is applied. The term “oral pain” as used herein means discomfort or pain in a subject within or about a subject's oral cavity. Examples of oral pain include, but are not limited to, pain and/or discomfort in a subject resulting from dry socket, herpes labialis (cold sores), aphthous stomatitis (canker sores), oral mucositis (stomatitis), thermal burns, pressure sores (decubitus ulcers), tooth sensitivity (dentin hypersensitivity), oral cancer, and discomfort or pain caused from dentures, teething, orthodontics, and inflammation. The term “subject” as used herein means a human or other mammal. As used herein, “treatment” means that administration of the composition that prevents, alleviates, ameliorates, inhibits, or mitigates one or more symptoms of a condition in a subject. Disclosed herein are compositions for oral pain relief. In an exemplary composition, the composition comprises a dissolvable carrier comprising xylitol and aloe and active ingredients comprising guaiacol, eugenol, glycerin, menthol oil, peppermint oil, oregano oil, and cardamom oil. In some embodiments, the guaiacol, eugenol, and glycerin collectively comprise about 70% by weight of the active ingredients, menthol oil and peppermint oil each comprise about 10% by weight of the active ingredients, and oregano oil and cardamom oil each comprise about 5% by weight of the active ingredients. In some embodiments, the guaiacol, eugenol, and glycerin collectively comprise about 77.5% by weight of the active ingredients, menthol oil and peppermint oil each comprise about 16% by weight of the active ingredients, and oregano oil and cardamom oil each comprise about 6.5% by weight of the active ingredients. Other carriers and active ingredients, or other percentages by weight of the components thereof, that possess sufficient viscosity and/or adherence may be used as known to those of skill in the art. For example, the carrier may be a surgical sponge, such as the absorbable gelatin sponge Gelita-Spon® available from Gelita Medical, and the one or more active ingredients can be soaked into or absorbed by the carrier. The compositions disclosed herein may include an effective amount of one or more active ingredients, the amount of which will vary depending on the particular active ingredient. The amount of active ingredient(s) in the carrier according to the present disclosure is about from about 0.01% to about 80% by weight, preferably from about 2.5% to about 40% by weight, and more preferably from about 5% to about 30% by weight. The composition may further comprise one or more flavoring agents. Suitable flavoring agents include both natural and artificial sweeteners such as: Water-soluble sweeteners including but not limited to monosaccharides, disaccharides, and polysaccharides; water-soluble artificial sweeteners including but not limited to soluble saccharin salts; dipeptide-based sweeteners such as L-aspartic acid derived sweeteners including but not limited to aspartame, and neotame; derivatives of naturally-occurring water-soluble sweeteners including but not limited to chlorinated derivatives of sucrose, and sucralose; protein-based sweeteners including but not limited toThaumatoccous danielli(Thaumatin I and II), and combinations thereof. In general, an effective amount of flavoring agent is utilized to provide the level of sweetness or taste desired for a particular composition, and this amount will vary with the particular flavoring agent selected. The effective amount will normally be from about 0.01% to about 10% by weight of the composition. Water-soluble sweeteners are typically used in amounts of from about 0.01% to about 10% by weight, and preferably in amounts of from about 2.0% to about 5.0% by weight of the composition. The other sweeteners described above, other than water-soluble sweeteners are generally used in amounts of from about 0.01% to about 10% by weight, preferably from about 2% to about 8% by weight, and more preferably from about 3% to about 6% by weight of the composition. The compositions disclosed herein may further comprise one or more preservatives. The one or more preservatives are added in amounts from about 0.001% to about 5%, preferably from about 0.01% to about 1% by weight of the composition. Preferred preservatives include sodium benzoate, potassium sorbate, EDTA, other preservatives known to those of skill in the art, and combinations thereof. Embodiments of the compositions disclosed herein can be manufactured by mixing one or more active ingredients with equal parts filtered water and heating the resulting mixture to a rolling boil at an approximate temperature of 205° F. While maintaining an approximate temperature of 205° F., the one or more active ingredients and the filtered water are stirred for approximately 24 hours to evaporate the water and to increase the viscosity of the mixture. The temperature is then reduced and the one or more active ingredients are then mixed with glycerin and one or more thickening agents, such as Aloe Vera and xylitol, to achieve the desired texture. The composition may be packaged as a kit, comprising a composition within a delivery device. The delivery device may include an aperture through which the composition may be dispensed by the subject or other individual applying the composition to the subject's oral cavity. Non-limiting examples of delivery devices include tubes and syringes. Other containers for the composition may be used, including a resealable tub and other containers known to those of skill in the art. Alternatively, the composition may be packaged in individual units, for example a composition packaged in multiple units of approximately 7 grams or other amount suitable for a single application to a site of oral pain. Also disclosed herein are methods for treatment of oral pain in a subject by applying the compositions disclosed herein topically to a site in the subject's oral cavity. The compositions disclosed herein may be used to prevent, reduce, suppress or eliminate oral pain. Prevention of oral pain, for example, occurs when the compositions disclosed herein are used as local anesthetics, typically prior to a dental procedure. Suppression or elimination of oral pain, for example, occurs when the compositions disclosed herein are applied to a surface in a subject's oral cavity, such as a canker sore or ulcer such that the application of the composition reduces or eliminates the oral pain. When applied to a surface, the compositions disclosed herein can be molded on and about the location of the oral pain. The compositions disclosed herein, for example, can be used to treat a dry socket by applying the composition within, on or about the dry socket. Similarly, the compositions disclosed herein may be applied on or about a canker sore or other source of oral pain in a subject. The amount of the active ingredient in the composition may be adjusted to deliver a predetermined dose of the active ingredient over a predetermined period of time, which may typically vary from 4 to 24 hours. For example, a composition may be administered every 12 hours to deliver an effective amount of the active ingredient(s) over a period of 12 hours to a subject in need of such administration. An exemplary adult dose of each active ingredient of the disclosed composition may contain from about 1 to 100 mg, and preferably from about 5 to 20 mg, of each active ingredient (e.g., guaiacol). Administration may be on an as-needed or as-desired basis, for example, once-monthly, once-weekly, or daily, including multiple times daily, for example, at least once daily, from one to about six times daily, from about two to about four times daily, or about three times daily. The amount of respiratory composition administered may be dependent on a variety of factors, including the general quality of health of the subject, and the subject's age, gender, weight, or severity of symptoms. Treatment of oral pain using the compositions and methods disclosed herein is not limited to human subjects. Domestic animals, such as dogs, cats, and horses often suffer from oral pain and may be treated with the compositions disclosed herein. Compared to conventional compositions for oral pain relief, the compositions disclosed herein have several advantages. The compositions disclosed herein offer significant and fast-acting pain relief and also are sufficiently tacky and malleable to be easily applied and adhered to the desired location within a subject's oral cavity. The compositions disclosed herein are also resistant to the moist environment of a subject's oral cavity, allowing prolonged contact with the site of oral pain. Further, the composition may serve as a protective barrier at the site of oral pain to prevent the site from further irritations and insults that would otherwise result in additional oral pain or prolong the time required for such oral pain to subside. The following examples, applications, descriptions and content are exemplary and explanatory, and are non-limiting and non-restrictive in any way. EXAMPLES Pain Relief Study The applicants performed a trial regarding the efficacy of an embodiment of the composition disclosed herein, described in greater detail in “Exemplary Composition” below. In the trial, 180 subjects who presented with oral pain were examined. They collectively had an initial average self-assessed oral pain of 6.95 out of a maximum of 10 on a visual analog scale. The subjects were administered the composition and oral pain was reassessed by the subjects at 0, 3, 6 and 9 minutes after administrations. The level of oral pain was reduced to a collective average of 2.43, 1.06, and 0.62 out of 10 within 3, 6, and 9 minutes, respectively, as shown inFIG.1. Exemplary Composition The ingredients in Table 1 were combined to provide a dissolvable composition, wherein the active ingredients comprised guaiacol, eugenol, glycerin, menthol oil, peppermint oil, oregano oil, and cardamom oil. TABLE 1Ingredient%/Weight of TotalXylitol3%Active Ingredients77%Aloe (powder)20%TOTAL100% In an alternative composition, the ingredients in Table 2 were combined to provide a dissolvable composition, comprising aloe powder, xylitol, filtered water, guaiacol, eugenol, menthol crystals, peppermint oil, oregano oil, cardamom oil, and vegetable glycerin. TABLE 2Ingredient%/Weight of TotalAloe Powder55%Xylitol21.3%Eugenol2.55%Filtered Water8.7%Menthol Crystals1.45%Peppermint Oil0.95%Vegetable Glycerin7.52%Guaiacol1.55%Oregano Oil0.53%Cardamom Oil0.45%TOTAL100% The compositions, methodologies, and the various embodiments thereof described herein are exemplary. Various other embodiments of the compositions and methodologies described herein are possible. | 13,431 |
11857677 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Various embodiments will now be described more fully hereinafter with reference to the accompanying examples and experiments, in which illustrative embodiments of the invention are shown. The novel concepts may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. As used herein “melphalan” refers to the pharmaceutically acceptable salts, solvates, hydrates and anhydrous forms thereof. The examples herein all refer to melphalan hydrochloride, but the invention is not limited in this regards. As used herein “ready-to-use” when used in connection with a melphalan formulation refers to a formulation that includes melphalan, optionally including one or more pharmaceutically acceptable excipients, in the form of a solution, suspension or emulsion, wherein the formulation does not require any reconstitution or dilution with parenterally acceptable diluent and can be directly administered to the patient. As used herein, the phrases “ready-to-dilute” and “concentrate ready for dilution” refer to any liquid formulation of melphalan, optionally including one or more pharmaceutically acceptable excipients, in the form of a solution, suspension or emulsion, in which the liquid formulation can be diluted with a suitable diluent for parenteral administration before administering to the patient. As used herein, the term “storage-stable” refers to any liquid melphalan-containing formulation having sufficient physical and chemical stability to allow storage at a convenient temperature and relative humidity (RH), such as from about 0° C. to about 60° C. and about 20% to 75% RH, for a reasonable period of time. “Physical stability” refers to maintenance of colour or colourless state, dissolved oxygen level, head space oxygen level and particulate matter and “chemical stability” relates to formation of drug-related impurities in terms of total impurities, single maximum individual impurity, or maximum individual unknown impurity. For pharmaceutical products, stability is required for commercially relevant times after manufacturing, such as for about 6, 12, 18, 24, or 36 months, during which time a product is kept in its original packaging under specified storage conditions. In one embodiment a liquid melphalan formulation may be considered storage-stable if after a predetermined period of time, such as but not limited to one week, at least one month, at least three months, at least six months, at least one year, or at least 2 years, no precipitation is observed, the composition remains clear and has sufficient chemical stability to permit safe administration to a patient. The term “parenteral” or “injectable” refers to routes selected from subcutaneous (SC), intravenous (IV), intramuscular (IM), intradermal (ID), intraperitoneal (IP) and the like. The ready-to-use or ready-to-dilute formulations disclosed herein may be formulated as aqueous or non-aqueous solutions, suspensions, or emulsions. The ready-to-use parenteral formulations disclosed herein include melphalan, preferably melphalan hydrochloride, and one or more pharmaceutically acceptable solvents, cosolvents and/or solubilizing agents. In other embodiments, ready-to-use liquid parenteral formulations of melphalan include melphalan, one or more pharmaceutically acceptable solvents, co-solvents, and/or solubilizing agents, and one or more pharmaceutically acceptable excipients such as but are not limited to surfactants, wetting agents, emulsifiers, preservatives, chelating agents, antioxidants, polymers, anti-foaming agents, buffering agents, pH adjusting agents, channel forming agents, osmotic adjustment agents and the like and mixtures thereof. Suitable pharmaceutically acceptable solvents include but are not limited to water, water immiscible solvents, water miscible solvents, oily components, hydrophilic solvents, and hydrophobic solvents. Specific examples of pharmaceutically acceptable solvents include but are not limited to dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), N-methylpyrolidone, dimethylisosorbide, ethanol, propylene glycol, glycerine, polyethylene alcohol, propylene glycol esters, polyethylene glycols and the like. Preferred solvents are dimethylacetamide (DMA), ethanol, polyethylene glycols (PEG), glycerine, benzyl alcohol and propylene glycol. Suitable pharmaceutically acceptable co-solvents include but are not limited to ethanol, polyethylene glycol, glycerine, polyethylene glycol and glycofurol. Suitable pharmaceutically acceptable solubilizing agents include but are not limited to cyclodextrin derivatives, alpha-cyclodextrin, beta-cyclodextrin, for example, hydroxypropyl beta cyclodextrin (HPBCD), sulfobutylether-betacyclodextrin, randomly methylated beta-cyclodextrin and the like, gamma-cyclodextrin, modified alpha-cyclodextrin, modified beta cyclodextrin, modified gamma cyclodextrin or any combination thereof. Examples of pharmaceutically acceptable surfactants include but are not limited to amphoteric, non-ionic, cationic and anionic molecules. For example, suitable surfactants include but are not limited to polysorbates, sodium lauryl sulfate, lauryl dimethyl amine oxide, docusate sodium, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, polyoxyl lauryl ether, Brij® surfactants (polyoxyethylene vegetable-based fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols), bile salts (such as sodium deoxycholate and sodium cholate), polyoxyl castor oil, nonylphenol ethoxylate, lecithin, polyoxyethylene surfactants, polyethylene glycol esters, glycol esters of fatty acids, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, polyoxyethylene alkyl and alicyclic amines, polyoxyethylene, sorbitan monolaurate, sorbitan stearate, Cremophor® (polyethoxylated castor oil), Solutol® (ethylene oxide/12-hydroxy stearic acid), tyloxapol, etc. and combinations thereof. Examples of pharmaceutically acceptable wetting agents include but are not limited to sulphate, sulphonate, carboxylates, ethoxylates, alkylphenol ethoxlates, esters of glycerol, esters of sorbitol, amine oxides, sulphoxides, phosphine oxides and combinations thereof. Examples of pharmaceutically acceptable emulsifiers include but are not limited to soybean oil, safflower oil, sesame oil, castor oil, lecithin, PEG-PE, Pluronic, sorbitol, xylitol, tocopherol, deferoxamine mesylate, benzyl alcohol and combinations thereof. Examples of pharmaceutically acceptable preservatives include but are not limited to chlorobutanol, benzalkonium chloride, methyl paraben, propyl paraben, benzoic acid, sodium benzoate, sorbic acid, benzethonium chloride, cetyl pyridinium chloride, benzyl bromide, benzyl alcohol, phenylmercury nitrate, phenylmercury acetate, thiomersal, merthiolate, chlorhexidine, phenylethyl alcohol, quaternary ammonium chloride, sodium benzoate, sodium propionate, etc. and combinations thereof. Pharmaceutically acceptable chelating agents include but are not limited to citric acid and derivatives thereof, for example, anhydrous citric acid and the like, ethylenediaminetetraacetic acid (EDTA), disodium EDTA or derivatives thereof, niacinamide or derivatives thereof, sodium deoxycholate or derivatives thereof, pentetic acid or derivatives thereof, etc. and combination thereof. Examples of pharmaceutically acceptable anti-oxidants include but are not limited to butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), monothioglycerol, ascorbic acid, sodium ascorbate, erythorbic acid, potassium metabisulfite, sodium metabisulfite, propionic acid, sodium formaldehyde sulphoxylate, reduced glutathione, thiourea, cysteine, n-aceticysteine, methionine, sodium sulfite, alkyl gallate, vitamin E or other tocopherol analogs such as tocopherol acetate and TPGS, etc. and combinations thereof. Examples of pharmaceutically acceptable polymers include but are not limited to carbomer, polycarbophil, gellan gum, cellulose derivatives, acrylates, etc. and combinations thereof. Examples of pharmaceutically acceptable anti-foaming agents include but are not limited to alkyl polyacrylates, castor oil, fatty acids, fatty acid esters, fatty acid sulphates, fatty alcohol, fatty alcohol esters, olive oil, paraffin oil, silicone oil, paraffin wax and combinations thereof. Examples of pharmaceutically acceptable buffering agents include but are not limited to hydrochloric acid, citrate buffer, acetate buffer, Sorensens's phosphate buffer, sodium bicarbonate, sodium carbonate, sodium hydroxide, and combinations thereof. Examples of pharmaceutically acceptable pH adjusting agents include but are not limited to sodium hydroxide, hydrochloric acid, boric acid, citric acid, acetic acid, phosphoric acid, succinic acid, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicates, malic acid, potassium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, fumaric acid, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, triethanolamine, etc. and combinations thereof. Examples of pharmaceutically acceptable osmotic adjustment agents include but are not limited to sodium chloride, potassium chloride, calcium chloride and magnesium chloride, glucose, glycerol, etc. and combinations thereof. Generally, a process for preparing formulations of melphalan and the resulting formulations are provided. The process generally involves first adding melphalan to a vessel containing a mixture of one or more solvents, to form a melphalan solution. In a preferred embodiment, the weight % of melphalan I the solution is between about 0.2% to about 0.8%, or more preferably about 0.4% to about 0.6%. In at least one embodiment, a plurality of solvents are used, such as, propylene glycol (PG) polyethylene glycol (PEG) and ethanol, or water and ethanol, or any combination thereof. In some embodiments, a chelating agent, such as EDTA, is mixed with the one or more solvents, at a weight % of from about 0.1% to about 2%, or more preferably from about 1% to about 1.5%, and the melphalan is added to the chelating agent solution. In some embodiments, povidone is added to the solution with the melphalan, at a weight % from about 0.2% to about 2%, or more preferably from about 0.5% to about 1.5%. Optionally, a sufficient amount of Polaxomer 188 or other surfactants may be added to clarify the solution. The pH of the solution may be adjusted, using an appropriate buffering agent, from about 3.0 to about 6.0, or more preferably about 3.5 to about 5.5. In certain embodiments, the pH of the solution may be adjusted from about 7.0 to about 9.5, or more preferably from about 7.5 to about 9.0. Suitable pharmaceutically acceptable solvents may then be added to make up the volume of the solution. The resulting solution may be filtered and used to fill vials for parenteral administration. The resulting formulation has a purity of at least 90 at 25° C./60% RH for a period of 43 days, or preferably at least 99 after 50 days, or more preferably at least 99 after 5 months of storage at 2-8° C. EXAMPLES The following examples are for the illustration of the invention only and are not intended in any way to limit the scope of the present invention. Example 1 TABLE 1IngredientsQty/vialMelphalan50mgPoloxamer 1885mgPolyethylene glycol (PEG)4mlEthanol6ml0.1N HClQS Melphalan was added to a manufacturing vessel containing a mixture of solvents polyethylene glycol (PEG) and ethanol. Polaxomer 188 was added to obtain a clear solution. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using a mixture of PEG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The melphalan formulation was tested for stability at 25° C./60% RH for a period of 43 days. Stability data is summarized in Table 1A. TABLE 1AStability at Day 43Day 43Purity90.41Maximum Individual Impurity3.3Total Impurities9.59 Example 2 TABLE 2IngredientsQty/vialMelphalan50EDTA10mgWater for injection (WFI)1mlEthanol9ml0.1N HClQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of ethanol and water to obtain a clear solution. Melphalan was added to the above solution mixture. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using water and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 25° C./60% RH for a period of 43 days. Stability data is summarized in Table 2A. TABLE 2AStability at Day 43Day 43Purity91.81Maximum Individual Impurity3.01Total Impurities8.2 Example 3 TABLE 3IngredientsQty/vialMelphalan50mgEDTA5mgPEG5mlWater0.2mlEthanol4.8ml0.1N HClQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PEG and ethanol to obtain a clear solution. Melphalan was added to the above solution mixture. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using PEG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 50 days. Stability data is summarized in Table 3A. TABLE 3AStability at Day 50Day 50Purity99.48Maximum Individual Impurity0.12Total Impurities0.52 Example 4 TABLE 4IngredientsQty/vialMelphalan50mgEDTA5mgPEG2mlWater0.4mlEthanol7.6ml0.1N HClQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PEG and ethanol to obtain a clear solution. Melphalan was added to the above solution mixture. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using mixture of PEG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 5 months. Stability data is summarized in Table 4A. TABLE 4AStability at 5 Months5 MonthsPurity99.05Maximum Individual impurity0.25Total Impurities0.95 Example 5 TABLE 5IngredientsQty/vialMelphalan50mgPovidone5mgEDTA5mgPEG5mlWater0.2mlEthanol4.8ml0.1N HClQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PEG and ethanol. Melphalan and povidone were added to the above solution mixture. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using mixture of PEG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 25° C./60% RH for a period of 22 days. Stability data is summarized in Table 5A. TABLE 5AStability at Day 22Day 22Purity92.04Maximum Individual Impurity1.76Total Impurities7.96 Example 6 TABLE 6IngredientsQty/vialMelphalan50mgPovidone5mgEDTA5mgPropylene glycol (PG)5mlWater0.2mlEthanol4.8ml0.1N HClQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PG and ethanol. Melphalan and povidone were added to the above mixture. pH was adjusted to 3.5-5.5 using 0.1N HCl. Volume was made up using PG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 25° C./60% RH for a period of 22 days. Stability data is summarized in Table 6A. TABLE 6AStability at Day 22Day 22Purity93.62Maximum Individual Impurity1.66Total Impurities6.38 Example 7 TABLE 7IngredientsQty/vialMelphalan50mgPovidone5mgEDTA5mgPG5mlWater0.2mlEthanol4.8ml0.1N NaOHQS EDTA and povidone was dissolved in water and added to a manufacturing vessel containing a mixture of PG and ethanol. Melphalan and povidone were added to the above solution mixture. pH was adjusted to 7.5-9.0 using 0.1 N NaOH. Volume was made up using PG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 25° C./60% RH for a period of 22 days. Stability data is summarized in Table 7A. Table 7A TABLE 7AStability at Day 22Day 22Purity92.79Maximum Individual Impurity1.82Total Impurities7.21 Example 8 TABLE 8IngredientsQty/vialMelphalan50mgPovidone5mgEDTA5mgPEG5mlWater0.2mlEthanol4.8ml0.1N NaOHQS EDTA and povidone were dissolved in water and added to a manufacturing vessel containing a mixture of PEG and ethanol. Melphalan was added to the above solution mixture. pH was adjusted between pH 7.5-9.0 using 0.1N NaOH. Volume was made up using PEG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 25° C./60% RH for a period of 22 days. Stability data is summarized in Table 8A. TABLE 8AStability at Day 22Day 22Purity88.36Maximum Individual impurity3.00Total Impurities11.64 Example 9 TABLE 9IngredientsQty/vialMelphalan50mgEDTA5mgPG1mlWater0.2mlEthanol8.8ml0.1N NaOHQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PG and ethanol. Melphalan was added to the above solution mixture. pH was adjusted between pH 3.5-5.5 using 0.1N HCl. Volume was made up using PG and ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 22 days. Stability data is summarized in Table 9A. TABLE 9AStability at Day 22Day 22Purity92.79Maximum Individual impurity1.82Total Impurities7.2 Example 10 TABLE 10IngredientsQty/vialMelphalan50mgEDTA5mgPG1mlWater0.2mlBenzyl alcohol8.8ml0.1N NaOH/0.1N HCLQS EDTA was dissolved in water and added to a manufacturing vessel containing a mixture of PG and benzyl alcohol. Melphalan was added to the above solution mixture. pH was adjusted between pH 3.5-5.5 using 0.1N HCl/0.1N NaOH. Volume was made up using PG and benzyl alcohol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 22 days. Stability data is summarized in Table 10A. TABLE 10AStability at Day 22Day 22Purity99.38Maximum Individual impurity0.38Total Impurities0.62 Example 11 TABLE 11IngredientsQty/vialMelphalan50mgEDTA5mgPG2mlWater0.4mlEthanol7.6ml0.1N NaOH/0.1N HCLQS EDTA was dissolved in water and added to a manufacturing vessel containing PG. 80% of Ethanol was added to above solution. Melphalan was added to the above solution mixture. pH was adjusted between pH 3.5-5.5 using 0.1N NaOH/0.1N HCL. Volume was made up using remaining quantity of Ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 6 Months. Stability data is summarized in Table 11A. TABLE 11AStability at 6 Months14 monthsPurity99.03Maximum Individual impurity0.21Total Impurities0.97 Example 12 TABLE 12IngredientsQty/vialMelphalan50mgEDTA5mgPEG2mlWater0.4mlEthanol7.6ml0.1N NaOH/0.1N HCLQS EDTA was dissolved in water and added to a manufacturing vessel containing PEG. 80% of ethanol was added to above solution. Melphalan was added to the above solution mixture. pH was adjusted between pH 3.5-5.5 using 0.1N NaOH/0.1N HCL. Volume was made up using remaining quantity of ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 5 Months. Stability data is summarized in Table 12A. TABLE 12AStability at 5 Month5 MonthsPurity99.05Maximum Individual impurity0.25Total Impurities0.95 Example 13 IngredientsQty/vialMelphalan50mgPG5mlEthanol5ml0.1N NaOH/0.1N HCLQS NLT 80% of batch volume ethanol was transferred into a manufacturing vessel. Propylene glycol was added to a vessel containing ethanol. Melphalan was added to the above mixture. Check the pH of the sample and if required adjust the pH to 3.5-5.5 using 0.1N NaOH/0.1N HCL. Final batch volume was made up using ethanol. The obtained solution was filtered and filled in vials followed by capping and sealing. The formulation was tested for stability at 2-8° C. for a period of 1 Month. Stability data is summarized13A. TABLE 13AStability at 1 Month1 MonthPurity99.48Maximum Individual impurity0.1Total Impurities0.52 Although the formulations, compositions, schemes and methods of the present disclosure have been described with reference to exemplary embodiments thereof, the present disclosure is not limited thereby. Indeed, the exemplary embodiments are implementations of the disclosed methods are provided for illustrative and non-limitative purposes. Changes, modifications, enhancements and/or refinements to the disclosed methods may be made without departing from the spirit or scope of the present disclosure. Accordingly, such changes, modifications, enhancements and/or refinements are encompassed within the scope of the present invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety. | 22,209 |
11857678 | DETAILED DESCRIPTION OF THE INVENTION The detailed description of exemplary embodiments herein makes reference to the accompanying drawings which show exemplary embodiments by way of illustration and their best mode. While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the inventions, it should be understood that other embodiments may be realized and that logical, chemical, and mechanical changes may be made without departing from the spirit and scope of the inventions. Thus, the detailed description herein is presented for purposes of illustration only and not of limitation. For example, the steps herein recited in any of the method of process descriptions may be executed in any order and are not necessarily limited to the order presented. Furthermore, any reference to singular includes plural embodiments, and any reference to more than one component or step may include a singular embodiment or step. Also, any reference to attached, fixed, connected or the like may include permanent, removable, temporary, partial, full and/or any other possible attachment option. Additionally, any reference to without contact (or similar phrases) may also include reduced contact or minimal contact. Applicant unexpectedly found that the presence of particular surfactants in a cannabidiol composition form an emulsion with an average globule size capable of passing through the gastrointestinal tract when dispersed in an aqueous medium. In one embodiment, the present invention is directed to a self-emulsifying cannabidiol composition comprising from about 1 to about 40% w/w cannabidiol and from about 40% to about 99% w/w of one or more surfactants selected from polyethylene glycol (“PEG”) 40 hydrogenated castor oil, caprylocaproyl polyoxyl-8 glycerides, lauoryl polyoxylglycerides, oleoyl polyoxyl-6 glycerides, linoleoyl polyoxyl-6 glycerides, lauroyl polyoxyl-6 glycerides, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, a polysorbate and sorbitan monooleate. In a preferred embodiment, the present invention is further directed to a self-emulsifying cannabidiol composition comprising:from about 5% to about 35% w/w cannabidiol;from about 2% to about 60% w/w PEG 40 hydrogenated castor oil;from about 2% to about 50% w/w of a surfactant selected from caprylocaproyl polyoxyl-8 glycerides, linoleoyl polyoxyl-6 glyceride or a combination thereof; andfrom about 0.1% to about 2% w/w alpha tocopherol. Cannabidiol may be present in compositions of the present invention at a concentration from about 0.1% to about 50% w/w, preferably from about 1% to about 40% w/w, more preferably from about 5% to about 35% w/w, even more preferably from about 10% to about 30% w/w or about 10% to about 20% w/w. Surfactants suitable for use in the present invention include, but are not limited to, PEG 40 hydrogenated castor oil, caprylocaproyl polyoxyl-8 glycerides, lauoryl polyoxylglycerides, oleoyl polyoxyl-6 glycerides, linoleoyl polyoxyl-6 glycerides, lauroyl polyoxyl-6 glycerides, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, a polysorbate and sorbitan monooleate. In a preferred embodiment, surfactants may be selected from PEG 40 hydrogenated castor oil, caprylocaproyl polyoxyl-8 glycerides, linoleoyl polyoxyl-6 glycerides, polyglyceryl-3 dioleate, polysorbate 80 or a combination thereof. The one or more surfactants may be present in compositions of the present invention at a concentration from about 1% to about 99% w/w, preferably from about 40% to about 99% or from about 2% to about 50% w/w, even more preferably from about 40% to about 80% w/w or from about 20% to about 40% w/w and yet even more preferably from about 44% to about 78% w/w. Polyethylene glycol (“PEG”) 40 hydrogenated castor oil may be present in compositions of the present invention at a concentration from about 2% to about 60% w/w, preferably from about 10% to about 50% w/w and more preferably from about 10% to about 40% w/w. Polyglyceryl-3 dioleate may be present in compositions of the present invention at a concentration from about 1% to about 15% w/w, preferably from about 2% to about 12% w/w. Caprylocaproyl polyoxyl-8 glycerides may be present in compositions of the present invention at a concentration from about 1% to about 30% w/w, more preferably from about 3% to about 26% w/w. Linoleoyl polyoxyl-6 glycerides may be present in compositions of the present invention at a concentration from about 1% to about 30% w/w, more preferably from about 4% to about 23% w/w. Polysorbate 80 may be present in compositions of the present invention at a concentration from about 1% to about 10% w/w, more preferably from about 2% to about 6% w/w. Oils suitable for use in compositions of the present invention include, but are not limited to, glyceryl monolinoleate, glyceryl monooleate, propylene glycol dicaprylocaprate, glycerol monostearate 40-55, a medium chain triglyceride or a combination thereof. In a preferred embodiment, the oil is a medium chain triglyceride, preferably a C8/C10 medium chain triglyceride. The one or more oils may be present in compositions of the present invention at a concentration from about 1% to about 50% w/w, preferably from about 5% to about 30% w/w, more preferably from about 5% to about 25% w/w. Medium chain triglycerides may be present in compositions of the present invention at a concentration from about 1% to about 30% w/w, preferably from about 9% to about 24% w/w. Cosolvents suitable for use in the present invention include, but are not limited to, propylene glycol, polyethylene glycol, ethanol or a combination thereof. The one or more cosolvents may be present in compositions of the present invention at a concentration from about 1% to about 50% w/w, preferably from about 5% to about 30% w/w, even more preferably from about 12% to about 21% w/w. Ethanol may be present in compositions of the present invention at a concentration from about 1% to about 20% w/w, preferably from about 5% to about 15% w/w, even more preferably from about 10% to about 15% w/w and most preferably from about 12% to about 14% w/w. Propylene glycol may be present in compositions of the present invention at a concentration from about 1% to about 20% w/w, preferably from about 5% to about 15% w/w, even more preferably from about 5% to about 10% w/w and most preferably from about 8% to about 10% w/w. Antioxidants suitable for use in the present invention include, but are not limited to, alpha tocopherol, butylated hydroxy anisole, butylated hydroxy toluene, ascorbyl palmitate, ascorbic acid, sodium ascorbate, sodium metabisulfite, EDTA, citric acid, sodium bisulfite, sodium thiosulfate, thioglycerol, propyl gallate or a combination thereof. In a preferred embodiment, the antioxidant is alpha tocopherol, ascorbyl palmitate or a combination thereof. The one or more antioxidants may be present in compositions of the present invention at a concentration from about 0.01% to about 2% w/w, preferably from about 0.05% to about 1% w/w and even more preferably from about 0.1% to about 0.5% w/w. Alpha tocopherol may be present in compositions of the present invention at a concentration from about 0.01% to about 2% w/w, preferably from about 0.01% to about 1% w/w, even more preferably from about 0.05% to about 0.5% w/w and yet more preferably from about 0.05% to about 0.4% w/w. Ascorbyl palmitate may be present in compositions of the present invention at a concentration from about 0.01% to about 2% w/w, preferably from about 0.01% to about 1% w/w and even more preferably from about 0.05% to about 0.2% w/w. In another embodiment, the composition of the present invention does not contain sesame oil, castor oil, olive oil or water. In a preferred embodiment, the compositions of the present invention form an emulsion having an average globule size from about 20 to about 5,000 nanometers when dispersed in an aqueous medium, preferably from about 30 to about 600 nanometers, even more preferably from about 100 to about 1,000 nanometers and yet more preferably from about 100 to about 300 nanometers. In a more preferred embodiment, the aqueous medium is gastric fluid. In another preferred embodiment, the compositions of the present invention emulsifies in less than 30 minutes upon contact with an aqueous medium including gastric fluid. In another preferred embodiment, the compositions of the present invention are contained in a soft or a hard gelatin capsule. In a most preferred embodiment, the present invention is directed to a self-emulsifying cannabidiol composition comprising:about 20.5% w/w cannabidiol;about 12.0% w/w ethanol;about 9.0% w/w propylene glycol;about 17.0/w/w polyethylene glycol 40 hydrogenated castor oil;about 26.0% w/w caprylocaproyl polyoxyl-8 glycerides;about 4.0% w/w linoleoyl polyoxyl-6 glycerides; andabout 0.4% w/w alpha tocopherol, wherein the composition forms an emulsion having an average globule size of about 201 nanometers when dispersed in an aqueous medium. In another embodiment, the present invention is directed to a method of treating a disease selected from Prader-Willi syndrome, obesity, graft versus host disease, gelastic seizures/hypothalamic hamartoma, neonatal seizures, dystonia, central pain syndromes, phantom limb pain, multiple sclerosis, traumatic brain injury, radiation therapy, acute graft versus host disease, chronic graft versus host disease, T-cell autoimmune disorders, colitis, Dravet Syndrome, Lennox Gastaut Syndrome, mycolonic seizures, juvenile mycolonic epilepsy, refractory epilepsy, childhood absence epilepsy, schizophrenia, juvenile spasms, West syndrome, infantile spasms, refractory infantile spasms, tuberous sclerosis complex, brain tumors, neuropathic pain,cannabisuse disorder, post-traumatic stress disorder, anxiety, early psychosis, Alzheimer's Disease, autism, acne, Parkinson's disease, social anxiety disorder, depression, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, ischemic injury of heart, ischemic injury of brain, chronic pain syndrome, and rheumatoid arthritis comprising administering a composition of the present invention to a subject in need thereof. In another embodiment, the present invention is directed to a method of treating withdrawal symptoms comprising administering a composition of the present invention to a subject in need thereof, wherein the withdrawal symptoms are caused by the subject reducing or quitting use of an opioid, cocaine, heroin, an amphetamine or nicotine. As used herein, all numerical values relating to amounts, weights, and the like, that are defined as “about” each particular value is plus or minus 10%. For example, the phrase “about 10% w/w” is to be understood as “9% w/w to 11% w/w.” Therefore, amounts within 10% of the claimed value are encompassed by the scope of the claims. As used herein “% w/w” and “percent w/w” refer to the percent weight of the total formulation. The disclosed embodiments are simply exemplary embodiments of the inventive concepts disclosed herein and should not be considered as limiting, unless the claims expressly state otherwise. The following examples are intended to illustrate the present invention and to teach one of ordinary skill in the art how to use the formulations of the invention. They are not intended to be limiting in any way. EXAMPLES Example 1—Preparation of Compositions of the Invention TABLE 1Compositions of the Invention% w/w12345Cannabidiol20.530.520.410.210.0Ethanol12.012.012.012.012.0Propylene Glycol9.08.010.010.0—PEG 40 hydrogenated castor oil17.113.017.213.040.0Polyglyceryl-3 dioleate4.75.22.32.312.0Caprylocaproyl polyoxyl-8 glycerides26.019.525.526.03.0Linoleoyl polyoxyl-6 glycerides4.36.5——23.0C8/C10 medium chain triglycerides——9.523.4—Alpha-tocopherol (Vitamin E)0.40.30.30.3—Polysorbate 806.05.02.82.8— Cremophor® RH40 was used as the source for polyethylene glycol 40 hydrogenated castor oil and is a registered trademark of and available from BASF SE corporation. Plurol® Oleique CC 497 was used as the source of polyglyceryl-3 dioleate and is a registered trademark of and available from Gattefosse SAS. Labrasol® was used as the source of caprylocaproyl polyoxyl-8 glycerides and is a registered trademark of and available from Gattefosse SAS. Labrafil® M 2125 CS was used as the source of linoleoyl polyoxyl-6 glycerides and is a registered trademark of and available from Gattefosse SAS. Miglyol® 812 was used as the source of C8/C10 medium chain triglycerides and is a registered trademark of and available from Cremer Oleo GMBH & Co. Method Alpha-tocopherol and cannabidiol were dissolved in ethanol to create a mixture while mixing. Propylene glycol was then added to this mixture followed by rest of the excipients and mixed well. Polyethylene glycol (“PEG”) 40 hydrogenated castor oil was melted before being added to the mixture. Emulsification time and globule size was measured. Emulsification time is the time it takes 1 gram of the composition to completely disperse in about 200 milliliters of 0.1 N HCl solution while stirring. Globule size is measured using Nicomp ZLS Z3000. TABLE 2Emulsification time and globule sizeEmulsification TimeGlobule Size(min)(nm)Composition 1<1201.1 ± 112.65Composition 2<1563.7 ± 525.95Composition 3<1291.9 ± 196.36Composition 4<1216.1 ± 66.32Composition 51234.5 ± 9.8 Results Compositions 1-4 emulsified in less than 1 minute. Composition 5 took 12 minutes to emulsify. Compositions 1, 3 and 4 created an emulsion having an average globule size of from 201.1 to 291.9 nanometers upon emulsification. Composition 2 created an emulsion having an average globule size of 563.7 nanometers upon emulsification. Composition 5 created an emulsion having an average globule size of 34.5 nanometers. Example 2—Stability of Composition 6 TABLE 3Composition 6% w/w6Cannabidiol18.18Ethanol14.0PEG 40 hydrogenated castor oil34.67Polyglyceryl-3 dioleate9.0Caprylocaproyl polyoxyl-8 glycerides3.0Linoleoyl polyoxyl-6 glycerides21.0Alpha-tocopherol (Vitamin E)0.05Ascorbyl palmitate0.1 Method Composition 6 from Table 3, above, was prepared as in Example 1, above, and subjected to 40° C.±2° C. and 75±5% relative humidity (“RH”) for 2 months and 25° C.±2° C. and 60±5% RH for 3 months. Results can be seen in Tables 4 and 5, below. TABLE 4Stability of Composition 6 at 40° C. ± 2° C. and 75 ± 5% RHRRTT = 01 Month2 MonthPhysical appearanceClear,Clear,Clear,yellowyellowyellowcoloredcoloredcoloredAssay (% of Initial100.0096.3794.24Conc.)Delta 9-tetra-1.7610.01%0.01%0.01%hydrocannabinolTrans-(1R,6R)-3′-1.8650.04%0.03%0.03%methyl-cannabidiolUnknown Impurity0.319ND0.01%0.02%0.373NDND0.01%0.390NDND0.01%0.436NDND0.02%0.459ND0.02%0.05%0.479ND0.02%0.06%0.5000.01%0.05%0.16%0.592ND0.01%ND0.681NDND0.01%0.7710.05%0.05%0.05%0.789ND0.02%0.06%0.8190.02%0.02%0.01%0.825NDND0.01%0.848NDND0.01%2.075ND0.01%NDTotal Impurities0.08%0.21%0.48%RRT denotes relative retention time TABLE 5Stability of Composition 6 at 25° C. ± 2° C. and 60 ± 5% RHRRTT = 01 Month2 Month3 MonthPhysical appearanceClear, PaleClear, PaleClear, PaleClear, PaleyellowyellowyellowyellowAssay (% of Initial Conc.)100.0098.5896.9096.31Delta 9-tetra-hydrocannabinol1.7610.01%0.01%0.01%0.01%Trans-(1R,6R)-3′-methyl-1.8650.04%0.03%0.03%0.03%cannabidiolCis-cannabidiol1.4530.01%0.01%0.01%0.01%Unknown Impurity0.313NDND0.01%0.01%0.374NDNDND0.02%0.396ND0.01%NDND0.452NDNDND0.01%0.481NDNDND0.01%0.5000.01%0.01%0.01%0.01%0.7710.05%0.05%0.05%0.06%0.8190.02%0.02%0.02%0.02%2.075ND0.01%NDNDTotal Impurities0.08%0.10%0.09%0.14%ND denotes not detected As seen in Tables 4 and 5, Composition 6 had only 0.48% total impurities after 2 months at 40° C. and 0.14% total impurities after 3 months at 25° C. Thus, compositions of the present invention are stable. Example 3. Pharmacokinetic Study in Dogs Method In-vivo bioavailability and pharmacokinetics of Composition 5 was evaluated in Beagle dogs. Specifically, five male Beagle dogs weighing from about 5 to about 11 kilograms were fasted overnight before dosing. Each Beagle dog was then administered 200 milligrams of cannabidiol in the form of Composition 5. Blood samples were collected at 0, 15 and 30 minutes and 1, 2, 3, 4, 8, 12, 24, 48, 72 and 96 hours after dosing. The following pharmacokinetic parameters were calculated: peak concentration in plasma (“Cmax”), time to peak concentration (“Tmax”) and area under the concentration-time curve (“AUC”). Results of this study can be seen in Table 6, below and inFIGS.1and2. TABLE 6Pharmacokinetic parametersCmax (ng/mL)672.84 ± 400.68Tmax (h)3AUC (ng/mL*h)0-1 h175.0 ± 76.80-4 h1881 ± 952.70-12 h3448.9 ± 1936.30-24 h3971.0 ± 2250.90-48 h4446.5 ± 2488.80-96 h4910.8 ± 2750.1 Results As seen in Table 6, Composition 5 provided a Cmaxof 672.84 nanograms per milliliter (“ng/mL”) and a Tmaxof 3 hours. Further, Composition 6 provided an AUC of 175.0 at 1 hour, 1,881 at 4 hours, 3448.9 at 12 hours, 3971.0 at 24 hours, 4446.5 at 48 hours and 4910.8 at 96 hours. Benefits, other advantages, and solutions to problems have been described herein with regard to specific embodiments. Furthermore, the connecting lines shown in various figures contained herein are intended to represent exemplary functional relationships and/or physical couplings between the various elements. It should be noted that many alternative or additional functional relationships or physical connections may be present in a practical system. However, the benefits, advantages, solutions to problems, and any elements that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as critical, required, or essential features or elements of the inventions. The scope of the inventions is accordingly to be limited by nothing other than the appended claims, in which reference to an element in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.” Moreover, where a phrase similar to “at least one of A, B, or C” is used in the claims, it is intended that the phrase be interpreted to mean that A alone may be present in an embodiment, B alone may be present in an embodiment, C alone may be present in a single embodiment; for example, A and B, A and C, B and C, or A and B and C. Different cross-hatching is used throughout the figures to denote different parts but not necessarily to denote the same or different materials. Systems, methods and apparatus are provided herein. In the detailed description herein, references to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. After reading the description, it will be apparent to one skilled in the relevant art(s) how to implement the disclosure in alternative embodiments. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed under the provisions of 35 U.S.C. 112(f), unless the element is expressly recited using the phrase “means for.” As used herein, the terms “comprises”, “comprising”, or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method or article, or apparatus. | 20,233 |
11857679 | DETAILED DISCLOSURE OF THE INVENTION As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional terms/phrases (and any grammatical variations thereof) “comprising,” “comprises,” “comprise,” “consisting essentially of,” “consists essentially of,” “consisting,” and “consists” can be used interchangeably. The phrases “consisting essentially of” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, where the term “about” is used to describe compositions containing amounts of ingredients or a temperature or a rate of stirring, these parameters can be varied between 0% and 10% around the stated value (X±10%). In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are intended to be explicitly included. “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the antigen in the vaccine, its use in the vaccine compositions of the invention is contemplated. “Treatment” (and grammatical variants of these terms), as used herein, are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. The term “therapeutically effective amount” refers to that amount of a drug that is sufficient to effect the intended application including but not limited to disease treatment. The therapeutically effective amount may vary depending upon the intended application or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. “Subject” refers to an animal, such as a mammal, for example a human. The methods described herein can be useful in both humans and non-human animals. In some embodiments, the subject is a mammal (such as an animal model of disease), and in some embodiments, the subject is a human. Typical subjects include canine, feline, porcine, bovine, equine, and primate. A homopolymer refers to a polymer consisting of only one type of monomer. A co-polymer refers to a polymer containing more than one type of monomers. This disclosure provides polymers and nanoparticles containing such polymers. Certain polymers disclosed herein are produced by polymerization of therapeutic monomers. The polymers and nanoparticles containing such polymers can be produced in the form of an emulsion with a surfactant, such as sodium dodecyl sulfate (SDS). Accordingly, certain embodiments of the invention provide a homopolymer of an acrylolated drug as a monomer. Acrylolation of a drug can be performed on a suitable atom in the drug, such as, C, S, O, and N, preferably, N. In preferred embodiments, the drug is an antibiotic, such as ciprofloxacin. The antibiotic compounds can belong to a class of penicillins, N-thiolated β-lactams, or fluoroquinolones. The β-lactam antibiotic can be a penicillin, penams, cephalosporin, monobactam, or carbapenem. Non-limiting examples of β-lactam antibiotics include benzylpenicillin, benzathine benzylpenicillin, procaine benzylpenicillin, phenoxymethylpenicillin (V), propicillin, pheneticillin, azidocillin, clometocillin, penamecillin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, methicillin, amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, ticarcillin, carbenicillin, carindacillin, temocillin, piperacillin, azlocillin, mezlocillin, mecillinam, pivmecillinam, sulbenicillin, faropenem, ritipenem, ertapenem, antipseudomonal, doripenem, imipenem, meropenem, biapenem, panipenem, cefazolin, cefalexin, cefadroxil, cefapirin, cefazedone, cefazaflur, cefradine, cefroxadine, ceftezole, cefaloglycin, cefacetrile, cefalonium, cfaloridine, cefalotin, cefatrizine, cefaclor, cefotetan, cephamycin, cefoxitin, cefprozil, cefuroxime, cefuroxime axetil, cefamandole, cefminox, cefonicid, ceforanide, cefotiam, cefbuperazone, cefuzonam, cefmetazole, carbacephem, loracarbef, cefixime, ceftriaxone, antipseudomonal, ceftazidime, cefoperazone, cefdinir, cefcapene, cefdaloxime, ceftizoxime, cefmenoxime, cefotaxime, cefpiramide, cefpodoxime, ceftibuten, cefditoren, cefetamet, cefodizime, cefpimizole, cefsulodin, cefteram, ceftiolene, oxacephem, flomoxef, latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftaroline fosamil, ceftolozane, ceftobiprole, ceftiofur, cefquinome, cefovecin, aztreonam, tigemonam, carumonam, and nocardicin A. Non-limiting examples of fluoroquinolone include enoxacin, ciprofloxacin, norfloxacin, ofloxacin, levofloxacin, trovafloxacin, gatifloxacin, or moxifloxacin. A person of ordinary skill in the art can envision that any drug that can be acrylolated to produce the homopolymers according to this disclosure and such embodiments are within the purview of the invention. In preferred embodiments, a homopolymer of an acrylolated drug is in the form of nanoparticles. In preferred embodiments, the nanoparticle have a size of between 600 nm and 1000 nm, preferably, between 700 nm and 1000 nm, even more preferably, between 800 nm and 1000 nm, and most preferably, between 900 nm and 1000 nm. In a particular embodiment, the nanoparticles have a size of about 970 nm. In other embodiments, the nanoparticles further comprise a detergent. In more preferred embodiments, the nanoparticles containing a detergent are in the form of an aqueous emulsion. Non-limiting examples of detergents that can be included in the nanoparticles or the emulsions disclosed herein include sodium dodecyl sulfate, cetyltrimethylammonium bromide, 3-(N,N-dimethylmyristylammonio)propanosulfonate, dedecanoic acid 2-(2-hydroxyethoxy)ethyl ester, sodium 11-(acrylolyloxyundecan-1-yl) sulfate, N-(11-Acryloyloxyundecyl)-N-(2-hydroxyethyl)-N,N-dimethylammoniuim bromide, N-(11-Acryloyloxyundecyl)-N,N-dimethyl-N-ethylammonium bromide, 3-[N,N-Diethyl-N-(3-sulfopropyl)ammonio] acrylate, 2 (2-Acryloyloxyethoxy)ethyl dodecanoate, or any mixture thereof. Certain other embodiments of the invention provide a co-polymer of two or more acrylolated drugs. Such co-polymers can be useful for administering a combination of drugs in one composition. A person of ordinary skill in the art can select appropriate combination of two or more drugs that can be acrylolated and formed into a co-polymer. Much like the homopolymers described herein, such co-polymers can also be in the form of nanoparticles comprising the co-polymers of acrylolated drugs, or emulsions containing the nanoparticles of the co-polymers of acrylolated drugs of the invention. Further, the methods described herein for producing homopolymers of acrylolated drugs can be readily modified to produce the co-polymers of two or more acrylolated drugs by simply mixing appropriate amounts of monomers of the corresponding acrylolated drugs. Specific embodiments of the invention provide a method of producing an aqueous emulsion of a homopolymer of an acrylate monomer of a drug. Such method comprises the steps of: a) dissolving the acrylolated monomer of the drug in an organic solvent; b) dissolving a detergent in water; c) mixing the solution of detergent in water with the solution of the acrylolated monomer of the drug in the organic solvent; d) increasing the temperature of the mixture produced in step c); e) contacting the mixture produced in step d) to an oxidant that initiates polymerization of the acrylate monomers of the drug; and optionally, additional water; f) stirring the mixture produced in step e) to produce the aqueous emulsion of the homopolymer of the acrylate monomer of the drug. In certain embodiments, the step of dissolving a detergent in water is performed at a temperature between 25° C. and 45° C., preferably, at a temperature between 30° C. and 40° C., more preferably, at a temperature of about 35° C. In preferred embodiments, the step of increasing the temperature of the mixture produced in step c) is performed under constant stirring. The temperature of the mixture produced in step c) can be increased to between 80° C. and 110° C., preferably, between 85° C. and 105° C., even more preferably, between 90° C. and 100° C., and most preferably, to about 95° C. A person of ordinary skill in the art can select a suitable organic solvent to dissolve an acrylolated drug. Organic solvents that can be used include methanol, ethanol, propylene glycol, hexane, glycerol, ethyl acetate, dichloromethane, or any mixture thereof. An organic solvent is selected that would evaporate at a temperature of between 80° C. and 110° C. and thus, can be removed from the mixture during the process of making the aqueous emulsion. Additional examples of organic solvents that can be used in the methods of the invention are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention. A detergent suitable for use in the methods of the invention include sodium dodecyl sulfate, cetyltrimethylammonium bromide, 3-(N,N-dimethylmyristylammonio)propanosulfonate, dedecanoic acid 2-(2-hydroxyethoxy)ethyl ester, sodium 11-(acrylolyloxyundecan-1-yl) sulfate, N-(11-Acryloyloxyundecyl)-N-(2-hydroxyethyl)-N,N-dimethylammoniuim bromide, N-(11-Acryloyloxyundecyl)-N,N-dimethyl-N-ethylammonium bromide, 3-[N,N-Diethyl-N-(3-sulfopropyl)ammonio] acrylate, 2(2-Acryloyloxyethoxy)ethyl dodecanoate, or any mixture thereof. Additional examples of detergents that can be used in the methods of the invention are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention. In preferred embodiments, dissolving an acrylate monomer of a drug in an organic solvent is performed at a temperature of between 35° C. and 45° C., i.e., at a temperature higher than the room temperature. Such temperature facilitates dissolution of the drug in the organic solvent. The step of increasing the temperature of the mixture produced in step c) to between 80° C. and 100° C. is performed at a rate of between 5° C. and 15° C. per hour, preferably, at a rate of between 6° C. and 14° C. per hour, more preferably, at a rate of between 7° C. and 13° C. per hour, even more preferably, at a rate of between 8° C. and 12° C. per hour, and most preferably, at a rate of between 9° C. and 11° C. per hour, and particularly, at a rate of about 10° C. per hour. Such slow increase in temperature ensures proper formation of homopolymers while slowly evaporating the organic solvent out of the mixture, thus producing an aqueous suspension of the homopolymeric nanoparticles. Evaporation of the organic solvent from the mixture produced in step d) can be further facilitated by constant stirring, for example, at a rate of between 800 rpm to 1300 rpm, preferably, at a rate of between 900 rpm to 1200 rpm, more preferably, at a rate of between 1000 to 1100 rpm, and even more preferably, at a rate of 1100 rpm. Preferably, the oxidant that initiates polymerization is a radical initiator. Examples of oxidant that initiates polymerization of the acrylate monomers include potassium persulfate. In preferred embodiments, the step of polymerization and stirring (steps e) and f) above) are carried out for between 36 to 60 hours, preferably, between 40 to 56 hours, more preferably, between 44 to 52 hours, and most preferably, for about 48 hours. In certain embodiments, the drug used in the methods of the invention is an antibiotic. Examples of antibiotics mentioned above in connection with the homopolymers of the invention can also be used in the methods of the invention. Also, additional drugs or antibiotics that can be used in the methods of the invention can be readily identified by a person of ordinary skill in the art and such embodiments are within the purview of the invention. Further, for co-polymers containing a combination of drugs or a combination of antibiotics, a person of ordinary skill in the art can select appropriate combinations of drugs based on intended applications. The homopolymers, nanoparticles containing such homopolymers, and emulsions containing the nanoparticles disclosed herein exhibit the activity of the drug, for example, the antibiotic used to produce the homopolymer. Accordingly, certain embodiments of the invention provide a method of treating a disease in a subject by administering a therapeutically effective amount of a homopolymers, nanoparticles containing such homopolymers, or emulsions containing the nanoparticles disclosed herein. Co-polymers of a combination of acrylolated drugs, nanoparticles containing such co-polymers, and emulsions containing the nanoparticles of such co-polymers can also be in the methods of treating a disease disclosed herein. In preferred embodiments, the disease is an infection caused by an infectious agent and the homopolymer is produced from an acrylolated antibiotic. Accordingly, certain embodiments of the invention provide methods of treating an infection in a subject caused by an infectious agent, the method comprising administering to the subject the homopolymers, nanoparticles containing such homopolymers, or emulsions containing the nanoparticles disclosed herein. The homopolymers, nanoparticles, or emulsions disclosed herein can be administered in the form of a pharmaceutical composition comprising pharmaceutically acceptable carriers. The polymers (including homopolymers and co-polymers disclosed herein), nanoparticles, or emulsions can be administered via, for example, oral, pulmonary, buccal, suppository, intravenous, intraperitoneal, intranasal, intramuscular or subcutaneous routes. Additional routes of administration are well known to a skilled artisan and such embodiments are within the purview of this invention. The appropriate route of administration depends on the type of disease being treated, the subject being treated, the stage and severity of the disease, etc. A person of ordinary skill in the art can determine an appropriate route of administration based on specific parameters. In certain embodiments, the disease is an infection caused by a virus, bacterium, protozoan, helminth, archaebacterial, or a fungus. A person of ordinary skill in the art can select an appropriate drug or combination of drugs to treat an infection and produce the corresponding polymers, nanoparticles, or emulsions based on this disclosure. Routes of Administration and Dosage Forms In certain embodiments, the polymers, nanoparticles, or emulsions can be administered intramuscularly, subcutaneously, intrathecally, intravenously or intraperitoneally by infusion or injection. Solutions of the polymers, nanoparticles, or emulsions can be prepared in water, optionally mixed with a nontoxic surfactant. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions comprising the polymers, nanoparticles, or emulsions, that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. Preferably, the ultimate dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained by, for example, the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the polymers, nanoparticles, or emulsions in the required amount in the appropriate solvent as described herein with various of the other ingredients enumerated herein, as required, preferably followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder the polymers, nanoparticles, or emulsions plus any additional desired ingredient present in the previously sterile-filtered solutions. The compositions of the subject invention may also be administered orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the subject's diet. For oral therapeutic administration the polymers, nanoparticles, or emulsions can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the polymers, nanoparticles, or emulsions present in such compositions and preparations can be varied can be conveniently be between about 2% to about 60% of the weight of a given unit dosage form. The amount of the polymers, nanoparticles, or emulsions in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose, or aspartame, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or for otherwise modifying the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar, and the like. A syrup or elixir can contain the polymers, nanoparticles, or emulsions and sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition the polymers, nanoparticles, or emulsions can be incorporated into sustained-release preparations and devices. For example, the polymers, nanoparticles, or emulsions can be incorporated into time release capsules, time release tablets, time release pills, and time release polymers or nanoparticles. Pharmaceutical compositions for topical administration of the polymers, nanoparticles, or emulsions to the epidermis (mucosal or cutaneous surfaces) can be formulated as ointments, creams, lotions, gels, or as a transdermal patch. Such transdermal patches can contain penetration enhancers such as linalool, carvacrol, thymol, citral, menthol, t-anethole, and the like. Ointments and creams can, for example, include an aqueous or oily base with the addition of suitable thickening agents, gelling agents, colorants, and the like. Lotions and creams can include an aqueous or oily base and typically also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, coloring agents, and the like. Gels preferably include an aqueous carrier base and include a gelling agent such as cross-linked polyacrylic acid polymer, a derivatized polysaccharide (e.g., carboxymethyl cellulose), and the like. Pharmaceutical compositions suitable for topical administration in the mouth (e.g., buccal or sublingual administration) include lozenges comprising the composition in a flavored base, such as sucrose, acacia, or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. The pharmaceutical compositions for topical administration in the mouth can include penetration enhancing agents, if desired. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Other solid carriers include nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols, or glycols, or water/alcohol/glycol blends, in which the polymers, nanoparticles, or emulsions can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions that can be used to deliver the polymers, nanoparticles, or emulsions to the skin are known in the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508), all of which are hereby incorporated by reference. The concentration of the polymers, nanoparticles, or emulsions in such formulations can vary widely depending on the nature of the formulation and intended route of administration. For example, the concentration of the homopolymers, nanoparticles, or emulsions in a liquid composition, such as a lotion, can preferably be from about 0.1-25% by weight, or, more preferably, from about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder can preferably be about 0.1-5% by weight, or, more preferably, about 0.5-2.5% by weight. Pharmaceutical compositions for spinal administration or injection into amniotic fluid can be provided in unit dose form in ampoules, pre-filled syringes, small volume infusion, or in multi-dose containers, and can include an added preservative. The compositions for parenteral administration can be suspensions, solutions, or emulsions, and can contain excipients such as suspending agents, stabilizing agents, and dispersing agents. A pharmaceutical composition suitable for rectal administration comprises the polymers, nanoparticles, or emulsions in combination with a solid or semisolid (e.g., cream or paste) carrier or vehicle. For example, such rectal compositions can be provided as unit dose suppositories. Suitable carriers or vehicles include cocoa butter and other materials commonly used in the art. According to one embodiment, pharmaceutical compositions of the present invention suitable for vaginal administration are provided as pessaries, tampons, creams, gels, pastes, foams, or sprays containing the polymers, nanoparticles, or emulsions in further combination with carriers known in the art. Alternatively, compositions suitable for vaginal administration can be delivered in a liquid or solid dosage form. Pharmaceutical compositions suitable for intra-nasal administration are also encompassed by the present invention. Such intra-nasal compositions comprise the polymers, nanoparticles, or emulsions in a vehicle and suitable administration device to deliver a liquid spray, dispersible powder, or drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays are conveniently delivered from a pressurized pack, an insufflator, a nebulizer, or other convenient means of delivering an aerosol comprising the polymers, nanoparticles, or emulsions. Pressurized packs comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as is well known in the art. Aerosol dosages can be controlled by providing a valve to deliver a metered amount of the homopolymers, nanoparticles, or emulsions. The polymers, nanoparticles, or emulsions can be combined with an inert powdered carrier and inhaled by the subject or insufflated. Pharmaceutical compositions for administration by inhalation or insufflation can be provided in the form of a dry powder composition, for example, a powder mix of the homopolymers, nanoparticles, or emulsions and a suitable powder base such as lactose or starch. Such powder composition can be provided in unit dosage form, for example, in capsules, cartridges, gelatin packs, or blister packs, from which the powder can be administered with the aid of an inhalator or insufflator. The exact amount (effective dose) of the polymers, nanoparticles, or emulsions administered can vary from subject to subject, depending on, for example, the species, age, weight, and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. Methods for the extrapolation of effective dosages in mice and other animals to humans are known to the art; for example, see U.S. Pat. No. 4,938,949, which is hereby incorporated by reference. A therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg/kg of body weight per day, preferably from about 0.01 to about 100 mg/kg of body weight per day, more preferably, from about 0.1 to about 50 mg/kg of body weight per day, or even more preferred, in a range of from about 1 to about 10 mg/kg of body weight per day. For example, a suitable dose may be about 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per day. The polymers, nanoparticles, or emulsions can be conveniently administered in unit dosage form, containing for example, about 0.05 to about 10000 mg, about 0.5 to about 10000 mg, about 5 to about 1000 mg, or about 50 to about 500 mg of the homopolymers, nanoparticles, or emulsions. The polymers, nanoparticles, or emulsions can be administered to achieve peak plasma concentrations of, for example, from about 0.25 to about 200 μM, about 0.5 to about 75 μM, about 1 to about 50 μM, about 2 to about 30 μM, or about 5 to about 25 μM of each of the drug. Exemplary desirable plasma concentrations include at least 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100 or 200 μM. For example, plasma levels may be from about 1 to about 100 micromolar or from about 10 to about 25 micromolar. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the polymers, nanoparticles, or emulsions in saline, or orally administered as a bolus containing about 1 to about 100 mg of the homopolymers, nanoparticles, or emulsions. Desirable blood levels may be maintained by continuous or intermittent infusion. The polymers, nanoparticles, or emulsions can be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include the polymers, nanoparticles, or emulsions at a concentration in the range of at least about 1 mg/ml, preferably at least about 4 mg/ml, more preferably at least 5 mg/ml and most preferably at least 6 mg/ml. The polymers, nanoparticles, or emulsions can conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as one dose per day or as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator. Optionally, the pharmaceutical compositions of the present invention can include one or more other therapeutic agents, e.g., as a combination therapy. The additional therapeutic agent(s) will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. The concentration of any particular additional therapeutic agent may be in the same range as is typical for use of that agent as a monotherapy, or the concentration may be lower than a typical monotherapy concentration if there is a synergy when combined with the homopolymers, nanoparticles, or emulsions. Following example illustrates procedures for practicing the invention. This example should not be construed as limiting. Example 1—Polyacrylate Nanoparticle Emulsions: Forming Homo Poly (N-Acryloylciprofloxacin) as an Antibacterial Polymer Emulsion The possibility of removing all non-bioactive monomers from nanoparticle construction was explored. Avoiding the use of co-monomers during the emulsion polymerization procedure allows for greater amount of loading of the bioactive antibacterial monomer, producing a homopolymer nanoparticle emulsion composed solely of the antibiotic monomer. This Example delves into tackling the issue of limited loading of bioactive compounds, and the need for a better carrier polymer to bind or encapsulate the drug for delivery. The surfactant has a limit of how many organic/hydrophobic compounds it can contain within the micelle during emulsion polymerization. As a result, the maximum amount of organic content of the final emulsion is typically in the range of 15-20% by weight. This restricts the usefulness of the nanoparticle as an effective drug carrier to 20% or less of the emulsion amount. Polyacrylate nanoparticle emulsions can be easily prepared through radical-induced emulsion polymerization of butyl acrylate/styrene mixtures (7:3 w/w) in water at 78° C., using sodium dodecyl sulfate (SDS) as an emulsifying agent and potassium persulfate as a radical initiator (FIG.1). The reactions led to the formation of a homogeneous, stable aqueous emulsion containing uniformly-sized nanoparticles of 45-50 nm in diameter. The method was successfully applied to penicillins and N-thiolated β-lactams, in which the antibacterial agents could be introduced into the nanoparticle either by non-covalent entrapment as a free drug, or covalently via their acryloyl derivative. While these earlier nanoparticle emulsions provided increased water solubility and, in some cases, improved bioactivity of the β-lactam antibacterial agent, the polyacrylate backbone was largely comprised of the non-bioactive monomers (butyl acrylate-styrene or methyl methacrylate-styrene (20% by weight of the emulsion), and thus only 1-3% (by weight) of the nanoparticle framework was the antibacterial acrylate.FIG.1shows the general scheme for the formation of the nanoparticle emulsion, and the amount of drug loading into the nanoparticle during the assembly process was limited by how much surfactant could be used, given that amounts exceeding 3 mole % of SDS caused unwanted cytotoxicity. The final crude nanoparticle emulsions contained up to 20% of solid content (a mixture of nanoparticles and a small amount of non-nanoparticle polymer), and only 0.2-0.6% of active antibacterial agent inside of the nanoparticles. The resulting emulsions are typically milky in consistency and somewhat sticky when exposed to air, causing films to form when dried, and forming coagulants within syringes, micro-porous filters, and gel columns that made it very difficult to purify and use for in vivo testing. Purification techniques that enable the removal of residual unreacted monomers and non-nanoparticle oligomers within the cloudy emulsion were used. Other surfactant combinations were used to try to enhance the amount of antibiotic that could be entrapped, or to alter nanoparticle sizes, without increasing overall cytotoxicity or instability of the emulsion. FIG.2depicts the polyacrylate polymer that was formed that allows for the incorporation of the bioactive drug either through covalently binding to the polymer backbone or encapsulating within the hydrophobic environment of the micelle. This in turn limits the amount of bioactive drug that can be contained in the particle. FIG.3shows that removing all acrylates except for the acrylolated bioactive drug (or other compound) for the emulsion polymerization would allow for an increase in the ability to load the desired drugs/compounds within the micelles, and thus the final concentration in the nanoparticle emulsion. If the same limit of 15-20% of organic material entrapped by the surfactant inside the micelles is maintained, then the final concentration of the drug incorporated into the nanoparticle would be considerably more than the typical 0.2%-0.6% achieved using the butyl acrylate/styrene polyacrylate nanoparticles. The use of only N-acryloylciprofloxacin as the sole monomer then would afford an advanced polyacrylate nanoparticle emulsion, which allows for the delivery of higher drug content. This would in return require much smaller volumes of the emulsion to be synthesized and used for drug delivery. The avoidance of using other monomers for the nanoparticle formation additionally removes the issue of unwanted coagulation and film formation previously observed for the poly(butyl acrylate/styrene) nanoparticle emulsions. The residual styrene and butyl acrylate and non-particle polymers that are not encapsulated within the surfactant could be removed by centrifugation and dialysis, however, the resulting emulsions after purification still continued to formed rubbery films when dehydrated, which clogged syringe needles and filtration membranes. The use of these particular monomers was problematic in this regard and not using them might eliminate the need to purify the ciprofloxacin acrylate emulsions. In this Example, a new approach to preparing antibiotic-bound polyacrylate nanoparticle emulsions is described that completely obviates the restriction of using butyl acrylate and styrene (or other co-monomers) to form the nanoparticle framework, and instead, uses the antibiotic compound itself as the sole acrylate monomer for the polymerization. This technique has never been reported and is thus an important advance in the polymer-based nanoparticle field. Ciprofloxacin was chosen as the antibiotic for the formation of the polyacrylate nanoparticles. The N-acryloyl derivative of commercial ciprofloxacin hydrochloride was prepared for this purpose according to N-acylation procedure. Synthesis of N-Acryloylciprofloxacin FIG.4shows the synthetic scheme for preparing N-acryloyl ciprofloxacin, and follows as such: To a round bottom flask was added 120 ml of dichloromethane, then 3.0 g (9.0 mmol) of ciprofloxacin and 1.8 ml (13.5 mmol) of triethylamine. The mixture was left stirring at 0° C. for 1 hour then acryloyl chloride (1.1 ml, 13 mmol) was added dropwise. The ice bath was removed and the reaction was left stirring overnight. The dichloromethane was added dropwise to a flask of hexane (200 ml) to cause a precipitate to form. The solid was collected by filtration and allowed to air dry. Yielded 2.90 g (83.7%) as a pale yellow solid. Melting point above 250° C.1H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 8.03 (d, J=12.8 Hz, 1H), 7.36 (d, J=7.1 Hz, 1H), 6.60 (dd, J=16.8, 10.5 Hz, 1H), 6.35 (dd, J=16.8, 1.7 Hz, 1H), 5.76 (dd, J=10.5, 1.7 Hz, 1H), 3.86 (m, 4H), 3.52 (br. s., 1H), 3.33 (m, 4H), 1.38 (d, J=6.2 Hz, 2H), 1.19 (br. s., 2H). Formation of Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsion One of the main challenges with polymerizing the desired acryloyl analog of the bioactive drug was that most of the previous antibiotics that were acrylolated and loaded into the nanoparticle emulsions were solids, and thus the liquid organic monomers of styrene and butyl acrylate could be used to pre-dissolve the small amount of the solid acrylolated antibiotic. This was also the case with the poly(menthyl acrylate) nanoparticle emulsions, in that the non-bioactive monomer menthyl acrylate was a liquid that allowed for the dissolution of the solid N-acryloyl ciprofloxacin antibiotic in order to be incorporated into micelles during emulsion polymerization. Attempts to use the same procedure for emulsion polymerization of the N-acrylolated ciprofloxacin monomer failed, however. Thus it was necessary to pre-dissolve the N-acryloyl ciprofloxacin into an organic solvent that could easily be evaporated off during the polymerization process or after the formation of the emulsions. It was considered important to use a solvent of very low cytotoxicity to aid in the dissolution of the bioactive compounds, in case it would also load into the micelles along with the bioactive compound. After experimentation with various common organic solvents, including methanol, ethanol, propylene glycol, glycerol, and ethyl acetate; dichloromethane was chosen. Attempted Preparation of Homo Poly(N—N-Acryloylciprofloxacin) Nanoparticle Emulsions Using Water-Soluble Organic Solvents Two liquid organic solvents were first used to aid the dissolution of N-acryloylciprofloxacin. Propylene glycol and glycerin have very low cytotoxicity and due to their hydrophobic nature would likely load into the surfactant-formed micelles, and thus potentially carry in with it the N-acryloylciprofloxacin. Though this technique would result in a co-solvent also being incorporated into the micelles, it would still possibly allow for formation of the poly(N-acryloylciprofloxacin) emulsion. However, the resulting emulsions formed from the use of these solvents were not homogeneous. Due to glycerin's high viscosity, it was very difficult to distribute and stir properly in the aqueous media. This led to a bilayer, preventing homogeneous mixing of the resulting emulsion. The mixture was heated up to 90° C. in order to reduce the viscosity and allow for more uniform stirring and mixing with water. However, the resulting emulsions remained non-homogeneous. Propylene glycol provided a much better carrier solvent due to its lower viscosity. It was able to form a more uniform emulsion and would require no modification in procedure compared to the typical one used to make polyacrylate nanoparticle emulsions. However, the resulting emulsions were unstable and formed a bilayer within minutes of being removed from the polymerization conditions. The DLS data did confirm multiple populations of particles within the emulsion and very low zeta potential values (−5 mV to −10 mV), which confirmed the inherent instability of the emulsions. So these attempts did not prove effective. Preparation of Homo Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsions Using a Water-insoluble Solvent. The other method investigated for polymerization of the solid N-acryloylciprofloxacin to be evenly distributed within the aqueous mixture was to pre-dissolve the compound in an organic solvent, and then remove the organic solvent via evaporation during the emulsion process or after the emulsion formation. It was critical to completely remove the organic solvent, because most organic solvents produce cytotoxicity. This method was attempted using methanol, ethanol, ethyl acetate, and dichloromethane. The main problem was the poor solubility of the N-acryloylciprofloxacin in most organic solvents, except for dichloromethane. Up to 500 mg/ml of N-acryloylciprofloxacin could be dissolved into dichloromethane. However, there was a critical issue that resulted with the polymerization procedure. Typically, organics were stirred at 75° C., then the surfactant and water were added. This would cause the dichloromethane to rapidly evaporate. Thus the starting temperature was adjusted to 25° C., and water and surfactant were added to the stirring dichloromethane solution, however this resulted in an uneven distribution and clumping of the surfactant. The result was a very sticky material that separated from the water layer. To solve this, the surfactant and water were added first at 75° C. so that the surfactant may form micelles initially, and the dichloromethane solution was added dropwise. However, this resulted in the near instant evaporation of the dichloromethane solvent, leaving clumps of solid N-acryloylciprofloxacin unincorporated into the micelles. The final adjustment of the procedure is discussed in the following section, resulting in successful formation of the emulsion. Preparation of Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsions As seen inFIG.5, the polyacrylate emulsions were prepared using a modified protocol of the usual nanoparticle emulsion technique used in our lab. The method to form the poly(N-acryloylciprofloxacin) emulsion required the following procedure: to a round bottom flask was added 4 ml of deionized water, which was then stirred using a 1.25 cm (300 mg) Teflon-coated magnetic stir bar at 1000 rpm on a Corning PC-420D magnetic stirrer at 30° C. using a self-regulated oil bath. To this was added 30 mg of SDS. N-Acryloylciprofloxacin (500 mg) was dissolved in 1 ml of warm dichloromethane, and this solution was added dropwise to the deionized water-SDS mixture. A vent was placed on top of the flask by inserting a small stainless steel syringe needle through a rubber septum on the flask, under dry nitrogen, and the temperature of the mixture was increased at a rate of 5° C. per 30 min until reaching 90° C. The mixture was left stirring overnight at this temperature, under an atmosphere of dry nitrogen. Potassium persulfate (10 mg) was added with an additional 0.5 ml of deionized water to the stirring mixture, and left stirring for 24 hours. The stirred emulsion was then removed from the oil bath and decanted into a storage vial for analysis. FIG.6shows an example of a successful emulsion (on the left), forming a uniform single layer emulsion, while previous attempted emulsions (the two on the right) show the results of an unsuccessful emulsion polymerization. Dynamic Light Scattering (DLS) Analysis Dynamic light scattering measurements were performed to test if any nanoparticles were being formed in the emulsion polymerization process. The average size and surface charge of the emulsion was analyzed on a Malvern Zetasizer nano-ZS instrument. To prepare the samples for the analyses, the freshly-made emulsion was subjected to centrifugation at 10,000 rpm for 5 min using an Eppendorf Centrifuge 5424. An aliquot of the liquid emulsion was then drawn and deposited into a Malvern disposable folded capillary cell DTS-1070. Each sample was analyzed in triplicate, and each data collection consisted of 1 run of 20 scans (for size analysis) and 3 runs of 100 scans (for zeta potential determination). The size distribution shows a single narrow peak indicating the uniformity of the emulsion with a single population centered on average at approximately 970 nm. Similarly, surface charge measurements indicated a highly stable emulsion, with an average of −63 (±5.6) mV. Dynamic Light Scattering (DLS) Analysis Results AsFIG.7demonstrates, the dynamic light scattering experiments confirmed the presence of a major population of nanoparticles in the emulsion, measuring on average approximately 970 nm in diameter. A general trend of increasing size was observed as the amount of N-acryloylciprofloxacin is increased in forming the polymer emulsions. In addition, the zeta potential measurements showed that the particles carry a high surface charge of −63 (+5.6) mV. This indicates the long-term stability of the emulsion. It is notable that these poly(N-acryloylciprofloxacin) nanoparticles are much larger than those previously constructed with butyl acrylate-styrene co-monomers, which routinely measured 45-50 nm in diameter. The basis for this 20-fold increase in size is not apparent at this time but deserves further investigation. In Vitro Antibacterial Testing To investigate whether the nanoparticles possess antibiotic capabilities, each crude emulsion was tested againstStaphylococcus aureus(ATCC 25923) andEscherichia coli(K12) using a 96-well plate broth assay to determine the minimum inhibitory concentration (MIC). Each assay was done in triplicate. The original stock emulsion was diluted using the Trypticase Soy Broth solution to an initial concentration of 1.28 mg/ml of the N-acryloylciprofloxacin, then serial diluted with TSB to half the concentration each time. A volume of 10 μl of each emulsion dilution was added to a well in series, resulting in a final concentration run of 64 μg/ml to 0.012 μg/ml. The MIC was done in triplicates for each bacterium, with ciprofloxacin hydrochloride being used as a positive control and a blank of broth medium was used as a negative control. Bacteria were grown overnight at 37° C. on an agar plate composed of BBL TSA II Trypticase Soy Agar (TSA) and BBL Trypticase Soy Broth (TSB) in a 1:2 ratio at 4.4% concentration. A broth solution of 2.4% TSB was inoculated using the bacteria from the agar plates, and incubated at 37° C. to reach a 0.5 McFarland standard. The bacteria were then further diluted by a factor of 1000 using a broth solution of 2.4% TSB, and 190 μl of the diluted bacterial solution was transferred by micropipette into each well. The inoculated plates were incubated at 37° C. for 16-20 hours and the resulting plates were observed for growth and MIC values recorded. The MIC was the lowest concentration of the antibiotic that completely inhibited bacterial growth (visually) within that series of dilutions. Antibacterial Data for Poly (N-Acryloylciprofloxacin) Emulsions TABLE 1MIC values of ciprofloxacin and ciprofloxacin emulsion vsS.aureusandE.coli.SampleS.aureus(ATCC 25923)E.coli(K12)Control Ciprofloxacin0.5 μg/ml0.012 μg/mlPoly (N-acryloyl-ciprofloxacin)0.5 μg/ml0.012 μg/mlemulsion The in vitro antibacterial studies showed that the nanoparticle emulsion was bioactive, with an MIC of 0.5 μg/ml forS. aureusand 0.012 μg/ml againstE. coli, identical to those of ciprofloxacin itself (Table 1). The finding that these nanoparticles show antibacterial capabilities against both the gram-positiveS. aureusand the gram-negativeE. coliwas surprising, given that particles of such large dimensions would not be expected to be antibacterially active. Ciprofloxacin must enter the bacterial cell to arrive at its target, bacterial DNA gyrase. Attachment of the molecule to the polymer backbone of the nanoparticle requires it be released through hydrolysis of the amide. This occurs either outside of the cell or within the bacterium itself if the nanoparticle can enter through the membrane. Most likely this requires enzymatic release, as the amide functionality is a difficult one to cleave otherwise. In Vitro Cytotoxicity of the Nanoparticle Emulsions In vitro cell cytotoxicity was tested on two human cell lines, human colorectal carcinoma cells HCT-116, and human embryonic kidney cells HEK 293. HCT-116 cells were grown in Dulbeco's Minimum Essential Medium (DMEM) with 10% fetal bovine serum and 0.1% penicillin/streptomycin as a growth medium for several days at 37° C. under an atmosphere of 5% CO2to reach confluence. HEK 293 cells were grown in Eagle Minimum Essential Medium (EMEM) with 10% fetal bovine serum and 0.1% penicillin/streptomycin as a growth medium for several days at 37° C. under an atmosphere of 5% CO2to reach confluence. Each cell type was then plated onto 96-well plates, at 50,000 cells per well at a volume of 150 μl with the respective growth medium. The cells were counted using a hemocytometer and then incubated for 24 hours at 37° C. under an atmosphere of 5% CO2. The test emulsion was diluted using the growth medium for each cell type, and added into the wells of each test plate to give a final concentration of N-acryloyl ciprofloxacin of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml within a series. The testing was done in triplicate and one well in each triplicate was left untreated as the negative control for 100% growth. The plates were further incubated and monitored for 48 hours at 37° C. under an atmosphere of 5% CO2. A 5 mg/ml solution of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) in sterile phosphate-buffered saline (PBS) was added to give a 10% final concentration in each well. The plates were then further incubated for 4 hours at 37° C. under an atmosphere of 5% CO2to allow for the formation of the purple crystals of 1-(4,5-dimethylthiazol)2-yl)-3,5-diphenylformazan. The liquid was then aspirated from each well and 100 μl of dimethylsulfoxide (DMSO) was added to each well, and gently shaken for 1 minute to allow for complete dissolution of the crystals. The IC50value for the assay was determined using a BioTek Synergy H1 hybrid plate reader at both 595 nm and 630 nm. The IC50was determined as the well with at least 50% cell viability compared to the untreated control cell with 100% cell growth. Cytotoxicity Results for Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsions The observed IC50was 500 μg/ml for both the HCT-116 and HEK-293 cell lines, a 1000-fold difference over the bacterial MIC value forS. aureusand greater than 40,000 forE. coli. Imaging Nanoparticle Emulsions Using a Scanning Electron Microscope A sample of poly(N-acryloyl ciprofloxacin) nanoparticle emulsion was prepared for imaging using scanning electron microscope. The samples were initially prepared by lyophilization of the emulsion which resulted in a dry powder that could be added to the sample holder for the scanning electron microscope instrument. The samples were placed onto an aluminum-coated sample holding tape, mounted onto a copper tape, placed onto the scanning electron microscope sample holder. The sample was diluted 1000× with deionized water, and a drop of the diluted emulsion was placed on the conductive aluminum-coated sample holding tape. The sample was placed in the −80° C. freezer for a few hours, then immediately lyophilized to dry the sample right onto the sample holding tape to produce a more even distribution of the material. In addition, the sample-containing tape was also sputter-coated with gold-palladium in order to increase the conductivity of the resulting sample, thus preventing or reducing the accumulation of electrons on the surface of the sample, and resulting in distortions. As observed inFIGS.8and9, the images from the scanning electron microscope do not provide clear images of the spheres within the emulsion as were previously observed with butyl acrylate/styrene and poly(menthyl acrylate) emulsions. This was thought to be the result of the material continuing to building up charge on the surface, thus giving a distorted image. Attempts to overcome this effect by ensuring a smooth and conductive surface for the sample holding tape, and sputter-coating with conductive gold-palladium coating, did not improve results. In addition, during the lyophilization process the spheres were dehydrated and deformed, thus resulting in the spheres binding to each other and not remaining separate. This led to the increase of the overall size when viewed from top down with the scanning electron microscope. Poly(N-acryloylciprofloxacin) nanoparticle emulsions were successfully prepared by modification of the previously reported emulsion polymerization methodology. The main difference with this new method was the need to dissolve the water-insoluble antibacterial agent in an organic solvent to permit more uniform addition into the aqueous solution to form homogeneous emulsions. Dichloromethane provided the best combination of solubilizing the ciprofloxacin monomer and being volatile enough to evaporate from the media during emulsion polymerization at 90° C. The increased temperature of 90° C. rather than 75° C., an increased stir speed, and the addition of sodium dodecyl sulfate before the organic monomers were added, provided more optimal results. Additionally, it was advantageous to let the reactions run for 48 hours rather than the 6 hours required for the butyl acrylate-styrene co-monomer systems. These new procedures are required mainly due to the physical properties of the compounds involved, and are pushing the limits and capabilities of the existing available equipment. Higher loading of the drug could perhaps be possible if the mixture could be heated in a pressurized system that would allow for a higher temperature to be achieved without boiling off the water. In addition, a mechanical stirrer able to achieve a higher spin rate that the existing magnetic stir bar method would most likely allow for additional loading of the monomer, since it would provide more uniform distribution of large quantities of the solid monomer. Lyophilization of the nanoparticle emulsion produced an amorphous powder that could not be reformulated to its original emulsified state through addition of water. Moreover, the resulting powder remained insoluble in organic solvents including methanol, ethanol, dichloromethane, hexane, acetone, ethyl acetate, and dimethylformamide. Extraction of the solid material with methanol, ethanol, dichloromethane, hexane, acetone, or ethyl acetate failed to show any trace of unreacted N-acryloylciprofloxacin upon evaporation and analysis by proton NMR spectroscopy. This confirms that the polymerization is complete, and thus all of the N-acryloylciprofloxacin is incorporated into the framework of the nanoparticle. Attempts to perform the emulsion polymerization procedure on the free ciprofloxacin instead of the N-acryloyl derivative led to a bilayer mixture, not an emulsion, with the layers separating within seconds after stirring was stopped. Additionally, the same procedure was attempted using N-acetyl ciprofloxacin as an analog similar in structure but without the requisite olefin. Once again, only an unstable mixture was formed, which separated into layers with a few seconds after stirring was stopped. Therefore, the acryloyl group is a prerequisite for emulsification and subsequent nanoparticle formation. This Example provides an aqueous nanoparticle polymer emulsion being formed from a monomer that is the antibiotic agent itself. The emulsion is formed via a one pot reaction in water and the final antibiotic polymer is suspended in water. The emulsified nano-cipro particles are antimicrobially-active towards gram positiveS. aureusand gram negativeE. coli. The methods and nanoparticle emulsions described herein can be used to produce other emulsions containing other antibiotics including water-insoluble antibiotics for delivery and effective treatment of drug-resistant bacterial infections. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. REFERENCES 1) Turos, E., Reddy, G. S. K., Greenhalgh, K., Ramaraju, P., Abeylath, S. C., Jang, S., et al. Penicillin-bound polyacrylate nanoparticles: Restoring the activity of β-lactam antibiotics against MRSA. Bioorg. Med. Chem. Lett. 2007; 17:3468-72.2) Turos, E., Shim, J. Y., Wang, Y., Greenhalgh, K., Reddy, G. S., Dickey, S., et al. Antibiotic-conjugated polyacrylate nanoparticles: New opportunities for development of anti-MRSA agents. Bioorg. Med. Chem. Lett. 2007; 17:53-6.3) Greenhalgh, K., Turos, E. In vivo studies of polyacrylate nanoparticle emulsions for topical and systemic applications. Nanomedicine: Nanotechnology, Biology, and Medicine, 2009; 5:46-54.4) Garay-Jimenez, J. C., Turos, E. A convenient method to prepare emulsified polyacrylate nanoparticles from powders for drug delivery applications. Bioorg. Med. Chem. Lett. 2011; 21:4589-91.5) Abeylath, S., Turos, E. Glycosylated polyacrylate nanoparticles by emulsion polymerization. Carb. Polym. 2007; 70:32-7.6) Abeylath, S., Turos, E., Dickey, S., Lim, D. V. Novel carbohydrated nanoparticle antibiotics for MRSA andBacillus anthracis. Bioorg. Med. Chem. 2008; 16:2412-8.7) Labruère, R., Turos, E. Attenuating the size and molecular carrier capabilities of polyacrylate nanoparticles by a hydrophobic fluorine effect. Bioorg. Med. Chem. 2012; 20:5042-5.8) Garay, J., Gergeres, D., Young, A., Dickey, S., Lim, D., Turos, E. Physical properties and biological activity of poly(butyl acrylate-styrene) nanoparticle emulsions prepared with conventional and polymerizable surfactants. Nanomedicine. 2009; 5:443-51.9) Abeylath, S. C., Turos, E. Nanobiotics to combat bacterial drug resistance. In Antibiotic Resistance: Causes and Risk Factors, Mechanisms and Alternatives. Adriel R. Bonilla and Kaden P. Muniz (eds.), Nova Science Publishers. 2009; 425-65.10) Cormier, R., Burda, W., Harrington, L., Edlinger, J., Kodigepalli, K., Thomas, J., Kapolka, R., Roma, G., Anderson, B., Turos, E., Shaw, L., Studies on the antimicrobial properties of N-acylated ciprofloxacins. Bioorg. Med. Chem. Lett. 2012; 22:6513-20.11) Thamizharasi, S., Vasantha, J. Synthesis, characterization and pharmacologically active sulfamethoxazole polymers. Eur. Polym. J. 2002; 38: 551-9.12) Moon, W. S., Chung, K., H. Antimicrobial effect of monomers and polymers with azole moieties. J. Appl. Polym. Sci. 2003; 90:2933-7.13) Kanazawa, A., Ikeda, T. J. Antibacterial activity of polymeric sulfonium salts. Polym. Sci., Part A: Polym. Chem. 1993; 31:2873-6. | 62,100 |
11857680 | DETAILED DESCRIPTION OF THE INVENTION The present invention provides herein, compositions consisting essentially of a therapeutically effective amount of docetaxel and an excipient that facilitates intravenous administration, and which will be used to target the tumour site. It relates to increase the drug's residence time in blood. The present invention also includes to achieve more than 90% drug loading using combination of various solvents, preferably methanol and tertiary butanol (T-butanol or tertiary butyl alcohol) in ratio of 1:1. In a preferred embodiment, the pharmaceutical composition of the invention is liposomal injection. In the most preferred embodiment, the pharmaceutical liposomal injection composition comprises docetaxel and pharmaceutically acceptable excipients. Docetaxel, preferably used in the present pharmaceutical liposomal composition is of about 0.8% w/w to about 1% w/w based on the total weight of the composition. The preferred concentration of docetaxel in composition is about 0.85% w/w to about 0.95% w/w based on the total weight of the composition. Most preferably, the docetaxel is used in the composition of about 0.9% w/w based on total weight of the composition. In further embodiment, the pharmaceutical liposomal composition comprises docetaxel, phospholipids, cholesterol, lyoprotectant and a pH adjusting agent, wherein the pH of liposomal composition is less than 3.5. In yet another embodiment, the present invention further provides the docetaxel liposomal composition comprising docetaxel, phospholipids, cholesterol, solubilizer, lyoprotectant, and a pH adjusting agent. Examples of the phospholipids are selected form the group consisting of a natural phospholipid, a synthetic phospholipid, and combinations thereof. Lecithin is one of the natural resources for phospholipid. Lecithin is a mixture found in egg yolk and soya. It comprises a number of phospholipids including phosphatidylcholine (PC, Soya Phosphatidyl Choline), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Natural phospholipids also include, e.g. soy phosphatidyl choline (SPC), sphingomyelin, and phosphatidylglycerol (PG). Synthetic phospholipids include, but are not limited to, derivatives of phosphocholine (for example, DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), derivatives of phosphoglycerol (for example, DMPG, DPPG, DSPG, POPG, DSPG-NA, DSPG-NH4), derivatives of phosphatidic acid (for example, DMPA, DPPA, DSPA), derivatives of phosphoethanolamine (for example, DMPE, DPPE, DSPE DOPE), derivatives of phosphoserine (for example, DOPS), PEG derivatives of phospholipid (for example, mPEG-phospholipid, mPEG 2000-DSPE, polyglycerin-phospholipid, functionalized-phospholipid, and terminal activated-phospholipid) and any mixtures thereof. Preferably, phospholipid is selected from soy phosphatidyl choline (SPC) and mixture phospholipids are selected from soy phosphatidyl choline (SPC) and N-Carbonylmethoxypolyethylenglycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE). Phospholipids preferably used in the pharmaceutical liposomal composition of present invention is Soya Phosphatidyl Choline. Soya Phosphatidyl Choline used in the present invention is from about 30% w/w to about 40% w/w based on the total weight of the composition, preferably from about 30% w/w to about 38% w/w based on the total weight of composition, even more preferably, of about 31% w/w to about 36% w/w based on total weight of the composition and most preferably of about 32% w/w based on the total weight of the composition. Examples of the cholesterol is selected from the group consisting of cholesterol, cholesteryl sulfate and its salts (e.g., sodium salt), cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone and calciferol. Preferably, the cholesterol is selected from Sodium Cholesteryl Sulfate. Sodium Cholesteryl sulfate is preferably used in the range from about 0.2% w/w to about 0.8% w/w based on the total weight of the composition, more preferably, of about 0.4% w/w to about 0.6% w/w based on total weight of the composition, and most preferably of about 0.5% w/w based on total weight of composition. Examples of the solubilizer is selected from the group consisting vitamin E TPGS, polyethylene glycol (PEG) 400, and propylene glycol (PG) tween 80, tween 20, glycerol span 80 and glycofurol. Preferably, the solubilizer is selected from vitamin E TPGS. Solubilizer preferably used in the pharmaceutical liposomal injection composition of about 0.1% to about 1.5% based on the total weight of the composition. Examples of the lyoprotectant are selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture thereof. Preferably, the lyoprotectant is selected from sucrose. Sucrose preferably used in the pharmaceutical liposomal composition is about 61% to about 68% based on the total weight of the composition, more preferably of about 62% to about 66% based on total weight of the composition and most preferably of about 66.5% w/w based on the total weight of composition. Examples of pH adjusting agents used in the present liposomal composition is hydrochloric acid. pH adjusting agent is used to adjust the pH of the liposomal composition to about less than pH 3.5, more preferably pH of about 3. The inventors of the present invention have surprisingly found that with the pH of less than 3.5, there was high drug loading of more than 90% docetaxel into liposomes (more 90% of docetaxel is encapsulated into liposomes as bound drug and less than about 10% of docetaxel is present as free drug). In embodiments of the present invention, the present invention provides a pharmaceutical liposomal composition comprising of about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w to about 38% w/w of Soya Phosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of Sodium Cholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is less than 3.5. In further embodiment of the present invention, the present invention provides a pharmaceutical liposomal composition consisting essentially of about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w to about 38% w/w of Soya Phosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of Sodium Cholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is less than 3.5. In another embodiment of the present invention, the present invention provides a pharmaceutical liposomal composition consisting of about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w to about 38% w/w of Soya Phosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of Sodium Cholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is less than 3.5. In specific embodiment, the present invention provides a pharmaceutical liposomal composition comprising of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is about 3. In another embodiment, the present invention provides a pharmaceutical liposomal composition consisting essentially of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is about 3. In further embodiment, the present invention provides a pharmaceutical liposomal composition consisting essentially of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is about 3. In specific embodiment, the present invention provides a pharmaceutical liposomal composition comprising of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is of about 2.5 to about 3.2. In another embodiment, the present invention provides a pharmaceutical liposomal composition consisting essentially of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is of about 2.5 to about 3.2. In further embodiment, the present invention provides a pharmaceutical liposomal composition consisting essentially of about 0.9% w/w of docetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of Sodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is of about 2.5 to about 3.2. The pharmaceutical liposomal composition of present invention comprises the liposomes of d90less than 200 nm, d50less than 150 nm and d10less than 100 nm. The docetaxel liposomes of the present invention are prepared by a process comprising the steps of comprising the steps of:a. dispersing Soya Phosphatidyl Choline in solvent mixture to solubilize Soya Phosphatidyl Choline;b. adding sodium cholesteryl sulfate to the solubilized Soya Phosphatidyl Choline;c. adding docetaxel to contents of step b;d. preparing sucrose solution by dissolving sucrose in purified water and adding the pH adjusting agent to form the sucrose solution, wherein the pH of sucrose solution is about 3.e. adding contents of step c to step d and mixing with high hear at 8000 RPM for 15 minutesf. rota evaporation;g. addition of pH adjusting agent to pH of about 3;h. extrusion of liposomes containing docetaxel to the particle size d90of less than 200 nm;i. filtration andj. lyophilization. Examples of the solvents are selected from the group consisting of methanol, ethanol (anhydrous alcohol), propanol, butanol (t-butanol, tertiary butyl alcohol), chloroform, isoamyl alcohol, isopropanol, 2-methoxy ethanol, Tetrahydrofuran, DMSO acetone, acetonitrile and any combinations thereof. The solvents preferably used for the preparation of liposomal composition is tertiary butyl alcohol and methanol in the ratio of 1:1. In a preferred embodiment, the present invention relates to the method of preparing liposomal composition comprising the steps of:a. dispersing Soya Phosphatidyl Choline in solvent mixture of methanol and tertiary butyl alcohol to solubilize Soya Phosphatidyl Choline;b. adding sodium cholesteryl sulfate to the solubilized Soya Phosphatidyl Choline;c. adding docetaxel to contents of step b;d. preparing sucrose solution by dissolving sucrose in purified water and adding the pH adjusting agent to form the sucrose solution, wherein the pH of sucrose solution is about 3.e. adding contents of step c to step d and mixing with high hear at 8000 RPM for 15 minutesf. rota evaporation;g. addition of pH adjusting agent to pH of about 3;h. extrusion of liposomes containing docetaxel to the particle size d90of less than 200 nm;i. filtration andj. lyophilization. In a more preferred embodiment, the present invention relates to the method of preparing liposomal composition comprising the steps of:a. dispersing Soya Phosphatidyl Choline in solvent mixture of methanol and tertiary butyl alcohol in the ratio of 1:1 to solubilize Soya Phosphatidyl Choline;b. adding sodium cholesteryl sulfate to the solubilized Soya Phosphatidyl Choline;c. adding docetaxel to contents of step b;d. preparing sucrose solution by dissolving sucrose in purified water and adding the pH adjusting agent to form the sucrose solution, wherein the pH of sucrose solution is about 3.e. adding contents of step c to step d and mixing with high hear at 8000 RPM for 15 minutesf. rota evaporation;g. addition of pH adjusting agent to pH of about 3;h. extrusion of liposomes containing docetaxel to the particle size d90of less than 200 nm;i. filtration andj. lyophilization. In another embodiment liposomal docetaxel liquid filtrate is lyophilized by comprising the steps of freezing the filtrate at temperature ranging from about −5° C. to about −50° C. for the time duration ranging from about 10 hours to about 20 hours; drying under vacuum at a temperature ranging from about −50° C. to about 40° C. for time duration ranging from about 40 hours to about 80 hours. In a further specific embodiment liposomal docetaxel liquid filtrate is lyophilized by comprising the steps ofa. Loading the filtrate filled vials at −5° C.±2° C.;b. Freezing the filtrate formulation at −5° C.±2° C. for 100 minutes±20 minutesc. Maintaining the freezing temperature for another 300 minutes±20 minutesd. Reducing the temperature up to −25° C.±2° C. for 50 minutes±10 minutese. Maintaining the reduced temperature for another 90 minutes±10 minutesf. Reducing the temperature up to −50° C.±2° C. for 60 minutes±10 minutesg. Maintaining the reduced temperature for another 300 minutes±10 minutesh. Evacuating the filtrate by creating vacuum of 750 m Torr to obtain frozen formulationi. Drying the frozen formulation at −50° C.±2° C. by creating vacuum at 750 m Torr for 30 minutes±10 minutesj. Drying the frozen formulation at −35° C.±2° C. by creating vacuum at 400 m Torr for 120 minutes±10 minutesk. Maintaining the drying for another 1255 minutes±20 minutes at −35° C.±2° C. and 400 m Torr vacuum.l. Drying the frozen formulation at −25° C.±2° C. by creating vacuum at 300 m Torr for 150 minutes±10 minutesm. Maintaining the drying for another 600 minutes±20 minutes at −25° C.±2° C. and 300 m Torr vacuum.n. Drying the frozen formulation at −5° C.±2° C. by creating vacuum at 200 m Torr for 150 minutes±10 minuteso. Maintaining the drying for another 900 minutes±20 minutes at −5° C.±2° C. and 200 m Torr vacuum.p. Drying the frozen formulation at 20° C.±2° C. by creating vacuum at 100 m Torr for 150 minutes±10 minutesq. Maintaining the drying for another 300 minutes±20 minutes at 20° C.±2° C. and 100 m Torr vacuum.r. Drying the frozen formulation at 25° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutess. Maintaining the drying for another 150 minutes±20 minutes at 25° C.±2° C. and 100 m Torr vacuum.t. Drying the frozen formulation at 40° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutesu. Maintaining the drying for another 120 minutes±20 minutes at 40° C.±2° C. and 100 m Torr vacuumv. Drying the frozen formulation at 25° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutesw. Maintaining the drying for another 60 minutes±20 minutes at 25° C.±2° C. and 100 m Torr vacuum. In the embodiments of the present invention, the lyophilized composition of present invention is administered intravenously to the patients for the treatment of breast cancer, Non-small cell lung cancer, Castration resistant prostate cancer, gastric adenocarcinoma and Squamous Cell Carcinoma of the Head and Neck Cancer. In embodiments of the invention for intravenous administration, the lyophilized composition is reconstituted with purified water and further diluted with either 0.9% sodium chloride solution or 5% dextrose solution. In embodiments of the invention the docetaxel liposomal composition of present invention is used for the treatment of breast cancer, Non-small cell lung cancer, Castration resistant prostate cancer, gastric adenocarcinoma and Squamous Cell Carcinoma of the Head and Neck Cancer, wherein the pre-medication with prednisone is not required. In embodiments of the invention the recommended dose of liposomal composition of present invention is 60 mg/m2to 100 mg/m2administered intravenously over 1 hour every 3 weeks. The following examples are provided to illustrate the present invention. It is understood, however, that the invention is not limited to the specific conditions or details described in the examples below. The examples should not be construed as limiting the invention as the examples merely provide specific methodology useful in the understanding and practice of the invention and its various aspects. While certain preferred and alternative embodiments of the invention have been set forth for purposes of disclosing the invention, modification to the disclosed embodiments can occur to those who are skilled in the art. Examples 1 to 3 Liposomal injection with the following compositions are prepared. Example 1Example 2Example 3Ingredients(% w/w)(% w/w)(% w/w)Docetaxel anhydrous0.84%-1%0.84%-0.87%0.84%-0.87%Soya Phosphatidyl Choline30%-38%30%-38%30%-38%Sodium Cholesteryl Sulfate0.2%-0.8%0.2%-0.8%0.2%-0.8%MPEG2000-DSPE—0.5%-2%—(N-(Carbonylmethoxy-polyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine)Vitamin E TPGS——0.5%-1.5%Sucrose61%-68%61%-68%61%-68%Solvent mixtureQ.SQ.SQ.S0.1N Hydrochloric acid asQ.SQ.SQ.SpH adjuster These Liposomal injections are prepared as follows:1. Prepare solvent mixture of solubilization of Lipids and Drug by mixing 50:50 v/v of Methanol and T-Butanol mixture.2. To 4 mL of the solvent mixture of step 1, add and solubilize, weighed quantity of Soya Phosphatidyl Choline, at 60-65° C.3. To the Lipid solution of step 2, add and solubilize, weighed quantity of Sodium Cholesteryl Sulfate, at 60-65° C.4. To the Lipid solution of step 3, add and solubilize, weighed quantity of MPEG 2000 DSPE/Vitamin E TPGS, at 60-65° C. (optionally).5. To the Lipid mixture of step 4, add and solubilize, weighed quantity of Docetaxel at 60-65° C.6. Mix the contents of step 4 for 10 minutes at 60-65° C. for uniform binding of docetaxel with the lipids.7. Prepare sucrose solution by dissolving the weighed quantity of sucrose in purified water equivalent to 75% of the batch size.8. Prepare 0.1 N Hydrochloric acid by diluting the required quantity of 37% concentrated HCl.9. Adjust the pH of the sucrose solution prepared in the step 7 between pH 3.0 to 4.0 using the 0.1N HCl.10. Heat the Sucrose solution of step 8 to 60-65° C.11. Add the Drug Lipid Mixture to Sucrose solution under High Shear Mixing at 5000-20000 RPM by Ethanol Injection method and rinse the container containing Lipid Drug mixture with 1 mL of Solvent Mixture and run the mixture for 2 to 30 minutes.12. Volume was made up to the mark of the liposomal formulation of step 11, with purified water and if required pH was adjusted to between 3.0 to 4.0.13. Size reduction of liposomal formulation of step 12, was done using extrusion with 400 nm, 200 nm, 100 nm and 50 nm polycarbonate filters.14. Post size reduction, the liposomal formulation is sterile filtered using 0.22-micron filter.15. Post sterile filtration, the samples are freeze dried to get the dry liposomal cake for Injection. Example 4 Example 4Ingredients(% w/w)Docetaxel anhydrous0.91Soya Phosphatidyl Choline32.05Sodium Cholesteryl Sulfate0.5Sucrose66.54MethanolQ.STertiary butyl alcoholQ.S0.1N Hydrochloric acid as pH adjusterQ.S to pH 3Purified waterQ.S Process for Preparation of Liposomes Encapsulated with Docetaxel:1. 14.46 g (32.05% w/w) of soya phosphatidyl choline was dispersed in 8 mL of solvent mixture (4 mL of tertiary butyl alcohol and 4 mL of methanol in ratio of 1:1) and mixed with magnetic stirrer in water bath at 47° C. for 25 minutes to solubilize soya phosphatidyl choline.2. To the solubilized sodium phosphatidyl choline solution of step 1, 226.6 mg (0.5% w/w) of sodium cholesteryl sulfate was added and solubilised with magnetic stirrer in water bath at 49° C. for 45 minutes to form the dispersed liposomes.3. To the contents of step 3, 412.1 mg (0.91% w/w) of solid docetaxel anhydrous was added and solubilized with magnetic stirrer in water bath at 46° C. for 10 minutes to form docetaxel containing dispersed liposomes.4. The contents of step 3, as added to sucrose solution (sucrose solution was prepared by dissolving 30.02 g [66.54% w/w] of sucrose in required quantity of purified water with magnetic stirrer in water bath at 45° C. for 2 minutes and further pH is adjusted to 2.7 using 0.1N hydrochloric acid solution) using high shear mixing ultra-turrax T-25 digital at 8000 RPM for 15 minutes.5. The contents of step 4, was subjected to rota evaporation with chiller temperature of 2° C., bath temperature of 46° C. with vacuum.6. After rota evaporation, to contents of step 5, required quantity of purified water was added and pH was adjusted to 3 using 0.1N hydrochloric acid solution.7. The contents of step 6, was extruded with 200 nm, 100 nm, 80 nm and 50 nm polycarbonate membranes using lipex extruder at 47° C. Three cycles of passing resulted in a liposome with particle size d90 of less than 200 nm (i.e 178 nm), d50 of less than 150 nm (i.e 113 nm) and d10 of less than 100 nm (i.e 73 nm)8. The contents of step 7, was filtered using 0.2 μm membrane filter.9. The filtrate of step 8, was filled into 30 mL moulded vials and lyophilized using the following lyo cycle.a. Loading the filtrate filled vials at −5° C.±2° C.;b. Freezing the filtrate formulation at −5° C.±2° C. for 100 minutes±20 minutesc. Maintaining the freezing temperature for another 300 minutes±20 minutesd. Reducing the temperature up to −25° C.±2° C. for 50 minutes±10 minutese. Maintaining the reduced temperature for another 90 minutes±10 minutesf. Reducing the temperature up to −50° C.±2° C. for 60 minutes±10 minutesg. Maintaining the reduced temperature for another 300 minutes±10 minutesh. Evacuating the filtrate by creating vacuum of 750 m Torr to obtain frozen formulationi. Drying the frozen formulation at −50° C.±2° C. by creating vacuum at 750 m Torr for 30 minutes±10 minutesj. Drying the frozen formulation at −35° C.±2° C. by creating vacuum at 400 m Torr for 120 minutes±10 minutesk. Maintaining the drying for another 1255 minutes±20 minutes at −35° C.±2° C. and 400 m Torr vacuum.l. Drying the frozen formulation at −25° C.±2° C. by creating vacuum at 300 m Torr for 150 minutes±10 minutesm. Maintaining the drying for another 600 minutes±20 minutes at −25° C.±2° C. and 300 m Torr vacuum.n. Drying the frozen formulation at −5° C.±2° C. by creating vacuum at 200 m Torr for 150 minutes±10 minuteso. Maintaining the drying for another 900 minutes±20 minutes at −5° C.±2° C. and 200 m Torr vacuum.p. Drying the frozen formulation at 20° C.±2° C. by creating vacuum at 100 m Torr for 150 minutes±10 minutesq. Maintaining the drying for another 300 minutes±20 minutes at 20° C.±2° C. and 100 m Torr vacuum.r. Drying the frozen formulation at 25° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutess. Maintaining the drying for another 150 minutes±20 minutes at 25° C.±2° C. and 100 m Torr vacuum.t. Drying the frozen formulation at 40° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutesu. Maintaining the drying for another 120 minutes±20 minutes at 40° C. 2° C. and 100 m Torr vacuumv. Drying the frozen formulation at 25° C.±2° C. by creating vacuum at 100 m Torr for 30 minutes±10 minutesw. Maintaining the drying for another 60 minutes±20 minutes at 25° C.±2° C. and 100 m Torr vacuum. Comparative Example 1 Comparative Example 1Ingredients(% w/w)Docetaxel anhydrous0.91Soya Phosphatidyl Choline32.05Sodium Cholesteryl Sulfate0.5Sucrose66.54MethanolQ.SEthanolQ.S0.1N Hydrochloric acid as pH adjusterQ.S to pH 4Purified waterQ.S The process for preparation is same as that of Example 4, with the changes in solvent mixture of Methanol and Ethanol in ratio of 1:1, pH of 4, without rota evaporation process (step 5) and change in lyophilization cycle (without 40° C. drying step t and u). Comparative Example 2 Comparative Example 2Ingredients(% w/w)Docetaxel anhydrous0.91Soya Phosphatidyl Choline32.05Sodium Cholesteryl Sulfate0.5Sucrose66.54MethanolQ.STertiary butyl alcoholQ.S0.1N Hydrochloric acid as pH adjusterQ.S to pH 4.5Purified waterQ.S The process for preparation is same as that of Example 4, with the changes in pH of formulation adjusted to 4.5 Comparative Example 3 Comparative Example 3Ingredients(% w/w)Docetaxel anhydrous0.91Soya Phosphatidyl Choline32.05Sodium Cholesteryl Sulfate0.5Sucrose66.54MethanolQ.STertiary butyl alcoholQ.S0.1N Hydrochloric acid as pH adjusterQ.S to pH 3Purified waterQ.S The process for preparation is same as that of Example 4, with change in lyophilization cycle (without 40° C. drying step t and u). Example 5: Free Drug, Entrapped Drug, Assay, pH, Residual Solvents of Example-4, Comparative Example 1, 2 & 3 ComparativeComparativeComparativeTestExample 4Ex.1Ex.2Ex.3Free Drug11.1%29.1%56.3%8.8%Entrapped Drug94.8%63.7%47.3%88.1%Assay103.6%93.5%103.3%96.0%pH3.14.14.73Residual SolventsMethanol384 ppm——1766 ppmT-Butanol741 ppm——5672 ppmEthanol———— For the measurement of pH, the lyophilized vial of inventive example 4 was reconstituted with purified water to produce 2 mg/mL liposomal formulation of docetaxel. The Free Drug, Entrapped Drug, Assay of docetaxel liposomal formulation was performed by HPLC and Residual Solvent analysis was performed by Gas chromatography as per the available literature to the personal skilled in the art. The inventors of present invention have surprisingly found that the example 4 formulation has high drug loading efficiency (about 95%) with the solvents of methanol and tertiary butanol in the ratio of 1:1, rota evaporated, with the formulation pH of about 3 and further the residual solvents (methanol and tertiary butanol) are less (within the limits of 1CH) in comparison to comparative example 1 (containing solvent mixture of ethanol and methanol in ratio of 1:1 at pH of 4.1), comparative example 2 (containing methanol and T-butanol in ratio of 1:1 at pH of 4.7) and comparative example 3 (containing methanol and T-butanol in ratio of 1:1 at pH of 3, without the drying step at 40° C. in lyophilization step t and u of example 4). | 26,311 |
11857681 | MODES FOR CARRYING OUT THE INVENTION RNA Replicons Various replicons are used below. In general these are based on a hybrid alphavirus genome with non-structural proteins from Venezuelan equine encephalitis virus (VEEV), a packaging signal from sindbis virus, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10kb long and has a poly-A tail. Plasmid DNA encoding alphavirus replicons (named: pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for synthesis of RNA in vitro. The replicons contain the alphavirus genetic elements required for RNA replication but lack those encoding gene products necessary for particle assembly; the structural proteins are instead replaced by a protein of interest (either a reporter, such as SEAP or GFP, or an immunogen, such as full-length RSV F protein) and so the replicons are incapable of inducing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity. Following linearization of the plasmid DNA downstream of the HDV ribozyme with a suitable restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). Following transcription the template DNA was digested with TURBO DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was capped post-transcriptionally with Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G Capping System (Epicentre Biotechnologies) as outlined in the user manual; replicons capped in this way are given the “v” prefix e.g. vA317 is the A317 replicon capped by VCE. Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring OD260nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis. Liposomal Encapsulation RNA was encapsulated in liposomes made by the method of references 11 and 42. The liposomes were made of 10% DSPC (zwitterionic), 40% DLinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % moles in the total liposome. DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 6. DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids. 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol was obtained from NOF Corporation (catalog #GM-020). Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 hour of equilibration. The mixture was diluted with 6 ml buffer then filtered. The resulting product contained liposomes, with ˜95% encapsulation efficiency. For example, in one particular method, fresh lipid stock solutions were prepared in ethanol. 37 mg of DLinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used has a 2 mm internal diameter and a 3 mm outer diameter). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of FEP tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation). Before using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through it. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using by tangential flow filtration before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes with a 100 kD pore size cutoff and 8 cm2surface area were used. For in vitro and in vivo experiments formulations were diluted to the required RNA concentration with 1×PBS. FIG.2shows an example electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number). The percentage of encapsulated RNA and RNA concentration were determined by Quant-iT RiboGreen RNA reagent kit (Invitrogen), following manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10× or 100× in 1×TE buffer (from kit) before addition of the dye. Separately, liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% Triton X before addition of the dye (to disrupt the liposomes and thus to assay total RNA). Thereafter an equal amount of dye was added to each solution and then ˜180 μL of each solution after dye addition was loaded in duplicate into a 96 well tissue culture plate. The fluorescence (Ex 485 nm, Em 528 nm) was read on a microplate reader. All liposome formulations were dosed in vivo based on the encapsulated amount of RNA. Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8mAU of RNase A per microgram of RNA, incubated for 30 minutes at room temperature. RNase was inactivated with Proteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to 25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added to extract the RNA from the lipids into the aqueous phase. Samples were mixed by vortexing for a few seconds and then placed on a centrifuge for 15 minutes at 12 k RPM. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Prior to loading (400 ng RNA per well) all the samples were incubated with formaldehyde loading dye, denatured for 10 minutes at 65° C. and cooled to room temperature. Ambion Millennium markers were used to approximate the molecular weight of the RNA construct. The gel was run at 90 V. The gel was stained using 0.1% SYBR gold according to the manufacturer's guidelines in water by rocking at room temperature for 1 hour.FIG.1shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4), and no change is seen if these liposomes are treated with RNase (lane 4). After RNase-treated liposomes are subjected to phenol extraction, undigested RNA is seen (lane 6). Even after 1 week at 4° C. the RNA could be seen without any fragmentation (FIG.4, arrow). Protein expression in vivo was unchanged after 6 weeks at 4° C. and one freeze-thaw cycle. Thus liposome-encapsulated RNA is stable. To assess in vivo expression of the RNA a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1× Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50 μl per leg with 0.1 μg or 1 μg RNA dose. The same vector was also administered without the liposomes (in RNase free 1×PBS) at 1 μg. Virion-packaged replicons were also tested. Virion-packaged replicons used herein (referred to as “VRPs”) were obtained by the methods of reference 43, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the genome of VEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbis virus packaging signal (PS), packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes. As shown inFIG.5, encapsulation increased SEAP levels by about ½ log at the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulated dose matched levels seen with 1 μg unencapsulated dose. By day 3 expression levels exceeded those achieved with VRPs (squares). Thus expressed increased when the RNA was formulated in the liposomes relative to the naked RNA control, even at a 10×lower dose. Expression was also higher relative to the VRP control, but the kinetics of expression were very different (seeFIG.5). Delivery of the RNA with electroporation resulted in increased expression relative to the naked RNA control, but these levels were lower than with liposomes. To assess whether the effect seen in the liposome groups was due merely to the liposome components, or was linked to the encapsulation, the replicon was administered in encapsulated form (with two different purification protocols, 0.1 μg RNA), or mixed with the liposomes after their formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as naked RNA (1 μg).FIG.10shows that the lipoplex gave the lowest levels of expression, showing that shows encapsulation is essential for potent expression. In vivo studies using liposomal delivery confirmed these findings. Mice received various combinations of (i) self-replicating RNA replicon encoding full-length RSV F protein (ii) self-replicating GFP-encoding RNA replicon (iii) GFP-encoding RNA replicon with a knockout in nsP4 which eliminates self-replication (iv) full-length RSV F-protein. 13 groups in total received: A——B0.1 μg of (i), naked—C0.1 μg of (i), encapsulated in—liposomeD0.1 μg of (i), with separate—liposomesE0.1 μg of (i), naked10 μg of (ii), nakedF0.1 μg of (i), naked10 μg of (iii), nakedG0.1 μg of (i), encapsulated in10 μg of (ii), nakedliposomeH0.1 μg of (i), encapsulated in10 μg of (iii), nakedliposomeI0.1 μg of (i), encapsulated in1 μg of (ii), encapsulated inliposomeliposomeJ0.1 μg of (i), encapsulated in1 μg of (iii), encapsulated inliposomeliposomeK5 μg F protein—L5 μg F protein1 μg of (ii), encapsulated inliposomeM5 μg F protein1 μg of (iii), encapsulated inliposome Results inFIG.18show that F-specific IgG responses required encapsulation in the liposome rather than mere co-delivery (compare groups C & D). A comparison of groups K, L and M shows that the RNA provided an adjuvant effect against co-delivered protein, and this effect was seen with both replicating and non-replicating RNA. Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1 ng RNA (FIG.6). Further experiments comparing expression from encapsulated and naked replicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-fold higher expression at day 6; at a 0.1p g dose levels were 24-fold higher at day 6. Rather than looking at average levels in the group, individual animals were also studied. Whereas several animals were non-responders to naked replicons, encapsulation eliminated non-responders. Further experiments replaced DLinDMA with DOTAP. Although the DOTAP liposomes gave better expression than naked replicon, they were inferior to the DLinDMA liposomes (2- to 3-fold difference at day 1). Whereas DOTAP has a quaternary amine, and so have a positive charge at the point of delivery, DLinDMA has a tertiary amine. To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (106IU; “VRP”) at days 0 and 21.FIG.7shows anti-F IgG titers 2 weeks after the second dose, and the liposomes clearly enhance immunogenicity.FIG.8shows titers 2 weeks later, by which point there was no statistical difference between the encapsulated RNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group. Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) were not significantly different in these three groups 2 weeks after the second dose (FIG.9).FIG.12shows both IgG and PRNT titers 4 weeks after the second dose. FIG.13confirms that the RNA elicits a robust CD8 T cell response. Further experiments compared F-specific IgG titers in mice receiving VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. Titer ratios (VRP:liposome) at various times after the second dose were as follows: 2 weeks4 weeks8 weeks0.1 μg2.91.01.11 μg2.30.90.9 Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery. Further experiments showed superior F-specific IgG responses with a 10 μg dose, equivalent responses for fig and 0.1 μg doses, and a lower response with a 0.01 μg dose.FIG.11shows IgG titers in mice receiving the replicon in naked form at 3 different doses, in liposomes at 4 different doses, or as VRP (106IU). The response seen with 1 μg liposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the higher response seen with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups. A further study confirmed that the 0.1 μg of liposome-encapsulated RNA gave much higher anti-F IgG responses (15 days post-second dose) than 0.1 μg of delivered DNA, and even was more immunogenic than 20 μg plasmid DNA encoding the F antigen, delivered by electroporation (Elgen™ DNA Delivery System, Inovio). A further study was performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose liposome encapsulation increased F-specific IgG titers by 8.3-fold compared to naked RNA and increased PRNT titers by 9.5-fold. The magnitude of the antibody response was equivalent to that induced by 5×106IU VRP. Both naked and liposome-encapsulated RNA were able to protect the cotton rats from RSV challenge (1×105plaque forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction by about 2-fold. A large-animal study was performed in cattle. Cows were immunised with 66 μg of replicon encoding full-length RSV F protein at days 0 and 21, formulated inside liposomes. PBS alone was used as a negative control, and a licensed vaccine was used as a positive control (“Triangle 4” from Fort Dodge, containing killed virus).FIGS.14A and14Bshow F-specific IgG titers over 63 day and 210 day periods, respectively, starting from the first immunisation. The RNA replicon was immunogenic in the cows, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose, and titers were very stable from the period of 2 to 6 weeks after the second dose (and were particularly stable for the RNA vaccine). Mechanism of Action Bone marrow derived dendritic cells (pDC) were obtained from wild-type mice or the “Resq” (rsq1) mutant strain. The mutant strain has a point mutation at the amino terminus of its TLR7 receptor which abolishes TLR7 signalling without affecting ligand binding as disclosed in reference 44. The cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000 or inside a liposome. As shown inFIGS.19A and19B, IL-6 and INFα, respectively, were induced in WT cells but this response was almost completely abrogated in mutant mice. These results shows that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines. pKa Measurement The pKa of a lipid is measured in water at standard temperature and pressure using the following technique:2 mM solution of lipid in ethanol is prepared by weighing the lipid and dissolving in ethanol. 0.3 mM solution of fluorescent probe 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) in ethanol:methanol 9:1 is prepared by first making 3 mM solution of TNS in methanol and then diluting to 0.3 mM with ethanol.An aqueous buffer containing sodium phosphate, sodium citrate sodium acetate and sodium chloride, at the concentrations 20 mM, 25 mM, 20 mM and 150 mM, respectively, is prepared. The buffer is split into eight parts and the pH adjusted either with 12N HCl or 6N NaOH to 4.44-4.52, 5.27, 6.15-6.21, 6.57, 7.10-7.20, 7.72-7.80, 8.27-8.33 and 10.47-11.12. 400 L of 2 mM lipid solution and 800 L of 0.3 mM TNS solution are mixed.7.5 μL of probe/lipid mix are added to 242.5 μL of buffer in a 1 mL 96 well plate. This is done with all eight buffers. After mixing, 100 L of each probe/lipid/buffer mixture is transferred to a 250 L black with clear bottom 96 well plate (e.g. model COSTAR 3904, Corning). A convenient way of performing this mixing is to use the Tecan Genesis RSP150 high throughput liquid handler and Gemini Software.Fluorescence of each probe/lipid/buffer mixture is measured (e.g. with a SpectraMax M5 spectrophotometer and SoftMax pro 5.2 software) with 322 nm excitation, 431 nm emission (auto cutoff at 420 nm).After the measurement, the background fluorescence value of an empty well on the 96 well plate is subtracted from each probe/lipid/buffer mixture. The fluorescence intensity values are then normalized to the value at lowest pH. The normalized fluorescence intensity is then plotted against pH and a line of best fit is provided.The point on the line of best fit at which the normalized fluorescence intensity is equal to 0.5 is found. The pH corresponding to normalized fluorescence intensity equal to 0.5 is found and is considered the pKa of the lipid. This method gives a pKa of 5.8 for DLinDMA. The pKa values measured by this method for cationic lipids of reference 5 are included below. Encapsulation in Liposomes Using Alternative Cationic Lipids As an alternative to using DlinDMA, the cationic lipids of reference 5 are used. These lipids can be synthesised as disclosed in reference 5. The liposomes formed above using DlinDMA are referred to hereafter as the “RV01” series. The DlinDMA was replaced with various cationic lipids in series “RV02” to “RV12” as described below. Two different types of each liposome were formed, using 2% PEG2000-DMG with either (01) 40% of the cationic lipid, 10% DSPC, and 48% cholesterol, or (02) 60% of the cationic lipid and 38% cholesterol. Thus a comparison of the (01) and (02) liposomes shows the effect of the neutral zwitterionic lipid. RV02 liposomes were made using the following cationic lipid (pKa>9, without a tertiary amine): RV03 liposomes were made using the following cationic lipid (pKa 6.4): RV04 liposomes were made using the following cationic lipid (pKa 6.62): RV05 liposomes were made using the following cationic lipid (pKa 5.85): RV06 liposomes were made using the following cationic lipid (pKa 7.27): RV07 liposomes were made using the following cationic lipid (pKa 6.8): RV08 liposomes were made using the following cationic lipid (pKa 5.72): RV09 liposomes were made using the following cationic lipid (pKa 6.07): RV10 liposomes were made for comparison using the following cationic lipid (pKa 7.86): RV11 liposomes were made using the following cationic lipid (pKa 6.41): RV12 liposomes were made using the following cationic lipid (pKa 7): RV16 liposomes were made using the following cationic lipid (pKa 6.1), as disclosed in reference 45: RV17 liposomes were made using the following cationic lipid (pKa 6.1), as disclosed in reference 45: RV18 liposomes were made using DODMA. RV19 liposomes were made using DOTMA, and RV13 liposomes were made with DOTAP, both having a quaternary amine headgroup. These liposomes were characterised and were tested with the SEAP reporter described above. The following table shows the size of the liposomes (Z average and polydispersity index), the % of RNA encapsulation in each liposome, together with the SEAP activity detected at days 1 and 6 after injection. SEAP activity is relative to “RV01(02)” liposomes made from DlinDMA, cholesterol and PEG-DMG: Lipid%SEAPSEAPRVpKaZav (pdI)encapsulationday 1day 6RV01 (01)5.8154.6 (0.131)95.580.971.1RV01 (02)5.8162.0 (0.134)85.3100100RV02 (01)>9133.9 (0.185)96.55745.7RV02 (02)>9134.6 (0.082)97.654.24.3RV03 (01)6.4158.3 (0.212)62.065.744.9RV03 (02)6.4164.2 (0.145)8662.239.7RV04 (01)6.62131.0 (0.145)74.091154.8RV04 (02)6.62134.6 (0.117)81.590.4142.6RV05 (01)5.85164.0 (0.162)76.076.9329.8RV05 (02)5.85177.8 (0.117)72.867.1227.9RV06 (01)7.27116.0 (0.180)79.825.512.4RV06 (02)7.27136.3 (0.164)74.924.823.1RV07 (01)6.8140.6 (0.184)7726.5163.3RV07 (02)6.8138.6 (0.122)8729.774.8RV 08 (01)5.72176.7 (0.185)5076.5187RV08 (02)5.72199.5 (0.191)46.382.4329.8RV09 (01)6.07165.3 (0.169)72.265.1453.9RV09 (02)6.07179.5 (0.157)6568.5658.2RV10 (01)7.86129.7 (0.184)78.4113.447.8RV10 (02)7.86147.6 (0.131)80.978.210.4RV11 (01)6.41129.2 (0.186)71113.6242.2RV11 (02)6.41139 (0198)75.271.8187.2RV12 (01)7135.7 (0.161)78.86510RV12 (02)7158.3 (0.287)69.478.88.2 FIG.15plots the SEAP levels at day 6 against the pKa of the cationic lipids. The best results are seen where the lipid has a pKa between 5.6 and 6.8, and ideally between 5.6 and 6.3. These liposomes were also used to deliver a replicon encoding full-length RSV F protein. Total IgG titers against F protein two weeks after the first dose (2wp1) are plotted against pKa inFIG.16. The best results are seen where the pKa is where the cationic lipid has a pKa between 5.7-5.9, but pKa alone is not enough to guarantee a high titer e.g. the lipid must still support liposome formation. RSV Immunogenicity Further work was carried out with a self-replicating replicon (vA317) encoding RSV F protein. BALB/c mice, 4 or 8 animals per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon (1 μg) alone or formulated as liposomes with the RV01 or RV05 lipids (see above; pKa of 5.8 or 5.85) or with RV13. The RV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2% PEG-DMG, but with differing amounts of RNA. The RV05(01) liposomes had 40% cationic lipid, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; the RV05(02) liposomes had 60% cationic lipid, 38% cholesterol, and 2% PEG-DMG. The RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol and 2% PEG-DMG. For comparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigen was delivered either using electroporation or with RV01(10) liposomes (0.1 μg DNA). Four mice were used as a naïve control group. Liposomes were prepared by method (A) or method (B). In method (A) fresh lipid stock solutions in ethanol were prepared. 37 mg of cationic lipid, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 75 μg RNA to give an 8:1 nitrogen to phosphate ratio (except that in RV01 (08) and RV01 (09) this ratio was modified to 4:1 or 16:1). A 2 mL working solution of RNA (or, for RV01(10), DNA) was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc syringes. 2 mL of citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction) using FEP tubing. The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of FEP tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 hour. Then the mixture was loaded in a 5 cc syringe, which was fitted to a piece of FEP tubing and in another 5 cc syringe with equal length of FEP tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using a syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using TFF before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs and were used according to the manufacturer's guidelines. Polyethersulfone (PES) hollow fiber filtration membranes (part number P-C1-100E-100-O1N) with a 100 kD pore size cutoff and 20 cm2surface area were used. For in vitro and in vivo experiments, formulations were diluted to the required RNA concentration with 1×PBS. Preparation method (B) differed in two ways from method (A). Firstly, after collection in the 20 mL glass vial but before TFF concentration, the mixture was passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation, Ann Arbor, MI, USA). This membrane was first washed with 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) in turn, and liposomes were warmed for 10 min at 37° C. before being filtered. Secondly, the hollow fiber filtration membrane was Polysulfone (part number P/N: X1AB-100-20P). The Z average particle diameter, polydispersity index and encapsulation efficiency of the liposomes were as follows: RVZav (nm)pdI% encapsulationPreparationRV01 (10)158.60.08890.7(A)RV01 (08)156.80.14488.6(A)RV01 (05)136.50.13699(B)RV01 (09)153.20.06776.7(A)RV05 (01)1480.12780.6(A)RV05 (02)177.20.13672.4(A)RV01 (10)134.70.14787.8 *(A)RV13 (02)128.30.17997(A)* For this RV01(10) formulation the nucleic acid was DNA not RNA Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were harvested from mice at day 49 for T cell analysis. F-specific serum IgG titers (GMT) were as follows: RVDay 14Day 36Naked DNA plasmid4396712Naked A317 RNA782291RV01 (10)302026170RV01 (08)23269720RV01 (05)535254907RV01 (09)442851316RV05 (01)13565346RV05 (02)9616915RV01 (10) DNA513RV13 (02)6443616 The proportion of T cells which are cytokine-positive and specific for RSV F51-66 peptide are as follows, showing only figures which are statistically significantly above zero: CD4+CD8−CD4−CD8+RVIFNγIL2IL5TNFαIFNγIL2IL5TNFαNaked DNA plasmid0.040.070.100.570.290.66Naked A317 RNA0.040.050.080.570.230.67RV01 (10)0.070.100.131.300.591.32RV01 (08)0.020.040.060.460.300.51RV01 (05)0.080.120.151.900.681.94RV01 (09)0.060.080.091.620.671.71RV05 (01)0.060.040.19RV05 (02)0.050.070.110.640.350.69RV01 (10) DNA0.030.08RV13 (02)0.030.040.061.150.411.18 Thus the liposome formulations significantly enhanced immunogenicity relative to the naked RNA controls, as determined by increased F-specific IgG titers and T cell frequencies. Plasmid DNA formulated with liposomes, or delivered naked using electroporation, was significantly less immunogenic than liposome-formulated self-replicating RNA. The RV01 and RV05 RNA vaccines were more immunogenic than the RV13 (DOTAP) vaccine. These formulations had comparable physical characteristics and were formulated with the same self-replicating RNA, but they contain different cationic lipids. RV01 and RV05 both have a tertiary amine in the headgroup with a pKa of about 5.8, and also include unsaturated alkyl tails. RV13 has unsaturated alkyl tails but its headgroup has a quaternary amine and is very strongly cationic. These results suggest that lipids with tertiary amines with pKas in the range 5.0 to 7.6 are superior to lipids such as DOTAP, which are strongly cationic, when used in a liposome delivery system for RNA. Further Alternatives to DLinDMA The cationic lipid in RV01 liposomes (DLinDMA) was replaced by RV16, RV17, RV18 or RV19. Total IgG titers are shown inFIG.17. The lowest results are seen with RV19 i.e. the DOTMA quaternary amine. BHK Expression Liposomes with different lipids were incubated with BHK cells overnight and assessed for protein expression potency. From a baseline with RV05 lipid expression could be increased 18× by adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposome, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× by instead using RV01. RSV Immunogenicity in Different Mouse Strains Replicon “vA142” encodes the full-length wild type surface fusion (F) glycoprotein of RSV but with the fusion peptide deleted, and the 3′ end is formed by ribozyme-mediated cleavage. It was tested in three different mouse strains. BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 22. Animals were divided into 8 test groups (5 animals per group) and a naïve control (2 animals):Group 1 were given naked replicon (1 μg).Group 2 were given 1 μg replicon delivered in liposomes “RV01(37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-conjugated DMG.Group 3 were given the same as group 2, but at 0.1 g RNA.Group 4 were given 1 μg replicon in “RV17(10)” liposomes (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG).Group 5 were 1 μg replicon in “RV05(11)” liposomes (40% RV07 lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG).Group 6 were given 0.1 μg replicon in “RV17(10)” liposomes.Group 7 were given 5 μg RSV-F subunit protein adjuvanted with aluminium hydroxide.Group 8 were a naïve control (2 animals) Sera were collected for antibody analysis on days 14, 35 and 49. F-specific serum IgG GMTs were: Day1234567814822463178924961171129512935351538341812560523579137188887738095 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG1234567IgG1946238483674258288181778604IgG2a5386770645908433749144371762424 RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group): Day1234567835<20143201013230111<2049<20139<208341321009<20 Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD4+ or CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F51-66, F164-178, F309-323 for CD4+, or for peptides F85-93 and F249-258 for CD8+): CD4+CD8−CD4−CD8+GroupIFNγIL2IL5TNFαIFNγIL2IL5TNFα10.030.060.080.470.290.4820.050.100.081.350.521.1130.030.070.060.640.310.6140.050.090.071.170.651.0950.030.080.070.650.280.5860.050.070.070.740.360.6670.020.040.048 C57BL/6 mice were immunised in the same way, but a 9th group received VRPs (1×106IU) expressing the full-length wild-type surface fusion glycoprotein of RSV (fusion peptide deletion). Sera were collected for antibody analysis on days 14, 35 & 49. F-specific IgG titers (GMT) were: Day123456789141140213310262792304513302975511013517215532318438829525240939251512139 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG12345678IgG16624714328468925625879IgG2a2170768550556161157329443514229 RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group): Day12345678935<2027292236<2028<20<2049<2044302336<2033<2037 Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F85-93 and F249-258): CD4−CD8+GroupIFNγIL2IL5TNFα10.420.130.3721.210.371.0231.010.260.7741.260.230.9352.130.701.7760.590.190.4970.100.05892.830.722.26 Nine groups of C3H/HeN mice were immunised in the same way. F-specific IgG titers (GMT) were: Day12345678914520491666110229898435195806351522775419008176933424610062297517249 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG12345678IgG1513231702111363483114189IgG2a30213694178424673851566727085380072727 RSV serum neutralizing antibody titers at days 35 and 49 were as follows: Day12345678935<205392606510195443<2059549<2045629635821251148<20387 Thus three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) were tested in three different inbred mouse strains. For all 3 strains RV01 was more effective than RV17; for BALB/c and C3H strains RV05 was less effective than either RV01 or RV17, but it was more effective in B6 strain. In all cases, however, the liposomes were more effective than two cationic nanoemulsions which were tested in parallel. CMV Immunogenicity RV01 liposomes with DLinDMA as the cationic lipid were used to deliver RNA replicons encoding cytomegalovirus (CMV) glycoproteins. The “vA160” replicon encodes full-length glycoproteins H and L (gH/gL), whereas the “vA322” replicon encodes a soluble form (gHsol/gL). The two proteins are under the control of separate subgenomic promoters in a single replicon; co-administration of two separate vectors, one encoding gH and one encoding gL, did not give good results. BALB/c mice, 10 per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0, 21 and 42 with VRPs expressing gH/gL (1×106IU), VRPs expressing gHsol/gL (1×106IU) and PBS as the controls. Two test groups received 1 g of the vA160 or vA322 replicon formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; made using method (A) as discussed above, but with 150 μg RNA batch size). The vA160 liposomes had a Zav diameter of 168 nm, a pdI of 0.144, and 87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, a pdI of 0.131, and 90% encapsulation. The replicons were able to express two proteins from a single vector. Sera were collected for immunological analysis on day 63 (3wp3). CMV neutralization titers (the reciprocal of the serum dilution producing a 50% reduction in number of positive virus foci per well, relative to controls) were as follows: gH/gL VRPgHsol/gL VRPgH/gL liposomegHsol/gL liposome45762393424010062 RNA expressing either a full-length or a soluble form of the CMV gH/gL complex thus elicited high titers of neutralizing antibodies, as assayed on epithelial cells. The average titers elicited by the liposome-encapsulated RNAs were at least as high as for the corresponding VRPs. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. REFERENCES [1] Johanning et al. (1995)Nucleic Acids Res 23:1495-1501.[2] WO2005/121348.[3] WO2008/137758.[4] WO2009/086558.[5] WO2011/076807.[6] Heyes et al. 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(eds) (2002)Short protocols in molecular biology,5th edition (Current Protocols).[39] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press)[40] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag)[41] Yoneyama & Fujita (2007)Cytokine&Growth Factor Reviews18:545-51.[42] Maurer et al. (2001)Biophysical Journal,80: 2310-2326.[43] Perri et al. (2003)J Virol77:10394-10403.[44] Iavarone et al. (2011)J Immunol186;4213-22.[45] WO2011/057020. | 39,755 |
11857682 | The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. DETAILED DESCRIPTION Dermal filler compositions containing HA such as Juvederm® are well-known in the art. Commercially available HA-based dermal fillers have greater than 20%, even greater than 40% of a soluble HA fraction. Because these products are under-hydrated, they can swell to greater than 300% of their volume post-injection, which causes injection site swelling and discomfort. The compositions and methods described herein avoid these problems of the prior art. By minimizing the weight percent of the soluble fraction with maximized crosslinking, post-injection swelling can be controlled. In addition, prior art HA dermal filler compositions are typically prepared, stored and injected in phosphate buffer solution such as phosphate buffered saline (PBS). Without being held to theory, it is believed that preparation of crosslinked HA in phosphate buffer results in aggregation of HA chains, and in particular high molecular weight aggregates. As shown herein, preparation of HA in 135 mM to 200 mM NaCl provides a reduction in self-association of HA segments which is expected to provide a higher quality dermal filler than the prior art compositions prepared in phosphate buffer. In addition, removal of low molecular weight HA fractions (<50, <100 and <250 kDa), which are prevalent in the soluble HA fraction, will alleviate FDA concerns that low molecular weight HA may be inflammatory and/or pro-fibrotic. In order to provide an improved modified HA preparation, the inventor has studied the relationship of fluid (soluble or non-modified hyaluronate) and gel (modified or crosslinked hyaluronate). The use of combined Dynamic Light Scattering (DLS) with Multi-angle (three or greater) laser light scattering (MALS) in-line with a refractive index detector (RI) provides excellent analytical tools for comparing Radius of Hydration (Rh) with Root Mean Square Radius (Rz). The relationship of Rh to Rz provides conformation information on the HA compositions. An Rh greater than Rz suggests a spherical structure of the HA. The composition that results in an Rz greater than Rh suggests an elongated, branched, or linear HA structure. Measuring the Rz and Rh allows one to manufacture a modified HA composition in a controlled manner to favor the spherical structure. For example:Rh>Rz=Globular and Spherical (Purified/retained modified hyaluronate network of polymer)Rh<Rz=Elongation and linear (Reduced non-modified hyaluronate) FIG.1is a schematic illustrating the purification of crude crosslinked hyaluronate to provide removed non-modified (linear and elongated) hyaluronate and retained modified spherical hyaluronate according to an aspect of the present disclosure. The use of combined Dynamic Light Scattering (DLS) with Multi-angle (three or greater) laser light scattering (MALS) in-line with a refractive index detector (RI) has allowed the inventor to identify modified HA preparations with improved properties as soft tissue/dermal fillers. Modified hyaluronate, as used herein, refers to hyaluronate having a high degree of crosslinking (e.g., greater than 10% crosslinking, defined as the percent weight ratio of the crosslinking agent to HA-monomeric units within the crosslinked portion of the HA) in a crosslinked portion and a low weight percentage of soluble, that is uncrosslinked, hyaluronate (e.g., <10% by weight of the HA is uncrosslinked, soluble HA). In an aspect, a method of making a modified hyaluronate composition comprises providing an aqueous solution of uncrosslinked hyaluronic acid or uncrosslinked sodium hyaluronate in unbuffered 135 mM to 200 mM NaCl, wherein the uncrosslinked hyaluronic acid or uncrosslinked sodium hyaluronate has a weight averaged molecular weight from 500-2000 kDa, and wherein the aqueous solution includes no phosphates. The uncrosslinked hyaluronic acid or uncrosslinked sodium hyaluronate in the aqueous solution is then crosslinked with a dialdehyde or disulfide crosslinking agent to provide crude modified hyaluronate having a soluble fraction and a crosslinked fraction with a 10% to 98% degree of crosslinking, preferably a 10% to 60% degree of crosslinking. The crude modified hyaluronate is then centrifuged to remove at least a portion of the soluble fraction. The centrifuging and removing is repeated until the modified hyaluronate has less than 10%, less than 5%, or less than 2% by weight of the soluble fraction based on the total weight of hyaluronate. The modified hyaluronate further has a soluble fraction polydispersity (Mw/Mn) of 1 to 1.6, an Rh of the crosslinked fraction of 150 nm to 2000 nm, and an Rz of the crosslinked fraction of 50 nm to 700 nm, wherein Rh>Rz. As used herein, the term “crosslinked” refers to the intermolecular bonds joining the individual polymer molecules, or monomer chains, into a more stable structure like a gel. As such, a crosslinked HA has at least one intermolecular bond joining at least one individual polymer molecule to another one. The crosslinking of HA typically result in the formation of a hydrogel. Degree of crosslinking as used herein refers to the intermolecular junctions joining the individual HA polymer molecules, or monomer chains, into a permanent structure, or as disclosed herein the soft tissue filler composition. Moreover, degree of crosslinking for purposes of the present disclosure is further defined as the percent weight ratio of the crosslinking agent to HA-monomeric units within the crosslinked portion of the HA based composition. It is measured by the weight ratio of crosslinker to HA monomers (crosslinker:HA monomers). Soluble HA, sometimes referred to as free HA, refers to individual HA polymer molecules that are not crosslinked, or very lightly crosslinked (very low degree of crosslinking). The highly crosslinked (higher degree of crosslinking) macromolecular structure makes up most of the soft tissue filler composition. Soluble HA generally remains water soluble. Soluble HA can alternatively be defined as the “uncrosslinked,” or lightly crosslinked component of the macromolecular structure making up the soft tissue filler composition disclosed herein. Based on the prior art such as U.S. Pat. No. 10,391,202, it is believed that a high percentage of soluble HA is required to provide sufficient injectability for use as a soft tissue filler. Specifically, the prior art HA gels can be considered as crosslinked HA in a relatively fluidic medium of free HA. The inventor of the present application has found that HA with an Rh greater than Rz, that is, HA with a spherical structure as illustrated inFIG.1, provides improved injectability by reducing swelling of the gel composition. Thus, a high amount of soluble HA is not required to provide injectability. In an aspect, the Rh:Rz of the crosslinked fraction is 3 to 1, favoring a substantially spherical structure. The underhydrated HA with Rh greater than Rz and a low amount of soluble fraction provides a reduction in excess fluid that, in addition to providing an injectable composition, also decreases epoxide degradation which is seen in prior art compositions. Thus, when the crosslinking agent comprises a dialdehyde crosslinking agent, the modified HA exhibits reduced hydraulic swelling measured as water uptake, no visible phase separation or viscosity reduction, less than 30% hydrolysis of ether linkages upon storage at 4 to 37° C. for 730 days, or a combination thereof. Another advantage of the compositions disclosed herein is the small particle size of the modified HA. In prior art compositions, the crosslinked HA particles would have diameters of greater than 200 μm. In aspects, the modified HA described herein is nanosized having an average particle size of less than about 2 μm (2000 nm). The modified hyaluronate further has a soluble fraction polydispersity (Mw/Mn) of 1 to 1.6, an Rh of the crosslinked fraction of 150 nm to 2000 nm, and an Rz of the crosslinked fraction of 50 nm to 700 nm, wherein Rh>Rz. In an aspect, the soluble fraction comprises less than 1%, 2% or 4% by weight of hyaluronate having a molecular weight less than 250 kDa based on the weight of the hyaluronate. In an aspect, the crosslinking is performed at a temperature greater than 20° C., preferably 25-40° C. Exemplary crosslinking agents include pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, 1,2-bis(2,3-epoxy propoxy)ethylene and 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), (phenylenebis-(ethyl)-carbodiimide and 1,6 hexamethylenebis (ethylcarbodiimide), adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (NMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, lysine, lysine methylester, or a combination thereof. In an aspect, after centrifuging and removing, the modified hyaluronate can be washed with a solution having a pH of 4.5 to 5.5. This optional washing step can increase the flow of the fluid comprising the modified hyaluronate as a single resultant mass even after the pH is subsequently increased to 5.5 to 7 and even up to pH 8. Without being held to theory, it is believed that washing provides for different structural properties to be obtained, such as formation of a co-joined mass of volumetric size, that is, an aggregate of nanostructured particles providing a larger overall mass. Thus, while the modified hyaluronate remains nanostructured, it can be conveniently grouped as a large homogenous mass. In an aspect, the method further comprises adding 200 to 590 ppm of a local anesthetic, e.g., lidocaine, to the aqueous solution of uncrosslinked hyaluronic acid or uncrosslinked sodium hyaluronate prior to crosslinking. As described in U.S. Pat. No. 10,391,202, lidocaine is added to an HA composition after crosslinking of the HA. This addition of lidocaine after crosslinking results in a lack of control of the lidocaine in the solution. Lidocaine addition to the non-crosslinked fraction advantageous because: 1) lidocaine is a separate entity that is best served in a least manipulated state during processing, thus inclusion with non-crosslinked hyaluronate and then crosslinking provides a composition with defined components; and 2) addition to the non-modified HA component makes it easier to determine that the soluble fraction is taken advantage of for its limited residence time post-injection, and it is best suited to accommodate and facilitate the solubilization of the lidocaine, specifically lidocaine monohydrate hydrochloride. Exemplary local anesthetics include ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and salts thereof. In an aspect, the local anesthetic agent is lidocaine, such as in the form of lidocaine HCl. The concentration of lidocaine in the compositions described herein can be therapeutically effective meaning the concentration is adequate to provide a therapeutic benefit without inflicting harm to the patient. In an aspect, the lidocaine is added as lidocaine hydrochloride monohydrate, CAS 6108-05-0, average molecular weight 288.81 g/mol, and not anhydrous lidocaine hydrochloride, CAS 73-78-9, average molecular weight 270.8 g/ml. Advantageously, adding the lidocaine hydrochloride in the form of the monohydrate and not anhydrous is predicted to improve the solubility of the lidocaine in the HA and eliminate the dose-dumping observed with prior art compositions. The use of lidocaine hydrochloride monohydrate provides a solubilized lidocaine that does not freely release in solution, rather remaining associated with the HA. Also included herein the product of the processes to make modified HA as described herein. While the modified HA described herein can be used as a soft tissue/dermal filler composition, soft tissue/dermal filler compositions can also include collagen. In an aspect, a collagen composition can be added to the modified HA, the modified HA can be disposed on collagen particles, or a combination thereof. The collagen can be in the form of collagen hollow spheres, collagen microparticles, collagen fibers, or a combination thereof. Collagen particles can have average diameters of 20 um to 250 um. In an aspect, the collagen comprises collagen fibers and the collagen fibers are comingled with the modified hyaluronate. Comingling can be done by mixing collage fibers with crosslinked HA and entangling, such as by freeze-drying, or by use of crosslinking using the crosslinking agents described above. In an aspect, an injectable soft tissue filler composition, comprises a modified hyaluronate comprising less than 10%, less than 5% or less than 2% by weight based on the total weight of hyaluronate of a soluble fraction with a soluble fraction polydispersity (Mw/Mn) of 1 to 1.6, and a crosslinked fraction with a 10% to 98% degree of crosslinking, preferably a 10% to 60% degree of crosslinking, an Rh of 150 nm to 2000 nm, and an Rz of 50 nm to 700 nm, wherein Rh>Rz, wherein the injectable dermal filler composition comprises no phosphate buffer. In an aspect, the injectable soft tissue filler composition comprises 35 to 200 nM NaCl, preferably 150 mM NaCl as a non-buffering suspending solution. The injectable soft tissue filler can be in the form of a reconstituted lyophilized foam, or an aqueous gel-like material. When the injectable soft tissue filler is in the form of a reconstituted lyophilized foam, the foam form can be freeze dried without topical anesthetic. The foam can them be rehydrated with NaCl and topical anesthetic for administration. Storage as a foam advantageously reduced the potential for hydrolytic degradation of crosslinked HA and provides a storage option for bulk production. In an aspect, the modified HA has an average particle size of less than about 2 μm (2000 nm). The injectable soft tissue filler composition can include 200 to 590 ppm topical anesthetic, e.g., lidocaine. Because the lidocaine is added during the crosslinking step, the lidocaine can be solubilized in the HA, preventing dos dumping of free lidocaine. In an aspect, the viscosity of the soft tissue filler composition is about 50 Pa*s to about 450 Pa*s. In other embodiments, the viscosity can be from about 50 Pa*s to about 300 Pa*s, from about 100 Pa*s to about 400 Pa*s, or about 250 Pa*s to about 400 Pa*s, or about 50 Pa*s to about 250 Pa*s. Generally, the concentration of HA in soft tissue filler composition is preferably at least 10 mg/mL and up to about 40 mg/mL, specifically 15 mg/ml to 30 mg/ml. For example, the concentration of HA in some of the compositions is in a range between about 20 mg/mL and about 30 mg/mL. Further, for example, in some embodiments, the compositions have a HA concentration of about 22 mg/mL, about 24 mg/mL, about 26 mg/mL, or about 28 mg/mL. Concentrations of 5 to 10 mg/ml can be used for sensitive injection areas such as thin skin and less dynamic movement regions. The soft tissue filler compositions can be introduced into syringes and optionally sterilized. Syringes are those capable of delivering viscous dermal filler compositions. The syringes generally have an internal volume of about 0.4 mL to about 3 mL, more preferably between about 0.5 mL and about 1.5 mL or between about 0.8 mL and about 2.5 mL. This internal volume is associated with an internal diameter of the syringe which plays a key role in the extrusion force needed to inject high viscosity dermal filler compositions. The internal diameters are generally about 4 mm to about 9 mm, more preferably from about 4.5 mm to about 6.5 mm or from about 4.5 mm to about 8.8 mm. Further, the extrusion force needed to deliver the HA/lidocaine compositions from the syringe is dependent on the needle gauge. The gauges of needles used generally include gauges between about 18 G and about 40 G, more preferably about 27 G to about 31 G or from about 25 G to about 27 G. A person of ordinary skill in the art can determine the correct syringe dimensions and needle gauge required to arrive at a particular extrusion force requirement. For example, 27 to 31 G needles are desirable for minimal injection site trauma, while deeper and high dosage delivery can utilize 25 to 27 G needles. The extrusion forces displayed by the soft tissue filler compositions described herein using the needle dimensions described above are at an injection speeds that are comfortable to a patient. Comfortable to a patient is used to define a rate of injection that does not injure or cause excess pain to a patient upon injection to the soft tissue. One skilled in the art will appreciate that comfortable as used herein includes not only patient comfort, but also comfort and ability of the physician or medical technician injecting the soft tissue filler compositions. Although certain extrusion forces may be achievable with the soft tissue filler compositions of the present disclosure, one skilled in the art understands that high extrusion forces can lead to lack of control during injection and that such lack of control may result in additional pain to the patient. Extrusion forces of the present soft tissue filler compositions should not exceed 20 N, such as about 8 N to about 15 N, or more preferably from about 10 N to about 13 N, or about 11 N to about 12 N, for example, at an extrusion rate of about 12.5 mm/min. For example, an exemplary extrusion force is not to exceed 20N continuous nor peak force (newtons) from 25.4 mm through 50 or more mm per min extrusions with 27 G or 30 G needles. Sterilization, as used herein comprises any method known in the art to effectively kill or eliminate transmissible agents, preferably without substantially altering of degrading the soft tissue filler compositions. A lyophilized form can be effectively sterilized with dry heat autoclaving, and similar sterilization assurance can be gained by steam or dry autoclaving for syringe enclosure gel compositions. In specific applications, such as fine lines and large dose delivery, pulsed light methods of xenon or LED can be utilized. A method of filling soft tissue comprises injecting into soft tissue of a subject in need thereof modified HA or a soft tissue filler composition as described herein. In an aspect, the injected composition comprises no phosphate buffer. In another aspect, wherein the injected composition comprises 135 mM to 200 mM NaCl. Exemplary soft tissues include comprises laugh lines, smile lines, crow's feet, wrinkles, and facial creases. Use of the modified hyaluronate as described herein is expected to improve predictable implant performance, provide repeatable metrics for injection force, and reduce unwanted biological responses once implanted. The invention is further illustrated by the following non-limiting examples. EXAMPLES Methods Example 1: Analysis of As-Received HA A SEC MALS RI analysis was performed on as-received HA. The as-received HA includes various manufactured HA composites that had been crosslinked. These composites still contain significant soluble fractions of hyaluronate, even after processing with crosslinker has been completed. The soluble fractions are then removed. The flow rate was 0.500 mL/min, the calibration constant of the DAWN light scattering instrument was 5.7653×10−51/(V/cm), the RI instrument was from Optilab, the solvent was aqueous PBS with a refractive index of 1.331. The light scattering model was Berry with a fit degree of 2 and a do/dc (mL/g) of 1.650. The fitted results are shown inFIG.2and Tables 1 and 2 include the numerical data. TABLE 1Peak 1 Results for As-Received HAParameterAs-received HACalculated mass (μg)396.14Mass fraction (%)100Mw, Da4.221 × 105Mw/Mn6.217Rz, nm107.9 TABLE 2Distribution Analysis for As-Received HARangeMolar MassCumulative %10-50,000Da24.420-100,000Da32.230-250,000Da41.84100,000-200,000Da9.250-300,000Da100 This example illustrates that:The as-received HA includes a large percentage of low molecular weight HA which the FDA has determined to be undesirable.The as-received HA is labeled as 150 kDa by the manufacturer, but does not specify polydispersity. Per the analysis, the Mw is 422 kDa and the polydispersity of the as-received HA is high at 6.2. Thus, the molecular weight is higher than reported and the polydispersity is extremely high.Only 9.2% of the as-received HA is in the target molecular weight range of 100-200 kDa. Example 2: SEC MALS RI Analysis of Crosslinked, Purified HA A SEC MALS RI analysis was performed on as-received HA after crosslinking and after two rounds of purification by centrifugation. Instrument parameters were the same as in Example 1. The fitted results are shown in Tables 3 and 4. TABLE 3Peak results for Crosslinked HA, And After TwoRounds of CentrifugationAs-receivedAfterAfterCrosslinkedfirst passsecond passParameterHApurificationpurificationMn, Da6.789 × 1041.74 × 1054.453 × 105Mw, Da4.221 × 1051.338 × 1062.371 × 106Mw/Mn6.2177.6915.325Rz, nm107.9103.7110.5Rh (avg, nm)36.936.9236.9 TABLE 4Distribution results for Crosslinked HA, And After TwoRounds of CentrifugationCumulativeCumulativeCumulative %% As% AfterAfter SecondMolarreceivedFirst PassPassRangeMassHAPurificationPurification10-50,000Da24.46.65020-100,000Da32.224.03.57530-250,000Da41.840.719.3234100,000-200,000Da9.331.77932.428 This example clearly shows a reduction in the 0-50 and 1-100 kDA fractions and enrichment of the 100-200 kDa fraction after two rounds of centrifugation. Example 3: SEC MALS RI Analysis of Crosslinked HA As-received Sodium HA 1.5M Da was hydrated in distilled water. The solution was adjusted to 150 mM NaCl. The HA was crosslinked with BDDE in the 150 mM NaCl solution at 23 to 37° C. for 20 to 60 min. After crosslinking, the sample was centrifuged at 10 G for 15 minutes. Instrument parameters were the same as in Example 1. TABLE 5Peak results for Crosslinked, Purified HAAs-receivedParameterCrosslinked HACalculated mass17.54(μg)Rz, nm132Rh (avg, nm)236 TABLE 6Distribution results for Crosslinked, Purified HACumulative %RangeMolar MassAs received HA10-50,000Da020-100,000Da030-250,000Da04250,000-1,000,000Da68.35100,000-200,000Da25.66200,000-3,122,9656.1 Importantly, the fractions <50 k, <100 k, and <250 kDa fractions are all essentially zero. An Rh>Rz (236 vs 132 nm) shows that globular structures dominate the material. A tight distribution of nanometer globular structures (approx. 230 nm) with Rh>Rz was achieved. Example 4: Comparison of NaCl and Phosphate Buffer, As Received HA 4 MDa raw material sodium hyaluronate powder was put into solution with PBS (no calcium, no magnesium to provide no disruption to the conformation of HA) to provide a 20 mg/ml concentration. The 20 mg/ml solution was used to provide 2 aliquots.Aliquot 1: diluted 1:10 with PBS (no calcium, no magnesium)Aliquot 2: diluted 1:10 with 150 mM NaCl (no calcium, no magnesium) The samples were vortexed and 500 μl of a 0.2 mg/ml solution was injected. Instrument conditions were as in Example 1. TABLE 7Peak results for NaCl compared to phosphate buffer, as-received HAPhosphateNaClMn1.562 × 1061.682 × 106Mw1.887 × 1061.822 × 106Mw/Mn1.2081.083Rh (Q)z69.4149.88Rz175.8168.6 TABLE 8Distribution results for NaCl compared to phosphate buffer,as-received HAPhosphateNaCl0-50kDa000-100kDa000-250kDa00250-2,000kDa30.665.4992,000-3,000kDa68.234.530-4,000kDa99.599.987 When the raw material was dissolved in NaCl vs phosphate buffer, there was a significant difference in Rh values, with Rz and Mw being similar. Example 5: Comparison of NaCl and Phosphate Buffer, Crosslinked HA A large sample of HA (7 liters) was prepared and diluted with either PBS or NaCl. The samples were then crosslinked, and centrifuged to remove the soluble fraction. The results are provides in Tables 7 and 8. TABLE 9Peak results for NaCl compared to phosphate buffer,crosslinked HAPBSNaClHydrodynamic radius44.6541.46Rh (nm)Rz (nm)105.4103.8Polydispersity Mw/Mn6.591.503Mn4.832 × 1043.605 × 105Mw3.187 × 1055.418 × 105 TABLE 10Distribution results for NaCl compared to phosphate buffer,crosslinked HAPBSNaCl0-50,000g/mol35.500-100,000g/mol40.300-250,000g/mol54.4919.4250,000-2,000,000g/mol45.480.5350-3,000,000g/mol99.9799.98 The analysis is the finished/final product residual soluble fractions.Identical Peak Limits (min) 11.350-22.00035% reduction in 0 to 50 k Da soluble fraction in NaCl processing40% reduction in 0 to 100 k Da soluble fraction in NaCl processing35% reduction in 0 to 250 k Da soluble fraction in NaCl processingReduced polydispersity in NaCl process—1.5 vs 6.5 (Mw/Mn) Example 6: Use of HPLC/SEC/MALS/RI to Verify Reduction of the Soluble HA Component Starting material HA was crosslinked with homobifunctional 1,4-butanediol diglycidyl ether (BDDE). Two centrifugation steps were performed under conditions 10 to 12 G for 15 minutes. The analytical method of HPLC/SEC/MALS/RI (High Pressure Liquid Chromatography-Size Exclusion Chromatography with Multi-Angle Light Scattering-Refractive Index Detection) was validated for determining the number average molecular weight, weight average molecular weight, Rz and wt % soluble HA. Mobile Phase was 150 mM NaCl or 150 mM NaNO3, flowrates of 0.3 to 0.8 ml/min, with Shodex™ LB 805 and 804 columns run in tandem, or dual Shodex™ LB 806M (mixed bed columns—run in tandem). Table 9 andFIGS.3-5show the original crosslinked material, first centrifugation and second successive centrifugation, respectively. TABLE 11Parameters for reduction of soluble HAOriginalFirstSecondParametersamplecentrifugationcentrifugationMn, kDa140130130Mw, kDa460430420Mw/Mn3.33.33.2Rz, nm135123115wt %21%15%10%soluble HA The amount of soluble HA was reduced by more than 50% using two centrifugation steps. The material with 10 wt % soluble HA has a much lower wt % HA than commercially available HA-based dermal fillers, which have more than 20 wt % and even more than 40 wt % soluble HA. The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. | 29,430 |
11857683 | DETAILED DESCRIPTION OF THE INVENTION Definitions The “phenobarbital or salts thereof” that may be used according to the present disclosure may be phenobarbital base or a phenobarbital salt. The particular salt form of phenobarbital is not particularly limited, and in non-limiting examples, may be, for example, phenobarbital sodium, phenobarbital potassium, phenobarbital benzathine, phenobarbital betaine, or phenobarbital choline. Preferred embodiments utilize a phenobarbital sodium salt. The term “reconstitution” as used herein, includes the addition of vehicle/diluent in to the lyophilized phenobarbital lyophilized phenobarbital. In preferred embodiments, the vehicle/diluent is water for injection, an aqueous saline solution or an aqueous dextrose solution. In additional preferred embodiments, the vehicle/diluent is water for injection or a 0.9% aqueous saline solution. The terms “about” as used herein refers to as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value. The term “about” when used in the present application preceding a number and referring to it, is meant to designate any value which lies within the range of ±10%. The term “total impurities” as used herein, includes known and unknown impurities, either present from the active pharmaceutical ingredient (API) or generated by the degradation of phenobarbital or salts thereof during the manufacturing or stability of the pharmaceutical compositions of the present disclosures. These total impurities includes but not limited to 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid, and can be represented by following structural formulas: The term “correctable abnormalities” as used herein is defined as any abnormality which can be corrected by medication. The correctable abnormalities include hypoglycemia or hypocalcemia. The term “C1-C3alcohol” as used herein means an alkanol having 1-3 carbon atoms which include methanol, ethanol, 1-propanol, or isopropyl alcohol. In the first aspect, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition of phenobarbital or salts thereof is an aqueous solution for injection. In one embodiment, the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. In one embodiment, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition of phenobarbital or salts thereof is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition of phenobarbital or salts thereof is reconstituted with 0.9% of aqueous saline. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment the osmolality of said pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of said pharmaceutical composition is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. In one embodiment, the newborn infants is of 2 weeks of age or younger. In one embodiment, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized phenobarbital or salts thereof, wherein the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. In one embodiment, the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected. In one embodiment, the said correctable abnormalities are hypoglycemia or hypocalcemia. In one embodiment, wherein the pharmaceutical composition is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes. In one embodiment, the method comprises administration of the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures, wherein if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg. Electrographic seizures can be measured by any means and instruments known in the art like electroencephalogram (EEG) or using 2-channel EEG with amplitude-integrated EEG. In one embodiment, wherein the method for the treatment of neonatal seizure in newborn infants comprising administering the pharmaceutical composition of the first aspect. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the pharmaceutical composition of phenobarbital or salts thereof is an aqueous solution for injection. In one embodiment, the lyophilized pharmaceutical composition of phenobarbital or salts thereof is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition of phenobarbital or salts thereof is reconstituted with 0.9% of aqueous saline. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment the osmolality of said pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of said pharmaceutical composition is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the second aspect, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In another embodiment of the second aspect, the pharmaceutical composition is a lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In another embodiment of the second aspect, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In another embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% of aqueous saline. In one embodiment, the aqueous solution comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the aqueous solution is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the aqueous solution is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of said composition is below 500 mOsm/kg. In one embodiment, the osmolality of said aqueous solution is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the newborn infants is 2 weeks of age or younger. In one embodiment, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected. In one embodiment, the said correctable abnormalities are hypoglycemia or hypocalcemia. In one embodiment, wherein the pharmaceutical composition of the second aspect is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes. In one embodiment, wherein the method comprises administration of the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures, wherein if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg. In one embodiment, wherein the method for the treatment of neonatal seizure in newborn infants comprising administering the pharmaceutical composition of the second aspect. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another embodiment, the pharmaceutical composition is a lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In another embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In another embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% of aqueous saline. In one embodiment, the aqueous solution comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the aqueous solution is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the aqueous solution is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the osmolality of said composition is below 500 mOsm/kg. In one embodiment, the osmolality of said aqueous solution is about 300-400 mOsm/kg. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In the third aspect, the present disclosure relates to a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the lyophilized pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants in need thereof, comprising administering the lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm. In one embodiment, the newborn infants is of 2 weeks of age or younger. In one embodiment, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm. In one embodiment, the method comprises reconstituting the lyophilized pharmaceutical composition of phenobarbital or salts thereof immediately prior to the administration. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution to obtain the aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the method comprises administering the aqueous solution to neonates in whom correctable abnormalities have been excluded or corrected. In one embodiment, the said correctable abnormalities are hypoglycemia or hypocalcemia. In one embodiment, wherein the aqueous solution is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes. In one embodiment, wherein the method comprises administration of the aqueous solution at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures, wherein if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg. In one embodiment, wherein the method for the treatment of neonatal seizure in newborn infants comprising administering the lyophilized pharmaceutical composition of the third aspect. In one embodiment, the lyophilized pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the fourth aspect, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of said pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of said aqueous solution is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the newborn infants is of 2 weeks of age or younger. In one embodiment, the present disclosure relates to a method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected. In one embodiment, the said correctable abnormalities are hypoglycemia or hypocalcemia. In one embodiment, wherein the pharmaceutical composition is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes. In one embodiment, wherein the method comprises administration of the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures, wherein if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg. In one embodiment, wherein the method for the treatment of neonatal seizure in newborn infants comprising administering the pharmaceutical composition of the fourth aspect. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of the pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of the pharmaceutical composition is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the fifth aspect, the present disclosure relates to a process of preparing the lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution of phenobarbital or salts thereof in a concentration of 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process comprises measuring the alcohol content of an aqueous solution of phenobarbital or salts thereof. In one embodiment, if the alcohol content of the aqueous solution is below 5000 ppm then the process further comprises a step of adding an alcohol in a quantity that is sufficient to achieve the alcohol content of at least about 5000 ppm. In one embodiment, the process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process may comprises repeating steps of lyophilization to achieve the alcohol content not more than about 66000 ppm or about 70000 ppm. In one embodiment, if the alcohol content is above 66000 ppm or 70000 ppm (whichever is desired), the process further comprises repeating the lyophilization step till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than about 70000 ppm. It is understood that even a single additional cycle of lyophilization would be sufficient or it has to be repeated n number of times to obtain the desired alcohol level. In another aspect, the present disclosure relates to a process of preparing the lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; measuring the alcohol content of aqueous solution; if the alcohol content is below 5000 ppm, adding an alcohol to achieve the alcohol content of at least about 5000 ppm; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition; measuring the alcohol content of the lyophilized pharmaceutical composition; if the alcohol content is above 66000 ppm or above about 70000 ppm (whichever is desired), repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or is not more than about 70000 ppm. In one embodiment, the process comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 8-12, preferably in a range of 9-11, more preferably in a range of 9-10.5. In one embodiment, the pH modifier is selected from HCl and/or NaOH. In one embodiment, the pH modifier is aqueous HCl solution. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the lyophilized pharmaceutical composition has an alcohol content of at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the sixth aspect, the present disclosure relates to a process of preparing the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof; and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process comprises measuring the alcohol content of an aqueous solution of phenobarbital or salts thereof. In one embodiment, if the alcohol content of the aqueous solution is below 5000 ppm then the process further comprises a step of adding an alcohol in a quantity that is sufficient to achieve the alcohol content of at least about 5000 ppm. In one embodiment, the process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process may comprises additional steps of lyophilization to achieve the alcohol content not more than about 66000 ppm or not more than about 70000 ppm (whichever is desired). In one embodiment, if the alcohol content is above 66000 ppm or about 70000 ppm, the process further comprises repeating the lyophilization step till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than about 70000 ppm. It is understood that even a single additional cycle of lyophilization would be sufficient or it has to be repeated n number of times to obtain the desired alcohol level. In another aspect, the present disclosure relates to a process of preparing the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; measuring the alcohol content of aqueous solution; if the alcohol content is below 5000 ppm, adding an alcohol to achieve the alcohol content of at least about 5000 ppm; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition; measuring the alcohol content of the lyophilized pharmaceutical composition; if the alcohol content is above 66000 ppm or about 70000 ppm (whichever is desired), repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than about 70000 ppm (whichever is desired); and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 8-12, preferably in a range of 9-11, more preferably in a range of 9-10.5. In one embodiment, the pH modifier is selected from HCl and/or NaOH. In one embodiment, the pH modifier is aqueous HCl solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities in the pharmaceutical composition present following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of the pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of the pharmaceutical composition is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the seventh aspect, the present disclosure relates to a lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the lyophilized pharmaceutical composition is obtained by a process comprising: dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process comprises measuring the alcohol content of an aqueous solution of phenobarbital or salts thereof. In one embodiment, if the alcohol content of the aqueous solution is below 5000 ppm then the process further comprises a step of adding an alcohol in a quantity that is sufficient to achieve the alcohol content of at least about 5000 ppm. In one embodiment, the process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process may comprises an additional step of lyophilization to achieve the alcohol content not more than about 66000 ppm or not more than 70000 ppm (whichever is desired). In one embodiment, if the alcohol content is above 66000 ppm or about 70000 ppm, the process further comprises repeating the lyophilization step till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than about 70000 ppm (whichever is desired). It is understood that even a single additional cycle of lyophilization would be sufficient or it has to be repeated n number of times to obtain the desired alcohol level. In one embodiment, the process comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 8-12, preferably in a range of 9-11, more preferably in a range of 9-10.5. In one embodiment, the pH modifier is selected from HCl and/or NaOH. In one embodiment, the pH modifier is aqueous HCl solution. In one embodiment, the lyophilized pharmaceutical composition has an alcohol content of at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In order to achieve a desired stability, the lyophilized pharmaceutical composition should have an alcohol content in the range from about 5000 ppm to 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, and thus the process may include the steps as discussed above for measuring the alcohol content, adding alcohol or removing additional alcohol by repeating the lyophilization step and ensuring that the alcohol content is achieved as described above. In the eight aspect, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the pharmaceutical composition is obtained by a process comprising: dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof; and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process comprises measuring the alcohol content of an aqueous solution of phenobarbital or salts thereof. In one embodiment, if the alcohol content of the aqueous solution is below 5000 ppm then the process further comprises a step of adding an alcohol in a quantity that is sufficient to achieve the alcohol content of at least about 5000 ppm. In one embodiment, the process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the process may comprises an additional step of lyophilization to achieve the alcohol content not more than about 66000 ppm or not more than about 70000 ppm (whichever is desired). In one embodiment, if the alcohol content is above about 66000 ppm or above about 70000 ppm, the process further comprises repeating the lyophilization step till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than about 70000 ppm (whichever is desired). It is understood that even a single additional cycle of lyophilization would be sufficient or it has to be repeated n number of times to obtain the desired alcohol level. In one embodiment, the process comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 8-12, preferably in a range of 9-11, more preferably in a range of 9-10.5. In one embodiment, the pH modifier is selected from HCl and/or NaOH. In one embodiment, the pH modifier is aqueous HCl solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the pharmaceutical composition following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of the pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of the pharmaceutical composition is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In order to achieve a desired stability, the pharmaceutical composition should have an alcohol content in the range from about 5000 ppm to 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, and thus the process may include the steps as discussed above for measuring the alcohol content, adding alcohol or removing additional alcohol by repeating the lyophilization step and ensuring that the alcohol content is achieved as described above. In the ninth aspect, the present disclosure relates to a lyophilized pharmaceutical composition comprising phenobarbital or salts thereof and an alcohol. In one embodiment, the alcohol is present in an amount sufficient to inhibit phenobarbital degradation, such that the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the amount of alcohol sufficient to inhibit phenobarbital degradation is in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In one embodiment, the present disclosure relates to a lyophilized pharmaceutical composition comprising phenobarbital sodium and ethanol, wherein ethanol is present in an amount sufficient to inhibit degradation of phenobarbital sodium, such that the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%; wherein the amount of ethanol sufficient to inhibit degradation of phenobarbital sodium is in the range from about 12000 ppm to about 25000 ppm; and wherein the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In the tenth aspect, the present disclosure relates to an aqueous solution for injection comprising phenobarbital or salts thereof and an alcohol. In one embodiment, the aqueous solution for injection is reconstituted from the lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the alcohol is present in an amount sufficient to inhibit phenobarbital degradation, such that the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the alcohol is present in an amount sufficient to inhibit phenobarbital degradation, such that the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the amount of alcohol sufficient to inhibit phenobarbital degradation is in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the aqueous solution comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the aqueous solution is stable up to 12 hours of storage at 20-25° C. In one embodiment, the aqueous solution is stable up to 36 hours of storage at 2-8° C. In one embodiment, the osmolality of the aqueous solution is below 500 mOsm/kg. In one embodiment, the osmolality of the aqueous solution is about 300-400 mOsm/kg. In one embodiment, the aqueous solution is free of benzyl alcohol. In one embodiment, the aqueous solution is also free of propylene glycol. In a preferred embodiment, the aqueous solution is free of benzyl alcohol and propylene glycol. In one embodiment, the present disclosure relates to an aqueous solution for injection comprising phenobarbital sodium and ethanol, wherein ethanol is present in an amount sufficient to inhibit degradation of phenobarbital sodium, such that the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. or following 36 hours of storage at 2-8° C. does not exceed 0.2%; wherein the amount of ethanol sufficient to inhibit degradation of phenobarbital sodium is in the range from about 12000 ppm to about 25000 ppm; wherein phenobarbital sodium is present in a concentration from 10-200 mg/ml; wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital sodium; and wherein the aqueous solution is free of benzyl alcohol and propylene glycol. In the eleventh aspect, the present disclosure relates to a process for the preparation of the pharmaceutical composition of phenobarbital or salts thereof, wherein the process comprises dissolving phenobarbital or salt thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition, wherein phenobarbital or salts thereof has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the process comprises phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the active pharmaceutical ingredient of phenobarbital or salts thereof for the purpose of this aspect is having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the process further comprises reconstitution of the lyophilized pharmaceutical composition. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, phenobarbital or salts thereof has an alcohol content of at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In one embodiment, the pharmaceutical composition is lyophilized pharmaceutical composition of phenobarbital or salts thereof. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the lyophilized pharmaceutical composition following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the aqueous solution comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the aqueous solution is stable up to 12 hours of storage at 20-25° C. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 12 hours of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the aqueous solution is stable up to 36 hours of storage at 2-8° C. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.5%. In one embodiment, the amount of total impurities present in the aqueous solution following 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the osmolality of the aqueous solution is below 500 mOsm/kg. In one embodiment, the osmolality of the aqueous solution is about 300-400 mOsm/kg. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In another aspect, the present disclosure relates to a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the lyophilized pharmaceutical composition has: (i) an amount of alcohol present following 36 months of storage at 20-25° C. in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm; or (ii) an amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%. In one embodiment, the lyophilized pharmaceutical composition has an amount of alcohol present following 36 months of storage at 20-25° C. in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol. In one embodiment, the lyophilized pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the lyophilized pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has: (i) an amount of alcohol present following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm; or (ii) an amount of total impurities present following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. does not exceed 0.2%. In one embodiment, the pharmaceutical composition has an amount of alcohol present following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the alcohol content is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution. In one embodiment, the lyophilized pharmaceutical composition is reconstituted with 0.9% aqueous saline. In one embodiment, the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, the pharmaceutical composition comprise phenobarbital or salts thereof in a concentration of 10-200 mg/ml. In one embodiment, phenobarbital or salts thereof is present in a concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml and 200 mg/ml. In one embodiment, the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C. In one embodiment, the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C. In one embodiment, the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid. In one embodiment, the osmolality of said pharmaceutical composition is below 500 mOsm/kg. In one embodiment, the osmolality of said aqueous solution is about 300-400 mOsm/kg. In one embodiment, the pharmaceutical composition is free of benzyl alcohol. In one embodiment, the pharmaceutical composition is also free of propylene glycol. In a preferred embodiment, the pharmaceutical composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a lyophilized pharmaceutical composition of phenobarbital sodium, wherein the composition has an ethanol content in the range from about 12000 ppm to about 25000 ppm; wherein the composition is stable up to 36 months of storage at 20-25° C. such that the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%; and wherein the composition is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to an aqueous solution for injection of phenobarbital sodium, wherein the aqueous solution has an ethanol content in the range from about 12000 ppm to about 25000 ppm; wherein the aqueous solution is stable up to 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. such that the amount of total impurities present following 12 hours of storage at 20-25° C. or following 36 hours of storage at 2-8° C. does not exceed 0.2%; wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital sodium; and wherein the aqueous solution is free of benzyl alcohol and propylene glycol. In another aspect, the present disclosure relates to a method of preventing degradation of phenobarbital or salts thereof, wherein the method comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the method comprises presence of alcohol, in an amount sufficient to inhibit the degradation of phenobarbital or salts thereof. In one embodiment, the present disclosure relates to reconstituting the lyophilized pharmaceutical composition to obtain the aqueous solution for injection of phenobarbital or salts thereof. In one embodiment, wherein the amount of alcohol sufficient to inhibit degradation of phenobarbital or salts thereof is in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm. In one embodiment, the amount of alcohol is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. In yet another aspect, the present invention relates to phenobarbital or salts thereof having an alcohol in an amount sufficient to inhibit degradation of phenobarbital sodium. In one embodiment, the amount of alcohol sufficient to inhibit degradation of phenobarbital or salts thereof is such that when said phenobarbital or salts thereof having an alcohol is stored for 36 months at 20-25° C., the amount of total impurities does not exceed 0.5%, preferably does not exceed 0.2%. In an alternate embodiment, the amount of alcohol sufficient to inhibit degradation of phenobarbital or salts thereof is such that when said phenobarbital or salts thereof is dissolved in an aqueous media and is stored for 12 hours at 20-25° C. or 36 hours at 2-8° C., the amount of total impurities does not exceed 0.5%, preferably does not exceed 0.2%. In one embodiment, the amount of alcohol sufficient to inhibit degradation of phenobarbital or salts thereof is from about 5000 ppm to about 70000 ppm. In one embodiment, the phenobarbital or salts thereof having an alcohol content in a range from about 5000 ppm to about 70000 ppm, is stable up to 36 months of storage at 20-25° C. such that the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%, preferably does not exceed 0.2%. In another embodiment, the phenobarbital or salts thereof having an alcohol content in a range from about 5000 ppm to about 70000 ppm, when dissolved in an aqueous media, the resulting aqueous solution of phenobarbital or salts thereof remains stable up to 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. such that the amount of total impurities present following 12 hours of storage at 20-25° C. or following 36 hours of storage at 2-8° C. does not exceed 0.5%, preferably does not exceed 0.2%. In one embodiment, the amount of alcohol is at least about 5000 ppm, about 10000 ppm, about 15000 ppm, about 20000 ppm, about 25000 ppm, about 30000 ppm, about 35000 ppm, about 40000 ppm, about 45000 ppm, about 50000 ppm, about 55000 ppm, about 60000 ppm, about 65000 ppm, about 66000 ppm, or about 70000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 70000 ppm, about 12000 ppm to about 66000 ppm, about 12000 ppm to about 65000 ppm, about 12000 ppm to about 60000 ppm, about 12000 ppm to about 55000 ppm, about 12000 ppm to about 50000 ppm, about 12000 ppm to about 45000 ppm, about 12000 ppm to about 40000 ppm, about 12000 ppm to about 35000 ppm, about 12000 ppm to about 30000 ppm, about 12000 ppm to about 25000 ppm, about 12000 ppm to about 20000 ppm or about 12000 ppm to about 15000 ppm. In another embodiment, the alcohol content is in the range from about 15000 ppm to about 70000 ppm, about 15000 ppm to about 66000 ppm, about 25000 ppm to about 66000 ppm, about 35000 to about 66000 ppm, about 45000 ppm to about 66000 ppm, about 55000 ppm to about 66000 ppm, about 35000 ppm to about 55000 ppm or about 30000 ppm to about 50000 ppm. In another embodiment, the alcohol content is in the range from about 12000 ppm to about 25000 ppm. In one embodiment, the alcohol content is in the range from about 35000 to about 66000 ppm. In one embodiment, the alcohol is a C1-C3alcohol. In one embodiment, the alcohol is ethanol. In one embodiment, phenobarbital or salts thereof is phenobarbital base or phenobarbital sodium, preferably phenobarbital sodium. Through lyophilization, the present inventors have found that the alcohol content of phenobarbital sodium may be reduced to less than 50000 ppm. The present inventors have found that in some embodiments, the alcohol content may be reduced to a level of about 5000 ppm. The present inventors have found that the low temperature vacuum drying conditions afforded by lyophilization provide a safe and effective mechanism for converting the tested material to a solid state. Lyophilization, also known as freeze drying, may consist of three separate, unique, and interdependent process; freezing, primary drying (sublimation) and secondary drying (desorption). In certain preferred embodiments, the step of freezing the aqueous solution may occur at a temperature range of −20 to −50° C. In certain preferred embodiments, the step drying the aqueous solution may occur at primary and/or secondary temperature ranges of −35 to 25° C. However, it should be understood that these temperatures are exemplary only, and temperatures may be chosen so as to optimize the lyophilized powder. Where secondary temperature ranges are used, the step of drying may be split into two separate steps, each occurring at a different temperature and a different time. In certain preferred embodiments, there may also be performed a step of annealing the aqueous solution, which may occur at a temperature range of −15 to −25°. In certain embodiments, a desired level of residual water content after lyophilization may be less than 3% w/w. As illustrated in the Examples below, the present inventors have observed that the use of certain level of alcohol and lyophilization in the development of the pharmaceutical composition of phenobarbital or slats thereof provides for greatly improved stability in the resulting product. EXAMPLES HPLC Method:HPLC instrument with UV detector or PDA detector;Reagents/Solvents: Potassium dihydrogen phosphate, Orthophosphoric acid, Acetonitrile, Methanol, Water, Water for Injection;Needle wash solutions—Water:Methanol (20:80 v/v) & Column wash solution—Water Acetonitrile (50:50 v/v);Preparation of diluted orthophosphoric acid: Dilute 1 ml of concentrated orthophosphoric acid to 10 ml with water;Preparation of Buffer: Weigh about 2.9 g (2.85 g to 2.95 g) of potassium dihydrogen phosphate and dissolve in 1000 ml of Milli-Q water, adjust pH to 3.5±0.05 with dilute orthophosphoric acid.Filter the solution through 0.45 PVDF Merck Durapore membrane or equivalent filter. Preparation of Mobile Phase Solutions: Mobile Phase A: Buffer to Acetonitrile (80:20) & Mobile Phase B: Buffer to Acetonitrile (50:50); Diluent: water:methanol (20:80) Chromatographic Conditions:Column Zorbax Eclipse XDB Phenyl, 150×4.6 mm, 5 m (Agilent)Column temperature 40° C.Sample Cooler 10° C.Flow rate 1.2 ml/minInjection Volume 10 μlWavelength 210 nm (UV/PDA)Run Time 15 minutes HPLC Gradient: Time (minute)Mobile phase A %Mobile phase B %08020480207208012208012.18020158020 Example 1 In a clean and dry flask, 1000 mg of phenobarbital base and 1.67 ml of 96% ethanol were combined and mixed for 1 minute. To this solution, 1.722 ml of the 10% NaOH solution was added and was mixed for 15 minutes. Water for injection sufficient to raise the volume to 25 ml was then added and the resulting solution was mixed for 2 minutes and filtered through a 0.2 micron PES filter. Example 2 The procedures of Example 1 were followed, with the exception that ethanol was not added and the batch size was 600 ml. The bulk solutions of both the examples were analyzed and the results are shown in Table 1 below: TABLE 1Example 1Example 2With additionalWithout additionalEthanolEthanolPhenobarbital baseLess than 5000 ppm ethanolInitial analysis0M0MTotal Impurity (%)0.060.314 Table 1 illustrates that the impurity profile in the composition with additional ethanol (example 1) is better than the composition without ethanol (example 2). It is to be noted that the phenobarbital base API as used in these examples had an ethanol content of less than 5000 ppm. Thus, the composition of example 1, comprises an ethanol content above 5000 ppm. Whereas, the composition of example 2 comprises no additional ethanol and thus the amount of ethanol in the composition of example 2 is equal to the amount present in phenobarbital base API (less than 5000 ppm) or less. Example 3 In a clean and dry vessel, 3.64 g phenobarbital base and water for injection were mixed by stirring until the phenobarbital base was dissolved. 6.3 ml of an aqueous 10% NaOH solution was then added while stirring. Water for injection was then added to bring the volume up to 100 ml and the resulting mixture was stirred for 2 minutes. Example 4 The procedures of Example 3 were followed, with the exception that following the step of dissolving phenobarbital in water for injection, 6.3 mL of 96% ethanol was added to the mixture. As with Example 3, 6.3 mL of an aqueous 10% NaOH solution was then added while stirring. Water for injection was then added to bring the volume up to 100 ml and the resulting mixture was stirred for 2 minutes. The results for Examples 3 and 4 are shown in Table 2 below: TABLE 2Example 3Example 4Without additional ethanolWith additional ethanolPhenobarbitalLess than 5000 ppm ethanolbaseTotal Impurities (%)Stage of Analysis2-8° C.20-25° C.2-8° C.20-25° C.0 hr0.3860.3860.3440.3448 hr0.9692.5260.7351.9324 hr5.0657.4233.9825.586 Table 2 illustrates that adding ethanol to the phenobarbital composition in the amounts tested above provided for fewer impurities than the composition that did not have additional ethanol. Ethanol content of 5000 ppm is not sufficient to prevent formation of impurities. The composition wherein additional ethanol was added, afforded a product with improved stability. Degradation of such composition when subjected to stability testing was found to be significantly lower than the composition wherein there was no additional ethanol addition. Example 5 In a clean and dry vessel, 4 g of phenobarbital sodium was dissolved via stirring in water for injection. 6.3 ml of 96% ethanol was added to the solution while stirring, and water for injection was then added in an amount sufficient to raise the volume to 100 ml. The resulting solution was stirred for 2 minutes. Example 6 The procedures of Example 5 were reproduced, with the exception that the pH of the final solution was adjusted to 9.0 by using 0.1N HCl. Example 7 The procedures of Example 5 were reproduced, with the exception that the ethanol was not added. Example 8 The procedures of Example 6 were reproduced, with the exception that the ethanol was not added It is to be noted that the phenobarbital sodium API as used in these examples had an ethanol content of about 66000 ppm. The results of Examples 5-8 are shown in Table 3 below: TABLE 3Composition with Phenobarbital SodiumExample 5Example 6Example 7Example 8PhenobarbitalAbout 66000 ppm ethanolNa40 mg40 mg40 mg40 mgEthanol 96%0.063 ml0.063 ml——pH of Solution9.789.029.619.04Total Impurities (%) (2-8° C.)0 h0.0040.0050.0070.0051 h0.0120.0110.0160.0112 h0.0250.0140.0280.0174 h0.0260.0170.0310.0188 h0.0280.0160.0320.0224 h0.040.0150.0520.022 Table 3 shows that when the amount of ethanol present in phenobarbital sodium composition was above 5000 ppm then the formation of total impurities was minimized. Example 9 In a clean and dry vessel, 60 g of phenobarbital sodium was dissolved via stirring in 1000 ml water for injection and further added 94.5 mL of 96% ethanol. The clarity of the solution was reviewed to ensure that it was clear, and then water for injection sufficient to raise the volume to 1500 ml was added and the solution was stirred followed by filtered through a 0.2 micron PES filter and lyophilization. The lyophilized phenobarbital sodium was kept on stability at 2-8° C. for 36 months. The lyophilized phenobarbital sodium was analyzed for ethanol content at regular intervals. Example 10 The procedures of Example 9 were reproduced, with the exception that the pH was adjusted to 9 by the use of 5% HCl and then lyophilized. The lyophilized phenobarbital sodium was kept on stability at 2-8° C. for 36 months. The lyophilized phenobarbital sodium was analyzed for ethanol content at regular intervals. It is to be noted that the phenobarbital sodium API as used in these examples had an ethanol content of about 66000 ppm. The results of Examples 9 and 10 are shown in Table 4 below: TABLE 4Example 9Example 10Initial38474.753078.71M52188.366539.33M46489.6544576M34028.549584.112M37039.54544018M42227.553834.124M380354680536M4513952043 Table 4 shows that when additional ethanol is added in the compositions followed by lyophilizing the solution, the amount of ethanol present in the tested compositions were within the range of from about 34000 ppm to about 66000 ppm. Example 11 In a clean and dry vessel, 64 g of phenobarbital sodium was dissolved via stirring in 1200 ml water for injection and the clarity of the solution was observed to ensure that it was clear. Water for injection was then added to the solution in an amount sufficient to raise the volume to 1600 ml and the solution was stirred, filtered through a 0.2 micron PES filter and lyophilized. The lyophilized phenobarbital sodium was kept on stability at 20-25° C. for 36 months. The lyophilized phenobarbital sodium was analyzed for ethanol content at regular intervals. Example 12 The procedures of Example 11 were reproduced, with the exception that the batch size was 1800 ml. The lyophilized phenobarbital sodium was kept on stability at 20-25° C. for 36 months. The lyophilized phenobarbital sodium was analyzed for ethanol content at regular intervals. It is to be noted that the phenobarbital sodium API as used in these examples had an ethanol content of about 66000 ppm. The results of Examples 11 and 12 were are shown in Table 5 below: TABLE 5West-ward'sExample 11Example 12Composition*Phenobarbital sodiumAbout 66000 ppm ethanolEthanol—0.1 ml/mlStage of Analysis24M36M24M36Manalysis after 2.1 yearSolvents (Ethanol in ppm)1880216680170301382666436.1Unknown Impurities0.0570.0290.0190.0332.113(Highest Unspecified) (%)Total Impurities (%)0.1380.0470.0390.0672.187Osmolality (mOsm/kg)3703713613571341*West-ward’s composition is Phenobarbital sodium in ethanol, benzyl alcohol and propylene glycol Table 5 illustrates that despite the use of a higher level of ethanol in a West-ward's product of phenobarbital sodium at 65 mg/ml, the impurities formation therein is higher than the pharmaceutical composition of preferred embodiments of the present disclosure. Accordingly, the optimum amount of ethanol and lyophilization may be achieved so as to provide for the greatest control of impurities in the composition. During storage, the amount of ethanol present in the composition may decrease to about 12000 ppm, and thus composition containing ethanol in a range of 12000 to 66000 ppm provide for controlled and lowered levels of impurities. The products of example 11 and 12 were subjected to stability testing. The results of the stability tests are shown in Table 6 below: TABLE 6Example 11Example 12Initial2433315510.71M1832115639.73M22172.2155406M23154.31503412M228911501818M184961230724M188021703036M1668013826 Table 6 shows that the amount of ethanol present in the tested compositions was within the range of about 12000 ppm to about 25000 ppm. Examples 13 & 14 In a clean and dry vessel, ˜50 L of water for injection was added and maintained at 2-8° C. Nitrogen purging was performed for 30 min. ˜2.0 kg of phenobarbital sodium was dissolved via stirring for 5-10 min. in to the water for injection and the clarity of the solution was observed to ensure that it was clear. Solution was filtered through a 0.2 micron PES filter and lyophilized. Ethanol content in the lyophilized phenobarbital sodium was measured to be about 13114 ppm. The lyophilized phenobarbital sodium was kept on stability at 20-25° C. for 36 months. Manufactured batch was also evaluated for reconstitution (in-use) stability. Drug product was reconstituted in 0.9% sodium chloride injection and stored at 20-25° C. and 2-8° C. for its stability evaluation. Reconstituted solution was analyzed at predefined time intervals. It is to be noted that the phenobarbital sodium API as used in these examples had an ethanol content of about 70000 ppm. The results of examples 13 were as shown in Table 7 below: TABLE 7Storage20-25° C.2-8° C.Stage of analysis0 h4 h8 h12 h0 h12 h24 h36 hUnknownBQLBQL0.0530.101BQL0.072BQLBQLimpurity(highestunspecified)(%)Total impurityBQLBQL0.0530.101BQL0.072BQLBQL(%)Osmolality365371366366371366364368mOsm/kgBQL: Below Quantitation Limit (0.05%) The results of examples 14 were as shown in Table 8 below: TABLE 8Storage20-25° C.2-8° C.Stage of analysis0 h4 h8 h12 h0 h12 h24 h36 hUnknownBQLBQLBQL0.088BQL0.063BQLBQLimpurity(highestunspecified)(%)Total impurityBQLBQLBQL0.088BQL0.063BQLBQL(%)Osmolality367369369365367367369365mOsm/kgBQL: Below Quantitation Limit (0.05%) Examples 15 and 16 Two 10% NaOH solutions were prepared in 50 ml flasks by mixing 5 g NaOH with water for injection sufficient to make 30 ml of solution. The resulting solution was mixed and shaken well. To prepare a bulk solution containing alcohol (example 15), 36.4 mg phenobarbital was placed in a clean and dry vessel. 0.045 ml of a 96% ethanol was added and the resultant was shaken and subject to a vortex for 1 minute. To this solution 0.063 ml of a 10% NaOH solution prepared previously was added and the resultant was subject to a vortex for 15 minutes. Water for injection sufficient to make 1 ml solution was then added and the resultant was subject to a vortex for 10 minutes. The resulting solution was transferred to a measuring cylinder and the pH was checked and adjusted by 0.1N HCl to be 9.78. Water for injection was added to obtain 1 ml of solution and the solution was stirred for 2 minutes. The resulting solution was filtered through a 0.2 micron PES filter, and the filtrate was collected and kept at room temperature (20-25° C.) or 2-8° C. until analysis. To prepare a bulk solution free from alcohol (example 16), 36.4 mg phenobarbital was placed in a clean and dry vessel. 0.045 ml of a 10% NaOH solution prepared previously was added and the resultant was subject to a vortex for 15 minutes. Water for injection sufficient to make 1 ml solution was then added and the resultant was subject to a vortex for 10 minutes. The resulting solution was transferred to a measuring cylinder and the pH was checked and adjusted by 0.1N HCl to be 9.74. Water for injection was added to obtain 1 ml of solution and the solution was stirred for 2 minutes. The resulting solution was filtered through a 0.2 micron PES filter, and the filtrate was collected and kept at room temperature (20-25° C.) or 2-8° C. until analysis. The two bulk solutions were analyzed at set time periods and Table 9 shows the results. TABLE 9ExampleImpurity20-25° C.2-8° C.150 h12 h24 h36 h0 h24 h36 hUnknown0.0420.3710.7081.0020.0420.1510.219impurity(highestunspecified)(%)Total0.0640.3980.7641.0910.0640.1730.236Impurity (%)ExampleUnknown0.0650.6341.2071.7240.0650.2690.38416impurity(highestunspecified)(%)Total0.0780.6741.2881.8590.0780.2930.404Impurity(%) Even though certain specific embodiments are thoroughly described in the present application, it should be understood that the same concepts disclosed with respect to those specific embodiments are also applicable to other embodiments. Furthermore, individual elements of the compositions and methods disclosed herein are described with reference to particular embodiments only for the sake of convenience. It should be understood that individual elements of the compositions and methods disclosed herein are applicable to embodiments other than the specific embodiments in which they are described. In addition, it should be understood that the scope of the present disclosure is not limited to the above-described embodiments, and those skilled in the art will appreciate that various modifications and alterations are possible without departing from the scope of the present disclosure. For example, the batch sizes may be altered by a person having ordinary skill in the art while staying within the present disclosure. Features of the Invention A1. A pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C.A2. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A3. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition has an alcohol content is in the range from about 12000 ppm to about 66000 ppm.A4. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition has an alcohol content is in the range from about 12000 ppm to about 25000 ppm.A5. The pharmaceutical composition of features A2-4, wherein alcohol is a C1-C3alcohol, such as ethanol.A6. The pharmaceutical composition of feature A1, wherein phenobarbital or salts thereof is phenobarbital sodium.A7. The pharmaceutical composition of feature A1, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A8. The pharmaceutical composition of feature A1, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A9. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A10. The pharmaceutical composition of feature A1, wherein phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml.A11. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition is free of benzyl alcohol.A12. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition is free of propylene glycol.A13. The pharmaceutical composition of feature A1, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A14. A method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the amount of total impurities in the pharmaceutical composition does not exceed 0.2% following 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C.A15. The method of feature A14, wherein the pharmaceutical composition is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes.A16. The method of feature A14, wherein the method comprises administering the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures; and if electrographic seizures persist or recur after completion of the initial loading dose, a second dose of 20 mg/kg is administered over subsequent 15 minutes for a total loading dose of 40 mg/kg.A17. The method of feature A14, wherein the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected.A18. The method of feature A17, wherein the correctable abnormalities are selected from hypoglycemia or hypocalcemia.A19. The method of feature A14, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A20. The method of feature A14, wherein the pharmaceutical composition has an alcohol content is in the range from about 12000 ppm to about 66000 ppm.A21. The method of feature A14, wherein the pharmaceutical composition has an alcohol content is in the range from about 12000 ppm to about 25000 ppm.A22. The method of features A19-21, wherein alcohol is a C1-C3alcohol, such as ethanol.A23. The method of feature A14, wherein phenobarbital or salts thereof is phenobarbital sodium.A24. The method of feature A14, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A25. The method of feature A14, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A26. The method of feature A14, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A27. The method of feature A14, wherein phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml.A28. The method of feature A14, wherein the pharmaceutical composition is free of benzyl alcohol.A29. The method of feature A14, wherein the pharmaceutical composition is free of propylene glycol.A30. The method of feature A14, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A31. A pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A32. The pharmaceutical composition of feature A31, wherein the composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A33. The pharmaceutical composition of feature A31, wherein the composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A34. The pharmaceutical composition of features A31-33, wherein alcohol is a C1-C3alcohol, such as ethanol.A35. The pharmaceutical composition of feature A31, wherein phenobarbital or salts thereof is phenobarbital sodium.A36. The pharmaceutical composition of feature A31, wherein the pharmaceutical composition is a lyophilized pharmaceutical composition of phenobarbital or salts thereof.A37. The pharmaceutical composition of feature A36, wherein the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C.A38. The pharmaceutical composition of feature A37, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A39. The pharmaceutical composition of feature A37, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A40. The pharmaceutical composition of feature A31, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A41. The pharmaceutical composition of feature A40, wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital or salts thereof.A42. The pharmaceutical composition of feature A41, wherein the lyophilized pharmaceutical composition of phenobarbital or salts thereof is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A43. The pharmaceutical composition of feature A40, wherein phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml.A44. The pharmaceutical composition of feature A40, wherein the aqueous solution is stable up to 12 hours of storage at 20-25° C.A45. The pharmaceutical composition of feature A44, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.5%.A46. The pharmaceutical composition of feature A44, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A47. The pharmaceutical composition of feature A40, wherein the aqueous solution is stable up to 36 hours of storage at 2-8° C.A48. The pharmaceutical composition of feature A47, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.5%.A49. The pharmaceutical composition of feature A47, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A50. The pharmaceutical composition of feature A40, wherein the aqueous solution has an osmolality below 500 mOsm/kg.A51. The pharmaceutical composition of features A38-39, A45-46 and A48-49, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A52. The pharmaceutical composition of feature A31, wherein the pharmaceutical composition is free of benzyl alcohol.A53. The pharmaceutical composition of feature A31, wherein the pharmaceutical composition is free of propylene glycol.A54. A method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A55. The method of feature A54, wherein the pharmaceutical composition is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes.A56. The method of feature A54, wherein the method comprises administering the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures; and if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg.A57. The method of feature A54, wherein the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected.A58. The method of feature A57, wherein the correctable abnormalities are selected from hypoglycemia or hypocalcemia.A59. The method of feature A54, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A60. The method of feature A54, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A61. The method of features A54, A59-60, wherein alcohol is a C1-C3alcohol, such as ethanol.A62. The method of feature A54, wherein phenobarbital or salts thereof is phenobarbital sodium.A63. The method of feature A54, wherein the pharmaceutical composition is a lyophilized pharmaceutical composition of phenobarbital or salts thereof.A64. The method of features A63, wherein the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C.A65. The method of features A64, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A66. The method of features A64, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A67. The method of feature A54, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A68. The method of features A67, wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital or salts thereof.A69. The method of features A68, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A70. The method of features A67, wherein the aqueous solution is stable up to 12 hours of storage at 20-25° C.A71. The method of features A70, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A72. The method of features A67, wherein the aqueous solution is stable up to 36 hours of storage at 2-8° C.A73. The method of features A72, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A74. The method of features A67, wherein the aqueous solution has an osmolality below 500 mOsm/kg.A75. The method of feature A54, wherein the pharmaceutical composition is free of benzyl alcohol.A76. The method of feature A54, wherein the pharmaceutical composition is free of propylene glycol.A77. A lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A78. The lyophilized pharmaceutical composition of feature A77, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A79. The lyophilized pharmaceutical composition of feature A77, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A80. The lyophilized pharmaceutical composition of features A77-79, wherein alcohol is a C1-C3alcohol, such as ethanol.A81. The lyophilized pharmaceutical composition of feature A77, wherein phenobarbital or salts thereof is phenobarbital sodium.A82. The lyophilized pharmaceutical composition of feature A77 is stable up to 36 months of storage at 20-25° C.A83. The lyophilized pharmaceutical composition of feature A82, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A84. The lyophilized pharmaceutical composition of feature A82, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A85. The lyophilized pharmaceutical composition of features A83-84, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A86. The lyophilized pharmaceutical composition of feature A77, wherein the lyophilized pharmaceutical composition is free of benzyl alcohol.A87. The lyophilized pharmaceutical composition of feature A77, wherein the lyophilized pharmaceutical composition is free of propylene glycol.A88. A method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering the lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A89. The method of feature A88 comprises reconstituting the lyophilized pharmaceutical composition of phenobarbital or salts thereof immediately prior to the administration.A90. The method of feature A89, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution to obtain the aqueous solution for injection of phenobarbital or salts thereof.A91. The method of feature A90, wherein the aqueous solution is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes.A92. The method of features A88 and A89, wherein the method comprises administering the aqueous solution at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures; and if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg.A93. The method of feature A88, wherein the method comprises administering to neonates in whom correctable abnormalities have been excluded or corrected.A94. The method of feature A93, wherein the correctable abnormalities are selected from hypoglycemia or hypocalcemia.A95. The method of feature A88, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A96. The method of feature A88, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A97. The method of features A88, A95-96, wherein alcohol is a C1-C3alcohol, such as ethanol.A98. The method of feature A88, wherein phenobarbital or salts thereof is phenobarbital sodium.A99. The method of feature A88, wherein the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C.A100. The method of feature A99, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A101. The method of feature A99, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A102. The method of features A100-101, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A103. The method of feature A88, wherein the lyophilized pharmaceutical composition is free of benzyl alcohol.A104. The method of feature A88, wherein the lyophilized pharmaceutical composition is free of propylene glycol.A105. A pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A106. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A107. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A108. The pharmaceutical composition of feature A105-107, wherein alcohol is a C1-C3alcohol, such as ethanol.A109. The pharmaceutical composition of feature A105, wherein phenobarbital or salts thereof is phenobarbital sodium.A110. The pharmaceutical composition of feature A105, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A111. The pharmaceutical composition of feature A105 is an aqueous solution for injection of phenobarbital or salts thereof.A112. The pharmaceutical composition of feature A105, wherein phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml.A113. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C.A114. The pharmaceutical composition of feature A113, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.5%.A115. The pharmaceutical composition of feature A113, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A116. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C.A117. The pharmaceutical composition of feature A116, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.5%.A118. The pharmaceutical composition of feature A116, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A119. The pharmaceutical composition of features A114-115 and A117-118, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A120. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A121. The pharmaceutical composition of feature A105, wherein the pharmaceutical composition is free of benzyl alcohol.A122. The pharmaceutical composition of feature AA105, wherein the pharmaceutical composition is free of propylene glycol.A123. A method for the treatment of neonatal seizure in newborn infants of 2 weeks of age or younger in need thereof, comprising administering a pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, and wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm.A124. The method of feature A123, wherein the pharmaceutical composition is administered intravenously by infusion at a dose of 20 mg/kg over a course of 15 minutes.A125. The method of feature A123, wherein the method comprises administering the pharmaceutical composition at an initial loading dose of 20 mg/kg over a course of 15 minutes and measuring the electrographic seizures; and if the electrographic seizures persist or recur after completion of the initial loading dose, a second dose 20 mg/kg is administered over the subsequent 15 minutes for a total loading dose of 40 mg/kg.A126. The method of feature A123, wherein the method comprises administering the pharmaceutical composition to neonates in whom correctable abnormalities have been excluded or corrected.A127. The method of feature A126, wherein the correctable abnormalities are selected from hypoglycemia or hypocalcemia.A128. The method of feature A123, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A129. The method of feature A126, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A130. The method of features A123, A128-129, wherein alcohol is a C1-C3alcohol, such as ethanol.A131. The method of feature A123, wherein phenobarbital or salts thereof is phenobarbital sodium.A132. The method of feature A123, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A133. The method of feature A123, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A134. The method of feature A123, wherein phenobarbital or salts thereof is present in a concentration of 10-200 mg/ml.A135. The method of feature A123, wherein the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C.A136. The method of feature A135, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A137. The method of feature A123, wherein the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C.A138. The method of feature A137, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A139. The method of features A136 and A138, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A140. The method of feature A123, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A141. The method of feature A123, wherein the pharmaceutical composition is free of benzyl alcohol.A142. The method of feature A123, wherein the pharmaceutical composition is free of propylene glycol.A143. A process of preparing the lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution of phenobarbital or salts thereof in a concentration of 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof.A144. The process of feature A143, wherein the process comprises measuring the alcohol content of the aqueous solution of phenobarbital or salts thereof and if the alcohol content is below 5000 ppm then the process further comprises a step of adding alcohol to achieve the alcohol content of at least about 5000 ppm.A145. The process of features A143, wherein the process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof and if the alcohol content is above 66000 ppm or about 70000 ppm (whichever is desired), the process further comprises repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or about 70000 ppm.A146. The process of feature A143 further comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 9-10.5.A147. The process of feature A143, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A148. The process of feature A143, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A149. The process of features A143, A147-148, wherein alcohol is a C1-C3alcohol, such as ethanol.A150. The process of feature A143, wherein phenobarbital or salts thereof is phenobarbital sodium.A151. The process of feature A143, wherein the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C.A152. The process of feature A151, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A153. The process of feature A151, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A154. The process of features A152-153, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A155. The process of feature A143, wherein the lyophilized pharmaceutical composition is free of benzyl alcohol.A156. The process of feature A143, wherein the lyophilized pharmaceutical composition is free of propylene glycol.A157. A process of preparing the lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; measuring the alcohol content of aqueous solution; if the alcohol content is below 5000 ppm, adding an alcohol to achieve the alcohol content of at least about 5000 ppm; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition; measuring the alcohol content of the lyophilized pharmaceutical composition; if the alcohol content is above about 66000 ppm or above about 70000 ppm (whichever is desired), repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or about 70000 ppm.A158. The process of feature A157 further comprises addition of a pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 9-10.5.A159. The process of feature A157, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A160. The process of feature A157, wherein the lyophilized pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A161. The process of features A157, A159-160, wherein alcohol is a C1-C3alcohol, such as ethanol.A162. The process of feature A157, wherein phenobarbital or salts thereof is phenobarbital sodium.A163. The process of feature A157, wherein the lyophilized pharmaceutical composition is stable up to 36 months of storage at 20-25° C.A164. The process of feature A163, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.5%.A165. The process of feature A163, wherein the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%.A166. The process of features A164-165, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A167. The process of feature A157, wherein the lyophilized pharmaceutical composition is free of benzyl alcohol.A168. The process of feature A157, wherein the lyophilized pharmaceutical composition is free of propylene glycol.A169. A process of preparing the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof; and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof.A170. The process of feature A169, wherein the process comprises measuring the alcohol content of the aqueous solution of phenobarbital or salts thereof and if the alcohol content is below 5000 ppm then the process further comprises a step of adding alcohol to achieve the alcohol content of at least about 5000 ppm.A171. The process of features A169, wherein said process comprises measuring the alcohol content of the lyophilized pharmaceutical composition of phenobarbital or salts thereof and if the alcohol content is above about 66000 ppm or about 70000 ppm (whichever is desired), the process further comprises repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or about 70000 ppm.A172. The process of feature A169 further comprises addition of pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 9-10.5.A173. The process of feature A169, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A174. The process of feature A169, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A175. The process of feature A169, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A176. The process of feature A169, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A177. The process of feature A169, A175-176, wherein alcohol is a C1-C3alcohol, such as ethanol.A178. The process of feature A169, wherein phenobarbital or salts thereof is phenobarbital sodium.A179. The process of feature A169, wherein the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C.A180. The process of feature A179, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A181. The process of feature A169, wherein the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C.A182. The process of feature A181, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A183. The process of features A180 and A182, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A184. The process of feature A169, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A185. The process of feature A169, wherein the pharmaceutical composition is free of benzyl alcohol.A186. The process of feature A169, wherein the pharmaceutical composition is free of propylene glycol.A187. A process of preparing the pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof, wherein the pharmaceutical composition has an alcohol content in the range from about 5000 ppm to about 66000 ppm or alternatively from about 5000 ppm to about 70000 ppm, wherein the process comprises dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; measuring the alcohol content of aqueous solution; if the alcohol content is below 5000 ppm, adding an alcohol to achieve the alcohol content of at least about 5000 ppm; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition; measuring the alcohol content of the lyophilized pharmaceutical composition; if the alcohol content is above 66000 ppm, repeating the lyophilization step multiple times till the alcohol content of the lyophilized pharmaceutical composition is not more than about 66000 ppm or not more than 70000 ppm (whichever is desired); and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof.A188. The process of feature A187 further comprises addition of pH modifier to the aqueous solution of phenobarbital or salts thereof to achieve a pH in a range of 9-10.5.A189. The process of feature A187, wherein the lyophilized pharmaceutical composition is reconstituted with water for injection, an aqueous saline or an aqueous dextrose solution.A190. The process of feature A187, wherein the pharmaceutical composition is an aqueous solution for injection of phenobarbital or salts thereof.A191. The process of feature A187, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 66000 ppm.A192. The process of feature A187, wherein the pharmaceutical composition has an alcohol content in the range from about 12000 ppm to about 25000 ppm.A193. The process of feature A187, A191-192, wherein alcohol is a C1-C3alcohol, such as ethanol.A194. The process of feature A187, wherein phenobarbital or salts thereof is phenobarbital sodium.A195. The process of feature A187, wherein the pharmaceutical composition is stable up to 12 hours of storage at 20-25° C.A196. The process of feature A195, wherein the amount of total impurities present following 12 hours of storage at 20-25° C. does not exceed 0.2%.A197. The process of feature A187, wherein the pharmaceutical composition is stable up to 36 hours of storage at 2-8° C.A198. The process of feature A197, wherein the amount of total impurities present following 36 hours of storage at 2-8° C. does not exceed 0.2%.A199. The process of features A196 and A198, wherein the total impurities are selected from 2-phenyl-2-ethyl acetyl urea, 2-phenyl-2-ethyl-malonamide, α-phenylbutyrylguanidine, 2-phenylbutyric acid and 5-methyl-5-phenylbarbituric acid.A200. The process of feature A187, wherein the pharmaceutical composition has an osmolality below 500 mOsm/kg.A201. The process of feature A187, wherein the pharmaceutical composition is free of benzyl alcohol.A202. The process of feature A187, wherein the pharmaceutical composition is free of propylene glycol.A203. A lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm, wherein the lyophilized pharmaceutical composition is obtained by a process comprising: dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof.A204. A pharmaceutical composition of phenobarbital or salts thereof that has been reconstituted from a lyophilized pharmaceutical composition of phenobarbital or salts thereof having an alcohol content in the range from about 5000 ppm to about 66000 ppm, wherein the pharmaceutical composition is obtained by a process comprising: dissolving phenobarbital or salts thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml; lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition of phenobarbital or salts thereof; and reconstituting the lyophilized pharmaceutical composition to obtain the pharmaceutical composition of phenobarbital or salts thereof.A205. A lyophilized pharmaceutical composition comprising phenobarbital sodium and ethanol, wherein ethanol is present in an amount sufficient to inhibit degradation of phenobarbital sodium, such that the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%;wherein the amount of ethanol sufficient to inhibit degradation of phenobarbital sodium is in the range from about 12000 ppm to about 25000 ppm;and wherein the pharmaceutical composition is free of benzyl alcohol and propylene glycol.A206. An aqueous solution for injection comprising phenobarbital sodium and ethanol, wherein ethanol is present in an amount sufficient to inhibit degradation of phenobarbital sodium, such that the amount of total impurities present following 12 hours of storage at 20-25° C. or following 36 hours of storage at 2-8° C. does not exceed 0.2%;wherein the amount of ethanol sufficient to inhibit degradation of phenobarbital sodium is in the range from about 12000 ppm to about 25000 ppm;wherein phenobarbital sodium is present in a concentration from 10-200 mg/ml;wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital sodium;and wherein the aqueous solution is free of benzyl alcohol and propylene glycol.A207. A process for the preparation of the pharmaceutical composition of phenobarbital or salts thereof, the process comprises dissolving phenobarbital or salt thereof in water to obtain an aqueous solution having a concentration 10-200 mg/ml and lyophilizing the aqueous solution to obtain lyophilized pharmaceutical composition, wherein phenobarbital or salts thereof has an alcohol content in the range from about 5000 ppm to about 66000 ppm.A208. A lyophilized pharmaceutical composition of phenobarbital sodium, wherein the composition has an ethanol content in the range from about 12000 ppm to about 25000 ppm; wherein the composition is stable up to 36 months of storage at 20-25° C. such that the amount of total impurities present following 36 months of storage at 20-25° C. does not exceed 0.2%; and wherein the composition is free of benzyl alcohol and propylene glycol.A209. An aqueous solution for injection of phenobarbital sodium, wherein the aqueous solution has an ethanol content in the range from about 12000 ppm to about 25000 ppm; wherein the aqueous solution is stable up to 12 hours of storage at 20-25° C. or 36 hours of storage at 2-8° C. such that the amount of total impurities present following 12 hours of storage at 20-25° C. or following 36 hours of storage at 2-8° C. does not exceed 0.2%; wherein the aqueous solution is reconstituted from the lyophilized pharmaceutical composition of phenobarbital sodium; and wherein the aqueous solution is free of benzyl alcohol and propylene glycol. | 153,666 |
11857684 | DETAILED DESCRIPTION Ailments like the common cold and flu often bring with them a multitude of symptoms, which are not all readily treated by a single active pharmaceutical ingredient. Fixed-dose combination formulations are one means of ameliorating multiple symptoms simultaneously to provide therapeutic relief with a single compact dosage form. However, the preparation of fixed-dose combinations is not a simple or straightforward process. Formulations containing a single active pharmaceutical ingredient are typically optimized with respect to a number of criteria, including but not limited to storage stability and drug dissolution and/or absorption rate, for the particular active pharmaceutical ingredient to be delivered. The combination of two or more active pharmaceutical ingredients requires careful thought as to how to accommodate the specific chemical instabilities of each component and mitigate any new formulation incompatibilities that may arise between the two or more active components. For example, acetylsalicylic acid may be susceptible to hydrolysis degradation to form less stable free salicylic acid as well as other unwanted and/or inactive byproducts in the presence of water (such as residual moisture content introduced by excipients). Similarly, certain environmental and/or chemical conditions may degrade pseudoephedrine over time to produce less active or inactive derivatives. In addition to their individual susceptibilities to degradation, acetylsalicylic acid and pseudoephedrine may accelerate existing degradation pathways of the other active ingredients when combined, which may result in reduced therapeutic efficacy overall. Therefore, a major obstacle in preparing a fixed-dose combination is striking a balance of chemical and storage stability with the desired delivery properties while maintaining or improving the overall efficacy of both individual active ingredients in a single dosage form. An additional concern with fixed-dose combinations, as with individual active pharmaceutical ingredients, is ensuring that the fixed-dose combinations demonstrate similar dissolution properties and rapid disintegration times in order to provide fast-acting relief from symptoms. While modifications may be made to address the storage stability and chemical compatibility of two or more active pharmaceutical ingredients, if the onset of action or symptomatic relief is not comparably as quick as would be provided with the individual dosage forms taken alone, consumers may be discouraged from using the fixed-dose combination. For example, acetylsalicylic acid is also known to have limited solubility in water and under acidic conditions. The solubility of acetylsalicylic acid is increased under basic conditions, as in the gastrointestinal tract. The addition of basic dissolution aids to acetylsalicylic acid formulations may improve the solubility of acetylsalicylic acid within a given environment but can further expose acetylsalicylic acid to additional degradation and/or chemical rearrangement pathways to form less therapeutically active degradation products. For fixed-dose combinations, the concern is compounded by the difficulty in selecting suitable dissolution aids that are chemically compatible with each active ingredient and any excipients, achieving congruent dissolution times for the two or more active ingredients, and, thus also, a single dosage form having uniform dissolution and stability properties throughout. Thus, there remains a need for fixed-dose combination dosage forms that achieve both storage stability and dissolution rate on par with the individual dosage forms of the relevant active pharmaceutical ingredients. The present disclosure addresses this need by providing a fixed-dose combination dosage form that succeed in preserving the storage stability and dissolution rate for its component active pharmaceutical ingredients as observed in corresponding individual dosage forms. More specifically, provided herein is a bilayer tablet, comprising acetylsalicylic acid, pseudoephedrine or a pharmaceutically acceptable salt thereof, and a dissolution aid. The bilayer tablet possesses a unique internal architecture that ultimately provides a quick-acting, but storage stable formulation of acetylsalicylic acid and pseudoephedrine in a single dosage form. The properties of the bilayer tablets described in the present disclosure are made possible by a combination of specific structural aspects utilized in the bilayer tablets. In order to achieve the desired stability and dissolution properties of both acetylsalicylic acid and pseudoephedrine in a single dosage form, the bilayer tablets employ two layers to separate the two actives from one another, a distribution of dissolution aids across the two layers, modification to the physical forms (e.g., granulated and non-granulated) of the acetylsalicylic acid present within the acetylsalicylic acid layer, and particular mass ratios of acetylsalicylic acid to the dissolution aid in the granules and the bilayer tablet as a whole. As suggested by the term “bilayer”, the bilayer tablet of the present disclosure utilizes two discrete layers, with one layer containing acetylsalicylic acid and the other containing pseudoephedrine. The separation of the two active pharmaceutical ingredients into two layers reduces the physical proximity and, thus, any chemical interaction between the two active components. With the incompatible active ingredients isolated from one another, the risk of accelerated degradation that may be caused by the combination of the two components is mitigated. The bilayer tablet further employs a distribution of dissolution aid sodium carbonate across the two layers of the tablet and, within the acetylsalicylic acid layer, an admixture of granulated acetylsalicylic acid and non-granulated powder acetylsalicylic acid in the acetylsalicylic acid layer. As described above, dissolution aids such as sodium carbonate modulate the local environment of the dosage form and thus promote dissolution of the active pharmaceutical ingredient, e.g., acetylsalicylic acid. However, in the case of dissolution aids and acetylsalicylic acid, chemical reactions between the two components over time may also lead to the increased presence of degradation products. In the bilayer tablets provided herein, the dissolution aid is distributed across both layers of the tablet, e.g., with the majority of the dissolution aid in the pseudoephedrine layer. The allocation of the dissolution aid in this manner improves the dissolution profile of acetylsalicylic acid during administration (i.e., dissolves more rapidly) but reduces the extent of direct physical contact of acetylsalicylic acid with the dissolution aid in the acetylsalicylic acid layer itself and decreases the potential for undesired chemical interactions to occur. Within the acetylsalicylic acid layer, the bilayer tablet utilizes a combination of acetylsalicylic acid in a non-granulated powder form with acetylsalicylic acid granules, wherein the dissolution aid present in the acetylsalicylic acid layer is restricted to the acetylsalicylic acid granules. The percentage of intragranular acetylsalicylic acid (provided in the granules) out of the total acetylsalicylic acid present is maintained within specific ranges (e.g., 10-50% w/w) to aid dissolution. Additionally, the ratio of acetylsalicylic acid to the dissolution aid(s) are controlled within certain mass ratio ranges, e.g., between 1:1 and 7:1, in both the granules and the bilayer tablet taken as a whole. The control over the quantities of acetylsalicylic acid and dissolution aids in the tablets as described above optimizes the effect of the dissolution aid where it directly contacts the acetylsalicylic acid by achieving minimal loss of the acetylsalicylic acid to degradation associated with the dissolution aids during storage with maximal dissolution of acetylsalicylic acid once administered. By virtue of the compartmentalization of the active ingredients and dissolution aid(s), the bilayer tablets described herein provide fast-acting therapeutic benefit in a single, storage stable fixed-dose combination dosage form. The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. It is understood that aspects and variations described herein also include “consisting” and/or “consisting essentially of” aspects and variations. Bilayer Tablet Provided herein is a fixed combination dosage form comprising two active pharmaceutical ingredients in a bilayer tablet, wherein each layer comprises one of the two active pharmaceutical ingredients, and both layers comprise one or more dissolution aids. As described herein, a primary aspect of the bilayer tablets provided herein is the separation of active pharmaceutical ingredients, which may be chemically incompatible (e.g., reactive), into discrete layers. Reduction of the physical contact between active pharmaceutical ingredients that could induce or accelerate degradation enhances the storage stability of the active ingredients and the shelf life of the bilayer tablet. In one aspect, provided herein is a bilayer tablet, wherein the bilayer tablet comprises a first layer and a second layer, wherein the first layer comprises a first active pharmaceutical ingredient, and wherein the second layer comprises a second active pharmaceutical ingredient. In some embodiments, the bilayer tablet comprises a first layer and a second layer, wherein the first layer comprises acetylsalicylic acid, and the second layer comprises pseudoephedrine or a pharmaceutically acceptable salt thereof. With reference toFIG.1, schematic100is an exemplary schematic of a bilayer tablet comprising a first layer (acetylsalicylic acid (ASA) layer)102and a second layer (pseudoephedrine hydrochloride (PSEH) layer)104. The acetylsalicylic acid layer102comprises a combination of granulated acetylsalicylic acid (e.g. roller-compacted granules) and non-granulated acetylsalicylic acid external to the granules (e.g., in a direct blend). As shown inFIG.1, sodium carbonate is used as a dissolution aid and is incorporated in both layers102and104. The sodium carbonate in the acetylsalicylic acid layer102is contained entirely within the granules. With reference again toFIG.1, in some embodiments, the bilayer tablet further comprises an external lubricant or coating106. In certain embodiments, the coating comprises a combination of hypromellose, zinc stearate and carnauba wax. In still further embodiments, the coating comprises a flavorant. It should be recognized that the bilayer tablets of the present disclosure may also be suitable to deliver combinations of active pharmaceutical ingredients, wherein one or both analgesic/antipyretic and nasal decongestant active pharmaceutical ingredients is substituted with a therapeutically equivalent active ingredient. Such combinations may include, for example, naproxen or ibuprofen as analgesics/antipyretics in place of acetylsalicylic acid, phenylephrine as a nasal decongestant in lieu of pseudoephedrine, or any combinations thereof. It should be further recognized that the bilayer tablets of the present disclosure may further comprise one or more additional active pharmaceutical ingredients in addition to acetylsalicylic acid and pseudoephedrine or a pharmaceutically salt thereof, provided the additional active ingredient is compatible with one or both of the acetylsalicylic acid and pseudoephedrine, and may be incorporated into one or both of the layers in the bilayer tablet. The inclusion of additional active pharmaceutical ingredients may be appropriate for the treatment of or provision of relief from other symptoms associated with the common cold and/or flu, such as sneezing or coughing. In still further embodiments, the bilayer tablets of the present disclosure may be suitable to deliver any two active pharmaceutical ingredients for a fixed-dose combination treatment, especially combinations of active pharmaceutical ingredients that may exhibit similar potential for degradation due to external variables (moisture content) and/or share similar incompatibilities as acetylsalicylic acid and pseudoephedrine. First Layer, or Acetylsalicylic Acid Layer In some embodiments, the bilayer tablet of the present disclosure comprises a first layer, wherein the first layer comprises a first active pharmaceutical ingredient. In certain embodiments, the bilayer tablet comprises a first layer comprising an analgesic. In still further embodiments, the analgesic is acetylsalicylic acid. Alternatively, in some embodiments wherein the bilayer tablet comprises acetylsalicylic acid in the first layer, the first layer may be described as an acetylsalicylic acid layer. Acetylsalicylic acid is a pain-relieving (analgesic) and fever-reducing (antipyretic) agent. Acetylsalicylic acid may also be used as an anti-inflammatory agent. As a result of its myriad effects, acetylsalicylic acid is widely used to treat various ailments, including those associated with the common cold and/or flu. Depending on the desired therapeutic effect to be provided by the bilayer tablet—that is, the extent to which the cold and flu symptoms are alleviated and how long relief is provided, the quantities of the active pharmaceutical ingredients, such as acetylsalicylic acid, contained in the bilayer tablet may vary. The quantity of the acetylsalicylic acid present in the bilayer tablet may be expressed in terms of absolute milligram amounts. It should be recognized that the absolute milligram amounts of the present disclosure are intended to indicate the quantity of the acetylsalicylic acid in an individual bilayer tablet. In some embodiments, the bilayer tablet comprises at least 50 mg, at least 75 mg, at least 100 mg, at least 150 mg, or at least 250 mg acetylsalicylic acid. In other embodiments, the bilayer tablet comprises less than or equal to 1000 mg, less than or equal to 500 mg, less than or equal to 325 mg, or less than or equal to 250 mg acetylsalicylic acid. Alternatively, the quantity of the acetylsalicylic acid may also be expressed as a weight percentage of the total weight of the bilayer tablet. In some embodiments, the bilayer tablet comprises at least 25% w/w, at least 30% w/w, at least 40% w/w, or at least 50% w/w acetylsalicylic acid by total weight of the bilayer tablet. In other embodiments, the bilayer tablet comprises less than or equal to 80% w/w, less than or equal to 75% w/w, less than or equal to 70% w/w, or less than or equal to 60% w/w acetylsalicylic acid by total weight of the bilayer tablet. In still further embodiments, the bilayer tablet comprises acetylsalicylic acid, wherein the entirety of acetylsalicylic acid present in the bilayer tablet is contained with the first (acetylsalicylic acid) layer. In certain embodiments wherein the entirety of acetylsalicylic acid present in the bilayer tablet is contained with the first (acetylsalicylic acid) layer, the amount of acetylsalicylic acid present may be characterized by a weight percentage of the weight of the acetylsalicylic acid layer. For example, in some embodiments, the acetylsalicylic acid layer comprises at least 50% w/w, at least 60% w/w, at least 70% w/w, or at least 75% w/w acetylsalicylic acid by weight of the acetylsalicylic acid layer. In other embodiments, the bilayer tablet comprises less than or equal to 95% w/w, less than or equal to 90% w/w, or less than or equal to 85% w/w acetylsalicylic acid by weight of the acetylsalicylic acid layer. As described above, the bilayer tablet of the present disclosure employs a combination of granulated and non-granulated acetylsalicylic acid within one of the layers of the bilayer tablet in order to achieve its observed storage stability and dissolution properties. In some embodiments, the acetylsalicylic acid present in the bilayer tablet may be further characterized by total quantity of acetylsalicylic acid, which is the sum of acetylsalicylic acid in granulated and non-granulated forms. In other embodiments, the bilayer tablet comprises granules, wherein at least a portion of the total acetylsalicylic acid present is contained within the granules (i.e., intragranular) and the remaining portion of the total acetylsalicylic acid is contained within the first layer external to the granules (i.e., extragranular). Granules (Acetylsalicylic Acid Composite) As described above, the bilayer tablets of the present disclosure comprise granules, wherein the granules comprise acetylsalicylic acid, the inclusion of which granules may lead to a faster absorption profile and improvement in the pharmacokinetic profile. The granules, which may collectively be referred to as an acetylsalicylic acid composite, also include one or more dissolution aids. It was observed that the incorporation of the acetylsalicylic acid composite comprising acetylsalicylic acid in intimate contact with one or more dissolution aids provided improvement in dissolution rate of the acetylsalicylic acid layer, particularly with respect to matching the dissolution profile of the active ingredient pseudoephedrine (or phenylephrine) in the second layer of the tablet. Without being bound to a particular theory of the invention, the envelopment of the acetylsalicylic acid by the dissolution aid and the smaller particle sizes of the ingredients are believed to lead to observed, significant improvement in the dissolution profile for the active pharmaceutical ingredient. Additional benefits of the envelopment of acetylsalicylic acid by the dissolution aid include the protection of the granulated acetylsalicylic acid from adventitious moisture and the secondary behavior of dissolution aid to act like a starch or binder, thereby removing the need for additional fillers, binders and or stabilizers in the granules. The quantity of acetylsalicylic acid present in the granules may be described as a weight percentage of the weight of the granules. In some embodiments, the granules comprise at least 50% w/w, at least 60% w/w, or at least 70% w/w acetylsalicylic acid by weight of the granules. In other embodiments, the granules comprise less than or equal to 90% w/w, less than or equal to 80% w/w, or less than or equal to 75% w/w acetylsalicylic acid by weight of the granules. In certain embodiments, the acetylsalicylic acid present in the granules may be referred to as intragranular acetylsalicylic acid. In still other embodiments, the quantity of intragranular acetylsalicylic acid present in the granules may be described as a weight percentage of the weight of the acetylsalicylic acid layer. In some embodiments, the acetylsalicylic acid layer comprises at least 10% w/w, at least 15% w/w, or at least 20% w/w intragranular acetylsalicylic acid by weight of the acetylsalicylic acid layer. In other embodiments, the granules comprise less than or equal to 50% w/w, less than or equal to 40% w/w, or less than or equal to 30% w/w intragranular acetylsalicylic acid by weight of the acetylsalicylic acid layer. In some embodiments, the granules comprise one or more dissolution aids. Dissolution aids, which may also be referred to as solubilizing agents or solubility enhancers, may enhance the dissolution profile of active pharmaceutical ingredients in a formulation. Suitable dissolution aids may include but are not limited to surfactants (e.g., sodium lauryl sulfate), magnesium hydroxide, magnesium oxide, aluminum oxide, calcium carbonate, sodium carbonate, sodium bicarbonate, or any combinations thereof. As with intragranular acetylsalicylic acid above, the dissolution aid present in the granules may be referred to as intragranular dissolution aid and characterized by a weight percentage of the weight of the granules. In some embodiments, the granules comprise at least 5% w/w, at least 10% w/w, at least 15% w/w, or at least 20% w/w dissolution aid by weight of the granules. In other embodiments, the granules comprise less than or equal to 50% w/w, less than or equal to 40% w/w, or less than or equal to 30% w/w dissolution aid by weight of the granules. In still further embodiments, the amount of intragranular dissolution aid may be described as a weight percentage of the weight of the acetylsalicylic acid layer. In some embodiments, the acetylsalicylic acid layer comprises at least 1% w/w, at least 2% w/w, at least 5% w/w, or at least 7% w/w intragranular dissolution aid by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 15% w/w, less than or equal to 12% w/w, or less than or equal to 10% w/w intragranular dissolution aid by weight of the acetylsalicylic acid layer. In some embodiments, the granules comprise sodium carbonate. In some embodiments, the granules comprise at least 5% w/w, at least 10% w/w, at least 15% w/w, or at least 20% w/w sodium carbonate by weight of the granules. In other embodiments, the granules comprise less than or equal to 50% w/w, less than or equal to 40% w/w, or less than or equal to 30% w/w sodium carbonate by weight of the granules. In some embodiments, the acetylsalicylic acid layer comprises at least 1% w/w, at least 2% w/w, at least 5% w/w, or at least 7% w/w intragranular sodium carbonate by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 15% w/w, less than or equal to 12% w/w, or less than or equal to 10% w/w intragranular sodium carbonate by weight of the acetylsalicylic acid layer. In other embodiments, the granules comprises one or more intragranular excipients in addition to the dissolution aid described above. Such intragranular excipients may be included in order to facilitate manufacture of the granules themselves or to modulate certain physical properties of the granules produced. Additional intragranular excipients apart from the dissolution aid(s) described above may include, but are not limited to, flow aids, diluents, binders, and disintegrants. In some embodiments, the granules comprise a flow aid. Flow aids, also known as glidants, may be employed to reduce friction between powder or granular material and increase flowability. Exemplary flow aids include but are not limited to silicon dioxide, colloidal silicon dioxide, talc, magnesium stearate, zinc stearate, and stearic acid. In some embodiments, the granules comprise colloidal silicon dioxide. In some embodiments, the granules comprise at least 0.1% w/w, at least 0.2% w/w, or at least 0.3% w/w colloidal silicon dioxide by weight of the granules. In other embodiments, the granules comprise less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w colloidal silicon dioxide by weight of the granules. In other embodiments, the acetylsalicylic acid layer comprises at least 0.03% w/w, at least 0.06% w/w, or at least 0.1% w/w intragranular colloidal silicon dioxide by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 0.5% w/w, less than or equal to 1% w/w, or less than or equal to 2% w/w intragranular colloidal silicon dioxide by weight of the acetylsalicylic acid layer. Extragranular Acetylsalicylic Acid and Other Excipients In some embodiments, the first (acetylsalicylic acid) layer comprises additional active pharmaceutical ingredient outside of the granules described herein. Active pharmaceutical ingredient outside of the granules but contained within the first layer may be referred to herein as extragranular active pharmaceutical ingredient. As described above, the bilayer tablets of the present disclosure utilize an admixture of acetylsalicylic acid in granulated and non-granulated form. The use of acetylsalicylic acid in both granulated and non-granulated forms contributes to the observed storage stability as well as the fast dissolution profile of the acetylsalicylic acid in the bilayer tablets. For example, in some embodiments wherein acetylsalicylic acid is the active pharmaceutical ingredient in the first layer (acetylsalicylic acid layer), the first layer comprises extragranular acetylsalicylic acid. In some embodiments, the first layer comprises extragranular acetylsalicylic acid. In some embodiments, the acetylsalicylic acid layer comprises at least 30% w/w, at least 40% w/w, or at least 50% w/w extragranular acetylsalicylic acid by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 80% w/w, less than or equal to 75% w/w, less than or equal to 70% w/w, or less than or equal to 60% w/w extragranular acetylsalicylic acid by weight of the acetylsalicylic acid layer. In some embodiments, the first layer further comprises additional active pharmaceutical excipients outside of the granules described herein. As with acetylsalicylic acid described above, excipients present outside of the granules but contained within the first layer may be referred to herein as extragranular excipients. In some embodiments wherein the first layer is an acetylsalicylic acid layer, the acetylsalicylic acid layer comprises one or more extragranular excipients. Similar to the intragranular excipients described above, extragranular excipients may be included in order to facilitate manufacture of the acetylsalicylic acid layer (e.g., flowability, ejection from tablet press) or to modulate certain physical properties of the acetylsalicylic acid layer produced (e.g., dissolution rate). In some embodiments, the one or more extragranular excipients comprise one or more extragranular binders (e.g, cellulose), one or more extragranular disintegrants (e.g., cornstarch), one or more extragranular glidants/flow aids (e.g., colloidal silicon dioxide), or any combinations thereof. In some embodiments, the acetylsalicylic acid layer comprises one or more extragranular binders. Binders may be incorporated in the acetylsalicylic acid layer to help adhere the extragranular acetylsalicylic acid, granules and other extragranular excipients together. Binder excipients may also contribute to the overall volume or mechanical properties of the resulting formulation, that is, the acetylsalicylic acid layer. In certain embodiments, the one or more extragranular binders comprises cellulose. In certain embodiments, the acetylsalicylic acid layer comprises at least 1% w/w, at least 2% w/w, or at least 5% w/w cellulose by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprise less than or equal to 15% w/w, less than or equal to 12% w/w, less than or equal to 10% w/w, or less than or equal to 7% w/w cellulose by weight of the acetylsalicylic acid layer. In some embodiments, the acetylsalicylic acid layer comprises one or more extragranular disintegrants. Disintegrants are typically included in pharmaceutical formulations to aid in the dissolution process. Upon contact with moisture, disintegrants promote breakage of a solid dosage form into smaller pieces for more rapid solubilization. In certain embodiments, the one or more extragranular disintegrants comprises cornstarch. In some embodiments, the acetylsalicylic acid layer comprises at least 1% w/w, at least 2% w/w, or at least 5% w/w cornstarch by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprise less than or equal to 15% w/w, less than or equal to 12% w/w, less than or equal to 10% w/w, or less than or equal to 7% w/w cornstarch by weight of the acetylsalicylic acid layer. In still further embodiments, the acetylsalicylic acid layer comprises one or more extragranular glidants. Glidants may be incorporated to improve the flowability of extragranular acetylsalicylic acid, granules, and other extragranular excipients for easier manufacture and tableting. In certain embodiments, the one or more extragranular glidants comprises colloidal silicon dioxide. In certain embodiments, the acetylsalicylic acid layer comprises at least 0.1% w/w, at least 0.2% w/w, or at least 0.3% w/w colloidal silicon dioxide by weight of the acetylsalicylic acid layer. In other embodiments, the acetylsalicylic acid layer comprise less than or equal to 5% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w colloidal silicon dioxide by weight of the acetylsalicylic acid layer. Distribution of Acetylsalicylic Acid in First Layer As described above, the bilayer tablets of the present invention may comprise a total quantity of acetylsalicylic acid distributed within the first (acetylsalicylic acid) layer in either granulated or non-granulated form. The distribution of acetylsalicylic acid between granulated and non-granulated forms may influence the dissolution profile as well as the storage stability of the resulting acetylsalicylic acid layer, and thus, also, the final bilayer tablet. As described herein, it was observed that certain ratios of extragranular acetylsalicylic acid to intragranular acetylsalicylic acid in combination with distribution of a dissolution aid across two layers of a bilayer tablet resulted in rapid dissolution rate without compromising storage stability. The amounts of granulated and non-granulated acetylsalicylic acid in the acetylsalicylic acid layer may be described as a percentage of the total acetylsalicylic acid present, or as a weight ratio of acetylsalicylic acid contained in the granules (that is, intragranular acetylsalicylic acid) to acetylsalicylic acid external to the granules (extragranular acetylsalicylic acid), or, conversely, a weight ratio of extragranular acetylsalicylic acid to intragranular acetylsalicylic acid. In some embodiments, the distribution of acetylsalicylic acid in the first layer of the tablet is such that at least 40% w/w, at least 50% w/w, at least 60% w/w, or at least 70% w/w of the total acetylsalicylic acid present in the bilayer tablet is extragranular acetylsalicylic acid. In other embodiments, less than or equal to 90% w/w, less than or equal to 80% w/w, less than or equal to 70% w/w, or less than or equal to 60% w/w of the total acetylsalicylic acid present in the bilayer tablet is extragranular acetylsalicylic acid. As described another way, in some embodiments, at least 10% w/w, at least 20% w/w, at least 30% w/w or at least 40% w/w of the total acetylsalicylic acid present in the bilayer tablet is intragranular acetylsalicylic acid. In other embodiments, the granules comprise less than or equal to 60% w/w, less than or equal to 50% w/w, less than or equal to 40% w/w, or less than or equal to 30% w/w of the total acetylsalicylic acid present in the bilayer tablet is intragranular acetylsalicylic acid. In further embodiments, the ratio of extragranular acetylsalicylic acid to intragranular acetylsalicylic acid is at least about 40:60, at least about 50:50, at least about 60:40, or at least about 70:30. In still other embodiments, the ratio of extragranular acetylsalicylic acid to intragranular acetylsalicylic acid is less than or equal to 60:40, less than or equal to 70:30, less than or equal to 80:20, or less than or equal to 90:10. Second Layer, or Pseudoephedrine Layer In some embodiments, the bilayer tablet comprises a second layer, wherein the second layer comprises a second active pharmaceutical agent. In some embodiments, the bilayer tablet comprises a second layer, wherein the second layer comprises a decongestant. In certain embodiments, the decongestant is pseudoephedrine or a pharmaceutically acceptable salt thereof. In still certain other embodiments, the second layer comprises pseudoephedrine hydrochloride, or pseudoephedrine HCl. Alternatively, in some embodiments wherein the bilayer tablet comprises pseudoephedrine in the second layer, the second layer may be described as a pseudoephedrine layer. The quantity of the pseudoephedrine (or other decongestant) present in the bilayer tablet may be expressed in terms of absolute milligram amounts. It should be recognized that the absolute milligram amounts of the present disclosure are intended to indicate the quantity of the pseudoephedrine in an individual bilayer tablet. In some embodiments, the bilayer tablet comprises at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, or at least 30 mg pseudoephedrine or a pharmaceutically acceptable salt thereof. In other embodiments, the bilayer tablet comprises less than or equal to 240 mg, less than or equal to 120 mg, less than or equal to 100 mg, less than or equal to 80 mg, or less than or equal to 60 mg pseudoephedrine or a pharmaceutically acceptable salt thereof. Alternatively, the quantity of the pseudoephedrine or a pharmaceutically acceptable salt thereof may also be expressed as a weight percentage of the total weight of the bilayer tablet. In some embodiments, the bilayer tablet comprises at least 1% w/w, at least 2% w/w, or at least 3% w/w pseudoephedrine or a pharmaceutically acceptable salt thereof. In other embodiments, the bilayer tablet comprises less than or equal to 24% w/w, less than or equal to 12% w/w, less than or equal to 10% w/w, less than or equal to 8% w/w, or less than or equal to 6% w/w pseudoephedrine or a pharmaceutically acceptable salt thereof. In still further embodiments, the bilayer tablet comprises pseudoephedrine or a pharmaceutically acceptable salt thereof, wherein the entirety of pseudoephedrine or a pharmaceutically acceptable salt thereof present in the bilayer tablet is contained with the second (pseudoephedrine) layer. In certain embodiments wherein the entirety of pseudoephedrine or a pharmaceutically acceptable salt thereof present in the bilayer tablet is contained with the second (pseudoephedrine) layer, the amount of pseudoephedrine or a pharmaceutically acceptable salt thereof present may be characterized by a weight percentage of the weight of the pseudoephedrine layer. For example, in some embodiments, the pseudoephedrine layer comprises at least 2% w/w, at least 5% w/w, or at least 7% w/w pseudoephedrine or a pharmaceutically acceptable salt thereof by weight of the pseudoephedrine layer. In other embodiments, the bilayer tablet comprises less than or equal to 15% w/w, less than or equal to 12% w/w, or less than or equal to 10% w/w pseudoephedrine or a pharmaceutically acceptable salt thereof by weight of the pseudoephedrine layer. As described above, the bilayer tablets of the present disclosure may utilize alternative nasal decongestants in lieu of pseudoephedrine. For example, in some embodiments, the second layer comprises phenylephrine or a pharmaceutically acceptable salt thereof in place of pseudoephedrine in a therapeutically equivalent amount. In other embodiments, the decongestant is phenylephrine or a pharmaceutically acceptable salt thereof. In certain embodiments, the second layer comprises phenylephrine hydrochloride, or phenylephrine HCl. In some embodiments, the bilayer tablet comprises at least 5 mg, at least 10 mg, at least 20 mg, or at least 30 mg phenylephrine or a pharmaceutically acceptable salt thereof. In other embodiments, the bilayer tablet comprises less than or equal to 240 mg, less than or equal to 120 mg, less than or equal to 100 mg, less than or equal to 80 mg, or less than or equal to 60 mg phenylephrine or a pharmaceutically acceptable salt thereof. Alternatively, the quantity of the phenylephrine or a pharmaceutically acceptable salt thereof may also be expressed as a weight percentage of the total weight of the bilayer tablet. In some embodiments, the bilayer tablet comprises at least 1% w/w, at least 2% w/w, or at least 3% w/w phenylephrine or a pharmaceutically acceptable salt thereof. In other embodiments, the bilayer tablet comprises less than or equal to 12% w/w, less than or equal to 10% w/w, less than or equal to 8% w/w, or less than or equal to 6% w/w phenylephrine or a pharmaceutically acceptable salt thereof. In certain embodiments wherein the second layer comprises phenylephrine or a pharmaceutically acceptable salt thereof, the amount of phenylephrine or a pharmaceutically acceptable salt thereof present may be characterized by a weight percentage of the weight of the phenylephrine (second) layer. For example, in some embodiments, the phenylephrine layer comprises at least 2% w/w, at least 5% w/w, or at least 7% w/w phenylephrine or a pharmaceutically acceptable salt thereof by weight of the phenylephrine layer. In other embodiments, the bilayer tablet comprises less than or equal to 15% w/w, less than or equal to 12% w/w, or less than or equal to 10% w/w phenylephrine or a pharmaceutically acceptable salt thereof by weight of the phenylephrine layer. As described above, the bilayer tablets incorporate a dissolution aid in the second (or pseudoephedrine) layer to accelerate the dissolution rate of the active ingredient (such as acetylsalicylic acid) in the first layer. By separating a fraction of the dissolution aid from acetylsalicylic acid, possible degradation induced by the interaction of the dissolution aid with acetylsalicylic acid is reduced significantly. In some embodiments, the pseudoephedrine layer comprises a dissolution aid. Suitable dissolution aids may include but are not limited to magnesium hydroxide, magnesium oxide, aluminum oxide, calcium carbonate, sodium carbonate, sodium bicarbonate, or any combinations thereof. In some embodiments, the pseudoephedrine layer comprises at least 10% w/w, at least 20% w/w, or at least 30% w/w dissolution aid by weight of the pseudoephedrine layer. In other embodiments, the pseudoephedrine layer comprises less than or equal to 60% w/w, less than or equal to 50% w/w, or less than or equal to 40% w/w dissolution aid by weight of the pseudoephedrine layer. In some embodiments, the pseudoephedrine layer comprises sodium carbonate. In some embodiments, the pseudoephedrine layer comprise at least 10% w/w, at least 20% w/w, or at least 30% w/w sodium carbonate. In other embodiments, the pseudoephedrine layer comprises less than or equal to 60% w/w, less than or equal to 50% w/w, or less than or equal to 40% w/w sodium carbonate. In still further embodiments, the pseudoephedrine layer comprises one or more excipients in addition to sodium carbonate. In some embodiments, the one or more excipients in the pseudoephedrine layer comprise one or more diluents/fillers, one or more binders, one or more disintegrants, one or more glidants/flow aids, or any combinations thereof. In some embodiments, the pseudoephedrine layer comprises one or more diluents. Diluents, which may also be known as fillers or thinners, are inactive ingredients that can be incorporated into the pseudoephedrine layer to improve flow and cohesion of formulations during manufacture, add to the bulk weight and improve content uniformity of the final formulation. Suitable diluents may include, for example, mannitol, lactose, microcrystalline cellulose, calcium phosphate, and pregelatinized starch. In some embodiments, the pseudoephedrine layer comprises mannitol. In some embodiments, the pseudoephedrine layer comprises at least 10% w/w, at least 20% w/w, or at least 30% w/w by weight of the pseudoephedrine layer. In other embodiments, the pseudoephedrine layer comprises less than or equal to 60% w/w, less than or equal to 50% w/w, or less than or equal to 40% w/w mannitol by weight of the pseudoephedrine layer. In some embodiments, the pseudoephedrine layer comprises one or more binders. Binders may be incorporated in the pseudoephedrine layer to help adhere the pseudoephedrine or pharmaceutically acceptable salt thereof and other excipients together within the layer. Binder excipients may also contribute to the overall volume or mechanical properties of the resulting formulation, that is, the pseudoephedrine layer. Suitable binders for use in the pseudoephedrine layer may include, for example, microcrystalline cellulose, cellulose and starch. In certain embodiments, the one or more binders comprises microcrystalline cellulose (MCC). In certain embodiments, the pseudoephedrine layer comprises at least 5% w/w, at least 7% w/w, at least 10% w/w, or at least 12% w/w microcrystalline cellulose by weight of the pseudoephedrine layer. In other embodiments, the pseudoephedrine layer comprise less than or equal to 20% w/w, less than or equal to 17% w/w, or less than or equal to 15% w/w microcrystalline cellulose by weight of the pseudoephedrine layer. In some embodiments, the pseudoephedrine layer comprises one or more disintegrants. As described above, disintegrants, such as cornstarch, crospovidone, and croscarmellose sodium, are typically included in pharmaceutical formulations to aid in the dissolution process. In certain embodiments, the one or more disintegrants comprises cornstarch. In some embodiments, the pseudoephedrine layer comprises at least 5% w/w, at least 7% w/w, at least 10% w/w, or at least 12% w/w cornstarch by weight of the pseudoephedrine layer. In other embodiments, the pseudoephedrine layer comprise less than or equal to 20% w/w, less than or equal to 17% w/w, or less than or equal to 15% w/w cornstarch by weight of the pseudoephedrine layer. In still further embodiments, the pseudoephedrine layer comprises one or more glidants, such as colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, zinc stearate, and stearic acid. In certain embodiments, the one or more glidants comprises colloidal silicon dioxide. In certain embodiments, the pseudoephedrine layer comprises at least 0.1% w/w, at least 0.2% w/w, or at least 0.3% w/w colloidal silicon dioxide by weight of the pseudoephedrine layer. In other embodiments, the pseudoephedrine layer comprise less than or equal to 5% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w colloidal silicon dioxide by weight of the pseudoephedrine layer. Total Dissolution Aid and Distribution in the Bilayer Tablet As described herein, the bilayer structure of the tablets provided herein enables use of the acetylsalicylic acid and pseudoephedrine or a pharmaceutically acceptable salt thereof in a single dosage form, which mitigates or minimizes any unfavorable chemical interactions between the two actives in the tablet itself. The bilayer tablets of the present disclosure further utilize the bilayer structure of the tablet to take advantage of the improved solubilization of acetylsalicylic acid in basic media by using sodium carbonate as a dissolution aid but mitigating degradation pathways of acetylsalicylic acid while in storage. More specifically, the storage stability and dissolution rate achieved in the bilayer tablets of the present disclosure arise from the combination of the bilayer architecture with a distribution of dissolution aids across the two layers. The total amount of dissolution aid present in the bilayer tablet may be considered as the sum of the amounts of dissolution aid present in each layer (i.e., intragranular dissolution aid in the acetylsalicylic acid layer and dissolution aid in the pseudoephedrine layer). The total dissolution aid present in the tablet may be described in absolute milligram quantities or in relative weight percentages of the total bilayer tablet weight. In some embodiments, the bilayer tablet comprises at least 15 mg, at least 20 mg, at least 30 mg, or at least 50 mg dissolution aid. In other embodiments, the bilayer tablet comprises less than or equal to 300 mg, less than or equal to 150 mg, less than or equal to 100 mg, or less than or equal to 75 mg dissolution aid. In certain embodiments wherein the dissolution aid is sodium carbonate, the bilayer tablet comprises at least 15 mg, at least 20 mg, at least 30 mg, or at least 50 mg sodium carbonate. In certain other embodiments, the bilayer tablet comprises less than or equal to 300 mg, less than or equal to 150 mg, less than or equal to 100 mg, or less than or equal to 75 mg sodium carbonate. In other embodiments, the bilayer tablet comprises at least 5% w/w, at least 10% w/w, or at least 15% w/w dissolution aid by total weight of the bilayer tablet. In yet other embodiments, the bilayer tablet comprises less than or equal to 25% w/w, less than or equal to 22% w/w, less than or equal to 20% w/w, or less than or equal to 17% w/w dissolution aid by total weight of the bilayer tablet. In yet further embodiments, the bilayer tablet comprises at least 5% w/w, at least 10% w/w, or at least 15% w/w sodium carbonate by total weight of the bilayer tablet. In still yet other embodiments, the bilayer tablet comprises less than or equal to 25% w/w, less than or equal to 22% w/w, less than or equal to 20% w/w, or less than or equal to 17% w/w sodium carbonate by total weight of the bilayer tablet. The total dissolution aid is distributed within the bilayer tablet such that a fraction of the total sodium carbonate is directly combined with acetylsalicylic acid in granulate form and the remaining fraction is supplied in the second layer containing pseudoephedrine or a pharmaceutically acceptable salt thereof. In some embodiments, the bilayer tablet comprises a dissolution aid, wherein at least 10%, at least 20%, at least 30%, or at least 40% of the total dissolution aid present is intragranular dissolution aid. In other embodiments, less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50% of the total dissolution aid present in the bilayer tablet is intragranular dissolution aid. In some embodiments, at least 10%, at least 20%, at least 30%, or at least 40% of the total dissolution aid present is present in the acetylsalicylic acid layer. In other embodiments, less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50% of the total dissolution aid present in the bilayer tablet is present in the acetylsalicylic acid layer. In certain embodiments wherein the dissolution aid is sodium carbonate, at least 10%, at least 20%, at least 30%, or at least 40% of the total sodium carbonate present is intragranular sodium carbonate. In other embodiments, the less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50% of the total sodium carbonate present in the bilayer tablet is intragranular sodium carbonate. In some embodiments, at least 10%, at least 20%, at least 30%, or at least 40% of the total sodium carbonate present is present in the acetylsalicylic acid layer. In other embodiments, less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50% of the total sodium carbonate present in the bilayer tablet is present in the acetylsalicylic acid layer. In some embodiments, the distribution of dissolution aid in bilayer tablet is such that the acetylsalicylic acid layer comprises at least 10%, at least 20%, at least 30%, or at least 40% of the total dissolution aid present in the bilayer tablet. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, or less than or equal to 70% of the total dissolution aid present in the bilayer tablet. In certain embodiments wherein the dissolution aid is sodium carbonate, the acetylsalicylic acid layer comprises at least 10%, at least 20%, at least 30%, or at least 40% of the total sodium carbonate present in the bilayer tablet. In other embodiments, the acetylsalicylic acid layer comprises less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, or less than or equal to 70% of the total sodium carbonate present in the bilayer tablet. In other embodiments, the pseudoephedrine layer comprises at least 10%, at least 20%, or at least 30% w/w of the total dissolution aid present in the bilayer tablet. In some embodiments, the pseudoephedrine layer comprises less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, or less than or equal to 60% w/w of the total dissolution aid present in the bilayer tablet. In still other embodiments wherein the dissolution aid is sodium carbonate, the pseudoephedrine layer comprises at least 10%, at least 20%, or at least 30% w/w of the total sodium carbonate present in the bilayer tablet. In some embodiments, the pseudoephedrine layer comprises less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, or less than or equal to 60% w/w of the total sodium carbonate present in the bilayer tablet. Weight Ratio of Acetylsalicylic Acid to Dissolution Aid As briefly described above and further detailed below, the amount of intragranular acetylsalicylic acid relative to the total amount of acetylsalicylic acid and intragranular dissolution aid (such as sodium carbonate) relative to the total amount of dissolution aid may be modulated to achieve the desired stability and dissolution properties. Although the two parameters may be adjusted independently, it was observed that certain ratios, e.g., between about 1:1 and 7:1, of the total amount of acetylsalicylic acid to the total amount of dissolution aid (in both layers) produced the desired balance of reduced chemical instability and improved dissolution rates. In some embodiments, the weight ratio of the total acetylsalicylic acid to total dissolution aid present in the bilayer tablet is at least about 1:1, at least about 2:1, at least about 3:1, or at least about 4:1. In other embodiments, the weight ratio of the total acetylsalicylic acid to total dissolution aid present in the bilayer tablet is less than or equal to about 7:1, less than or equal to about 6:1, less than or equal to about 5:1, or less than or equal to about 4:1. In still further embodiments, the weight ratio of the total acetylsalicylic acid to total dissolution aid present in the bilayer tablet is between about 1:1 and 7:1, between about 1:1 and 6:1, between about 1:1 and 5:1, between about 1:1 and 4:1, between about 2:1 and 7:1, between about 2:1 and 6:1, between about 2:1 and 5:1, between about 2:1 and 4:1, between about 3:1 and 7:1, between about 3:1 and 6:1, between about 3:1 and 5:1, or between about 3:1 and 4:1. In certain embodiments wherein the dissolution aid is sodium carbonate, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet is at least about 1:1, at least about 2:1, at least about 3:1, or at least about 4:1. In other embodiments, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet is less than or equal to about 7:1, less than or equal to about 6:1, less than or equal to about 5:1, or less than or equal to about 4:1. In still further embodiments, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet is between about 1:1 and 7:1, between about 1:1 and 6:1, between about 1:1 and 5:1, between about 1:1 and 4:1, between about 2:1 and 7:1, between about 2:1 and 6:1, between about 2:1 and 5:1, between about 2:1 and 4:1, between about 3:1 and 7:1, between about 3:1 and 6:1, between about 3:1 and 5:1, or between about 3:1 and 4:1. As also described above, the dissolution aid present in the acetylsalicylic acid layer is provided intragranularly, i.e., within the granules. In still further embodiments, the bilayer tablet comprises granules comprising acetylsalicylic acid and a dissolution aid, wherein the weight ratio of acetylsalicylic acid present in the granules to the dissolution aid present in the granules is at least about 1:1, at least about 2:1, at least about 3:1 or at least about 4:1. In other embodiments, the weight ratio of the acetylsalicylic acid present in the granules to the dissolution aid present in the granules is less than or equal to about 7:1, less than or equal to about 6:1, less than or equal to about 5:1, or less than or equal to about 4:1. In still further embodiments, the weight ratio of the acetylsalicylic acid present in the granules to the dissolution aid present in the granules is between about 1:1 and 7:1, between about 1:1 and 6:1, between about 1:1 and 5:1, between about 1:1 and 4:1, between about 2:1 and 7:1, between about 2:1 and 6:1, between about 2:1 and 5:1, between about 2:1 and 4:1, between about 3:1 and 7:1, between about 3:1 and 6:1, between about 3:1 and 5:1, or between about 3:1 and 4:1. In certain embodiments wherein the dissolution aid is sodium carbonate, the bilayer tablet comprises granules comprising acetylsalicylic acid and sodium carbonate, wherein the weight ratio of acetylsalicylic acid present in the granules to the sodium carbonate present in the granules is at least about 1:1, at least about 2:1, at least about 3:1, or at least about 4:1. In other embodiments, the weight ratio of the acetylsalicylic acid present in the granules to the sodium carbonate present in the granules is less than or equal to about 7:1, less than or equal to about 6:1, less than or equal to about 5:1, or less than or equal to about 4:1. In still further embodiments, the weight ratio of the acetylsalicylic acid present in the granules to the sodium carbonate present in the granules is between about 1:1 and 7:1, between about 1:1 and 6:1, between about 1:1 and 5:1, between about 1:1 and 4:1, between about 2:1 and 7:1, between about 2:1 and 6:1, between about 2:1 and 5:1, between about 2:1 and 4:1, between about 3:1 and 7:1, between about 3:1 and 6:1, between about 3:1 and 5:1, or between about 3:1 and 4:1. It should be recognized that the weight ratio of acetylsalicylic acid to dissolution aid present in the granules may be the same or different from the weight ratio of acetylsalicylic acid to dissolution aid in the bilayer tablet. For example in some embodiments, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet is between about 1:1 and 7:1 and the weight ratio of acetylsalicylic acid present in the granules to the sodium carbonate present in the granules is between about 1:1 and 7:1. In certain embodiments, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet and the weight ratio of acetylsalicylic acid present in the granules to the sodium carbonate present in the granules are the same. In other embodiments, the weight ratio of the total acetylsalicylic acid to total sodium carbonate present in the bilayer tablet and the weight ratio of acetylsalicylic acid present in the granules to the sodium carbonate present in the granules are the different. As described an alternative manner, in some embodiments, the weight percentage of intragranular acetylsalicylic acid out of the total acetylsalicylic acid present is 10-50% and the weight percentage of the intragranular sodium carbonate out of the total sodium carbonate is 10-50%. In certain embodiments, the weight percentage of intragranular acetylsalicylic acid out of the total acetylsalicylic acid present is equal to the weight percentage of the intragranular sodium carbonate out of the total sodium carbonate. Properties of the Bilayer Tablet As described herein, the bilayer tablets of the present disclosure may provide similar release profile, compatibility of active ingredients for storage, and long-term storage stability against degradation. Dissolution Profile In some embodiments, the bilayer tablet of the present disclosure may be characterized as immediate release. In some embodiments, the acetylsalicylic acid layer is immediate release. In other embodiments, the pseudoephedrine layer is immediate release. In certain embodiments, the acetylsalicylic acid layer and the pseudoephedrine layer are both immediate release. The tablet dissolution profiles were determined in accordance with the USP dissolution test (Apparatus 1, basket, 500 mL of pH 4.5, 50 mM sodium acetate buffer, at 37±0.5° C., rotation speed 50 rpm) (USP34-NF29 Chapter <711> Dissolution, Stage 6 Harmonization Bulletin dated Dec. 1, 2011). In some embodiments, the bilayer tablet has a dissolution profile wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the total acetylsalicylic acid present in the tablet is dissolved after 10 minutes as determined by the USP Dissolution Test Apparatus-1 in 50 mM sodium acetate buffer at pH 4.5 and 37±0.5° C. In other embodiments, the bilayer tablet has a dissolution profile wherein at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the total pseudoephedrine or a pharmaceutically acceptable salt thereof is dissolved after 10 minutes as determined by the USP Dissolution Test Apparatus-1 in 50 mM sodium acetate buffer at pH 4.5 and 37±0.5° C. In some embodiments, the bilayer tablet has a dissolution profile wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the total acetylsalicylic acid is dissolved and at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the total pseudoephedrine or a pharmaceutically acceptable salt thereof is dissolved after 10 minutes as determined by the USP Dissolution Test Apparatus-1 in 50 mM sodium acetate buffer at pH 4.5 and 37±0.5° C. In still further embodiments wherein phenylephrine or a pharmaceutically acceptable salt is employed in lieu of pseudoephedrine or a pharmaceutically acceptable salt thereof, the bilayer tablet has a dissolution profile wherein at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the total phenylephrine or a pharmaceutically acceptable salt is dissolved after 10 minutes as determined by the USP Dissolution Test Apparatus-1 in 50 mM sodium acetate buffer at pH 4.5 and 37±0.5° C. Stability to Degradation As described herein, the bilayer tablets of the present disclosure combine a mixture of granulated and non-granulated acetylsalicylic acid with a unique distribution of sodium carbonate in the bilayer tablets to achieve a balanced dissolution profile and storage stability. The bilayer tablets incorporate sodium carbonate or other dissolution aids into the pseudoephedrine layer in order to facilitate quick dissolution of acetylsalicylic acid in dissolution media while minimizing intimate contact of acetylsalicylic acid with the dissolution air to reduce the extent of chemical degradation that may occur during storage. The stability of the bilayer tablets to degradation may be characterized variously by the amount of the initial active pharmaceutical ingredients that remain or the quantity of degradation by-products that are observed within the tablet as a function of various storage conditions (e.g., temperature, humidity, and/or time). Evaluation of the stability of the bilayer tablets described herein may be carried out by various methods, which may include subjecting the tablets to precise temperature and/or humidity conditions for a duration of time and subsequently determining the presence and quantity of active pharmaceutical ingredients as well as any potential degradation products that may have formed under the evaluation conditions. For example, in some embodiments, the bilayer tablets may be subjected to a storage temperature of at least about 20° C., at least about 25° C., at least about 30° C., at least about 40° C. or at least about 50° C. In other embodiments, the bilayer tablets are subjected to a storage temperature of less than or equal to 80° C., less than or equal to 75° C., less than or equal to 70° C., or less than or equal to 60° C. In still other embodiments, the bilayer tablets are subjected to a storage temperature of about 20° C., about 25° C., about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 75° C., or about 80° C. In some embodiments, any of which may be combined with any of the foregoing embodiments, the bilayer tablets are subjected to a storage humidity of at least about 30% relative humidity, at least about 40% relative humidity, at least about 50% relative humidity, or at least about 60% relative humidity. In other embodiments, the bilayer tablets are subjected to a storage humidity of less than or equal to about 100% relative humidity, less than or equal to about 90% relative humidity, less than or equal to about 80% relative humidity, less than or equal to about 75% relative humidity, or less than or equal to about 70% relative humidity. In still further embodiments, the bilayer tablets are subjected to a storage at a temperature of about 25° C. and humidity of about 60% relative humidity (RH). In certain embodiments, the bilayer tablets are subjected to a storage at a temperature of about 40° C. and humidity of about 75% relative humidity. In other embodiments, the bilayer tablets are the bilayer tablets are subjected to a storage at a temperature of about 50° C. and humidity of between about 50% and about 70% relative humidity. In certain embodiments, the bilayer tablets are subjected to a storage at a temperature of about 50° C. and humidity of between about 50% and about 60% relative humidity or between about 60% and about 70% relative humidity. In still other embodiments, the bilayer tablets are subjected to a storage at a temperature of about 50° C. and humidity of 60% or 65% relative humidity. In yet other embodiments, the bilayer tablets may be subjected to certain storage temperatures and relative humidities for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 10 days, at least about 20 days, at least 1 month, at least 1.5 months, at least 2 months, at least 3 months, or at least 6 months. In still other embodiments, the bilayer tablets are stored with a desiccant. In other embodiments, the bilayer tablets are stored without a desiccant. The storage stability and/or degree of degradation of the bilayer tablets provided herein may be characterized by the content of the two active pharmaceutical ingredients remaining after storage as well as the presence and content of degradation products after storage. The contents of the acetylsalicylic acid, pseudoephedrine or a pharmaceutically acceptable salt thereof, and their respective degradation products may be characterized in absolute quantities (such as total milligrams present) or in relative quantities (such as by weight percentage of the total weight of the tablet or as a molar percentage converted from the original acetylsalicylic acid and/or pseudoephedrine contents). The stability of the bilayer tablet may be characterized by the preservation of acetylsalicylic acid and/or pseudoephedrine content remaining in the bilayer tablet after exposure to the aforementioned storage conditions. In some embodiments, the stability of the bilayer tablet may be characterized by the content of acetylsalicylic acid preserved after storage. In some embodiments, the bilayer tablet comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% acetylsalicylic acid of the initial acetylsalicylic acid content after storage. In other embodiments, the bilayer tablet comprises less than or equal to 100%, less than or equal to 99%, less than or equal to 97%, or less than or equal to 95% acetylsalicylic acid of the initial acetylsalicylic acid content after storage. In still further embodiments, the bilayer tablet comprises between 75 and 100%, between 75 and 99%, between 75 and 97%, between 75 and 95%, between 80 and 100%, between 80 and 99%, between 80 and 97%, between 80 and 95%, between 85 and 100%, between 85 and 99%, between 85 and 97%, between 85 and 95%, between 90 and 100%, between 90 and 99%, between 90 and 97%, between 90 and 95%, between 95 and 100%, between 95 and 99%, between 95 and 97%, between 97 and 100%, between 97 and 99%, or between 99 and 100% acetylsalicylic acid of the initial acetylsalicylic acid content after storage. In other embodiments, the bilayer tablet may be characterized by the absolute quantity of acetylsalicylic acid remaining after storage. For example, in some embodiments wherein the bilayer tablet has an initial acetylsalicylic acid content of 500 mg, the bilayer tablet comprises at least 375 mg, at least 400 mg, at least 425 mg, at least 450 mg, at least 475 mg, at least 485 mg, or at least 495 mg acetylsalicylic acid after storage. In other embodiments wherein the bilayer tablet has an initial acetylsalicylic acid content of 500 mg, the bilayer tablet comprises less than or equal to 495 mg, less than or equal to 485 mg, less than or equal to 475 mg, or less than or equal to 450 mg acetylsalicylic acid after storage. In still further embodiments wherein the bilayer tablet has an initial acetylsalicylic acid content of 500 mg, the bilayer tablet comprises between 375 mg and 500 mg, between 375 mg and about 495 mg, between 375 mg and 485 mg, between 375 mg and 475 mg, between 375 mg and 450 mg, between 400 mg and 500 mg, between 400 mg and about 495 mg, between 400 mg and 485 mg, between 400 mg and 475 mg, between 400 mg and 450 mg, between 425 mg and 500 mg, between 425 mg and about 495 mg, between 425 mg and 485 mg, between 425 mg and 475 mg, between 425 mg and 450 mg, between 450 mg and 500 mg, between 450 mg and about 495 mg, between 450 mg and 485 mg, between 450 mg and 475 mg, between 475 mg and 500 mg, between 475 mg and about 495 mg, between 475 mg and 485 mg, between 485 mg and 500 mg, between 485 mg and about 495 mg, or between 495 mg and 500 mg acetylsalicylic acid after storage. In other embodiments, the stability of the bilayer tablet may be characterized by the content of pseudoephedrine (or a pharmaceutically acceptable salt thereof) preserved after storage. In some embodiments, the bilayer tablet comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% pseudoephedrine of the initial pseudoephedrine content after storage. In other embodiments, the bilayer tablet comprises less than or equal to 100%, less than or equal to 99%, less than or equal to 97%, or less than or equal to 95% pseudoephedrine of the initial pseudoephedrine content. In still further embodiments, the bilayer tablet comprises between 75 and 100%, between 75 and 99%, between 75 and 97%, between 75 and 95%, between 80 and 100%, between 80 and 99%, between 80 and 97%, between 80 and 95%, between 85 and 100%, between 85 and 99%, between 85 and 97%, between 85 and 95%, between 90 and 100%, between 90 and 99%, between 90 and 97%, between 90 and 95%, between 95 and 100%, between 95 and 99%, between 95 and 97%, between 97 and 100%, between 97 and 99%, or between 99 and 100% pseudoephedrine of the initial pseudoephedrine content after storage. As with acetylsalicylic acid, the content of pseudoephedrine or a pharmaceutically acceptable salt thereof may be characterized in absolute quantities after storage. In some embodiments wherein the bilayer tablet comprises an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises at least 22 mg, at least 24 mg, at least 26 mg, at least 28 mg, or at least 29 mg pseudoephedrine or a pharmaceutically acceptable salt thereof after storage. In other embodiments wherein the bilayer tablet comprises an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises less than or equal to 30 mg, less than or equal to 29 mg, less than or equal to 28 mg, or less than or equal to 26 mg pseudoephedrine or a pharmaceutically acceptable salt thereof after storage. In still further embodiments wherein the bilayer tablet has an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises between 22 mg and 30 mg, between 22 mg and 29 mg, between 22 mg and 28 mg, between 22 mg and 26 mg, between 22 mg and 24 mg, 24 mg and 30 mg, between 24 mg and 29 mg, between 24 mg and 28 mg, between 24 mg and 26 mg, 26 mg and 30 mg, between 26 mg and 29 mg, between 26 mg and 28 mg, 28 mg and 30 mg, between 28 mg and 29 mg, or between 29 mg and 30 mg pseudoephedrine or a pharmaceutically acceptable salt thereof after storage. It should be recognized that, when alternative active pharmaceutical ingredients are employed in lieu of acetylsalicylic acid and/pseudoephedrine or the pharmaceutically acceptable salt thereof or further active pharmaceutical ingredients are used in combination with acetylsalicylic acid and pseudoephedrine or a pharmaceutically acceptable salt thereof, the alternative and/or additional active ingredients may be similarly characterized by the remaining content present after storage. For example, in some embodiments wherein the bilayer tablet comprises phenylephrine or a pharmaceutically acceptable salt in lieu of pseudoephedrine, the bilayer tablet comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% phenylephrine of the initial phenylephrine content after storage. In other embodiments, the bilayer tablet comprises less than or equal to 100%, less than or equal to 99%, less than or equal to 97%, or less than or equal to 95% phenylephrine of the initial phenylephrine content. In still further embodiments, the bilayer tablet comprises between 75 and 100%, between 75 and 99%, between 75 and 97%, between 75 and 95%, between 80 and 100%, between 80 and 99%, between 80 and 97%, between 80 and 95%, between 85 and 100%, between 85 and 99%, between 85 and 97%, between 85 and 95%, between 90 and 100%, between 90 and 99%, between 90 and 97%, between 90 and 95%, between 95 and 100%, between 95 and 99%, between 95 and 97%, between 97 and 100%, between 97 and 99%, or between 99 and 100% phenylephrine of the initial phenylephrine content after storage. In some embodiments wherein the bilayer tablet comprises an initial phenylephrine content of 30 mg, the bilayer tablet comprises at least 22 mg, at least 24 mg, at least 26 mg, at least 28 mg, or at least 29 mg phenylephrine or a pharmaceutically acceptable salt thereof after storage. In other embodiments wherein the bilayer tablet comprises an initial phenylephrine content of 30 mg, the bilayer tablet comprises less than or equal to 30 mg, less than or equal to 29 mg, less than or equal to 28 mg, or less than or equal to 26 mg phenylephrine or a pharmaceutically acceptable salt thereof after storage. In still further embodiments wherein the bilayer tablet has an initial phenylephrine content of 30 mg, the bilayer tablet comprises between 22 mg and 30 mg, between 22 mg and 29 mg, between 22 mg and 28 mg, between 22 mg and 26 mg, between 22 mg and 24 mg, 24 mg and 30 mg, between 24 mg and 29 mg, between 24 mg and 28 mg, between 24 mg and 26 mg, 26 mg and 30 mg, between 26 mg and 29 mg, between 26 mg and 28 mg, 28 mg and 30 mg, between 28 mg and 29 mg, or between 29 mg and 30 mg phenylephrine or a pharmaceutically acceptable salt thereof after storage. In still further embodiments, the stability of the bilayer tablet may be characterized by the presence and quantity of degradation products of acetylsalicylic acid and/or pseudoephedrine (or phenylephrine if present) in the tablet after exposure to the storage conditions. As described above, acetylsalicylic acid and pseudoephedrine may undergo various degradation processes to produce less active or inactive chemical compounds. The degradation products of acetylsalicylic acid and pseudoephedrine may be characterized on an individual basis as discrete chemical byproducts (e.g., salicylic acid or acetylsalicylsalicylic acid), on a combined basis as the byproducts of a single active pharmaceutical ingredient (e.g., total degradation products of acetylsalicylic acid, which may include but are not limited to salicylic acid and acetylsalicylsalicylic acid), or on as full basis as the total degradation products of both active pharmaceutical ingredients (e.g., the full set of degradation products observed for both acetylsalicylic acid and pseudoephedrine or a pharmaceutically acceptable salt thereof). Methods known in the art may be utilized to identify and quantify the degradation products present in the bilayer tablet including, for example, high performance liquid chromatography (HPLC) and ultraviolet (UV) absorption spectrometry. The table below shows the chemical structures of acetylsalicylic acid and a selection of its degradation products. Name of CompoundStructure of CompoundAcetylsalicylic Acid (ASA)(Free) Salicylic acid (FSA)Acetylsalicylsalicylic acid (ASSA) A common hydrolytic degradation product of acetylsalicylic acid is free salicylic acid (also referred to herein as FSA, or as salicylic acid). The quantity of salicylic acid formed after exposure to particular storage and/or degradation conditions may be characterized as a weight or molar percentage of acetylsalicylic acid converted to salicylic acid from the original or initial acetylsalicylic acid (molar) content prior to being subjected to the specified degradation conditions. For example, a bilayer tablet as-prepared may contain 500 mg acetylsalicylic acid (initial acetylsalicylic acid content, 100 mol %) and, following a period of storage, a fraction of the initial acetylsalicylic acid content may have been converted to one or more degradation products. Following storage at a specified time, temperature, and/or humidity, the bilayer tablet may be observed to contain 95 mol % acetylsalicylic acid (475 mg) and 5 mol % salicylic acid (19.2 mg) of the initial acetylsalicylic acid content. Alternatively, the same degradation products may be described in terms of their weight percentage by weight of the initial acetylsalicylic acid content. For example, the bilayer tablet may be observed to contain 95% w/w acetylsalicylic acid (475 mg) by weight of the initial acetylsalicylic acid content and 3.8% w/w salicylic acid (19.2 mg) by weight of the initial acetylsalicylic acid content. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % salicylic acid of the initial acetylsalicylic acid content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % salicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w salicylic acid of the initial acetylsalicylic acid content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w salicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. In still other embodiments, the stability of the bilayer tablet may be characterized by the absolute quantity of free salicylic acid (FSA) present after storage. In some embodiments, the bilayer tablet comprises less than or equal to 5 mg, less than or equal to 10 mg, less than or equal to 15 mg, less than or equal to 20 mg, or less than or equal to 25 mg salicylic acid after storage. Another degradation product of acetylsalicylic acid that may be observed is acetylsalicylsalicylic acid (also referred to herein as ASSA). Acetylsalicylsalicylic acid may be similarly characterized In some embodiments, the bilayer tablet comprises less than or equal to 1 mol %, less than or equal to 2 mol %, less than or equal to 3 mol %, less than or equal to 4 mol %, or less than or equal to 5 mol % acetylsalicylsalicylic acid of the initial acetylsalicylic acid content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 1 mol %, less than or equal to 2 mol %, less than or equal to 3 mol %, less than or equal to 4 mol %, or less than or equal to 5 mol % acetylsalicylsalicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. In yet other embodiments, the bilayer tablet comprises less than or equal to 1% w/w, less than or equal to 2% w/w, less than or equal to 3% w/w, less than or equal to 4% w/w, or less than or equal to 5% w/w acetylsalicylsalicylic acid of the initial acetylsalicylic acid content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 1% w/w, less than or equal to 2% w/w, less than or equal to 3 mol %, less than or equal to 4% w/w, or less than or equal to 5% w/w acetylsalicylsalicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. In still further embodiments wherein the bilayer tablet has an initial acetylsalicylic acid content of 500 mg, the bilayer tablet comprises less than or equal to 5 mg, less than or equal to 10 mg, less than or equal to 15 mg, less than or equal to 20 mg, or less than or equal to 25 mg acetylsalicylsalicylic acid (ASSA). Additional byproducts of acetylsalicylic acid may form including free salicylic acid and acetylsalicylsalicylic acid as described herein and may contribute to the total amount of acetylsalicylic acid degradation byproducts formed in the bilayer tablet after exposure to certain storage conditions. It should be recognized that the bilayer tablets may be characterized by the amount of total degradation products, which can include but are not limited to salicylic acid, acetylsalicylsalicylic acid and others. In some embodiments, the bilayer tablet comprises less than or equal to 1 mol %, less than or equal to 2 mol %, less than or equal to 3 mol %, less than or equal to 4 mol %, less than or equal to 5 mol %, less than or equal to 6 mol %, less than or equal to 7 mol %, less than or equal to 8 mol %, less than or equal to 9 mol %, or less than or equal to 10 mol % total degradation byproducts of acetylsalicylic acid of the initial acetylsalicylic acid content after storage. In other embodiments, the bilayer tablet comprises less than or equal to 1 mol %, less than or equal to 2 mol %, less than or equal to 3 mol %, less than or equal to 4 mol %, less than or equal to 5 mol %, less than or equal to 6 mol %, less than or equal to 7 mol %, less than or equal to 8 mol %, less than or equal to 9 mol %, or less than or equal to 10 mol % total degradation byproducts of acetylsalicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. In some embodiments, the bilayer tablet comprises less than or equal to 1% w/w, less than or equal to 2% w/w, less than or equal to 3% w/w, less than or equal to 4% w/w, less than or equal to 5% w/w, less than or equal to 6% w/w, less than or equal to 7% w/w, less than or equal to 8% w/w, less than or equal to 9% w/w, or less than or equal to 10% w/w total degradation byproducts of acetylsalicylic acid of the initial acetylsalicylic acid content after storage. In other embodiments, the bilayer tablet comprises less than or equal to 1% w/w, less than or equal to 2% w/w, less than or equal to 3% w/w, less than or equal to 4% w/w, less than or equal to 5% w/w, less than or equal to 6% w/w, less than or equal to 7% w/w, less than or equal to 8% w/w, less than or equal to 9% w/w, or less than or equal to 10% w/w total degradation byproducts of acetylsalicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 60% relative humidity for at least 20 days. The stability of the bilayer tablet to degradation may be further characterized by the presence and/or quantity of pseudoephedrine degradation byproducts. It should also be recognized that bilayer tablets comprising phenylephrine or a pharmaceutically acceptable salt thereof in lieu of pseudoephedrine may also be characterized as described below but for phenylephrine and its degradation products. Pseudoephedrine or pharmaceutically acceptable salts thereof may undergo degradation pathways to form, for example, N-acetyl pseudoephedrine (PSEH N-acetyl or PSEH N-ester), O-acetyl pseudoephedrine (PSEH O-acetyl or PSEH O-ester) and N,O-diacetyl pseudoephedrine (PSEH N,O-diacetyl or PSEH diester). The table below shows the chemical structure of pseudoephedrine hydrochloride and a selection of its degradation products. Name of CompoundStructure of CompoundPseudoephedrine Hydrochloride (PSEH)O-Acetyl pseudoephedrine (PSEH O-acetyl)N-Acetyl pseudoephedrine (PSEH N-acetyl)N,O-Diacetyl pseudoephedrine (PSEH N,O-diacetyl) In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % N-acetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % N-acetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w N-acetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w N-acetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In other embodiments wherein the bilayer tablet has an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises less than or equal to 10 mg, less than or equal to 8 mg, less than or equal to 6 mg, less than or equal to 4 mg, or less than or equal to 2 mg N-acetyl pseudoephedrine after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % O-acetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % O-acetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w O-acetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w O-acetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In other embodiments wherein the bilayer tablet has an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises less than or equal to 10 mg, less than or equal to 8 mg, less than or equal to 6 mg, less than or equal to 4 mg, or less than or equal to 2 mg O-acetyl pseudoephedrine after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % N,O-diacetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % N,O-diacetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6% w/w, less than or equal to 4 mol %, less than or equal to 2% w/w, or less than or equal to 1% w/w N,O-diacetyl pseudoephedrine of the initial pseudoephedrine content after storage. In some embodiments, the bilayer tablet comprises less than or equal to 10% w/w, less than or equal to 8% w/w, less than or equal to 6 mol %, less than or equal to 4% w/w, less than or equal to 2% w/w, or less than or equal to 1% w/w N,O-diacetyl pseudoephedrine of the initial pseudoephedrine content after storage at 50° C. and 60% relative humidity for at least 10 days. In other embodiments wherein the bilayer tablet has an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises less than or equal to 10 mg, less than or equal to 8 mg, less than or equal to 6 mg, less than or equal to 4 mg, or less than or equal to 2 mg N,O-diacetyl pseudoephedrine after storage. The various degradation byproducts of pseudoephedrine or a pharmaceutically acceptable salt thereof may be considered in aggregate as the total degradation byproducts of pseudoephedrine. In still further embodiments, the bilayer tablet comprises less than or equal to 10 mol %, less than or equal to 8 mol %, less than or equal to 6 mol %, less than or equal to 4 mol %, less than or equal to 2 mol %, or less than or equal to 1 mol % total degradation byproducts of pseudoephedrine or a pharmaceutically acceptable salt thereof of the initial pseudoephedrine content after storage. In other embodiments wherein the bilayer tablet has an initial pseudoephedrine content of 30 mg, the bilayer tablet comprises less than or equal to 10 mg, less than or equal to 8 mg, less than or equal to 6 mg, less than or equal to 4 mg, or less than or equal to 2 mg total degradation byproducts of pseudoephedrine or a pharmaceutically acceptable salt thereof after storage. Methods of Preparing the Bilayer Tablet In another aspect, provided herein are methods for preparing the bilayer tablets as described herein. With reference toFIG.2, process200is an exemplary process for preparing a bilayer tablet as described herein. In step202, acetylsalicylic acid is compacted and milled with sodium carbonate and colloidal silicon dioxide to form granules. The resulting granules are further combined with (non-granulated) acetylsalicylic acid and extragranular excipients in step204, thereby providing an acetylsalicylic acid blend. The acetylsalicylic acid blend corresponds to the acetylsalicylic acid layer in the final bilayer tablet. In parallel step206, pseudoephedrine, sodium carbonate and additional excipients are combined to provide a pseudoephedrine blend. The pseudoephedrine blend corresponds to the pseudoephedrine layer in the bilayer tablet. Following preparation of the acetylsalicylic acid blend and the pseudoephedrine blend, the two blends are passed to a tablet press to be compressed. The tablet press may optionally be treated with external lubrication to facilitate the pressing of the tablet as provided in step208. The acetylsalicylic acid blend and pseudoephedrine blend are subsequently compressed to form the bilayer tablet in step210. It should be recognized that the exemplary process200may be adapted to accommodate alternative active pharmaceutical ingredients, dissolution aids and/or excipients as described herein. It should also be understood that, in other variations, process200may include additional processing steps. In yet other variations, certain steps in process200may be omitted. In one aspect, the present disclosure provides a method for preparing a bilayer tablet, comprising: compacting and milling acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide to provide granules; combining the granules with acetylsalicylic acid, cornstarch, powdered cellulose, and colloidal silicon dioxide to provide an acetylsalicylic acid blend; combining pseudoephedrine or a pharmaceutically acceptable salt thereof, cornstarch, mannitol, sodium carbonate, microcrystalline cellulose and colloidal silicon dioxide to provide a pseudoephedrine blend; and compressing the acetylsalicylic acid blend and pseudoephedrine blend to form the bilayer tablet. In step202, granules comprising acetylsalicylic acid, also referred to herein as an acetylsalicylic acid composite, are prepared. As described previously, the incorporation of the acetylsalicylic acid composite in the acetylsalicylic acid layer confers a number of advantageous pharmacokinetic and stability properties to the resulting bilayer tablet, including for example a fast dissolution rate that provides an enhanced onset of therapeutic action. The granules comprising acetylsalicylic acid contain the active pharmaceutical ingredient in a micronized form that, due to surface area effects, may contribute to the observed enhanced dissolution profile of the bilayer tablet. The inclusion of one or more dissolution aids into the granules further accelerates the rapid dissolution rate of the bilayer tablet. Moreover, the dissolution aids in the granules can serve as a protective, enveloping coating around the micronized acetylsalicylic acid, thereby protecting the acetylsalicylic acid from moisture during downstream processing and in the final tablet. Acetylsalicylic acid is combined with a dissolution aid and one or more intragranular excipients in a pre-blend mixture, which is compacted and subsequently milled to provide granules comprising acetylsalicylic acid. In some embodiments, prior to being combined with the dissolution aid and intragranular excipients, the acetylsalicylic acid has an average particle size of less than or equal to 50 μm, less than or equal to 40 μm, less than or equal to 30 μm, less than or equal to 20 μm, or less than or equal to 10 μm. In certain embodiments, the acetylsalicylic acid has an average particle size of less than or equal to 40 μm. In some embodiments, prior to being combined with the dissolution aid and intragranular excipients, the acetylsalicylic acid has an average particle size of greater than or equal to 1 μm, greater than or equal to 2 μm, greater than or equal to 5 μm, or greater than or equal to 10 μm. In other embodiments, the acetylsalicylic acid has an average particle size of between 1 μm and 50 μm, between 1 μm and 40 μm, between 1 μm and 30 μm, between 1 μm and 20 μm, between 1 μm and 10 μm, between 2 μm and 50 μm, between 2 μm and 40 μm, between 2 μm and 30 μm, between 2 μm and 20 μm, between 2 μm and 10 μm, between 5 μm and 50 μm, between 5 μm and 40 μm, between 5 μm and 30 μm, between 5 μm and 20 μm, between 5 μm and 10 μm, between 10 μm and 50 μm, between 10 μm and 40 μm, between 10 μm and 30 μm, between 10 μm and 20 μm, between 20 μm and 50 μm, between 20 μm and 40 μm, between 20 μm and 30 μm, between 30 μm and 50 μm, between 30 μm and 40 μm, or between 40 μm and 50 μm. In some embodiments, the method comprises compacting and milling acetylsalicylic acid, sodium carbonate (or other suitable dissolution aid), and colloidal silicon dioxide to provide granules. In some embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction. In certain embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with variable roller size, roller speeds, roller gaps, and/or roller pressures. In some embodiments of the foregoing, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller speed of at least 5 rpm, at least 6 rpm, at least 7 rpm, at least 8 rpm, or at least 9 rpm. In other embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller speed of less than or equal to 20 rpm, less than or equal to 17 rpm, less than or equal to 15 rpm, or less than or equal to 12 rpm. In other embodiments, which may be combined with any of the preceding embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller gap of at least 1 mm, at least 1.2 mm, at least 1.4 mm, or at least 1.6 mm. In still other embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller gap of less than or equal to 3 mm, less than or equal to 2.8 mm, less than or equal to 2.6 mm, or less than or equal to 2.4 mm. In some embodiments, which may be combined with any of the foregoing embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller pressure of at least 10 bar, at least 12 bar, at least 15 bar, at least 17 bar or at least 20 bar. In other embodiments, acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide are compacted by roller compaction with a roller pressure of less than or equal to 50 bar, less than or equal to 45 bar, less than or equal to 40 bar, or less than or equal to 35 bar. Following compaction, the resulting compacted material (acetylsalicylic acid, sodium carbonate or other dissolution aid, and colloidal silicon dioxide) are milled to provide granules. The milling step may include but is not limited to dry milling techniques, such as ball milling, air-jet milling, or techniques employing mechanical mills (such as hammer mills or conical mills). In some embodiments, the compacted material is milled with a milling speed of at least 50 rpm, at least 75 rpm, or at least 100 rpm. In other embodiments, the compacted material is milled with a milling speed of less than or equal to 150 rpm, less than or equal to 125 rpm, or less than or equal to 110 rpm. It should be recognized that the parameters of the compaction and/or milling steps described above as well as the residence time for the milling step may be modified accordingly to achieve suitable particle sizes of the resulting granules. Furthermore, it should be recognized that the parameters employed for the compaction and milling steps may vary depending on the compacting or milling equipment utilized. Accordingly, in some embodiments, after the compacting and milling steps, the resulting acetylsalicylic acid composite, or granules, may be characterized by their particle size distribution and/or other particle size properties. In some embodiments, the granules have a particle size distribution wherein at least 5% w/w, at least 10% w/w, or at least 15% w/w of the granules out of the total granule weight has a particle size of less than 150 μm. In other embodiments, the granules have a particle size distribution wherein less than or equal to 30% w/w, less than or equal to 25% w/w less than or equal to 20% w/w of the granules out of the total granule weight has a particle size of less than 150 μm. In some embodiments, the granules have a particle size distribution wherein at least 50% w/w, at least 55% w/w, or at least 60% w/w of the granules out of the total granule weight has a particle size of greater than 400 μm. In other embodiments, the granules have a particle size distribution wherein less than or equal to 80% w/w, less than or equal to 75% w/w less than or equal to 70% w/w of the granules out of the total granule weight has a particle size of greater than 400 μm. In some embodiments, the granules have a particle size distribution wherein at least 10% w/w, at least 15% w/w, or at least 20% w/w of the granules out of the total granule weight have a particle size between 150 μm and 400 μm. In other embodiments, the granules have a particle size distribution wherein less than or equal to 40% w/w, less than or equal to 35% w/w, less than or equal to 30% w/w, or less than or equal to 25% w/w of the granules out of the total granule weight have a particle size between 150 μm and 400 μm. In still other embodiments, the granules have a particle size distribution wherein at least 5% w/w, at least 10% w/w, or at least 15% w/w of the granules out of the total granule weight has a particle size of less than or equal to 150 μm and less than or equal to 80% w/w, less than or equal to 75% w/w less than or equal to 70% w/w of the granules out of the total granule weight has a particle size of greater than or equal to 400 μm. In yet other embodiments, the granules have a particle size distribution wherein less than or equal to 30% w/w, less than or equal to 25% w/w less than or equal to 20% w/w of the granules out of the total granule weight has a particle size of less than or equal to 150 μm and at least 50% w/w, at least 55% w/w, or at least 60% w/w of the granules out of the total granule weight has a particle size of greater than or equal to 400 μm. In certain embodiments, the granules have a particle size distribution wherein between 5% w/w and 30% w/w, between 10% w/w and 30% w/w, between 15% w/w and 30% w/w, between 5% w/w and 25% w/w, between 10% w/w and 25% w/w, between 15% w/w and 25% w/w, between 5% w/w and 20% w/w, between 10% w/w and 20% w/w, or between 15% w/w and 20% w/w of the granules out of the total granule weight have a particle size of less than 150 μm; and wherein between 50% w/w and 80% w/w, between 55% w/w and 80% w/w, between 60% w/w and 80% w/w, between 50% w/w and 75% w/w, between 55% w/w and 75% w/w, between 60% w/w and 75% w/w, between 50% w/w and 70% w/w, between 55% w/w and 70% w/w, or between 60% w/w and 70% w/w, of the granules out of the total granule weight have a particle size of greater than 400 μm. In certain embodiments, the granules have a particle size distribution wherein between 5% w/w and 30% w/w of the granules out of the total granule weight have a particle size of less than 150 μm and wherein between 50% w/w and 80% w/w of the granules out of the total granule weight have a particle size of greater than 400 μm. In still further embodiments, the granules produced by the milling step may be further sieved to provide granules having a particular particle size distribution. In some embodiments, the method further comprises sieving the granules prior to combining the granules with extragranular components. In some embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 5% w/w, at least 10% w/w, or at least 15% w/w of the total granule weight has a particle size of less than or equal to 150 μm. In other embodiments, the granules have a particle size distribution wherein less than or equal to 30% w/w, less than or equal to 25% w/w less than or equal to 20% w/w of the total granule weight has a particle size of less than or equal to 150 μm. In some embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 50% w/w, at least 60% w/w, or at least 70% w/w of the total granule weight has a particle size of greater than or equal to 400 μm. In other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein less than or equal to 95% w/w, less than or equal to 90% w/w less than or equal to 85% w/w of the total granule weight has a particle size of greater than or equal to 400 μm. In some embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 5% w/w, at least 10% w/w, or at least 15% w/w of the granules out of the total granule weight has a particle size of less than 150 μm. In other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein less than or equal to 30% w/w, less than or equal to 25% w/w less than or equal to 20% w/w of the granules out of the total granule weight has a particle size of less than 150 μm. In some embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 50% w/w, at least 55% w/w, or at least 60% w/w of the granules out of the total granule weight has a particle size of greater than 400 μm. In other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein less than or equal to 80% w/w, less than or equal to 75% w/w less than or equal to 70% w/w of the granules out of the total granule weight has a particle size of greater than 400 μm. In some embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 10% w/w, at least 15% w/w, or at least 20% w/w of the granules out of the total granule weight have a particle size between 150 μm and 400 μm. In other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein less than or equal to 40% w/w, less than or equal to 35% w/w, less than or equal to 30% w/w, or less than or equal to 25% w/w of the granules out of the total granule weight have a particle size between 150 μm and 400 μm. In still other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein at least 5% w/w, at least 10% w/w, or at least 15% w/w of the granules out of the total granule weight has a particle size of less than or equal to 150 μm and less than or equal to 80% w/w, less than or equal to 75% w/w less than or equal to 70% w/w of the granules out of the total granule weight has a particle size of greater than or equal to 400 μm. In yet other embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein less than or equal to 30% w/w, less than or equal to 25% w/w less than or equal to 20% w/w of the granules out of the total granule weight has a particle size of less than or equal to 150 μm and at least 50% w/w, at least 55% w/w, or at least 60% w/w of the granules out of the total granule weight has a particle size of greater than or equal to 400 μm. In certain embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein between 5% w/w and 30% w/w, between 10% w/w and 30% w/w, between 15% w/w and 30% w/w, between 5% w/w and 25% w/w, between 10% w/w and 25% w/w, between 15% w/w and 25% w/w, between 5% w/w and 20% w/w, between 10% w/w and 20% w/w, or between 15% w/w and 20% w/w of the granules out of the total granule weight have a particle size of less than 150 μm; and wherein between 50% w/w and 80% w/w, between 55% w/w and 80% w/w, between 60% w/w and 80% w/w, between 50% w/w and 75% w/w, between 55% w/w and 75% w/w, between 60% w/w and 75% w/w, between 50% w/w and 70% w/w, between 55% w/w and 70% w/w, or between 60% w/w and 70% w/w, of the granules out of the total granule weight have a particle size of greater than 400 μm. In certain embodiments, the granules produced by the milling step are sieved to provide granules having a particle size distribution wherein between 5% w/w and 30% w/w of the granules out of the total granule weight have a particle size of less than 150 μm and wherein between 50% w/w and 80% w/w of the granules out of the total granule weight have a particle size of greater than 400 μm. With reference to step204, the method comprises combining the granules with additional acetylsalicylic acid and one or more extragranular excipients. In some embodiments, the method comprises combining the granules with acetylsalicylic acid, cornstarch, powdered cellulose, and colloidal silicon dioxide to provide an acetylsalicylic acid blend. In some embodiments, the additional extragranular acetylsalicylic acid may have the same or different particle size properties as the acetylsalicylic acid used in the preparation of the granules. In some embodiments, the acetylsalicylic acid has an average particle size of less than or equal to 50 μm, less than or equal to 40 μm, less than or equal to 30 μm, less than or equal to 20 μm, or less than or equal to 10 μm. In certain embodiments, the acetylsalicylic acid has an average particle size of less than or equal to 40 μm. In other embodiments, the additional acetylsalicylic acid has an average particle size of greater than or equal to 1 μm, greater than or equal to 2 μm, greater than or equal to 5 μm, or greater than or equal to 10 μm. In other embodiments, the acetylsalicylic acid has an average particle size of between 1 μm and 50 μm, between 1 μm and 40 μm, between 1 μm and 30 μm, between 1 μm and 20 μm, between 1 μm and 10 μm, between 2 μm and 50 μm, between 2 μm and 40 μm, between 2 μm and 30 μm, between 2 μm and 20 μm, between 2 μm and 10 μm, between 5 μm and 50 μm, between 5 μm and 40 μm, between 5 μm and 30 μm, between 5 μm and 20 μm, between 5 μm and 10 μm, between 10 μm and 50 μm, between 10 μm and 40 μm, between 10 μm and 30 μm, between 10 μm and 20 μm, between 20 μm and 50 μm, between 20 μm and 40 μm, between 20 μm and 30 μm, between 30 μm and 50 μm, between 30 μm and 40 μm, or between 40 μm and 50 μm. Acetylsalicylic acid is susceptible to hydrolytic degradation pathways to form various inactive and/or less stable byproducts, such as free salicylic acid. Minimization of hydrolysis may be achieved by drying the acetylsalicylic acid blend prior to passing the acetylsalicylic acid blend to the tablet press for compression. In some embodiments, prior to compressing, the method comprises drying the acetylsalicylic acid blend prior to compressing. In certain embodiments, the method comprises drying the acetylsalicylic acid blend to a moisture content or water activity below a certain threshold level. Water activity may be determined as described in USP42-NF37 Chapters <922> Water Activity and <1112> Application Of Water Activity Determination To Nonsterile Pharmaceutical Products. In some embodiments, the method comprises drying the acetylsalicylic acid blend to a water activity of less than or equal to 0.4, less than or equal to 0.3, less than or equal to 0.2, less than or equal to 0.1, or less than or equal to 0.08. In some embodiments, the method comprises drying the acetylsalicylic acid blend to a water activity of between 0.01 and 0.3, between 0.02 and 0.2, or between 0.05 and 0.1. With reference to step206, the method comprises combining pseudoephedrine or a pharmaceutically acceptable salt thereof, such as pseudoephedrine hydrochloride, with one or more excipients, including for example, cornstarch, mannitol, sodium carbonate, microcrystalline cellulose and colloidal silicon dioxide, to provide a pseudoephedrine blend. With further reference toFIG.2, in step208, external lubricants may be added to the tablet press or other tableting equipment prior to forming the bilayer tablet with the two blends. The use of external lubricants may facilitate the ejection of the final tablet by reducing sticking of material to the tablet press. In still further embodiments of the foregoing, the method comprises optionally applying one or more external lubricants to the tablet press prior to compressing. In some embodiments, the one or more external lubricants comprise hypromellose, zinc stearate, carnauba wax, or any combinations thereof. Depending upon the tablet press utilized, the compression of the two blends in step210to form the bilayer tablet may be carried out in a single compression step or in a two-step process comprising first (pre-)compressing one of the blends to form one layer, subsequently loading the remaining blend into the press with the already prepared layer, and compressing the remaining blend and prepared layer to form the bilayer tablet. It should be recognized that, in instances wherein the two-step process for tableting is utilized, the sequence of compression may be ordered with either the acetylsalicylic acid blend or pseudoephedrine blend may be subjected to the pre-compression. In some embodiments wherein the acetylsalicylic acid blend and pseudoephedrine blend are compressed in a single compression step, the acetylsalicylic acid blend and pseudoephedrine blend are compressed at a compression force of at least 1 kN, at least 2 kN, at least 3 kN, at least 4 kN, at least 5 kN, at least 10 kN, at least 15 kN, at least 20 kN, or at least 25 kN. In other embodiments, the acetylsalicylic acid blend and pseudoephedrine blend are compressed at a compression force of less than or equal to 45 kN, less than or equal to 40 kN, less than or equal to 35 kN, less than or equal to 30 kN, less than or equal to 25 kN, or less than or equal to 20 kN. In certain embodiments, the acetylsalicylic acid blend and the pseudoephedrine blend are compressed at a compression force of between 5 kN and 45 kN, between 5 kN and 40 kN, between 5 kN and 35 kN, between 5 kN and 30 kN, between 5 kN and 25 kN, between 5 kN and 20 kN, between 5 kN and 15 kN, between 5 kN and 10 kN, between 10 kN and 45 kN, between 10 kN and 40 kN, between 10 kN and 35 kN, between 10 kN and 30 kN, between 10 kN and 25 kN, between 10 kN and 20 kN, between 10 kN and 15 kN, between 15 kN and 45 kN, between 15 kN and 40 kN, between 15 kN and 35 kN, between 15 kN and 30 kN, between 15 kN and 25 kN, between 15 kN and 20 kN, between 20 kN and 45 kN, between 20 kN and 40 kN, between 20 kN and 35 kN, between 20 kN and 30 kN, between 20 kN and 25 kN, between 25 kN and 45 kN, between 25 kN and 40 kN, between 25 kN and 35 kN, between 25 kN and 30 kN, between 30 kN and 45 kN, between 30 kN and 40 kN, between 30 kN and 35 kN, between 35 kN and 45 kN, between 35 kN and 40 kN, or between 40 kN and 45 kN. In some embodiments wherein the acetylsalicylic acid blend and the pseudoephedrine blend are compressed in a two-step process, the acetylsalicylic acid blend or pseudoephedrine blend may be compressed at a first compression force to form a first layer, followed by compression of the pseudoephedrine blend or acetylsalicylic acid blend on top of the first layer at a second compression force to form the bilayer tablet. In some embodiments, the method comprises compressing the acetylsalicylic acid blend to provide an acetylsalicylic acid layer; and compressing the pseudoephedrine blend on top of the acetylsalicylic acid layer to provide the bilayer tablet. In certain embodiments, the method comprises compressing the acetylsalicylic acid blend to provide an acetylsalicylic acid layer at a first compression force; and compressing the pseudoephedrine blend on top of the acetylsalicylic acid layer at a second compression force to provide the bilayer tablet. In other embodiments, the method comprises compressing the pseudoephedrine blend to provide a pseudoephedrine layer; and compressing the acetylsalicylic acid blend on top of the pseudoephedrine layer to provide the bilayer tablet. In certain other embodiments, the method comprises compressing the pseudoephedrine blend at a first compression force to provide a pseudoephedrine layer; and compressing the acetylsalicylic acid blend on top of the pseudoephedrine layer at a second compression force to provide the bilayer tablet. In some embodiments, the first compression force is at least 1 kN, at least 2 kN, at least 3 kN, at least 4 kN, at least 5 kN, at least 10 kN, at least 15 kN, at least 20 kN, or at least 25 kN. In other embodiments, the first compression force is less than or equal to 45 kN, less than or equal to 40 kN, less than or equal to 35 kN, less than or equal to 30 kN, less than or equal to 25 kN, or less than or equal to 20 kN. In some embodiments, the second compression force is at least 5 kN, at least 10 kN, at least 15 kN, at least 20 kN, or at least 25 kN. In other embodiments, the second compression force is less than or equal to 45 kN, less than or equal to 40 kN, less than or equal to 35 kN, less than or equal to 30 kN, less than or equal to 25 kN, or less than or equal to 20 kN. In some embodiments, the first compression force and the second compression force are the same. In other embodiments, the first compression force and the second compression force are different. In still further embodiments, the first compression force is less than or equal to the second compression force. Methods of Use and Articles of Manufacture The bilayer tablets as described herein may be suitable for use in the treatment of or provision of relief from symptoms associated with the common cold and/or flu, including nasal congestion experienced in conjunction with fever and/or pain. In one aspect, the present disclosure provides methods for using the bilayer tablets of the present disclosure. In some embodiments, provided herein is a method for treating nasal/sinus congestion (rhinosinusitis) with pain and fever associated with the common cold and/or flu-like symptoms in a subject in need thereof, comprising administering a bilayer tablet as described herein to the subject. As described herein a subject may include but is not limited to a mammal, or more particularly a human. In certain embodiments, provided herein is a method for treating nasal/sinus congestion (rhinosinusitis) with pain and fever associated with the common cold and/or flu-like symptoms in a human in need thereof, comprising administering a bilayer tablet as described herein to the human. In certain embodiments of the foregoing methods, the bilayer tablet is administered orally. In still other embodiments, the bilayer tablet is formulated for oral administration. In other aspects, provided is an article of manufacture, such as a container comprising a bilayer tablet as described herein, and a label containing instructions for use of the bilayer tablet. In some embodiments, provided herein is a package comprising a bilayer tablet as described herein. In certain embodiments, the package further comprises a desiccant. In still further embodiments, the package further comprises a package insert or a package label containing instructions for use of the bilayer tablet. In yet other aspects, provided is a kit comprising a bilayer tablet as described herein; and instructions for use of such a bilayer tablet. In certain embodiments of the foregoing aspects, the instructions for use are instructions for use of the bilayer tablet in treatment of nasal/sinus congestion with pain and fever associated with the common cold and/or flu-like symptoms. ENUMERATED EMBODIMENTS The following enumerated embodiments are representative of some aspects of the disclosure. 1. A bilayer tablet, comprising: an acetylsalicylic acid layer, comprising:granules, wherein the granules comprise intragranular acetylsalicylic acid, intragranular sodium carbonate, and one or more intragranular excipients;extragranular acetylsalicylic acid; andone or more extragranular excipients; anda pseudoephedrine layer, comprising:pseudoephedrine or a pharmaceutically acceptable salt thereof;sodium carbonate; andone or more excipients; wherein 10-50% w/w of the total acetylsalicylic acid present in the tablet is intragranular acetylsalicylic acid, and wherein the tablet has a weight ratio of the acetylsalicylic acid to sodium carbonate of between 1:1 and 5:1. 2. The tablet of embodiment 1, wherein the tablet has a weight ratio of acetylsalicylic acid to sodium carbonate between 2:1 and 4:1. 3. The tablet of embodiment 1 or embodiment 2, wherein 10-50% w/w of the total sodium carbonate present in the tablet is intragranular sodium carbonate. 4. The tablet of any one of embodiments 1 to 3, wherein 20-40% w/w of the total acetylsalicylic acid present is intragranular acetylsalicylic acid. 5. The tablet of any one of embodiments 1 to 4, wherein 20-40% w/w of the total sodium carbonate present in the tablet is present in the acetylsalicylic acid layer. 6. The tablet of any one of embodiments 1 to 5, wherein the weight percentage of intragranular acetylsalicylic acid out of the total acetylsalicylic acid present is equal to the weight percentage of the intragranular sodium carbonate out of the total sodium carbonate present in the tablet. 7. The tablet of any one of embodiments 1 to 6, comprising between 250 mg and 1000 mg acetylsalicylic acid. 8. The tablet of any one of embodiments 1 to 7, comprising between 15 mg and 60 mg pseudoephedrine or a pharmaceutically acceptable salt thereof. 9. The tablet of any one of embodiments 1 to 8, wherein the pseudoephedrine or a pharmaceutically acceptable salt thereof is pseudoephedrine hydrochloride. 10. The tablet of any one of embodiments 1 to 9, wherein the one or more intragranular excipients in the granules comprises colloidal silicon dioxide. 11. The tablet of any one of embodiments 1 to 10, wherein the one or more extragranular excipients in the acetylsalicylic acid layer comprises colloidal silicon dioxide, cornstarch, and cellulose. 12. The tablet of any one of embodiments 1 to 11, wherein the one or more excipients in the pseudoephedrine layer comprises mannitol, microcrystalline cellulose, cornstarch, and colloidal silicon dioxide. 13. The tablet of any one of embodiments 1 to 12, further comprising a coating. 14. The tablet of any one of embodiments 1 to 13, wherein the tablet comprises less than or equal to 4% w/w salicylic acid of the initial acetylsalicylic acid content after storage at 50° C. and 65% relative humidity for at least 10 days. 15. The tablet of any one of embodiments 1 to 14, wherein the tablet comprises less than or equal to 4% w/w salicylic acid of the initial acetylsalicylic acid content after storage at 40° C. and 75% relative humidity for at least 1 month. 16. The tablet of any one of embodiments 1 to 15, wherein the tablet comprises at least 95% acetylsalicylic acid of the initial acetylsalicylic acid content after storage at 40° C. and 75% relative humidity for at least 1 month. 17. The tablet of any one of embodiments 1 to 16, wherein the tablet has a dissolution profile wherein at least 85% acetylsalicylic acid and at least 85% pseudoephedrine are dissolved at 10 minutes as determined by USP Dissolution Test (Apparatus 1) in 50 mM sodium acetate buffer at pH 4.5 at 37±0.5° C. 18. A method for preparing a bilayer tablet according to any one of embodiments 1 to 17, comprising: compacting and milling acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide to provide granules; combining the granules with acetylsalicylic acid, cornstarch, powdered cellulose, and colloidal silicon dioxide to provide an acetylsalicylic acid blend; combining pseudoephedrine or a pharmaceutically acceptable salt thereof, cornstarch, mannitol, sodium carbonate, microcrystalline cellulose and colloidal silicon dioxide to provide a pseudoephedrine blend; and compressing the acetylsalicylic acid blend and pseudoephedrine blend to form the bilayer tablet. 19. The method of embodiment 18, comprising compacting the acetylsalicylic acid, sodium carbonate, and colloidal silicon dioxide by roller compaction. 20. The method of embodiment 18 or 19, wherein the granules have a particle size distribution wherein between 5% w/w and 30% w/w of the granules out of the total granule weight have a particle size of less than 150 μm, and wherein between 50% w/w and 80% w/w of the granules out of the total granule weight have a particle size of greater than 400 μm. 21. The method of any one of embodiments 18 to 20, further comprising drying the acetylsalicylic acid blend prior to compressing. 22. The method of embodiment 21, comprising drying the acetylsalicylic acid blend to a water activity of less than or equal to 0.2. 23. The method of any one of embodiments 18 to 22, wherein the pseudoephedrine or a pharmaceutically acceptable salt thereof is pseudoephedrine hydrochloride. 24. The method of any one of embodiments 18 to 23, comprising compressing the acetylsalicylic acid blend and pseudoephedrine blend at a compression force between 1 kN and 30 kN. 25. The method of any one of embodiments 18 to 24, comprising compressing the acetylsalicylic acid blend and pseudoephedrine blend at a compression force between 10 kN and 20 kN. 26. The method of any one of embodiments 18 to 23, wherein compressing the acetylsalicylic acid blend and pseudoephedrine blend to form the bilayer tablet comprises: compressing the acetylsalicylic acid blend at a first compression force to provide an acetylsalicylic acid layer; and compressing the pseudoephedrine blend on top of the acetylsalicylic acid layer at a second compression force to form the bilayer tablet. 27. The method of any one of embodiments 18 to 23, wherein compressing the compressing the acetylsalicylic acid blend and pseudoephedrine blend to form the bilayer tablet comprises: compressing the pseudoephedrine blend at a first compression force to provide a pseudoephedrine layer; and compressing the acetylsalicylic acid blend on top of the pseudoephedrine layer at a second compression force to form the bilayer tablet. 28. The method of embodiment 26 or embodiment 27, wherein the first compression force is between 1 kN and 30 kN and the second compression force is between 5 kN and 30 kN. 29. The method of any one of embodiments 18 to 28, further comprising coating the tablet. 30. A method for treating nasal congestion and pain or fever a in a human in need thereof, comprising administering a bilayer tablet according to any one of embodiments 1 to 17 to the human. 31. A package comprising a bilayer tablet according to any one of embodiments 1 to 17. 32. The package according to embodiment 31, further comprising a desiccant. EXAMPLES The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation. Example 1: Preparation of a Bilayer Tablet Comprising Acetylsalicylic Acid and Pseudoephedrine HCl The present example describes a method for preparing a bilayer tablet as described herein.FIG.1depicts a schematic of the bilayer tablet produced in this example. Table 1 below details the ingredient list for the separate layers of the bilayer tablet. TABLE 1Ingredient%mg/Functionw/wtabletACETYLSALICYLIC ACID-LAYER I1Acetylsalicylic acid Granulation32.16200.41aAcetylsalicylic acidActive24.07150.01bSodium CarbonateDiluent7.9449.51cColloidal Silicon DioxideFlow Aid0.140.92Acetylsalicylic acid (0-180), USPActive56.17350.03Powdered Cellulose, NFBinder5.6235.04Cornstarch, NFDisintegrant5.6235.05Colloidal Silicon Dioxide, NFFlow Aid0.432.7Acetylsalicylic acid Layer Sub-total100.00623.1PSEUDOEPHEDRINE HCL-LAYER II6Pseudoephedrine HCl, USPActive8.2130.07Mannitol, USPFiller/32.50118.8Diluent8Sodium Carbonate anhydrous, USPDiluent31.60115.59Microcrystalline Cellulose, NFBinder13.6850.010Cornstarch, NFDisintegrant13.6850.011Colloidal Silicon Dioxide, NFFlow Aid0.331.2Pseudoephedrine Layer Sub-total100.00365.5Bilayer TabletAcetylsalicylic acid Layer (Layer I)63.03623.1Pseudoephedrine HCl Layer36.97365.5(Layer II)12Dry Coating Powder BlendExternalTraceTrace(Hypromellose/Zinc Stearate/LubricationCarnauba Wax Blend,Blend50%/40%/10% w/w)*Bilayer Tablet Total100.00988.6 Acetylsalicylic acid Layer. As provided in the present disclosure, the bilayer tablet comprising an acetylsalicylic acid layer contains acetylsalicylic acid in granulated and non-granulated forms. Granulated acetylsalicylic acid was prepared by combining acetylsalicylic acid with sodium carbonate and colloidal silicon dioxide. Acetylsalicylic acid, sodium and colloidal silicon dioxide were combined in the mass proportions shown in Table 1 above for ingredients 1a, 1b and 1c, passed through roller compactors (at roller speed of 9 rpm, a roller pressure of 20-35 bar, and a roller gap of 1.4-3.0 mm) to provide compressed material in the form of rectangular ribbons, and the resulting compacted ribbons milled (at a mill speed of 107 rpm). The acetylsalicylic acid granules were then combined with additional powdered acetylsalicylic acid and extragranular excipients (powdered cellulose, cornstarch and colloidal silicon dioxide) at the mass proportions shown in Table 1 for ingredients 2-5. The resulting acetylsalicylic acid blend containing the granulated acetylsalicylic acid and powdered acetylsalicylic acid was dried to a moisture content of 8% wt. and held in storage until the pseudoephedrine blend was prepared. Pseudoephedrine Layer. Pseudoephedrine hydrochloride was combined with mannitol, anhydrous sodium carbonate, microcrystalline cellulose, cornstarch and colloidal silicon dioxide in the mass proportions indicated in Table 1 above to provide a pseudoephedrine blend. Tableting. Following preparation of the acetylsalicylic acid and pseudoephedrine blends, the two blends were fed into a bilayer tablet press sequentially for compression. The tablet press was pre-treated with an application of external lubrication (hypromellose/zinc stearate/carnauba wax blend, 50%/40%/10% w/w) to reduce material sticking and aid ejection of the tablets from the press. The acetylsalicylic acid blend was fed into the tablet first and compressed at a compression force of 3 kN to provide the acetylsalicylic acid layer. The pseudoephedrine blend was then added to the acetylsalicylic acid layer and the pseudoephedrine blend was compressed at a compression force of 26 kN to provide the pseudoephedrine layer and acetylsalicylic acid layer in the final bilayer tablet form. Example 2: Ratio of Granulated to Non-Granulated Acetylsalicylic Acid in Acetylsalicylic Acid Layer In the present example, the effect of varying ratios of granulated acetylsalicylic acid (roller-compacted, RC) to non-granulated acetylsalicylic acid (powdered, direct compression, DC) on the stability of the acetylsalicylic acid layer to degradation was evaluated. Six separate acetylsalicylic acid monolayer tablet formulations having one of six ratios of granulated to non-granulated (0:100, 10:90, 20:80, 30:70, 40:60 and 50:50) were prepared largely in accordance with the protocol described in Example 1 above. In each sample formulation, the ratio of total acetylsalicylic acid to total sodium carbonate in the bilayer tablet was maintained at 3:1; the ratio of acetylsalicylic acid present in the granules (if present) to the sodium carbonate present in the granules was also maintained at 3:1, with the remainder of sodium carbonate included in the second layer of pseudoephedrine. Approximately 10 individual tablets of each formulation ratio were subjected to accelerated stability conditions (storage at 50° C. at a 65% relative humidity for 20 days) in order to evaluate the stability of each tablet to degradation of acetylsalicylic acid into salicylic acid (or “free salicylic acid”, FSA) and other degradation products. Following storage, the quantity of free salicylic acid formed and total acetylsalicylic acid-derived degradation products (including salicylic acid) were determined by HPLC analysis. HPLC Analysis. The concentrations of acetylsalicylic acid (ASA) and pseudoephedrine hydrochloride (PSEH) present in the tablets after storage were determined by HPLC analysis, as calibrated an HPLC chromatogram of a known sample of acetylsalicylic acid and pseudoephedrine hydrochloride as a standard solution. After storage, HPLC injection samples were prepared by dissolving the tablets in a solution of 0.01N sulfuric acid:acetonitrile 80:20, v/v. Aliquots of the sample solutions were injected into an HPLC column under the following parameters and conditions: injection volume 15 μL; column: Waters XSelect®, HSS PFP 2.5 μm, 100 Å, length: 100 mm, ID: 4.6 mm; column temperature 40±2° C.; Mobile Phase A: 50 mM NaClO4, pH 2.5, Mobile Phase B: ACN:MeOH, 60:40 v/v; UV detection wavelengths: 257 nm for acetylsalicylic acid and 214 nm for pseudoephedrine hydrochloride. The determination of the concentrations of two major acetylsalicylic acid degradation products—free salicylic acid (FSA) and acetylsalicylsalicylic acid (ASSA)—were carried out as measured against the response of a known sample of acetylsalicylsalicylic acid (ASSA) (detection wavelength 257 nm). The quantitation of acetylsalicylic acid-derived degradation products were measured as calibrated against the response of the known sample of acetylsalicylsalicylic acid, with the application of relative response factors for each degradation product [relative response factor as compared to ASSA, for: free salicylic acid (FSA) 0.329× factor]. All pseudoephedrine-derived degradation products were measured as calibrated against the response known sample of pseudoephedrine hydrochloride, with the application of relative response factors for each degradation product [relative response factor as compared to PSEH, for: N-acetyl pseudoephedrine (PSEH-N ester) 2.207×; O-acetyl pseudoephedrine (PSEH-O ester) 0.883×; and N,O-acetyl pseudoephedrine (PSEH diester) 1.834×]. As shown inFIG.3, increased quantities of granulated acetylsalicylic acid resulted in increasing quantities of free salicylic acid and other degradation products. Example 3: Bilayer Tablet Dissolution Profile Testing The present example describes experiments to evaluate the dissolution profile of bilayer tablets formed with varying ratios of granulated acetylsalicylic acid to non-granulated acetylsalicylic acid within the acetylsalicylic acid layer as well as differing distributions of sodium carbonate throughout the acetylsalicylic acid and pseudoephedrine layers. Six separate bilayer tablet formulations were prepared as described Example 1 above with the distributions of sodium carbonate and ratios of granulated acetylsalicylic acid (intragranular, “INTRA”) to non-granulated acetylsalicylic acid (extragranular, “EXTRA”) as shown in Table 2 below. All six formulations contained a fixed quantity of 500 mg acetylsalicylic acid (ASA), 30 mg pseudoephedrine hydrochloride (PSEH), and 165 mg sodium carbonate (Na2CO3), in which all sodium carbonate in the acetylsalicylic acid layer (Layer I) was contained within the granulated acetylsalicylic acid. TABLE 2Na2CO3ASAPSEHAcetylsalicylic acidLayerLayerINTRAEXTRAIntra:Extra(I)(II)Layer I:Layer IISample #(mg)(mg)Ratio(mg)(mg)Ratio115035030:701650100:0215035030:701650100:035045010:901650100:0415035030:7049.5115.530:7055045010:9082.582.550:50615035030:7082.582.550:50 The dissolution profiles of each active ingredient in the bilayer tablet—the acetylsalicylic acid and pseudoephedrine hydrochloride—were evaluated in a dual dissolution test performed as described below. In brief, a single bilayer tablet is placed in a basket apparatus (Apparatus 1) containing 50 mM sodium acetate buffer of pH 4.5 (500 mL, equilibrated to 37±0.5° C.), at rotation speed of 50 rpm. Aliquots of the dissolution medium were taken at 2 minutes, 8 minutes, 15 minutes and 30 minutes. The quantities of each of the acetylsalicylic acid and pseudoephedrine hydrochloride dissolved in the dissolution medium were determined by UV absorption spectrometry at 257 nm and 214 nm, respectively. The dissolution measurements were taken for three bilayer tablets (n=3) for each bilayer tablet formulation. Table 3 and Table 4 below respectively show the observed average percentages of acetylsalicylic acid dissolved and pseudoephedrine hydrochloride dissolved at each time point. TABLE 3% Acetylsalicylic acid Dissolved at Time PointSample #2 min8 min15 min30 min173%93%96%95%235%92%98%98%367%95%97%97%438%89%96%97%555%92%98%98%635%94%99%100% TABLE 4% Pseudoephedrine HCl Dissolved at Time PointSample #2 min8 min15 min30 min1104%109%110%110%2102%102%102%102%3100%105%105%105%416%66%97%112%515%54%92%104%613%55%87%100% In further studies, the acetylsalicylic acid and pseudoephedrine hydrochloride dissolution profiles of bilayer tablets (Sample #4 formulation) prepared under different tableting compression forces were evaluated. The observed average percentage dissolution of acetylsalicylic acid and pseudoephedrine hydrochloride are shown in Table 8 after 60 minutes. The dissolution profiles for each active ingredient at time points 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes are depicted inFIGS.4A and4B. Table 5 below shows the percentages of acetylsalicylic acid and pseudoephedrine hydrochloride dissolved at 60 minutes. As shown inFIGS.4A and4Band Table 5, the dissolution profile of acetylsalicylic acid was similar across all compression forces but pseudoephedrine hydrochloride showed slightly slower dissolution rates in the first 30 minutes with increasing compression force. TABLE 5Compression% ASA Dissolved% PSEH DissolvedForce (kN)at 60 minat 60 min1597.00100.702097.96101.232598.98100.963098.03103.153597.65102.83Average97.93101.77% RSD0.71.1 Example 4: Short-Term and Long-Term Storage Stability and Dissolution Studies The present examples describes stability and dissolution studies of various bilayer tablet formulations after short-term and long-term storage. Each of the six formulations in Table 2 were subjected to accelerated stability test conditions, kept at 50° C. and 65% relative humidity for twenty (20) days, with the quantities of acetylsalicylic acid (ASA), pseudoephedrine hydrochloride (PSEH), and their respective major degradation products measured at specific time points (days 0 [initial], 5, 10 and 20). The amounts of acetylsalicylic acid, pseudoephedrine hydrochloride, and their respective major degradation products were determined by HPLC analysis as described in Example 2 above. Degradation products observed in the accelerated stability tests include free salicylic acid (FSA), acetylsalicylsalicylic acid (ASSA), N-acetyl pseudoephedrine (PSEH-N ester), O-acetyl pseudoephedrine (PSEH-O ester), and N,O-acetyl pseudoephedrine (PSEH diester). Table 6 below shows the average percentages of acetylsalicylic acid, pseudoephedrine hydrochloride, and a selection of degradation products observed at each time point for three individual tablets for each bilayer formulation sample. TABLE 6% w/wSampleTotal ASAPSEH-N andPSEHTotal PSEH#TimePSEHASAFSAASSADegsO-esterDiesterDegs1Initial110.597.20.840.141.00.150.020.2Day 5108.793.63.000.533.90.670.491.2Day 10114.092.73.800.534.80.950.941.9Day 20109.590.44.710.535.71.211.813.02Initial100.998.40.760.100.90.11ND0.1Day 5105.792.72.070.362.60.700.431.1Day 10104.494.52.490.373.20.900.861.8Day 20108.790.33.140.363.81.451.673.13Initial104.099.80.760.080.90.17ND0.2Day 5103.796.62.860.493.70.730.541.3Day 10106.395.93.550.484.40.911.172.1Day 20103.292.34.560.475.51.102.023.14Initial111.197.10.780.090.90.170.020.2Day 5109.996.21.850.242.31.560.812.4Day 10107.794.32.100.242.51.881.343.2Day 20110.693.72.650.293.22.392.434.85Initial108.1100.40.720.070.90.19ND0.2Day 5102.698.12.070.292.51.890.952.8Day 10100.398.02.510.333.12.161.894.0Day 20102.395.63.310.364.02.683.055.76Initial102.6101.20.560.100.70.05ND0.1Day 5100.799.42.120.362.71.540.752.3Day 1098.099.52.750.363.42.111.683.8Day 2098.095.93.690.394.42.772.955.7 As compared to the samples #1-3, in which sodium carbonate was localized in the acetylsalicylic acid layer, a distribution of sodium carbonate across both layers of the bilayer tablet in samples #4-6 resulted in higher quantities of remaining acetylsalicylic acid in the tablet after storage for 20 days. In a series of further experiments, the same six formulations of Table 2 were subjected to additional long-term stability studies (either at 40° C. and 75% relative humidity or 25° C. and 60% relative humidity) with and without desiccant. Following storage for a fixed period of time, the bilayer tablets tested were evaluated for the amounts of acetylsalicylic acid and pseudoephedrine hydrochloride remaining and for the presence of degradation products in accordance with the protocol described in Example 4 above. Table 7 shows the variable stability conditions and the corresponding table(s) (Tables 8, 10-14, and 16) of degradation results. Dissolution profiles were recorded for the six formulations after storage at one month and 3 months at 40° C. and 75% relative humidity, without desiccant. The dissolution test results observed after 1 month and 3 months in storage are shown in Table 9 and Table 15, respectively. TABLE 7DesiccantStorageStorage(with-yes;DissolutionTable #DurationConditionswithout-no)ProfileTable 81 month40° C., 75% RHwithoutTable 9Table 101 month,40° C., 75% RHwith andn/a3 weekswithoutTable 111 month,25° C., 60% RHwith andn/a3 weekswithoutTable 123 months40° C., 75% RHwith andn/awithoutTable 133 months25° C., 60% RHwith andn/awithoutTable 143 months40° C., 75% RHwithoutTable 15Table 163 months25° C., 60% RHwithoutn/a TABLE 8% w/wSampleTotal ASAPSEH-N andPSEHTotal PSEH#PSEHASAFSAASSADegsO-esterDiesterDegs1114.694.42.900.363.40.670.521.22100.596.31.860.182.10.730.511.23105.896.22.490.292.90.730.581.34110.495.81.720.161.91.160.731.95103.699.22.040.212.31.491.032.5695.2100.82.110.232.41.340.872.2 TABLE 9% ASA Dissolution at% PSEH Dissolution atSampleTime Point (average)Time Point (average)#2 min8 min15 min30 min2 min8 min15 min30 min1658992929911311411425091949399105105105373919493891061071074418692929388510755092969610448710763492979711387095 As shown in Table 8, the sodium carbonate distribution of 30:70 in sample #4 as compared to 50:50 in samples #5 and 6 resulted in less formation of free salicylic acid and other major acetylsalicylic acid-derived degradation products after storage. The dissolution profiles of the bilayer tablets after storage in Table 9 were comparable to the dissolution profiles observed in Table 3 and Table 4, which recorded for tablets that were not subjected to storage conditions. TABLE 10% w/wDesiccantTotal ASAPSEH-esterPSEHTotal PSEHSample #Yes/NoPSEHASAFSAASSADegsNODiesterDegs1N113.194.53.330.363.90.360.410.661.4Y111.497.61.650.652.60.200.400.351.02N103.396.22.200.182.50.410.470.691.6Y105.197.91.270.291.70.110.410.310.83N107.994.82.900.283.30.350.340.781.5Y103.398.61.370.592.20.240.410.371.04N109.595.11.980.162.21.31<LOQ1.042.4Y111.796.91.240.251.60.97<LOQ0.331.35N102.299.02.390.212.71.55<LOQ1.392.9Y104.7100.81.350.361.91.35<LOQ0.471.86N99.799.92.480.232.91.44<LOQ1.222.7Y101.1102.21.460.392.01.19<LOQ0.331.5*LOQ = Limit of quantification TABLE 11% w/wDesiccantTotal ASAPSEH-esterPSEHTotal PSEHSample #Yes/NoPSEHASAFSAASSADegsNODiesterDegs1N113.196.91.570.211.80.230.110.140.5Y112.996.71.070.191.30.100.120.080.32N100.398.41.140.131.30.140.110.110.4Y104.698.20.860.121.00.050.120.050.23N107.698.81.250.171.40.240.100.160.5Y104.8100.70.830.151.00.140.130.080.44N112.497.71.030.121.20.44<LOQ0.110.6Y113.296.00.850.111.00.36<LOQ0.090.55N103.2100.41.070.111.20.61<LOQ0.170.8Y104.099.60.830.100.90.50<LOQ0.120.66N100.0101.91.170.151.30.57<LOQ0.140.7Y102.7100.80.910.141.10.44<LOQ0.090.5*LOQ = Limit of quantification TABLE 12% w/wDesiccantTotal ASAPSEH-esterPSEHTotal PSEHSample #Yes/NoPSEHASAFSAASSADegsNODiesterDegs1N111.693.13.830.344.40.320.530.621.5Y111.393.71.750.863.10.220.570.301.12N104.595.02.530.182.80.420.620.651.7Y98.896.91.240.361.80.200.640.241.13N103.395.43.290.293.80.330.510.691.5Y102.096.61.460.782.60.270.470.311.04N106.895.52.430.172.71.570.041.032.6Y112.495.91.320.311.81.190.040.321.55N101.897.62.890.223.31.720.061.233.0Y102.298.41.440.492.21.470.060.401.96N98.698.52.970.243.41.670.061.062.8Y100.998.41.420.472.21.210.060.281.5 TABLE 13% w/wDesiccantTotal ASAPSEH-esterPSEHTotal PSEHSample #Yes/NoPSEHASAFSAASSADegsNODiesterDegs1N113.696.61.750.252.00.300.130.060.5Y114.596.51.020.231.30.150.140.000.32N104.795.41.170.141.30.260.150.030.4Y109.896.20.780.140.90.130.140.020.33N103.898.91.470.191.70.300.110.060.5Y105.699.40.810.201.00.160.140.010.34N111.296.41.120.131.30.66ND0.060.7Y114.797.50.830.131.00.530.010.010.55N98.3100.51.240.141.40.86ND0.101.0Y103.6101.20.790.130.90.520.010.010.56N102.7101.61.320.171.50.75ND0.070.8Y102.7100.80.840.171.00.17ND0.010.2*ND = not detected Table 10, Table 11, Table 12, and Table 13 show the observed amounts of degradation products after storage with or without desiccant. As shown in the Tables, the inclusion of desiccant during storage appeared to reduce the formation of several major degradation products, including free salicylic acid, total acetylsalicylic acid degradation products, N-acetyl pseudoephedrine, N,O-acetyl pseudoephedrine diester, and total pseudoephedrine degradation products. Desiccant did not appear to affect the formation of acetylsalicylsalicylic acid under storage conditions of 25° C. and 60% RH. TABLE 14% w/wTotal ASAPSEH-esterTotal PSEHSample #PSEHASAFSAASSADegsNOPSEH DiesterDegs1109.392.04.460.335.20.870.084.695.62102.591.13.220.183.71.030.144.846.03102.493.74.040.274.700.810.085.035.94109.591.63.030.203.520.052.136.338.5598.996.23.510.244.130.062.067.439.5695.396.63.570.254.20.061.896.868.8 TABLE 15% ASA Dissolution at% PSEH Dissolution atSampleTime Point (average)Time Point (average)#2 min8 min15 min30 min2 min8 min15 min30 min1577580848810710911124576818473939799346747881771011021024288087881027549852472767783982936238085877316689 Table 14 shows that the acetylsalicylic acid-derived degradation products formed after 3 months of storage at 40° C. and 75% RH, were present at nearly similar weight percentages as observed under accelerated storage conditions on Day 20 (in Table 6). However, the pseudoephedrine diester and total pseudoephedrine degradation products were present in significantly higher quantities as compared to amounts observed the accelerated storage conditions on Day 20 (in Table 6). As shown in Table 15, the total dissolution of acetylsalicylic acid at 30 minutes was on average reduced as compared to the dissolution at the same time point in Table 3 (not subjected to storage conditions) and in Table 9 (subjected to 1 month at same storage conditions, 40° C., 75% RH, without desiccant). Table 16 shows observed degradation products after 3 months of storage at storage conditions of 25° C. and 60% RH. After storage at conditions of 25° C. and 60% RH, the quantities of free salicylic acid, pseudoephedrine diester, and total pseudoephedrine degradation products were increased relative to the same amounts present in tablets not subjected to storage (in Table 6, initial time point). TABLE 16% w/wTotal ASAPSEH-esterTotal PSEHSample #PSEHASAFSAASSADegsNOPSEH DiesterDegs197.8102.31.590.201.9ND0.150.640.82106.499.21.510.161.8ND0.100.470.63109.198.21.340.141.6ND0.130.640.84103.696.71.780.222.10.050.760.791.65104.896.41.420.161.70.040.970.992.06115.093.82.120.272.60.050.770.881.7*ND = not detected Example 5: Dissolution Profile Comparison of Various Acetylsalicylic Acid Formulations In this example, various commercial acetylsalicylic acid formulations were assessed for their acetylsalicylic acid dissolution profiles as compared to the bilayer tablets provided herein to evaluate the effects of various physical formulation parameters (granulated vs non-granulated; single layer vs bilayer) on dissolution rate. Five separate samples were subjected to the dissolution test protocol described in Example 3 above. The samples included: (A) the bilayer tablet formulation described in Table 1 (Example 1) including both acetylsalicylic acid and pseudoephedrine HCl layers; (B) a single layer tablet formulated identically to the acetylsalicylic acid sub-layer described in Table 1 (Example 1); (C) a single layer tablet comprising only granulated acetylsalicylic acid (74.85% w/w acetylsalicylic acid, 24.7% w/w sodium carbonate, 0.45% w/w colloidal silicon dioxide, trace coating); (D) granules comprising acetylsalicylic acid and pseudoephedrine HCl (500 mg acetylsalicylic acid; 30 mg pseudoephedrine HCl; citric acid anhydrous, sucrose, hypromellose, saccharin, flavor, maltodextrin); and (E) acetylsalicylic acid, corn starch, hypromellose, powdered cellulose, triacetin. FIG.5shows the dissolution profiles of acetylsalicylic acid prepared in various formulations. Example 6: Phenylephrine Substitution for Pseudoephedrine In additional experiments, phenylephrine hydrochloride was employed as a substitute for pseudoedphedrine hydrochloride as a decongestant in Layer II. Four sample bilayer tablet formulations were prepared using phenylephrine hydrochloride in accordance with the preparation protocol of Example 1, with the ratios of granulated acetylsalicylic acid (intragranular, “INTRA”) to non-granulated acetylsalicylic acid (extragranular, “EXTRA”) within the acetylsalicylic acid layer and distributions of sodium carbonate throughout the acetylsalicylic acid and phenylephrine layers as described in Table 17 below. All four formulations contained a fixed quantity of 500 mg acetylsalicylic acid (ASA), 30 mg phenylephrine hydrochloride (PEH), and 165 mg sodium carbonate (Na2CO3). In sample #9, all sodium carbonate in the acetylsalicylic acid layer (Layer I) was contained within the granulated acetylsalicylic acid. TABLE 17Na2CO3ASAPEHAcetylsalicylic acidLayerLayerSampleINTRAEXTRAIntra:Extra(I)(II)Layer I:Layer II#(mg)(mg)Ratio(mg)(mg)Ratio705000:10082.582.5050:50805000:10001650:100915035030:7049.5115.530:701005000:10049.5115.530:70 The same dissolution test parameters as described in Example 2 were utilized to evaluate the bilayer tablets comprising phenylephrine. Aliquots were taken at time points of 5 minutes, 15 minutes, 30 minutes and 45 minutes. Three bilayer tablet trials were carried out for each sample formulation (n=3). Table 18 and Table 19 respectively show the observed average dissolution of acetylsalicylic acid and phenylephrine hydrochloride at each time point. The quantity of acetylsalicylic acid dissolved at each time point was determined by UV absorption spectrometry at detection wavelength 257 nm. The quantity of phenylephrine hydrochloride dissolved at each time point was determined by UV absorption spectrometry at detection wavelength 214 nm. TABLE 18% Acetylsalicylic acid Dissolved at Time PointSample #5 min15 min30 min45 min789%96%96%95%853%85%97%99%984%94%94%93%1084%92%91%91% TABLE 19% Phenylephrine HCl Dissolved at Time PointSample #5 min15 min30 min45 min771%107%108%108%841%85%98%98%955%102%108%107%1054%102%108%108% Further to the dissolution assays, the four phenylephrine-containing bilayer tablets (Samples #7-10) and four additional samples (Samples #11-14), the compositions of which are detailed in Table 20, were subjected to accelerated stability test conditions at (storage at 50° C. at a 65% relative humidity for 20 days) to gauge susceptibility to degradation. The formation of degradation products was determined using the protocol described in Examples 2 and 4 above. TABLE 20Na2CO3ASAPEHAcetylsalicylic acidLayerLayerSampleINTRAEXTRAIntra:Extra(I)(II)Layer I:Layer II#(mg)(mg)Ratio(mg)(mg)Ratio1115035030:7049.5115.530:701215035030:7001650:1001315035030:7049.5115.530:701415035030:7001650:100 Table 21 presents the observed quantity of free salicylic acid for each tablet formulation after storage under accelerated stability test conditions. As shown in Table 18, Table 19, and Table 21, sample #8, prepared with no intragranular acetylsalicylic acid and all sodium carbonate localized in the phenylephrine layer, resulted in the lowest formation of free salicylic acid but also gave the slowest dissolution profile relative to samples #7 and 9-10. Similarly, in samples #11-14, the samples with sodium carbonate localized to the phenylephrine layer resulted in less free salicylic acid formation. TABLE 21Sample #% w/w free salicylic acid71.2680.3591.10101.00112.62121.67131.86141.02 | 149,663 |
11857685 | DETAILED DESCRIPTION OF THE INVENTION Collagen is the major structural constituent of mammalian organisms and makes up a large portion of the total protein content of skin and other parts of the animal body. Various skin traumas such as burns, surgery, infection and accident are often characterized by the erratic accumulation of fibrous tissue rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfiguring deposits of new tissue sometimes form during the healing process. Some diseases and conditions are associated with excess collagen deposition and the erratic accumulation of fibrous tissue rich in collagen. Such diseases and conditions are collectively referred to herein as “collagen-mediated diseases”. It has now been found that uterine fibroids are a collagen-mediated disease, associated with excess collagen deposition and the erratic accumulation of fibrous tissue rich in collagen. The considerable variation in growth rates over time of individual fibroids, and microarray studies revealing that genes encoding for ECM proteins or related to ECM synthesis and secretion account for a large portion of changes in gene expression in fibroids compared with myometrium make dysregulation of ECM (extracellular matrix) a possible contributing factor to this condition. Transforming growth factor (TGF) plays a role in fibroid development. Fibroids grow by deposition of altered collagen. The expression of other molecules is likewise altered in fibroids. For example, dermatopontin expression is decreased, fibronectin and glycosaminoglycans (GAG) are increased, alpha 11 integrin, a collagen-binding integrin is expressed. In addition, fibroids are resistant to apoptosis. Recent studies indicate that fibroids are formed by the accumulation of extracellular matrix (ECM) as well as by cellular proliferation. SeeFIG.1, noting the disordered collagen fibrils in the fibroid tissue. The appearance and spatial orientation of collagen fibrils in uterine fibroids were shorter, randomly aligned and widely dispersed compared with those of the myometrium. They were non-aligned and not parallel whereas in the adjacent myometrium the fibrils were well packed and parallel in orientation to each other, a finding that is characteristic of collagen containing tissue. Myofibroblast type cells (elongated appearance, notched nucleus) also have been found in uterine fibroids. The notched appearance of the fibroid cell nucleus represents folding and envaginations of the nuclear membrane due to cell contraction by stress fibers. Therefore, the present invention takes advantage of collagenase, an enzyme that has the specific ability to digest collagen, to treat uterine fibroids. Degradation of the collagen not only causes collagenolysis, it also reduces the increased cell compression leading to mechanotransduction. Thereby, the cycle of increased collagen secretion and enlargement of the uterine fibroid is broken. In summary, uterine fibroids contain an abundance of altered collagen consistent with fibrosis and stiffness. A stiff extracellular matrix (ECM) exerts force against individual cells. Mechanotransduction alters cell signaling and prevents apoptosis, and thus collagen accumulation continues. (See,FIG.15.) Uterine fibroids grow at individual rates suggesting that mechanical transduction of tumors is responsible for variation in growth rates. This specification describes embodiments of an invention for treatment to reduce the symptoms of uterine fibroids, shrink uterine fibroids, reduce the stiffness and mechanical stress of fibroid tissue on the uterus and/or eliminate uterine fibroids by local delivery of a purified collagenase composition to avoid systemic side-effects and harm to other tissues. In general, some of the preferred methods use a syringe and needle under ultrasound or other visualization for guided injection of purified collagenase directly into the uterine fibroid tissue to be treated. The collagenase product preferably is in a vehicle for delivery, such as a nanocarrier or other protective or sustained release carrier. Because the center of fibroids is more fibrotic and contains smaller vascular capillary beds than the periphery, and due to a dense vascular capsule which surrounds the fibroid tumor, systemic therapy is not likely to provide therapeutic tissue levels of a drug in the fibroid center while leaving the likely possibility of systemic effects. Thus, pharmacotherapy has not been successful for uterine fibroids. The local injection of a treatment agent under imaging guidance allows for exact tissue placement of the drug and greatly reduces the chance of systemic effects. Uterine fibroids are classified into several types, based on their location, including subserosal, intramural, submucosal, pedunculated submucosal, fibroid in statu nascendi, and fibroid of the broad ligament. Any and all of these uterine fibroids are contemplated for treatment using the invention. Myometrial Hyperplasia is a condition which can mimic uterine fibroid symptoms and may be a precursor lesion of these tumors. It is structural variation with irregular zones of hypercellularity and increased nucleus/cell ratio, causing a bulging, firm, enlarged uterus. The condition often leads to hysterectomy. Deeper MMH has lower cellularity, and tends to have increased collagen. Therefore, this condition also may be treated using the methods and compositions of the invention. The local treatment of uterine fibroids by injection of collagenase can be conducted in an office or clinic visit under ultrasound guidance with minimal chance for sequelae. This method can be used to treat small to moderate size fibroids or asymptomatic fibroids, which currently are not treated at all, allowing the clinician to prevent potentially debilitating symptoms and preservation of fertility in women of child-bearing years, and also larger fibroids, eliminating the need for hysterectomy for this disease. Thus, the methods of this invention are contemplated to be useful to treat any stage or type of uterine fibroid disease. Collagenase for use according to the invention may be obtained from any convenient source, including mammalian (e.g., human, porcine), crustacean (e.g., crab, shrimp), fungal, and bacterial (e.g., from the fermentation ofClostridium, Streptomyces, Pseudomonas, VibrioorAchromobacter iophagus). Collagenase can be isolated from a natural source or can be genetically engineered/recombinant. One common source of crude collagenase is from a bacterial fermentation process, specifically the fermentation ofClostridium histolyticum. The crude collagenase obtained fromC. histolyticumcan be purified using any of a number of techniques known in the art of protein purification, including chromatographic techniques. Collagenase compositions useful for the invention also can be prepared using any commercially available or isolated collagenase activity, or by mixing such activities. For example, purified collagenase can be provided by Biospecifics Technologies, Lynbrook, N.Y. Preferred collagenases for use in the invention are fromC. histolyticum, i.e., collagenase class I and class II. A practical advantage of usingC. histolyticumfor the production of collagenases is that it can be cultured in large quantities in simple liquid media, and it regularly produces amounts of proteolytic enzymes which are secreted into the culture medium. Bovine products have been used in culture media in the fermentation ofC. histolyticum, but these run the risk of contamination by agents which cause transmissible spongiform encephalopathies (TSEs; e.g., prions associated with bovine spongiform encephalopathy or “mad cow disease”). Therefore, it is preferred to avoid such bovine products. An animal-product-free system is preferred. The H4 strain ofClostridium histolyticum, originally developed in 1956 can serve as a source for cells for culture. This strain, and a strain derived from the H4 strain, named the ABCClostridium histolyticummaster cell bank (deposited as ATCC 21000) were developed using animal products, but are suitable to use in the invention. U.S. Pat. No. 7,811,560, which is incorporated herein by reference in its entirety, discloses methods of producing collagenases. Using soybean derived fermentation medium, the methods described therein generated separately highly purified collagenase I and II. This patent also discloses methods of producing highly purified collagenases using culture media containing porcine-derived products. Any of these methods are suitable for use with the invention. U.S. Patent Publication 2010/0086971, which is also incorporated herein by reference in its entirety, discloses numerous fermentation recipes which are based on vegetable peptone, including soybean-derived peptone, or vegetable-derived peptone plus fish gelatin. The methods described in this publication are suitable to produce growth ofClostridiumand collagenase activities. These methods also are suitable and contemplated for use with the invention, however any method known in the art of producing collagenase enzyme activity may be used. In preferred culture methods, the peptone is from a plant source selected from the group consisting of soy bean, broad bean, pea, potato, and a mixture thereof. The peptone may be selected from the group consisting of Oxoid VG100 Vegetable peptone No. 1 from pea (VG100), Oxoid VG200 Vegetable peptone phosphate broth from Pea (VG200), Merck TSB CASO-Bouillion animal-free (TSB), Invitrogen Soy bean peptone No 110 papainic digest (SP6), Fluka Broad bean peptone (BP), Organotechnie Plant peptone E1 from potato (E1P), BBL Phytone™ peptone and BD Difco Select Phytone™. In a preferred embodiment of the invention, a single type of peptone is present in the nutrient composition of the invention, whereby the peptone is selected from the group consisting of BP, E1P, Soy bean peptone E110, VG100, and VG200, and whereby the concentration of the peptone in the composition is about 5% weight by volume. In yet another very much preferred embodiment of the invention, a single type of peptone is present in the nutrient composition of the invention, whereby the peptone is BBL phytone peptone or Difco Select Phytone™ UF, and whereby the concentration of the peptone in the composition is about 10-13% weight by volume. Preferred methods of isolating collagenase avoid undesirable contaminating proteases such as clostripain. Clostripain, a cysteine protease, is believed to be a major cause of collagenase degradation and instability, and is present inClostridiumculture. When such proteases are present in a crude collagenase mixture, one must take extra precautions to neutralize the proteases, including using protease inhibitors, such as leupeptin, and performing all of the purification steps in specially designed cold rooms with chilled solutions to reduce protease activity. Preferred methods of isolation therefore take advantage of one of two approaches to avoid clostripain: remove clostripain as early as possible in the purification method or reduce clostripain production during the fermentation stage. Preferred collagenase compositions are produced by fermentingC. histolyticumin medium free of animal material-derived ingredients and are substantially free of clostripain, and thus are highly stable. “Substantially free” indicates that the collagenase contains less than 10 U clostripain per mg total collagenase, more preferably less than 5 U/mg, and most preferably about 1 U/mg or less, and/or that no visible band appears representing clostripain and/or degraded collagenase on SDS-PAGE gel compared to a reference standard. Preferred methods for purifying collagenase involve using a “low glucose” medium as described herein, which contains less than about 5 g/L glucose, more preferably less than about 1 g/L, even more preferably less than about 0.5 g/L glucose, or is glucose-free, for culture ofC. histolyticum. High salt concentrations in the growth media can reduce the amount of clostripain produced in culture, thus preferred media forC. histolyticumculture contain greater than about 5 g/L (or 0.5% w/v) total salt, more preferably greater than about 7.5 g/L (or 7.5%) total salt, and more preferably about 9 g/L (or 9%) or more. It is contemplated that any salt known to be suitable for use in microbiological fermentation media may be used in the current invention. In a preferred embodiment, chloride, phosphate or sulfate salts may be used. In a more preferred embodiment, the salts may be sodium chloride, potassium chloride, monosodium phosphate, disodium phosphate, tribasic sodium phosphate, potassium monophosphate, potassium diphosphate, tripotassium phosphate, calcium chloride, magnesium sulfate or various combinations thereof. In certain embodiments, potassium diphosphate may be about 0.1-0.3%, potassium phosphate may be about 0.75% to 0.175%, sodium phosphate may be about 0.2-0.5%, and/or sodium chloride may be about 0.15-0.35%. Preferably, the medium further comprises magnesium sulfate and vitamins, including, riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxine and thiamine. In another preferred embodiment, the nutrient composition may contain 0.5-5% yeast extract, more preferably about 1-4%, and most preferably about 1.5-2.5%. Yeast extract is available from a variety of suppliers, including Cole Parmer (Vernon Hills, Ill.) and Fisher Scientific (Pittsburgh, Pa.). In yet a preferred embodiment of the invention, the pH of the media is between pH 7 and pH 8. Even more preferred is a pH between about pH 7.2 and about pH 7.7, most preferably about 7.4. The collagenase contemplated for use with the invention can be any collagenase which is active under the necessary conditions. However, preferred compositions contain a mass ratio of collagenase I and collagenase II which is modified or optimized to produce a desired or even a maximal synergistic effect. Preferably, collagenase I and collagenase II are purified separately from the crude collagenase mixture produced in culture, and the collagenase I and collagenase II are recombined in an optimized fixed mass ratio. Preferred embodiments contain a collagenase I to collagenase II mass ratio of about 0.5 to 1.5, more preferably 0.6 to 1.3, even more preferably 0.8 to 1.2, and most preferably, 1 to 1, however any combination or any single collagenase activity may be used. A preferred method of producing collagenase which is contemplated for use with the invention involves fermentingC. histolyticumin a non-mammalian or non-animal medium, wherein the culture supernatant is substantially clostripain-free. The collagenases so produced can be isolated, purified, and combined to provide a composition for use in the invention which comprises a mixture of collagenase I and collagenase II in an optimized fixed mass ratio which is substantially clostripain-free. The crude collagenase obtained from fermentation ofC. histolyticummay be purified by a variety of methods known to those skilled in the art, including dye ligand affinity chromatography, heparin affinity chromatography, ammonium sulfate precipitation, hydroxylapatite chromatography, size exclusion chromatography, ion exchange chromatography, and/or metal chelation chromatography. Additionally, purification methods for collagenases are known, such as, for example, those described in U.S. Pat. No. 7,811,560, which is hereby incorporated by reference in its entirety. Both collagenase I and collagenase II are metalloproteases and require tightly bound zinc and loosely bound calcium for their. Both collagenases have broad specificity toward all types of collagen. Collagenase I and Collagenase II digest collagen by hydrolyzing the triple-helical region of collagen under physiological conditions. Each collagenase shows different specificity (e.g. each have a different preferred target amino sequence for cleavage), and together they have synergistic activity toward collagen. Collagenase II has a higher activity towards all kinds of synthetic peptide substrates than collagenase I as reported for class II and class I collagenase in the literatures. The preferred collagenase consists of two microbial collagenases, referred to as Collagenase ABC I and Collagenase ABC II. The terms “Collagenase I”, “ABC I”, and “collagenase ABC I” mean the same and can be used interchangeably. Similarly, the terms “Collagenase II”, “ABC II”, and “collagenase ABC II” refer to the same enzyme and can also be used interchangeably. These collagenases are secreted by bacterial cells. Preferably, they are isolated and purified fromClostridium histolyticumculture supernatant by chromatographic methods. Both collagenases are special proteases and share the same EC number (E.C 3.4.24.3). However, a collagenase or a combination of collagenases from other sources are contemplated for use with the invention. Collagenase ABC I has a single polypeptide chain consisting of approximately 1000 amino acids with a molecular weight of 115 kDa. Collagenase ABC II has also a single polypeptide chain consisting of about 1000 amino acids with a molecular weight of 110 kDa. Collagenase acts by hydrolyzing the peptide bond between Gly-Pro-X, wherein X is often proline or hydroxyproline. Collagenase I acts at loci at ends of triple-helical domains, whereas Collagenase II cleaves internally. Hydrolysis continues over time until all bonds are cleaved. Preferably, the collagenase product is at least 95% pure collagenase(s) and is substantially free of any contaminating proteases. More preferably, the collagenase product is 97% pure and most preferably 98% pure or more as determined by one or more of the following: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); high performance liquid chromatography (HPLC); reverse-phase HPLC; or by enzymatic assays. The preferred collagenase product is essentially clostripain-free, and the purification preferably is performed in the absence of leupeptin. The preferred collagenase product for use with the invention has at least one specification selected from Table 1 below. TABLE 1Preferred Specifications for Collagenase ProductsSpecificationTestABC-IABC-IIAppearanceClear colorless and essentially free fromparticulate matterEndotoxin<10EU/mLIdentity (and purity) byMajor collagenaseMajor collagenaseSDS-PAGE (Reducedband between 98-band between 97-conditions, Coomasie)188 kDa200 kDa≥95%≥95%SRC assay (ABC-I)1967-3327 SRCNAunits/mgGPA assay (ABC-II)NA81934-119522GPA units/mgAnalysis of Proteins≥98% main peak; ≤2% aggregates by areaHPLC System(Aggregation by sizeexclusionchromatography)Identity and purity byMajor peak (ABC I or ABC II), ≥95% byreverse phase liquidarea; Retention times of ABC-I and ABC-IIchromatography)within 5% of referenceClostripain assay (BAEE≤1U/mgassay)Bioburden<1cfu/mL The collagenase products described for use herein are useful for the treatment of collagen-mediated disease, including uterine fibroids. Examples of other collagen mediated-diseases that may be treated by the compositions of the invention include but are not limited to: Dupuytren's disease; Peyronie's disease; frozen shoulder (adhesive capsulitis), keloids; tennis elbow (lateral epicondylitis); scarred tendon; glaucoma; herniated discs; adjunct to vitrectomy; hypertrophic scars; depressed scars such as those resulting from inflammatory acne; post-surgical adhesions; acne vulgaris; lipomas, and disfiguring conditions such as wrinkling, cellulite formation and neoplastic fibrosis. In addition to its use in treating specific collagen-mediated diseases, the compositions of the invention also are useful for the dissociation of tissue into individual cells and cell clusters as is useful in a wide variety of laboratory, diagnostic and therapeutic applications. These applications involve the isolation of many types of cells for various uses, including microvascular endothelial cells for small diameter synthetic vascular graft seeding, hepatocytes for gene therapy, drug toxicology screening and extracorporeal liver assist devices, chondrocytes for cartilage regeneration, and islets of Langerhans for the treatment of insulin-dependent diabetes mellitus. Enzyme treatment works to fragment extracellular matrix proteins and proteins which maintain cell-to-cell contact. In general, the compositions of the present invention are useful for any application where the removal of cells or the modification of an extracellular matrix, are desired. The collagenase compositions according this invention are designed to administer to a patient in need thereof a therapeutically effective amount of a collagenase composition as described, or a therapeutically effective amount of a pharmaceutical collagenase formulation as described. A “therapeutically effective amount” of a compound, composition or formulation is an amount of the compound which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. A therapeutic effect includes but is not limited to a shrinkage or reduction in the size of one or more uterine fibroids (including elimination of the fibroid), liquification, partial liquification, or reduction in stiffness (increase in softness) or pressure in or around a uterine fibroid, a change in viscoelastic properties, or reduction in symptoms such as pain, hemorrhage and the like. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect), and may be determined by the clinician or by the patient. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts. The term “patient” or “patient in need” encompasses any mammal having a uterus and uterine fibroids or symptoms thereof. Such “patients” or “patients in need” include humans or any mammal, including farm animals such as horses and pigs, companion animals such as dogs and cats, and experimental animals such as mice, rats and rabbits. Preferred patients are human females of child-bearing age. The pharmaceutical compositions of this invention preferably are administered by injection, insertion or implantation directly into or onto the uterine fibroid tissue to be treated, i.e. local administration to the tissue to be treated. Other modes of administration contemplated included, but are not limited to transvaginal instillation or application onto the affected tissues, instillation or application during surgery (such as laparoscopy or hysteroscopy) onto the affected tissues, i.e. topical administration to the fibroid tissue, by spray or other application of a liquid, fluid or gel formulation. Formulations of the present invention are injected/inserted into uterine tissue in a variety of forms, by a variety of routes, using a variety of apparatuses. In some embodiments, the formulation is injected/inserted using an apparatus consisting of a simple needle (e.g., a 10 gauge or smaller needle) and sample pusher (e.g., a mandrel or modified obturator). For example, according to one embodiment, a formulation (e.g., a rod-shaped or other shaped solid or semi-solid formulation, beads, suspension, gel, polymer or the like) is placed in the needle or in a syringe or other chamber affixed to the needle. Once the needle is placed at the desired depth and location in the tissue, the pusher is used to push the sample from the needle and into the tissue. In some embodiments, the sample pusher is provided with a holding clip or it is provided with a hollow end to secure the sample up to the time of delivery. In still other embodiments, formulations in accordance with the present invention are injected/inserted via jet injection without a physical delivery channel such as a needle, as is known in the art. Typically, a compression system (e.g., a mechanical system or a gas, such as helium, nitrogen, carbon dioxide, etc.) is used to accelerate the formulations to a high enough velocity so that the formulation can penetrate the tissue to a desired depth. Jet injector devices can be, for example, disposable, or reusable with medication cartridges that are prefilled or non-prefilled medication cartridges. Examples of jet injectors include Biojector from Bioject, N.J., USA and the PowderJect® System from PowderJect, UK. In other embodiments, a device is employed that cores out a section of the fibroid (e.g., a biopsy device or tissue morcellator or laser radiation), thereby leaving behind a void for insertion of a dosage form. The formulations for collagenase delivery to a patient generally are contemplated to comprise injectable or implantable formulations, or any fluid, liquid, solid, semi-solid, gel, or other composition which is suitable to administer the collagenase to the tissue to be treated as described herein. Formulations in accordance with the present invention may be formulated by any method known in the pharmaceutical arts. Thus, any injectable or implantable formulation known in the art and consistent with collagenase activity may be used. Formulations which create a depot or extended release of the active collagenase agent are contemplated. In particular, injectable extended or sustained release compositions are preferred, however any implantable formulation can be used. Such compositions produce or form a depot effect, where active agent is present in the tissue where administered and release active agent over a period of time to continuously treat the tissue. Immediate release injectable formulations, where the active agent is immediately released for activity upon administration, also are contemplated for use with the invention. These formulations are known in the art and can be adapted for use with the present invention by any person of skill. In some embodiments, the injectable or insertable formulations of the present invention are solids, semi-solids or high-viscosity fluids. This improves dosage retention in the tissue, thereby improving delivery efficiency of the treatment agents and/or minimizing the adverse effects such as unintended, nonspecific tissue damage. “High viscosity” and other such terms are used herein to describe fluids having viscosities greater than 1000 centipoise as measured by any of a number of standard techniques, including, for example, a Brookfield Kinematic Viscometer, model HBDV-II+CP with a CPE-40 cone spindle, set at 37° C. and using a 0.5 rpm speed setting. “Low viscosity” fluids have viscosities less than this value. In some embodiments, a formulation in accordance with the present invention is injected into a patient in a fluid state, whereupon it converts (or is converted) in vivo into a more readily retained form, for example, into a solid form (including conversion of an injected liquid into a solid, conversion of an injected semi-solid into a solid and conversion of a liquid into a gel), into a semi-solid form (including conversion of an injected liquid into a semi-solid, conversion of an injected semi-solid into a semi-solid having increased yield stress and/or viscosity and conversion of a liquid into a gel), or into a high-viscosity fluid (including conversion of a low-viscosity fluid into a high-viscosity fluid, and conversion of a high-viscosity fluid into a higher-viscosity fluid). Preferred formulations for injection into a uterine fibroid use a carrier or nanocarrier. Appropriate carriers include solid or semi-solid pellets, beads or gel-forming polymers, high-viscosity liquids and the like to maintain the active collagenase in the tissue, protecting the active enzyme from action of the tissue or tissue components which could inactivate the collagenase, and allow steady release of the enzyme to the tissue for treatment. Any injectable dosage form which can protect and contain the active compound(s) in place may be used. In mammals,C. histolyticumcollagenase is inhibited rapidly in the blood stream by serum. Therefore, systemic administration, or administration under conditions where the collagenase can be deactivated, or orally, where the collagenase can be degraded by digestive enzymes, is problematic. Nanocarriers are designed to deliver and protect drug therapeutics (e.g. proteins, for example) from degradation. A nanocarrier formulation also is preferred because this method impedes diffusion and distribution of the drug away from the injected fibroid, prolongs release, delays inactivation, and therefore reduces the frequency of repeat injections. Any such nanocarrier known in the art can be used with the invention. Some of these nanocarriers also are referred to as thermoresponsive delivery systems. Atrigel•comprises a water-insoluble biodegradable polymer (e.g., poly(lactic-co-glycolic acid, PLGA) dissolved in a bio-compatible, water-miscible organic solvent (e.g., N-methyl-2-pyrrolidone, NMP). In use, collagenase is added to form a solution or suspension. Both the PLGA molecular weight and lactide-glycolide molar ratio (L:G ratio) governs drug delivery. Using an L:G ratio of from 50:50 to 85:15 and a polymer concentration of from 34 to 50%, clinical studies have demonstrated a depot which was maintained for more than 3 months. ReGel•is a 4000 Da triblock copolymer formed from PLGA and polyethylene glycol (PEG, 1000 Da or 1450 Da) in repetitions of PLGA-PEG-PLGA or PEG-PLGA-PEG. ReGel•is formulated as a 23 wt % copolymer solution in aqueous media. A drug is added to the solution and upon temperature elevation to 37° C. the whole system gels. Degradation of ReGel•to final products of lactic acid, glycolic acid and PEG occurs over 1-6 weeks depending on copolymer molar composition. Chemically distinct drugs like porcine growth hormone and glucagon-like peptide-1 (GLP-1) may be incorporated, one at a time, and released from ReGel•. LiquoGel™ can work by mechanistically independent drug delivery routes: entrapment and covalent linkage. Two or more drugs can be delivered to the tumor site using this carrier. LiquoGel™ is a tetrameric copolymer of thermogelling N-isopropylacrylamide; biodegrading macromer of poly(lactic acid) and 2-hydroxyethyl methacrylate; hydrophilic acrylic acid (to maintain solubility of decomposition products); and multi-functional hyperbranched polyglycerol to covalently attach drugs. LiquoGel™ generally is formulated as a 16.9 wt % copolymer solution in aqueous media. The solution gels under physiological conditions and degrades to release drug contents within 1-6 days. Any of the above carriers can be used as a nanocarrier with the invention. A preferred nanocarrier, however, contains hyperbranched polyglycerols (HPG), which have many desirable features. HPGs grow by imperfect generations of branched units and are produced in a convenient single step reaction. Previous problems of large polydispersities in molecular weight in their production have been overcome. The resulting polymers contain a large number of modifiable surface functional groups as well as internal cavities for drug interaction. Other polymer approaches cannot easily provide these properties without significant increases in the number of synthetic steps and, consequently, cost. HPG polymers are based on glycerol and because of structural similarity with polyethylene glycol, is biocompatible. Additional components optionally can be added to the polymer, therefore, modified HPG polymers and co-polymers of HPG are contemplated. These additional components or monomers can include, for example, crosslinks, biodegradable moieties, and thermoresponsive moieties. For example, thermally responsive hydrogels are attractive for injection therapy since it is possible to inject the necessary fluid volume from a syringe maintained below body temperature and upon warming, the mechanical properties are increased, thereby restraining the material at the injection site. Poly(N-isopropylacrylamide) (poly-NIPAAm) is a thermally responsive polymer with a lower critical solution temperature (LCST) of approximately 32° C. Copolymers of HPG with NIPAAm are therefore contemplated for use with the invention, and are preferred. This nanocarrier has a versatile mesh size and can be customized to entrap small drug molecules, large proteins, or a mixture of components, and gels at body temperature to permit slow release as the nanocarrier biodegrades. In preferred embodiments of the invention, formulations exist as a liquid at temperatures below body temperature and as a gel at body temperature. The temperature at which a transition from liquid to gel occurs is sometimes referred to as the LCST, and it can be a small temperature range as opposed to a specific temperature. Materials possessing an LCST are referred to as LCST materials. Typical LCST's for the practice of the present invention range, for example, from 10 to 37° C. As a result, a formulation injected below the LCST warms within the body to a temperature that is at or above the LCST, thereby undergoing a transition from a liquid to a gel. Suitable LCST materials for use with the invention include polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymers. Two acceptable compounds are Pluronic acid F127 and F108, which are PEO-PPO block copolymers with molecular weights of 12,600 and 14,600, respectively. Each of these compounds is available from BASF (Mount Olive, N.J.). Pluronic acid F108 at 20-28% concentration concentration, in phosphate buffered Saline (PBS) is an example of a suitable LCST material. One beneficial preparation is 22.5% Pluronic acid F108 in PBS. A preparation of 22% Pluronic acid F108 in PBS has an LCST of 37° C. Pluronic acid F127 at 20-35% concentration in PBS is another example of a suitable LCST material. A preparation of 20% Pluronic acid F127 in PBS has an LCST of 37° C. Typical molecular weights are between 5,000 and 25,000, and, for the two specific compounds identified above are 12,600 and 14,600. More generally, materials, including other PEO-PPO block copolymers, which are biodisintegrable, and which exist as a gel at body temperature and as a liquid below body temperature can also be used according to the present invention. Further information regarding LCST materials can be found in U.S. Pat. Nos. 6,565,530 B2 and 6,544,227 B2, each of which is hereby incorporated by reference. Pharmaceutical formulations of the collagenase compounds for the invention include a collagenase composition formulated together with one or more pharmaceutically acceptable vehicles or excipients. As used herein, the term “pharmaceutically acceptable carrier or excipient” means a non-toxic, inert, solid, semi-solid or liquid filler, diluent, encapsulating material, vehicle, solvent, or formulation auxiliary of any type, and may be made available in individual dosage forms or in bulk. Other dosage forms designed to create a depot of the active compound also are contemplated for use with the invention. Dosage forms for collagenase suitable for use with the invention include, but are not limited to lyophilized or other dried powder for reconstitution prior to injection, in multiple or single dose amounts, individual dosage units ready for injection (which preferably also include one or more preservatives), frozen unit dosage forms, or any mode of preparation known in the art. The formulations also may be provided in the form of a kit, which can contain the collagenase in solid form, liquid or solvent for reconstitution and injection, and any equipment necessary for administration, such as a syringe and needle, particularly a specialized syringe and/or needle for administration to a uterine fibroid. Preferably, the dosage form has a largest dimension between 1 mm and 20 mm. Preferably, the formulations are sterile. The products may be sterilized by any method known in the art, such as by filtration through a bacterial-retaining filter or are produced under aseptic conditions. Other methods include exposing the formulation or components thereof to heat, radiation or ethylene oxide gas. Some examples of materials which can serve as pharmaceutically acceptable carriers are solvents for injection as known in the art. Examples include, but are not limited to sterile water, buffering solutions, saline solutions such as normal saline or Ringer's solution, pyrogen-free water, ethyl alcohol, non-toxic oils, and the like, or any solvent compatible with injection or other forms of administration as described herein for use with the invention. In addition, any solid excipients known in the art for use in pharmaceutical products can be used with the invention as a vehicle or filler, for example. Sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; gums; talc; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar, and the like can be used. Buffering agents compatible with the active compounds and the methods of use are contemplated for use, including acid or alkali compounds, such as magnesium hydroxide and aluminum hydroxide, citric acid, phosphate or carbonate salts and the like. Non-toxic compatible excipients such as lubricants, emulsifiers, wetting agents, suspending agents, binders, disintegrants, preservatives or antibacterial agents, antioxidants, sustained release excipients, coating agents and the like (e.g., sodium lauryl sulfate and magnesium stearate) also may be used, as well as coloring agents, perfuming agents, viscosity enhancing agents, bioadhesives, and the like, according to the judgment of the formulator. For example, one or more biodisintegrable binders may be included in the formulations of the present invention, typically in connection with dosage forms having solid characteristics. Where employed, a wide range of biodisintegrable binder concentrations may be utilized, with the amounts varying based, for example, on the desired physical characteristics of the resulting dosage form and on the characteristics of the uterine fibroid treatment agent that is selected (e.g., the degree of dilution, release delay, etc. that is desired/tolerated), among other considerations. The concentration of biodisintegrable binder typically ranges are from about 1 to 80 wt % of biodisintegrable binder, more typically about 5 to 50 wt %. A “biodisintegrable” material is one that, once placed in tissue such as uterine tissue, undergoes dissolution, degradation, resorption and/or other disintegration processes. Where such materials are included, formulations in accordance with the present invention will typically undergo at least a 10% reduction in weight after residing in tissue such as uterine tissue for a period of 7 days, more typically a 50-100% reduction in weight after residing in the tissue for a period of 4 days. Suitable biodisintegrable binders for use in connection with the present invention include, but are not limited to biodisintegrable organic compounds, such as glycerine, and biodisintegrable polymers, or any known disintegrant compound known in the art of pharmaceutics. Where used, viscosity adjusting agent(s) are typically present in an amount effective to provide the formulation with the desired viscosity, for example, by rendering the formulation highly viscous, for example, in an amount effective to provide a viscosity between about 5,000 and 200,000 centipoise, more typically between about 10,000 and 100,000 centipoise, more typically between about 10,000 and 50,000 centipoise, and even more typically between about 20,000 and 40,000 centipoise. By providing formulations having viscosities within these ranges, the formulations can be injected into tissue, such as uterine tissue, using conventional injection equipment (e.g., syringes). However, due to their elevated viscosities, the formulations have improved retention within the tissue at the injection site. The concentration of the viscosity adjusting agent(s) that is (are) used can vary widely. Commonly, the overall concentration of the viscosity adjusting agent(s) is between about 1 and 20 wt %. In many embodiments, the viscosity adjusting agents are polymers, which may be of natural or synthetic origin and are typically biodisintegrable. The polymers are also typically water soluble and/or hydrophilic. However, in some embodiments, for instance where an organic solvent such as dimethylsulfoxide (DMSO) is used as a liquid component, the viscosity adjusting agent can be relatively hydrophobic. The polymeric viscosity adjusting agents include homopolymers, copolymers and polymer blends. Examples of viscosity adjusting agents for the practice of the present invention include, but are not limited to the following: cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, hydroxyethyl starch (HES), dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carrageenan, various gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, heparin, heparin sulfate, dermatan sulfate, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid/acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide and poly(ethylene oxide-propylene oxide) (e.g., Pluronic acid), polyoxyethylene (polyethylene glycol), polyethyleneamine and polypyrridine, poly-metaphosphate (Kurrol salts), polyvinyl alcohol, additional salts and copolymers beyond those specifically set forth above, and blends of the foregoing (including mixtures of polymers containing the same monomers, but having different molecular weights), and so forth. Many of these species are also useful as binders. In other embodiments of the invention, formulations or carriers are crosslinked, either prior to use or in vivo. Crosslinking is advantageous, for example, in that it acts to improve formulation retention (e.g., by providing a more rigid/viscous material and/or by rendering the polymer less soluble in a particular environment). Where the formulation is crosslinked in vivo, a crosslinking agent is commonly injected into tissue either before or after the injection or insertion of a formulation in accordance with the present invention. Depending on the nature of the formulation and the crosslinking agent, the formulation may be converted, for example, into a solid, into a semi-solid, or into a high-viscosity fluid. Crosslinking agents suitable for use in the present invention include, any non-toxic crosslinking agent, including ionic and covalent crosslinking agents. For example, in some embodiments, polymers are included within the formulations of the present invention, which are ionically crosslinked, for instance, with polyvalent metal ions. Suitable crosslinking ions include polyvalent cations selected from the group consisting of calcium, magnesium, barium, strontium, boron, beryllium, aluminum, iron, copper, cobalt, lead and silver cations ions. Polyvalent anions include phosphate, citrate, borate, succinate, maleate, adipate and oxalate anions. More broadly, crosslinking anions are commonly derived from polybasic organic or inorganic acids. Ionic crosslinking may be carried out by methods known in the art, for example, by contacting ionically crosslinkable polymers with an aqueous solution containing dissolved ions. In some embodiments, polymers are included, which are covalently crosslinkable, for example, using a polyfunctional crosslinking agent that is reactive with functional groups in the polymer structure. The polyfunctional crosslinking agent can be any compound having at least two functional groups that react with functional groups in the polymer. Various polymers described herein can be both covalently and ionically crosslinked. Suitable polymers for ionic and/or covalent crosslinking can be selected, for example, from the non-limiting list of the following: polyacrylates; poly(acrylic acid); poly(methacrylic acid); polyacrylamides; poly(N-alkylacrylamides); polyalkylene oxides; poly(ethylene oxide); poly(propylene oxide); poly(vinyl alcohol); poly(vinyl aromatics); poly(vinylpyrrolidone); poly(ethylene imine); poly(ethylene amine); polyacrylonitrile; poly(vinyl sulfonic acid); polyamides; poly(L-lysine); hydrophilic polyurethanes; maleic anhydride polymers; proteins; collagen; cellulosic polymers; methyl cellulose; carboxymethyl cellulose; dextran; carboxymethyl dextran; modified dextran; alginates; alginic acid; pectinic acid; hyaluronic acid; chitin; pullulan; gelatin; gellan; xanthan; carboxymethyl starch; hydroxyethyl starch; chondroitin sulfate; guar; starch; and salts, copolymers, mixtures and derivatives thereof. In one preferred embodiment, the collagenase is formulated as a lyophilized injectable composition formulated with lactose, sucrose or any suitable sugar. One preferred collagenase composition is a lyophilized injectable composition formulated with sucrose, Tris at a pH level of about 8.0. Most preferably, 1.0 mg of the drug substance of the invention is formulated in 60 mM sucrose, 10 mM Tris, at a pH of about 8.0 (e.g., about 20.5 mg/mL of sucrose and 1.21 mg/mL of Tris in the formulation buffer). Preferred collagenase compositions for use in the invention comprise a mixture of collagenase I and collagenase II has a specific activity of at least about 700 SRC units/mg, such as at least about 1000 SRC units/mg, more preferably at least about 1500 SRC units/mg. One SRC unit will solubilize rat tail collagen into ninhydrin reaction material equivalent to 1 nanomole of leucine per minute, at 25° C., pH 7.4. Collagenase has been described in ABC units as well. This potency assay of collagenase is based on the digestion of undenatured collagen (from bovine tendon) at pH 7.2 and 37° C. for 20-24 hours. The number of peptide bonds cleaved are measured by reaction with ninhydrin. Amino groups released by a trypsin digestion control are subtracted. One net ABC unit of collagenase will solubilize ninhydrin reactive material equivalent to 1.09 nanomoles of leucine per minute. One SRC unit equal approximate 6.3 ABC unit or 18.5 GPA unit. In one embodiment, each milligram of collagenase for injection will contain approximately 2800 SRC units. Doses contemplated for administration by direct injection to the uterine fibroid tissue will vary depending on the size of the tissue to be treated and the discretion of the treating physician. However, doses generally are about 0.06 mg collagenase to about 1 mg collagenase per cm3of tissue to be treated or about 0.1 mg collagenase to about 0.8 mg collagenase per cm3of tissue to be treated, or about 0.2 mg collagenase to about 0.6 mg collagenase per cm3of tissue to be treated. Formulations that contain an additional active agent or medication also are contemplated. Optional additional agents which can be included in the formulation for concomitant, simultaneous or separate administration include, for example, any pharmaceutical known in the art for shrinkage, treatment or elimination of uterine fibroids or their symptoms, or to assist in performance of the present treatment methods. For example, one or more fibroid treatment agents such as aromatase inhibitors (e.g., letrozole, anastrozole, and exemestande), progesterone receptor agonists and modulators (e.g., progesterone, progestins, mifepristone, levonoergestrel, norgestrel, asoprisnil, ulipristal and ulipristal acetate, telepristone), selective estrogen receptor modulators (SERMs) (e.g., benzopyran, benzothiophenes, chromane, indoles, naphtalenes, tri-phenylethylene compounds, arzoxifene, EM-652, CP 336,156, raloxifene, 4-hydroxytamoxifen and tamoxifen), gonadotrophin-releasing hormone analogs (GnRHa) (e.g., GnRH agonist peptides or analogs with D-amino acid alterations in position 6 and/or ethyl-amide substitutions for carboxyl-terminal Gly10-amide such as triptorelin or GnRH antagonists such as cetrorelix, ganirelix, degarelix and ozarelix), growth factor modulators (e.g., TGFb neutralizing antibodies), leuprolide acetate, non-steroidal anti-inflammatory drugs, inhibitors of the mTOR pathway, inhibitors of the WNT signaling pathway, vitamin D, vitamin D metabolites, vitamin D modulators, and/or an additional anti-fibrotic compound (e.g., pirfenidone and halofuginone) may be co-administered with collagenase in the same or a separate administration. Chemical ablation agents also can be included in the formulations of the present invention. In effective amounts, such compounds cause tissue necrosis or shrinkage upon exposure. Any known ablation agent can be used according to the art, in concentrations as appropriate to the conditions while avoiding inactivation of the collagenase, with the amounts employed being readily determined by those of ordinary skill in the art. Typical concentration ranges are from about 1 to 95 wt % of ablation agent, more typically about 5 to 80 wt %. Ablation agents suitable for use with the invention include, but are not limited to osmotic-stress-generating agents (e.g., a salt, such as sodium chloride or potassium chloride), organic compounds (e.g., ethanol), basic agents (e.g., sodium hydroxide and potassium hydroxide), acidic agents (e.g., acetic acid and formic acid), enzymes (e.g., hyaluronidase, pronase, and papain), free-radical generating agents (e.g., hydrogen peroxide and potassium peroxide), oxidizing agents (e.g., sodium hypochlorite, hydrogen peroxide and potassium peroxide), tissue fixing agents (e.g., formaldehyde, acetaldehyde or glutaraldehyde), and/or coagulants (e.g., gengpin). These agents may be combined with collagenase in the same formulation so long as they do not negatively affect the enzymatic activity of the collagenase, or they may be administered separately, at the same time or at different times. The methods according to the invention may be used in conjunction with any known treatments to control symptoms caused by fibroids. For example, NSAIDs or other analgesics can be used to reduce painful menses, oral contraceptive pills are may be prescribed to reduce uterine bleeding, and iron supplementation may be given to treat anemia. A levonorgestrel intrauterine device can be used to reduce hemorrhage and other symptoms if the condition of the uterus does not result in expulsion of the device. The ability to non-invasively image regions where the formulations of the present invention are being introduced and where they have been introduced is a valuable diagnostic tool for the practice of the present invention. Therefore, in addition to a uterine fibroid treatment agent and any of the various optional components discussed above, the uterine fibroid formulations of the present invention also optionally include one or more imaging contrast agents to assist with guiding the clinician to administer the collagenase compound to the fibroid or tissue to be treated or to determine that administration has been correctly located. Non-non-invasive imaging techniques include magnetic resonance imaging (MRI), ultrasonic imaging, x-ray fluoroscopy, nuclear medicine, and others. Any contrast agent suitable for use with such techniques and known in the art can be used as part of the inventive compositions and formulations. Any real-time imaging technology can be used to guide injection or insertion in the invention. For example, X-ray based fluoroscopy is a diagnostic imaging technique that allows real-time patient monitoring of motion within a patient. To be fluoroscopically visible, formulations are typically rendered more X-ray absorptive than the surrounding tissue. In various embodiments of the invention, this is accomplished by the use of contrast agents. Examples of contrast agents for use in connection with X-ray fluoroscopy include metals, metal salts and oxides (particularly bismuth salts and oxides), and iodinated compounds. More specific examples of such contrast agents include tungsten, platinum, tantalum, iridium, gold, or other dense metal, barium sulfate, bismuth subcarbonate, bismuth trioxide, bismuth oxychloride, metrizamide, iopamidol, iothalamate sodium, iodomide sodium, and meglumine. Ultrasound and magnetic resonance imaging can provide two- and/or three-dimensional images of a portion of the body. Ultrasound and MRI are advantageous, inter alia, because they do not expose the patient or medical practitioner to harmful radiation and they can provide detailed images of the observed area. These detailed images are valuable diagnostic aids to medical practitioners and can be used to more precisely control the quantity and location of the formulations of the present invention. Suitable ultrasonic imaging contrast agents for use in connection with the present invention include solid particles ranging from about 0.01 to 50 microns in largest dimension (e.g., the diameter, where spherical particles are used), more typically about 0.5 to 20 microns. Both inorganic and organic particles can be used. Examples include microparticles/microspheres of calcium carbonate, hydroxyapatite, silica, poly(lactic acid), and poly(glycolic acid). Microbubbles can also be used as ultrasonic imaging contrast agents, as is known in the imaging art. The ultrasonic imaging contrast agents for use in connection with the present invention are preferably biocompatible and stable in the formulation. Concentrations of the ultrasonic imaging contrast agents typically range from about 0.01 wt % to 10 wt % of the formulation, more typically about 0.05 to 2 wt %, where solid particles are used. For contrast-enhanced MRI, a suitable contrast agent has a large magnetic moment, with a relatively long electronic relaxation time. Based upon these criteria, contrast agents such as Gd(III), Mn(II) and Fe(III) can be used. Gadolinium(III) has the largest magnetic moment among these three and is, therefore, a widely-used paramagnetic species to enhance contrast in MRI. Chelates of paramagnetic ions such as Gd-DTPA (gadolinium ion chelated with the ligand diethylenetriaminepentaacetic acid) also are suitable. Further information can be found, for example, in U.S. Patent Application No. 2003-0100830 entitled “Implantable or insertable medical devices visible under magnetic resonance imaging,” the disclosure of which is incorporated herein by reference. The collagenase formulations described here preferably are injected into one or more individual uterine fibroid tumors using a hollow delivery channel, such as a hollow needle or cannula. For instance, administration can be performed using a needle in association with a conventional or specially designed syringe, cannula, catheter, and the like. A source of manual, mechanical, hydraulic, pneumatic or other means to apply pressure (e.g., a conventional syringe plunger, a pump, aerosol, etc.) can be used to inject the formulation into the fibroid. Alternatively, the formulations can be administered during surgery, for example via a trocar during laparoscopic surgery and during hysteroscopic treatment. Injection routes include, for example, transabdominal, transcervical and transvaginal routes. Where the formulations have fluid attributes, the injection volume will vary, depending, for example, on the size of the fibroid, the type and concentration of treatment agent, and so forth, and will typically range from 1.0 to 10.0 ml per injection. Similarly, where formulations having solid attributes (e.g., pellets or powders) are used, the amount of formulation injected/inserted will also depend, for example, on the size of the fibroid, the type and concentration treatment agent utilized, etc. Multiple pellets or doses of collagenase composition can be administered at a single injection site. Regardless of the physical attributes of the formulation, multiple injection/insertion sites may be established within a single fibroid, with the number of injections depending on the size and shape of the fibroid as well as the type and/or concentration of the treatment agent that is used. Multiple fibroids or a single fibroid can be treated. In various embodiments, the injection/insertion device is guided to the fibroid site under image guidance. Image guidance can include, for example, direct visual guidance (e.g., laparoscopic guidance in trans-abdominal procedures and hysteroscopic guidance in trans-vaginal procedures) and non-direct visual guidance (e.g., ultrasound guidance, fluoroscopic guidance, and/or MRI guidance). As a specific example, visual guidance of the injection/insertion device is conducted laparoscopically using a scope that is positioned in the abdomen (e.g., by insertion through a trocar). In this way, a device (e.g., a delivery needle or canula) can be inserted percutaneously into the abdomen and guided under laparoscopic vision to the uterine fibroid. Once the fibroid is reached, fluoroscopy, MRI or ultrasound (e.g., trans-vaginal ultrasound, trans-abdominal ultrasound, intra-abdominal ultrasound, etc.) preferably is used to guide the tip of the delivery needle to a desired position within the fibroid, at which point the formulation is injected or inserted into the fibroid. To the extent that there is sufficient contrast between the formulation and the surrounding tissue, the location of the formulation within the fibroid will also be viewed. The compositions and processes of the present invention will be better understood in connection with the following examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the processes, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims. EXAMPLES Example 1. General Collagenase Production To prepare an animal-material-free clostridia cell bank,Clostridium histolyticumcells are suspended in a medium containing a vegetable peptone and optionally yeast extract. For example, one general method for accomplishing this is as follows. TABLE 2General Method to Produce Clostridium Cell Bank.Step 1Starting cells: anyClostridiumhistolyticumculture which isconvenient and available, for exampleClostridiumhistolyticumATCC 21000, strain 004Step 2Inoculate 1 mL of step 1 into 300 mL of media containing15.45 g Phytone, 2.55 g yeast extract, and water sufficient toproduce 0.3 L (M#1);step 2 for 24 hours at 37° C. (1stculture);Step 4Transfer 3 mL of step 3 (1stculture) to 1000 mL of M#1;Step 5Incubate step 4 for 16 hours at 37° C. (2ndculture);Step 6Centrifuge the 2ndculture;Step 7Re-suspend the pellet with the 5 mL of media #1 and 5 mL of20% glycerol;Step 8Freeze the aliquot of cells gradually ;Step 9Store the aliquot at −80° C. Once an animal material-free cell bank is established, the cells can be grown or fermented in convenient media known in the art, preferably non-animal-derived medium. The medium can optionally contain yeast extract. Exemplary, non-limiting examples of such media are M #1, M #2, M #3, and M #4 as described in Table 3, below. In addition, see Table 4 for an exemplary, non-limiting general example of the steps of the fermentation process. TABLE 3Media recipes and preparation.M #1M #2M #3M #4Phytone15.45 g103 gVeggitone15.45 g103 gYeast extract2.55 g2.55 g17 g17 gKH2PO41.92 g1.92 gK2HPO41.25 g1.25 gNa2HPO43.5 g3.5 gNaCl2.5 g2.5 gvol of water0.3 L0.3 L1 L1 L TABLE 4Fermentation Process.Step 1Starting cells: Animal material free clostridia cell bankStep 2Inoculate 1 mL of step 1 into the 300 ml of M#1;Step 3Incubate step 2 for 16 to 24 hours at 37° C. (1stculture);Step 4Transfer 10 mL of step 3 (1stculture) and 10 mL Vitamin/Mg solution* to 1000 mL of M#3, or 4 respectively;Step 5Incubate step 4 for about 22 hours at 37° C. (2ndculture);Step 6Use 2ndculture for downstream isolation and purification.*Prepared separately by dissolving 8 g MgSO4, 1.2 g ferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 ml water, followed by sterilization by 0.22 um filtration. After preparation of “2ndculture,” the collagenase I and collagenase II can be isolated and purified using any method capable of producing each enzyme separately to at least 95% purity. The method may combine one or more of the steps of ammonium sulfate precipitation, dialysis, hydroxyapatite (HA) chromatography, gel filtration and ion-exchange, for example, preferably in that order. The gel filtration is preferably G75 gel filtration. The ion-exchange is preferably anion-exchange: Q-Sepharose chromatography. In addition, when the Clostridia have been cultured in medium containing less glucose and more salt compared to the majority of known bacterial culture, as preferred, protease inhibitors such as leupeptin are not required. Example 2. Preparation of Animal Material FreeClostridiumCell Bank The starter cell culture wasClostridium histolyticumATCC 21000, strain 004 which was originally created with bovine-derived materials. The cells were first grown in animal material free medium (M #1, Table 3). Briefly, the recipe includes: phytone, 51.5 g, yeast extract 8.5 g, 1000 mL water. The pH was adjusted to 7.30 with NaOH, and the medium sterilized at 121° C. for 20 minutes. One milliliter of the starting material was then inoculated into 300 mL of M #1 and incubated for 24 hours at 37° C. (1st culture). Three milliliters of the 1st culture was transferred to 1000 mL of M #1 and incubated for 16 hours (2nd culture). The 2nd culture was then centrifuged aseptically. The pellet was re-suspended in 5 mL M #1 with 5 mL 20% glycerol. The aliquots of cell suspension were frozen gradually and stored at −80° C. Example 3. Fermentation Process Clostridium histolyticumATCC 21000, strain 004 was inoculated into the starting culture with M #1 or M #2 and incubated at 37° C. for 16 hours. Ten milliliters of the starting culture (M #1 or M #2) and 10 mL Mg/vitamin solution (prepared separately by dissolving 8 g MgSO4, 1.2 g ferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water, followed by sterilization by 0.22 μm filtration) was then transferred to each liter of M #3 or M #4 (or a variation thereof), and incubated for 22 hours.Clostridium histolyticumgrew well with the OD600 reaching >2.5. Example 4. General Procedure for Isolation and Purification of Collagenase I and Collagenase II TABLE 5General Exemplary, Non-Limiting Isolation and PurificationProcedure for Collagenase I and Collagenase II.Stages of ProductOperationsFermentationCentrifugation or 1.0 μm filtration;brothClarifiedAdd ammonium sulfate (590 g/liter); centrifugation;fermentationbrothCrude collagenaseDissolve crude collagenase precipitate by addingprecipitatepurified water;Crude collagenaseDialyze crude collagenase solution against purifiedsolutionwater overnight with 10 kDa pore size dialysis(store at −20° C.)membrane;Dialyzed crudeClarify the dialyzed crude collagenase solutioncollagenasewith either centrifugation or filtration or thecombination of both;Clarified solutionAdd potassium phosphate buffer, pH 6.7 to a finalconc. of 0.1M;Collagenase inLoad collagenase solution to hydroxylapatitephosphate buffercolumn and elute column with gradient ofincreasing K2PO4conc. at ambient temp. (20° C.);Collagenase HAConcentrate the eluate with ultrafiltration (30 kDaeluateof pore size);ConcentratedLoad the concentrate onto a G75 gel filtrationcollagenasecolumn at ambient temperature (20° C.) and elutewith 20 mM Tris/150 mM NaCl;Collagenase G75Dialyze the eluate against a buffer (10 mM Tris, 3eluatemM calcium chloride (CaCl2), pH 8.0) overnight;Dialyzed G75Load dialyzed eluate on to a Q-Sepharose anion-eluateexchange column at ambient temperature (20° C.);elute using a gradient of 10 mM Tris HCl, 3 mMCaCl2, pH 8.0 buffer and 10 mM Tris HCl, 3 mMCaCl2, 1M NaCl, pH 8.0 buffer;Collagenase classStore separately at −20° C.I and class IIfractions Example 5. Ex Vivo Treatment of Uterine Fibroid Tissue Samples of fibroid tissue and myometrium were obtained post-hysterectomy from women with consent and identified by evaluation by a surgical pathologist. The tissue samples were transported to the laboratory and cut into 1 cm3cubes. SeeFIG.2. These cubes were injected with purified collagenase (0.06 or 0.2 mg in 100 μL) dissolved in media or serum and then incubated for 24, 48, 72, or 96 hours at 37° C. SeeFIG.3. Each treatment was carried out in tissues from three different patients with two tissue samples per treatment because fibroid tissue is extremely variable. Control fibroid and myometrium cubes were injected with vehicle or sham injected. At the end of the incubation, the tissue samples were photographed to document gross appearance. Degree of liquefaction and softening was observed and documented using a 4-point subjective scale. Samples were frozen for biomechanical assessment (compression analysis). Samples were fixed in formalin for histology and Masson trichrome and picrosirius red staining. They were analyzed by light microscopy for the presence or absence of collagen and assessed using computer morphometry to determine the extent of degradation. In the case of picrosirius red staining, polarized light microscopy was performed to determine collagen fiber orientation. Samples were fixed in glutaraldehyde and postfixed with osmium tetraoxide for electron microscopy to determine collagen fibril orientation and evidence of fibril degradation. Additional injections were done at a dose of 0.58 mg/injection (250 ul of 2.3 mg/ml). These ex-vivo studies have shown the efficacy of purified collagenase in softening and partial liquefaction of post-hysterectomy fibroid specimens, as well as a decrease in the collagen content. Treated fibroid-specimens were grossly softer and had partially liquefied centers. Masson trichrome and picrosirius red stains of theses tissues showed a dramatic subjective decrease in collagen content compared to fibroid tissue injected with vehicle. Example 6. Treatment of Whole Uterine Fibroids Ex Vivo Donated tissue was obtained from four female adult patients 18 years of age or older who can give legally effective consent and who were planning to undergo definitive treatment for fibroids by hysterectomy. After the removal of the hysterectomy specimen, the uterus was observed grossly by standard procedures by a surgical pathologist. Complete fibroids (submucosal (abutting the endometrium), intramural (within the myometrium), and subserosal (abutting the uterine serosa) fibroids, or pedunculated fibroids (attached to the uterus by a stalk) if they are present) from 1 to 4 cm (including the capsule) along with 1.5 cm of the surrounding adjacent myometrium and, if available, a 0.5 cm section of endometrium were dissected free from the specimen and placed in normal saline. Tissues were brought to the laboratory immediately, washed and injected with purifiedClostridium histolyticumcollagenase (PCHC) (0.1 mg/100 μl/cm3). Optionally, a higher concentration of the collagenase was used to decrease the volume of the injection. Purified collagenase was diluted in 0.3 mg/mL calcium chloride dihydrate in 0.9% sodium chloride, optionally combined with 1% methylene blue as a marker to visually assess the area of distribution of the injected material within the fibroid and uterus. Fibroids were injected with PCHC or vehicle in the center of the obtained specimen. SeeFIGS.4A and4B. The amount of collagenase injected depended on the size of the fibroids (1-4 cm). Generally, about 818 μL of material was injected into a fibroid with a diameter of about 2.5 cm. If injecting the entire treatment volume centrally was not feasible due to tissue resistance to the injection or other factors, multiple locations were injected within the fibroid. The fibroid tissue then was incubated in DMEM/F12 culture medium at 37° C. for 24 hours. At least one fibroid with attached myometrium served as the control. This specimen received an injection of 1% methylene blue in vehicle without collagenase as a non-randomized placebo injection, centrally into the fibroid. Color photographs were taken of the uterus and of the fibroid and myometrial pieces pre- and post-injection. Fibroid diameters were measured with a metric ruler. At the end of the incubation, the samples were reassessed grossly for size, consistency and firmness, and color photographs were obtained, as well as optional video recording to record fibroid manual distensibility and any liquefied portions upon sectioning. The degree of liquefaction and softening were observed and documented using a 4-point subjective scale. Whether the collagenase can penetrate the capsule and affect the nearby myometrium was determined. Samples were obtained, including tissue from the injected fibroid and adjacent tissue, plus a section that included fibroid and adjacent myometrium and/or endometrium still attached, and myometrium alone. Samples were fixed in formalin for histology and Masson trichrome, picrosirius red, and hematoxylin-eosin staining. The samples were analyzed by light microscopy for the presence or absence of collagen and using computer morphometry to assess the extent of degradation. Picrosirius red staining was used with polarized light microscopy to determine collagen fiber orientation. Exemplary treatment schemes for each patient: fibroid 1: inject 818 μL 1 mg/mL collagenase; fibroid 2: inject 818 μL 1 mg/mL collagenase; fibroid 3: inject 818 μL control vehicle; Injections were given through the fibroid capsule into the center of the fibroid, through the myometrium into the center of the fibroid, or through the endometrium into the center of the fibroid, simulating in vivo injection routes. The fibroids here were liquefied in the same manner as shown inFIG.5(see below). Example 7. Biomechanical Evaluation of Human Uterine Fibroids after Injection with Purified Clostridial Collagenase The two collagenases isolated fromClostridium histolyticum(ABC I and ABC II) were combined in a 1:1 mass ratio. Both collagenases are metalloproteases and have a broad hydrolyzing reactivity and degrade type I and III collagens. The biomechanical properties of uterine fibroid tissue were analyzed by rheometry in control and collagenase-treated specimens. Uterine fibroids have been shown to contain about 70% Type I collagen compared to about 80% in myometrium; about 28% Type III collagen compared to about 20% in myometrium; and about 5% Type V collagen compared to about 2% in myometrium. Type I/III is lower at the center and the edge of fibroids as compared to myometrium. (Feng et al, Fibroid tissue was obtained after surgery (hysterectomy or myomectomy) from 4 different patients and cut into cubes (1 cm3; n=43). Tissue cubes were injected into the center with 100 μL of purified collagenase (0, 0.25, 0.5, 1.0, 2.0 mg/mL; n=4-14 per dose) and incubated at 37° C. for 24, 48, or 96 hours. At the end of the incubation period, cubes were cut in half and snap-frozen in liquid nitrogen. Different degrees of softening and liquefaction at the center were noted. An AR-G2 rheometer was used to measure the sample stiffness dynamically (complex shear modulus (Pa) at 10 rad/sec), taking into account both the viscous and elastic behavior of the material. At least 2 specimens (5 mm diameter punch) from each tissue cube were measured. Data were analyzed by 2-way ANOVA and Dunnett's multiple comparisons test. Overall, stiffness in control fibroid cubes (6585±707 Pa; n=13) was greater than in treated cubes (2003±275 Pa; n=30; p<0.0001). More specifically, stiffness in fibroid tissues was reduced in a time and dose dependent manner. At 48 hours, treatment with 0.25 mg/mL did not reduce stiffness (5032±1796 Pa), but treatment with 0.5 mg/mL did (2014±1331 Pa; p 0.05). At 96 hours, both the 0.25 and the 0.5 doses were effective (1720±377 and 1072±160 Pa; p 0.01). The 1.0 and 2.0 mg/mL treatments reduced stiffness at 24 hours, but not significantly (2177±37 and 2480±984 Pa; n=4). However, doses of 1.0 and 2.0 mg/mL were effective at 48 hours (3588±637; p 0.05 and 1254±445 Pa; p 0.01; n=6) and at 96 hours (921±305 and 1350±571 Pa; p 0.0001; n=10). Using a torsional rheometer, tissue stiffness was quantitated over a wide range (very firm to liquefied). Our data indicate that treatment of the fibroid tissue with defined doses of purified clostridial collagenase significantly decreased the stiffness (modulus) of the tissue. SeeFIG.5, which shows collagenolysis in fibroid tissue after 48 hour incubation. The left photograph is tissue that was injected with vehicle (control) and the right photograph is tissue that was injected with collagenase.FIG.6shows micrographs of control (FIGS.6A and6B) and collagenase-treated (FIGS.6C and6D) tissue. Mason stain in Figures A and C (left) shows that collagen is decreased. Picrosirus red stain visualized under polarized light (FIG.6D) clearly shows in the bottom right that collagen fibers are degraded. Example 8. Treatment of Human Uterine Fibroids in Nude Mouse Model The xenograft mouse model, in which three-dimensional organotypic cultures of human uterine fibroid cells are implanted under the skin of female nude mice, has been successfully employed to study keloids, a fibrotic skin disorder with biology similar to fibroids. This model is used to demonstrate effects of PCHC injection, in an HPG nanocarrier formulation, on fibroid tissue in vivo. Polylactic acid sponges, other synthetic polylactic acid scaffolds, or any suitable commercially available scaffold is inoculated with human uterine fibroid cells to produce an organotypic 3-D culture of uterine fibroid cells that can be implanted into nude mice. These 3-dimensional organotypic cultures (3D-fibroids) are representative of human fibroids and produce and contain extracellular matrix. OPLA sponges (Open-Cell Polylactic Acid, BD Biosciences;FIG.7) are synthetic polymer scaffolds that are synthesized from D,D-L,L polylactic acid. This material has a facetted architecture which is effective for culturing high density cell suspensions. The cells will be seeded onto the 3D sponge-like scaffolds under dynamic conditions, leading to uniform cell population throughout the sponges and higher cell numbers per sponge than static seeding. Post-sterilization, the molecular weight of the OPLA is 100-135 kD. They have an approximate size of 5 mm×3 mm (0.04 cm3) with an average pore size of 100-200 μm. Cells and scaffolds are placed into cell culture chambers of a bioreactor consisting of a fluid (culture media)-filled, rotating chamber that allows for constant floating of cells while minimizing shearing forces and gravitational settlement of cells and/or scaffolds (Synthecon, Inc.). Cells inside the rotating bioreactor chamber are suspended in virtual weightlessness. Primary human fibroid cells from specimens obtained at hysterectomy are seeded statically or dynamically into OPLA sponges and grown for 30 days to allow for production and assembly of extracellular matrix. Cells grow throughout the scaffold and can be formalin fixed, paraffin embedded and thin sectioned for observation, optionally with staining for multiple markers. SeeFIG.8, which shows the formation of the cell lattice following the outlines of the sponge-like scaffold. FIG.9shows primary cultures of fibroid cells after static seeding. The cells are fixed on the scaffold and observed in situ. Scaffolds containing cells were fixed and were unstained (FIG.9A) or stained for f-actin with fluorescent phalloidin (FIG.9B). Cells were evenly distributed throughout the scaffold. The imaged scaffolds are >1 mm thick and therefore not all cells are in focus, indicating that the cells are growing not only on the surface, but also deep inside the scaffolds.FIG.10shows the population of cells throughout the sponge-like scaffolds using confocal microscopy (FIGS.10A and10B). High quality RNA is extracted from the 3D-cultures of fibroid cells on OPLA sponges and used to verify the expression of two genes of interest. Versican and TGFβ3 are known to be highly expressed in fibroid tissue and cells. Results in Table 6 show that both a fibroid cell line and primary cultures of fibroid cells in this 3D-culture system express these two genes in high amounts. TABLE 6Real Time PCR Assay ResultsCDNA (ng)Threshold Cycle Ct (mean ± SEM)per reactionVersicanTGFβ3Fibroid5022.1 ± 0.0726.8 ± 0.07CellLinePrimary2522.2 ± 0.2124.0 ± 0.04FibroidCells The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference in their entireties for all purposes. All published foreign patents and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference in their entireties for all purposes. 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11857686 | DETAILED DESCRIPTION The embodiment of the UV lamp module1ofFIG.1has a metallic housing2with a bottom side3, an outwardly curved top side4, two flat side walls5and two closing walls6opposite each other at the ends. The housing length is about 1,050 mm, the housing height about 300 mm and the maximum housing width on the bottom side3is about 160 mm. The largest part of the bottom side3is taken up by a rectangular opening, which is sealed in a waterproof manner by a beam exit window7in the form of a fused silica plate with dimensions of 856×142 mm. The curvature of the top side4has a radius of about 90 mm and extends over the entire housing length from one closing wall6to the other. The two flat side walls5extend from the bottom side3to the top side4and fit closely with the curvature thereof. They extend towards each other at an oblique angle, forming an angle of 14 degrees with each other in their imaginary elongation. The side walls5and the curved top side4are made from a piece of sheet metal. On the visible side of one side wall5, lettering8has been engraved by laser and then the side wall5has been completely electropolished. A lower connector9of an air supply duct (FIG.3; reference number31) for the supply of cooling air into the housing2and a further, upper, connector10of an exhaust air duct (FIG.3; reference number32) for the discharge of heated cooling air from the housing2protrude from one closing wall6. From the same closing wall6a cable is also fed out for the electrical connection of four UV lamps21, which are visible from the view ofFIG.2. The UV lamps21are low-pressure mercury lamps with a cylindrical lamp tube composed of fused silica and electrodes arranged opposite each other therein. The lamp tube has an outer diameter of 28 mm and is sealed in a gas-tight manner at both ends by a pinch seal, through which the power connections for the electrical bonding of the electrodes are fed in a conventional manner. The lamp tube is filled with a mercury amalgam and neon; each lamp tube has an amalgam deposit. The UV lamps21form a planar lamp arrangement20, in which the longitudinal axes of the lamps21extend parallel to one another and in a common plane (FIG.3; lamp plane E2). The lamp arrangement21extends evenly on both sides of a mirror plane M of the housing2(more readily visible inFIG.3). The distance between the longitudinal axes of the lamps21is 36 mm. The nominal connected load of the individual mercury vapor discharge lamps is 580 W. The radiant flux can be up to 150 W. The low-pressure mercury lamps21display an emission spectrum with high efficiency of the characteristic emission line at 254 nm. At a distance of 48 mm from the lamp plane E2(i.e., 20 mm below the bottom side3of the housing2), a UV irradiation intensity of 140 mW/cm2 is obtained. From the sectional illustration ofFIG.3, the vertical arrangement of the essential components of the UV lamp module1inside the housing2can readily be seen. The top side of the beam exit window7extends in the window plane E1and above it the planar arrangement20of the four UV lamps21, the longitudinal axes of which span the lamp plane E2. The distance between the planes E1and E2is 21 mm. The arrangement20of the UV lamps21is surrounded at the top and sides by a reflector sheet33with a trapeziform profile. Above them extends the air supply duct31for the cooling air, the central axis of which defines the horizontal plane E3. And above this extends the exhaust air duct32, which is configured only as a short connector with a length of 2 cm and the central axis of which defines the horizontal plane E4. The housing2is substantially symmetrical relative to the mirror plane M. The beam exit window7has a sheet thickness of 4 mm; it rests against a folded edge of the side walls5and is adhesively bonded thereto in a waterproof manner. The reflector sheet33extends over the entire length of the UV lamp arrangement20. The air supply duct31has an inner diameter of 85 mm. Its central axis lies in the mirror plane M. It extends along the housing length from the lower connector9to a gas distribution chamber41arranged in the middle of the housing2(FIG.4). The exhaust air duct32also has an inner diameter of 85 mm, and its central axis is likewise in the mirror plane M. It can be seen that the exhaust air duct32defines the curvature of the housing top side4and almost completely fills it. It can be seen from the view ofFIG.4that the air supply duct31leads into the gas distribution chamber41. The gas distribution chamber41is provided on its side facing the reflector sheet33with a plurality of openings43, through which the cooling air flows into the space in which the arrangement20of the UV lamps21is located. At the openings43of the gas distribution chamber41a first airflow zone terminates, which determines the airflow of the cold cooling air from the air supply duct lower connector9to the lamp arrangement20. The airflow of the heated cooling air to the exhaust air duct upper connector10is defined by a second airflow zone. Here, the heated cooling air volume, starting from the lamp arrangement20, is supplied by way of the free internal space44of the housing2to the end of the exhaust air duct32that protrudes into the housing2, and is removed completely from the housing2by way of the exhaust air duct upper connector10. No mixing with cold cooling air takes place here, since the second airflow zone is in particular fluidically separated from the first airflow zone. The cooling performance of the cooling air is designed such that a maximum temperature of less than 110° C. is obtained on the lamp arrangement20. And to achieve a distribution of the UV irradiation profile that is locally as homogeneous as possible, the cooling performance and the local distribution of the cooling air are designed such that a temperature difference of less than 10° C. is obtained between the maximum temperature and the minimum temperature at the mercury deposits of the individual low-pressure mercury lamps21of the lamp arrangement20. For the simple maintenance and replacement of the low-pressure mercury lamps21, the UV lamp module1can be opened in the manner of a drawer. In this case the metal housing2, including one of the two end closing walls6and the beam exit window7, remains firmly in place. It is the opposite end closing wall6provided with a connection cable11that is pulled out, with the mechanically connected components such as the low-pressure mercury lamps21, the gas distribution chamber41and the air supply duct31. The end of the “drawer” protruding into the housing2is provided with an electrical plug, which joins to a corresponding socket in a mount42when pushed back in to form an electrical plug connection. The lamp module1according to the invention also meets strict requirements of the above-mentioned hygiene standards and achieves the degree of sealing according to IP66. When structurally identical UV lamp modules1are arranged in a closely juxtaposed series (theoretically without a gap; although in practice a small gap is useful so that liquid can drain), as illustrated schematically inFIG.5, a clearance of only 13.7 mm is obtained between the UV lamps21of adjacent modules1and a distance between the central axes of 55.4 mm exists. The UV lamp module1according to the invention is therefore particularly suitable for use in a disinfection system for the ultraviolet irradiation of a packaging material51for food or medicines. In this case, multiple UV lamp modules1are arranged one after the other in a direction of transport52of the packaging material51to be irradiated, in such a way that the central axes of the low-pressure mercury lamps21extend parallel to one another and transverse to the direction of transport52. Although illustrated and described above with reference to certain specific embodiments and examples, the present disclosure is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the disclosure. | 8,148 |
11857687 | Persons skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and may not have been drawn to scale. For example, the dimensions of some of the elements in the figure may be exaggerated relative to other elements to help to improve understanding of various exemplary embodiments of the present disclosure. Throughout the drawings, it should be noted that like reference numbers are used to depict the same or similar elements, features, and structures. DETAILED DESCRIPTION OF DRAWINGS For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting. Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof. Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment. The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or subsystems or elements or structures or components proceeded by “comprises . . . a” does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components. Reference is made herein to some “embodiments.” It should be understood that an embodiment is an example of a possible implementation of any features and/or elements presented in the attached claims. Some embodiments have been described for the purpose of illuminating one or more of the potential ways in which the specific features and/or elements of the attached claims fulfil the requirements of uniqueness, utility and non-obviousness. Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. FIG.1is a perspective view of an apparatus for sanitising products. According to an aspect of the present invention, an apparatus (10) for sanitising products (12) is disclosed. In some embodiments, the apparatus (10) comprises a housing (14) comprising a bottom portion (14b), interior walls, an external portion and doors. In some embodiments, the apparatus (10) further comprises a pair of support beams. In some embodiments, the apparatus (10) further comprises a plurality of internal conveyor belts (3,4,5). In some embodiments, the apparatus (10) further comprises at least one UV-C light (1,2) connected inside the housing. In some embodiments, the apparatus (10) further comprises a first opening adapted to allow products (12) to enter the housing (14) for sanitisation. The first opening is configured with a first disinfection spray nozzle (not shown). In some embodiments, the apparatus (10) further comprises a second opening adapted to allow products to exit the housing (14) after sanitisation. The second opening is configured with a second disinfection spray nozzle (not shown). According to an embodiment of the present invention, the housing (14) may comprise the bottom portion (14b), the top portion (14t) and side portions. The housing (14) forms a closed body with two opening on a front portion and a read portion to allow entry and exit of the products before sanitisation and after sanitisation, respectively. The housing (14) may comprise the interior walls configured with UV-C lights (1,2) connected on the top portion and bottom portions of the interior walls. The UV-C lights (1,2) are focused on the plurality of conveyor belts (3,4,5) carrying the products (12). According to an embodiment of the present invention, the apparatus (10) further comprises the pair of support beams. When the apparatus (10) is used on a conveyor belt of a grocery store, the apparatus (10) will sit on support beams. The support beams will be placed horizontally across the existing conveyor belt of the grocery store or alike. The support beam will allow the apparatus (10) to sit above the existing conveyer belt as to not interfere with it. Thus, the apparatus (10) can be made portable such that it can be placed on any kind of conveyor belts in any market. In some embodiments, the apparatus is raised by an inch above the existing conveyor belt in order to not interfere with it. The apparatus (10) is raised by means of horizontal supports. According to an embodiment of the present invention, the bottom portion (14b) is adapted to be attached to the support beams. The support beams carrying the housing (4) are placed on an external conveyor belt. According to an embodiment of the present invention, the apparatus (10) further comprises a plurality of internal conveyor belts (3,4,5). The apparatus (10) does not require a separate conveyor belt. The apparatus (10) is provided with an internal conveyor belt system (3,4,5). In some embodiments, the plurality of internal belts (3,4,5) move at a constant speed so that the products are inside the apparatus for 16 seconds for neutralizing all pathogens. In some embodiments, the plurality of internal belts (3,4,5) is arranged apart each other to allow seamless transfer of products between the plurality of internal belts. In some embodiments, the plurality of internal belts (3,4,5) may comprise of an entry and exit conveyor belts that run at the same speed as the middle one. In some embodiments, the apparatus (12) operates using three conveyor belts (3,4,5) all of which operate independently of each other. The first conveyor belt is angled and allows products (12) to be brought up to the second conveyor belt. The second conveyor belt is made from polycarbonate and is clear, this allows the conveyor belt to remain durable and allows UV-C lights (1,2) to sanitize the bottom of products (12). The last conveyor belt is angled downward and allows groceries or products (12) to exit the apparatus (10). The conveyor belts (3,4,5) will be at least a centimeter apart to ensure a seamless transfer between belts (3,4,5). In some embodiments, the apparatus (10) further comprises at least one UV-C light (1,2) connected inside the housing. At least one effect can be that the apparatus (10) utilizes UV-C lights (1,2) to eliminate pathogens, germs, and microbes on grocery items. In some embodiments, once groceries enter the apparatus (10), they will be sanitized by means of UV-C lights (1,2) placed in the top portion (14t) and bottom portion (14b) and the products (12) will come out from the other side. This apparatus (12) is meant to sanitize the outer packaging of a product. According to an embodiment of the present invention, the apparatus (10) utilizes UV-C lights (1,2) to sanitize the products (12). In some embodiments, the apparatus (10) is provided with at least three UV-C light lamps installed on the ceiling (14t) of the apparatus (10), i.e., directly above the second conveyor belt. In some embodiments, the apparatus (10) is provided with at least two UV-C lights posted under the second conveyor belt. The conveyor belts (3,4,5) will move at a constant speed in order to ensure that the products (12) are inside the apparatus (10) for at least 16 seconds. At least one effect of this feature can be that all pathogens on the products (1) are neutralized. According to an embodiment of the present invention, the apparatus (10) further comprises a first opening adapted to allow products (12) to enter the housing (14) for sanitisation. The first opening is configured with a first disinfection spray nozzle (not shown). The effect of the spray nozzle is that the products (12), before entering the apparatus (10), are first sanitized with a disinfection liquid so that the neutralisation of the germs is made for effective. According to an embodiment of the present invention, the apparatus (10) further comprises a second opening adapted to allow products to exit the housing (14) after sanitisation. The second opening is configured with a second disinfection spray nozzle (not shown). The effect of the spray nozzle is that the products (12), exiting the apparatus (10) after UV-C sanitisation, are again sanitized with disinfection so that the neutralisation of the germs is optimised. FIG.2is a perspective view of exit and entry portions of an apparatus for sanitising products. In some embodiments, the interior walls comprise reflective strips to ensure that UV-C light can reach all angles and cavities of products. Thus, at least one effect of the reflective strips posted on the interior walls of the apparatus can be that UV-C light can reach all angles and cavities of any product that enters the apparatus. According to an embodiment of the present invention, the first opening and the second opening comprise black strips (19) adapted to block users from the vicinity of the UV-C lights and to prevent escape of the UV-C lights. In some embodiments, the external portion comprises angled covers (20) adapted to block UV-C lights that pass through the rubber strips (19). Thus, at least one effect of this feature can be that the safety of everyone around the apparatus is ensured. In particular, at the entrance and exit of the apparatus there are black rubber strips that block and UV-C light from escaping. Further, the usage of angled covers ensures the blockage of any light that makes it past the rubber strips. FIG.3is a perspective view of a remote-control operation for an apparatus for sanitising products. According to an embodiment of the present invention, the external portion comprising at least one switch (16) for controlling lights. In some embodiments, the external portion further comprises switches (17) for moving the internal conveyor belts. In some embodiments, the external portion further comprises switches (18) for opening/closing the doors. According to an embodiment of the present invention, the at least one light (16), switches (17) for moving the internal conveyor belts and switches (18) for opening/closing the doors are operated via network. In some embodiments, the network may be formed to provide an effect of remote control. In some embodiments, the network may be formed to provide an effect of automatic control. Moreover, the actions of any components in the block diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims. Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims. While specific language has been used to describe the present subject matter, any limitations arising on account thereto, are not intended. As would be apparent to a person in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein. The drawings and the foregoing description give examples of embodiments. | 13,080 |
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